Urinalysis and Body Fluids [7 ed.] 0803675828, 9780803675827

Table of contents :
Cover
Title Page
Copyright Page
Dedication
Preface
Contributor and Special Content Reviewer
Reviewers
Acknowledgments
Contents
Atlas of Images
Part One: Basic Principles
Chapter 1: Safety and Quality Management
Safety
Biological Hazards
Infectious Agent (Pathogen)
Reservoir
Portal of Exit
Means of Transmission
Portal of Entry
Susceptible Host
Standard Precautions
Sharp Hazards
Chemical Hazards
Chemical Spills and Exposure
Chemical Handling
Chemical Hygiene Plan
Chemical Labeling
Safety Data Sheets
The Globally Harmonized System of Classification and Labeling of Chemicals
Chemical Waste Disposal
Radioactive Hazards
Electrical Hazards
Fire/Explosive Hazards
Physical Hazards
Quality Management
Urinalysis Procedure Manual
Preexamination (Preanalytical) Variables
Examination (Analytical) Variables
Postexamination Variables
Chapter 2: Urine and Body Fluid Analysis Automation
Urinalysis Automation
Reflectance Photometry
Analyzers
Fully Automated Urine Chemistry Analyzers
Automated Microscopy Analyzers
Automated Urinalysis Systems
Body Fluid Analysis Automation
Chapter 3: Introduction to Urinalysis
History and Importance
Urine Formation
Urine Composition
Urine Volume
Specimen Collection
Containers
Labels
Requisition Form
Specimen Rejection
Specimen Handling
Specimen Integrity
Specimen Preservation
Types of Specimens
Random Specimen
First Morning Specimen
24-Hour (or Timed) Specimen
Catheterized Specimen
Midstream Clean-Catch Specimen
Suprapubic Aspiration
Prostatitis Specimen
Pediatric Specimens
Drug Specimen Collection
Chapter 4: Renal Function
Introduction
Renal Physiology
Renal Blood Flow
Glomerular Filtration
Tubular Reabsorption
Tubular Secretion
Renal Function Tests
Glomerular Filtration Tests
Tubular Reabsorption Tests
Tubular Secretion and Renal Blood Flow Tests
Part Two: Urinalysis
Chapter 5: Physical Examination of Urine
Introduction
Color
Normal Urine Color
Abnormal Urine Color
Clarity
Normal Clarity
Nonpathological Turbidity
Pathological Turbidity
Specific Gravity
Refractometer
Osmolality
Reagent Strip Specific Gravity
Odor
Chapter 6: Chemical Examination of Urine
Introduction
Reagent Strips
Reagent Strip Technique
Handling and Storing Reagent Strips
Quality Control of Reagent Strips
Confirmatory Testing
pH
Clinical Significance
Reagent Strip Reactions
Protein
Clinical Significance
Prerenal Proteinuria
Renal Proteinuria
Postrenal Proteinuria
Reagent Strip Reactions
Reaction Interference
Glucose
Clinical Significance
Reagent Strip (Glucose Oxidase) Reaction
Reaction Interference
Copper Reduction Test (Clinitest)
Clinical Significance of Clinitest
Ketones
Clinical Significance
Reagent Strip Reactions
Reaction Interference
Blood
Clinical Significance
Reagent Strip Reactions
Reaction Interference
Bilirubin
Bilirubin Production
Clinical Significance
Reagent Strip (Diazo) Reactions
Reaction Interference
Urobilinogen
Clinical Significance
Reagent Strip Reactions and Interference
Reaction Interference
Nitrite
Clinical Significance
Reagent Strip Reactions
Reaction Interference
Leukocyte Esterase
Clinical Significance
Reagent Strip Reaction
Reaction Interference
Specific Gravity
Reagent Strip Reaction
Reaction Interference
Chapter 7: Microscopic Examination of Urine
Introduction
Macroscopic Screening
Specimen Preparation
Specimen Volume
Centrifugation
Sediment Preparation
Volume of Sediment Examined
Commercial Systems
Examining the Sediment
Reporting the Microscopic Examination
Correlating Results
Sediment Examination Techniques
Sediment Stains
Cytodiagnostic Urine Testing
Microscopy
Types of Microscopy
Urine Sediment Constituents
Red Blood Cells
White Blood Cells
Epithelial Cells
Bacteria
Yeast
Parasites
Spermatozoa
Mucus
Casts
Urinary Crystals
Urinary Sediment Artifacts
Chapter 8: Renal Disease
Introduction
Glomerular Disorders
Glomerulonephritis
Nephrotic Syndrome
Tubular Disorders
Acute Tubular Necrosis
Hereditary and Metabolic Tubular Disorders
Interstitial Disorders
Urinary Tract Infection
Acute Pyelonephritis
Chronic Pyelonephritis
Acute Interstitial Nephritis
Renal Failure
Renal Lithiasis
Chapter 9: Urine Screening for Metabolic Disorders
Introduction
Overflow Versus Renal Disorders
Newborn Screening Tests
Amino Acid Disorders
Phenylalanine-Tyrosine Disorders
Branched-Chain Amino Acid Disorders
Tryptophan Disorders
Cystine Disorders
Porphyrin Disorders
Mucopolysaccharide Disorders
Purine Disorders
Carbohydrate Disorders
Part Three: Other Body Fluids
Chapter 10: Cerebrospinal Fluid
Introduction
Formation and Physiology
Specimen Collection and Handling
Appearance
Traumatic Collection (Tap)
Uneven Blood Distribution
Clot Formation
Xanthochromic Supernatant
Cell Count
Methodology
Total Cell Count
WBC Count
Quality Control of CSF and Other Body Fluid Cell Counts
Differential Count on a CSF Specimen
Cytocentrifugation
CSF Cellular Constituents
Chemistry Tests
Cerebrospinal Protein
CSF Glucose
CSF Lactate
CSF Glutamine
Microbiology Tests
Gram Stain
Immunologic Assays
PCR Molecular Diagnostic Testing
Serological Testing
Chapter 11: Semen 277
Introduction
Physiology
Specimen Collection
Specimen Handling
Semen Analysis
Appearance
Liquefaction
Volume
Viscosity
pH
Sperm Concentration and Sperm Count
Sperm Motility
Automated Semen Analysis Systems
Sperm Morphology
Additional Testing
Sperm Vitality
Seminal Fluid Fructose
Antisperm Antibodies
Microbial and Chemical Testing
Postvasectomy Semen Analysis
Sperm Function Tests
Semen Analysis Quality Control
Chapter 12: Synovial Fluid
Physiology
Specimen Collection and Handling
Color and Clarity
Viscosity
Cell Counts
Differential Count
Crystal Identification
Types of Crystals
Slide Preparation
Crystal Polarization
Chemistry Tests
Glucose
Total Protein
Uric Acid
Lactate
Enzymes
Microbiological Tests
Serological Tests
Chapter 13: Serous Fluid
Introduction
Formation
Specimen Collection and Handling
Transudates and Exudates
General Laboratory Procedures
Pleural Fluid
Appearance
Hematology Tests
Chemistry Tests
Microbiological and Serological Tests
Pericardial Fluid
Appearance
Laboratory Tests
Peritoneal Fluid
Transudates Versus Exudates
Appearance
Laboratory Tests
Chapter 14: Bronchoalveolar Lavage Fluid
Clinical Significance
Specimen Collection
Specimen Handling and Transport
Color and Clarity
Cell Counts
Leukocytes
Macrophages
Lymphocytes
Neutrophils
Eosinophils
Mast Cells
Erythrocytes
Epithelial Cells
Microbiological Tests
Cytology
Chapter 15: Amniotic Fluid
Introduction
Physiology
Function
Volume
Chemical Composition
Differentiating Maternal Urine From Amniotic Fluid
Specimen Collection
Indications for Amniocentesis
Collection
Specimen Handling and Processing
Color and Appearance
Tests for Fetal Distress
Hemolytic Disease of the Fetus and Newborn
Neural Tube Defects
Tests for Fetal Maturity
Fetal Lung Maturity
Lecithin-Sphingomyelin Ratio
Phosphatidyl Glycerol
Foam Stability Index
Lamellar Bodies
Chapter 16: Fecal Analysis
Introduction
Physiology
Diarrhea and Steatorrhea
Diarrhea
Steatorrhea
Specimen Collection
Macroscopic Screening
Color
Appearance
Microscopic Examination of Feces
Fecal Leukocytes
Muscle Fibers
Qualitative Fecal Fats
Chemical Testing of Feces
Occult Blood
Quantitative Fecal Fat Testing
APT Test (Fetal Hemoglobin)
Fecal Enzymes
Carbohydrates
Chapter 17: Vaginal Secretions
Introduction
Specimen Collection and Handling
Color and Appearance
Diagnostic Tests
pH
Microscopic Procedures
Other Diagnostic Tests
Vaginal Disorders
Bacterial Vaginosis
Trichomoniasis
Candidiasis
Desquamative Inflammatory Vaginitis
Atrophic Vaginitis
Additional Procedures for Vaginal Secretion Associated With Pregnancy
Fetal Fibronectin Test
AmniSure Test
Actim PROM
ROM Plus
Answers to Study Questions, Case Studies, and Clinical Situations
Abbreviations
Glossary
Index

Citation preview

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Urinalysis and Body Fluids SEVENTH EDITION

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Urinalysis and Body Fluids SEVENTH EDITION Susan King Strasinger, DA, MT(ASCP) Retired Faculty Associate Clinical Laboratory Sciences Program The University of West Florida Pensacola, Florida

Marjorie Schaub Di Lorenzo, BS, MT(ASCP)SH Phlebotomy Technician Program Coordinator Nebraska Methodist College Omaha, Nebraska

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F. A. Davis Company 1915 Arch Street Philadelphia, PA 19103 www.fadavis.com Copyright © 2021 by F. A. Davis Company Copyright © 2021 by F. A. Davis Company. All rights reserved. This product is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America Last digit indicates print number: 10 9 8 7 6 5 4 3 2 1 Publisher: Christa A. Fratantoro Manager of Content Development: George W. Lang Developmental Editor: Stephanie Kelly Content Project Manager: Megan Suermann Art and Design Manager: Carolyn O’Brien

As new scientific information becomes available through basic and clinical research, recommended treatments and drug therapies undergo changes. The author(s) and publisher have done everything possible to make this book accurate, up to date, and in accord with accepted standards at the time of publication. The author(s), editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of the book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised always to check product information (package inserts) for changes and new information regarding dose and contraindications before administering any drug. Caution is especially urged when using new or infrequently ordered drugs. Library of Congress Cataloging-in-Publication Data Names: Strasinger, Susan King, author. | Di Lorenzo, Marjorie Schaub, 1953author. Title: Urinalysis and body fluids / Susan King Strasinger, Marjorie Schaub Di Lorenzo. Description: Seventh edition. | Philadelphia: F. A. Davis Company, [2021] | Includes bibliographical references and index. Identifiers: LCCN 2020022498 (print) | LCCN 2020022499 (ebook) | ISBN 9780803675827 (paperback) | ISBN 9780803675834 (ebook) Subjects: MESH: Urinalysis—methods | Body Fluids—chemistry Classification: LCC RB53 (print) | LCC RB53 (ebook) | NLM QY 185 | DDC 616.07/566—dc23 LC record available at https://lccn.loc.gov/2020022498 LC ebook record available at https://lccn.loc.gov/2020022499

Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by F. A. Davis Company for users registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that the fee of $.25 per copy is paid directly to CCC, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payment has been arranged. The fee code for users of the Transactional Reporting Service is: 978-0-80367582-7/20 0 + $.25.

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To Harry—you will always be my editor-in-chief —SKS To my husband, Scott, for his support and encouragement —MSD

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Preface As will be apparent to readers, the seventh edition of Urinalysis and Body Fluids has been revised and enhanced substantially. However, the objective of the text—to provide concise, comprehensive, and carefully structured instruction in the analysis of nonblood body fluids—remains the same. This seventh edition has been redesigned to meet the changes occurring in both laboratory medicine and instructional methodology. The book is organized into three parts: Basic Principles, Urinalysis, and Other Body Fluids. To meet the expanding technical information required by students in laboratory medicine, all of the chapters have been updated. Part One focuses on basic principles associated with urinalysis and body fluids. Chapter 1 covers overall laboratory safety, precautions relating to analysis of urine and body fluids, and the importance of quality assessment and management in the urinalysis laboratory. Preexamination (preanalytical), examination (analytical), and postexamination (postanalytical) variables; procedure manuals; and current regulatory issues are stressed. Chapter 2 is new and introduces the student to the most up-to-date automation tools for analysis of urine and body fluids. This chapter covers the ever-increasing variety of automated instrumentation available by different manufacturers for the analysis of urine and body fluids. The basic principles used in urine chemical automation, such as reflectance photometry, light scatter, fluorescence light, and refractometry, are explained. Included in this chapter are the significant advancements in the automated microscopic analysis of urine that cover the principles of flow cytometry and digital image analysis. Chapter 3 discusses the importance of urine specimen collections, handling, and preservation. Chapter 4 covers renal physiology and the various renal function tests. These chapters provide the background for discussing the three components of a complete urinalysis and associated diseases that are discussed in the next part. In Part Two, Chapter 5 covers the physical examination of urinalysis, whereas Chapter 6 describes the chemical examination and includes the chemical reaction principle, clinical significance, and reaction interference for each chemical test. Chapter 7 includes numerous additional images showing the various urine microscopic components. In Chapters 8 and 9, the diseases of glomerular, tubular, interstitial, vascular, and hereditary origin that are encountered most frequently are related to their associated laboratory tests. Part Three is dedicated to a discussion of other body fluids. To accommodate advances in laboratory testing of those fluids, including cerebrospinal, seminal, synovial, serous,

bronchoalveolar lavage, and amniotic fluids, Chapters 10 through 17 have been enhanced. Sections covering anatomy and physiology; specimen collection, handling, and processing; current testing methodology; and associated clinical significance have been added for each of these fluids. A new chapter (Chapter 14) discusses the collection, analysis, and significance of specimens for bronchoalveolar lavage. Each chapter opens with learning outcomes and key terms and concludes with multiple choice questions, case studies, and clinical situations for student review. In response to readers’ suggestions, the number of color images and figures have been increased significantly. The text has been supplemented extensively with tables, summaries, and procedure boxes. Case studies in the traditional format, as well as clinical situations relating to technical considerations included at the end of the chapters, offer students an opportunity to think critically about the material. A feature titled Historical Notes provides a reference for topics or tests that are no longer performed routinely. Another feature, Technical Tips, emphasizes information important to performing procedures. Procedures have been reformatted into step-by-step instructions. The F. A. Davis logo icon that appears in various chapters will direct the students to the procedural videos found at www.fadavis.com under the Instructor Resources. Reagent Strip Color Charts enhance Chapter 6, Chemical Examination of Urine, highlighting the timing, specificity, and sensitivity of each chemical reaction. New to this edition is a two-part, full-color Atlas of Images with images for urine and body fluid structures. Over 130 new images are included in the atlas for use as a reference in the identification of cellular components in both urine and other body fluids. Images showing real urine sediments with mixed field components can be used for identifying cells that are often misidentified and serve as an excellent teaching resource. The atlas provides a description; clinical significance; physical, chemical, and microscopic correlations; and tips for identification for student reference. Boxes and Tables summarize important information and correlations. An Answer Key for the study questions, case studies, and clinical situations is included at the end of the book. Key terms appear in boldface blue color within the chapters. General medical terms appear in boldface black in the text and also are included in the Glossary. The abbreviations noted in boldface red color throughout the book have been collected in a convenient Abbreviations list at the back of the book. The online Instructor Resources at www.fadavis.com include example lecture and laboratory schedules, procedures for student laboratory simulated

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Preface

specimens, class activities, chapter outlines, case studies and clinical situations with answers, study questions and answers, a test question bank, image bank, chapter-by-chapter PowerPoints, an electronic version of the textbook, the Instructor’s Guide, animations, and videos. For self-study, students will

have access to online review questions, animations, videos, interactive exercises, and robust case studies at www.fadavis.com. As always, we thank our readers for their valuable suggestions, which have guided us in creating this exciting new edition as well as the student and instructor resources.

Susan King Strasinger Marjorie Schaub Di Lorenzo

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Contributor and Special Content Reviewer Kathleen Finnegan, MS, MT(ASCP)SH Clinical Associate Professor Clinical Laboratory Sciences Stony Brook University Stony Brook, New York

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Reviewers Wayne Aguiar, MS, MLS(ASCP) Director of Health Sciences and Medical Laboratory Science College of Arts and Sciences University of Bridgeport Bridgeport, Connecticut

Linda Bau, MPA, MT(ASCP) MLT Program Director Allied Health Moraine Park Technical College Fond du Lac, Wisconsin

Annette E. Bednar, MSE, MT(ASCP) Laura Ahonen, MS, MT(ASCP) MLT Program Director Phlebotomy Program Director Northcentral Technical College Wausau, Wisconsin

Assistant Professor of Clinical Laboratory Sciences College of Nursing and Health Professions Arkansas State University Jonesboro, Arkansas

Angela R. Bell, MS, MLS(ASCP)SM, DLM Alison Albertson, MS, MT(ASCP) MLT Program Director Medical Laboratory Lake Area Technical Institute Watertown, South Dakota

Lois C. Anderson, MA, MT(ASCP) Associate Professor Minnesota State University, Mankato Mankato, Minnesota

Medical Laboratory Technology and Phlebotomy Program Director Health Professions Tidewater Community College Norfolk, Virginia

Jan Bradley, MT(ASCP) Program Director and Instructor Medical Laboratory Technology Wayne Community College Goldsboro, North Carolina

Beverly J. Barham, PhD, MPH, MT(ASCP) Professor Medical Laboratory Science Program Department of Health Sciences Illinois State University Normal, Illinois

Brenda C. Barnes, PhD, MT(ASCP)SBB Director, Medical Laboratory Science Program Director, EdD Health Professions Education Program Professor Allen College – UnityPoint Health Waterloo, Iowa

Crystal E. Barrett, MAED/T, MLS(ASCP)CM Adjunct Faculty Nursing and Allied Health J. Sargeant Reynolds Community College Richmond, Virginia

Nicholas Brehl, MLS(ASCP)CM Assistant Clinical Professor Pathology and Laboratory Medicine Indiana University School of Medicine Indianapolis, Indiana

Carol Brennan, BS, MT(ASCP) Clinical Instructor, Microbiology and Serology Medical Laboratory Science Program Methodist Hospital Pathology Omaha, Nebraska

Michelle Briski, MEd, MT(ASCP) MLT Program Director Saint Paul College Saint Paul, Minnesota

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Reviewers

Keri Brophy-Martinez, MT(ASCP) Department Chair/Medical Laboratory Technology Professor Medical Laboratory Technology Austin Community College Austin, Texas

Christy A. Cole, MT(ASCP) Instructor/Phlebotomy Advisor Clinical Laboratory Technology West Georgia Technical College Waco, Georgia

Janet E. Cooper, MSNS, BS, MT(ASCP)H Karen Brown, MS, MLS(ASCP)CM Professor Pathology University of Utah Salt Lake City, Utah

Program Director Medical Laboratory Technology Mississippi Delta Community College Moorhead, Mississippi

Rhonda Craver, MLS(ASCP) Renelle Brun, MLT Clinical Biochemistry Instructor Medical Laboratory Sciences CCNB Dieppe Campus Dieppe, New Brunswick Canada

Jennifer Bushnell, MEd, MLS(ASCP) MLS Program Director RMLS McNeese State University Lake Charles, Louisiana

Tracy Camara, MBA, MT(ASCP)SM Program Director, School of Medical Laboratory Science System Director, Laboratory Education and Performance Improvement Baptist Health – South Montgomery, Alabama

Leslie Cameron, MLT Program Instructor Medical Laboratory Technology Chemistry Nova Scotia Community College Halifax, Nova Scotia Canada

Katie Cavnar, MS, MT(ASCP) Chair Medical Laboratory Sciences University of West Florida Pensacola, Florida

Carol E. Church, MAT, MT(ASCP)SM Assistant Professor Clinical Laboratory Technologies SUNY Broome Community College Binghamton, New York

Director Montana Medical Laboratory Science Program Montana State University Bozeman, Montana

Diane Davis, PhD, MLS (ASCP)CM, SCCM, SLSCM Professor and Chair Medical Laboratory Science Program Salisbury University Salisbury, Maryland

Linda J. Dickson, MSPH, MT(ASCP) MLT Program Director Medical Clinical Laboratory Technician Erwin Technical College Tampa, Florida

Patricia Ellinger, MSEd, MLS(ASCP)CM, SBBCM MLS Faculty Medical Laboratory Science Program St. Cloud State University St. Cloud, Minnesota

Mary Emes, MMedSc (Pathology) Certificate in Adult Education, MLT, ART (CC, MI, TS) Professor Medical Laboratory Science and Continuing Education The Michener Institute of Education at University Health Network Toronto, Ontario Canada

Julie Foley, CLS, MT(ASCP) Education Coordinator CLS/CGMBS Training Programs California State University, Los Angeles Los Angeles, California

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Reviewers

Kristin A. Forsberg, MEd, MLS(ASCP)CM MLT Program Director MLT Program Laurel Technical Institute Hermitage, Pennsylvania

Karen Gatewood, MEd, MLS(ASCP)CM MLT Assistant Program Director MLT Program Harcum College Bryn Mawr, Pennsylvania

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Virginia Haynes, MS, MLS(ASCP)CM Program Director MLT Lake Superior College Duluth, Minnesota

Tamara Henderson, CLS(ASCP) Medical Laboratory Technology Pearl River Community College Poplarville, Mississippi

Candy Hill, MAEd, MT(ASCP) Lynette K. Gayle, MLS(ASCP), JD, MA Ed CLS Program Director/Assistant Professor Allied Health Tuskegee University Tuskegee, Alabama

Jeffrey S. Gillette, PhD, MT(ASCP) Program Director Professor, Medical Laboratory Technology Virginia Western Community College Roanoke, Virginia

Karen Gordon, MS, MLS(ASCP)CM, SLS Professor Emeritus Retired MLT Program Head/Assistant Dean Northern Virginia Community College – Medical Education Campus Springfield, Virginia

Cheri Goretti, MA, MT(ASCP), CMA(AAMA) Professor Emeritus Medical Assisting and Allied Health Quinebaug Valley Community College Danielson, Connecticut

Nancy Grandbois, MS, MLS(ASCP)CM Instructor Biomedical and Nutritional Sciences University of Massachusetts Lowell Lowell, Massachusetts

Tammy Hardie, BA, MLT, RT Instructor Medical Laboratory Sciences British Columbia Institute of Technology Burnaby, British Columbia Canada

MLT Program Coordinator Center for Professional, Career and Technical Education Jefferson State Community College Birmingham, Alabama

Elizabeth Hoffman, MA Ed, CMA(AAMA), CPT(ASPT) Medical Assistant Program Director Henry Ford College Dearborn, Michigan

Ruth Jacobs, MT(ASCP) Assistant Professor/Department Chair Allied Health BridgeValley Community and Technical College South Charleston, West Virginia

Haywood B. Joiner Jr., EdD, MLS(ASCP) Chair Department of Allied Health Louisiana State University of Alexandria Alexandria, Louisiana

Minh Do Kosfeld, PhD, MLT(ASCP)CM Investigative and Medical Sciences Program Director Assistant Professor Department of Clinical Health Sciences Saint Louis University St. Louis, Missouri

Heather Lai, MLS(ASCP)SH Clinical Instructor of Hematology, Coagulation, Urinalysis, and Body Fluids Unity Point Health – St. Luke’s Medical Laboratory Science Program Cedar Rapids, Iowa

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xiv Reviewers

Karlyn Lange, MT(ASCP) Program Director Mercy Integrated CLS Program Toledo, Ohio

Lori Moore, MEd, MT(ASCP) Program Director, Instructor School of Medical Technology Berkshire Medical Center Pittsfield, Massachusetts

Laura R. Link, MS, MLS(ASCP)CM Program Director and Assistant Professor Medical Laboratory Science Radford University Radford, Virginia

Janis P. Livingston, MT(ASCP) MLT Clinical Education Coordinator Medical Laboratory Technology Midlands Technical College Columbia, South Carolina

Patricia Longpre, MLT Professor Health Sciences Cambrian College of Applied Arts and Technology Sudbury, Ontario Canada

Deirdre Parsons, MS, MT(ASCP)SBB Program Director University of Maryland Baltimore, Maryland

Srinivas Pentyala, PhD Professor and Director of Translational Research of Anesthesiology Faculty Member in Urology, Health Sciences, Physiology and Biophysics Stony Brook Medical Center Stony Brook, New York

Lori Rager, CMA (AAMA), COC, CPC (AAPC) Lead MA Instructor Medical Assistant Utah State University Eastern Price, Utah

Nina Maniotis, MT(ASCP) Phlebotomy Program Director/Instructor Health and Human Sciences Weatherford College Weatherford, Texas

Mary Sadlowski, BS, MT Adjunct Teacher Health Department Community College of Baltimore County Baltimore, Maryland

Teresa Maxwell, BS, MT(ASCP) Technical Coordinator Methodist Hospital Pathology Omaha, Nebraska

Ann C. McConnell, MLS(ASCP)CM Chair and Program Director Clinical Associate Professor East Carolina University Greenville, North Carolina

Carol McCoy, PhD, MLS(ASCP)CM Adjunct Professor Oklahoma Christian University Oklahoma City, Oklahoma

Dana McGuire, MPH, BS, MLS(ASCP)CM Technical Coordinator Methodist Women’s Hospital Pathology Omaha, Nebraska

Salle Sappington, MLS(ASCP) Teacher Monroe Technology Center Loudoun County Schools Leesburg, Virginia

Rebbecca Silva, MS, MT(ASCP) Assistant Professor and Chair Medical Laboratory Technology New England Institute of Technology East Greenwich, Rhode Island

Rosalyn H. Singleton, MHS, MT(ASCP), BS Clinical Coordinator Medical Laboratory Technology Northeast Mississippi Community College Booneville, Mississippi

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Reviewers

C. Thomas Somma, EdD Associate Professor Clinical Laboratory Sciences Old Dominion University Norfolk, Virginia

Rose Marie Spangler, MPH, MLS(ASCP) Program Director/Assistant Professor Health-Related Professions Northeast State Community College Blountville, Tennessee

Donna Spannaus-Martin, PhD, MLS(ASCP)CM Professor Medical Laboratory Sciences University of Minnesota Minneapolis, Minnesota

Brian Spezialetti, MS, MT(ASCP) Program Director School of Medical Laboratory Sciences Guthrie Robert Packer Hospital Sayre, Pennsylvania

John Strous, MS, MLS(ASCP)CM Emeritus Program Director Medical Technology Program University of Wisconsin Oshkosh Oshkosh, Wisconsin

Harvey Suski, MLT Instructor Medical Laboratory Sciences Red River College Winnipeg, Manitoba Canada

MaryEllen Tancred, PhD, MLS(ASCP)CMSHCM NAACLS Program Director/Coordinator Associate Professor Allied Health – Medical Laboratory Technology Columbus State Community College Columbus, Ohio

Angela Tarrant, MEd, MT(ASCP)SMCM Program Director Medical Laboratory Technician Otero Junior College La Junta, Colorado

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Karlien Smith Tebbetts, MLS(ASCP) Faculty Medical Technology Houston Community College Houston, Texas

M. Lorraine Torres, EdD, MT(ASCP) Program Director Clinical Laboratory Sciences Program Director The University of Texas at El Paso El Paso, Texas

Maria Torres-Pillot, MA, MLS (ASCP)CM Program Director of Medical Laboratory Technology Health Sciences Argosy University – Dallas Farmers Branch, Texas

Misty D. Turner, MT(ASCP) Education Coordinator School of Clinical Laboratory Science Augusta Health Fishersville, Virginia

Meridee Van Draska, MSMHA, MLS (ASCP), AHI (AMT) Associate Professor Medical Laboratory Science Program Director Illinois State University Normal, Illinois

WenFang Wang, PhD, C(ASCP)CM Clinical Assistant Professor Department of Clinical Laboratory Sciences Kansas University Medical Center Kansas City, Kansas

Lanette Hall Washington, MSM, MLS(ASCP)CM, MT (AMT) MLT Program Director Education Fortis College – Baton Rouge Baton Rouge, Louisiana

Tiffany H. Whittle, MSHS, MLS(ASCP)CM MLT Associate Professor Medical Laboratory Technology Greenville Technical College Greenville, South Carolina

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xvi Reviewers

Marie L. Wilson, MT(ASCP) Laboratory Supervisor Daviess Community Hospital Washington, Indiana

Kathi Winker, MS, MT(ASCP) MLT Program Director Health and Public Safety Blackhawk Technical College Janesville, Wisconsin

Kimberly G. Yarborough, MLS(ASCP)CMSHCM Medical Laboratory Science Carolinas College of Health Sciences Charlotte, North Carolina

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Acknowledgments Many people deserve credit for the help and encouragement they have provided us in the preparation of this seventh edition. Our continued appreciation is also extended to all of the people who were instrumental in the preparation of previous editions. We want to especially thank Kathleen Finnegan of Stony Brook, New York, for her countless hours capturing urine and body fluid images for the Atlas of Images that is new to the seventh edition. We are forever indebted to her for her expertise, enthusiasm, and commitment in the development of this project. The valuable suggestions from previous readers and the support from our colleagues at Methodist Hospital, Omaha, Nebraska, have been a great asset to us in the production of this new edition. We thank each and every one of you. We extend special thanks to the people who have provided us with so many beautiful photographs for the text over the years: Donna L. Canterbury, BA, MT(ASCP)SH; Joanne M. Davis, BS, MT(ASCP)SH; M. Paula Neumann, MD; Gregory J. Swedo, MD; and Scott Di Lorenzo, DDS. We also remember and appreciate Sherman Bonomelli, MS, who had contributed original visual concepts that became the foundation for many of the line

illustrations, and the students from the University of West Florida who worked under the guidance of Sherman Bonomelli to produce many of those images. Images for Chapter 16 had been provided previously by Carol Brennan, MT(ASCP); Diane Siedlik, MT(ASCP); Christian Herdt, MT(ASCP); and Teresa Karre, MD, from Methodist Hospital. Images for the atlas were provided by Dawn Plank, MLS(ASCP)CM, Methodist Hospital, Omaha, Nebraska, and Kathleen Finnegan, MS, MT(ASCP)SH, Clinical Associate Professor, Stony Brook University, New York, as well as the Stony Brook University Clinical Laboratory Science 2020 senior students, who helped find the urinalysis elements that were photographed for the atlas. We would like to express our gratitude for the help, patience, guidance, and understanding of our editors at F. A. Davis: Christa Fratantoro, Publisher; Megan Suermann, Content Project Manager; and Stephanie Kelly, Developmental Editor. We thank all the members of the F. A. Davis team who were instrumental in bringing this edition to fruition: George Lang, Manager of Content Development; Carolyn O’Brien, Art and Design Manager; Sharon Lee, Production Manager; Kate Margeson, Illustrator Coordinator; and Julie Mangoff of Graphic World, Inc.

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Contents Automated Microscopy Analyzers 79 Automated Urinalysis Systems 86 Body Fluid Analysis Automation 87

Atlas of Images 1 PART ONE:

Basic Principles

48

CHAPTER 1

CHAPTER 3

Safety and Quality Management 49

Introduction to Urinalysis

Safety 50 Biological Hazards 50 Infectious Agent (Pathogen) 50 Reservoir 50 Portal of Exit 51 Means of Transmission 51 Portal of Entry 51 Susceptible Host 51 Standard Precautions 51 Sharp Hazards 56 Chemical Hazards 56 Chemical Spills and Exposure 56 Chemical Handling 57 Chemical Hygiene Plan 57 Chemical Labeling 57 Safety Data Sheets 57 The Globally Harmonized System of Classification and Labeling of Chemicals 58 Chemical Waste Disposal 58 Radioactive Hazards 58 Electrical Hazards 59 Fire/Explosive Hazards 60 Physical Hazards 61 Quality Management 61 Urinalysis Procedure Manual 62 Preexamination (Preanalytical) Variables 62 Examination (Analytical) Variables 64 Postexamination Variables 68 CHAPTER 2

Urine and Body Fluid Analysis Automation

75

Urinalysis Automation 76 Reflectance Photometry 76 Analyzers 76 Fully Automated Urine Chemistry Analyzers 78

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History and Importance 92 Urine Formation 93 Urine Composition 93 Urine Volume 93 Specimen Collection 94 Containers 94 Labels 95 Requisition Form 95 Specimen Rejection 95 Specimen Handling 95 Specimen Integrity 95 Specimen Preservation 96 Types of Specimens 96 Random Specimen 96 First Morning Specimen 96 24-Hour (or Timed) Specimen 97 Catheterized Specimen 98 Midstream Clean-Catch Specimen 98 Suprapubic Aspiration 98 Prostatitis Specimen 98 Pediatric Specimens 100 Drug Specimen Collection 100 CHAPTER 4

Renal Function 105 Introduction 106 Renal Physiology 106 Renal Blood Flow 106 Glomerular Filtration 106 Tubular Reabsorption 110 Tubular Secretion 111 Renal Function Tests 113 Glomerular Filtration Tests 113 Tubular Reabsorption Tests 116 Tubular Secretion and Renal Blood Flow Tests 119

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Contents

PART TWO: Urinalysis

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CHAPTER 5

Physical Examination of Urine

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Introduction 126 Color 126 Normal Urine Color 126 Abnormal Urine Color 127 Clarity 129 Normal Clarity 129 Nonpathological Turbidity 129 Pathological Turbidity 130 Specific Gravity 131 Refractometer 131 Osmolality 132 Reagent Strip Specific Gravity 133 Odor 134 CHAPTER 6

Chemical Examination of Urine 139 Introduction 140 Reagent Strips 140 Reagent Strip Technique 140 Handling and Storing Reagent Strips 141 Quality Control of Reagent Strips 142 Confirmatory Testing 142 pH 142 Clinical Significance 143 Reagent Strip Reactions 143 Protein 143 Clinical Significance 144 Prerenal Proteinuria 144 Renal Proteinuria 144 Postrenal Proteinuria 145 Reagent Strip Reactions 146 Reaction Interference 146 Glucose 148 Clinical Significance 148 Reagent Strip (Glucose Oxidase) Reaction 149 Reaction Interference 150 Copper Reduction Test (Clinitest) 150 Clinical Significance of Clinitest 151 Ketones 151 Clinical Significance 151 Reagent Strip Reactions 152 Reaction Interference 152 Blood 152 Clinical Significance 153 Reagent Strip Reactions 153 Reaction Interference 154

Bilirubin 154 Bilirubin Production 154 Clinical Significance 155 Reagent Strip (Diazo) Reactions 155 Reaction Interference 156 Urobilinogen 156 Clinical Significance 157 Reagent Strip Reactions and Interference 157 Reaction Interference 157 Nitrite 157 Clinical Significance 157 Reagent Strip Reactions 158 Reaction Interference 158 Leukocyte Esterase 159 Clinical Significance 159 Reagent Strip Reaction 160 Reaction Interference 160 Specific Gravity 160 Reagent Strip Reaction 160 Reaction Interference 160 CHAPTER 7

Microscopic Examination of Urine 167 Introduction 168 Macroscopic Screening 168 Specimen Preparation 168 Specimen Volume 168 Centrifugation 168 Sediment Preparation 169 Volume of Sediment Examined 169 Commercial Systems 169 Examining the Sediment 169 Reporting the Microscopic Examination 170 Correlating Results 170 Sediment Examination Techniques 170 Sediment Stains 171 Cytodiagnostic Urine Testing 173 Microscopy 173 Types of Microscopy 176 Urine Sediment Constituents 178 Red Blood Cells 179 White Blood Cells 181 Epithelial Cells 182 Bacteria 187 Yeast 189 Parasites 189 Spermatozoa 189 Mucus 190 Casts 191

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Contents Urinary Crystals 199 Urinary Sediment Artifacts

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CHAPTER 8

Renal Disease 219 Introduction 220 Glomerular Disorders 220 Glomerulonephritis 220 Nephrotic Syndrome 222 Tubular Disorders 222 Acute Tubular Necrosis 222 Hereditary and Metabolic Tubular Disorders 225 Interstitial Disorders 226 Urinary Tract Infection 226 Acute Pyelonephritis 226 Chronic Pyelonephritis 227 Acute Interstitial Nephritis 228 Renal Failure 228 Renal Lithiasis 229 CHAPTER 9

Urine Screening for Metabolic Disorders 235 Introduction 236 Overflow Versus Renal Disorders 236 Newborn Screening Tests 236 Amino Acid Disorders 236 Phenylalanine-Tyrosine Disorders 236 Branched-Chain Amino Acid Disorders 239 Tryptophan Disorders 241 Cystine Disorders 242 Porphyrin Disorders 243 Mucopolysaccharide Disorders 246 Purine Disorders 247 Carbohydrate Disorders 247 PART THREE:

Other Body Fluids

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CHAPTER 10

Cerebrospinal Fluid 253 Introduction 254 Formation and Physiology 254 Specimen Collection and Handling 254 Appearance 255 Traumatic Collection (Tap) 256 Uneven Blood Distribution 256 Clot Formation 257 Xanthochromic Supernatant 257 Cell Count 257 Methodology 257 Total Cell Count 258

WBC Count 258 Quality Control of CSF and Other Body Fluid Cell Counts 258 Differential Count on a CSF Specimen 258 Cytocentrifugation 259 CSF Cellular Constituents 259 Chemistry Tests 266 Cerebrospinal Protein 266 CSF Glucose 268 CSF Lactate 268 CSF Glutamine 268 Microbiology Tests 268 Gram Stain 269 Immunologic Assays 270 PCR Molecular Diagnostic Testing 270 Serological Testing 271 CHAPTER 11

Semen 277 Introduction 278 Physiology 278 Specimen Collection 278 Specimen Handling 280 Semen Analysis 280 Appearance 280 Liquefaction 280 Volume 280 Viscosity 281 pH 281 Sperm Concentration and Sperm Count 281 Sperm Motility 283 Automated Semen Analysis Systems 283 Sperm Morphology 283 Additional Testing 285 Sperm Vitality 286 Seminal Fluid Fructose 286 Antisperm Antibodies 286 Microbial and Chemical Testing 287 Postvasectomy Semen Analysis 287 Sperm Function Tests 288 Semen Analysis Quality Control 288 CHAPTER 12

Synovial Fluid 293 Physiology 294 Specimen Collection and Handling 294 Color and Clarity 295 Viscosity 296 Cell Counts 296 Differential Count 296

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Contents Crystal Identification 297 Types of Crystals 297 Slide Preparation 297 Crystal Polarization 298 Chemistry Tests 300 Glucose 300 Total Protein 300 Uric Acid 301 Lactate 301 Enzymes 301 Microbiological Tests 301 Serological Tests 301

CHAPTER 13

Serous Fluid

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Introduction 306 Formation 306 Specimen Collection and Handling 306 Transudates and Exudates 307 General Laboratory Procedures 307 Pleural Fluid 308 Appearance 308 Hematology Tests 309 Chemistry Tests 310 Microbiological and Serological Tests 312 Pericardial Fluid 312 Appearance 312 Laboratory Tests 312 Peritoneal Fluid 314 Transudates Versus Exudates 314 Appearance 314 Laboratory Tests 314 CHAPTER 14

Bronchoalveolar Lavage Fluid 321 Clinical Significance 322 Specimen Collection 322 Specimen Handling and Transport 322 Color and Clarity 322 Cell Counts 322 Leukocytes 323 Macrophages 323 Lymphocytes 323 Neutrophils 323 Eosinophils 323 Mast Cells 324 Erythrocytes 324 Epithelial Cells 324 Microbiological Tests 324 Cytology 324

CHAPTER 15

Amniotic Fluid 327 Introduction 328 Physiology 328 Function 328 Volume 328 Chemical Composition 329 Differentiating Maternal Urine From Amniotic Fluid 329 Specimen Collection 329 Indications for Amniocentesis 329 Collection 330 Specimen Handling and Processing 330 Color and Appearance 331 Tests for Fetal Distress 331 Hemolytic Disease of the Fetus and Newborn 331 Neural Tube Defects 332 Tests for Fetal Maturity 333 Fetal Lung Maturity 333 Lecithin-Sphingomyelin Ratio 333 Phosphatidyl Glycerol 334 Foam Stability Index 334 Lamellar Bodies 334 CHAPTER 16

Fecal Analysis 339 Introduction 340 Physiology 340 Diarrhea and Steatorrhea 341 Diarrhea 341 Steatorrhea 342 Specimen Collection 342 Macroscopic Screening 342 Color 343 Appearance 343 Microscopic Examination of Feces 343 Fecal Leukocytes 343 Muscle Fibers 344 Qualitative Fecal Fats 344 Chemical Testing of Feces 345 Occult Blood 345 Quantitative Fecal Fat Testing 347 APT Test (Fetal Hemoglobin) 348 Fecal Enzymes 349 Carbohydrates 349

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Contents CHAPTER 17

Vaginal Secretions 355 Introduction 356 Specimen Collection and Handling 357 Color and Appearance 357 Diagnostic Tests 357 pH 357 Microscopic Procedures 358 Other Diagnostic Tests 363 Vaginal Disorders 364 Bacterial Vaginosis 364 Trichomoniasis 364 Candidiasis 364

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Desquamative Inflammatory Vaginitis 365 Atrophic Vaginitis 365 Additional Procedures for Vaginal Secretion Associated With Pregnancy 365 Fetal Fibronectin Test 365 AmniSure Test 366 Actim PROM 366 ROM Plus 366 Answers to Study Questions, Case Studies, and Clinical Situations 371 Abbreviations 381 Glossary 385 Index 393

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Atlas of Images Kathleen Finnegan

URINALYSIS 2 Cells 2 Red Blood Cells 2 White Blood Cells 3 Epithelial Cells 5 Transitional Epithelial (Urothelial) Cell Renal Tubular Epithelial Cells 8 Oval Fat Bodies 10 Microorganisms 11 Bacteria 11 Yeast 12 Parasites 14 Trichomonads 14 Pinworm 14 Schistosoma Haematobium 15 Miscellaneous Structures 16 Sperm 16 Mucus 16 Casts 17 Hyaline Casts 17 Red Blood Cell Casts 18 White Blood Cell Casts 19 Bacterial Casts 20 Epithelial Cell Casts 20 Granular Casts 21 Mixed Cell Casts 23 Fatty Casts 23 Waxy Casts 24 Broad Casts 25

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Crystals/Acid 26 Uric Acid Crystals 26 Amorphous Urate Crystals 27 Calcium Oxalate Crystals 28 Sodium Urate Crystals 29 Crystals/Alkaline 30 Amorphous Phosphate Crystals 30 Triple Phosphate Crystals 30 Calcium Phosphate Crystals 32 Calcium Carbonate Crystals 32 Ammonium Biurate Crystals 33 Abnormal Crystals 34 Cystine Crystals 34 Leucine Crystals 34 Tyrosine Crystals 35 Cholesterol Crystals 35 Bilirubin Crystals 36 Sulfonamide Crystals 37 Ampicillin Crystals 37 Artifacts 38 Radiographic Dye Crystals 38 Starch 38 Fibers 39 Oil Droplets or Air Bubbles 40 BODY FLUIDS 41 Cerebrospinal Fluid 41 Peritoneal Fluid 43 Pleural Fluid 44 Synovial Fluid 46

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URINALYSIS Cells Red Blood Cells

Description • Normal: Pale nonnucleated biconcave discs, membrane regular, 6 to 8 µm in size • Hypotonic urine: Red blood cells (RBCs) appear colorless with membrane intact and regular; often lose their hemoglobin in a dilute urine with low specific gravity • Hypertonic urine: RBCs have lost their biconcave shape, the membrane becomes irregular, and the cells appear crenated; they appear like small spheres with spicules in a urine with high specific gravity • Dysmorphic: RBCs vary in size and shape, are often fragmented, or have cellular protrusions (burr cells or acanthrocytes)

Clinical Significance • Increased RBCs are seen with urinary tract infections (UTIs), trauma to the kidneys, or menstrual contamination • Dysmorphic RBCs are associated with glomerular membrane damage UA Correlations • Color: Pale pink to bright red • Specific gravity • Blood Tips • RBCs can be confused with yeast cells (round to oval and smaller in shape and tend to bud) or oil droplets and air bubbles (tend to be refractile)

A-01. Normal RBCs. Note uric acid crystals.

A-03. Ghost RBCs.

A-02. Normal and crenated RBCs.

A-04. Crenated RBCs.

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A-05. Dysmorphic RBCs.

White Blood Cells

Description • Colorless, 12 µm in size • Neutrophils are granular and multinucleated; the majority of white blood cells (WBCs) in the peripheral blood are neutrophils • Lymphocytes and monocytes are agranular • Hypotonic urine: Sparkling cytoplasm in the neutrophils associated with Brownian movement are called glitter cells; they absorb water and swell Clinical Significance • Neutrophils are associated with bacterial infection, cystitis, and inflammation • Eosinophils are associated with drug-induced interstitial nephritis

A-06. One-segmented WBC. Note the multilobed nucleus.

• Lymphocytes are associated with renal transplant • Monocytes are associated with chronic inflammation UA Correlations • Clarity • Leukocyte esterase • Nitrite with bacterial infection Tips • Neutrophils lose their nuclear detail in dilute urine and can be confused with renal tubular epithelial (RTE) cells, which tend to be larger with an eccentric nucleus • During active inflammation of the urinary tract, the WBCs may have extended pseudopodia

A-07. Clump of WBCs.

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A-08. WBCs and a single transitional epithelial cell.

A-11. WBCs with three RBCs.

A-09. WBCs with a fine granular cast.

A-12. Numerous WBCs, bacteria, and two RBCs.

A-10. Segmented WBCs.

A-13. WBCs, RBCs, squamous epithelial cells, and two calcium oxalate crystals.

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A-14. Glitter cell. Observe the granules. RBCs are also present.

Epithelial Cells

Description • Large cells, 60 to 100 µm in size, with abundant irregular cytoplasm and wrinkled borders • Nucleus is in the center and is the size of an RBC; can be binucleated, tend to have multiridge borders that tend to fold or curl • Clue cell: Variation of squamous epithelial cells; covered with Gardnerella coccobacillus, which gives the cell a granular appearance Clinical Significance • Originate from the mucous membrane around the urethra • Squamous epithelial cells can be associated with wear and tear from urethra or vaginal mucosa; seen more frequently in females

A-15. Clump of squamous epithelial cells. Note normal RBCs and one crenated RBC.

• Clue cells are pathological and indicative of vaginal infection caused by Gardnerella vaginalis UA Correlations • Clarity Tips • Squamous epithelial cells tend to resemble a fried egg • When rolled or curled, they tend to look like casts • Casts do not have a nucleus • When seen in high numbers, they can indicate contamination from vaginal secretions or poor collection

A-16. Rolled squamous epithelial cell. Note bacteria.

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A-17. Clue cell. Note bacteria covering the epithelial cell.

A-20. Three squamous epithelial cells and one WBC.

A-18. Squamous epithelial cells with WBCs and one calcium oxalate crystal. Note refractile fiber.

A-21. Squamous epithelial cell. Note the abundant cytoplasm and prominent centrally located nucleus.

A-19. Two squamous epithelial cells, one calcium oxalate crystal, and one RBC.

A-22. Squamous epithelial cell and transitional epithelial cell. The amount of cytoplasm is much larger in the squamous epithelial cell.

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Transitional Epithelial (Urothelial) Cell Description • Smaller than squamous epithelial cells • Can be spherical, pear-shaped, polyhedral, or caudate shaped with a centrally located nucleus • In elongated cells, the nucleus tends to be off-center • Cells contain a moderate amount of cytoplasm Clinical Significance • Originate from mucosa of renal pelvis, ureter, and bladder • Appear in urine with infection, inflammation, renal stones, bladder cancer, and catheterization

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UA Correlations • Clarity • Blood, if malignancy associated Tips • Spherical forms resemble RTE cells • When seen in clusters or sheets, they are associated with catheterized specimens • Transitional epithelial cells tend to take on water and can look like large round balloons

A-23. Pear-shaped transitional epithelial cell. Note bacteria.

A-25. Two spherical transitional epithelial cells. WBCs, RBCs, and yeast (refractile) are also present.

A-24. Clump of transitional epithelial cells. Note RBC.

A-26. Squamous and transitional epithelial cells. Notice the central location of the nucleus of the epithelial cells. The squamous epithelial cell has a greater amount of cytoplasm. Many WBCs and bacteria are also present.

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A-27. A clump of transitional epithelial cells.

A-28. Spherical transitional epithelial cell with a hyaline cast in the left lower corner.

Renal Tubular Epithelial Cells (RTE)

UA Correlations • Clarity • Color • Protein • Blood • Bilirubin (hepatitis) • Leukocyte esterase (pyelonephritis) • Nitrite (pyelonephritis)

Description • Originate from proximal renal tubules, loop of Henle, distal convoluted renal tubules, and collecting ducts; vary in size and shape due to different origins • Proximal tubular cells are large in size and oblong in shape with grainy cytoplasm; nucleus is eccentric • Distal tubular cells are smaller and more round to oval, with small, round eccentric nucleus • Collecting duct cells are polygonal or cuboidal with a large nucleus Clinical Significance • Increased amounts are indicative of necrosis of the renal tubules, renal tubular damage, exposure to heavy metals, druginduced toxicity, hemoglobin and myoglobin toxicity, ischemic events, and viral infections

A-29. Single and double RTE cells. Note fiber.

Tips • RTEs from the collecting ducts have a flat edge to one side of the membrane; cells from the proximal convoluted tubules are more rectangular and can be mistaken for casts • Cells from the distal convoluted tubule are small and can be mistaken for WBCs

A-30. One RTE cell with two squamous epithelial cells.

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A-31. One RTE cell. Note bacteria.

A-34. Three RTE cells. Notice the eccentrically placed nuclei.

A-32. Clump of RTE cells.

A-35. RTE cells. Fiber artifact present.

A-33. RTE cell.

A-36. RTE cell and WBCs.

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Oval Fat Bodies

Description • Oval fat bodies are renal tubular cells laden with fat • They are highly refractile and globular in shape, with a nucleus that is difficult to see • Polarizing microscopy demonstrates a Maltese cross formation Clinical Significance • Oval fat bodies can be associated with nephrotic syndrome, glomerular dysfunction, and renal tubular cell death UA Correlations • Clarity • Protein

A-37. Oval fat body.

• Blood • Free fat droplets • Fatty casts Tips • Oval fat bodies can be seen in conjunction with free floating fat droplets • Lubricants used in specimen collection can be confused with fat droplets

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Microorganisms Bacteria

Description • Small, spherical cocci and rod-shaped (bacilli) structures • Vary in size from short to long • Can appear single or in pairs or chains Clinical Significance • Increased bacteria indicate a UTI • Bacteria should be seen with increased WBCs, positive leukocyte esterase, an alkaline pH, and a +/– nitrite UA Correlations • Alkaline pH • Leukocyte esterase

A-38. Bacteria in chains with WBCs and RBCs.

A-39. Rod-shaped bacteria with WBCs.

• Nitrite • WBCs • WBCs in clumps Tips • Motility will help distinguish bacteria from amorphous urates (pink-colored sediment) or amorphous phosphates (whitecolored sediment) or calcium carbonate crystals, which can look like bacteria • Bacteria multiply in urine if left at room temperature for several hours

A-40. Many bacteria and WBCs.

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Yeast

Description • Appear as colorless, small, refractile, round to oval structures that may be single, budding, or with pseudohyphae and branching Clinical Significance • Candida albicans is yeast found commonly in urine of women, it most often indicates vaginal secretion contamination or vaginal infection • It can also be seen in patients who are diabetic or immunocompromised

Tips • Yeast can be confused with RBCs • Budding yeast can help differentiate • Yeast does not dissolve when acetic acid is added • As with bacteria, yeast will multiply in urine if left at room temperature for several hours

UA Correlations • Glucose • Increased WBCs • Leukocyte esterase

A-41. Packed field of budding yeast and pseudohyphae.

A-42. Budding yeast with WBCs and one RBC.

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A-43. Budding yeast with RBCs, WBCs, and one squamous epithelial cell. Note the halo around RBCs.

A-44. Clump of budding yeast and WBCs.

A-45. Budding yeast with pseudohyphae and RBCs.

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Parasites Trichomonads

Description • A trophozoite that is a pear-shaped flagellate with an underlying membrane that causes a rapid flitting motion; resembles WBC with tail-like flagella Clinical Significance • Trichomonas vaginalis is a sexually transmitted pathogen associated with vaginal or urethral inflammation • In males, it is asymptomatic

• WBC clumps may be present • Leukocyte esterase Tips • T. vaginalis must be identified in fresh urine • They begin to die, and when not moving, they resemble WBCs • Organisms must be moving or have motility not to be confused with WBCs and RTE cells

UA Correlations • Increased WBCs • Increased epithelial cells

A-46. A trichomonad. Notice the flagella and undulating membrane.

Pinworm

Description • Eggs or ova are football shaped with one flattened side and one round side • Eggs are transparent, and developing larva can be seen sometimes Clinical Significance • Can be found as a result of fecal contamination • Usually seen with school-age children

UA Correlations • N/A Tips • Eggs or ova tend to be quite large compared with WBCs and epithelial cells

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A-47. Enterobius vermicularis worm (pinworm).

A-48. Enterobius vermicularis ova.

Schistosoma Haematobium

UA Correlation • Increased RBCs • Increased WBCs

Description • Very large in size and football shaped with a terminal spine • Cell wall looks thick Clinical Significance • Eggs can be found in urine and are endemic to Africa and the Middle East

A-49. Schistosoma haematobium ova.

Tips • Sometimes the terminal spine can be small and hard to see • Examine several ova for confirmation

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Miscellaneous Structures Sperm

Description • Spermatozoa have tapered oval heads with long, delicate, flagella-like tails

UA Correlations • Protein Tips • Urine is very toxic to sperm, so it rarely shows motility in urine

Clinical Significance • Sperm can be found in the urine of both males and females after sexual intercourse, masturbation, and nocturnal emission; rarely have any clinical significance

A-50. Sperm and bacteria in urine sediment.

A-51. Two sperm and a hyaline cast.

Mucus

UA Correlations • Clarity

Description • Single or clumped, ribbon-like or thread-like structures that have irregular or serrated ends with low refractive index • Subdued light is required when using bright-field microscopy Clinical Significance • No clinical significance • Mucus can be abundant in the presence of inflammation or irritation to the urinary tract

A-52. Mucus with sperm present.

Tips • Mucus is hard to see with increased light due to its low refractive index • Heavy mucus threads have WBCs among them • These structures can be misidentified as hyaline casts • Casts have rounded edges and are more cylindrical

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Casts Hyaline Casts

Description • Composed of uromodulin protein and appear colorless and transparent under low power (100×) • Low refractive index similar to urine, making them difficult to see using bright-field microscopy • Consist of parallel sides with rounded ends, with a variety of sizes and shapes • May contain incisions or have adhering cells or granules under them Clinical Significance • Can be found in normal individuals; increased numbers with strenuous exercise, stress, fever, and dehydration

• Can be seen in patients with chronic renal disease and congestive heart failure UA Correlations • Protein • Blood Tips • Hyaline casts have a low refractive index and should be viewed under low light • Under bright-light microscopy, they can be missed • Hyaline casts can be confused with mucus and fibers • Mucus is thin and ribbon-like, whereas fibers do not have parallel sides or rounded edges and tend to have dark edges

A-53. Hyaline cast.

A-55. Hyaline cast. Note refractile fiber in the top left corner.

A-54. Hyaline cast. Several RBCs present.

A-56. Hyaline cast and numerous squamous epithelial cells.

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A-57. Cylindroid.

Red Blood Cell Casts

Description • There are few to many RBCs dispersed in the protein matrix • They appear red to brown but vary in size and shape Clinical Significance • RBC casts are associated with damage to the glomerulus or glomerulonephritis, bleeding into the nephron, and lupus UA Correlations • Increased RBCs • Protein • Blood

A-58. Hemoglobin cast.

Tips • RBC clumps can be confused with RBC casts • Casts have parallel sides with rounded edges • The RBCs should be distinct and inside the protein matrix to be called a cast • If the RBCs have degenerated to a yellow-brown homogenous structure, it is called a hemoglobin cast

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White Blood Cell Casts

Description • WBCs with prominent nuclei are found in a cylindrical matrix with parallel sides and rounded edges that vary in size and length Clinical Significance • The presence of WBC casts can indicate renal inflammation, infection, acute pyelonephritis, or interstitial nephritis • The presence of WBC casts can distinguish pyelonephritis or upper UTI from cystitis or lower UTI

A-59. WBC cast. Notice the WBCs within the matrix of the cast and free-floating in the urine sediment.

A-60. Degenerating WBC cast. WBCs, RBCs, a hyaline cast, a RTE, and an artifact fiber are also present.

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UA Correlations • Protein • Leukocyte esterase • Increased WBCs Tips • WBC clumps can be confused with WBC casts • To identify the cast, the WBCs are intact with visible nuclei within the protein matrix

A-61. Broad WBC cast with many free-floating WBCs, two RBCs, and budding yeast present.

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Bacterial Casts

Description • Casts contain bacilli within the protein matrix Clinical Significance • Diagnostic for pyelonephritis or upper UTI UA Correlations • WBCs • Bacteria

• Protein • Leukocyte esterase • Nitrite Tips • These casts can also contain WBCs and are called mixed casts • These casts can be confused with granular casts; confirmation is through a Gram stain

A-62. Mixed granular cast with bacteria embedded in the matrix. Note bacteria surrounding in cast. Bacterial casts are not common.

Epithelial Cell Casts

Description • RTE cells with large nuclei are seen within protein matrix; they have a high refractive index • As the RTE cells undergo degenerating changes, they become smaller and round resembling WBCs Clinical Significance • Most often associated with acute tubular necrosis, advanced tubular destruction, and heavy metal or drug toxicity

UA Correlations • Protein • RTE cells Tips • Epithelial cell casts can be confused with WBC casts • A negative leukocyte esterase can help distinguish the epithelial cell cast from a WBC cast

A-64. Cast with oval fat bodies. A-63. Renal epithelial cell cast.

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Granular Casts

Description • Casts are a degeneration of cellular casts; granules begin large and are coarse and granular • As stasis continues, granules break down further and are called fine granular casts • Coarse granules are dark in color, and fine granules tend to be pale • Casts can be of various sizes and shapes Clinical Significance • These casts can indicate urinary stasis, tubular disease, glomerulonephritis, pyelonephritis, or may be seen in stress or after exercise

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UA Correlations • Protein • RBCs • WBCs • Cellular casts Tips • Examine under low power, but casts are identified under high power • These casts can be reported as coarse, fine, or just granular • They can be confused with fibers or clumps of sediment • For it to be a cast, granules must be in a surrounding protein matrix

A-65. Granular cast.

A-67. Granular cast with a squamous epithelial cell and RBC.

A-66. Granular cast. Note RBCs, WBCs, and bacteria.

A-68. One granular cast. Numerous sperm present.

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A-69. Granular cast.

A-72. Granular cast.

A-70. Granular cast. RBCs are also present.

A-73. One granular cast and two hyaline casts.

A-71. One granular cast with four squamous epithelial cells. Note the highly refractile artifact.

A-74. Granular cast. Sperm present at the top of image. Amorphous is also present.

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Mixed Cell Casts

Description • Contain multiple cellular constituents, which can include RBCs, WBCs, RTE cells, and bacteria Clinical Significance • Can indicate renal disease and are usually seen with other cellular casts

• RBCs • WBCs • RTE cells Tips • Mixed casts should have separate free-floating cellular elements present

UA Correlations • Protein • Leukocyte esterase

A-75. Mixed-cell cast. Note free-floating RBCs and WBCs.

Fatty Casts

Description • Highly refractile • Casts may contain fat droplets and intact oval fat bodies of varying sizes within the protein matrix Clinical Significance • Associated with nephrotic syndrome, acute tubular necrosis, diabetes mellitus, toxic tubular necrosis, and crushing injuries

A-76. Fatty cast. Note intact oval fat bodies within the matrix.

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UA Correlations • Protein • Free fat droplets • Oval fat bodies Tips • Maltese cross formations can be seen with cholesterol under polarization

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Waxy Casts

Description • Highly refractile with broken, jagged, or blunt ends with cracks on the margin of the matrix • May be colorless to gray Clinical Significance • Casts indicate urinary stasis, chronic renal failure, or end-stage renal disease

• Cellular casts • Granular casts Tips • These casts tend to look like paraffin shavings sitting on top of the urine • They are often broad in size due to renal stasis

UA Correlations • Protein • Increased RBCs • Increased WBCs

A-77. Waxy cast with a fragmented end and notches on its side. WBCs are also present.

A-78. A waxy cast is located in the center with four granular casts to the side. Also present are RTE cells, RBCs, and squamous epithelial cells.

A-79. Waxy cast.

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Broad Casts

Description • Named for their wide size • Formed due to distention of the collecting tubules where they are formed Clinical Significance • Represent extreme urinary stasis and renal failure UA Correlations • Protein • Increased RBCs • Increased WBCs

A-80. Broad granular cast.

• Granular casts • Waxy casts Tips • Granular or waxy casts may be classified as broad

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Crystals/Acid Uric Acid Crystals

Description • Yellow to brown in color; can occur in many shapes, including rhombic, four-sided flat plates, rosettes, barrels, and wedges Clinical Significance • Can be found in normal individuals • Can be associated with pathological conditions, such as urinary stones, gout, high purine metabolism, acute febrile conditions, newborns, and patients with leukemia receiving chemotherapy

UA Correlations • Acid pH Tips • These crystals can be confused with cystine crystals • Uric acid crystals are birefringent under polarized light • Crystals have four sides and look more like a football or lemon wedge • Cystine have six sides or look like a stop sign

A-81. Uric acid crystals, with barrel shape.

A-83. Uric acid crystals. Note the rosette and wedge shapes.

A-82. Uric acid crystal clump.

A-84. Two uric acid crystals observed using high-power magnification. One hyaline cast, WBCs, and bacteria are also present.

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A-85. Uric acid crystals observed using low-power magnification. Note the numerous hyaline casts.

Amorphous Urate Crystals

Description • Appear as yellow to reddish granules Clinical Significance • No clinical significance • These are salts of sodium, potassium, magnesium, and calcium; usually appear in older urine UA Correlations • Acid pH • Sediment has a pink to brick dust–like color after centrifugation

A-86. Amorphous urates distinguished by acid pH.

Tips • When amorphous urates clump, they tend to look like a granular cast • Always look for the protein matrix to define a cast • They also can hide bacteria and RBCs • Amorphous urates dissolve when heated to 60°C

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Calcium Oxalate Crystals

Description • Often appear as colorless, envelope-shaped crystals with an X or cross in the middle • Can appear as dumbbell- or small oval–shaped Clinical Significance • Frequently can be found in normal urine after ingesting oxalate-rich food, such as tomatoes, spinach, oranges, fruit juice, vitamin C, and chocolate • Pathological conditions include ethylene glycol poisoning, liver disease, and renal calculi

UA Correlations • Acid or neutral pH Tips • The more common calcium oxalate crystals have the shape of an emerald-cut diamond • The oval shapes tend to look like RBCs, but they are more elliptical and tend to have a dark outline along their edge

A-87. Calcium oxalate crystals. Note RBCs and budding yeast.

A-89. Monohydrate calcium oxalate crystals.

A-88. Clump of calcium oxalate crystals.

A-90. One monohydrate calcium crystal and two classic dihydrate calcium oxalate crystals.

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A-91. Calcium oxalate crystal.

A-92. Monohydrate calcium oxalate crystals.

Sodium Urate Crystals

Tips • These are usually seen when urine has been standing • When found in clusters, they look like “pick-up sticks”

Description • Colorless or yellowish, slender needles with blunt ends that can be found singly or in clusters Clinical Significance • No clinical significance UA Correlations • Acid pH

A-93. Sodium urate crystals.

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Crystals/Alkaline Amorphous Phosphate Crystals

Description • Fine colorless granules that can be hard to distinguish from amorphous urates; differentiated by sediment color and urine pH Clinical Significance • No clinical significance

Tips • Amorphous phosphates can interfere with the microscopic examination • RBCs and bacteria may not be visualized • Amorphous phosphates do not dissolve when heated at 60°C • Refrigeration increases their presence • They are distinguished from urates by urine pH

UA Correlations • Urine is cloudy when many crystals are present • Alkaline or neutral pH • White sediment after centrifugation

A-94. Amorphous phosphate crystals.

Triple Phosphate Crystals

Description • Most common form is colorless prisms with three to six sides referred to as coffin covers • Vary in size • When they denature, they become feathery or leaf-like Clinical Significance • Can be seen in healthy individuals with no clinical significance • Can be associated with urinary stones and UTI UA Correlations • Alkaline or neutral pH

Tips • Triple phosphate crystals vary in size and sometimes have the shape and size of a calcium oxalate • The cross on top of the crystal overlaps or meets in the middle of the triple phosphate crystal • For calcium oxalate crystals, the X crosses in the middle

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A-95. Triple phosphate crystals.

A-98. Square triple phosphate crystals with bacteria and yeast present in urine sediment.

A-96. Clump of triple phosphate crystals.

A-99. Triple phosphate crystals.

A-97. Triple phosphate crystal with RBCs.

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Calcium Phosphate Crystals

Description • Colorless to grayish-white thin needles • Arranged in bundles or thin, long prisms and can be arranged in rosettes or can be flat plates • Tend to float on the top of the urine

UA Correlations • Alkaline or neutral pH Tips • Sometimes the sheets resemble large degenerating squamous cells

Clinical Significance • Can be found in normal urine and are associated with renal stones

A-100. Calcium phosphate crystals.

Calcium Carbonate Crystals

Description • Small, colorless, dumbbell-shaped; appear in pairs or may have a spherical shape Clinical Significance • No clinical significance

A-101. Calcium carbonate crystals.

UA Correlations • Alkaline pH Tips • Calcium carbonate can appear in clumps and resemble amorphous phosphates or be mistaken for bacteria • They tend to be a little larger than amorphous phosphates

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Ammonium Biurate Crystals

Description • Appear as yellow to brown spheres with long spicules surrounding the crystal; resemble a “thorny apple”

UA Correlations • Alkaline pH Tips • Usually encountered in old specimens

Clinical Significance • Usually seen in urines that have not been stored properly • May be associated with the presence of ammonia produced by urea-splitting bacteria

A-102. Two ammonium biurate crystals.

A-103. Ammonium biurate crystal. Note bacteria.

A-104. Ammonium biurate crystals.

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Abnormal Crystals Cystine Crystals

Description • Appear as colorless hexagonal plates with sides that are not always even, resembling a stop sign • Can be found single or overlapping each other to form rosettes Clinical Significance • Clinically significant and indicate a metabolic hereditary disorder called cystinosis or cystinuria

Tips • Cystine crystals can be distinguished from uric acid crystals using polarization • Uric acid crystals are birefringent under polarization, whereas cystine crystals are not • Cystine crystals appear as a six-sided stop sign, whereas uric acid crystals appear as a four-sided stop sign

UA Correlations • Acid pH

A-105. Cystine crystals.

Leucine Crystals

Description • Amino acid crystals • Appear as oily or highly refractile yellow to brown spheres with radial striations Clinical Significance • Can appear in patients with severe liver disease, terminal cirrhosis, viral hepatitis, and maple syrup disease • Can also be seen in patients with tyrosine crystals UA Correlations • Acid or neutral pH • Bilirubin

Tips • These crystals can sometimes resemble fat globules • Under polarization, they produce a pseudo–Maltese cross pattern • They also can resemble wagon wheels or concentric circles resembling a tree’s growth pattern or striations of a tree • These crystals are rarely seen in urinalysis

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A-106. Leucine crystals (×400). Note the concentric circles.

Tyrosine Crystals

Description • Amino acid crystals • Appear as fine needles occurring in shelves or clusters that are colorless or yellow Clinical Significance • Associated with liver disease and with inherited disorders of amino acid metabolism

UA Correlations • Acid or neutral pH • Bilirubin Tips • This is a rare crystal not seen very often in urine sediment • They are often observed with leucine crystals

A-107. Tyrosine crystal.

Cholesterol Crystals

Description • Appear as irregular, rectangular, large transparent plates with a notch in one or more corners • Often appear in stacks Clinical Significance • Can be seen in patients with nephrotic syndrome, chyluria, and disorders producing lipiduria

UA Correlations • Acid pH • Protein • Oval fat bodies • Fatty casts • Fat globules Tips • These crystals are seen after refrigeration • They are highly birefringent with polarized light

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A-108. Cholesterol crystals.

Bilirubin Crystals

Description • Appear as small clusters of fine clumped needles or spheres • Dark yellow in color Clinical Significance • Associated with hepatic disease, producing large amounts of bilirubin

A-109. Bilirubin crystal. Note the classic bright yellow color.

UA Correlations • Acid pH • Bilirubin Tips • A positive result for bilirubin must be present for these crystals to be identified

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Sulfonamide Crystals

Description • Can appear in many different forms; most shapes are bundles of needles that resemble stacks of wheat or have a fan shape • Can be brown in color

37

UA Correlations • Acid pH Tips • These crystals can be confused with ammonium biurate • Ammonium biurate crystals are found with an alkaline pH

Clinical Significance • With the development of more soluble sulfur drugs, these crystals are not found often • Patients with UTIs are treated with sulfonamides • Also can be associated with renal calculi or tubular damage

A-110. Sulfa crystal.

Ampicillin Crystals

Description • Appear as colorless long needles that tend to form bundles after refrigeration Clinical Significance • Indicative of large doses of ampicillin without adequate hydration

A-111. Ampicillin crystals.

UA Correlations • Acid or neutral pH Tips • Knowledge of patient history is helpful to identify these crystals

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Artifacts Radiographic Dye Crystals

Description • When ionic radiographic crystals precipitate out in urine, they appear as flat, elongated rectangles that are strongly birefringent Clinical Significance • X-ray procedures, such as a pyelogram, use dye to visualize the urinary tract

UA Correlations • High specific gravity when using a refractometer Tips • These have a similar appearance to cholesterol crystals • Cholesterol crystals are accompanied by other lipid elements • May persist for 3 days after the procedure

A-112. Radiographic dye crystals.

Starch

Description • Vary in size • Have a Y indentation in the center • Not perfectly round, but have more scalloped edges

UA Correlations • N/A Tips • Starch or pollen granules can be confused with fat droplets • Under polarized light, starch can appear as the Maltese cross

Clinical Significance • Caused by contamination from baby powder or powder from health care worker gloves

A-113. Starch granule.

A-114. Starch granules. Notice the dimpled centers.

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Fibers

Description • Large with distinct, flat, dark edges • Refractile Clinical Significance • Considered contaminants

Tips • Fibers can be plant, vegetable, fabric threads, toilet paper, cotton, or diaper • They can resemble hyaline casts • Fibers are thicker at their margins and polarize

UA Correlations • N/A

A-115. Fiber. Note RBCs.

A-117. Fiber. Note refractibility.

A-116. Fiber.

A-118. Fiber.

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A-119. Refractile fiber, dysmorphic RBCs, and an RTE cell.

Oil Droplets or Air Bubbles

Description • Highly refractile; may resemble RBCs Clinical Significance • Contamination; oil droplets from immersion oil and air bubbles formed when the specimen is placed under a cover slip are highly refractile using low power

UA Correlations • N/A Tips • Oil droplets and air bubbles can look like RBCs; however, their high refractibility distinguishes them

A-121. Note the oil droplet indicated by the arrow. A-120. Air bubble. Note high refractibility.

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BODY FLUIDS Cerebrospinal Fluid

A-122. Bloody CSF with neutrophils, lymphocytes, and one monocyte.

A-124. Bloody CSF with neutrophils, with cytoplasmic vacuoles resulting from cytoplasmic centrifugation.

A-123. Bloody CSF with neutrophils.

A-125. Ependymal cells in CSF. Notice the nucleoli and less distinct borders.

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A-126. Neutrophils with intracellular and extracellular bacteria in CSF.

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Peritoneal Fluid

A-127. Two large mesothelial cells and three monocytes in peritoneal fluid.

A-128. Two monocytes and one lymphocyte in peritoneal fluid.

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Pleural Fluid

A-129. Two monocytes, one eosinophil, and one neutrophil in pleural fluid.

A-131. Lymphocytes, neutrophils, and monocytes in pleural fluid. Note the large reactive lymphocyte.

A-130. Reactive lymphocytes in pleural fluid.

A-132. Reactive lymphocytes in pleural fluid.

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A-133. Mesothelial cells in pleural fluid.

A-134. Eosinophil and RBCs in pleural fluid.

A-135. RBCs, lymphocyte, and neutrophil in pleural fluid.

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Synovial Fluid

A-136. Wright-stained neutrophils containing CPPD crystals in synovial fluid.

A-138. Wright-stained eosinophil in synovial fluid.

A-139. Polarized urate crystals in synovial fluid.

A-137. Numerous Wright-stained neutrophils with vacuoles in synovial fluid, indicative of inflammation.

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PART ONE

Basic Principles Chapter 1:

Safety and Quality Management

Chapter 2:

Urine and Body Fluid Analysis Automation

Chapter 3:

Introduction to Urinalysis

Chapter 4:

Renal Function

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1

CHAPTER

Safety and Quality Management LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 1-1 Define and give an example of the types of safety hazards encountered in the laboratory.

1-9 Describe the use of the Globally Harmonized System (GHS).

1-2 List the six components of the chain of infection and the laboratory safety precautions that break the chain.

1-10 Recognize standard hazard warning symbols.

1-3 State the purpose of the Standard Precautions policy, and describe its guidelines.

1-11 State and interpret the components of the National Fire Protection Association (NFPA) hazardous material labeling system.

1-4 State the requirements mandated by the Occupational Exposure to Bloodborne Pathogens Compliance Directive.

1-12 Describe precautions that laboratory personnel should take with regard to radioactive, electrical, fire, and physical hazards.

1-5 Describe the types of personal protective equipment (PPE) that laboratory personnel wear, including when, how, and why each article is used.

1-13 Explain the RACE and PASS actions to be taken when a fire is discovered.

1-6 Correctly perform hand-hygiene procedures following guidelines provided by the Centers for Disease Control and Prevention (CDC). 1-7 Describe the acceptable methods for handling and disposing of biological waste and sharp objects in the urinalysis laboratory. 1-8 Discuss the components and purpose of a chemical hygiene plan and a Safety Data Sheet (SDS).

1-14 Explain the role of quality management (QM) in the urinalysis laboratory. 1-15 Define the preexamination (preanalytical), examination (analytical), and postexamination (postanalytical) components of QM. 1-16 Distinguish among internal, external, and electronic quality control, as well as external quality assessment (proficiency testing) in a QM program.

KEY TERMS Accreditation Accuracy Autoverification Biohazardous Chain of infection

Clinical and Laboratory Standards Institute (CLSI)

Globally Harmonized System (GHS)

Delta check

Internal quality control

Electronic quality control Examination variable

Chemical hygiene plan (CHP)

External quality assessment (EQA)

Clinical Laboratory Improvement Amendments (CLIA)

Fomite

External quality control

Infection control Occupational Safety and Health Administration (OSHA) Personal protective equipment (PPE) Postexamination variable Continued

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Part One | Basic Principles

K E Y T E R M S —cont’d Postexposure prophylaxis (PEP)

Quality management (QM)

Specificity

Precision

Quality management system (QMS)

Standard Precautions (SP)

Preexamination variable

Radioisotope

Test utilization

Preventive maintenance (PM)

Reliability

Turnaround time (TAT)

Proficiency testing (PT)

Root cause analysis (RCA)

Universal Precautions (UP)

Quality assessment (QA)

Safety Data Sheet (SDS)

Quality control (QC)

Sensitivity

Safety The clinical laboratory contains a variety of safety hazards, many capable of producing serious injury or life-threatening disease. To work safely in this environment, laboratory personnel must learn what hazards exist, the basic safety precautions associated with them, and how to apply the basic rules of common sense required for everyday safety for patients, coworkers, and themselves. As seen in Table 1-1, some hazards are unique to the healthcare environment, whereas others are encountered routinely

Table 1–1

Types of Safety Hazards

Type

Source

Possible Injury

Biological

Infectious agents

Sharps

Needles, lancets, broken glass

Chemical

Preservatives and reagents

Bacterial, fungal, viral, or parasitic infections Cuts, punctures, or exposure to bloodborne pathogens Exposure to toxic, carcinogenic, or caustic agents Exposure to radiation Burns or shock

Radioactive Electrical

Fire/ explosive Physical

Equipment and radioisotopes Ungrounded or wet equipment; frayed cords Open flames, organic chemicals Wet floors, heavy boxes, patients

throughout life. Safety procedure manuals that describe the safety policies mandated by the Centers for Disease Control and Prevention (CDC) and the Occupational Safety and Health Administration (OSHA) must be readily available in the laboratory, and it is essential for laboratory personnel to adhere closely to these guidelines. The laboratory director must update and review the manual annually. The Clinical and Laboratory Standards Institute (CLSI) provides the guidelines for writing these procedures and policies.1-3

Biological Hazards The health-care setting provides abundant sources of potentially harmful microorganisms. These microorganisms are frequently present in the specimens received in the clinical laboratory. Understanding how microorganisms are transmitted (chain of infection) is essential to preventing infection. All health-care facilities have developed procedures to control and monitor infections occurring within their facilities, which is referred to as infection control. The chain of infection requires a continuous link among six elements: an infectious agent, a reservoir, a portal of exit, a means of transmission, a portal of entry, and a susceptible host.4

Infectious Agent (Pathogen) Infectious agents can include bacteria, fungi, parasites, and viruses. The chain of infection is broken by early detection of one of these agents. The treatment of infectious agents can reduce their opportunity for growth.

Reservoir Burns or dismemberment Falls, sprains, or strains

From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, p. 44, with permission.

The reservoir is the location of potentially harmful microorganisms, such as a contaminated clinical specimen or an infected patient. It is the place where the infectious agent can live and possibly multiply. Humans and animals make excellent reservoirs. Equipment and other soiled inanimate objects, called fomites, will serve as reservoirs, particularly if they contain blood, urine, or other body fluids. Some microorganisms form spores or become inactive when conditions are not ideal and wait until a suitable reservoir is available. Disinfecting the work area kills the infectious agent and eliminates the reservoir, thereby breaking the chain of infection.

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Chapter 1 | Safety and Quality Management 51

Portal of Exit The infectious agent must have a way to exit the reservoir to continue the chain of infection. This can be through the mucous membranes of the reservoir’s nose, mouth, and eyes, as well as in blood or other body fluids. The chain of infection is broken when contaminated materials are placed in biohazard containers and/or when tubes and specimen containers are sealed. When contaminated materials are in the appropriate containers that remain sealed, the infectious agent still has a reservoir but no means of exit.

Means of Transmission Once the infectious agent has left the reservoir, it must have a way to reach a susceptible host. Means of transmission include: 1. Direct contact: the unprotected host touches the patient, specimen, or a contaminated object (reservoir) 2. Airborne: the host inhales dried aerosol particles circulating on air currents or attached to dust particles 3. Droplet: the host inhales infected aerosol droplets from the reservoir (e.g., aerosol droplets from a patient or an uncapped centrifuge tube, or when specimens are aliquoted or spilled) 4. Vehicle: the host ingests a contaminated substance (e.g., food, water, specimen) 5. Vector: from an animal or insect bite Hand sanitizing and adhering to Standard Precautions (SP) are methods to break the chain of infection.

Portal of Entry After the infectious agent has been transmitted to a new reservoir, it must have a means to enter the reservoir. The portal of entry can be the same as the portal of exit, which includes the mucous membranes of the reservoir’s nose, mouth, and eyes, as well as breaks in the skin and open wounds. Disinfection and sterilization and strict adherence to SPs block the portal of entry, thereby breaking the chain of infection.

Susceptible Host The susceptible host can be another patient during invasive procedures, visitors, and health-care personnel when exposed to infectious specimens or needlestick injuries. Immunocompromised patients, newborns and infants, and the elderly are often more susceptible hosts. Stress, fatigue, and lack of proper nutrition depress the immune system and contribute to the susceptibility of patients and health-care providers. Once the chain of infection is complete, the infected host becomes another source able to transmit the microorganisms to others.1 To break the chain of infection, health-care personnel must sanitize hands often, wear gloves, stay current with the required immunizations and tests, and maintain a healthy lifestyle. In the clinical laboratory, the most direct contact with a source of infection is through contact with patient specimens, although contact with patients and infected objects also occurs.

Preventing completion of the chain of infection is a primary objective of biological safety. Figure 1-1 illustrates the universal symbol for biohazardous material and demonstrates how following prescribed safety practices can break the chain of infection. Figure 1-1 places particular emphasis on laboratory practices.

Standard Precautions Proper hand hygiene, correct disposal of contaminated materials, and wearing personal protective equipment (PPE) are of major importance in the laboratory. Concern over exposure to bloodborne pathogens, such as hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV), caused the CDC and OSHA to draft guidelines and regulations to prevent exposure. In 1987, the CDC instituted Universal Precautions (UP), in which all patients are considered to be possible carriers of bloodborne pathogens. The guideline recommends wearing gloves when collecting or handling blood and body fluids contaminated with blood and wearing face shields when there is danger of blood splashing on mucous membranes and when disposing of all needles and sharp objects in puncture-resistant containers. The CDC excluded urine and body fluids not visibly contaminated by blood from UP, although many specimens can contain a considerable amount of blood before it becomes visible. UP was modified for body substance isolation (BSI) to help alleviate this concern. BSI guidelines are not limited to bloodborne pathogens; they consider all body fluids and moist body substances to be potentially infectious. According to BSI guidelines, personnel must wear gloves at all times when encountering moist body substances. A major disadvantage of BSI guidelines is that they do not recommend hand sanitizing after removing gloves unless visual contamination is present. In 1996, the CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC) combined the major features of UP and BSI guidelines and called the new guidelines Standard Precautions. Although SP, as described next, stress patient contact, the principles also can be applied to handling patient specimens in the laboratory.5 SP are as follows: 1. Hand hygiene: Hand hygiene includes both hand washing and the use of alcohol-based antiseptic cleansers. Sanitize hands after touching blood, body fluids, secretions, excretions, and contaminated items, whether or not gloves are worn. Sanitize hands immediately after removing gloves, between patient contacts, and when otherwise indicated to avoid transferring microorganisms to other patients or environments. Sanitizing hands may be necessary between tasks and procedures on the same patient to prevent crosscontamination of different body sites. 2. Gloves: Wear gloves (clean, nonsterile, latex-free gloves are adequate) when touching blood, body fluids, secretions, excretions, and contaminated items. Put on gloves just before touching mucous membranes and nonintact skin. Change gloves between tasks and procedures on

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52

Part One | Basic Principles Break the link • Immunizations • Patient isolation • Nursery precautions • Healthy lifestyle Susceptible host • Patients • Elderly • Newborns • Immunocompromised • Health-care workers

Infectious agent • Bacteria • Fungi • Parasites • Viruses Reservoir • Humans • Animals • Insects • Fomites • Blood/body fluids

Portal of exit • Nose • Mouth • Mucous membranes • Specimen collection

Portal of entry • Nose • Mouth • Mucous membranes • Skin • Unsterile equipment

Break the link • Hand hygiene • Standard precautions • PPE • Sterile equipment

Break the link • Disinfection • Hand hygiene

Means of transmission • Droplet • Airborne • Contact • Vector • Vehicle

Break the link • Sealed biohazardous waste containers • Sealed specimen containers • Hand hygiene • Standard precautions

Break the link • Hand hygiene • Standard precautions • PPE • Patient isolation

Figure 1–1 Chain of infection and safety practices related to the biohazard symbol. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2011, with permission.)

the same patient after contact with material that may contain a high concentration of microorganisms. Remove gloves promptly after use, before touching noncontaminated items and environmental surfaces, and between patients. Always sanitize your hands immediately after glove removal to avoid transferring microorganisms to other patients or environments. 3. Mouth, nose, and eye protection: Wear a mask and eye protection or a face shield to protect mucous membranes of the eyes, nose, and mouth during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. A specially fitted respirator (N95) must be

used during patient care activities related to suspected Mycobacterium exposure. 4. Gown: Wear a gown (a clean, nonsterile gown is adequate) to protect skin and to prevent soiling of clothing during procedures and patient care activities that are likely to generate splashes or sprays of blood, body fluids, secretions, or excretions. Select a gown that is appropriate for the activity and the amount of fluid likely to be encountered (e.g., fluid-resistant laboratory coat in the laboratory). Remove a soiled gown as promptly as possible, and sanitize hands to avoid transferring microorganisms to other patients or environments.

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Chapter 1 | Safety and Quality Management 53 5. Patient care equipment: Handle used patient care equipment soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposure, clothing contamination, and transfer of microorganisms to other patients or environments. Ensure that reusable equipment is not used for the care of another patient until it has been cleaned and reprocessed appropriately. Ensure that single-use items are discarded properly. 6 Environmental control: Ensure that the hospital has adequate procedures for the routine care, cleaning, and disinfection of environmental surfaces, beds, bed rails, bedside equipment, and other surfaces that are touched frequently. Ensure that these procedures are being followed. 7. Linen: Handle, transport, and process linen soiled with blood, body fluids, secretions, and excretions in a manner that prevents skin and mucous membrane exposures and clothing contamination and that avoids the transfer of microorganisms to other patients and environments. 8. Occupational health and bloodborne pathogens: Take care to prevent injuries when using needles, scalpels, and other sharp instruments or devices; when handling sharp instruments after procedures; when cleaning used instruments; and when disposing of used needles. Never recap used needles or otherwise manipulate them using both hands or use any other technique that involves directing the point of a needle toward any part of the body; rather, use self-sheathing needles or a mechanical device to conceal the needle. Do not remove used unsheathed needles from disposable syringes by hand, and do not bend, break, or otherwise manipulate used needles by hand. Place used disposable syringes and needles, scalpel blades, and other sharp items in appropriate puncture-resistant containers, which are located as close as practical to the area in which the items were used, and place reusable syringes and needles in a puncture-resistant container for transport to the reprocessing area. Use mouthpieces, resuscitation bags, or other ventilation devices as an alternative to mouth-tomouth resuscitation methods in areas where the need for resuscitation is predictable. 9. Patient placement: Place a patient in a private room if he or she contaminates the environment or does not (or cannot be expected to) assist in maintaining appropriate hygiene or environment control. If a private room is not available, consult with infection control professionals regarding patient placement or other alternatives. 10. Respiratory hygiene/cough etiquette: Educate health-care personnel, patients, and visitors to contain respiratory secretions to prevent droplet and fomite transmission of respiratory pathogens. Offer masks to coughing patients, distance symptomatic patients from others, and practice good hand hygiene to prevent the transmission of respiratory pathogens.

The Occupational Exposure to Bloodborne Pathogens Standard is a law monitored and enforced by OSHA.6,7 OSHA requires these controls to be provided by or mandated by the employer for all employees. Specific requirements of this OSHA standard include the following:

Engineering Controls 1. Providing sharps disposal containers and needles with safety devices 2. Requiring discarding of needles with the safety device activated and the holder attached 3. Labeling all biohazardous materials and containers

Work Practice Controls 4. Requiring all employees to practice SP and documenting training on an annual basis 5. Prohibiting eating, drinking, smoking, and applying cosmetics in the work area 6. Establishing a daily work surface disinfection protocol

Personal Protective Equipment 7. Providing laboratory coats, gowns, face shields, and gloves to employees and laundry facilities for nondisposable protective clothing

Medical 8. Providing immunization for HBV free of charge 9. Providing medical follow-up to employees who have been exposed accidentally to bloodborne pathogens

Documentation 10. Documenting annual training of employees in safety standards 11. Documenting evaluations and implementation of safer needle devices 12. Involving employees in the selection and evaluation of new devices and maintaining a list of those employees and the evaluations 13. Maintaining a sharps injury log, including the type and brand of safety device, location and description of the incident, and confidential employee follow-up Any accidental exposure to a possible bloodborne pathogen through needlestick, mucous membranes, or nonintact skin must be immediately reported to a supervisor. A confidential evaluation of the incident must begin right away to ensure appropriate postexposure prophylaxis (PEP) is initiated within 24 hours. If it is determined that a significant exposure occurred, an incident report will be completed; blood testing for HIV, HBV, and HCV on both the employee and the source patient is performed; and employee treatment and counseling are initiated. The CDC provides periodically updated guidelines for the management of exposures and recommended PEP.8,9

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Part One | Basic Principles

Personal Protective Equipment

Hand Hygiene

PPE used in the laboratory includes gloves, fluid-resistant gowns, eye and face shields, and Plexiglas countertop shields. Shoes must be closed-toed and cover the entire foot. Gloves should be worn when in contact with patients, specimens (urine, blood, body fluids, secretions, excretions), contaminated items, and laboratory equipment or fixtures. When specimens are collected, gloves must be changed between every patient. In the laboratory, they are changed whenever they become noticeably contaminated or damaged and are always removed when leaving the work area. Wearing gloves is not a substitute for hand hygiene, and hands must be sanitized after gloves are removed. A variety of gloves is available, including sterile and nonsterile, powdered and unpowdered, and latex and nonlatex. Allergy to latex is increasing among health-care workers, and laboratory personnel should be alert for symptoms of reactions associated with latex. Reactions to latex include irritant contact dermatitis, which produces patches of dry, itchy irritation on the hands; delayed hypersensitivity reactions resembling poison ivy that appear 24 to 48 hours after exposure; and true, immediate hypersensitivity reactions often characterized by facial flushing and breathing difficulties. Hand sanitizing immediately after removing gloves and avoiding powdered gloves may aid in preventing the development of latex allergies. Replacing latex gloves with nitrile or vinyl gloves provides an alternative. Any symptoms of latex allergy should be reported to a supervisor because true latex allergy can be life-threatening.10

Hand hygiene is emphasized in Figure 1-1 and in the SP guidelines. Hand contact is the primary method of infection transmission. Laboratory personnel must always sanitize hands in the following situations: • Before patient contact • After gloves are removed • Before leaving the work area • At any time when hands have been knowingly contaminated • Before going to designated break areas • Before and after using bathroom facilities Hand hygiene includes both hand washing and using alcohol-based antiseptic cleansers. Alcohol-based cleansers can be used when hands are not visibly contaminated, but those cleansers are not recommended after contact with sporeforming bacteria, including Clostridium difficile and Bacillus sp. because the spores formed by these bacteria are not killed by alcohol-based cleanser. Hand washing with soap and water and scrubbing to create a friction can remove these spores. When using alcohol-based cleansers, apply the cleanser to the palm of one hand. Rub your hands together and over the entire cleansing area, including the area between the fingers and thumbs. Continue rubbing until the alcohol dries. The CDC has developed guidelines to be followed for correct hand washing.1,11 Procedure 1-1 demonstrates CDC routine hand washing guidelines.4 More stringent procedures are used in surgery and in areas with patients who are highly susceptible, such as patients who are immunocompromised and/or burn patients.

Laboratory Coats Full-length, fluid-resistant laboratory coats with wrist cuffs are worn to protect clothing and skin from exposure to patients’ body substances. These coats should always be completely buttoned, and gloves should be pulled over the cuffs. They are worn at all times when working with patient specimens and are removed before leaving the work area. They are changed when they become visibly soiled. Disposable coats are placed in containers for biohazardous waste, and nondisposable coats are placed in designated laundry receptacles. Masks, Goggles, and Face Shields The mucous membranes of the eyes, nose, and mouth must be protected from specimen splashes and aerosols. A variety of protective equipment is available, including masks and goggles, full-face plastic shields that cover the front and sides of the face, masks with attached shields, and Plexiglas countertop shields. Particular care should be taken to avoid splashes and aerosols when removing container tops, pouring specimens, and centrifuging specimens. Transfer pipettes should be used when aliquoting samples from a specimen container. Specimens must never be centrifuged in uncapped tubes or in uncovered centrifuges. When specimens are received in containers with contaminated exteriors, the exterior of the container must be disinfected or, if necessary, a new specimen may be requested.

Biological Waste Disposal All biological waste, except urine, must be placed in appropriate leakproof containers labeled with the biohazard symbol (Fig. 1-2) or with red or yellow color coding. This includes both specimens and the materials with which the specimens come in contact. Then waste is decontaminated following facility policy: incineration, autoclaving, or pickup by a certified hazardous waste company. Urine may be discarded by pouring it into a laboratory sink under a Plexiglas countertop shield. Care must be taken to avoid splashing, and the sink should be flushed with water after specimens are discarded. Disinfection of the sink using a 1:5 or 1:10 dilution of sodium hypochlorite (bleach) should be performed daily. Sodium hypochlorite dilutions stored in plastic bottles are effective for 1 month if protected from light after preparation.12 Empty urine containers can be discarded as nonbiologically hazardous waste (Fig. 1-3).

Decontamination Regular disinfection is needed for contaminated nondisposable equipment, blood spills, and laboratory areas that process blood and body fluids. Laboratory countertops should be disinfected after every work shift and after any spill occurs. The

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Chapter 1 | Safety and Quality Management CDC recommends the use of a 1:10 dilution of sodium hypochlorite for routinely disinfecting countertops and accidental spills. The solution should be allowed to air-dry on the contaminated area. Absorbent materials used for cleaning countertops and removing spills must be discarded in biohazard containers. Other products are commercially available both in prefilled spray bottles and single-use wipes. However, it

55

must be confirmed that the product is effective enough to eliminate most bacteria, including Mycobacterium tuberculosis, fungi, and viruses. It is also important to know the contact time needed for disinfectant chemical products to work effectively on laboratory surfaces as prescribed by the manufacturer. When spills occur, do not mop or wipe the fluid; instead, use an absorbent powder (e.g., Zorbitrol) or paper towels to

PROCEDURE 1-1 Hand Washing Procedure Visit www.fadavis.com for Phlebotomy Video 3-1 and Video 3-2 (Hand Washing).

fingernails for at least 20 seconds; include your thumbs and wrists in the cleaning.

Equipment Antimicrobial soap Paper towels Running water Waste container Procedure 1. Wet your hands with warm water. Do not allow parts of your body to touch the sink. 4. Rinse your hands in a downward position to prevent recontamination of your hands and wrists.

2. Apply soap, preferably antimicrobial.

5. Obtain a paper towel from the dispenser.

3. Rub to form a lather, create friction, and loosen debris. Thoroughly clean between your fingers and under your

Continued

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PROCEDURE 1-1—cont’d 6. Dry your hands with a paper towel.

7. Turn off the faucets with a clean paper towel to prevent recontamination.

remove as much as possible before disinfecting. When using an absorbent powder, the liquid will solidify and then can be scooped up. If paper towels are used to absorb the liquid, pour bleach over the towels. Dispose of absorbent material and paper towels used for disinfection in the appropriate biohazard container. Disinfect the spill area with bleach or a phenol solution.

Sharp Hazards Sharp objects in the laboratory, including needles, lancets, and broken glassware, present a serious biological hazard, particularly for the transmission of bloodborne pathogens. All sharp objects must be disposed in puncture-resistant, leakproof container with the biohazard symbol. Puncture-resistant containers should be conveniently located within the work area. The biohazard sharp containers should not exceed three-fourths full and must always be replaced when the level of waste inside reaches the safe capacity mark.

Chemical Hazards The same general rules for handling biohazardous materials apply to chemically hazardous materials; that is, to avoid getting these materials in or on bodies, clothes, or the work area. Every chemical in the workplace should be presumed to be hazardous.

Chemical Spills and Exposure Figure 1–2 Biohazard symbol. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, p. 60, with permission.)

When skin or eye contact occurs, the best first aid is to flush the area with large amounts of water for at least 15 minutes and then seek medical attention. For this reason, all laboratory personnel should know the location and proper use of emergency showers and eye wash stations. Contaminated clothing should be removed as soon as possible. No attempt should be made to neutralize chemicals that come in contact with the skin.

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Chemical Handling Chemicals should never be mixed together unless specific instructions are followed, and they must be added in the order specified. This is particularly important when combining acid and water. Acid should always be added to water to avoid the possibility of sudden splashing caused by the rapid generation of heat in some chemical reactions. Wearing goggles and preparing reagents under a fume hood are recommended safety precautions. Chemicals should be used from containers that are of an easily manageable size. Pipetting by mouth is unacceptable in the laboratory.

Chemical Hygiene Plan OSHA also requires all facilities that use hazardous chemicals to have a written chemical hygiene plan (CHP) available to employees.13 The purpose of the plan is to detail the following: 1. Appropriate work practices 2. Standard operating procedures 3. PPE 4. Engineering controls, such as fume hoods and flammables safety cabinets 5. Employee training requirements 6. Medical consultation guidelines Each facility must appoint a chemical hygiene officer, who is responsible for implementing and documenting compliance with the plan. Examples of required safety equipment and information are shown in Figure 1-4.

A

Chemical Labeling

B Figure 1–3 Technologist disposing of urine (A) sample and (B) container.

Chemical spill kits containing protective apparel, nonreactive absorbent material, and disposable bags and waste tags for disposing of contaminated materials should be available for cleaning up spills. Liquids are absorbed using a neutralizer, such as sodium bicarbonate, and a sand mixture or ground clay. After the liquid is absorbed, it is scooped up and placed into a waste bag and labeled appropriately for disposal. Then the area is cleaned thoroughly. A chemical spill or exposure must be reported to the employee health department and an incident (variance) report submitted that documents the exposure according to the facility policy. Examples of chemical exposure include any time a chemical was breathed in or touched the skin, two or more chemicals that came in contact with each other and created an odor or fume, or an identified substance spill.

Hazardous chemicals should be labeled with a description of their particular hazard, such as poisonous, corrosive, flammable, explosive, teratogenic, or carcinogenic (Fig. 1-5). The National Fire Protection Association (NFPA) has developed the Standard System for the Identification of the Fire Hazards of Materials, NFPA 704.14 This symbol system is used to inform firefighters of the hazards that they may encounter with fires in a particular area. The diamond-shaped, color-coded symbol contains information relating to health (blue), flammability (red), reactivity (yellow), and specific hazards (white). Each category is graded on a scale of 0 to 4, based on the extent of concern. These symbols are placed on doors, cabinets, and containers. The federal Department of Transportation (DOT) regulations state that all chemicals are required to have the NFPA 704-M Hazard Identification System warnings on their shipping containers. An example of this system is shown in Figure 1-6.

Safety Data Sheets The OSHA Federal Hazard Communication Standard requires that all employees have a right to know about all chemical hazards present in their workplace. All chemicals and reagents containing hazardous ingredients in a concentration greater than 1% are required by OSHA to have a Safety Data Sheet (SDS) on file in the workplace. By law, vendors are required

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Part One | Basic Principles to provide these sheets to purchasers; however, the facility itself is responsible for obtaining and making SDSs available to employees. Information contained in an SDS includes the following: 1. Physical and chemical characteristics 2. Fire and explosion potential 3. Reactivity potential 4. Health hazards and emergency first aid procedures 5. Methods for safe handling and disposal 6. Primary routes of entry 7. Exposure limits and carcinogenic potential

The Globally Harmonized System of Classification and Labeling of Chemicals

A

The Globally Harmonized System (GHS) of Classification and Labeling of Chemicals is an international effort to standardize both the classification of hazardous chemicals and the symbols used to communicate these hazards on labels and in SDS documentation (Fig. 1-7). It includes criteria for the classification of health, physical, and environmental hazards. The standard label elements include GHS hazard pictograms, signal words, and a GHS hazard statement. The SDS under GHS provides a clear description of the data used to identify hazards and has 16 sections in a specified order (Box 1-1). OSHA aligned its Hazard Communication Standard with the GHS and requires that all employees be trained on the new label elements and SDS format. The adoption of the GHS in the United States has increased awareness and understanding of hazards in the workplace.

Chemical Waste Disposal State and federal regulations are in place for the disposal of chemicals and should be consulted. Any hazardous chemical waste should be disposed of per current Environmental Protection Agency (EPA) regulations. Most reagents used in the laboratory come with an SDS, which provides specific information for the disposal of particular chemicals. All chemicals should be disposed of by following the SDS directions. Sinks and drains should be flushed with large amounts of water after disposal of chemical waste.

Radioactive Hazards

B Figure 1–4 Chemical safety aids. A. Emergency shower. B. Eye wash station. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, p. 64, with permission.)

Radioactivity may be encountered in the clinical laboratory when procedures using radioisotopes are performed. The amount of radioactivity present in the clinical laboratory is very small and represents little danger; however, the effects of radiation are cumulative related to the amount of exposure. The amount of radiation exposure is related to a combination of time, distance, and shielding. People working in a radioactive environment are required to wear measuring devices to determine the amount of radiation that they are accumulating.

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A

59

B

C Figure 1–5 Chemical hazard symbols. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, p. 65, with permission.)

Laboratory personnel should be familiar with the radioactive hazard symbol shown here. This symbol must be displayed on the doors of all areas where radioactive material is present. Exposure to radiation during pregnancy presents a danger to the fetus; personnel who are pregnant or think they may be should avoid areas with this symbol.

Electrical Hazards The laboratory setting contains electrical equipment with which workers have frequent contact. The same general rules of electrical safety observed outside the workplace apply. The danger of water or fluid coming in contact with equipment is greater in the laboratory

setting. Equipment should not be operated with wet hands. Designated hospital personnel monitor electrical equipment closely; however, laboratory personnel should continually observe for any dangerous conditions, such as frayed cords and overloaded circuits, and report them to the supervisor. Equipment that has become wet should be unplugged and allowed to dry completely before reusing. Equipment also should be unplugged before cleaning. All electrical equipment must be grounded with three-pronged plugs. When an accident involving electrical shock occurs, the electrical source must be removed immediately. This must be done without touching the person or the equipment involved to avoid transferring the current. Turning off the circuit breaker, unplugging the equipment, or moving the equipment using a nonconductive glass or wood object are safe procedures

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Part One | Basic Principles to follow. The victim should receive immediate medical assistance after discontinuation of the electricity. Cardiopulmonary resuscitation (CPR) may be necessary.

HAZARDOUS MATERIALS CLASSIFICATION FIRE HAZARD Flash Point

HEALTH HAZARD 4 3 2 1 0

Deadly Extreme danger Hazardous Slightly hazardous Normal material

2

3 SPECIFIC HAZARD Oxidizer Acid Alkali Corrosive Use No Water Radiation

4 3 2 1 0

Below 73°F Below 100°F Below 200°F Above 200°F Will not burn

Fire/Explosive Hazards

1 W

REACTIVITY 4 May deteriorate 3 Shock and heat may deteriorate 2 Violent chemical change 1 Unstable if heated 0 Stable

OXY ACID ALK COR W

Figure 1–6 NFPA hazardous material symbols.

Health hazard

• Carcinogen • Mutagenicity • Reproductive toxicity • Respiratory sensitizer • Target organ toxicity • Aspiration toxicity

Gas cylinder

• Gases under pressure

Flame over circle

• Oxidizers

The Joint Commission (TJC) requires that all healthcare facilities post evacuation routes and detailed plans to follow in the event of a fire. Laboratory personnel should be familiar with these procedures. When a fire is discovered, all employees are expected to take the actions to protect themselves and others, following the acronym RACE: Rescue—rescue anyone in immediate danger Alarm—activate the institutional fire alarm system Contain—close all doors to potentially affected areas Extinguish/Evacuate—attempt to extinguish the fire, if possible, or evacuate, closing the door As discussed previously, laboratory workers often use potentially volatile or explosive chemicals that require special procedures for handling and storage. Flammable chemicals should be stored in safety cabinets and explosion-proof refrigerators,

Flame

• Flammables • Pyrophorics • Self-heating • Emits flammable gas • Self-reactives • Organic peroxides

Corrosion

• Skin corrosion/burns • Eye damage • Corrosive metals Environment (nonmandatory)

• Aquatic toxicity

Exclamation mark

• Irritant (skin and eye) • Skin sensitizer • Acute toxicity (harmful) • Narcotic effects • Respiratory tract irritant • Hazardous to ozone layer (nonmandatory) Exploding bomb

• Explosives • Self-reactives • Organic peroxides Skull and crossbones

• Acute toxicity (fatal or toxic)

Figure 1–7 GHS pictograms and hazards chart. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, Figure 3-16, p. 66, with permission.)

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Chapter 1 | Safety and Quality Management 61 Box 1–1

Globally Harmonized System (GHS) and Safety Data Sheet (SDS) Format

Sixteen-section SDS that includes the following headings in the order specified:

9. Physical and chemical properties 10. Chemical stability and reactivity

1. Identification

11. Toxicological information

2. Hazard(s) identification

12. Ecological information

3. Composition/information on ingredients

13. Disposal considerations

4. First-aid measures

14. Transport information

5. Firefighting measures

15. Regulatory information

6. Accidental release measures

16. Other information

7. Handling and storage From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, p. 67, with permission.

8. Exposure control/personal protection

and cylinders of compressed gas should be located away from heat and securely fastened to a stationary device to prevent accidental capsizing. Fire blankets may be present in the laboratory. Persons with burning clothes should be wrapped in the blanket to smother the flames. The NFPA classifies fires with regard to the type of burning material. It also classifies the type of fire extinguisher that is used to control them. This information is summarized in Table 1-2. The multipurpose ABC fire extinguishers are the most common, but the label should always be checked before using. It is important to be able to operate the fire extinguishers. The acronym PASS is about handling a fire extinguisher and can be used to remember the steps in the operation: 1. Pull pin 2. Aim at the base of the fire 3. Squeeze handles 4. Sweep nozzle side to side

Physical Hazards Physical hazards are not unique to the laboratory, and routine precautions observed outside the workplace apply. General precautions to consider are to avoid running in rooms and hallways; watch for wet

Table 1–2

floors; bend the knees when lifting heavy objects; keep long hair pulled back; avoid dangling jewelry; and maintain a clean, organized work area. Closed-toed shoes that provide maximum support are essential for safety and comfort.

Quality Management The term quality management (QM) refers to the overall process of guaranteeing quality patient care, and it is regulated throughout the total testing system. A quality management system (QMS) refers to all of the laboratory’s policies, processes, procedures, and resources needed to achieve quality testing.15 In a clinical laboratory, a QMS includes not only testing controls, referred to as quality control (QC), but also encompasses preexamination (preanalytical) variables (e.g., specimen collection, handling, and storage), examination (analytical) variables (e.g., reagent and test performance, instrument calibration and maintenance, personnel requirements, and technical competence), postexamination (postanalytical) variables (e.g., reporting of results and interpretation), and documentation that the program is being followed meticulously. The original terms preanalytical, analytical, and postanalytical have been replaced with the standard terms of preexamination, examination, and postexamination from the International Organization for Standardization (ISO).

Types of Fires and Fire Extinguishers

Fire Type

Extinguishing Material

Type/Composition of Fire

Extinguisher

Class A Class B Class C Class D

Wood, paper, clothing Flammable organic chemicals Electrical Combustible metals

Class K

Grease, oils, fats

Class A Class B Class C None Class ABC Class K

Water Dry chemicals, carbon dioxide, foam, or halon Dry chemicals, carbon dioxide, or halon Sand or dry powder Dry chemicals Liquid designed to prevent splashing and cool the fire

From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, with permission.

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All of the following are included in a QMS: • Procedure manuals • Internal quality control • External quality control • Electronic quality control • Calibration or calibration verification • Standardization • Proficiency testing (PT), more formally known as external quality assessment (EQA)16 • Record-keeping • Equipment maintenance • Safety programs • Training • Education and competency assessment of personnel • A review process that is scheduled and documented Essentially, QM is the continual monitoring of the entire test process, from test ordering and specimen collection through reporting and interpreting results. Written policies and documented actions as they relate to the patient, the laboratory, ancillary personnel, and the health-care provider are required. Having written remedial actions mandating the steps to take when any part of the system fails is essential to a QM program. QM in the urinalysis laboratory—or any other laboratory department—is an integration of many factors used to monitor, evaluate, and improve the quality of laboratory services through all three phases of patient testing. This section of the text will provide a collection of the procedures essential for providing quality urinalysis. In the following chapters, the methods of ensuring accurate results will be covered on an individual basis for each of the tests. Documentation of QM procedures is required by all laboratory accreditation agencies, including TJC, College of American Pathologists (CAP), American Association of Blood Banks (AABB), American Osteopathic Association (AOA), American Society of Histocompatibility and Immunogenetics (ASHI), American Association for Laboratory Accreditation (A2LA), and the Commission on Laboratory Assessment (COLA); it is also required for Medicare and Medicaid reimbursement. Guidelines published by CAP and the CLSI provide very complete instructions for documentation and are used as a reference for the ensuing discussion of the specific areas of urinalysis QC and QM.16-18 Documentation in the form of a procedure manual is required in all laboratories, and this format is used as the basis for the following discussion.

Urinalysis Procedure Manual A procedure manual containing all the procedures performed in the urinalysis section must be available for reference in the working area and must comply with the CLSI guidelines. The following information is included for each procedure: • Principle or purpose of the test • Clinical significance

• • • • • •

Patient preparation Specimen type and method of collection Specimen acceptability and criteria for rejection Reagents Standards and controls Instrument calibration and maintenance protocols and schedules • Step-by-step procedure • Calculations • Frequency and tolerance limits for controls and corrective actions • Reference values and critical values • Interpretation of results • Specific procedure notes • Limitations of the method • Method validation • Confirmatory testing • Recording of results • References • Effective date • Author • Review schedule Current package inserts should be reviewed and available at the workplace. Electronic manuals are acceptable and must be readily available to all personnel. As with written procedural manuals, electronic versions must be subjected to proper document control (i.e., only authorized persons may make changes, changes are dated/signed [manually or electronically], and there is documentation of periodic review).17,18 Evaluating procedures and adopting new methodologies is an ongoing process in the clinical laboratory. Whenever changes are made (Fig. 1-8), a person with designated authority, such as the laboratory director or section supervisor, should review and reference the procedure and then sign off on the change, and personnel should be notified of the changes. There must be documentation of an annual review of all procedures by the designated authority.

Preexamination (Preanalytical) Variables Preexamination (preanalytical) variables occur before the actual testing of the specimen and include test requests, patient preparation, timing, specimen collection, handling, and storage. Health-care personnel outside the clinical laboratory control many of these factors, such as ordering tests and specimen collection. Communication between departments and adequate training on the correct procedures for ordering a test (test utilization), collecting, and transporting the specimen improves the turnaround time (TAT) of results, avoids duplication of test orders, and ensures a high-quality specimen. TAT is defined as the amount of time required from the point at which a test is ordered by the health-care provider until the results are reported to the health-care provider. Laboratories determine the TAT for tests, including both stat and routine

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tests, as appropriate. Then the laboratory can monitor the TATs to determine areas in the process that need improvement. This can be determined by creating a cause-and-effect diagram, as shown in Figure 1-9.

URINALYSIS SECTION SPECIMEN ACCEPTABILITY/LABELING

Specimen Collection and Handling

Prepared by: Initial approval: Procedure placed in use: Revised: Reason for revision: Effective Date Supervisor Approval Medical Director Approval Reviewed Reviewed Reviewed Reviewed Reviewed

Figure 1–8 Example of procedure review documentation. (Adapted from the Department of Pathology, St. Joseph Hospital, Omaha, NE.)

Specific information on specimen collection and handling should be stated at the beginning of each procedure listed in the manual. Requisition forms and computerized entry forms should designate the type of urine specimen to be collected and the date and time of collection. The form should include space for recording 1. The actual date and time of specimen collection 2. Whether the specimen was refrigerated before transporting 3. The time the specimen was received in the laboratory and the time the test was performed 4. Tests requested 5. An area for specific instructions that might affect the results of the analysis 6. Patient identification information18 The patient’s sex, age or date of birth, and, when appropriate, the source of the specimen and the time it was collected must be documented.15 Along with the specific procedure, the requisition form must detail patient preparation (e.g., fasting or elimination of interfering medications), type and volume of specimen required, and the need for sterile or opaque containers. All

CAUSE-AND-EFFECT DIAGRAM

URINALYSIS TURN-AROUND-TIME (TAT)

Collection

Transport

Missed collection

Incorrect specimen type

Delayed delivery Contaminated specimen container

Insufficient urine volume Patient misidentification Special conditions not followed

Lost label

Insufficient container

No requisition Leaking closure lid

Missed pick-up Specimen misplaced Incorrect storage

Time of collection not indicated

Transport staff unavailable

No chemical preservative

Patient preparation

Incorrect container

Distance

Not refrigerated

Not fasting Medications

Incomplete requisition UA results TAT

Interfering substances Instrument malfunction

Report not charted Procedure failure

Computer down

Wrong specimen tested

Printer error Failure to send report

Inadequate volume

Test analysis

Figure 1–9 Cause-and-effect diagram for analyzing urinalysis TAT.

Reporting

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urine specimens should be examined within 2 hours. If this is not possible, written instructions for preserving the specimen must be available.18 The manual should also include instructions of a general nature, such as procedures for collecting clean-catch midstream and timed specimens, as well as information on specimen processing and printed instructions that are given to patients. Criteria for specimen rejection for both physical characteristics and labeling errors must be present. Box 1-2 gives an example of a policy for handling mislabeled specimens. Written criteria for rejecting specimens must be documented and available to the health-care provider and nursing staff.18 Box 1-3 lists the criteria for urine specimen rejection. Laboratory personnel must determine the suitability of a specimen and document any problems and corrective actions taken. An example of an internal laboratory quality improvement form is shown in Figure 1-10. It is used as a tool to document a problem at the point of discovery, describing what happened and the immediate corrective action taken. This enables the laboratory director to capture the information to

Box 1–2

Policy for Handling Mislabeled Specimens

Do NOT assume any information about the specimen or patient. Do NOT relabel an incorrectly labeled specimen. Do NOT discard the specimen until investigation is complete. Leave specimen EXACTLY as you receive it; put in the refrigerator for preservation until errors can be resolved. Notify nursing station, doctor’s office, etc. of problem and why it must be corrected for analysis to continue. Identify problem on specimen requisition form with date, time, and your initials. Make person responsible for specimen collection participate in solution of problem(s). Any action taken should be documented on the requisition slip. Report all mislabeled specimens to the appropriate supervisor. From Schweitzer, SC, Schumann, JL, and Schumann, GB: Quality assurance guidelines for the urinalysis laboratory. Journal of Medical Technology 3(11): 568, 1986, with permission.

determine the root cause analysis (RCA) and develop a preventive or corrective action plan. Laboratory information systems have the capability to electronically generate these forms for review. An acceptable specimen requires verification of the patient’s identification information on the requisition form and the container label, timely transport to the laboratory, the presence of refrigeration or recommended preservative if transport was delayed, and collection of an adequate amount of the correct urine specimen type in a noncontaminated, tightly closed container.18

Examination (Analytical) Variables The examination (analytical) variables are the processes that directly affect the testing of specimens. They include reagents, instrumentation and equipment, testing procedure, QC, preventive maintenance (PM), access to procedure manuals, and competency of personnel performing the tests.

Reagents The manual should state the name and chemical formula of each reagent used; instructions for preparation, when necessary, or company source of prepared materials; storage requirements; and procedures for reagent QC. The type of water used for preparing reagents and controls must be specified. Distilled or deionized water or clinical laboratory reagent water (CLRW) must be available. CLRW quality must be monitored regularly for microbial contamination as well as ionic and organic impurities.19 A written policy must be available with the procedures to follow when the CLRW quality is not within the tolerance limits. A bold-type statement of any safety or health precautions associated with reagents should be present. An example of this would be the heat produced in the Clinitest reaction. All reagents and reagent strips must be properly labeled with the date of preparation or opening, purchase and received date, expiration date, and appropriate safety information. Reagent strips should be checked against known negative and positive control solutions on each shift or, at a minimum, once a day and whenever a new bottle is opened. Reagents are checked daily or when tests requiring their use are requested. After checking is performed, laboratory personnel should properly record the results of all reagent checks. Reagent strips must never be refrigerated, must be protected from light, and must be recapped immediately after removing each strip.

Instrumentation and Equipment Box 1–3

Criteria for Urine Specimen Rejection

Unlabeled containers Nonmatching labels and requisition forms Specimens contaminated with feces or toilet paper Containers with contaminated exteriors Insufficient volume of urine Specimens that were transported or preserved improperly Delay between time of collection and receipt in the laboratory

The procedure manual should clearly state the instructions regarding the operation, performance, and frequency of calibration; limitations; and procedures to follow when limitations or linearity are exceeded, such as dilution procedures. Instructions detailing the appropriate recording procedures must be included. The instruments encountered in the urinalysis laboratory most frequently are refractometers, osmometers, automated reagent strip readers, and automated microscopy instruments. Refractometers are calibrated daily or when used against deionized water (1.000) and a known control,

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Quality Improvement Follow-up Report CONFIDENTIAL Instructions: Section I should be completed by the individual identifying the event. Date of report: Date of incident: Patient MR#

Reported by: Date/time of discovery: Patient accession#

Section I. Summary of incident — describe what happened

What immediate corrective action was taken?

Provide the ORIGINAL to team leader/technical specialist within 24 hours of incident discovery Date: To: Forwarded for follow-up: Date: To: Section II. Management investigation: Tracking # Instructions: Section II should be completed by laboratory management within 72 hours Check appropriate problem category Unacceptable patient samples (Due to hemolysis, QNS, or contaminated)

Wrong tube type

Equipment-related event

Misidentified sample

Standard operating procedure deviation

Wrong location

Communication problem/complaint

Other (explain)

Accident Explain answers:

Preventive/corrective action recommendations:

Technical specialist/team leader:

Date:

Medical director review:

Date:

Quality assurance review:

Date:

FDA reportable: Yes or no

Date reported:

Figure 1–10 Sample of quality improvement follow-up report form. (From Danville Regional Medical Center Laboratory, Danville, VA, with permission.)

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such as 5% saline (1.022 ± 0.001) or 9% sucrose (1.034 ± 0.001). Two levels of commercial controls are available for the osmometer, urine reagent strip tests, and human chorionic gonadotropin (hCG) kit tests. All control values must be recorded. Automated urinalysis systems and reagent strip readers are calibrated using manufacturer-supplied calibration materials, following the protocol specified by the manufacturer. Both positive and negative control values must be run and recorded (Fig. 1-11). There must be documentation showing evidence of corrective action for any failed QC tests. No patient’s testing may be performed until QC is acceptable. Other common equipment found in the urinalysis laboratory includes refrigerators, freezers, pipettes, centrifuges, microscopes, and water baths. Temperatures of refrigerators and water baths should be taken daily and recorded. Calibration of centrifuges and accuracy of timers are customarily performed every 3 months, and there should be a record of the appropriate relative centrifugal force for each setting. Disinfection of centrifuges should be done routinely on a weekly basis or after spills. Microscopes should be kept clean at all times and have an annual professional cleaning. Automatic pipettes are initially and periodically checked for accuracy and reproducibility. As mandated by the TJC or CAP guidelines, a routine PM schedule for instruments and equipment should be prepared and records kept of all routine and nonroutine maintenance performed. Deionized water used for reagent preparation is quality controlled by checking pH and purity meter resistance on a weekly basis and the bacterial count on a monthly schedule. All results must be recorded on the appropriate forms.

Testing Procedure Detailed, concise testing instructions are written in a step-bystep manner. Instructions should begin with specimen preparation, such as time and speed of centrifugation, and include types of glassware needed, time limitations and stability of specimens and reagents, calculation formulas and a sample calculation, health and safety precautions, QC checks, reference ranges, and procedures. Additional procedure information,

including reasons for special precautions; sources of error and interfering substances; criteria for the correlation of the physical, chemical, and microscopic urinalysis results; helpful hints; clinical situations that influence the test; alternative procedures; and acceptable TATs for stat tests are listed under the title of Procedure Notes following the step-by-step procedure. Reference sources should be listed. Manufacturer’s package inserts may be included but cannot replace the written procedure. The laboratory director must sign and date new procedures and all modifications of procedures before they are used.15

Quality Control QC refers to the materials, procedures, and techniques that monitor the accuracy, precision, and reliability of a laboratory test. QC procedures are performed to ensure that acceptable standards are met during the process of patient testing. The step-by-step instructions for each test should include specific QC information regarding the type of control specimen preparation and handling, frequency of use, tolerance levels, and methods of recording. QC is performed at scheduled times, such as at the beginning of each shift or before testing patient specimens, and it must always be performed if reagents are changed, an instrument malfunction has occurred, or test results are questioned by the health-care provider. Control results must be recorded in a log, either paper or electronic. Patient test results may not be reported until the QC is verified. Both external quality control monitoring and internal and electronic quality control processes are practiced in the urinalysis laboratory. External Quality Control External quality controls are used to verify the accuracy (ability to obtain the expected result) and precision (ability to obtain the same result on the same specimen) of a test, and they are exposed to the same conditions as the patient specimens. Reliability is the ability to maintain both precision and accuracy. Commercial controls are available for the urine chemistry tests, specific gravity, and certain microscopic constituents. Analysis of two levels of control material is required. The concentration

QUALITY CONTROL GLU mg/dL

BIL

Positive control SP BLD PH PROT NIT GR mg/dL 1.0 mg/dL KET

Month:__________20_____ Negative control LEU GLU BIL KET SP BLD PH PROT NIT EST GR mg/dL mg/dL 1.0 mg/dL

LEU EST

Control values Reagent Lot # EXP

TECH DATE

Figure 1–11 Sample instrument QC recording sheet. (Adapted from the Department of Pathology, Methodist Hospital, Omaha, NE, with permission.)

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Chapter 1 | Safety and Quality Management of controls should be at medically significant levels and should be as much like the human specimen as possible. Documentation of QC includes dating and initialing the material when it is first opened and recording the manufacturer’s lot number and the expiration date each time a control is tested and the test result is obtained. Food and Drug Administration (FDA) standards require that control material test negative for HIV and HBV. The same person who is performing the patient testing should perform the testing and interpretation of external controls in the laboratory. Control data are evaluated before releasing patient results. Data obtained from repeated measurements have a Gaussian distribution or spread in the values that indicate the ability to repeat the analysis and obtain the same value. The laboratory, after repeated testing, establishes the value for each analyte, and then it should calculate the mean and standard deviation (SD). The control mean is the average of all data points, and the SD is a measurement statistic that describes the average distance each data point in a normal distribution is from the mean. The coefficient of variation (CV) is the SD expressed as a percentage of the mean. The CV indicates whether the distribution of values about the mean is in a narrow versus broad range and should be less than 5%. Confidence intervals are the limits between which the specified proportion or percentage of results will lie. Control ranges are determined by setting confidence limits that are within ±2 SD or ±3 SD of the mean, which indicates that 95.5% to 99.7% of the values are expected to be within that range. Values are plotted on Levy-Jennings control charts to visually monitor control values. Immediate decisions about patient results are based on the ability of control values to remain within a preestablished limit. Changes in the accuracy of results are indicated by either a trend, which is a gradual changing in the mean in one direction, or a shift, which is an abrupt change in the mean (Fig. 1-12). Changes in precision are shown by a large amount of scatter about the mean and an uneven distribution above and below the mean that are most often caused by errors in technique. Corrective action, including the use of new reagents, reagent strips, or controls and the verification of lot numbers and expiration dates, must be taken when control values are outside the tolerance limits. All corrective actions taken are documented. A protocol for corrective action is shown in Figure 1-13. A designated supervisor reviews all QC results. Laboratories may participate in a commercial QC program. Participating laboratories return results from the same lot of QC material sent by the manufacturer back to the manufacturer for statistical analysis and comparison with other laboratories using the same methodology. Internal Quality Control Internal quality control consists of internal monitoring systems built in to the test system and are called internal or procedural controls.16,20 Internal controls monitor the sufficient addition of a patient sample or reagent, the instruments/reagents interaction, and, for lateral flow test methods, whether the sample migrated through the test strip properly.16

2

4

6

8

Day 10 12

14

16

18

67

20 + 2 SD + 1 SD X – 1 SD – 2 SD

In Control

+ 2 SD + 1 SD X – 1 SD – 2 SD Shift

+ 2 SD + 1 SD X – 1 SD – 2 SD Trend

Figure 1–12 Levy-Jennings charts showing in-control, shift, and trend results.

Electronic Controls Electronic controls use a mechanical or electrical device in place of a liquid QC specimen. This type of QC can be an internal or an external component inserted into a point-of-care (POC) instrument. Electronic controls verify the functional ability of a testing device, but they do not verify the integrity of the testing supplies. Many test systems use a combination of external and internal controls to verify that the entire test system is working properly. Proficiency Testing (External Quality Assessment) PT, or EQA, is the testing of unknown specimens received from an outside agency and provides unbiased validation of the quality of patient test results. Several commercial vendors, such as CAP, provide PT. Laboratories subscribing to these programs receive lyophilized or ready-to-use specimens for routine urinalysis and digital images or photomicrographs for sediment constituent identification. Personnel in subscribing laboratories test these proficiency survey specimens in the same manner as patient specimens. No communication with other laboratories is permitted. The subscribing laboratory returns the results to the PT vendors, where they are statistically analyzed with those from all participating laboratories. Then the director of the subscribing laboratory receives a report that evaluates laboratory accuracy and compares it with other laboratories using the same method of analysis. Corrective action must be taken for unacceptable results.16 The Clinical Laboratory Improvement Amendments (CLIA) mandates comparison testing for laboratory accreditation.20

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Part One | Basic Principles A. Record all actions taken and the resolution of any problems B. Use the flow diagram below: 1. Run control In control

Out of control

Proceed with testing

Go to Step 2

2. Inspect control for: Outdate (age), proper storage, correct lot number, signs of contamination Yes, there is a problem

No obvious explanation

Make new control and retest

Retest In control

Out of control

Proceed with testing

Go to Step 3

An IQCP requires risk assessment (RA), a quality control plan (QCP), and quality assessment (QA).21 RA identifies and evaluates potential problems that may occur in the entire testing process from preexamination through the examination and postexamination processes. Components that are assessed for potential errors are the specimen, test system, reagents, environment, and testing personnel. QCP establishes control procedures to reduce the possibility of reporting an inaccurate patient test result. It must include the number, type, and frequency of testing, as well as criteria for acceptable results of the QCs. IQCP QA involves selecting quality monitors that include: • QC records • PT records • Review of patient results • Specimen rejection logs • TAT reports • Records of preventive measures, corrective actions, and follow-up • Personnel competency records

3. Make up new bottle of control In control

Out of control

Discard old control and proceed with testing

Go to Step 4

4. Open new can of reagent strips and test with new control In control

Out of control

Discard bad reagent strips and proceed with testing

Switch lot numbers and retest

In control Discard entire lot, notify manufacturer, and proceed with testing

Out of control Notify supervisor or resource person: do not proceed with testing

Figure 1–13 “Out-of-control” procedures. (From Schweitzer, SC, Schumann, JL, and Schumann, GB: Quality assurance guidelines for the urinalysis laboratory. Journal of Medical Technology 3(11): 567–572, 1986, with permission.)

Individualized Quality Control Plan An individualized quality control plan (IQCP) is an alternative CLIA QC option that provides equivalent quality testing to meet the CLIA regulations for moderate- and highcomplexity tests and provider-performed microscopy procedures (PPMP) tests. IQCP considers the entire testing process— preexamination, examination, and postexamination—to ensure quality testing, not just the frequency of QC and number of QC materials. With this option, the laboratory can determine the frequency of QC testing based on information about the test, the risk assessment, and the accreditation agencies’ requirements.

Personnel and Facilities QC is only as good as the personnel performing and monitoring it. Personnel assessment includes education and training, continuing education, competency assessment, and performance appraisals. Each new employee must have documentation of training during orientation to the laboratory. This is a checklist of procedures and must include the date and initials of the person doing the training and of the employee being trained. Up-to-date reference materials and atlases should be readily available, and documentation of continuing education must be maintained.16 An adequate, uncluttered, safe working area is also essential for both quality work and personnel morale. Standard precautions for handling body fluids must be followed at all times.

Postexamination Variables Postexamination variables are processes that affect the reporting of results and correct interpretation of data. Reporting of test results to the appropriate health-care provider in an efficient and accurate manner is essential to quality patient care. Reports may be handwritten or instrument printouts. Reports may be delivered by telephone or fax or transmitted electronically to the requesting health-care provider. Laboratory reports must be present in the patient’s record. Required information includes: • Patient’s first and last name • Patient’s unique identification number • Specimen collection date and time • Specimen source (if pertinent to the test) • Condition of unsatisfactory specimen

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Chapter 1 | Safety and Quality Management • Tests performed, the results, and the reference ranges of the tests • Date and time of the final results generated • Facility where the test was performed

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instrument to the designated health-care provider. It is essential that the operator carefully review results before transmittal. The operator also may enter the results manually into the laboratory computer system and then transmit them to the health-care provider.

Written Reports

Telephone (Verbal) Results

Each procedure covered in the procedure manual should include standardized reporting formats and, when applicable, reference ranges. There must be a written procedure for reporting, reviewing, and correcting errors. Forms for reporting results should provide adequate space for writing and should present the information in a logical sequence. Standardized reporting methods minimize health-care provider confusion when interpreting results (Fig. 1-14).

The telephone is frequently used to transmit results of stat tests and critical values. Calls requesting additional results may be received from personnel on hospital units and from health-care providers. When telephoning results, confirm that the results are being reported to the appropriate person. The time of the call and the name of the person receiving the results must be documented according to the facility’s policy.

Electronic Results

Errors may be discovered in the laboratory through a QM procedure known as the delta check that compares a patient’s test results with the previous results. Variation outside the established parameters alerts laboratory personnel to the possibility of an error that occurred during the testing procedure or during patient identification. Many laboratory analyzers now have autoverification, an automated comparison of patient results with predetermined preapproved criteria, programmed into them.4 Erroneous results must be corrected in a timely manner to assure that the patient does not receive treatment based on incorrect results. Errors can occur in patient identification, specimen labeling, or result transcription. The patient’s record should be corrected as soon as the error is detected; however, the original result must not be erased in the event that the health-care provider treated the patient based on the erroneous results. An incident (variance) report documenting the problem and corrective action may need to be submitted. Appropriate documentation of erroneous results should follow facility protocol.

Electronic transmission is now the most common method for reporting results. Many urinalysis instruments have the capability for the operator to transmit results directly from the

MICROSCOPIC QUANTITATIONS Quantitate an average of 10 representative fields. Do not quantitate budding yeast, mycelial elements, Trichomonas, or sperm, but do note their presence with the appropriate LIS code. Epithelial cells/LPF None: Rare: Few: Moderate: Many: Casts/LPF None: Numerical ranges: RBCs/HPF None: Numerical ranges: WBCs/HPF None: Numerical ranges: Crystals/HPF None: Rare: Few: Moderate: Many: Bacteria/HPF None: Rare: Few: Moderate: Many: Mucous threads Rare: Few: Moderate: Many:

0 0–5 5–20 20–100 >100 0 0–2, 2–5, 5–10, >10 0 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 0 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100 0 0–2 2–5 5–20 >20 0 0–10 10–50 50–200 >200 0–1 1–3 3–10 >10

Figure 1–14 Sample standardized urine microscopic reporting format. (From University of Nebraska Medical Center, Omaha, NE, with permission.)

Result Errors

Critical Results There should be written procedures available for the reporting of critical values. Critical values are test results that are significantly lower or higher than the normal reference range. They can be life-threatening and must be relayed to the health-care provider immediately, following facility policy. Figure 1-15 is an example of the procedure instructions for reporting critical values in the urinalysis department analyzing pediatric specimens; this should include the presence of ketones or reducing substances in newborns.

Interpreting Results The specificity and the sensitivity for each test should be included in the procedure manual for correct interpretation of results. Sensitivity (the lowest level of an analyte that a test can detect) and specificity (the likelihood of measuring the analyte desired) vary among manufacturers. The manual should list all known interfering substances for evaluation of patient test data. A well-documented QM program ensures quality test results and patient care data.

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SUMMARY 1-1

Quality Management Errors

Preexamination

Instrument malfunction

Patient misidentification

Interfering substances present

Wrong test ordered

Misinterpretation of QC data

Incorrect urine specimen type collected

Postexamination

Insufficient urine volume

Patient misidentification

Delayed transport of urine to the laboratory

Poor handwriting

Incorrect storage or preservation of urine

Transcription error

Examination

Poor quality of instrument printer

Sample misidentification

Failure to send report

Erroneous instrument calibration

Failure to call critical values

Reagent deterioration

Inability to identify interfering substances

Poor testing technique

Reporting Critical Results In Urinalysis POSITIVE KETONES For children age 2 years and younger: All results positive for ketones should be telephoned to the appropriate nursing unit. Document the following information in the computer as a chartable footnote appended to the result: Time of telephone call Initials of the person making the call Name of the person receiving the telephone call POSITIVE CLINITEST For children age 2 years and younger: All Clinitest results should be telephoned to the appropriate nursing unit. Document the following information in the computer as a chartable footnote appended to the result: Time of telephone call Initials of the person making the call Name of the person receiving the telephone call

Figure 1–15 An example of procedure instructions for reporting critical values in the urinalysis section. A procedure review document similar to that shown in Figure 1–8 would accompany this instruction sheet.

For additional resources please visit www.fadavis.com

References 1. Clinical and Laboratory Standards Institute: Protection of Laboratory Workers from Occupationally Acquired Infections: Approved Guideline, ed 4, CLSI Document M29-A4, Clinical and Laboratory Standards Institute, Wayne, PA, 2014, CLSI. 2. Clinical and Laboratory Standards Institute: Clinical Laboratory Safety: Approved Guideline, ed 3, CLSI Document GP17-A3, Clinical and Laboratory Standards Institute, Wayne, PA, 2012, CLSI. 3. Clinical and Laboratory Standards Institute: Clinical Laboratory Waste Management: Approved Guideline, ed 3, CLSI Document GP05-A3, Clinical and Laboratory Standards Institute, Wayne, PA, 20011, CLSI. 4. Strasinger, SK, Di Lorenzo, MS: The Phlebotomy Textbook, ed 4. F.A. Davis, Philadelphia, 2019. 5. Centers for Disease Control and Prevention. Guidelines for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings, 2007. Web site: https://www.cdc.gov/ infectioncontrol/guidelines/isolation/index.html. Accessed April 18, 2019. 6. Occupational Exposure to Blood-Borne Pathogens, Final Rule. Federal Register 29 (Dec 6), 1991. 7. Occupation Safety and Health Administration. Revision to OSHA’s Bloodborne Pathogens Standard. Department of Labor. Web site: https://www.osha.gov/SLTC/bloodbornepathogens/ standards.html. Accessed April 18, 2019. 8. Centers for Disease Control and Prevention. Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposures to HBV, HCV and HIV and Recommendations for Post-exposure Prophylaxis. MMWR 50(RR11):1–42, 2001. Web site: https://www.cdc.gov/mmwr/preview/mmwrhtml/ rr5011a1.htm. Accessed April 18, 2019. 9. Centers for Disease Control and Prevention. Updated U.S. Public Health Service Guidelines for the Management of Occupational Exposure to HIV and Recommendations for Post-exposure Prophylaxis. MMWR 54(RR09):1–17, 2005. Web site: https://www.cdc.gov/mmwr/preview/mmwrhtml/ rr5409a1.htm. Accessed April 18, 2019.

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Chapter 1 | Safety and Quality Management 10. NIOSH Alert. Preventing Allergic Reactions to Natural Rubber Latex in the Workplace. DHHS (NIOSH) Publication 97-135. National Institute for Occupational Safety and Health, Cincinnati, OH, 1997. 11. Centers for Disease Control and Prevention. Guideline for Hand Hygiene in Health-Care Settings: Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force, MMWR 51(pages 21-26), 2002. Web site: https://www.cdc.gov/ mmwr/preview/mmwrhtml/rr5116a1.htm. Accessed April 18, 2019. 12. Centers for Disease Control and Prevention. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. Web site: https://www.cdc.gov/infectioncontrol/pdf/guidelines/ disinfection-guidelines.pdf. Accessed April 18, 2019. 13. Occupational Exposure to Hazardous Chemicals in Laboratories (Non-Mandatory Appendix); Technical Amendment. January 22, 2013. Office of the Federal Register. Web site: https://www. federalregister.gov/documents/2013/01/22/2013-00788/ occupational-exposure-to-hazardous-chemicals-in-laboratoriesnon-mandatory-appendix-technical. Accessed April 18, 2019. 14. National Fire Protection Association: Hazardous Chemical Data, No. 49. Boston, NFPA, 1991. 15. Centers for Medicare & Medicaid Services, Department of Health and Human Services: Clinical Laboratory Improvement Amendments. Web site: www.cms.hhs.gov/CLIA/05_CLIA_ Brochures.asp Accessed April 18, 2019.

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16. Clinical and Laboratory Standards Institute (CLSI): Quality Practices in Noninstrumented Point of Care Testing: An Instructional Manual and Resources for Health Care Workers. Approved Guideline. CLSI Document POCT08-A, Wayne, PA, 2010. 17. College of American Pathologists: Commission on Laboratory Accreditation, Urinalysis Checklist. College of American Pathologists, Skokie, IL, 2007. Web site: http://webapps.cap. org/apps/docs/laboratory_accreditation/checklists/urinalysis_ clinical_microscopy_sep07.pdf. Accessed April 18, 2019. 18. Clinical and Laboratory Standards Institute (CLSI), Urinalysis: Approved Guideline – Third Edition, CLSI Document GP16-A3, Wayne, PA, 2009. 19. Clinical and Laboratory Standards Institute (CLSI): Preparation and Testing of Reagent Water in the Clinical Laboratory: Approved Guideline, Fourth Edition, CLSI Document GP40-A4AMD, Wayne, PA 2006. 20. Centers for Medicare & Medicaid Services, Clinical Laboratory Improvement Amendments (CLIA). Proficiency Testing and PT Referral. Dos and Don’ts. Web site: https://www.cms.gov/ Regulations-and-Guidance/Legislation/CLIA/Downloads/ CLIAbrochure8.pdf. Accessed April 18, 2019. 21. Centers for Medicare & Medicaid Services (CMS). Clinical Laboratory Improvement Amendments (CLIA). Individualized Quality Control Plan (IQCP). Web site: https://www.cms.gov/ Regulations-and-Guidance/Legislation/CLIA/Individualized_ Quality_Control_Plan_IQCP.html. Accessed April 15, 2019.

Study Questions 1. Which of the following organizations publishes guidelines for writing procedures and policies in the urinalysis? A. CDC B. OSHA C. CLSI D. CLIA 2. Exposure to toxic, carcinogenic, or caustic agents is what type of laboratory safety hazard? A. Biological B. Sharps C. Chemical D. Fire/explosive 3. In the urinalysis laboratory, the primary source in the chain of infection would be: A. Patients B. Needlesticks C. Specimens D. Biohazardous waste

4. The best way to break the chain of infection is: A. Hand sanitizing B. Personal protective equipment C. Aerosol prevention D. Decontamination 5. The current routine infection control policy developed by CDC and followed in all health-care settings is: A. Universal Precautions B. Isolation Precautions C. Blood and Body Fluid Precautions D. Standard Precautions 6. An employee who is accidentally exposed to a possible bloodborne pathogen should immediately: A. Report to a supervisor B. Flush the area with water C. Clean the area with disinfectant D. Receive HIV prophylaxis

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72 Part One | Basic Principles 7. Personnel in the urinalysis laboratory should wear laboratory coats that: A. Do not have buttons B. Are fluid-resistant C. Have short sleeves D. Have full-length zippers 8. All of the following should be discarded in biohazardous waste containers except: A. Urine specimen containers B. Towels used for decontamination C. Disposable laboratory coats D. Blood collection tubes 9. An employer who fails to provide sufficient gloves for the employees may be fined by the: A. CDC B. NFPA C. OSHA D. FDA 10. An acceptable disinfectant for decontamination of blood and body fluids is: A. Sodium hydroxide B. Antimicrobial soap C. Hydrogen peroxide D. Sodium hypochlorite 11. Correct hand washing includes all of the following except: A. Using warm water B. Rubbing to create a lather C. Rinsing hands in a downward position D. Turning on the water with a paper towel 12. Centrifuging an uncapped specimen may produce a biological hazard in the form of: A. Vectors B. Sharps contamination C. Aerosols D. Specimen contamination 13. An employee who accidentally spills acid on his arm should immediately: A. Neutralize the acid with a base B. Hold the arm under running water for 15 minutes C. Consult the SDS D. Wrap the arm in gauze and go to the emergency department

14. When combining acid and water, ensure that: A. Acid is added to water B. Water is added to acid C. They are added simultaneously D. Water is slowly added to acid 15. An employee can learn the carcinogenic potential of potassium chloride by consulting the: A. Chemical hygiene plan B. Safety Data Sheet C. OSHA standards D. Urinalysis procedure manual 16. Employees should not work with radioisotopes if they are: A. Wearing contact lenses B. Allergic to iodine C. Sensitive to latex D. Pregnant 17. All of the following are safe to do when removing the source of an electric shock except: A. Pulling the person away from the instrument B. Turning off the circuit breaker C. Using a glass container to move the instrument D. Unplugging the instrument 18. The acronym PASS refers to: A. Presence of vital chemicals B. Operation of a fire extinguisher C. Labeling of hazardous material D. Presence of radioactive substances 19. The system used by firefighters to assess the risk potential when a fire occurs in the laboratory is: A. SDS B. RACE C. NFPA D. PASS 20. A class ABC fire extinguisher contains: A. Sand B. Water C. Dry chemicals D. Acid 21. The first thing to do when a fire is discovered is to: A. Rescue people in danger B. Activate the alarm system C. Close doors to other areas D. Extinguish the fire if possible

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Chapter 1 | Safety and Quality Management 73 22. If a red rash is observed after removing gloves, the employee: A. May be washing her hands too often B. May have developed a latex allergy C. Should apply cortisone cream D. Should not rub her hands so vigorously

30. During laboratory accreditation inspections, procedure manuals are examined for the presence of: A. Critical values B. Procedure references C. Procedures for specimen preservation D. All of the above

23. Pipetting by mouth is: A. Acceptable for urine but not serum B. Not acceptable without proper training C. Acceptable for reagents but not specimens D. Not acceptable in the laboratory

31. As the supervisor of the urinalysis laboratory, you have just adopted a new procedure. You should: A. Put the package insert in the procedure manual B. Put a complete, referenced procedure in the manual C. Notify the microbiology department D. Put a cost analysis study in the procedure manual

24. The NPFA classification symbol contains information on all of the following except: A. Fire hazards B. Biohazards C. Reactivity D. Health hazards 25. The GHS requires the following on a chemical label: A. Biohazard symbol, warning sign, environmental impact B. Hazard pictogram, signal words, hazard statement C. Biological symbol, hazard pictogram, long-term effects D. Signal words, hazard statement, biological symbol 26. The classification of a fire that can be extinguished with water is: A. Class A B. Class B C. Class C D. Class D 27. Employers are required to provide free immunization for: A. HIV B. HTLV-1 C. HBV D. HCV 28. A possible physical hazard in the hospital is: A. Wearing closed-toed shoes B. Not wearing jewelry C. Having short hair D. Running to answer the telephone 29. Quality management refers to: A. Analysis of testing controls B. Increased productivity C. Precise control results D. Quality of specimens and patient care

32. Indicate whether each of the following would be considered a 1) preexamination, 2) examination, or 3) postexamination factor by placing the appropriate number in the blank: Reagent expiration date Rejecting a contaminated specimen Constructing a Levy-Jennings chart Telephoning a positive Clinitest result on a newborn Calibrating the centrifuge Collecting a timed urine specimen 33. The testing of a specimen from an outside agency and the comparison of results with participating laboratories is called: A. External QC B. Electronic QC C. Internal QC D. Proficiency testing 34. A color change indicating that a sufficient amount of a patient’s specimen or reagent is added correctly to the test system would be an example of: A. External QC B. Equivalent QC C. Internal QC D. Proficiency testing 35. What steps are taken when the results of reagent strip QC are outside the stated confidence limits? A. Check the expiration date of the reagent strip B. Run a new control C. Open a new reagent strips container D. All of the above

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36. When a new bottle of QC material is opened, what information is placed on the label? A. The supervisor’s initials B. The lot number C. The date and the laboratory worker’s initials D. The time the bottle was opened

37. When a control is run, what information is documented? A. The lot number B. Expiration date of the control C. The test results D. All of the above

Case Studies and Clinical Situations 1. State a possible reason for an accreditation team to report a deficiency in the following situations: a. The urine microscopic reporting procedure has been recently revised. b. An unusually high number of urine specimens are being rejected because of improper collection. c. A key statement is missing from the Clinitest procedure. d. Open control bottles in the refrigerator are examined. 2. As the new supervisor of the urinalysis section, you encounter the following situations. Explain whether you would accept them or take corrective action. a. You are told that the supervisor always performs the CAP proficiency survey. b. QC is not performed daily on the reagent strips. c. The urinalysis section is primarily staffed by personnel assigned to other departments for whom you have no personnel data. 3. The medical laboratory scientist was assigned to test 10 urine specimens chemically. She removed 10 strips from the container and proceeded with testing. Several patients’ urine indicated a trace positive glucose in the urine. She then opened a new bottle of reagent dipsticks and proceeded to perform the QC. The negative control also tested as a trace positive for glucose. The medical laboratory scientist consulted the supervisor. The supervisor ran the QC, and the results were in the correct range. After observing the medical laboratory scientist’s technique, the supervisor realized that the medical laboratory

scientist was waiting too long to read the glucose results and therefore reporting erroneous results. a. What is wrong with this scenario? b. Who should run the QC for each test? Why? c. When should controls be run? d. What do you do when the QC is out of range? e. When can you report patient results? 4. An outpatient urine specimen was delivered to the laboratory at 0800 and placed on the counter in the urinalysis department. The medical laboratory scientist performed urinalysis on the specimen at 1130. The following results were abnormal: Clarity: Cloudy pH: 9.0 Nitrite: Positive The patient was a known diabetic; however, the glucose result was negative. a. What could be a possible cause for the abnormal results? b. Where would the information concerning what should have been done with this specimen be found, as well as the criteria for rejection? c. What QM procedure may have detected this error? d. What form will need to be completed for this scenario? e. How might this affect this patient’s care? f. How will the corrected results be documented?

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CHAPTER

2

Urine and Body Fluid Analysis Automation LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 2-1 Explain the principle of reflectance photometry. 2-2 Differentiate between semiautomated urine chemistry analyzers and fully automated urine chemistry analyzers. 2-3 State the advantages of automated microscopy methods over manual microscopy methods for analyzing urine sediment.

2-4 Describe laser-based flow cytometry, digital imaging, and auto particle recognition technologies used in automated urine microscopy analyzers. 2-5 Discuss the advantages of automated body fluid analyzers over the Neubauer hemocytometer for body fluid cell counts.

KEY TERMS Auto-checks

Digital imaging

Light-emitting diode (LED)

Auto particle recognition (APR)

Flow cytometry

Reflectance photometry

Autovalidated

Histograms

Scattergrams

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Urinalysis Automation Studies have shown that the major variable in urinalysis testing is the conscientiousness of the laboratory personnel in their timing and interpretations of the color reactions. Correct color readings depend on the accuracy of the timing. The ultimate goal of urinalysis automation is to improve reproducibility and color discrimination while increasing productivity and standardization for reporting urinalysis results.

Reflectance Photometry Subjectivity associated with visual discrimination among colors has been alleviated by the development of automated reagent strip readers that use a spectrophotometric measurement of light reflection, termed “reflectance photometry.” Reflectance photometry uses the principle that light reflection from the test pads decreases in proportion to the intensity of the color produced by the concentration of the test substance. In reflectance photometry, a monochromatic light source is directed toward the reagent pads by placing a filter between the light source and the reflective surface of the pad or by using a lightemitting diode (LED) to provide the specific wavelength needed for each test pad color reaction. The light is reflected to a photodetector, as well as a converter that is either analog or digital. The instruments compare the amount of light reflection with that of known concentrations and then display or print concentration units or transmit data to a laboratory information system (LIS). Several automated instruments are available that standardize sample processing, analyze chemistry test strips, perform urine sediment analysis, and report results with consistent quality and reduced hands-on time. The instruments are userfriendly and include visual and audio prompts for operation. The various manufacturers’ instruments include different features and principles for testing. See Table 2-1 for a breakdown of the measurement technologies used by the major urine analyzer manufacturers.1,2 Additional advantages to automation include online computer capability with an LIS interface; barcoding; manual entry of color, clarity, and microscopic results to be included on the

Table 2–1

printed report; flagging abnormal results; storing patient and control results; and minimal calibration, cleaning, and maintenance. Automated instruments in urinalysis include semiautomated and fully automated chemistry analyzers, automated urine cell microscopy analyzers, and systems that are completely automated. Semiautomated instruments still depend on an operator for specimen mixing, test strip dipping, and microscopic results input. In a fully automated chemistry analyzer, the tubes of urine are placed on a rack or a carousel and moved automatically through the instrument. Automated urine cell microscopy analyzers mix, aspirate, dilute, and stain urine to classify urine sediment particles. Automated urine systems perform a complete urinalysis that includes the physical, chemical, and microscopic parts of a routine urinalysis by integrating a fully automated chemistry analyzer with an automated urine cell microscopy analyzer. The automated urinalysis instruments currently available are listed in Table 2-2; however, new instruments are being developed continually.1,2

Analyzers Semiautomated Urine Chemistry Analyzers Semiautomated urine analyzers test for the chemical components of urine. The instruments read and interpret the reagent strip results consistently, thereby standardizing the interpretation of reagent strip results and eliminating personnel color bias and timing discrepancies. Depending on the instrument and the reagent strip used, the following tests can be performed: leukocyte, nitrite, protein, blood, glucose, ketone, bilirubin, urobilinogen, pH, specific gravity, color, creatinine, and protein-tocreatinine ratio. Semiautomated analyzers are well suited for small- and medium-volume laboratories and physicians’ offices and meet the Clinical Laboratory Improvement Amendments (CLIA)–waived standards. Semiautomated analyzers are self-calibrating, and some instruments perform automatic checks (auto-checks) to identify strip type and humidity exposure. For a semiautomated instrument, the reagent strips are manually dipped into the urine and placed on the strip reader, the reaction pads are read at the correct time, and the strip is moved to the waste container. The results

Measurement Technology Methods in Automated Urinalysis1,2 Urine Measurement Technology

Manufacturer

Color

Clarity

Specific Gravity

U.S. ARKRAY, Inc. Iris Diagnostics- Beckman Coulter, Inc. DIRUI Roche Diagnostics Siemens Healthcare Diagnostics, Inc. Sysmex Corporation

Reflectance photometry Light transmission/light scatter

Light scatter Light transmission/light scatter

Refractive index Refractive index

Reflectance photometry Reflectance photometry Reflectance photometry

Turbidity Turbidity Light transmission/light scatter

Refractometry Refractometry Refractive index

Reflectance photometry

Reflectance photometry

Refractometry

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Chapter 2 | Urine and Body Fluid Analysis Automation Table 2–2

Urinalysis Automation

Equipment

Manufacturer

Semiautomated Chemistry Instruments Clinitek Advantus Clinitek Status Urisys 1800 system COBAS u411 AUTION Eleven AE-4022

Siemens Healthineers Siemens Healthineers Roche Diagnostics Roche Diagnostics U. S. ARKRAY Inc.

Fully Automated Chemistry Instruments Clinitek Atlas Clinitek Novus Urisys 2400 system AUTION MAX AX-4030 IChemVELOCITY Cobas u 601 urine analyzer FUS-100 and FUS-200

Siemens Healthineers Siemens Healthineers Roche Diagnostics U.S. ARKRAY, Inc. Iris Diagnostics-Beckman Coulter, Inc. Roche Diagnostics DIRUI

Automated Microscopy UF-1000i Urine Cell Analyzer UN-2000 iQ 200 Automated Urine Microscopy (IQ200SELECT, IQ200ELITE, IQ200SPRINT) Urine Analyzer (iQ 200 Sprint) Cobus u 701 Microscopy Analyzer LabUMat 2

Sysmex Corporation Sysmex Corporation Iris Diagnostics-Beckman Coulter, Inc. Iris Diagnostics-Beckman Coulter, Inc. Roche Diagnostics 77 Elektronika

Automated Urinalysis Systems Iris Diagnostics-Beckman Coulter, Inc. iRICELL Urinalysis – IQ Series Urinalysis Workcell (iRICELL 3000plus, iRICELL 2000plus, iRICELL 3000pro, iRICELL 2000pro, iRICELL 1500) Siemens Healthineers CLINITEK AUWi Pro System Roche Diagnostics Cobas 6500 DIRUI Dirui FUS – 100/200 with H-800 77 Elektronika UriSed 2 or UriSed 3 with LabUMat2 Sysmex Corporation UX-2000 U.S. ARKRAY, Inc. AUTION HYBRID AU-4050 Body Fluid Analyzers ADVIA2120i with Body Fluids Software Sysmex XN-Series using Body Fluids mode iQ 200 using Body Fluids Software DxH 900 Hematology Analyzer

Siemens Healthcare Diagnostics, Inc. Sysmex Corporation Iris Diagnostics-Beckman Coulter, Inc. Beckman Coulter, Inc.

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are displayed, printed, or transmitted to an LIS. Patient identification and specimen color and clarity may be entered manually, or a barcode reader can be used to identify samples. Positive results are flagged to indicate a patient sample that requires additional confirmation testing or microscopic evaluation. The semiautomated instrument requires the operator to: 1. Dip the reagent strip into a urine sample that has been mixed well. 2. Blot the strip to remove excess urine. 3. Place the strip onto the reagent strip platform. 4. Press the analyze/enter button. The results are printed out, and abnormal results are flagged automatically. As the strip moves through the instrument, reflectance readings are taken at the correct time intervals. Some manufacturers’ test strips have a color compensation pad that adjusts results for urine color. This feature allows the instrument to subtract the urine color from the color developed on the reaction pad, providing an accurate result for each pad despite the interference. Then the strips are moved to the waste container. Results are stored in the analyzer, printed, or sent to the LIS. Examples of semiautomated instruments are shown in Figures 2-1 through 2-5. Daily maintenance is minimal and includes cleaning the reagent strip platform and emptying the reagent strip waste container.

Figure 2–2 Cobas u 411 urine chemistry analyzer. (Image courtesy of Roche Diagnostics.)

Fully Automated Urine Chemistry Analyzers Fully automated instruments are designed for a high-volume urinalysis laboratory with user walk-away capability. The various instruments can load many labeled tubes of urine on a carousel or rack at one time with the capability to insert a stat sample during the run. The “start” or “analyze” button is pressed to begin testing, and the sample moves through the instrument automatically. The sample is identified, mixed, and aspirated. A sample probe aspirates an exact amount of urine and dispenses it

Figure 2–1 AUTION ELEVEN™ AE-4022. (Image courtesy of U.S. ARKRAY, Inc.)

Figure 2–3 Urisys 1100 semiautomated urine chemistry analyzer. (Image courtesy of Roche Diagnostics.)

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Chapter 2 | Urine and Body Fluid Analysis Automation

A

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directly onto the reagent strip. The reagent strip advances automatically to the reflectance photometer to measure the color change of each reagent pad. Then the strip advances automatically to the disposal area. Tests are measured by the dry chemistry test strip, using reflectance photometry to detect color change and then taking readings at the appropriate time and wavelength for each specific test. Analytes measured vary by the instrument and may include leukocytes, ketones, protein, glucose, nitrite, blood, urobilinogen, pH, bilirubin, color, clarity, creatinine, and protein-low. Color is measured by either reflectance photometry or spectrophotometry at multiple wavelengths. Specific gravity is measured by the refractive index methodology, and clarity is a measurement of transmitted or scattered light. The instruments use integrated barcoded sample identification and allow abnormal ranges to be selected so that samples that require microscopic examination or confirmatory testing can be identified and flagged. Patient results and quality control results and calibrations are stored for visual display, printout, or transmission to an LIS. Standardized controls are run as set by laboratory protocol. Examples of fully automated urine chemistry analyzers are shown in Figures 2-6 through 2-9.

Automated Microscopy Analyzers

B

In a routine urinalysis, a test strip determines the chemical analytes and the formed elements are determined by microscopy. Manual microscopy is not easily standardized because of the high variation among operators even in the same facility. Routine specimen processing, such as centrifugation, can affect accuracy because rare elements, such as casts or cells, may be lost during handling. It has been demonstrated that recovery of formed elements in the sediment after centrifugation is highly variable. Results are not quantitative because they must be reported in

Figure 2–4 Clinitek Status + Analyzer. A. Clinitek Status Connect with barcode stand. B. Clinitek Status with test strip. (Images courtesy of Siemens Healthcare Diagnostics, Inc.)

Figure 2–5 Clinitek Advantus semiautomated urine chemistry analyzer. (Image courtesy of Siemens Healthcare Diagnostics, Inc.)

Figure 2–6 Urisys 2400 automated urine chemistry analyzer. (Image courtesy of Roche Diagnostics.)

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Part One | Basic Principles ranges or averages. Overall, manual microscopy is not cost effective because of the labor and time required to process and analyze the specimen, which ultimately increases turnaround times (TATs) for results. Automated urine cell microscopy analyzers provide efficient standardized results in less than 1 minute compared with approximately 6 minutes using the manual method, markedly improving TATs. A number of automated urine cell microscopy analyzers are currently available in the United States. Two types of technology are used for urine sediment analysis with these instruments: flow cytometry and digital imaging techniques.

Sysmex UF-1000i Fully Automated Urine Particle Analyzer and Sysmex UN-2000 Automated Urinalysis System Figure 2–7 Clinitek Atlas automated urine chemistry analyzer. (Image courtesy of Siemens Healthcare Diagnostics, Inc.)

Figure 2–8 AUTION MAX AX-4030 fully automated urine chemistry analyzer. (Image courtesy of U.S. ARKRAY, Inc.)

Figure 2–9 iChemVELOCITY automated urine chemistry analyzer. (Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.)

The Sysmex UF-1000i (Sysmex Corporation, Kobe, Japan) uses fluorescent flow cytometry to measure the forward-scattered, side-scattered, and fluorescence light characteristics of particles present in urine. The information obtained from these measurements is used to detect and identify stained urine sediment particles (Fig. 2-10). To perform an automated urine sediment analysis, 1.2 mL of uncentrifuged urine is aspirated into the instrument and divided into two channels: the sediment channel for urine particle analysis and the bacteria channel for bacteria staining and detection (Fig. 2-11). Each channel has a specific stain that targets surface and/or internal components of the cells. The stained urine sample passes through the flow cell, where it is hydrodynamically focused and presented to a red semiconductor laser (635 nm) (Fig. 2-12). Particles are identified by measuring the height and width of the fluorescent and light scatter signals, which are presented in scattergrams and histograms. In the bacteria channel, the diluent stabilizes the pH and lyses the nonbacterial particles, reducing interference from amorphous crystals. The stain is specific to the ribonucleic acid (RNA) in a bacterial cell, eliminating any nonspecific staining of debris. The width of the fluorescent signal measures cellular inclusions, and the width of forward light scatter measures the length of cells (Fig. 2-13).

Figure 2–10 Sysmex UF 1000i urine chemistry analyzer. Image courtesy of Sysmex Corporation, Kobe, Japan.)

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Chapter 2 | Urine and Body Fluid Analysis Automation Forward scatter signal amplifier

Fluorescence signal amplifier Red semiconductor laser

Sheath reagent

Side scatter signal amplifier Conductivity sensor

Dilution and staining for bacteria analysis

Dilution and staining for sediment analysis

Urine sample

Flow cytometry

Figure 2–11 Diagram of urine particle analysis in the Sysmex UF1000i. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Resulting values are presented in quantitative cells per microliter and cells per high- or low-powered field. Thresholds to be flagged for primary elements can be established, and abnormal results are flagged for confirmatory review. The main particles enumerated are red blood cells (RBCs), white blood cells (WBCs), squamous epithelial cells, hyaline casts, and bacteria. The results are displayed as scattergrams (Fig. 2-14). These parameters are reportable directly without technologist intervention and may be autovalidated. Flagged particles include pathological casts, crystals, small round cells (renal tubular epithelial cells or transitional epithelial cells), sperm, mucus, and yeastlike cells, and these particles must be

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confirmed by manual microscopy. A latex particle quality control system monitors performance, and quality control records can be viewed on the instrument screen in a LevyJennings graph. The analyzers can store up to 10,000 patient results, including scattergrams, histograms, and specimen characteristics, which can be saved and viewed by the user. A bidirectional interface is provided to download and report results. The UF-1000i can be used independently or integrated with an automated urine chemistry strip reader to create a complete urinalysis system. The newest Sysmex model, the UN-2000 (Fig. 2-15), is an automated, modular, and scalable urinalysis system that combines urine flow cytometry with digital image analysis by integrating the UF-5000 Fully Automated Urine Particle Analyzer (Fig. 2-16) and the UD-10 Fully Automated Urine Particle Digital Imaging Device (Fig. 2-17). The UF-5000 utilizes fluorescent flow cytometry coupled with a blue semiconductor laser to measure particle length, particle volume, particle internal complexity, and total amount of nucleic acid contained within the particle (Figs. 2-18 and 2-19). The shorter wavelength blue (488 nm) semiconductor laser offers the benefit of enhanced detection and differentiation of small particles. The sediment and bacteria channels used in the previous-generation UF-1000i have been replaced with two new analysis channels to further enhance the sensitivity and specificity of particle detection in urine sediment. The core channel (CR ch) stains elements with nuclear material, such as WBCs, epithelial cells, and bacteria (Fig. 2-20). In regard to the bacteria analysis in the CR ch, the degree of staining, and thus intensity, of the side fluorescence signal is dependent on the bacterial cell wall structure. The surface channel (SF ch) stains and measures anucleate elements, such as RBCs, crystals, and casts (Fig. 2-21).3 In addition to the two new measuring channels, the UF-5000 has a new depolarized side scatter (DSS) detector that provides information on the particles’ ability to depolarize

UF-1000i Technology

Diluents Sediments

Bacteria

Sediments

Bacteria

Incubation Detection unit Figure 2–12 Staining elements for the Sysmex UF1000i. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Stain Sediments

Bacteria

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Part One | Basic Principles UF-1000i Signal Waveforms for Cells Forward scattered Lateral fluorescent Lateral scattered light waveform light waveform light waveform

RBC

WBC

Bacteria

Epithelial cells

Figure 2–13 UF1000i signal waveform for cells. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Figure 2–15 UN-2000-110™ Sysmex Urinalysis System. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Figure 2–14 Scattergram showing Sysmex UF1000i microscopy results. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

light (Fig. 2-22). Through the detection of depolarized light, the DSS detector works to differentiate urine particles that are birefringent, such as crystals. An advantage of the Sysmex UF-5000 is that it requires a minimal volume of urine for analysis. The Sysmex UD-10 is a complementary digital imaging system designed to capture detailed images of urine particles in urine samples that require further review. All captured urine particle images are classified, based on size, into eight different classes. Then the technologist has the opportunity to review the captured images and identify the type(s) of urine particles present.3 The UN-2000 uses a single-screen data management system for monitoring analyzer status, result reporting, and reflex rule setting, as well as providing the connection point for interface

Figure 2–16 UF-5000™ Fully Automated Urine Particle Analyzer. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

with the LIS or middleware system. In the United States and Canada, this data management system is called the Urinalysis Data Manager (UDM). In regions outside the United States and Canada, U-WAM is the name of the data management system for UN-Series analyzers.

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Cell length Diameter of nucleus

Signal intensity

Nucleic acid content (fluorescence signal waveform area)

Figure 2–19 UF-5000™ measurement of the cell length, diameter, and nucleic acid content. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Figure 2–17 UD-10™ Fully Automated Urine Particle Digital Imaging Device. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Beckman Coulter’s iQ200 Microscopy Analyzer The iQ200 Automated Urine Microscopy Analyzer (Iris Diagnostics-Beckman Coulter, Brea, CA) uses digital flow morphology (imaging) and auto particle recognition (APR) to categorize and count urine particles automatically in uncentrifuged urine based on size, shape, texture, and contrast. The instrument also can be used for counts of body fluid cells by adding the optional body fluids software module (Fig. 2-23). The microscopy unit can be integrated with an automated urine chemistry analyzer to provide a complete urinalysis system.

The analyzer mixes the sample and aspirates 1.3 mL of urine. The sample is hydrodynamically focused in a planar flow cell and presented to a digital microscope in a thin moving sheet. Then a digital camera takes 500 images as the sample advances through the flow cell (Fig. 2-24). Individual urine particles are extracted from the raw images and automatically classified using a proprietary neural network algorithm into 12 major classification categories: RBCs, WBCs, WBC clumps, squamous epithelial cells, nonsquamous epithelial cells, unclassified casts, hyaline casts, unclassified crystals, bacteria, yeast, mucus, and sperm (Figs. 2-25 and 2-26).4 Results are either reviewed by a trained operator or autoreleased to the LIS based on user-defined parameters. Because the images are archived digitally, results can be reviewed easily and reclassified by the operator without the need for manual microscopy. In addition to the 12 major categories, the software allows the user to subclassify particles into 27 additional categories,

Figure 2–18 Diagram of the urine particle analysis in the Sysmex UF-5000™. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

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Figure 2–20 UF-5000 core channel. The CR channel analyzes nucleic acid–containing particles such as white blood cells, bacteria, and epithelial cells. Nucleic acid–containing cells are detected and classified through the use of a newly developed nucleic acid staining reagent. The nucleic acid–containing particles are further classified based on the area under the fluorescence signal waveform, which reflects the amounts of nucleic acid content and the known difference in nucleic acid content of different types of particles. A short-wavelength blue semiconductor laser is used to detect small particles, such as bacteria, with greater accuracy. In this channel, red blood cells are lysed and removed by a diluent, and crystals are removed by a chelating agent, contained in the diluent. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

refined strain

Figure 2–21 UF-5000 surface channel. The SF channel analyzes urine particles that do not contain nucleic acid, such as casts, red blood cells, and crystals. For differentiating casts from castlike elements (mucus threads, salt clumps, bacterial clumps, etc.), those particles are dispersed by reagents, and then the analyzer uses waveform information about particles stained with a newly developed staining reagent for more precise analysis. Besides this, amorphous salts are moved by the chelating action of a reagent and heating process. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

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Figure 2–22 UF-5000 depolarized side-scattered light (DSS) detector. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Figure 2–25 Auto particle recognition (APR) process. (Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.) Figure 2–23 iQ 200 microscopy analyzer. (Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.) s

o

l

c

c

Figure 2–24 Diagram of the iQ 200 digital flow capture process. (Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.)

such as specific types of crystals, casts, nonsquamous epithelial cells, yeast with pseudohyphae, trichomonas, and oval fat bodies. Additional user observations can be added to the report in the Edit Comment section.

77 Electronika UriSed 2 and 3Pro Automated Urine Sediment Analyzer The UriSed 2 and UriSed 3 Pro (77 Electronika, Budapest, Hungry) perform automated microscopy with digital imaging using auto image evaluation module (AIEM) software. The

Figure 2–26 iQ 200 urinalysis results display, showing particle categories available for analysis or counting. (Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.)

UriSed 2 requires a minimum of 2 mL of urine that is centrifuged in a special cuvette to produce a monolayer of urine sediment. The sediment is analyzed by a bright-field microscope and digital camera to capture and categorize 15 particle images based upon size and shape using AIEM software.1,5 An advantage to this instrument is the zoom capability to view images, and interpretation of the images is similar to that of manual microscopic smears. The UriSed 3 Pro incorporates phasecontrast microscopy in addition to bright-field microscopy to improve differentiation of elements, such as hyaline casts, RBC membranes, crystals, and yeast.1 Both the UriSed 2 and

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UriSed 3 can be linked with the chemistry analyzer LabUMat 2 to make a complete automated urinalysis laboratory system.1

FUS-100 and FUS-200 Analyzers The FUS-100 and FUS-200 urine sediment analyzers (DIRUI Industrial Co., Ltd., Changchun City, Jilin Province, China) identify cells using flat-flow cell digital imaging technology and a trained neural network. Artificial imaging identification (AII) software is used to classify and quantify the cells and formed particles in the uncentrifuged urine.6 A digital camera captures up to 820 photos, and the AII identifies and classifies 12 visible components in urine based on shape, contrast, texture, and frequency domain features.1,6 The FUS instruments can be integrated with the H800 chemistry analyzer for a total automated urinalysis system.6

Automated Urinalysis Systems Combining automated urine chemistry analyzers and automated urine cell analyzers to create completely automated urinalysis systems has improved TATs for urinalysis significantly. Technologists’ hands-on time has been reduced significantly as well. Using similar sample racks and moving on a conveyor system, samples are easily transferred from one instrument to the next, providing complete walkaway capability with minimal sample handling from sampling through results. By interfacing with the LIS, barcoded samples are identified automatically and processed according to the requested tests. The systems can independently perform both physical and chemical testing, microscopy analysis, and a combination of both. A complete urinalysis report can be sent directly to the LIS or printed out, thereby reducing clerical error. Autoverification of results and reflex testing can be validated according to laboratory protocol. Abnormal results are flagged for manual examination by laboratory personnel.

Clinitek AUWi System and AUWi Pro System The Clinitek Atlas System (Siemens Healthcare Diagnostics, Tarrytown, NY), an automated urine chemistry analyzer, and the Sysmex UF-1000i (Sysmex Corporation, Mundelein, IL), an automated urine cell analyzer, have been integrated to develop the Clinitek AUWi System (Siemens), which performs a completely automated urinalysis (Fig. 2-27). A minimum of 5 mL of urine is required in the automated mode. The barcoded tubes are racked and placed onto the system. The rack advances to the Atlas analyzer, where the sample is identified, mixed, aspirated, and tested for physical and chemical components. Then the sample travels across a connecting bridge to the UF-1000i for microscopic analysis. The instrument automatically reflexes samples requiring sediment analysis, reducing the time associated with manual microscopic analysis. Results are verified automatically and integrated into a complete urinalysis report to be sent to the LIS or printed. The Clinitek AUWi PRO Automated Urinalysis System integrates the Clinitek Novus Automated Urine Chemistry Analyzer and the Sysmex UF-1000i Urine Particle Analyzer. This automated system can load up to 200 sample tubes and complete up to 80 tests per hour. As with the AUWi system, the sample volume requirement is 5 mL in the automated

Figure 2–27 AUWi, a fully automated urinalysis system that combines the Siemens Clinitek Atlas Chemistry analyzer and the Sysmex UF-1000i particle analyzer. (Image courtesy of Siemens Healthcare Diagnostics, Inc.)

mode and 2 mL in the manual mode. Samples can be placed on the instrument with fewer manual steps, and no centrifugation is required. Autoverification of results to be reported to the LIS is based on laboratory protocol. In addition, the system is capable of automatically reflexing samples requiring sediment analysis based on rules defined by each laboratory.7

iRICELL Urinalysis Systems The iRICELL Automated Urinalysis System (Iris DiagnosticsBeckman Coulter, Brea, CA) consists of the iChemVELOCITY urine chemistry analyzer and the iQ200 urine microscopy analyzer (Fig. 2-28). A minimum of 4 mL of urine is required. Barcoded tubes are placed into the 10-position rack and are moved to the iChemVELOCITY. Upon completion of the physical and chemical analysis, the rack moves across the connecting bridge to the iQ200 for microscopy testing, where a reflex rule may be applied based on settings. Combined chemistry and microscopy urinalysis results are transmitted to the LIS or printed. The IQ series of urinalysis workcells (iQ2000 and iQ3000) pairs the Iris IRICELL series of urine microscopy instrumentation and the ARKRAY AUTION MAX AX4030 fully automated urine chemistry analyzer to provide a complete walk-away urinalysis system (refer back to Table 2-2).

Figure 2–28 iRICELL3000, a fully automated urinalysis system that combines the iChemVELOCITY urine chemistry analyzer and the iQ 200 microscopy analyzer. (Image courtesy of Iris DiagnosticsBeckman Coulter, Inc.)

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LabUMat 2 with UriSed 2 or UriSed 3 System The chemistry analyzer LabUMat 2 (77 Electronika) can be integrated with the UriSed 2 or UriSed 3 (77 Electronika) microscopic urine analyzers to make a complete automated urinalysis laboratory system. The minimum volume is 3 mL of uncentrifuged urine for the combined instruments, and the system can analyze up to 240 tests per hour. The samples are loaded into the 10-position sample racks where the physical (color, clarity, and specific gravity) and chemical tests (10 parameters) are performed by the LabUMat2. Then the sample racks are transferred across a connecting bridge to the UriSed 2 (bright-field microscopy) or the UriSed 3 (both bright-field and phase microscopy) for the urine microscopic analysis.

cobas 6500 Urine Analyzer The cobas 6500 Urine Analyzer (Roche Diagnostics, Indianapolis, IN) is another fully automated urine system. Its modular design integrates the cobas u 701 microscopy analyzer and the cobas u 601 fully automated urine chemistry analyzer into one platform. Cassettes with urine testing strips and sediment cuvettes are loaded onto the instrument, and 2.8 mL of urine is required. Urine is pipetted on the chemical strip, where 12 physical and chemical urine tests are performed using the cobas u 601 urine analyzer. After chemical testing, the sample is resuspended before pipetting and automatically centrifuged at 2000 rpm for 10 seconds to a monolayer of sediment. The cobas u 701 uses digital imaging to take 15 microscopic images of the sediment, and the images are displayed on the result screen. Particle recognition software determines the identification of RBCs, WBCs, bacteria, epithelial cells, casts, crystals, yeast, sperm, and mucus. Automated result validation and automated reflex testing are available.

UX-2000 Automated Urinalysis Analyzer The UX-2000 (Sysmex Corporation, Kobe, Japan) is a fully automated integrated urine analyzer. It consists of a chemical component for analyzing the physical and chemical part of urine as well as a flow cytometry component for microscopic examination of sediments contained in a single instrument.8 The system requires 5 mL of urine. For the physical examination, refractometry, reflectivity measurement, and light scattering are used to measure specific gravity, turbidity, and color. The chemical examination uses a test strip that is measured by the dualwavelength reflectance methods.9 The microscopic examination uses fluorescent-flow cytometry to measure RBCs, WBCs, hyaline casts, bacteria, and epithelial cells. Crystals, yeast, small round cells, spermatozoa, and casts are detected and flagged for laboratory personnel to review because this analyzer cannot differentiate between those types of particles in urine sediment.8 Visit www.fadavis.com for Video 2-1 (Automated urine chemical strip test).

Body Fluid Analysis Automation Traditionally body fluid counts for RBCs and WBCs, as well as WBC differentials, are performed manually using a hemocytometer and optical microscopy. Three types of hemocytometers

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are available—Fuchs-Rosenthal, Nageotte, and Neubauer—and each differs in the design of the calibrated counting area etched on the slide. An exact amount of fluid fills the chamber that provides a defined volume for cell enumeration. These procedures are labor-intensive and time-consuming, and often they are subject to technologist variability. Visit www.fadavis.com for Video 2-2 (Hemacytometer).

Automation brings quality control, precision, faster TATs, and standardization of results10 to a method that previously was uncontrolled. However, automated instruments have not eliminated completely the use of a manual hemacytometer count. Body fluids with low cell counts or malignant cells still require a manual differential using a stained cytospin smear. The laboratory must define the limits for the automated instrument and establish the lower limits for cell counting to determine when a manual procedure must be performed.5 For example, the iQ 200 is linear down to zero (0 to 10,000 cells/L). Linearity can be extended during method comparison. In addition, the laboratory must follow manufacturers’ recommended procedures for special treatment required for the specific body fluid analyzed, intended use, and reportable ranges.11 Visit www.fadavis.com for Video 2-3 (Making a cytospin smear).

Hematology analyzers that are used to perform body fluids cell counts include the ADVIA 2120i (Siemens), the Sysmex XN-Series analyzers (Sysmex America, Inc.), and the DxH 900 (Beckman Coulter, Inc.).

ADVIA 2120i The ADVIA 2120i uses flow cytometry, light scattering, and absorbance to count RBCs and WBCs, as well as to perform a WBC differential that includes percentages and absolute numbers of mononuclear cells and polymorphonuclear cells on specimens with more than 20 WBC/µL. The WBC differential includes the numbers of neutrophils, lymphocytes, monocytes, and eosinophils. A specimen of cerebrospinal fluid (CSF) is pretreated with CSF reagent to fix and spherize the cells. The prepared specimen remains stable for 4 minutes to 4 hours when stored at 18°C to 30°C. The specimen is aspirated into the instrument, and cells are differentiated and enumerated by three optical measurements. The signals are digitized and used to construct the CSF cytogram. With this system, more cells are counted, achieving increased accuracy and precision. The automated results for RBC, WBC, polymorphonuclear, mononuclear, and differential are available within 1 minute of sample aspiration. In addition, the ADVIA 2120i can provide a rapid automated diagnostic test for fetal lung maturity by counting lamellar bodies in amniotic fluid. Lamellar bodies are counted in the platelet channel using high and low laser light scattering. The analyzer is approved for counting cells in pleural fluids, peritoneal fluids, and peritoneal dialysates.

Sysmex XN-Series Analyzers The Sysmex XN-Series analyzer is the newest-generation hematology analyzer that includes a dedicated body fluid mode that

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is cleared for analysis of synovial fluid, pleural fluid, and peritoneal fluid, and the XN-10 analyzer is also cleared for CSF specimens in the body fluid mode.10 Bronchoalveolar lavage and amniotic fluid are not cleared for analysis on the XN-Series analyzers. Table 2-3 lists the fluid types cleared for analysis on the XN-Series analyzers. The body fluid mode has extended cell counting to increase precision in specimens with small numbers of cells. Body fluids can be analyzed without specimen preparation or pretreatment. The body fluid mode on the XN-Series reports both a body fluid white blood cell count (WBC-BF) and a total nucleated body fluid cell count (TC-BF) as well as a reportable two-part automated differential that differentiates mononuclear and polymorphonuclear cells using flow cytometry technology.10 The XN analyzer also identifies high-fluorescing body fluid cells (HF-BF), such as mesothelial cells, synovial cells, and malignant/tumor cells, or, rarely, large cell clusters. Digital imaging technology also may be used for body fluid differentials. The CellaVision DI-60 software classifies five different nucleated cell types found in body fluid. This software allows users to add reference cells to a digital library, enables tagging and sharing of cell images electronically, and offers a program to improve staff competency.10 The XN analyzer uses impedance counting principles for RBC-BF enumerations and flow cytometry for performing the WBC-BF counts and the two-part differential. The XN series uses Lysercell WDF as an RBC lysing reagent and Fluorocell WDF to stain RNA and DNA in the nucleated cells. In addition, the differential scatterplots should be inspected visually to detect noncellular particulate matter, such as bacteria, Cryptococcus, and interference from large cells (macrophages and mesothelial cells).

GloCyte Automated Cell Counter for CSF The GloCyte analyzer (Sysmex Corporation, Kobe, Japan) combines the principles of both technologies to accurately enumerate cells present in CSF, even at low numbers.10 A fluorochromelabeled antibody stains the RBCs, and a dye specific to nucleic acids in WBCs is used to treat aliquots of the CSF specimen.10 Digital imaging is used to count the cells as they are illuminated with a semiconductor laser. The instrument automatically enumerates each cell type and displays the stained cells on a screen.10

Beckman Coulter’s iQ200 The iQ200 (Iris Diagnostics – Beckman Coulter, Brea, CA) can be used for body fluid analysis using the iQ200 Body Fluids Table 2–3

Body Fluids Analyzed on the Sysmex XN-Series10

CSF Peritoneal fluid Pleural fluid Pericardial fluid Synovial fluid CSF, Cerebrospinal fluid.

XN-10

XN-11

XN-20

Yes Yes Yes No Yes

No Yes Yes No Yes

Yes Yes Yes No Yes

Module. The Body Fluid menu includes such fluids as CSF, synovial, pleural, peritoneal, peritoneal dialysate, peritoneal lavage, pericardial, and general serous. Two dilutions of the body fluid specimen are analyzed; one tube is diluted with Iris Diluent, and the other with the iQ Body Fluids Lysing Reagent. The instrument provides counts for total cells, nucleated cells, and RBCs. As with urine microscopy, results are either reviewed by a trained operator or auto-released to the LIS based on user-defined-parameters. Visit www.fadavis.com for Video 2-4 (Body fluid cell count automated analyzer).

For additional resources please visit www.fadavis.com

References 1. Kenyon, SM, and Cradic, KW: Automated urinalysis in the clinical lab. Clinical Issues – Urinalysis. Web site: http://www. mlo-online.com. Published October 24, 2017. Accessed February 19, 2020. 2. Halasey, S: Tech guide: Urinalysis. Clinical Laboratory Products (CLP). April 2019. 3. Godfrey, D: Advancing urinalysis. Thoughtful automation aims for a new level of standardization and workflow efficiency. Clinical Lab Products (CLP). Web site: http://www.clpmag.com/ 2019/05/advancing-urinalysis. Published May 22, 2019. Accessed July 31, 2019. 4. Clinical Diagnostic Products and Solutions/Urinalysis/iQ200 Series. Beckman Coulter, 2019. Web site: https://www. beckmancoulter.com/en/products/urinalysis/iq200. Accessed July 29, 2019. 5. Block, DR, and Lieske, JC: Automated urinalysis in the clinical lab. Medical Laboratory Observer. Web site: www.mlo-online. com. Published October 19, 2012. Accessed February 19, 2020. 6. Yuksel, H, Kilic, E, Ekinci, A, and Evliyaoglu, O: Comparison of fully automated urine sediment analyzers H800-FUS100 and Labumat-UriSed with manual microscopy. J Clin Lab Anal. 27:312–316, 2013. 7. AuWi Pro Automated Urinalysis System Brochure. Siemens Diagnostics, 2015. Web site: www.usa.siemens.com/ diagnostics. Accessed July 31, 2019. 8. Wesarachkitti, B, Khejonnit, V, Pratumvinit, B, Reesukumal, K, Meepanya, S, Pattanavin, C, and Wongkrajang, C: Performance evaluation and comparison of the fully automated urinalysis analyzers UX-2000 and cobas 6500. Lab Med 47(2):124–133, 2016. https://doi.org/10.1093/labmed/lmw002. Accessed August 1, 2019. 9. Laiweipithaya, S, Wongkraiang, P, Reesukumal, K, Bucha, C, Meepanva, S, Pattanavin, C, Kheionnit, V, and Chuntarut, A: UriSed 3 and UX-2000 automated urine sediment analyzers vs. manual microscopic method: A comparative performance analysis. J Clin Lab Anal. 2018 Feb;32(2). Doi: 10.1002/jcla. 22249. Epub 2017 May 2. Accessed February 19, 2020. 10. Sysmex: The Value-Driven Laboratory. White Paper. Automating Body Fluid Analysis for Increased Efficiency. 2019. Web site: www.sysmex.com/us. Accessed April 23, 2020. 11. Clinical and Laboratory Standards Institute: Body Fluid Analysis for Cellular Composition: Approved Guideline. CLSI Document H-56A. CLSI, Wayne, PA, 2006.

Additional Information Sources U.S. ARKRAY, Inc., Minneapolis, MN: www.arkrayusa.com DIRUI, Changchun, China: en.dirui.com.cn

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Roche Diagnostics, Indianapolis, IN: www.roche.com/products Siemens Healthcare Diagnostics Inc., Deerfield, IL: www.usa. siemens.com/diagnostics Sysmex Corporation, Kobe, Japan: www.sysmex.com/usa

Study Questions 1. The principle commonly used to measure the concentration of a particular analyte in the chemical examination of urine is: A. Reflectance photometry B. Digital imaging C. Flow cytometry D. Auto particle recognition 2. In automated urinalysis, the specific gravity is measured by: A. Light transmittance B. Light scattering C. Refractometry D. Turbidity 3. All of the following are true concerning fully automated urine chemistry analyzers, except: A. They are designed for a high-volume urinalysis laboratory. B. The reagent strip is dipped into the well-mixed urine. C. The urine tube moves through the instrument. D. A sample probe aspirates the urine. 4. The advantages of an automated urine microscopy analyzer over manual microscopy includes: A. Cost-effective B. Centrifugation not required C. Standardized results D. All of the above 5. Which of the following is a complete urinalysis automated urinalysis system? A. AUTION ELEVEN AE 4022 B. Clinitek Atlas C. iQ200 Automated Urine Microscopy D. Clinitek AUWi Pro System 6. What two technologies are used for urine sediment analysis? A. Light scattering and refractometry B. Light scattering and flow cytometry C. Flow cytometry and digital imaging D. Digital imaging and refractometry

7. Which automated urine particle counter combines urine flow cytometry with digital image analysis? A. UN-2000 B. iRICELL C. UF-1000i D. iQ 200 8. Which of the following urine sediment particles cannot be autovalidated but will be flagged and must be reviewed by laboratory personnel? A. RBCs B. WBCs C. RTEs D. Squamous epithelial cells 9. Which of the automated body fluid analyzers does not need to dilute or pretreat body fluids before analysis? A. ADVIA 2120i B. XN Series C. iQ 200 D. None of the above 10. What is a disadvantage of counting body fluid cells using an automated instrument versus a Neubauer hemocytometer? A. Less labor-intensive and time-consuming B. More precise C. Unable to count low WBC numbers and malignant cells D. Able to perform a WBC differential

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Case Studies and Clinical Situation 1. A medical laboratory scientist student is being trained on the laboratory automated urinalysis system that performs both the chemical and microscopic examination of urine. The student poured 12 mL of urine into the urine tube that was labeled correctly and placed it on the instrument. The reagent strip results indicated that there was a large amount of blood and leukocytes; however, the microscopic results reported 0 to 2 RBC/hpf and 0 to 2 WBC/hpf. a. Do the reagent strip and microscopic results correlate? b. What mistake in the procedure may have caused these results? c. What should be performed to ensure that the instrument was functioning properly?

2. A patient in the emergency department (ED) presented with a high temperature, headache, and stiff neck. The doctor suspected meningitis and performed a lumbar puncture to obtain a cerebrospinal fluid (CSF) specimen for analysis in the laboratory. a. What instruments would be used to count the number of white blood cells in the CSF? b. Which instrument would require that the specimen be pretreated? c. How long is the prepared CSF specimen stable?

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CHAPTER

3

Introduction to Urinalysis LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 3-1 List three major organic and three major inorganic chemical constituents of urine.

3-8 Discuss the actions of bacteria on a urine specimen that is unpreserved.

3-2 Describe a method for determining whether a fluid in question is urine.

3-9 Briefly discuss five methods for preserving urine specimens, including the advantages and disadvantages of each.

3-3 Recognize normal and abnormal daily urine volumes. 3-4 Describe the characteristics of the specimen containers recommended for urine. 3-5 Describe the correct procedure for labeling urine specimens. 3-6 State four possible reasons why a laboratory would reject a urine specimen. 3-7 List the changes that may take place in a urine specimen that remains at room temperature for more than 2 hours.

3-10 Instruct a patient in the correct procedure for collecting the following specimens: random, first morning, 24-hour timed, catheterized, midstream clean-catch, suprapubic aspiration, three-glass collection, four-glass collection, and pediatric. Identify a diagnostic use for each collection technique. 3-11 Describe the type of specimen needed for optimal results when a specific urinalysis procedure is requested.

KEY TERMS Albuminuria

Midstream clean-catch specimen

Random specimen

Anuria

Nocturia

Suprapubic aspiration

Catheterized specimen

Oliguria

Timed specimen

Chain of custody (COC)

Polydipsia

First morning specimen

Polyuria

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History and Importance Analyzing urine was actually the beginning of laboratory medicine. References to the study of urine can be found in the drawings of cavemen and in Egyptian hieroglyphics, such as the Edwin Smith Surgical Papyrus. Pictures of early physicians commonly showed them examining a bladder-shaped flask of urine (Fig. 3-1). Often these physicians never saw the patient, only the patient’s urine. Although these physicians lacked the sophisticated testing mechanisms now available, they were able to obtain diagnostic information from such basic observations as color, turbidity, odor, volume, viscosity, and even sweetness (by noting that certain specimens attracted ants or tasted sweet). These same urine characteristics are still noted by laboratory personnel. However, modern urinalysis has expanded beyond the physical examination of urine to include chemical analysis and microscopic examination of urinary sediment. Many well-known names in the history of medicine are associated with the study of urine, including Hippocrates, who, in the 5th century BCE, wrote a book on “uroscopy.” During the Middle Ages, physicians concentrated their efforts very intensively on the art of uroscopy, receiving instruction in urine examination as part of their training (Fig. 3-2). By 1140 CE, color charts had been developed that described the significance of 20 different colors (Fig. 3-3). Chemical testing progressed from “ant testing” and “taste testing” for glucose to Frederik Dekkers’s discovery in 1694 of albuminuria by boiling urine.1

Figure 3–2 Instruction in urine examination. (Courtesy of National Library of Medicine.)

Figure 3–1 Physician examines urine flask. (Courtesy of National Library of Medicine.)

The credibility of urinalysis became compromised when charlatans without medical credentials began offering their predictions to the public for a healthy fee. These charlatans, called “pisse prophets,” became the subject of a book published by Thomas Bryant in 1627. The revelations in this book inspired the passing of the first medical licensure laws in England— another contribution of urinalysis to the field of medicine. The invention of the microscope in the 17th century led to the examination of urinary sediment and to the development by Thomas Addis of methods for quantitating the microscopic sediment. Richard Bright introduced the concept of urinalysis as part of a doctor’s routine patient examination in 1827. By the 1930s, however, the number and complexity of the tests performed in a urinalysis had reached a point of impracticality, and urinalysis began to disappear from routine examinations. Fortunately, the development of modern testing techniques rescued routine urinalysis, which has remained an integral part of the patient examination. Two unique characteristics of a urine specimen account for this continued popularity: 1. A urine specimen is readily available and easily collected.

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for nearly half of the total dissolved solids in urine. Other organic substances include primarily creatinine and uric acid. The major inorganic solid dissolved in urine is chloride, followed by sodium and potassium. Small or trace amounts of many additional inorganic chemicals are also present in urine (Table 3-1). Dietary intake greatly influences the concentrations of these inorganic compounds, making it difficult to establish normal levels. Other substances found in urine include hormones, vitamins, and medications. Although not a part of the original plasma filtrate, the urine also may contain formed elements, such as cells, casts, crystals, mucus, and bacteria. Increased amounts of these formed elements are often indicative of disease. Sometimes it is necessary to determine whether a fluid is urine. The best way to do so is to consider the components of the specimen. Creatinine, urea, sodium, and chloride are significantly higher in urine than in other body fluids. Protein and glucose are not present in a normal urine specimen.

Urine Volume Figure 3–3 A chart used for urine analysis. (Courtesy of National Library of Medicine.)

2. Urine contains information, which can be obtained by inexpensive laboratory tests, about many of the body’s major metabolic functions. These characteristics fit in well with the current trends toward preventive medicine and lower medical costs. In fact, the Clinical and Laboratory Standards Institute (CLSI) defines urinalysis as “the testing of urine with procedures commonly performed in an expeditious, reliable, accurate, safe, and costeffective manner.” The reasons for performing urinalysis identified by CLSI include aiding in the diagnosis of disease, screening asymptomatic populations for undetected disorders, and monitoring the progress of disease and the effectiveness of therapy.2

Urine Formation The kidneys continuously form urine as an ultrafiltrate of plasma. Reabsorption of water and filtered substances essential to body function converts approximately 170,000 mL of filtered plasma to the average daily urine output of 1200 mL, depending on fluid intake. (Refer to Chapter 4.)

Urine Composition In general, urine consists of urea and other organic and inorganic chemicals dissolved in water. Urine is normally 95% water and 5% solutes, although considerable variations in the concentrations of these solutes can occur due to the influence of factors such as dietary intake, physical activity, body metabolism, and endocrine functions. Urea, a metabolic waste product produced in the liver from the breakdown of protein and amino acids, accounts

Urine volume depends on the amount of water that the kidneys excrete. Water is a major body constituent; therefore, the amount excreted is usually determined by the body’s state of hydration. Factors that influence urine volume include fluid intake, fluid loss from nonrenal sources, variations in the secretion of antidiuretic hormone (ADH), and need to excrete increased amounts of dissolved solids, such as glucose or salts. Taking these factors into consideration, although the normal daily urine output is usually 1200 to 1500 mL, a range of 600 to 2000 mL is considered normal.

Technical Tip 3-1. Should it be necessary to determine whether a particular fluid is urine, the specimen can be tested for its urea and creatinine content. Because both these substances are present in much higher concentrations in urine than in other body fluids, a fluid that is high in urea and creatinine content can be identified as urine. Oliguria, a decrease in urine output (less than 1 mL/kg/hr in infants, less than 0.5 mL/kg/hr in children, and less than 400 mL/day in adults), is seen commonly when the body enters a state of dehydration as a result of excessive water loss from vomiting, diarrhea, perspiration, or severe burns. Oliguria leading to anuria, cessation of urine flow, may result from any serious damage to the kidneys or from a decrease in the flow of blood to the kidneys. The kidneys excrete two to three times more urine during the day than during the night. An increase in the nocturnal excretion of urine is termed nocturia. Polyuria, an increase in daily urine volume (greater than 2.5 L/day in adults and 2.5 to 3 mL/kg/day in children), is often associated with diabetes mellitus and diabetes insipidus; however, it may be induced artificially by diuretics, caffeine, or alcohol, all of which suppress the secretion of ADH.

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Table 3–1

Primary Components in Normal Urine3

Component

Comment

Urea

Primary organic component. Product of metabolism of protein and amino acids Product of metabolism of creatine by muscles Product of breakdown of nucleic acid in food and cells Primary inorganic component. Found in combination with sodium (table salt) and many other inorganic substances Primarily from salt, varies by intake Combined with chloride and other salts Combines with sodium to buffer the blood Regulates blood and tissue fluid acidity Combines with chloride, sulfate, and phosphate

Creatinine Uric acid Chloride

Sodium Potassium Phosphate Ammonium Calcium

Diabetes mellitus and diabetes insipidus produce polyuria for different reasons, and analysis of the urine is an important step in the differential diagnosis (Fig. 3-4). Diabetes mellitus is caused by a defect either in the pancreatic production of insulin or in the function of insulin, which results in an increased concentration of body glucose. The kidneys do not reabsorb excess glucose, necessitating excretion of increased amounts of water to remove the dissolved glucose from the body. Although appearing to be dilute, a urine specimen from a Polydipsia

Polyuria

Specific gravity

Decreased SG

Increased SG

Decreased production or Function of ADH

Decreased insulin or Decreased function of insulin

Diabetes insipidus

Increased glucose

Diabetes mellitus

Figure 3–4 Differentiation between diabetes mellitus and diabetes insipidus.

patient with diabetes mellitus has a high specific gravity because of the increased glucose content. Diabetes insipidus results from a decrease in the production or function of ADH; thus, the water necessary for adequate body hydration is not reabsorbed from the plasma filtrate. In this condition, the urine is truly dilute and has a low specific gravity. Fluid loss in both diseases is compensated by increased ingestion of water (polydipsia), producing an even greater volume of urine. Polyuria accompanied by increased fluid intake is often the first symptom of either disease.

Specimen Collection As discussed in Chapter 1, urine is a biohazardous substance whose handling requires the observance of Standard Precautions (SP). Gloves should be worn at all times when in contact with the urine specimen. The type of urine collection container, as well as specimen preservation and storage, will affect the quality of urine test results. Written criteria for the collection, preservation, handling, storage, and labeling of urine must be available.

Containers Specimens must be collected in clean, dry, leakproof containers. Disposable containers should be used because they eliminate the chance of contamination due to improper washing. These disposable containers are available in a variety of sizes and shapes, including bags with adhesive for the collection of pediatric specimens and large containers for 24-hour specimens. Properly applied screw-top lids are less likely to leak than are snap-on lids. Containers for routine urinalysis should have a wide mouth to facilitate collections from female patients and a wide, flat bottom to prevent overturning. They should be made of a clear material to allow for determination of color and clarity. The recommended capacity of the container is 50 mL, which allows 12 mL of specimen needed for microscopic analysis, additional specimen for repeat analysis, and enough room for the specimen to be mixed by swirling the container. Individually packaged sterile containers with secure closures should be used for microbiological studies of urine. Sterile containers are also suggested if more than 2 hours elapse between specimen collection and analysis.2 Specially designed sterile containers are available that have a screw top containing an integrated transfer device. A BD Vacutainer Urine Transfer Straw is a nonsterile, plastic holder device that contains a needle with a straw attachment that can be used with the collection container to fill evacuation tubes. This device allows for the sterile transfer of urine to tubes containing preservatives for microbiology testing and tubes with conical bottoms for sediment analysis or round bottoms for automated reagent strip testing.4 Additional information and pictures can be found at https://www.bd.com/en-us/offerings/ capabilities/specimen-collection/urine-collection/bdvacutainer-collection-and-transfer-products.

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Labels All specimens must be labeled immediately after collection with the patient’s last and first name and identification number; the date and time of collection; and additional information such as the patient’s age and location, the health-care provider’s name, and the preservative used, if any, as required by facility protocol. Labels must be attached to the container, not to the lid, and should not become detached if the container is refrigerated or frozen.

Requisition Form A requisition form (manual or computerized) must accompany specimens delivered to the laboratory. The information on the form must match the information on the specimen label. Additional information on the form can include method of collection or type of specimen, possible interfering medications, and the patient’s clinical information. The time the specimen is received in the laboratory should be recorded on the form.

Specimen Rejection The laboratory should reject specimens that are improperly labeled and/or collected, and appropriate personnel should be notified to collect a new specimen. Situations leading to specimens that should be rejected can include: 1. Specimens in containers that are unlabeled or improperly labeled 2. Labels and requisition forms that do not match 3. Specimens contaminated with feces or toilet paper 4. Containers with contaminated exteriors 5. Specimens of insufficient quantity 6. Specimens that have been transported improperly Table 3–2

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7. Specimens that have not been preserved correctly during a time delay 8. Specimens for urine culture collected in a nonsterile container 9. Inappropriate collection for the type of testing needed (for example, midstream clean-catch specimen for bacterial culture)

Technical Tip 3-2. Never discard a specimen before checking with a supervisor. Laboratories should have a written policy detailing their conditions for specimen rejection. Visit www.fadavis.com for Video 3-1 (Preparing urine for urinalysis).

Specimen Handling The fact that a urine specimen is so readily available and easily collected often leads to laxity in the treatment of the specimen after its collection. Changes in urine composition take place not only in vivo but also in vitro, requiring correct handling procedures.

Specimen Integrity After collection, specimens should be delivered to the laboratory promptly and tested within 2 hours. A specimen that cannot be delivered and tested within 2 hours should be refrigerated or have an appropriate chemical preservative added. Table 3-2 describes the most significant changes that may occur in a specimen allowed to remain unpreserved at room temperature for longer than 2 hours. Notice that most of the changes are related to the presence and growth of bacteria.

Changes in Unpreserved Urine

Analyte

Change

Cause

Color Clarity Odor pH

Modified/darkened Decreased Increased ammonia smell Increased

Glucose Ketones Bilirubin Urobilinogen Nitrite Red and white blood cells and casts Bacteria Trichomonas

Decreased Decreased Decreased Decreased Increased Decreased

Oxidation or reduction of metabolites Bacterial growth and precipitation of amorphous material Bacterial multiplication causing breakdown of urea to ammonia Breakdown of urea to ammonia by urease-producing bacteria/loss of CO2 Glycolysis and bacterial use Volatilization and bacterial metabolism Exposure to light/photo oxidation to biliverdin Oxidation to urobilin Multiplication of nitrate-reducing bacteria Disintegration/lyse in dilute alkaline urine

Increased Decreased

Multiplication Loss of motility, death

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These variations are discussed again under the individual test procedures. At this point, it is important to realize that improper preservation can seriously affect the results of a routine urinalysis.

Specimen Preservation The method of preservation used most routinely is refrigeration at 2°C to 8°C, which decreases bacterial growth and metabolism. If the urine is to be cultured, it should be refrigerated during transit and kept refrigerated until cultured, up to 24 hours.2 The specimen must return to room temperature before chemical testing by reagent strips. Refrigeration also can cause precipitation of amorphous urate and phosphate crystals. When a specimen must be transported over a long distance and refrigeration is impossible, chemical preservatives may be added. Commercially prepared transport tubes with a lyophilized preservative are available that allow for the transport, testing, and storage of the urine specimens. The ideal preservative should be bactericidal, inhibit urease, and preserve formed elements in the sediment. At the same time, the preservative should not interfere with chemical tests. Unfortunately, as seen in Table 3-3, the ideal preservative does not exist; therefore, a preservative that best suits the needs of the required analysis should be chosen.

Technical Tip 3-3. Specimens must be returned to room temperature before chemical testing by reagent strips because the enzyme reactions on the strips perform best at room temperature. Table 3–3

Technical Tip 3-4. When preserving specimens that will be transported to another laboratory, confirm that the appropriate preservative is used.

Types of Specimens To obtain a specimen that is representative of a patient’s metabolic state, regulation of certain aspects of specimen collection is often necessary. These special conditions may include time, length, and method of collection, as well as the patient’s dietary and medicinal intake. It is important to instruct patients when they must follow special collection procedures. Frequently encountered specimens are listed in Table 3-4.

Random Specimen This is the specimen received most commonly because of its ease of collection and convenience for the patient. The random specimen may be collected at any time, but the actual time of voiding should be recorded on the container.2 The random specimen is useful for routine screening tests to detect obvious abnormalities. However, it also may show erroneous results resulting from dietary intake or physical activity just before collection. Then the patient will be requested to collect an additional specimen under more controlled conditions.

First Morning Specimen Although it may require the patient to make an additional trip to the laboratory, this is the ideal screening specimen. It is also essential for preventing false-negative pregnancy tests

Urine Preservatives

Preservatives

Advantages

Disadvantages

Additional Information

Refrigeration

Does not interfere with chemical tests Prevents bacterial growth and metabolism

Precipitates amorphous phosphates and urates Interferes with analysis of drugs and hormones

Excellent sediment preservative

Acts as a reducing agent, interfering with chemical tests for glucose, blood, leukocyte esterase, and copper reduction Inhibits reagent strip tests for glucose, blood, and leukocytes Check tablet composition to determine possible effects on desired tests

Prevents bacterial growth for 24 hours2 Keeps pH at about 6.0 Can be used for transport of urine cultures Rinse specimen container with formalin to preserve cells and casts

Acids (boric acid, HCL, acetic acid, tartaric acid) Formalin (formaldehyde)

Sodium fluoride

Good preservative for drug analyses

Commercial preservative tablets

Convenient when refrigeration not possible Have controlled concentration to minimize interference Contains collection cup, transfer straw, culture and sensitivity (C&S) preservative tube, or UA tube

Urine Collection Kits4 (Becton, Dickinson, Rutherford, NJ)

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Urine Preservatives—cont’d

Preservatives

Advantages

Disadvantages

Additional Information

Light gray and gray C&S tube

Specimen stable at room temperature (RT) for 48 hours; prevents bacterial growth and metabolism Use on automated instruments Specimen stable for 72 hours at RT; instrument-compatible

Do not use if urine is below minimum fill line

Preservative is boric acid, sodium borate, and sodium formate Keeps pH at about 6.0

Must refrigerate within 2 hours Must be filled to minimum fill line Bilirubin and urobilinogen may be decreased if specimen is exposed to light and left at RT

Round or conical bottom, no preservative Preservative is sodium propionate, ethyl paraben, and chlorhexidine Round or conical bottoms

Yellow UA Plus tube Cherry red/yellow Preservative Plus tube

Table 3–4

Types of Urine Specimens

Type of Specimen

Purpose

Random First morning

Routine screening Routine screening Pregnancy tests Orthostatic protein Quantitative chemical tests Bacterial culture Routine screening

24-hour (or timed) Catheterized Midstream cleancatch Suprapubic aspiration Three-glass collection Four-glass collection

Bacterial culture Bladder urine for bacterial culture

HISTORICAL NOTE

Glucose Tolerance Specimens Glucose tolerance specimens were sometimes collected to correspond with the blood specimens drawn during an oral glucose tolerance test (OGTT). The number of specimens varied with the length of the test. GTTs included fasting, half-hour, 1-hour, 2-hour, and 3-hour specimens and possibly 4-hour, 5-hour, and 6-hour specimens. The urine was tested for glucose and ketones, and the results were reported along with the blood test results as an aid to interpreting the patient’s ability to metabolize a measured amount of glucose. The results are correlated with the renal threshold for glucose. Collection of these specimens was a facility option.5

Cytology Prostatic infection Prostatic infection

and for evaluating orthostatic proteinuria (see Chapter 6). The first morning specimen is a concentrated specimen, thereby assuring detection of chemicals and formed elements that may not be present in a dilute random specimen. The patient should be instructed to collect the specimen immediately on arising and to deliver it to the laboratory within 2 hours or keep it refrigerated.

24-Hour (or Timed) Specimen Measuring the exact amount of a urine chemical is often necessary instead of just reporting its presence or absence. A carefully timed specimen must be used to produce accurate quantitative results. Many solutes exhibit diurnal variations

such as catecholamines, 17-hydroxysteroids, and electrolytes in which the lowest concentration is in the early morning and the highest concentration occurs in the afternoon.2 When the concentration of the substance to be measured changes with diurnal variations and with daily activities, such as exercise, meals, and body metabolism, 24-hour collection is required. If the concentration of a particular substance remains constant, the specimen may be collected over a shorter period. Care must be taken, however, to keep the patient adequately hydrated during short collection periods. Patients must be instructed on the procedure for collecting a timed specimen. To obtain an accurate timed specimen, the patient must begin and end the collection period with an empty bladder. The concentration of a substance in a particular period must be calculated from the urine volume produced during that time. On its arrival in the laboratory, a 24-hour specimen must be mixed thoroughly and the volume accurately measured and recorded. If only an aliquot is needed for testing, the amount

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saved must be adequate to permit repeat or additional testing. If a specimen is collected in two containers, the contents of the containers should be combined and thoroughly mixed before aliquoting. Consideration also must be given to the preservation of specimens collected over extended periods. All specimens should be refrigerated or kept on ice during the collection period and may require addition of a chemical preservative. The preservative chosen must be nontoxic to the patient and should not interfere with the tests to be performed. Appropriate collection information is included with test procedures and should be read before issuing a container and instructions to the patient. Box 3-1 lists the most common errors associated with timed urine collections.

Box 3–1

Common Errors Associated With Timed Urine Collections

• Loss of urine specimen • Inclusion of two first morning specimens • Inaccurate measurement of total urine volume • Inadequate urine preservation • Transcription error

PROCEDURE 3-1 24-Hour (Timed) Urine Specimen Collection Procedure Equipment Requisition form 24-hour urine specimen container with lid Label Container with ice, if required Preservative, if required Procedure 1. Provide the patient with written instructions, and explain the collection procedure. 2. Provide the patient with the collection container and preservative, if required. 3. Day 1: 7 a.m.: Patient voids and discards specimen; collects all urine for the next 24 hours. 4. Day 2: 7 a.m.: Patient voids and adds this urine to the previously collected urine. 5. Specimen is transported to the laboratory, where the entire 24-hour specimen is thoroughly mixed and the volume is measured and recorded. 6. The required amount of urine (approximately 50 mL) is aliquoted for testing. 7. The remaining specimen is discarded.

Technical Tip 3-5.

Adding urine formed before the start of the collection period will falsely elevate the results, and failure to include the urine produced at the end of the collection period will falsely decrease the results.

Catheterized Specimen This specimen is collected under sterile conditions by passing a hollow tube (catheter) through the urethra into the bladder. Urine passes from the bladder through the catheter into a plastic bag, where it accumulates. Urine specimens then can be collected from this urine bag. The test requested most commonly on a catheterized specimen is a bacterial culture.

Midstream Clean-Catch Specimen As an alternative to the catheterized specimen, the midstream clean-catch specimen provides a safer, less traumatic method for obtaining urine for bacterial culture and routine urinalysis. It provides a specimen that is less contaminated by epithelial cells and bacteria and therefore is more representative of the actual urine than the routinely voided specimen. Patients must be provided with appropriate cleansing materials, a sterile container, and instructions for cleansing and voiding. Strong bacterial agents, such as hexachlorophene or povidone-iodine, should not be used as cleansing agents. Mild antiseptic towelettes are recommended. Some urine collection transfer kits contain Castile Soap Towelettes. Procedures 3-2 and 3-3 provide instruction details for male and female patients.

Suprapubic Aspiration Occasionally urine may be collected by external introduction of a needle through the abdomen into the bladder. Because the bladder is sterile under normal conditions, suprapubic aspiration provides a specimen for bacterial culture that is completely free of extraneous contamination, particularly in infants or children. The specimen also can be used for cytological examination.

Prostatitis Specimen Several methods are available to detect the presence of prostatitis.

Three-Glass Collection Before collection, the area is cleansed using the male midstream clean-catch procedure. Then, instead of discarding the first urine passed, it is collected in a sterile container. Next, the midstream portion is collected in another sterile container. Then the prostate is massaged so that prostate fluid will be passed with the remaining urine into a third sterile container. Quantitative cultures are performed on all specimens, and the first and third specimens are examined microscopically. In prostatic infection, the third specimen will have a white blood cell/highpower field count and a bacterial count 10 times that of the first specimen. Macrophages containing lipids also may be

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PROCEDURE 3-2 Clean-Catch Specimen Collection: Female Cleansing Procedure2 Equipment Requisition form Sterile urine container with label Sterile antiseptic towelettes Written instructions for cleansing and voiding Procedure Instruct the patient to: 1. Wash her hands. 2. Remove the lid from the sterile container without touching the inside of the container or lid. 3. Separate the skin folds (labia).

4. Cleanse from front to back on either side of the urinary opening with an antiseptic towelette, using a clean one for each side. 5. Hold the skin folds apart and begin to void into the toilet. 6. Bring the urine container into the middle stream of urine, and collect an adequate amount of urine. Do not touch the inside of the container or allow the container to touch the genital area. 7. Finish voiding into the toilet. 8. Cover the specimen with the lid. Touch only the outside of the lid and container. 9. Confirm the container is labeled correctly with the patient’s first and last name and time of collection, and place it in the specified area, or follow facility policy.

PROCEDURE 3-3 Clean-Catch Specimen Collection: Male Cleansing Procedure2 Equipment Requisition form Sterile urine container with label Sterile antiseptic towelettes Written instructions for cleansing and voiding Procedure Instruct the patient to: 1. Wash his hands. 2. Remove the lid from the sterile container without touching the inside of the container or lid.

present. The second specimen is used as a control for bladder and kidney infection. If it is positive, the results from the third specimen are invalid because infected urine has contaminated the specimen.6

Technical Tip 3-6. When both a routine urinalysis and a culture are requested on a catheterized or midstream collection, the culture should be performed first to prevent contamination of the specimen. A collection transfer kit also can be used. Pre- and Postmassage Test In the pre- and postmassage test (PPMT), a clean-catch midstream urine specimen is collected. A second urine sample is collected after the prostate is massaged. A positive result is

3. Cleanse the tip of the penis with antiseptic towelette and let it dry. Retract the foreskin if uncircumcised. 4. Void into the toilet. Hold back the foreskin if necessary. 5. Bring the sterile urine container into the middle stream of urine, and collect an adequate amount of urine. Do not touch the inside of the container or allow the container to touch the genital area. 6. Finish voiding into the toilet. 7. Cover the specimen with the lid. Touch only the outside of the lid and container. 8. Confirm the container is labeled correctly with the patient’s first and last name and time of collection, and place it in the specified area, or follow facility policy.

significant bacteriuria in the postmassage specimen of greater than 10 times the premassage count.7

Stamey-Meares Test for Prostatitis The traditional four-glass urine collection technique, as described by Meares and Stamey,7,8 includes examination of four urine specimens as follows: • The first urine specimen is voided bladder (VB1), which is the first 10 mL of urine and represents the urethral specimen. • Then the patient voids another 100 to 150 mL of urine. • The second specimen, voided bladder 2 (VB2), is collected, which is another 10 mL of urine and represents the bladder specimen. • The third specimen is the expressed prostatic specimen (EPS),which is the fluid collected during prostatic massage.

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100 Part One | Basic Principles • The fourth specimen, voided bladder 3 (VB3), consists of the first 10 mL of urine collected after EPS; it contains any EPS trapped in the prostatic urethra. • All four specimens are sent for culture. • The three urine specimens are centrifuged, and the sediment is examined for white blood cells/aggregates, macrophages, oval fat bodies, bacteria, and fungal hypha. Urethral infection or inflammation is tested for by the VB1, and the VB2 tests for urinary bladder infection. The prostatic secretions are cultured and examined for white blood cells. Having more than 10 to 20 white blood cells per highpower field is considered abnormal.7

Pediatric Specimens Collection of pediatric specimens can present a challenge. Soft, clear plastic bags are available for collecting routine specimens; these bags have hypoallergenic skin adhesive to attach to the cleaned genital area of both boys and girls. Sterile specimens may be obtained by catheterization or by suprapubic aspiration. Care must be taken not to touch the inside of the bag when applying it to the patient’s skin. For routine specimen analysis, ensure the area is free of contamination. Attach the bag firmly over the cleaned genital area, avoiding the anus. A diaper is placed over the collection bag. When enough specimen has been collected, remove the bag and label it, or pour the specimen into a container and label the container following facility policy. For microbiology specimens, clean the area with soap and water and sterilely dry the area, removing any residual soap residue. Firmly apply a sterile bag. Sterilely transfer the collected specimen into a sterile container and label the container.2

Technical Tip 3-7. Check the applied bags approximately every 15 minutes until the needed amount of sample has been collected.

Drug Specimen Collection Urine specimen collection is the most vulnerable part of a drug-testing program. Correct collection procedures and documentation are necessary to ensure that the results are those

of the specific individual submitting the specimen. The chain of custody (COC) is the process that provides this documentation of proper specimen identification from the time of collection to the receipt of laboratory results. The COC is a standardized form that must document and accompany every step of drug testing, from collector to courier to laboratory to medical review officer to employer. For urine specimens to withstand legal scrutiny, it is necessary to prove that no tampering of the specimen occurred, such as substitution, adulteration, or dilution. All personnel handling the specimen must be noted. The specimen must be handled securely, with a guarantee that no unauthorized access to the specimen was possible. Proper identification of the individual whose information is indicated on the label is required. Either photo identification or positive identification by an employer representative with photo ID is acceptable. Urine specimen collections may be “witnessed” or “unwitnessed.” The decision to obtain a witnessed collection is indicated when it is suspected that the donor may alter or substitute the specimen or when it is the policy of the client ordering the test. If a witnessed specimen collection is ordered, a same-gender collector will observe the collection of 30 to 45 mL of urine. Witnessed and unwitnessed collections should be handed to the collector immediately. The urine temperature must be taken within 4 minutes from the time of collection to confirm the specimen has not been adulterated. The temperature should read within the range of 32.5°C to 37.7°C. If the specimen temperature is not within range, the temperature should be recorded and the supervisor or employer contacted immediately. Urine temperatures outside of the recommended range may indicate specimen contamination. Re-collection of a second specimen as soon as possible will be necessary. The urine color is also inspected to identify any signs of contaminants. The pH and specific gravity of the specimen also may be tested. A urine pH of greater than 9 suggests adulteration of the urine specimen and requires that the specimen be re-collected if clinically necessary. A specific gravity of less than 1.005 could indicate dilution of the urine specimen and requires re-collection. The specimen is labeled, packaged, and transported following laboratory-specific instructions. A typical urine drug specimen collection is described in Procedure 3-4.

PROCEDURE 3-4 Urine Drug Specimen Collection Procedure Equipment Requisition form Gloves Bluing agent (dye) Specimen container Label COC form Temperature strip

Procedure 1. The collector sanitizes his or her hands and wears gloves. 2. The collector adds bluing agent (dye) to the toilet water reservoir to prevent an adulterated specimen. 3. The collector eliminates any source of water other than toilet by taping the toilet lid and faucet handles. 4. The donor provides photo identification or positive identification from the employer’s representative.

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PROCEDURE 3-4—cont’d 5. The collector completes step 1 of the COC form and has the donor sign it. 6. The donor leaves his or her coat, briefcase, and/or purse outside the collection area to avoid the possibility of concealed substances contaminating the urine. 7. The donor sanitizes his or her hands and receives a specimen cup. 8. The collector remains in the restroom but outside the stall, listening for unauthorized water use, unless a witnessed collection is requested. 9. The donor hands the specimen cup to the collector. The transfer is documented. 10. The collector checks the urine for abnormal color and for the required amount (30 to 45 mL). 11. The collector checks that the temperature strip on the specimen cup reads 32.5°C to 37.7°C. The collector records the in-range temperature on the COC form (COC step 2). If the specimen temperature is out of range or the specimen is suspected of having been diluted or adulterated, a new specimen must be collected and a supervisor notified.

12. The specimen must remain in the sight of the donor and collector at all times. 13. With the donor watching, the collector peels off the specimen identification strips from the COC form (COC step 3) and puts them on the capped bottle, covering both sides of the cap. 14. The donor initials the specimen bottle seals. 15. The collector writes the date and time on the bottle seals. 16. The donor completes step 4 on the COC form. 17. The collector completes step 5 on the COC form. 18. Each time the specimen is handled, transferred, or placed in storage, every individual must be identified and the date and purpose of the change recorded. 19. The collector follows laboratory-specific instructions for packaging the specimen bottles and laboratory copies of the COC form. 20. The collector distributes the COC copies to appropriate personnel.

For additional resources please visit www.fadavis.com 5.

References 1. Herman, JR: Urology: A View Through the Retrospectroscope. Harper & Row, Hagerstown, MD, 1973. 2. Clinical and Laboratory Standards Institute, Urinalysis: Approved Guideline, ed 3, CLSI Document GP16-A3: Wayne, PA, 2009, CLSI. 3. Torora, GJ, and Anagnostakos, NP: Principles of Anatomy and Physiology, ed 6, Harper & Row, New York, 1990, p. 51. 4. Becton, Dickinson and Company: BD Vacutainer Urine Products for Collection, Storage, and Transport of Urine Specimens.

6. 7. 8.

Product Circular, 2014. Becton, Dickinson and Company, 1 Becton Drive, Franklin Lakes, NJ 07417. Web site: www.bd. com/vacutainer/referencematerial. ©2014 BD 8606531 Mfg by BD, USA 04/2014. Accessed April 25, 2019. Baer, DM: Glucose tolerance test: Tips from the clinical experts. Medical Laboratory Observer, Sept. 2003. Rous, SN: The Prostate Book. Consumers Union, Mt. Vernon, NY, 1988. Stevermer, JJ, and Easley, SK: Treatment of prostatitis. Am Fam Physician 61(10):2015–3022, 2000. Kraemer, Samantha, MD: Chronic Bacterial Prostatitis Workup. Medscape. Web site: https://emedicine.medscape.com/article/ 458391-workup. Published January 15, 2019. Accessed April 22, 2019.

Study Questions 1. The primary inorganic substance found in urine is: A. Sodium B. Phosphate C. Chloride D. Calcium

2. An unidentified fluid is received in the laboratory with a request to determine whether the fluid is urine or another body fluid. Using routine laboratory tests, which substances would determine that the fluid is most probably urine? A. Glucose and ketones B. Urea and creatinine C. Uric acid and amino acids D. Protein and amino acids

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3. The average daily output of urine is: A. 200 mL B. 500 mL C. 1200 mL D. 2500 mL 4. A patient presenting with polyuria, nocturia, polydipsia, and a low urine specific gravity is exhibiting symptoms of: A. Diabetes insipidus B. Diabetes mellitus C. Urinary tract infection D. Uremia 5. A patient with oliguria might progress to having: A. Nocturia B. Polyuria C. Polydipsia D. Anuria 6. All of the following are characteristics of recommended urine containers except: A. A flat bottom B. A capacity of 50 mL C. A snap-on lid D. Are disposable 7. Labels for urine containers are: A. Attached to the container B. Attached to the lid C. Placed on the container before collection D. Not detachable 8. A urine specimen may be rejected by the laboratory for all of the following reasons except the fact that the: A. Requisition form states the specimen is catheterized B. Specimen contains toilet paper C. Label and requisition form do not match D. Outside of the container has contamination from fecal material 9. A cloudy specimen received in the laboratory may have been preserved using: A. Boric acid B. Chloroform C. Refrigeration D. Formalin

10. For general screening, the specimen collected most frequently is a: A. Random one B. First morning C. Midstream clean-catch D. Timed 11. The primary advantage of a first morning specimen over a random specimen is that it: A. Is less contaminated B. Is more concentrated C. Is less concentrated D. Has a higher volume 12. If a routine urinalysis and a culture are requested on a catheterized specimen, then: A. Two separate containers must be collected B. The routine urinalysis is performed first C. The patient must be recatheterized D. The culture is performed first 13. If a patient fails to discard the first specimen when collecting a timed specimen, then the: A. Specimen must be re-collected B. Results will be falsely elevated C. Results will be falsely decreased D. Both A and B 14. The primary cause of unsatisfactory results in an unpreserved routine specimen not tested for 8 hours is: A. Bacterial growth B. Glycolysis C. Decreased pH D. Chemical oxidation 15. Prolonged exposure of a preserved urine specimen to light will cause: A. Decreased glucose B. Increased cells and casts C. Decreased bilirubin D. Increased bacteria 16. Which of the following would be least affected in a specimen that has remained unpreserved at room temperature for more than 2 hours? A. Urobilinogen B. Ketones C. Protein D. Nitrite

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Chapter 3 | Introduction to Urinalysis 103 17. Bacterial growth in an unpreserved specimen will: A. Decrease clarity B. Increase bilirubin C. Decrease pH D. Increase glucose 18. The most sterile specimen collected is a: A. Catheterized B. Midstream clean-catch C. Three-glass D. Suprapubic aspiration

19. Which of the following would not be given to a patient before the collection of a midstream clean-catch specimen? A. Sterile container B. Iodine cleanser C. Antiseptic towelette D. Instructions 20. Urine specimen collection for drug testing requires the collector to do all of the following except: A. Inspect the specimen color B. Perform reagent strip testing C. Read the specimen temperature D. Fill out a chain-of-custody form

Case Studies and Clinical Situations 1. A patient brings a first morning specimen to the laboratory at 1:00 p.m. a. How could this affect the urinalysis results? b. If the patient is a known diabetic, what results would be most suspect? c. What is the best course of action? d. What could the patient have said that would have made the specimen satisfactory for testing? 2. A patient collecting a midstream clean-catch specimen voids immediately into the container. a. How could this affect the clarity of the specimen? b. How could this affect the microscopic examination? 3. A patient brings a 24-hour-timed specimen to the laboratory and reports that he or she forgot to collect a specimen voided during the night. a. How will this affect the results of a quantitative test for creatinine? b. What should the patient be told to do?

4. The laboratory receives a urine preservative tube for culture containing a volume of specimen that is considerably below the minimum fill line. a. Could this affect the culture? b. Why? 5. A worker suspects that he or she will be requested to collect an unwitnessed urine specimen for drug analysis. He or she carries a substitute specimen in his or her pocket for 2 days before being told to collect the specimen. Shortly after the worker delivers the specimen to the collector, he or she is instructed to collect another specimen. a. What test was performed on the specimen to determine possible specimen manipulation? b. How was the specimen in this situation affected? c. If a specimen for drug analysis tests positive, state a possible defense related to specimen collection and handling that an attorney might employ. d. How can this defense be avoided?

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CHAPTER

4

Renal Function LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 4-1 Identify the components of the nephron, kidney, and excretory system. 4-2 Trace the flow of blood through the nephron, and state the physiological functions that occur. 4-3 Describe the process of glomerular ultrafiltration. 4-4 Discuss the functions and regulation of the renin–angiotensin–aldosterone system (RAAS). 4-5 Differentiate between active and passive transport in relation to renal concentration. 4-6 Explain the function of antidiuretic hormone in the concentration of urine. 4-7 Describe the role of tubular secretion in maintaining acid–base balance. 4-8 Identify the laboratory procedures used to evaluate glomerular filtration, tubular reabsorption and secretion, and renal blood flow. 4-9 Describe the creatinine clearance test.

4-10 Given hypothetical laboratory data, calculate a creatinine clearance and determine whether the result is normal. 4-11 Discuss the clinical significance of the glomerular filtration rate tests. 4-12 Describe and contrast the Modification of Diet in Renal Disease (MDRD), cystatin C, and beta2-microglobulin tests for performing estimated glomerular filtration rates (eGFR). 4-13 Define osmolarity, and discuss its relationship to urine concentration. 4-14 Describe the basic principles of freezing-point osmometers. 4-15 Given hypothetic laboratory data, calculate a free-water clearance and interpret the result. 4-16 Given hypothetic laboratory data, calculate a PAH clearance and relate this result to renal blood flow. 4-17 Describe the relationship of urinary ammonia and titratable acidity to the production of an acidic urine.

KEY TERMS Active transport

Creatinine clearance

Glomerular filtration rate (GFR)

Afferent arteriole

Cystatin C

Glomerulus

Aldosterone

Density

Juxtaglomerular apparatus

Antidiuretic hormone (ADH)

Distal convoluted tubule

Loops of Henle

Beta2-microglobulin (B2M)

Endogenous procedure

Macula densa

Clearance tests

Efferent arteriole

Maximal reabsorptive capacity (Tm)

Collecting duct

Exogenous procedure

Metabolic acidosis

Concentration tests

Fenestrated endothelium

Nephron

Countercurrent mechanism

Free water clearance

Osmolality

Creatinine

Glomerular filtration barrier

Osmolar clearance

Continued

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K E Y T E R M S —cont’d Osmolarity

Renal threshold

Titratable acidity

Passive transport

Renal tubular acidosis

Tubular reabsorption

Peritubular capillaries

Renin

Tubular secretion

Podocytes

Renin–angiotensin–aldosterone system (RAAS)

Vasa recta

Proximal convoluted tubule Renal plasma flow

Shield of negativity

Introduction This chapter reviews nephron anatomy and physiology and discusses their relationship to urinalysis and renal function testing. A section on laboratory assessment of renal function is included.

Renal Physiology Each kidney contains approximately 1 to 1.5 million functional units called nephrons. As shown in Figure 4-1, the human kidney contains two types of nephrons. Cortical nephrons, which make up approximately 85% of nephrons, are situated primarily in the cortex of the kidney. They are responsible primarily for removal of waste products and reabsorption of nutrients. Juxtamedullary nephrons have longer loops of Henle that extend deep into the medulla of the kidney. Their primary function is concentration of the urine. The ability of the kidneys to clear waste products selectively from the blood and simultaneously to maintain the body’s essential water and electrolyte balances is controlled in the nephron by the following renal functions: • Renal blood flow • Glomerular filtration • Tubular reabsorption • Tubular secretion The physiology, laboratory testing, and associated pathology of these four functions are discussed in this chapter.

Renal Blood Flow The renal artery supplies blood to the kidney. The human kidneys receive approximately 25% of the blood pumped through the heart at all times. Blood enters the capillaries of the nephron through the afferent arteriole. It then flows through the glomerulus and into the efferent arteriole. The varying sizes of these arterioles help create the hydrostatic pressure differential that is important for glomerular filtration and to maintain consistency of glomerular capillary pressure and renal blood flow within the glomerulus. Notice the smaller size of the efferent arteriole in Figure 4-2. This increases the glomerular capillary pressure. Renal blood flow is outlined in Box 4-1. Before returning to the renal vein, blood from the efferent arteriole enters the peritubular capillaries and the vasa recta

Vasopressin

and flows slowly through the cortex and medulla of the kidney close to the tubules. The peritubular capillaries surround the proximal and distal convoluted tubules, providing for the immediate reabsorption of essential substances from the fluid in the proximal convoluted tubule and final adjustment of the urinary composition in the distal convoluted tubule. The vasa recta are located adjacent to the ascending and descending loops of Henle in juxtamedullary nephrons. In this area, the major exchanges of water and salts take place between the blood and the medullary interstitium. This exchange maintains the osmotic gradient (salt concentration) in the medulla, which is necessary for renal concentration. Box 4-2 outlines the urinary filtrate flow. Based on an average body size of 1.73 m2 of surface, the total renal blood flow is approximately 1200 mL/min, and the total renal plasma flow ranges from 600 to 700 mL/min. Normal values for renal blood flow and renal function tests depend on body size. When dealing with body sizes that vary greatly from the average 1.73 m2 of body surface, a correction must be calculated to determine whether the observed measurements represent normal function. This calculation is covered in the discussion on tests for glomerular filtration rate (GFR) later in this chapter. Variations in normal values have been published for different age groups and should be considered when evaluating renal function studies.

Glomerular Filtration The glomerulus consists of a coil of approximately eight capillary lobes, the walls of which are referred to as the glomerular filtration barrier. The glomerulus is located within Bowman capsule, which forms the beginning of the renal tubule. Although the glomerulus serves as a nonselective filter of plasma substances with molecular weights less than 70,000, several factors influence the actual filtration process. These include the cellular structure of the capillary walls and Bowman capsule, hydrostatic pressure and oncotic pressure, and the feedback mechanisms of the renin–angiotensin–aldosterone system (RAAS).

Cellular Structure of the Glomerulus Plasma filtrate must pass through three glomerular filtration barrier cellular layers: the capillary wall membrane, the basement membrane (basal lamina), and the visceral epithelium of the Bowman capsule. The endothelial cells of the capillary wall

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Chapter 4 | Renal Function 107 Glomerulus

Renal cortex

Cortical nephron

Renal tubule

Renal medulla

Papilla of pyramid Loop of Henle

Renal artery

Juxtamedullary nephron

Collecting duct

Calyx Renal vein

Cortex Renal pelvis

Ureter

Figure 4–1 The relationship of the nephron to the kidney and excretory system. (From Scanlon, VC, and Sanders, T: Essentials of Anatomy and Physiology, ed 3. FA Davis, Philadelphia, PA, 1999, p 405, with permission.)

differ from those in other capillaries by containing pores and are referred to as fenestrated endothelium. The pores increase capillary permeability but do not allow the passage of large molecules and blood cells. Further restriction of large molecules occurs as the filtrate passes through the basement membrane and the thin membranes covering the filtration slits formed by the intertwining foot processes of the podocytes of the inner layer of the Bowman capsule (Fig. 4-3A). In addition to the structure of the glomerular filtration barrier that prohibits the filtration of large molecules, the barrier contains a shield of negativity that repels molecules with a negative charge even though they are small enough to pass through the three layers of the barrier. The shield is very

Urinary bladder

Urethra

important because it is the place where albumin (the primary protein associated with renal disease) has a negative charge and is repelled (Figs. 4-3B and 4-3C).

Glomerular Pressure As mentioned previously, the presence of hydrostatic pressure, resulting from the smaller size of the efferent arteriole and the glomerular capillaries, enhances filtration. This pressure is necessary to overcome the opposition of pressures from the fluid within the Bowman capsule and the oncotic pressure of unfiltered plasma proteins in the glomerular capillaries. By increasing or decreasing the size of the afferent and efferent arterioles,

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Part One | Basic Principles Juxtaglomerular apparatus

Afferent arteriole Efferent arteriole Bowman capsule Glomerulus

Distal convoluted tubule

Proximal convoluted tubule

Cortex

Collecting duct

Peritubular capillaries Vasa recta Thick descending loop of Henle Vasa recta Thin descending loop of Henle

Medulla Thick ascending loop of Henle Thin ascending loop of Henle

Figure 4–2 The nephron and its component parts.

Box 4–1

Renal Blood Flow

Box 4–2

Urinary Filtrate Flow

1. Renal artery

1. Bowman capsule

2. Afferent arteriole

2. Proximal convoluted tubule

3. Glomerulus

3. Descending loop of Henle

4. Efferent arteriole

4. Ascending loop of Henle

5. Peritubular capillaries

5. Distal convoluted tubule

6. Vasa recta

6. Collecting duct

7. Renal vein

7. Renal calyces 8. Ureter 9. Bladder

an autoregulatory mechanism within the juxtaglomerular apparatus maintains the glomerular blood pressure at a relatively constant rate, regardless of fluctuations in systemic blood pressure. Dilation of the afferent arterioles and constriction of the efferent arterioles when blood pressure drops prevent a marked decrease in blood flowing through the kidney, thus preventing an increase in the blood level of toxic waste products. Likewise, an increase in blood pressure results in constriction of the afferent arterioles to prevent overfiltration or damage to the glomerulus.

Technical Tip 4-1. If it were not for the shield of negativity, all routine urines would have positive readings on reagent strips for protein and albumin.

10. Urethra

Renin–Angiotensin–Aldosterone System The RAAS regulates the flow of blood to and within the glomerulus. The system responds to changes in blood pressure and plasma sodium content that are monitored by the juxtaglomerular apparatus, which consists of the juxtaglomerular cells in the afferent arteriole and the macula densa of the distal convoluted tubule (Fig. 4-4). Low plasma sodium content decreases water retention within the circulatory system, resulting in a decreased overall blood volume and subsequent decrease in blood pressure. When the macula densa senses such changes, a cascade of

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Chapter 4 | Renal Function 109 Afferent arteriole Efferent arteriole Glomerular basement membrane

Hydrostatic pressure Oncotic pressure (unfiltered plasma proteins)

Protein

Glomerular basement membrane

Foot processes of podocyte

B

Fenestrated epithelium

Slit diaphragm Podocyte foot process

Podocyte

Bowman’s space

Proximal convoluted tubule Blood

A C

Glomerular basement membrane Fenestrated endothelium

Figure 4–3 Factors affecting glomerular filtration in the renal corpuscle (A). Inset B, glomerular filtration barrier. Inset C, the shield of negativity.

Afferent arteriole

Distal tubule

Macula densa

Efferent arteriole

Juxtaglomerular cells

Glomerulus

Figure 4–4 Close contact of the distal tubule with the afferent arteriole, macula densa, and the juxtaglomerular cells within the juxtaglomerular apparatus. Note the smaller size of the afferent arteriole indicating increased blood pressure.

reactions within the RAAS occurs (Fig. 4-5). Renin, an enzyme produced by the juxtaglomerular cells, is secreted and reacts with the bloodborne substrate angiotensinogen to produce the inert hormone angiotensin I. As angiotensin I passes through the alveoli of the lungs, angiotensin-converting enzyme (ACE) changes it to the active form angiotensin II. Angiotensin II corrects renal blood flow in the following ways: • Causing vasodilation of the afferent arterioles and constriction of the efferent arterioles • Stimulating reabsorption of sodium and water in the proximal convoluted tubules • Triggering the release of the sodium-retaining hormone aldosterone by the adrenal cortex and antidiuretic hormone by the hypothalamus (Box 4-3). As systemic blood pressure and plasma sodium content increase, the secretion of renin decreases. Therefore, the actions of angiotensin II produce a constant pressure within the nephron. As a result of the glomerular mechanisms just discussed, every minute, approximately 2 to 3 million glomeruli filter approximately 120 mL of water-containing low-molecular-weight substances. Because this filtration is nonselective, the only difference between the compositions of the filtrate and the plasma is the absence of plasma protein, any protein-bound substances, and cells. Analysis of the fluid as it leaves the glomerulus shows the filtrate to have a specific gravity of 1.010 and confirms that it is chemically an ultrafiltrate of plasma. This information provides a useful baseline for evaluating the renal mechanisms involved in converting the plasma ultrafiltrate into the final urinary product.

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Part One | Basic Principles Low blood pressure Low plasma sodium

Renin secretion Angiotensinogen Angiotensin I Angiotensinconverting enzymes Angiotensin II

Vasoconstriction

Box 4–3

Proximal convoluted tubule Sodium reabsorption

Aldosterone

ADH

Distal convoluted tubule Sodium reabsorption

Collecting duct Water resorption

Actions of the RAAS

Table 4–1

1. Dilates the afferent arteriole and constricts the efferent arteriole 2. Stimulates sodium reabsorption in the proximal convoluted tubule 3. Triggers the adrenal cortex to release the sodium-retaining hormone aldosterone to cause sodium reabsorption and potassium excretion in the distal convoluted tubule and collecting duct

Active transport

Figure 4–5 Algorithm of the renin– angiotensin–aldosterone system.

Tubular Reabsorption Substance

Location

Glucose, amino acids, salts Chloride

Proximal convoluted tubule Ascending loop of Henle Proximal and distal convoluted tubules Proximal convoluted tubule Descending loop of Henle Collecting duct Proximal convoluted tubule Ascending loop of Henle Ascending loop of Henle

Sodium

4. Triggers the hypothalamus to release antidiuretic hormone to stimulate water reabsorption in the collecting duct

Passive transport

Water

Tubular Reabsorption The body cannot lose 120 mL of water-containing essential substances every minute. Therefore, when the plasma ultrafiltrate enters the proximal convoluted tubule, the nephrons, through cellular transport mechanisms, begin reabsorbing these essential substances and water (Table 4-1).

Reabsorption Mechanisms The cellular mechanisms involved in tubular reabsorption are termed active transport and passive transport. For active transport to occur, the substance to be reabsorbed must combine with a carrier protein contained in the membranes of the renal tubular epithelial cells. The electrochemical energy created by this interaction transfers the substance across the cell membranes and back into the bloodstream. Active transport is responsible for the reabsorption of the following substances: • Glucose, amino acids, and salts in the proximal convoluted tubule • Chloride in the ascending loop of Henle • Sodium in the distal convoluted tubule

Urea

Sodium

Passive transport is the movement of molecules across a membrane as a result of differences in their concentration or electrical potential on opposite sides of the membrane. These physical differences are called gradients. Passive reabsorption of water takes place in all parts of the nephron except the ascending loop of Henle, the walls of which are impermeable to water. Urea is passively reabsorbed in the proximal convoluted tubule and the ascending loop of Henle, and passive reabsorption of sodium accompanies the active transport of chloride in the ascending loop.

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Chapter 4 | Renal Function 111 Active transport, like passive transport, can be influenced by the concentration of the substance being transported. When the plasma concentration of a substance that is normally completely reabsorbed reaches a level that is abnormally high, the filtrate concentration exceeds the maximal reabsorptive capacity (Tm) of the tubules, and the substance begins appearing in the urine. The plasma concentration at which active transport stops is termed the renal threshold. For glucose, the plasma renal threshold is 160 to 180 mg/dL, and glucose appears in the urine when the plasma concentration reaches this level. Knowledge of the renal threshold and the plasma concentration can be used to distinguish between excess solute filtration and renal tubular damage. Active transport of more than two-thirds of the filtered sodium out of the proximal convoluted tubule is accompanied by the passive reabsorption of an equal amount of water. Therefore, as seen in Figure 4-6, the fluid leaving the proximal convoluted tubule still maintains the same concentration as the ultrafiltrate.

Technical Tip 4-2.

Glucose appearing in the urine of a person with a normal blood glucose level is the result of tubular damage and not diabetes mellitus. A nonfasting patient with high glucose intake would not have a normal blood glucose. The plasma glucose would reach the renal threshold and appear in the urine.

Tubular Concentration Renal concentration begins in the descending and ascending loops of Henle, where the filtrate is exposed to the high osmotic gradient of the renal medulla. Water is removed by osmosis in the descending loop of Henle, and sodium and chloride are reabsorbed in the ascending loop. Excessive reabsorption of water as the filtrate passes through the highly concentrated medulla is prevented by the water-impermeable walls of the ascending loop. This selective reabsorption process is called the countercurrent mechanism and serves to maintain the osmotic gradient of the medulla (see Fig. 4-6). The sodium and chloride leaving the filtrate in the ascending loop prevent dilution of the medullary interstitium by the water reabsorbed from the descending loop. Maintenance of this osmotic gradient is essential for the final concentration of the filtrate when it reaches the collecting duct. In Figure 4-6, the actual concentration of the filtrate leaving the ascending loop is quite low due to the reabsorption of salt and not water in that part of the tubule. Reabsorption of sodium continues in the distal convoluted tubule, but now it is under the control of the hormone aldosterone, which regulates reabsorption in response to the body’s need for sodium (Fig. 4-5).

Collecting Duct Concentration The final concentration of the filtrate through the reabsorption of water begins in the late distal convoluted tubule and continues in the collecting duct. Reabsorption depends on the

osmotic gradient in the medulla, as well as the hormone vasopressin (antidiuretic hormone [ADH]) that is released by the posterior pituitary gland when the amount of water in the body decreases. One would expect that as the dilute filtrate in the collecting duct comes in contact with the higher osmotic concentration of the medullary interstitium, passive reabsorption of water would occur. However, the process is controlled by the presence or absence of ADH, which renders the walls of the distal convoluted tubule and collecting duct permeable or impermeable to water. A high level of ADH increases permeability, resulting in increased reabsorption of water, and a low-volume concentrated urine. Likewise, absence of ADH renders the walls impermeable to water, resulting in a large volume of dilute urine. Just as the production of aldosterone is controlled by the body’s sodium concentration, the production of ADH is determined by the state of body hydration. Therefore, the chemical balance in the body is actually the final determinant of urine volume and concentration. The concept of ADH control can be summarized as follows: ↑ Body Hydration = ↓ ADH = ↑ Urine Volume ↓ Body Hydration = ↑ ADH = ↓ Urine Volume

Tubular Secretion In contrast to tubular reabsorption, in which substances are removed from the glomerular filtrate and returned to the blood, tubular secretion involves the passage of substances from the blood in the peritubular capillaries to the tubular filtrate (Fig. 4-7). Tubular secretion serves two major functions: eliminating waste products not filtered by the glomerulus and regulating the acid–base balance in the body through the secretion of hydrogen ions. Many foreign substances, such as medications, cannot be filtered by the glomerulus because they are bound to plasma proteins. When these protein-bound substances enter the peritubular capillaries, they develop a stronger affinity for the tubular cells and dissociate from their carrier proteins, which results in their transport into the filtrate by the tubular cells. The major site for removal of these nonfiltered substances is the proximal convoluted tubule.

Acid–Base Balance To maintain the normal blood pH of 7.4, the blood must buffer and eliminate the excess acid formed by dietary intake and body metabolism. The buffering capacity of the blood depends on bicarbonate (HCO3–) ions, which are readily filtered by the glomerulus and must be returned to the blood expediently to maintain the proper pH. As shown in Figure 4-8, the secretion of hydrogen ions (H+) by the renal tubular cells into the filtrate prevents the filtered bicarbonate from being excreted in the urine and causes the return of a bicarbonate ion to the plasma. This process, which occurs primarily in the proximal convoluted tubule, provides for almost 100% reabsorption of filtered bicarbonate. As a result of their small molecular size, hydrogen ions are readily filtered and reabsorbed. Therefore, the actual excretion of excess hydrogen ions also depends on tubular secretion.

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Cortex

Blood (vein) 300 mOsm/L

Efferent arteriole

Proximal convoluted tubule

300

Glomerulus

Na+Cl- H2O

300

H2O

100

Descending limb

Na+ClThick ascending limb

H2O 600

Na+

Na+

Na+ClH2O

H2O

H2O H2O H2O

900 Loop of Henle 1200

Medulla

Countercurrent mechanism Reabsorption Secretion Variable reabsorption

Figure 4–6 Renal concentration. Tubular lumen

Renal tubular cell

Peritubular plasma

HCO3– (filtered)

Efferent arteriole

HCO3 + H+

Afferent arteriole

H+

HCO3

–

HCO3

–

H2CO3

Bowman’s capsule Glomerular filtrate

Reabsorption

H2CO3

Carbonic anhydrase

H2O + CO2

H2O + CO2

CO2

Final urine To blood

Figure 4–8 Reabsorption of filtered bicarbonate.

Secretion

Tubule To urine

Figure 4–7 Summary of movement of substances in the nephron.

Figures 4-9 and 4-10 are diagrams of the two primary methods for the excretion of hydrogen ions in the urine. In Figure 4-9 the secreted hydrogen ion combines with a filtered phosphate ion instead of a bicarbonate ion and is excreted rather than reabsorbed. In the proximal convoluted tubule, ammonia is produced from the breakdown of the amino acid glutamine.

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Chapter 4 | Renal Function 113 Tubular lumen

Renal tubular cell

Peritubular plasma

Osmolarity

HPO4– (filtered) HPO4– + H+

PAH GFR tests

HCO3–

H+

HCO3–

H2CO3 Ammonia H2PO4

Carbonic anhydrase

H2O + CO2

CO2

Titratable acidity

Final urine

Figure 4–9 Excretion of secreted hydrogen ions combined with phosphate. Tubular lumen

Renal tubular cell

Free water clearance

Osmolarity

Peritubular plasma

NH3 NH3 + H+

H+

HCO3

–

HCO3

–

H2CO3 NH4+

Carbonic anhydrase

H2O + CO2

CO2

Final urine

Figure 4–10 Excretion of secreted hydrogen ions combined with ammonia produced by the tubules.

Figure 4–11 The relationship of nephron areas to renal function tests.

which the kidneys are able to remove (to clear) a filterable substance from the blood. To ensure that glomerular filtration is being measured accurately, the substance analyzed must be one that is neither reabsorbed nor secreted by the tubules. Other factors to consider in selecting a clearance test substance include the stability of the substance in urine during a possible 24-hour collection period, the consistency of the plasma level, the substance’s availability to the body, and the availability of tests to analyze the substance.

Clearance Tests The ammonia reacts with the H+ to form the ammonium ion (NH4+) (Fig. 4-10). The resulting ammonium ion is excreted in the urine. Should there be additional need for the elimination of hydrogen ions, the distal convoluted tubule and the collecting duct are also able to produce ammonium ions. All three of these processes occur simultaneously at rates determined by the acid–base balance in the body. A disruption in these secretory functions can result in metabolic acidosis or renal tubular acidosis, the inability to produce an acid urine.

Renal Function Tests This brief review of renal physiology shows that there are many metabolic functions and chemical interactions to be evaluated through laboratory tests of renal function. In Figure 4-11, the parts of the nephron are related to the laboratory tests used to assess their function.

Glomerular Filtration Tests The standard tests used to measure the filtering capacity of the glomeruli are termed clearance tests. As its name implies, a clearance test measures the rate in milliliters per minute at

A variety of substances has been used to measure the GFR. Newer methods that eliminate many of the problems mentioned previously have replaced some of these tests. They are summarized as Historical Notes. At present, creatinine, beta2-microglobulin (B2M), cystatin C, and, possibly, radioisotopes are the primary substances used in clearance tests. Each procedure has its advantages and disadvantages.

HISTORICAL NOTE

Urea Clearance The earliest glomerular filtration tests measured urea because of its presence in all urine specimens, as well as the existence of routinely used methods of chemical analysis. Because approximately 40% of the filtered urea is reabsorbed, normal values were adjusted to reflect the reabsorption, and patients were hydrated to produce a urine flow of 2 mL/min to ensure that no more than 40% of the urea was reabsorbed.

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HISTORICAL NOTE

Inulin Clearance Inulin, a polymer of fructose, is an extremely stable substance that is neither reabsorbed nor secreted by the tubules. It is not a normal body constituent, however, and must be infused by IV at a constant rate throughout the testing period. Therefore, although inulin was the original reference method for clearance tests, current methods are available that are endogenous and can provide accurate GFR results.

A test that requires an infused substance is termed an exogenous procedure and is seldom the method of choice if a suitable test substance is already present in the body (endogenous procedure). Creatinine Clearance Creatinine is a waste product of muscle metabolism that is produced enzymatically by creatine phosphokinase from creatine, which links with adenosine triphosphate (ATP) to produce adenosine diphosphate (ADP) and energy. Because creatinine is normally found at a relatively constant level in the blood, it provides the laboratory with an endogenous procedure for evaluating glomerular function. The use of creatinine has several disadvantages, and careful consideration should be given to them: 1. Some creatinine is secreted by the tubules, and secretion increases as blood levels rise. 2. Chromogens present in human plasma react in the chemical analysis. Their presence, however, may help counteract the falsely elevated rates caused by tubular secretion. 3. Medications, including gentamicin, cephalosporins, and cimetidine (Tagamet), inhibit tubular secretion of creatinine, thus causing serum levels that are falsely low.1 4. Bacteria will break down urinary creatinine if specimens are kept at room temperature for extended periods.2 5. A diet heavy in meat consumed during collection of a 24-hour urine specimen will influence the results if the plasma specimen is drawn before the collection period. The increased intake of meat can raise the creatinine levels in urine and plasma during the 24-hour collection period. 6. Measurement of creatinine clearance is not a reliable indicator in patients suffering from muscle-wasting diseases or those involved in heavy exercise or athletes supplementing with creatine. 7. Accurate results depend on the accurate completeness of a 24-hour collection. 8. Creatinine clearance values must be corrected for body surface area, unless normal is assumed, and must always be corrected for children.

Newer methods that do not require the collection of timed (24-hour) urine specimens have been developed using just the serum creatinine, cystatin C, or B2M values. The results of these tests are reported as estimated glomerular filtration rate (eGFR). The traditional procedure for creatinine clearance is included here because it is still being performed, and its principles apply to other clearance procedures using urine. Procedure By far, the greatest source of error in any clearance procedure using urine is the use of urine specimens that are improperly timed. The importance of using a specimen that is accurately timed (see Chapter 3) will become evident in the following discussion of the calculations involved in converting isolated laboratory measurements to the GFR. The GFR is reported in milliliters cleared per minute; therefore, it is necessary to determine the number of milliliters of plasma from which the clearance substance (creatinine) is removed completely during 1 minute. To calculate this information, one must know urine volume in mL/min (V), urine creatinine concentration in mg/dL (U), and plasma creatinine concentration in mg/dL (P). The urine volume is calculated by dividing the number of milliliters in the specimen by the number of minutes used to collect the specimen. EXAMPLE Calculate the urine volume (V) for a 2-hour specimen measuring 240 mL: 2 hours × 60 minutes = 120 minutes 240 mL/120 minutes = 2 mL/min V = 2 mL/min

The concentrations of plasma and urine are determined by chemical testing. The standard formula used to calculate the milliliters of plasma cleared per minute (C) is: C =

UV P

This formula is derived as follows: The milliliters of plasma cleared per minute (C) times the mg/dL of plasma creatinine (P) must equal the mg/dL of urine creatinine (U) times the urine volume in mL/min (V), because all of the filtered creatinine will appear in the urine. Therefore: CP = UV and C =

UV P

EXAMPLE Using urine creatinine of 120 mg/dL (U), plasma creatinine of 1.0 mg/dL (P), and urine volume of 1440 mL obtained from a 24-hour specimen (V), calculate the GFR. V =

1440 mL = 1 mL/min 60 minutes × 24 = 1440 minutes

C =

120 mg/dL × 1 mL/min (V) = 120 mL/dL 1.0 mg/dL (P)

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Chapter 4 | Renal Function 115 Plasma (1 mg/dL = 0.01 mg/mL creatinine)

6" 4" 2"

By analyzing this calculation and referring to Figure 4-12, at a concentration of 1 mg/dL, each milliliter of plasma contains 0.01 mg creatinine. Therefore, to arrive at a urine concentration of 120 mg/dL (1.2 mg/mL), it is necessary to clear 120 mL of plasma. Although the filtrate volume is reduced, the amount of creatinine in the filtrate does not change because the creatinine is not reabsorbed. Knowing that in the average person (1.73 m2 body surface), the approximate amount of plasma filtrate produced per minute is 120 mL, it is not surprising that normal creatinine clearance values approach 120 mL/min (men, 107 to 139 mL/min; women, 87 to 107 mL/min). The normal reference range of plasma creatinine is 0.6 to 1.2 mg/dL. These reference values consider variations in size and muscle mass. Values are considerably lower in older people; however, an adjustment also may have to be made to the calculation when dealing with body sizes that deviate greatly from 1.73 m2 of surface, such as with children. To adjust a clearance for body size, the formula is: C=

UV 1.73 × P A

with A being the actual body size in square meters of surface. The actual body size may be calculated as: log A = (0.425 × log weight) + (0.725 × log height) – 2.144

or it may be obtained from the nomogram shown in Figure 4-13.

Estimated Glomerular Filtration Rates In the past years, a variety of formulas for eGFR have been used, and those formulas continue to be revised. Because the formulas can be programmed into automated instruments, estimated clearances can be used for routinely screening patients as part of a metabolic profile and to monitor patients already diagnosed with renal disease or at risk for renal disease. In addition, the formulas are valuable when there is a need to prescribe medications that require adequate renal clearance.

10" 8" 6" Height in feet

Figure 4–12 Creatinine filtration and excretion.

5'

4" 2" 4'

10" 8" 6" 4"

160 155 150 145 140 135 130 125 120 115 110

1.10 1.05

95

.80

90

160 150

190 180 170

140 130 120 110 100 95 90 85 80 75

160 150 140

70 65

130

60

120

55

110

50

100

45

90

40

.90 .85

3'

180

1.00

100

2"

10"

1.15

.95 105

300 290 280 270 260 250 240 230 220 210 200

2.10

165

170

190

320

2.30 2.20 2.00 1.95 1.90 1.85 1.80 1.75 1.70 1.65 1.60 1.55 1.50 1.45 1.40 1.35 1.30 1.25 1.20

380 360 340

Weight in pounds

Urine (1 mL/min) Creatinine (1.2 mg/mL or 120 mg/dL)

6' 10" 8"

220 215 210 205 200 195 190 185 180 175 170

Surface area in square meters

Reabsorption (119 mL H2O)

7' 10" 8" 6" 4" 2"

Height in centimeters

Plasma filtrate (120 mL/min x 0.01 mg/mL = 1.2 mg)

3.00 2.90 2.80 2.70 2.60 2.50 2.40

200

Weight in kilograms

Glomerulus

440 420 400

80 35 70

.75

30

.70 60

85 .65

8"

25

80 .60 6"

50 75

.55 20 .50 40

15

Figure 4–13 A nomogram for determining body surface area. (From Boothby, WM, and Sandiford, RB: Nomogram for determination of body surface area. N Engl J Med 185:227, 1921, with permission.)

The formula used most frequently is called the Modification of Diet in Renal Disease (MDRD) study. The formula has been modified several times to make it more accurate and standardized. At present, the formula recommended by the National Kidney Disease Education Program (NKDEP) is called the MDRD-IDMS-traceable formula. A primary discrepancy in the previous formulas was found to be the methods used to measure serum creatinine. eGFR equations are superior to serum creatinine alone because they include variables for race, age, and gender.3 Current laboratory methods primarily use creatinine assays, such as enzyme assays, that do not have the same interference as the original Jaffe chemical method. These methods correspond more closely to the isotope dilution mass spectrophotometry (IDMS) reference method.

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Part One | Basic Principles The MDRD-IDMS traceable formula is: creatinine–1.154

age–0.203

GFR = 175 × serum × × 0.742 (if patient is female) × 1.212 (if patient is black)

The formula is designed to essentially equal the results that compare to the reference body size of 1.73 m2. Because eGFRs are calculated for an average body size, they are not accurate for pediatric patients. They also have been shown to be most accurate when results are lower than 60 mL/min.4 It is recommended that results be reported with numerical values below 60 mL/min (for example, 59 mL/min) and higher values reported as equal to or greater than 60 mL/min (for example, ≥60 mL/min). The formula recommended for use when serum creatinine methods do not compare to the IDSM standard is provided as a Historical Note. The eGFR is classified into the following stages based on the kidney disease. The Improving Global Outcomes (KDIGO) stages of chronic kidney disease (CKD) are3: • Stage 1 GFR greater than 90 mL/min/1.73 m • Stage 2 GFR between 60 and 89 mL/min/1.73 m • Stage 3 GFR between 45 and 59 mL/min/1.73 m • Stage 4 GFR between 15 and 29 mL/min/1.73 m • Stage 5 GFR less than 15 mL/min/1.73 m (end-stage renal disease)

Cystatin C The measurement of serum cystatin C has been shown to be a good procedure for screening and monitoring GFR. Cystatin C is a small protein (molecular weight 13,359) produced at a constant rate by all nucleated cells. It is filtered readily by the glomerulus and reabsorbed and broken down by the renal tubular cells. Therefore, no cystatin C is secreted by the tubules, and the serum concentration can be related directly to the GFR. Immunoassay procedures are available for measuring cystatin C.5 Monitoring levels of cystatin C is recommended for pediatric patients, people with diabetes, the elderly, and patients who are critically ill.6 An advantage of cystatin C is that it is independent of muscle mass. Recent studies also have shown that measuring both serum or plasma cystatin C and creatinine can provide even more accurate information on a patient’s GFR.7

HISTORICAL NOTE

Original MDRD Calculation The formula for MDRD calculation of GFR when the serum creatinine method is not standardized to IDMS. GFR = 173 × serum creatinine–1.154 × age–0.203 × 0.742 (if patient is female) × 1.212 (if patient is black)

Beta2-Microglobulin B2M (molecular weight 11,800) dissociates from human leukocyte antigens at a constant rate and is removed rapidly from the plasma by glomerular filtration. The B2M may be used to distinguish disorders of the kidney as either glomerular or tubular. Its excretion in urine is normally low, but when the renal tubules become damaged or diseased, the B2M concentration increases due to the decreased ability to reabsorb this protein. By measuring B2M concentrations of both blood and urine to evaluate kidney damage or disease, disorders that affect the glomerulus and renal tubules may be distinguished.8 The concentration of B2M in serum is normal, and its concentration in urine increases when tubular reabsorption is reduced. Increased levels of B2M in the blood combined with low levels in the urine indicate that the disorder is associated with glomeruli disorders. The B2M also is used to identify end-stage renal disease and early rejection of a kidney transplant. Sensitive methods using enzyme immunoassay are available for the measurement of B2M. A rise in the plasma level of B2M has been shown to be a more sensitive indicator of a decrease in GFR than creatinine clearance. However, the test is not reliable in patients who have a history of immunologic disorders or malignancy.9

Radionucleotides Although it is an exogenous procedure and more labor intensive and costly, injecting radionucleotides such as 125I-iothalamate provides a method for determining glomerular filtration through the plasma disappearance of the radioactive material and enables visualization of the filtration in one or both kidneys.10 This procedure can be valuable to measure the viability of a transplanted kidney. Other exogenous markers used are radioisotopes, such as chromium-51 ethylene-diamine-tetra-acetic acid (51 Cr-EDTA), and technetium-99-labeled diethylene-triaminepentaacetate (99-Tc-DTPA). A nonradioactive contrast agent, iohexol, is used for children.3 Clinical Significance When interpreting the results of a creatinine clearance test, the GFR is determined not only by the number of functioning nephrons but also by the functional capacity of these nephrons. In other words, even though half of the available nephrons may be nonfunctional, a change in the GFR will not occur if the remaining nephrons double their filtering capacity. This is evident in people who lead normal lives with only one kidney. Therefore, although the GFR is a frequently requested laboratory procedure, its value does not lie in the detection of early renal disease. Instead, it is used to determine the extent of nephron damage in known cases of renal disease, to monitor the effectiveness of treatment designed to prevent further nephron damage, and to determine the feasibility of administering medications, which can build up to dangerous blood levels if the GFR is markedly reduced.

Tubular Reabsorption Tests As discussed, although measurement of the GFR is not a useful indication of early renal disease, the loss of tubular reabsorption capability is often the first function affected in renal disease.

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Chapter 4 | Renal Function 117 This is not surprising when one considers the complexity of the tubular reabsorption process. Tests to determine the ability of the tubules to reabsorb the essential salts and water that have been nonselectively filtered by the glomerulus are called concentration tests. As mentioned, the ultrafiltrate that enters the tubules has a specific gravity of 1.010; therefore, after reabsorption, one would expect the final urine product to be more concentrated. However, many urine specimens do not have a specific gravity higher than 1.010, yet no renal disease is present. This is because urine concentration is largely determined by the body’s state of hydration, and the normal kidney will reabsorb only the amount of water necessary to preserve an adequate supply of body water. As seen in Figure 4-14, both urine specimens contain the same amount of solute; however, the urine density (specific gravity) of patient A will be higher. Therefore, control of fluid intake must be incorporated into laboratory tests that measure the concentrating ability of the kidney. Throughout the years, various methods have been used to produce water deprivation, including the Fishberg and Mosenthal concentration tests, which measured specific gravity. In the Fishberg test, patients were deprived of fluids for 24 hours before specific gravity was measured. The Mosenthal test compared the volume and specific gravity of urine specimens collected during the day and at night to evaluate concentrating ability. Neither test is used now because the information provided by specific gravity measurements is most useful as a screening procedure, and quantitative measurement of renal concentrating ability is best assessed through osmometry. Currently renal concentrating testing is performed after various periods of fluid deprivation, measuring urine, and often serum osmolality. Controlled intake procedures can include after-dinner overnight deprivation of fluid for 12 hours followed by collection of a urine specimen. A urine osmolality reading of 800 mOsm or higher is normal, and the test can be discontinued. If the urine test is abnormal, the fluid is restricted for another 2 hours, and specimens of both urine and serum are collected for osmolality testing. A urine-to-serum

ratio (U:S ratio) of 3:1 or greater or a urine osmolality of 800 mOsm or greater indicates normal tubular reabsorption. If the test continues to be abnormal, additional testing is performed to determine whether the failure to concentrate the urine is caused by diabetes insipidus that occurs as the result of a problem with the production of or the response of the kidney to ADH. The patient is injected with ADH, and specimens of serum and urine are collected in 2 and 4 hours. Normal test results indicate that the patient is not capable of producing ADH (neurogenic diabetes insipidus). If the test is abnormal, then the renal tubules are not responding to ADH (nephrogenic diabetes insipidus). See Figure 4-15.

Osmolality As will be discussed in Chapter 5, osmolality measures only the number of particles in a solution, whereas specific gravity is influenced by both the number and density (molecular weight) of the particles. Renal concentration is concerned with small particles, primarily sodium and chloride molecules. Large-molecular-weight molecules, such as glucose and urea, do not contribute to the evaluation of renal concentration. Therefore, osmolality is performed for a more accurate evaluation of renal concentrating ability.

Freezing-Point Osmometers Measurement of freezing-point depression was the first principle incorporated into clinical osmometers, and many instruments employing this technique are available now. These osmometers determine the freezing point of a solution by supercooling a measured amount of sample to approximately 27°C. The supercooled sample is vibrated to produce crystallization of water in the solution. The heat of fusion produced by the crystallizing water temporarily raises the temperature of the solution to its freezing point. A temperature-sensitive probe called a thermistor, in which resistance decreases as temperature increases, measures this temperature increase, which corresponds to the freezing point of the solution, and the information is converted into milliosmoles. Conversion is

Patient A

Patient B

Water (1 glass)

Water (4 glasses)

Glomerulus

Glomerulus

Ultrafiltrate

120 mL Water 300 mg Solute Reabsorption

Figure 4–14 The effect of hydration on renal concentration. Notice the decreased specific gravity in the morehydrated Patient B.

Urine

1 mL Water 200 mg Solute

Specific gravity 1.015

Ultrafiltrate

119 mL Water 100 mg Solute

120 mL Water 300 mg Solute Reabsorption

Urine

10 mL Water 200 mg Solute

Specific gravity 1.005

110 mL Water 100 mg Solute

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118 Part One | Basic Principles Polyuria Urine osmolality 100 WBCs/hpf 0–1 bacterial casts/lpf with clumps A repeat urinalysis a day later has the following results: Color: Yellow Ketones: Negative Clarity: Cloudy Blood: Small Sp. gravity: >1.035 Bilirubin: Negative pH: 7.5 Urobilinogen: Normal Protein: 2+ Nitrite: Positive Glucose: Negative Leukocyte: 2+

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Part Two | Urinalysis

Microscopic: 6–10 RBCs/hpf 0–2 WBC casts/lpf >100 WBCs/hpf

Many bacteria Moderate birefringent, flat crystals

0–1 bacterial casts/lpf a. What diagnostic procedure was performed on the patient that could account for the differences in the two urinalysis results? b. Considering the patient’s age and history, what is the most probable diagnosis? c. What microscopic constituent is most helpful to this diagnosis? d. What is the most probable cause of this disorder? e. How can the presence of the bacterial cast be confirmed? f. What is the most probable source of the crystals present in the sediment? g. Without surgical intervention, what is the patient’s prognosis? 8. A 35-year-old patient being treated for a sinus infection with methicillin develops fever, a skin rash, and edema. Urinalysis results are as follows: Color: Dark yellow Ketones: Negative Clarity: Cloudy Blood: Moderate Sp. gravity: 1.015 Bilirubin: Negative pH: 6.0 Urobilinogen: Normal Protein: 3+ Nitrite: Negative Glucose: Negative Leukocyte: 2+ Microscopic: 20–30 RBCs/hpf 1–2 WBC casts/lpf >100 WBCs/hpf 1–2 granular casts/lpf After receiving the urinalysis report, the physician orders a test for urinary eosinophils. The urinary eosinophil result is 10%. a. Is the urinary eosinophil result normal or abnormal? b. What is the probable diagnosis for this patient? c. Discuss the significance of the increased WBCs and WBC casts in the absence of bacteria. d. How can this condition be corrected? 9. After surgery to correct a massive hemorrhage, a 55-yearold patient exhibits oliguria and edema. Blood test results indicate increasing azotemia and electrolyte imbalance. The glomerular filtration rate is 20 mL/min. Urinalysis results are as follows: Color: Yellow Ketones: Negative Clarity: Cloudy Blood: Moderate

Sp. gravity: 1.010 pH: 7.0 Protein: 3+ Glucose: 2+ Microscopic: 50–60 RBCs/hpf 3–6 WBCs/hpf 3–4 RTE cells/hpf

a. b.

c. d. e.

Bilirubin: Negative Urobilinogen: Normal Nitrite: Negative Leukocyte: Negative

2–3 granular casts/lpf 2–3 RTE cell casts/lpf 0–1 waxy casts/lpf 0–1 broad granular casts/lpf What diagnosis do the patient’s history and laboratory results suggest? What is the most probable cause of the patient’s disorder? Is this considered to be of prerenal, renal, or postrenal origin? What is the significance of the specific gravity result? What is the significance of the RTE cells? State two possible reasons for the presence of the broad casts.

10. A 40-year-old man develops severe back and abdominal pain after dinner. The pain subsides during the night but recurs in the morning, and he visits his family physician. Results of a complete blood count and an amylase test are normal. Results of a routine urinalysis are as follows: Color: Dark yellow Ketones: Negative Clarity: Hazy Blood: Moderate Sp. gravity: 1.030 Bilirubin: Negative pH: 5.0 Urobilinogen: Normal Protein: Trace Nitrite: Negative Glucose: Negative Leukocytes: Negative Microscopic 15–20 RBCs/hpf 0–2 WBCs/hpf Few squamous epithelial cells a. What condition could these urinalysis results and the patient’s symptoms represent? b. What would account for the crenated RBCs? c. Is there a correlation between the urine color and specific gravity and the patient’s symptoms? d. Based on the primary substance that causes this condition, what type of crystals might have been present? e. What changes will the patient be advised to make in his lifestyle to prevent future occurrences?

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CHAPTER

9

Urine Screening for Metabolic Disorders LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 9-1 Explain abnormal accumulation of metabolites in the urine in terms of overflow and renal disorders.

9-10 State the significance of increased urinary 5-hydroxyindoleacetic acid.

9-2 Discuss the importance of and the MS/MS testing methods for newborn screening.

9-11 Differentiate between cystinuria and cystinosis, including the differences found during analysis of the urine and the disease processes.

9-3 Name the metabolic defect in phenylketonuria, and describe the clinical manifestations it produces. 9-4 State three causes of tyrosyluria. 9-5 Name the abnormal urinary substance present in alkaptonuria, and explain how its presence may be suspected. 9-6 Discuss the appearance and significance of urine that contains melanin. 9-7 Describe a basic laboratory observation that has relevance in maple syrup urine disease. 9-8 Discuss the significance of ketonuria in a newborn.

9-12 Describe the components in the heme synthesis pathway, including the primary specimens used for their analysis, and explain the cause and clinical significance of major porphyrias and the appearance of porphyrins in urine. 9-13 Define mucopolysaccharides, and name three syndromes in which they are involved. 9-14 State the significance of increased uric acid crystals in newborns’ urine. 9-15 Explain the reason for performing tests for urinaryreducing substances on all newborns.

9-9 Differentiate between the presence of urinary indican due to intestinal disorders and Hartnup disease.

KEY TERMS Alkaptonuria

Homocystinuria

Melituria

Aminoaciduria

Inborn error of metabolism (IEM)

Ochronosis

Cystinosis

Indicanuria

Organic acidemias

Cystinuria

Lesch-Nyhan disease

Phenylketonuria (PKU)

Galactosuria

Maple syrup urine disease (MSUD)

Porphyrinuria

Hartnup disease

Melanuria

Tyrosyluria

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Introduction As discussed in previous chapters, many of the abnormal results obtained in the routine urinalysis are related to metabolic rather than renal disease. Urine as an end product of body metabolism may contain additional abnormal substances not tested for by routine urinalysis. Often these substances can be detected or monitored by additional screening tests that also can be performed in the urinalysis laboratory. Then positive screening tests can be followed up with more sophisticated procedures performed in other sections of the laboratory (Table 9-1). The need to perform additional tests may be detected by the observations of alert laboratory personnel when performing the routine analysis or from observations of abnormal specimen color and odor by nursing staff and patients (Table 9-2). In other instances, clinical symptoms and family histories are the determining factors.

Overflow Versus Renal Disorders The appearance of abnormal metabolic substances in the urine can be caused by a variety of disorders that generally can be grouped into two categories, termed the overflow type and the

Table 9–1

Urine Screening Tests for Metabolic Disorders

Test

Disorder

Benedict test Ferric chloride test

Alkaptonuria Alkaptonuria MSUD Melanuria PKU Porphyria Tyrosinuria Alkaptonuria Porphyria

Hoesch test Nitrosonaphthol test Silver nitrate test Watson-Schwartz test

Table 9–2

Abnormal Metabolic Constituents or Conditions Detected in the Routine Urinalysis

Color

Odor

Crystals

Homogentisic acid Melanin

Phenylketonuria

Cystine

Maple syrup urine disease Isovaleric acidemia Cystinuria Cystinosis Homocystinuria

Leucine

Indican Porphyrins

Tyrosine Lesch-Nyhan disease

renal type. Overflow disorders result from disruption of a normal metabolic pathway that causes increased plasma concentrations of the nonmetabolized substances. These chemicals either override the reabsorption ability of the renal tubules or are not normally reabsorbed from the filtrate because they are present in only minute amounts. Abnormal accumulations of the renal type are caused by malfunctions in the tubular reabsorption mechanism, as discussed in Chapter 8. The abnormalities encountered most frequently are associated with metabolic disturbances that produce urinary overflow of substances involved in the metabolism of protein, fat, and carbohydrates. This is understandable when one considers the vast number of enzymes used in the metabolic pathways of proteins, fats, and carbohydrates and the fact that their function is essential for complete metabolism. Disruption of enzyme function can be caused by failure to inherit the gene to produce a particular enzyme, referred to as an inborn error of metabolism (IEM),1 or by organ malfunction from disease or toxic reactions. The abnormal urinary metabolites that are encountered most frequently are summarized in Table 9-3, and their appearance is classified according to functional defect. Table 9-3 also includes substances and conditions that are covered in this chapter.

Newborn Screening Tests Many of the urine tests discussed in this chapter traditionally were performed primarily to detect and monitor newborns for IEMs. In recent years, the screening of newborns has increased to include more sensitive detection methods and ever-increasing levels of state-mandated tests for IEMs. Many states currently require testing for as many as 31 core conditions and 25 secondary target conditions.2 As discussed later in this chapter, because many of these disorders cause the buildup of unmetabolized toxic food ingredients, it is important that the defects be detected early in life. Levels of these substances are elevated more rapidly in blood than in urine. Therefore, blood collected by infant heel puncture is tested initially. Testing for many substances is now performed using tandem mass spectrophotometry (MS/MS). MS/MS is capable of screening the infant blood specimen for specific substances associated with particular IEMs. Figure 9-1 shows the standard form collected for testing using MS/MS. Methods for specific gene testing also are being developed rapidly.

Amino Acid Disorders The amino acid disorders with urinary screening tests include phenylketonuria (PKU), tyrosyluria, melanuria, alkaptonuria, maple syrup urine disease (MSUD), organic acidemias, indicanuria, cystinuria, and cystinosis.

Phenylalanine-Tyrosine Disorders Major inherited disorders include PKU, tyrosyluria, and alkaptonuria. Metabolic defects cause overproduction of melanin. The relationship of these varied disorders is illustrated in Figure 9-2.

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Chapter 9 | Urine Screening for Metabolic Disorders Table 9–3

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Major Disorders of Protein and Carbohydrate Metabolism Associated With Abnormal Urinary Constituents, Classified by Functional Defect

Overflow Inherited

Metabolic

Renal

Phenylketonuria Tyrosinemia Alkaptonuria Maple syrup urine disease Organic acidemias Cystinosis Porphyria Mucopolysaccharidoses Galactosemia Lesch-Nyhan disease

Infantile tyrosinemia Melanuria Indicanuria 5-Hydroxyindoleacetic acid Porphyria

Hartnup disease Cystinuria

Figure 9–1 Specimen collection form for MS/MS newborn screening test. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, 4th ed, FA Davis.)

Phenylketonuria The most well-known of the aminoacidurias, phenylketonuria (PKU) is estimated to occur in 1 of every 10,000 to 20,000 births and, if undetected, results in severe intellectual disability. It was first identified in Norway by Ivan Følling in 1934, when a mother with other children who were intellectually delayed reported a peculiar mousy odor to her child’s urine and sweat. Analysis of the urine showed increased amounts of the keto acids, including phenylpyruvate. As shown in Figure 9-2, this occurs when the normal conversion of phenylalanine to tyrosine is disrupted. Interruption of the pathway also produces children with fair complexions and lighter hair and eyes—even in dark-skinned families—due to the decreased production of tyrosine and its pigmentation metabolite melanin. Seizures, hyperactivity, developmental delay, and psychiatric disturbances also may be present in some patients. PKU is caused by failure to inherit the gene to produce the enzyme phenylalanine hydroxylase. The gene is inherited as an autosomal recessive trait with no noticeable characteristics or defects exhibited by heterozygous carriers. Fortunately,

screening tests are available for early detection of the abnormality, and all states have laws that require the screening of newborns for PKU.2 Once discovered, dietary changes that eliminate phenylalanine, a major constituent of milk, from the infant’s diet can prevent excessive buildup of serum phenylalanine, thereby avoiding damage to the child’s mental capabilities. Many products that contain large amounts of phenylalanine, such as aspartame (added to medications, diet foods, and diet sodas), now features warning labels for people with PKU. The initial screening for PKU does not come under the auspices of the urinalysis laboratory because increased blood levels of phenylalanine must, of course, occur before urinary excretion of phenylpyruvic acid, which may take 2 to 6 weeks. State laws require that blood be collected between 24 and 48 hours after birth and before the newborn leaves the hospital. The increasing tendency to release newborns from the hospital as early as 24 hours after birth has caused concern about the ability to detect increased phenylalanine levels at that early stage. Studies have shown that in many cases phenylalanine can be detected as early as 4 hours after birth, and if the cutoff level for normal results is lowered from 4 mg/dL to 2 mg/dL, then the presence of PKU should be detected. Tests may need to be repeated during an early visit to the pediatrician. More girls than boys escape detection of PKU during early tests because of slower rises in levels of blood phenylalanine.1 Urine testing using ferric chloride may be used as a followup test to ensure proper dietary control in cases diagnosed previously and as a means of monitoring the dietary intake of pregnant women known to lack phenylalanine hydroxylase. Urine tests to screen for phenylpyruvic acid are based on the ferric chloride reaction performed by tube test. The addition of ferric chloride to urine containing phenylpyruvic acid produces a permanent blue-green color (see Procedure 9-1).

Tyrosyluria Tyrosyluria, the accumulation of excess tyrosine in the plasma (tyrosinemia) producing urinary overflow, may be due to several causes and is not well categorized. As seen in Table 9-2, disorders

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238 Part Two | Urinalysis Normal metabolism Metabolic disorders

Phenylalanine

Phenylketonuria

Enzymes

Phenylalanine hydroxylase Tyrosine

Phenylpyruvic acid Tyrosinemia type 2

Tyrosine aminotransferase

p-hydroxyphenylpyruvate oxidase Homogentisic acid

Homogentisic acid oxidase

Alkaptonuria

Homogentisic acid Tyrosinemia type 1b

Maleylacetoacetic acid Maleylacetoacetic acid isomerase Fumarylacetoacetic acid

Tyrosinemia type 1 Tyrosinemia type 1a

Normal byproducts

p-Hydroxyphenylpyruvic acid

Tyrosyluria p-hydroxyphenylpyruvic acid p-hydroxyphenyllactic acid Tyrosinemia type 3

Melanin Thyroxine Epinephrine

Fumarylacetoacetic acid hydrolase Fumaric acid and acetoacetic acid

Figure 9–2 Phenylalanine and tyrosine metabolic pathway, including the normal pathway (blue), enzymes (yellow), and disorders caused by failure to inherit particular enzymes (green).

PROCEDURE 9-1 Ferric Chloride Tube Test 1. Place 1 mL of urine in a tube. 2. Slowly add five drops of 10% ferric chloride. 3. Observe color for a permanent blue-green color.

of tyrosine metabolism may result from either inherited or metabolic defects. Also, because two reactions are directly involved in the metabolism of tyrosine, the urine may contain excess tyrosine or its degradation products, p-hydroxyphenylpyruvic acid and p-hydroxyphenyllactic acid. Most frequently seen is a transitory tyrosinemia in premature infants, which is caused by underdevelopment of the liver function required to produce the enzymes necessary to complete the tyrosine metabolism. Acquired severe liver disease also produces tyrosyluria resembling that of the transitory newborn variety and, of course, is a more serious condition. In both instances, rarely seen tyrosine

and leucine crystals may be observed during microscopic examination of the urine sediment. Hereditary disorders in which enzymes required in the metabolic pathway are not produced present serious and often fatal conditions that result in both liver and renal tubular disease, producing a generalized aminoaciduria. Based on the enzymes affected, the hereditary disorders can be classified into three types, all producing tyrosinemia and tyrosyluria. As shown in Figure 9-2, type 1 is caused by the deficiency of the enzyme fumarylacetoacetate hydrolase (FAH). Type 1 produces a generalized renal tubular disorder and progressive liver failure in infants soon after birth. Tyrosemia type 1 is diagnosed by the detection of tyrosine and succinylacetone in the urine and blood. Tyrosine and succinylacetone can be measured on the newborn screening by MS/MS. Molecular genetic testing for FAH gene mutations is available to confirm the diagnosis.3 Type 2 tyrosinemia is caused by lack of the enzyme tyrosine aminotransferase. People with type 2 tyrosinemia develop corneal erosions and lesions on the palms, fingers, and soles of the feet, believed to be caused by crystallization of tyrosine in the cells. Diagnosis of type 2 tyrosinemia is made by testing

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Chapter 9 | Urine Screening for Metabolic Disorders for plasma tyrosine and molecular diagnostic tests to determine genetic markers. Patients also may have elevated 4-hydroxy phenylpyruvic acid in urine. Type 3 tyrosinemia is caused by lack of the enzyme p-hydroxyphenylpyruvic acid dioxygenase. This can result in intellectual disability, seizures, and intermittent ataxia if dietary restrictions of phenylalanine and tyrosine are not implemented. Type 3 tyrosinemia is diagnosed by the presence of elevated levels of plasma tyrosine and the presence of the urine metabolites 4-hydroxyphenylpyruvic acid,4-hydroxyphenyllactic, and 4-hydroxyphenylacetic acid. Genetic studies include testing for mutations in the gene for 4-hydroxyphenylpyruvic acid dioxygenase.4 Screening tests using MS/MS and molecular diagnostic tests are available for tyrosinemia types 1, 2, and 3. See Procedure 9-2 for a qualitative urine screening test for tyrosyluria using nitroso-naphthol.

Melanuria The previous discussion focused on the major phenylalanine– tyrosine metabolic pathway illustrated in Figure 9-2; however, as also shown in Figure 9-2 and is the case with many amino acids, a second metabolic pathway also exists for tyrosine. This pathway is responsible for the production of melanin, thyroxine, epinephrine, protein, and tyrosine sulfate. Of these substances, the major concern of the urinalysis laboratory is melanin, the pigment responsible for the dark color of hair, skin, and eyes. Deficient production of melanin results in albinism. Increased urinary melanin (melanuria) darkens the urine. The darkening appears after the urine is exposed to air. Elevated urinary melanin is a serious finding that indicates proliferation of the normal melanin-producing cells (melanocytes), producing a malignant melanoma. These tumors secrete a colorless precursor of melanin, 5,6-dihydroxyindole, which oxidizes to melanogen and then to melanin, producing the characteristic dark urine.

Alkaptonuria Alkaptonuria was one of the six original IEMs described by Garrod in 1902. The name alkaptonuria was derived from the observation that urine from patients with this condition

PROCEDURE 9-2 Nitroso-Naphthol Test for Tyrosine 1. 2. 3. 4. 5. 6. 7.

Place five drops of urine in a tube. Add 1 mL of 2.63N nitric acid. Add one drop of 21.5% sodium nitrite. Add 0.1 mL 1-nitroso-2-napthol. Mix. Wait 5 minutes. Observe for an orange-red color, indicating tyrosine metabolites.

239

darkened after becoming alkaline from standing at room temperature. Therefore, the term “alkali lover,” or alkaptonuria, was adopted. This metabolic defect is actually the third major defect in the phenylalanine–tyrosine pathway and occurs from failure to inherit the gene to produce the enzyme homogentisic acid oxidase. Without this enzyme, the phenylalanine–tyrosine pathway cannot proceed to completion, and homogentisic acid accumulates in the blood, tissues, and urine. This condition usually does not manifest itself clinically in early childhood, but observations of brown-stained or black-stained cloth diapers and reddish-stained disposable diapers have been reported.5 In later life, the brown pigment becomes deposited in the body tissues, particularly noticeable in the ears (ochronosis). Deposits in connective tissue, especially the cartilage, eventually lead to arthritis. A high percentage of people with alkaptonuria develop liver and cardiac disorders.6 Homogentisic acid reacts in several of the screening tests historically used for metabolic disorders, including the ferric chloride test, in which a transient deep blue color is produced in the tube test. A yellow precipitate is produced in the Clinitest, indicating the presence of a reducing substance. Another screening test for urinary homogentisic acid is to add alkali to freshly voided urine and observe for darkening of the color; however, large amounts of ascorbic acid interfere with this reaction. Gas chromatography–mass spectrometry (GC-MS) procedures are available for quantitating homogentisic acid. Molecular genetic testing for the homogentisic acid oxidase gene is available on a clinical basis.7 The ferric chloride test and the silver nitrate test for homogentisic acid, provided in Procedure 9-3, are screening tests for homogentisic acid. Treatment for alkaptonuria is aimed at the specific symptoms that are present in each patient. Vitamin C has been used to treat alkaptonuria because it hinders the accumulation and deposition of homogentisic acid.7

Technical Tip 9-1. Careful observation during the physical component of urinalysis is helpful in cases of alkaptonuria. The appearance of black urine from a patient of any age should be reported to a supervisor. Technical Tip 9-2. Melanin may react with sodium nitroferricyanide (acetone reagent strip), producing a red color. Technical Tip 9-3. It is important to differentiate between the presence of homogentisic acid and melanin when urine is observed to have turned black upon standing.

Branched-Chain Amino Acid Disorders The branched-chain amino acids differ from other amino acids by having a methyl group that branches from the main aliphatic carbon chain (Fig. 9-3 A and B). Two major groups of disorders are associated with errors in the metabolism of the

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PROCEDURE 9-3 Homogentisic Acid Test 1. 2. 3. 4. 5.

Place 4 mL of 3% silver nitrate in a tube. Add 0.5 mL of urine. Mix. Add 10% NH4OH by drops. Observe for black color.

branched-chain amino acids. In one group, accumulation of one or more of the early amino acid degradation products occurs, as is seen in MSUD. Disorders in the other group are termed organic acidemias and result in accumulation of organic acids produced further down in the amino acid metabolic pathway. A significant laboratory finding in branched-chain amino acid disorders is the presence of ketonuria in a newborn.

urinalysis, notifying the physician about this unusual finding can prevent progression to severe intellectual disability and even death. Studies have shown that if MSUD is detected by the 11th day, dietary regulation and careful monitoring of urinary concentrations of keto acid can control the disorder.8 The 2,4-dinitrophenylhydrazine (DNPH) urine screening test for MSUD is provided in Procedure 9-4. Newborn screening for MSUD is performed with MS/MS. Plasma amino acids (PAA) testing should be performed to assess for elevated levels of branchedchain amino acids (BCAAs) and to detect alloisoleucine. A plasma alloisoleucine level greater than 5 µmol/L is the most specific and sensitive marker for MSUD.9 Molecular testing for the genes reported in patients with MSUD confirm the diagnosis, provide information about prognosis, and provide information for genetic counseling.9 Treatment of MSUD is the dietary restriction of foods that contain BCAAs. Nutritional therapies are prescribed to reduce toxic metabolites while promoting normal growth.

Technical Tip 9-4. As discussed in Chapter 5, as part of the physical examination of urine, the sweet odor of urine is a clue to MSUD.

Maple Syrup Urine Disease Although maple syrup urine disease (MSUD) is rare, a brief discussion is included in this chapter because the urinalysis laboratory can provide valuable information for the essential early detection of this disease. MSUD is also included in newborn screening profiles using MS/MS. MSUD is caused by an IEM, inherited as an autosomal recessive trait. The amino acids involved are leucine, isoleucine, and valine. The metabolic pathway begins normally, with the transamination of the three amino acids in the liver to the keto acids (␣-ketoisovaleric, ␣-ketoisocaproic, and ␣-ketoβ-methylvaleric). Failure to inherit the gene for the enzyme necessary to produce oxidative decarboxylation of these keto acids results in their accumulation in the blood and urine.1 Newborns with MSUD begin to exhibit clinical symptoms associated with failure to thrive after approximately l week. The presence of the disease may be suspected from these clinical symptoms; however, many other conditions have similar symptoms. Personnel in the urinalysis laboratory, nurses, or parents may detect the disease by noticing a urine specimen that produces a strong odor resembling maple syrup that is caused by the rapid accumulation of keto acids in the urine. Even though a report of urine odor is not a part of the routine

H N

H A

Generalized symptoms of the organic acidemias include early severe illness, often with vomiting accompanied by metabolic acidosis; hypoglycemia; ketonuria; and increased serum ammonia.10 The three disorders encountered most frequently are isovaleric, propionic, and methylmalonic acidemia. Isovaleric acidemia may be suspected when urine specimens, and sometimes even the patient, possess a characteristic odor of “sweaty feet.” This odor is caused by the accumulation of isovalerylglycine due to a deficiency of isovaleryl coenzyme A in the leucine pathway. Propionic and methylmalonic acidemias result from errors in the metabolic pathway converting isoleucine, valine, threonine, and methionine to succinyl coenzyme A. Propionic acid is the immediate precursor to methylmalonic acid in this pathway. The presence of isovaleric, propionic, and methylmalonic acidemias can be detected by newborn screening programs using MS/MS.

Alpha amino group

Alpha amino group

H

Organic Acidemias

C

R R group (variant)

O

Carboxyl group

H

H N

C H

OH

C

C

CH H 3C

B

O

CH3

Leucine group

OH

Figure 9–3 ␣-Alpha amino acid and branched chain amino acid structures. A. Structure of an ␣-amino acid. B. Structure of the branched chain amino acid leucine.

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Chapter 9 | Urine Screening for Metabolic Disorders

Except in cases of Hartnup disease, correction of the underlying intestinal disorder returns urinary indican levels to normal. The inherited defect in Hartnup disease affects not only the intestinal reabsorption of tryptophan but also the renal tubular reabsorption of other amino acids, resulting in a generalized aminoaciduria (Fanconi syndrome). The defective renal transport of amino acids does not appear to affect other renal tubular functions. Therefore, with proper dietary supplements, including niacin, people with Hartnup disease have a good prognosis.11

PROCEDURE 9-4 2,4-DNPH Test for MSUD 1. 2. 3. 4.

241

Place 1 mL of urine in a tube. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl. Wait 10 minutes. Observe for yellow turbidity or precipitate.

5-Hydroxyindoleacetic Acid

Tryptophan Disorders

As shown in Figure 9-4, a second metabolic pathway of tryptophan is for the production of serotonin used in the stimulation of smooth muscles. Serotonin is produced from tryptophan by the argentaffin cells in the intestine and is carried through the body primarily by the platelets. Normally, the body uses most of the serotonin, and only small amounts of its degradation product, 5-HIAA, are available for excretion in the urine. However, when carcinoid tumors involving the argentaffin (enterochromaffin) cells develop, excess amounts of serotonin are produced, resulting in the elevation of urinary 5-HIAA levels. Adding nitrous acid and 1-nitroso-2-naphthol to urine that contains 5-HIAA causes the urine to turn purple to black, depending on the amount of 5-HIAA present (Procedure 9-5). The normal daily excretion of 5-HIAA is 2 to 8 mg, and excretion of greater than 25 mg/24 h can be an indication of argentaffin cell tumors.12 The test can be performed on a random or first morning specimen; however, false-negative results can occur based on the specimen concentration and also because 5-HIAA may not be produced at a constant rate throughout the day. If a 24-hour specimen is used, it must be preserved with hydrochloric or boric acid. Also, a plasma method using high-performance liquid chromatography with fluorescence detection is available. Patients must be given explicit dietary instructions before collecting any specimen to be tested for 5-HIAA because serotonin

The major concern of the urinalysis laboratory in the metabolism of tryptophan is the increased urinary excretion of the metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA). Figure 9-4 shows a simplified diagram of the metabolic pathways by which these substances are produced. Other metabolic pathways of tryptophan are not included because they do not relate directly to the urinalysis laboratory.

Indicanuria Under normal conditions, most of the tryptophan that enters the intestine is either reabsorbed for use by the body in producing protein or is converted to indole by intestinal bacteria and excreted in the feces. However, in certain intestinal disorders (including obstruction; the presence of abnormal bacteria; malabsorption syndromes; and Hartnup disease, a rare inherited disorder) increased amounts of tryptophan are converted to indole. Then the excess indole is reabsorbed from the intestine into the bloodstream and circulated to the liver, where it is converted to indican and then excreted in the urine (indicanuria). Indican excreted in the urine is colorless until oxidized to the dye indigo blue by exposure to air. Sometimes early diagnosis of Hartnup disease is made when a parent reports a blue staining of the infant’s diapers, referred to as the “blue diaper syndrome.”

Tryptophan (normal) Abnormal

Abnormal Intestinal disorder

Malignant tumors

Excess indole

5-hydroxytryptophan

Reabsorbed from intestine into bloodstream Liver

Indican

Indole

Serotonin

Feces

5-hydroxyindoleacetic acid (5-HIAA)

Urine Exposure to air Indigo blue

Figure 9–4 Tryptophan metabolism.

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242 Part Two | Urinalysis

PROCEDURE 9-5 Silver Nitroprusside Test 1. 2. 3. 4. 5. 6.

Place 1 mL of urine in a tube. Add two drops concentrated NH4OH. Add 0.5 mL 5% silver nitrate. Wait 10 minutes. Add five drops sodium nitroprusside. Observe for purple-black color.

is a major constituent of numerous foods, such as avocados, bananas, pineapples, plums, kiwi fruit, eggplant, walnuts, and tomatoes. These foods should be avoided for 3 days before and during urine collection.13 Medications, including phenothiazines and acetanilides, also interfere with results. Patients should be directed by their physician to withhold medications for 72 hours before specimen collection.

Because cystine is much less soluble than the other three amino acids, laboratory screening determinations are based on observing cystine crystals in the sediment of concentrated or first morning specimens. Cystine is also the only amino acid found during the analysis of calculi from these patients. A chemical screening test for urinary cystine can be performed using cyanide–nitroprusside. Reduction of cystine by sodium cyanide followed by the addition of nitroprusside produces a red-purple color in a specimen that contains excess cystine (see Procedure 9-6). False-positive reactions occur in the presence of ketones and homocysteine, and additional tests may have to be performed. For patients with positive test results for cyanide–nitroprusside, ion-exchange chromatographic quantitative analysis of a 24-hour collected urine specimen is performed.16

Technical Tip 9-6. Positive confirmation of cystine crystals in the urine sediment is made using the cyanide–nitroprusside test and is diagnostic for cystinuria.

Cystine Disorders Two distinct disorders of cystine metabolism exhibit renal manifestations. Confusion as to their relationship existed for many years after the discovery of renal calculi consisting of cystine. However, now it is known that although both disorders are inherited, one is a defect in the renal tubular transport of amino acids (cystinuria) and the other is an IEM (cystinosis).

Technical Tip 9-5. A noticeable odor of sulfur may be present in the urine of people with cystine metabolism disorders. Cystinuria As the name implies, cystinuria is marked by elevated amounts of the amino acid cystine in the urine. The presence of increased urinary cystine is not due to a defect in the metabolism of cystine, but rather to the inability of the renal tubules to reabsorb cystine filtered by the glomerulus. The demonstration that not only cystine but also lysine, arginine, and ornithine are not reabsorbed has ruled out the possibility of an error in metabolism even though the condition is inherited.14 The disorder has two modes of inheritance: • One in which reabsorption of all four amino acids— cystine, lysine, arginine, and ornithine—is affected • One in which only cystine and lysine are not reabsorbed Genetic studies have grouped cystinuria into three types based on the two inherited genes and their heterozygous and homozygous inheritance. In general, people with any form of inheritance may form renal calculi, but the calculi are less common in people in whom only lysine and cystine are affected.15 Approximately 65% of the people in whom all four amino acids are affected can be expected to produce calculi early in life.

Cystinosis Regarded as a genuine IEM, cystinosis can occur in three variations, ranging from a severe fatal disorder developed in infancy to a benign form appearing in adulthood. The disorder has two general categories, termed nephropathic and nonnephropathic. The nephropathic category is subdivided into infantile and late-onset cystinosis. A defect in the lysosomal membranes prevents the release of cystine into the cellular cytoplasm for metabolism. The incomplete metabolism of cystine results in crystalline deposits of cystine in many areas of the body, including the cornea, bone marrow, lymph nodes, and internal organs. A major defect in the renal tubular reabsorption mechanism (Fanconi syndrome) also occurs. The renal tubules, particularly the proximal convoluted tubules, are affected by the cystine deposits that interfere with reabsorption. This is not an inherited disorder of renal tubular reabsorption, as seen in cystinuria. Continued deposition of cystine, if untreated, results in renal failure early in life. In infantile nephropathic cystinosis, there is rapid progression to renal failure. In late-onset nephropathic cystinosis, there is a gradual progression to total renal failure. Renal transplants and the use of cystine-depleting medications

PROCEDURE 9-6 Cyanide–Nitroprusside Test for Cystine 1. 2. 3. 4. 5.

Place 3 mL of urine in a tube. Add 2 mL sodium cyanide. Wait 10 minutes. Add five drops 5% sodium nitroprusside. Observe for red-purple color.

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Chapter 9 | Urine Screening for Metabolic Disorders to prevent the buildup of cystine in other tissues are extending lives. Nonnephropathic cystinosis is relatively benign but may cause some ocular disorders. Routine laboratory findings in infantile nephropathic cystinosis include polyuria, generalized aminoaciduria, positive Clinitest results for reducing substances, and lack of urinary concentration. A diagnosis of cystinosis can be confirmed by molecular genetic testing.

Homocystinuria Defects in the metabolism of the amino acid methionine produce an increase in homocysteine throughout the body. The increased homocysteine can result in failure to thrive, cataracts, intellectual disability, thromboembolic problems, stroke, and death. Therefore, screening for homocysteine is included in newborn screening programs. Newborn screening tests are performed using MS/MS testing. Early detection of this disorder (homocystinuria) and a change in diet that excludes foods high in methionine can alleviate the metabolic problems. As mentioned, increased urinary homocysteine gives a positive result with the cyanide–nitroprusside test. Therefore, an additional screening test for homocystinuria must be performed by following a positive cyanide–nitroprusside test result with a silver–nitroprusside test, in which only homocysteine will react. The use of silver nitrate in place of sodium cyanide reduces homocysteine to its nitroprusside-reactive form but does not reduce cystine. Consequently, a positive reaction in the silver– nitroprusside test confirms the presence of homocystinuria. Fresh urine should be used when testing for homocysteine (see Procedure 9-7).

Porphyrin Disorders Porphyrins are the intermediate compounds in the production of heme. The basic pathway for heme synthesis presented in Figure 9-5 shows the three primary porphyrins (uroporphyrin, coproporphyrin, and protoporphyrin) and the porphyrin precursors (␣-aminolevulinic acid [ALA] and porphobilinogen). As seen in the figure, the synthesis of heme can be blocked at a number of stages. Blockage of a pathway reaction results in the accumulation of the product formed just before the interruption. Then detection and identification of this product in the urine, bile, feces, or blood can aid in determining the cause of a specific disorder.

PROCEDURE 9-7 Silver–Nitroprusside Test for Homocysteine 1. 2. 3. 4. 5. 6.

Place 1 mL of urine in a tube. Add two drops concentrated NH4OH. Add 0.5 mL 5% silver nitrate. Wait 10 minutes. Add five drops sodium nitroprusside. Observe for red-purple color.

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The solubility of the porphyrin compounds varies with their structure. ALA, porphobilinogen, and uroporphyrin are the most soluble and readily appear in the urine. Coproporphyrin is less soluble but is found in the urine, whereas protoporphyrin is not seen in the urine. Usually fecal analysis has been performed to detect coproporphyrin and protoporphyrin. However, to avoid false-positive interference, bile is a more acceptable specimen.17 The Centers for Disease Control and Prevention (CDC) recommends analysis of whole blood for the presence of free erythrocyte protoporphyrin (FEP) as a screening test for lead poisoning. Disorders of porphyrin metabolism are collectively termed porphyrias. They can be inherited or acquired from erythrocytic and hepatic malfunctions or exposure to toxic agents. Common causes of acquired porphyrias include: • Lead poisoning • Excessive alcohol intake • Iron deficiency • Chronic liver disease • Renal disease Inherited porphyrias are much rarer than acquired porphyrias. They are caused by failure to inherit the gene that produces an enzyme needed in the metabolic pathway. The enzyme deficiency sites for some of the more common porphyrias are shown in Figure 9-5. Frequently the inherited porphyrias are classified by their clinical symptoms, either neurological/psychiatric or cutaneous photosensitivity or a combination of both (Table 9-4). An indication of the possible presence of porphyrinuria is the observation of a red or port wine color to the urine after exposure to air. The port wine urine color is more prevalent in the erythropoietic porphyrias, and staining of the teeth also may occur. As seen with other inherited disorders, the presence of congenital porphyria is suspected sometimes from a red discoloration of an infant’s diapers. The two screening tests for porphyrinuria use the Ehrlich reaction and fluorescence under ultraviolet light in the 550- to 600-nm range. The Ehrlich reaction can be used only for the detection of ALA and porphobilinogen. Acetyl acetone must be added to the specimen to convert the ALA to porphobilinogen before performing the Ehrlich test. The fluorescent technique must be used for the other porphyrins. The Ehrlich reaction that is included in the Multistix urobilinogen pad now was originally used for all urobilinogen testing. Variations of the Ehrlich reaction include the Watson-Schwartz test for differentiation between the presence of urobilinogen and porphobilinogen (see Procedures 9-8 and 9-9) and the Hoesch test (see Procedure 9-10). These procedures are performed rarely today. High-pressure liquid chromatography with fluorometric detection, ion exchange chromatography, and spectrometry methodologies are currently used for the evaluation of urine porphyrins. Testing for the presence of porphobilinogen is most useful when patients exhibit symptoms of an acute attack. This can be done with the Hoesch test. Increased porphobilinogen is associated with acute intermittent porphyria. A negative test result is obtained in the presence of lead poisoning unless ALA is first converted to porphobilinogen.

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244 Part Two | Urinalysis

HISTORICAL NOTE

Vampires in Old Europe Did you ever wonder how the legend of vampires got started? Think about the previous discussion on the symptoms and inheritance of porphyrias. Photosensitivity→Avoidance of sunlight Pale coloring→Anemia caused by heme disorder Port wine-colored urine, red-stained teeth→Drinking blood Psychiatric symptoms→Abnormal behavior Inherited disorder→Familial association, small gene pool

Dracula is associated with Transylvania, now Romania. Porphyria was a common disease of early royalty in Europe as a result of intermarriage among the royals of different countries. King George III reportedly died blind, deaf, and mad from porphyria.

HEME SYNTHESIS Glycine and succinyl-CoA Aminolevulinic acid synthetase γ-aminolevulinate (ALA) Aminolevulinic acid synthetase

Lead exposure

ALA hydratase deficiency porphyria Porphobilinogen Uroporphyrinogen synthase Acute intermittent porphyria Hydroxymethylbilane Uroporphyrinogen cosynthase Congenital erythropoietic porphyria Uroporphyrinogen (UPG) Uroporphyrinogen decarboxylase Porphyria cutanea tarda Coproporphyrinogen (CPG) Coproporphyrinogen oxidase Hereditary coproporphyria Protoporphyrinogen Protoporphyrinogen oxidase Variegate porphyria Protoporphyrin IX Ferrochelatase

Lead exposure

Erythropoietic protoporphyria Heme

Figure 9–5 Pathway of heme formation, including normal pathway (green), enzymes (orange), and stages affected by the major disorders (yellow) of porphyrin metabolism.

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Chapter 9 | Urine Screening for Metabolic Disorders Table 9–4

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Common Porphyrias

Porphyria Acute intermittent porphyria Porphyria cutanea tarda Congenital erythropoietic porphyria

Elevated Compound(s)

Clinical Symptoms

Laboratory Testing

ALA Porphobilinogen Uroporphyrin Uroporphyrin

Neurological/psychiatric Photosensitivity Photosensitivity

Urine/Ehrlich reaction Urine fluorescence Urine or feces fluorescence

Photosensitivity/neurological Photosensitivity

Bile or feces fluorescence Blood FEP Bile or feces fluorescence Acetoacetic acid + urine/Ehrlich reaction Blood FEP

Coproporphyrin Coproporphyrin Variegate porphyria Erythropoietic protoporphyria Protoporphyrin Lead poisoning

ALA

Neurological

Protoporphyrin

PROCEDURE 9-8 Watson-Schwartz Differentiation Test The classic test for differentiating among urobilinogen, porphobilinogen, and Ehrlich-reactive compounds is the WatsonSchwartz test. The test is performed as follows: 1. To each tube, add: Tube 1 Tube 2 2 mL urine 2 mL urine 2 mL chloroform 2 mL butanol 4 mL sodium acetate 4 mL sodium acetate 2. Observe the color of the layers. 3. Interpretation: The addition of chloroform to Tube 1 results in the extraction of urobilinogen into the chloroform (bottom) layer, producing a colorless urine (top) layer and a red chloroform layer on the bottom. Neither porphobilinogen nor other Ehrlich-reactive compounds are soluble in chloroform. Porphobilinogen also is not soluble in butanol; however, urobilinogen and other Ehrlich-reactive compounds are extracted into butanol. Therefore, the addition of butanol to Tube 2 produces a red (upper) butanol layer if urobilinogen or Ehrlich-reactive compounds are present and a colorless butanol layer if porphobilinogen is present. As shown in the figure, urobilinogen is soluble in both chloroform and butanol, and porphobilinogen is soluble in neither. If both urobilinogen and porphobilinogen are present, both layers appear red. Before reporting the test as positive for both

substances, an additional chloroform extraction should be performed on the red urine (upper) layer in Tube 1 to ensure that the red color is not due to excess urobilinogen.

UR

B

UR

B

UR

B

C

UR

C

UR

C

UR

Urobilinogen

Porphobilinogen

Ehrlich-reactive

UR

B

UR

UR

C

UR

C

C

Urobilinogen/porphobilinogen

Excess urobilinogen

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PROCEDURE 9-9 Watson-Schwartz Test 1. Label two tubes #1 and #2. 2. To each tube add: Tube 1 Tube 2 2 mL urine 2 mL urine 2 mL chloroform 2 mL butanol 4 mL sodium acetate 4 mL sodium acetate 3. Vigorously shake both tubes. 4. Place in a rack for layers to settle. 5. Observe both tubes for red color in the layers. Interpretation: Tube 1 Upper layer = urine; if colorless = porphobilinogen or Ehrlich-reactive compounds.

PROCEDURE 9-10 Hoesch Screening Test for Porphobilinogen The Hoesch test is used for rapid screening or monitoring of urinary porphobilinogen. 1. Two drops of urine are added to approximately 2 mL of Hoesch reagent (Ehrlich reagent dissolved in 6 M HCl). 2. Immediately observe the top of the solution for the appearance of a red color that indicates the presence of porphobilinogen. 3. Shake the tube. Interpretation: When the tube is shaken, the red color is seen throughout the solution. The test detects approximately 2 mg/dL of porphobilinogen, and urobilinogen is inhibited by the highly acidic pH. High concentrations of methyldopa and indican, as well as highly pigmented urines, may produce false-positive results.

Fluorescent screening for the other porphyrins uses their extraction into a mixture of glacial acetic acid and ethyl acetate. Then the solvent layer is examined. Negative reactions have a faint blue fluorescence. Positive reactions fluoresce as violet, pink, or red, depending on the concentration of porphyrins. If the presence of interfering substances is suspected, the organic layer can be removed to a separate tube, and 0.5 mL of hydrochloric acid should be added to the tube. Only porphyrins are extracted into the acid layer, which then produces a bright orange-red fluorescence. The fluorescence method does not distinguish among uroporphyrin, coproporphyrin, and protoporphyrin, but it rules out porphobilinogen and

Bottom layer = chloroform; if red = urobilinogen. If both layers are red, re-extract the urine layer from Tube 1. Place 2 mL of urine layer from Tube 1 and 2 mL chloroform and 4 mL sodium acetate into a new tube. Repeat procedure. Interpretation: Upper layer—urine colorless Bottom layer—chloroform—red = excess urobilinogen Both layers red = porphobilinogen and urobilinogen Tube 2 Upper layer = butanol If red = urobilinogen or Ehrlich-reactive compounds Bottom layer = urine If colorless = porphobilinogen

ALA. Identifying specific porphyrins requires additional techniques and the analysis of fecal and erythrocyte samples. Increased protoporphyrin is best measured in whole blood.

Mucopolysaccharide Disorders Mucopolysaccharides, or glycosaminoglycans (GAGs), are a group of large compounds located primarily in the connective tissue. They consist of a protein core with numerous polysaccharide branches. Inherited disorders in the metabolism of these compounds prevent complete breakdown of the polysaccharide portion of the compounds, resulting in accumulation of the incompletely metabolized polysaccharide portions in the lysosomes of the connective tissue cells and their increased excretion in the urine. The products found most frequently in the urine are dermatan sulfate, keratan sulfate, and heparan sulfate, with the appearance of a particular substance being determined by the specific metabolic error that was inherited.18 Therefore, identification of the specific enzyme deficiency is necessary to establish a specific diagnosis. There are many types of mucopolysaccharidoses (MPSs), but the best known are Hurler syndrome, Hunter syndrome, and Sanfilippo syndrome. In both Hurler and Hunter syndromes, the skeletal structure is abnormal and there is severe intellectual disability; in Hurler syndrome, mucopolysaccharides accumulate in the cornea of the eye. Hunter syndrome is inherited as sex-linked recessive and is seen rarely in females. Without treatment, both syndromes are usually fatal during childhood, whereas in Sanfilippo syndrome, the only abnormality is intellectual disability. Bone marrow transplants and gene replacement therapy are the most promising treatments for these disorders. Urinary screening tests for mucopolysaccharides may be requested either as part of a routine battery of tests performed on all newborns or on infants who exhibit symptoms of intellectual

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Chapter 9 | Urine Screening for Metabolic Disorders disability or failure to thrive. Historically, the screening tests used most frequently are the acid–albumin and cetyltrimethylammonium bromide (CTAB) turbidity tests and the metachromatic staining spot tests. In both the acid–albumin and the CTAB tests, a thick, white turbidity forms when these reagents are added to urine that contains mucopolysaccharides. Turbidity is usually graded on a scale of 0 to 4 after 30 minutes with acid–albumin and after 5 minutes with CTAB.19 (See Procedure 9-11.) A diagnostic workup for a patient with suspected MPSs includes both the quantitative analysis of urine for the total GAGs and the qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of the specific sulfates. Molecular analysis is used for confirmation.

Purine Disorders A disorder of purine metabolism known as Lesch-Nyhan disease that is inherited as sex-linked recessive results in massive excretion of urinary uric acid crystals. Failure to inherit the gene to produce the enzyme hypoxanthine guanine phosphoribosyltransferase is responsible for the accumulation of uric acid throughout the body. Patients suffer from severe motor defects, intellectual disability, a tendency toward self-destruction, gout, and renal calculi. Usually development is normal for the first 6 to 8 months, and often the first sign is uric acid crystals resembling orange sand in diapers.11 Laboratorians should be alert for the presence of increased uric acid crystals in pediatric urine specimens.

Carbohydrate Disorders The presence of increased urinary sugar (melituria) is due most frequently to an inherited disorder. In fact, pentosuria was one of Garrod’s original six IEMs.20 Fortunately, most meliturias cause no disturbance to body metabolism. However, as discussed in Chapter 6, pediatric urine should be screened routinely for the presence of reducing substances using the Clinitest procedure. The finding of a positive result on the copper reduction test combined with a negative reagent strip result on the glucose oxidase test is strongly suggestive of a disorder of carbohydrate metabolism. Of primary concern is the presence of galactosuria, indicating the inability to properly metabolize galactose to glucose. The resulting galactosemia with toxic intermediate metabolic products results in infant failure to thrive combined with liver disorders, cataracts, and severe intellectual disability. Early detection of galactosuria followed by removal of lactose (a disaccharide containing galactose and glucose) from the diet can prevent these symptoms.

PROCEDURE 9-11 CTAB Test for Mucopolysaccharides 1. Place 5 mL of urine in a tube. 2. Add 1 mL 5% CTAB in citrate buffer. 3. Read turbidity in 5 minutes.

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Technical Tip 9-7. Lesch-Nyhan disease should not be confused with uromodulin-associated kidney disease, in which the uric acid crystals appear later in life.

Galactosuria can be caused by a deficiency in any of three enzymes, galactose-1-phosphate uridyl transferase (GALT), galactokinase (GALK), and UDP-galactose-4-epimerase (GALE). Of these enzymes, it is GALT deficiency that causes the severe, possibly fatal, symptoms associated with galactosemia. Newborn screening protocols currently test for the presence of GALT deficiency. The enzyme is measured in the red blood cells as part of the protocol of the newborn heel puncture. As a result, people with deficiencies in the other two enzymes still may produce galactosuria but have negative newborn screening tests. Galactose kinase deficiency can result in cataracts in adulthood. UDP-galactose-4-epimerase deficiency may be asymptomatic or produce mild symptoms. Molecular genetic testing can be used to identify mutations in the GALT gene. Other causes of melituria include lactose, fructose, and pentose. Lactosuria may be seen during pregnancy and lactation. Fructosuria is associated with parenteral feeding and pentosuria with ingestion of large amounts of fruit. Additional tests, including chromatography, can be used to identify other nonglucose-reducing substances.

For additional resources please visit www.fadavis.com

References 1. Frimpton, GW: Aminoacidurias due to inherited disorders of metabolism. N Engl J Med 1289:835–901, 1973. 2. National newborn screening and global resource center (NNSGRC). Web site: http://genes-r-us.uthscsa.edu/sites/genes-rus/files/nbsdisorders.pdf. 2014. Accessed June 13, 2019. 3. National Organization for Rare Disorders (NORD). Tyrosinemia Type 1. Web site: https://rarediseases.org/rare-diseases/ tyrosinemia-type-1. Published October 12, 2005. Accessed June 13, 2019. 4. U.S. Department of Health & Human Services. National Institutes of Health. Tyrosinemia type 3. Web site: https:// rarediseases.info.nih.gov/diseases/10332/tyrosinemia-type-3. Published February 3, 2010. Accessed June 13, 2019. 5. Nyhan, WL, and Sakati, NO: Diagnostic Recognition of Genetic Disease. Lea & Febiger, Philadelphia, 1987. 6. Stanbury, JB: The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, 1983. 7. Introne, WJ: Alkaptonuria. National Organization for Rare Disorders (NORD). Web site: https://rarediseases.rg/rarediseases/alkaptonuria/. 2017. Accessed June 13, 2019. 8. Clow, CL, Reade, TH, and Scriver, CR: Outcome of early and long-term management of classical maple syrup urine disease. Pediatrics 68(6):856–862, 1981. 9. Defendi, GL: Maple Syrup Urine Disease (MSUD) Workup. Medscape. Web site: https://emedicine.medscape.com/article/ 946234-workup. Published May 2, 2018. Accessed June 13, 2019.

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248 Part Two | Urinalysis 10. Goodman, SI: Disorders of organic acid metabolism. In Emery, AEH, and Rimoin, DL: Principles and Practice of Medical Genetics. Churchill Livingstone, New York, 1990. 11. Jepson, JB: Hartnup’s disease. In Stanbury, JB, Wyngaarden, JB, and Fredrickson, DS (eds): The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, 1983. 12. Van Leeuwen, AM, Poelhuis-Leth, DJ, and Bladh, ML: Davis’s Comprehensive Laboratory and Diagnostics Handbook. 6th ed. Philadelphia, FA Davis Company; 2015. 13. American Association for Clinical Chemistry (AACC). Lab Tests online. 5-HIAA. Web site: https://labtestsonline.org/tests/5-hiaa. Published June 6, 2019. Accessed June 13, 2019. 14. Nyhan, WL: Abnormalities in Amino Acid Metabolism in Clinical Medicine. Appleton-Century-Crofts, Norwalk, CT, 1984. 15. Dello Strolongo, L, et al: Comparison between SLC3A1 and SLC7A9 cystinuria patients and carriers: A need for a new classification. J Am Soc Nephrol 13:2547–2553, 2002.

16. Biyani, CS: Cystinuria Work-up. Medscape. Web site: https://emedicinemedscape.com/article/435678-work-up. Published March 19, 2019. Accessed June 13, 2019. 17. Nuttall, KL: Porphyrins and disorders of porphyrin metabolism. In Burtis, CA, and Ashwood, ER: Tietz Fundamentals of Clinical Chemistry. WB Saunders, Philadelphia, 1996. 18. McKusick, VA, and Neufeld, EF: The mucopolysaccharide storage diseases. In Stanbury, JB, Wyngaarden, JB, and Fredrickson, DS (eds): The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, 1983. 19. Kelly, S: Biochemical Methods in Medical Genetics. Charles C. Thomas, Springfield, IL, 1977. 20. Garrod, AE: Inborn Errors of Metabolism. Henry Froude & Hodder & Stoughton, London, 1923.

Study Questions 1. Abnormal urine screening tests categorized as an overflow disorder include all of the following except: A. Alkaptonuria B. Galactosemia C. Melanuria D. Cystinuria 2. All states require newborn screening for PKU for early: A. Modifications of the diet B. Administration of antibiotics C. Detection of diabetes D. Initiation of gene therapy 3. All of the following disorders can be detected by newborn screening except: A. Tyrosyluria B. MSUD C. Melanuria D. Galactosemia 4. The best specimen for early newborn screening is a: A. Timed urine specimen B. Blood specimen C. First morning urine specimen D. Fecal specimen 5. Which of the following disorders is not associated with the phenylalanine–tyrosine pathway? A. MSUD B. Alkaptonuria C. Albinism D. Tyrosinemia

6. The least serious form of tyrosylemia is: A. Immature liver function B. Type 1 C. Type 2 D. Type 3 7. An overflow disorder of the phenylalanine–tyrosine pathway that would produce a positive reaction with the reagent strip test for ketones is: A. Alkaptonuria B. Melanuria C. MSUD D. Tyrosyluria 8. An overflow disorder that could produce a false-positive reaction with the Clinitest procedure is: A. Cystinuria B. Alkaptonuria C. Indicanuria D. Porphyrinuria 9. A urine that turns black after sitting by the sink for several hours could be indicative of: A. Alkaptonuria B. MSUD C. Melanuria D. Both A and C 10. Ketonuria in a newborn is an indication of: A. MSUD B. Isovaleric acidemia C. Methylmalonic acidemia D. All of the above

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Chapter 9 | Urine Screening for Metabolic Disorders 11. Urine from a newborn with MSUD will have a significant: A. Pale color B. Yellow precipitate C. Milky appearance D. Sweet odor 12. Hartnup disease is a disorder associated with the metabolism of: A. Organic acids B. Tryptophan C. Cystine D. Phenylalanine 13. 5-HIAA is a degradation product of: A. Heme B. Indole C. Serotonin D. Melanin 14. Elevated urinary levels of 5-HIAA are associated with: A. Carcinoid tumors B. Hartnup disease C. Cystinuria D. Platelet disorders 15. False-positive levels of 5-HIAA can be caused by a diet high in: A. Meat B. Carbohydrates C. Starch D. Bananas 16. Place the appropriate letter in front of the following statements. A. Cystinuria B. Cystinosis IEM Inherited disorder of tubular reabsorption Fanconi syndrome Cystine deposits in the cornea Early renal calculi formation 17. Blue diaper syndrome is associated with: A. Lesch-Nyhan syndrome B. Phenylketonuria C. Cystinuria D. Hartnup disease

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18. Homocystinuria is caused by failure to metabolize: A. Lysine B. Methionine C. Arginine D. Cystine 19. The Ehrlich reaction will detect only the presence of: A. Uroporphyrin B. Porphobilinogen C. Coproporphyrin D. Protoporphyrin 20. Acetyl acetone is added to the urine before performing the Ehrlich test when checking for: A. Aminolevulinic acid B. Porphobilinogen C. Uroporphyrin D. Coproporphyrin 21. The classic urine color associated with porphyria is: A. Dark yellow B. Indigo blue C. Pink D. Port wine 22. Which of the following specimens can be used for porphyrin testing? A. Urine B. Blood C. Feces D. All of the above 23. The two stages of heme formation affected by lead poisoning are: A. Porphobilinogen and uroporphyrin B. Aminolevulinic acid and porphobilinogen C. Coproporphyrin and protoporphyrin D. Aminolevulinic acid and protoporphyrin 24. Hurler, Hunter, and Sanfilippo syndromes are hereditary disorders affecting the metabolism of: A. Porphyrins B. Purines C. Mucopolysaccharides D. Tryptophan

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250 Part Two | Urinalysis 25. Many uric acid crystals in a pediatric urine specimen may indicate: A. Hurler syndrome B. Lesch-Nyhan disease C. Melituria D. Sanfilippo syndrome 26. Deficiency of the GALT enzyme will produce a: A. Positive Clinitest B. Glycosuria C. Galactosemia D. Both A and C

27. Match the metabolic urine disorders with their classic urine abnormalities. PKU A. Sulfur odor Indicanuria B. Sweaty feet odor Cystinuria C. Orange sand in diaper Alkaptonuria D. Mousy odor Lesch-Nyhan disease E. Black color Isovaleric acidemia F. Blue color

Case Studies and Clinical Situations 1. A premature infant develops jaundice. Laboratory tests are negative for hemolytic disease of the newborn, but the infant’s bilirubin level continues to rise. Abnormal urinalysis results include a dark yellow color, positive bilirubin, and needle-shaped crystals seen on microscopic examination. a. What is the most probable cause of the infant’s jaundice? b. Could these same urine findings be associated with an adult? Explain your answer. c. What kinds of crystals are present? Name another type of crystal with a spherical shape that is associated with this condition. d. When blood is drawn from this infant, what precaution should be taken to ensure the integrity of the specimen? 2. A newborn develops severe vomiting and symptoms of metabolic acidosis. Urinalysis results are positive for ketones and negative for glucose and other reducing substances. a. If the urine had an odor of “sweaty feet,” what metabolic disorder would be suspected? b. If the newborn was producing dark brown urine with a sweet odor, what disorder would be suspected? c. Would an MS/MS screen be helpful for the diagnosis? 3. A 13-year-old boy is awakened with severe back and abdominal pain and is taken to the emergency department by his parents. A complete blood count is normal. Family history shows that both his father and uncle are chronic formers of kidney stones. Results of a urinalysis are as follows: COLOR: Yellow KETONES: Negative APPEARANCE: Hazy BLOOD: Moderate SP. GRAVITY: 1.025 pH: 6.0 PROTEIN: Negative

BILIRUBIN: Negative UROBILINOGEN: Normal NITRITE: Negative

GLUCOSE: Negative Microscopic: >15–20 RBCs/hpf

LEUKOCYTE: Negative

Few squamous epithelial cells 0–3 WBCs/hpf Many cystine crystals a. What condition does the patient’s symptoms represent? b. What is the physiological abnormality causing this condition? c. If amino acid chromatography was performed on this specimen, what additional amino acids could be identified if present? d. Why are they not present in the microscopic constituents? e. Based on the family history, what genetic disorder should be considered? 4. An 8-month-old boy is admitted to the pediatric unit with a general diagnosis of failure to thrive. The parents have observed slowness in the infant’s development of motor skills. They also mention the occasional appearance of a substance resembling orange sand in the child’s diapers. Urinalysis results are as follows: COLOR: Yellow KETONES: Negative APPEARANCE: Slightly BLOOD: Negative hazy SP. GRAVITY: 1.025 BILIRUBIN: Negative pH: 5.0 UROBILINOGEN: Normal PROTEIN: Negative NITRITE: Negative GLUCOSE: Negative LEUKOCYTE: Negative Microscopic: Many uric acid crystals a. Is the urine pH consistent with the appearance of uric acid crystals?

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Chapter 9 | Urine Screening for Metabolic Disorders b. Is there any correlation between the urinalysis results and the substance observed in the child’s diapers? Explain your answer. c. What disorder do the patient’s history and the urinalysis results indicate? d. Is the fact that this is a male patient of any significance? Explain your answer. e. Name the enzyme that is missing. 5. Shortly after arriving for the day shift in the urinalysis laboratory, a technician notices that an undiscarded urine specimen has a black color. The report completed previously indicates the color to be yellow. a. Is this observation significant? Explain your answer. b. The original urinalysis report showed the specimen to be positive for ketones. Is this significant? Why or why not? c. If the ketones are negative and the pH is 8.0, is this significant? Why or why not? 6. An 8-year-old boy is admitted through the emergency department with a ruptured appendix. Although surgery is

251

successful, the patient’s recovery is slow, and the physicians are concerned about his health before the ruptured appendix. The patient’s mother states that he has always been noticeably underweight despite eating a balanced diet and having a strong appetite and that his younger brother exhibits similar characteristics. A note in his chart from the first postoperative day reports that the evening nurse noticed a blue coloration in the urinary catheter bag. a. Is the catheter bag color significant? b. What condition can be suspected from this history? c. What is the patient’s prognosis? 7. An anemic patient is suspected of having lead poisoning. a. Historically, what urine test was requested? b. What should be added to the urine before testing? c. What element of heme synthesis would this be testing for? d. Name another substance that can be tested for lead poisoning. e. What element of heme synthesis would this test for?

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PART THREE

Other Body Fluids Chapter 10: Cerebrospinal Fluid Chapter 11: Semen Chapter 12: Synovial Fluid Chapter 13: Serous Fluid Chapter 14: Bronchoalveolar Lavage Fluid Chapter 15: Amniotic Fluid Chapter 16: Fecal Analysis Chapter 17: Vaginal Secretions

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CHAPTER

10

Cerebrospinal Fluid LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 10-1 Describe the formation of cerebrospinal fluid (CSF), and state the three major functions of CSF. 10-2 Distribute CSF specimen tubes numbered 1, 2, 3, and possibly 4 to their appropriate laboratory sections, and correctly preserve them. 10-3 Describe the appearance of normal CSF and the causes of CSF not appearing normally. 10-4 Define xanthochromia, and state its significance. 10-5 Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap. 10-6 Calculate CSF total cell count, as well as counts for white blood cells and red blood cells, when given the number of cells seen, amount of specimen dilution, and squares counted in the Neubauer chamber. 10-7 Describe the leukocyte content of the CSF in various forms of meningitis, including bacterial, viral, tubercular, and fungal. 10-8 Describe and state the significance of macrophages in the CSF. 10-9 Differentiate between the appearance of normal choroidal cells and malignant cells. 10-10 State the reference values for CSF total protein, and name three pathological conditions that produce an elevated CSF protein.

10-11 Determine whether increased CSF albumin or immunoglobulin is the result of damage to the blood– brain barrier or central nervous system production. 10-12 Discuss the significance of findings of CSF electrophoresis, immunophoresis, and isoelectric focusing in multiple sclerosis and the identification of CSF. 10-13 State the reference values for CSF glucose, and name the possible pathological significance of a decreased CSF glucose. 10-14 Discuss the diagnostic value of CSF lactate and glutamine determinations. 10-15 Name the microorganism associated with a positive India ink preparation. 10-16 Discuss the diagnostic value of the bacterial and cryptococcal antigen tests. 10-17 Explain the advantage of molecular diagnostic methods for determining the causative organism in meningitis. 10-18 Determine whether a suspected case of meningitis is of bacterial, viral, fungal, or tubercular origin when presented with pertinent laboratory data. 10-19 Describe the Venereal Disease Research Laboratory test and the fluorescent treponemal antibodyabsorption test for syphilis in CSF testing.

KEY TERMS Arachnoid granulations

Meningitis

Traumatic tap

Blood–brain barrier

Oligoclonal bands

Xanthochromia

Choroid plexuses

Pleocytosis

Meninges

Subarachnoid space

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Introduction Cerebrospinal fluid (CSF) is a major fluid of the body. It provides a physiological system to supply nutrients to the nervous tissue, remove metabolic wastes, maintain intracranial pressure, and produce a mechanical barrier to cushion the brain and spinal cord against trauma.

Formation and Physiology The brain and spinal cord are lined by the meninges, which consist of three layers: the dura mater (Latin for “hard mother”), the arachnoid (“spiderweb-like”), and the pia mater (Latin for “gentle mother”). The outer layer is the dura mater that lines the skull and vertebral canal. The arachnoid is a filamentous (spiderweblike) inner membrane. The pia mater is a thin membrane lining the surfaces of the brain and spinal cord (Fig. 10-1). CSF is produced in the choroid plexuses of the two lumbar ventricles and the third and fourth ventricles. In adults, approximately 20 mL of fluid is produced every hour. The fluid flows through the subarachnoid space located between the arachnoid and pia mater (Fig. 10-2). To maintain a volume of 90 to 150 mL in adults and 10 to 60 mL in neonates, the circulating fluid is reabsorbed back into the blood capillaries in the arachnoid granulations/villae at a rate equal to its production. The cells of the arachnoid granulations act as one-way valves that respond to pressure within the central nervous system (CNS) and prevent reflux of the fluid.1 The choroid plexuses are capillary networks that form the CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion. Therefore, the chemical composition of the CSF does not resemble an ultrafiltrate of plasma. Capillary walls throughout the body are lined with endothelial cells that are connected loosely to allow

passage of soluble nutrients and wastes between the plasma and tissues. In the choroid plexuses, the endothelial cells have very tight-fitting junctures that prevent the passage of many molecules. This tight-fitting structure of the endothelial cells in the choroid plexuses is termed the blood–brain barrier. Maintaining the integrity of the blood–brain barrier is essential to protect the brain from chemicals and other substances circulating in the blood that could harm the brain tissue. In addition, the junctures prevent the passage of helpful substances, including antibodies and medications. Disruption of the blood–brain barrier by diseases, such as meningitis and multiple sclerosis, allows leukocytes, proteins, and additional chemicals to enter the CSF.

Specimen Collection and Handling CSF is collected routinely by lumbar puncture between the third and fourth or fourth and fifth lumbar vertebra. Although this procedure is not complicated, it does require certain precautions, including measurement of intracranial pressure and careful technique, to prevent infection or damage to neural tissue. The volume of CSF that can be removed is based on the volume available in the patient (adult vs. neonate) and the opening pressure of the CSF, measured when the needle first enters the subarachnoid space. Elevated pressure requires fluid to be withdrawn slowly, with careful monitoring of the pressure, and may prevent collection of a large volume. Specimens are collected in three sterile tubes, which are labeled 1, 2, and 3 in the order in which they are withdrawn: • Tube 1 is used for chemical and serological tests because these tests are least affected by blood or bacteria introduced as a result of the tap procedure.

Central canal

Gray matter White matter Spinal nerve roots Dorsal root ganglion

Arachnoid granulation

Superior sagittal sinus

Pia mater Skin

Spinal nerve

Periosteum Skull

Dura mater Arachnoid mater

Arachnoid Meninges membrane

Subarachnoid space

Pia mater Dura mater

Subarachnoid space Gray matter

A

White matter

Brain tissue

B

Figure 10–1 The layers of the meninges. A. The layers of the meninges in the brain. B. The layers of the meninges in the spinal cord. (A and B from Scanlon & Sanders, Essentials of Anatomy and Physiology, 6th ed., F. A. Davis Company, Philadelphia, 2011, with permission.)

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Choroid plexus of third ventricle Cerebrum

Subarachnoid space Arachnoid villus/granulation Pia mater

Choroid plexus of lateral ventricle

Arachnoid mater Dura mater Cerebellum Choroid plexus of fourth ventricle

Spinal cord

Figure 10–2 The flow of CSF through the brain and spinal column.

• Tube 2 usually is designated for testing in the microbiology laboratory. • Tube 3 is used for the cell count because it is the least likely to contain cells introduced by the spinal tap procedure. A fourth tube may be drawn for the microbiology laboratory to better exclude skin contamination or for additional serological tests. Supernatant fluid that is left over after each section has performed its tests also may be used for additional chemical or serological tests. Excess fluid should not be discarded and should be frozen until there is no further use for it (Fig. 10-3). Considering the discomfort to the patient and the possible complications that can occur during specimen collection, laboratory personnel should handle CSF specimens carefully. Ideally, tests are performed on a STAT basis. If this is not possible, specimens are maintained in the following manner: • Hematology tubes are refrigerated up to 4 hours. • Microbiology tubes remain at room temperature. • Chemistry and serology tubes are frozen.

report. The major terminology used to describe CSF appearance includes crystal-clear, cloudy or turbid, milky, xanthochromic, and hemolyzed/bloody (Fig. 10-4). A cloudy, turbid, or milky specimen can be the result of an increased concentration of protein or lipids, but also it may be indicative of infection, with the cloudiness being caused by the presence of white blood cells (WBCs). All specimens should be treated with extreme care

1

2

3

Chemistry serology

Microbiology

Hematology

Technical Tip 10-1. Cells must be counted within 1 hour of collection when the specimen is maintained at room temperature.

Appearance The initial appearance of the normally crystal-clear CSF can provide valuable diagnostic information. Examination of the fluid occurs first at the bedside and also is included in the laboratory

Additional tests

Figure 10–3 CSF specimen collection tubes.

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Clinical Significance of CSF Appearance

Appearance

Cause

Major Significance

Crystal clear Hazy, turbid, milky, cloudy

WBCs

Normal Meningitis

Microorganisms Protein

Figure 10–4 Tubes of CSF. Appearance left to right is normal, xanthochromic, hemolyzed, and cloudy.

because they can be highly contagious; gloves always must be worn and face shields or splash guards should be used while preparing specimens for testing. Fluid for centrifugation must be in capped tubes.

Bloody

Radiographic contrast media RBCs

Xanthochromic

Hemoglobin

Oily

Technical Tip 10-2. If only one tube can be collected, it must be tested first by microbiology.

Bilirubin

Technical Tip 10-3. It is not unusual for cell counts requested to be performed on both Tubes 1 and 4 to check for cellular contamination by the puncture.

Carotene

Xanthochromia is a term used to describe CSF supernatant that is pink, orange, or yellow. A variety of factors can cause xanthochromia, the most common being the presence of red blood cell (RBC) degradation products. Depending on the amount of blood and the length of time it has been present, the color will vary from pink (very slight amount of oxyhemoglobin) to orange (heavy hemolysis) to yellow (conversion of oxyhemoglobin to unconjugated bilirubin). Other causes of xanthochromia include elevated serum bilirubin, presence of the pigment carotene, markedly increased protein concentrations, and melanoma pigment. Xanthochromia that is caused by bilirubin due to immature liver function also is seen commonly in infants, particularly premature infants. The clinical significance of CSF appearance is summarized in Table 10-1.

Protein

Melanin Clotted

Protein

Clotting factors Pellicle

Protein

Clotting factors

Meningitis Disorders affecting blood–brain barrier Production of IgG within the CNS

Hemorrhage Traumatic tap Old hemorrhage Lysed cells from traumatic tap RBC degradation Elevated serum bilirubin level Increased serum levels Disorders affecting blood–brain barrier Meningeal melanosarcoma Disorders affecting blood–brain barrier Introduced by traumatic tap Disorders affecting blood–brain barrier Tubercular meningitis

Traumatic Collection (Tap) Grossly bloody CSF can be an indication of intracranial hemorrhage, but also it may be due to the puncture of a blood vessel during the spinal tap procedure. Usually, three visual examinations of the collected specimens can determine whether the blood is the result of hemorrhage or a traumatic tap.

Uneven Blood Distribution Blood from a cerebral hemorrhage will be distributed evenly throughout the three CSF specimen tubes, whereas a traumatic tap will leave the heaviest concentration of blood in tube 1, with amounts gradually diminishing in tubes 2 and 3. Performing RBC counts on all three tubes to measure decreasing or constant

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Chapter 10 | Cerebrospinal Fluid blood is not always reliable.2 Streaks of blood also may be seen in specimens acquired after a traumatic procedure.

Clot Formation Fluid collected from a traumatic tap may form clots due to the introduction of plasma fibrinogen into the specimen. Bloody CSF caused by intracranial hemorrhage does not contain enough fibrinogen to clot. Diseases in which damage to the blood–brain barrier allows increased filtration of protein and coagulation factors also cause clot formation but usually do not produce a bloody fluid. These conditions include meningitis, Froin syndrome, and blocked CSF circulation through the subarachnoid space. A classic web-like pellicle is associated with tubercular meningitis and can be seen after overnight refrigeration of the fluid.3

Xanthochromic Supernatant Usually RBCs must remain in the CSF for approximately 2 hours before noticeable hemolysis begins; therefore, a xanthochromic supernatant would be the result of blood that has been present longer than that introduced by the traumatic tap. Care should be taken, however, to consider this examination in conjunction with those previously discussed, because a very recent hemorrhage would produce a clear supernatant, and introduction of serum protein from a traumatic tap also could cause the fluid to appear xanthochromic. To examine a bloody fluid for the presence of xanthochromia, the fluid should be centrifuged in a microhematocrit tube and the supernatant examined against a white background. Additional testing for differentiation includes microscopic examination and the D-dimer test. The microscopic finding of macrophages containing ingested RBCs (erythrophagocytosis) or hemosiderin granules indicates intracranial hemorrhage. Detection of the fibrin degradation product D-dimer by latex agglutination immunoassay indicates fibrin formation at a hemorrhage site.

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to 200 WBCs or 400 RBCs/µL may appear clear, so it is necessary to examine all specimens microscopically.6 An improved Neubauer counting chamber (Fig. 10-5) is used routinely for performing CSF cell counts. Traditionally, electronic cell counters have not been used for performing CSF cell counts because of the instrument’s high background counts and poor reproducibility of low counts. Automation increases precision, standardization, and faster turnaround time for results. Various automated instruments, such as the ADVIA 2120i (Siemens Healthcare Diagnostics Incorporated, Deerfield, IL),7 Sysmex XN Series (Sysmex, Kobe, Japan), Iris iQ200 with Body Fluids Module (Iris DiagnosticsBeckman Coulter, Inc., Chatsworth, CA), DxH900 (Beckman Coulter, Inc.),8 and the Glocyte System (Sysmex, Kobe, Japan),9 are available for CSF cell counts. See Chapter 2 for more information on automated body fluid analyzers.

Calculating CSF Cell Counts The standard Neubauer calculation formula used for blood cell counts is also applied to CSF cell counts to determine the number of cells per microliter. Number of cells counted × dilution = cells/µL Number of cells counted × volume of 1 square

This formula can be used for both diluted and undiluted specimens and offers flexibility in the number and size of the squares counted. Many varied calculations are available, including condensations of the formula to provide single factors by which to multiply the cell count. Keep in mind that the purpose of any calculation is to convert the number of cells counted in a specific amount of fluid to the number of cells that would be

Cell Count The cell count that is performed routinely on CSF specimens is the leukocyte (WBC) count. Normally RBCs are not present in CSF. As discussed previously, the presence and significance of RBCs usually can be ascertained from the appearance of the specimen. Therefore, RBC counts are determined only when a traumatic tap has occurred and a correction for leukocytes or protein is desired. The RBC count can be calculated by performing a total cell count and a WBC count and subtracting the WBC count from the total count, if necessary. Any cell count should be performed immediately because WBCs (particularly granulocytes) and RBCs begin to lyse within 1 hour, and 40% of the leukocytes disintegrate after 2 hours.4 Specimens that cannot be analyzed immediately should be refrigerated up to 4 hours.

1

2

3

4

5

6

7

8

9

Methodology Normal adult CSF contains 0 to 5 WBCs/µL. The number is higher in children, and as many as 30 mononuclear cells/µL can be considered normal in newborns.5 Specimens that contain up

Figure 10–5 Neubauer counting chamber depicting the nine large square counting areas.

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present in 1 µL of fluid. Therefore, a factor can be used only when the dilution and counting area are specific for that factor. The methodology presented in this chapter eliminates the need to correct for the volume counted by counting the four large corner squares (0.4 µL) and the large center square (0.1 µL) on each side of the counting chamber.10 EXAMPLE Number of cells counted × dilution ×

1 μL = cells/μL 1 μL (0.1 × 10) (volume counted)

Total Cell Count Clear specimens may be counted undiluted, provided no overlapping of cells is seen during the microscopic examination. When dilutions are required, calibrated automatic pipettes, not mouth pipetting, are used. Dilutions for total cell counts are made with normal saline, mixed by inversion, and loaded into the hemocytometer with a Pasteur pipette. Cells are counted in the four corner squares and the center square on both sides of the hemocytometer. As shown in the preceding example, the number of cells counted multiplied by the dilution factor equals the number of cells per microliter.

PROCEDURE 10-1 CSF WBC Count Equipment 3% glacial acetic acid Pasteur pipette Hemocytometer Methylene blue stain, if necessary Procedure To prepare a clear specimen that does not require dilution for counting, follow the steps listed. 1. Place four drops of mixed specimen in a clean tube. 2. Rinse a Pasteur pipette with 3% glacial acetic acid, draining thoroughly, and draw the four drops of CSF into the rinsed pipette. 3. Allow the pipette to sit for 1 minute, mix the solution in the pipette, discard the first drop, and load the hemocytometer. 4. As in the total cell count, WBCs are counted in the four corner squares and the center square on both sides of the hemocytometer. Multiply that number by the dilution factor to obtain the number of WBCs per microliter.

WBC Count Lysis of RBCs must be obtained before performing the WBC count on either diluted or undiluted specimens. Specimens requiring dilution can be diluted in the manner described previously, substituting 3% glacial acetic acid to lyse the RBCs. Adding methylene blue to the diluting fluid stains the WBCs, providing better differentiation between neutrophils and mononuclear cells. Visit www.fadavis.com for Video 10-1 (Manual hemocytometer test).

Technical Tip 10-4. The number of squares counted for CSF varies between laboratories. If a different number of squares is counted, use the standard Neubauer formula to obtain the number of cells per microliter.

Quality Control of CSF and Other Body Fluid Cell Counts Liquid commercial controls are available from several manufacturers for spinal fluid counts of RBCs and WBCs. They can be purchased at two levels of concentration. Also, in-house controls can be prepared. All diluents should be checked biweekly for contamination by examining them in a counting chamber under 400× magnification. Contaminated diluents should be discarded and new solutions prepared. The speed of the cytocentrifuge should be checked monthly with a tachometer, and the timing should be checked with a stopwatch.

If nondisposable counting chambers are used, they must be soaked in a bactericidal solution for at least 15 minutes and then rinsed thoroughly with water and cleaned with isopropyl alcohol after each use.

Differential Count on a CSF Specimen Identifying the type or types of cells present in the CSF is a valuable diagnostic aid. The differential count should be performed on a stained smear and not from the cells in the counting chamber. Poor visualization of the cells as they appear in the counting chamber has led to the laboratory practice of reporting only the percentage of mononuclear and polynuclear cells present, which can result in overlooking abnormal cells with considerable diagnostic importance. To ensure that the maximum number of cells is available for examination, the specimen should be concentrated before preparing the smear. Methods available for specimen concentration include sedimentation, filtration, centrifugation, and cytocentrifugation. Sedimentation and filtration are not used routinely in the clinical laboratory, although they do produce less cellular distortion. Most laboratories that do not have a cytocentrifuge concentrate specimens with routine centrifugation. The specimen is centrifuged for 5 to 10 minutes, supernatant fluid is removed and saved for additional tests, and slides made from the suspended sediment are allowed to air dry and then are stained with Wright’s stain. When the differential count is performed, 100 cells should be

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Chapter 10 | Cerebrospinal Fluid counted, classified, and reported in terms of percentage. If the cell count is low and finding 100 cells is not possible, report only the numbers of the cell types seen. Visit www.fadavis.com for Video 10-2 (Body fluid differential cytocentrifuge).

Cytocentrifugation A diagrammatic view of the principle of cytocentrifugation is shown in Figure 10-6. Fluid is added to the conical chamber, and as the specimen is centrifuged, cells present in the fluid are forced into a monolayer within a 6-mm-diameter circle on the slide. Fluid is absorbed by the filter paper blotter, producing a more concentrated area of cells. As little as 0.1 mL of CSF combined with one drop of 30% albumin produces an adequate cell yield when processed with the cytocentrifuge. Adding albumin increases the cell yield and decreases the cellular distortion frequently seen on cytocentrifuged specimens. Positively charged coated slides to attract cells (Shandon, Inc., Pittsburgh, PA) are available also. Cellular distortion may include cytoplasmic vacuoles, nuclear clefting, prominent nucleoli, indistinct nuclear and cytoplasmic borders, and cellular clumping that resembles malignancy. Cells from both the center and periphery of the slide should be examined because cellular characteristics may vary between areas of the slide. A daily control slide for bacteria also should be prepared using 0.2 mL saline and two drops of the 30% albumin currently being used. The slide is stained and examined if bacteria are seen on a patient’s slide.

Stainless steel Cytoclip™ slide clip

Completed assembly Cytoslide™ microscope slide Cytofunnel™ disposable sample chamber with attached filter card In-load position

Table 10-2 presents a cytocentrifuge recovery chart for comparison with chamber counts. The chamber count should be repeated if too many cells are seen on the slide, and a new slide should be prepared if not enough cells are seen on the slide.10

CSF Cellular Constituents The cells found in normal CSF are primarily lymphocytes and monocytes (Figs. 10-7 and 10-8). Adults usually have a predominance of lymphocytes to monocytes (70:30), whereas the ratio is essentially reversed in children.5 Improved concentration methods also are showing occasional neutrophils in normal

Table 10–2

Cytocentrifuge Recovery Chart10

Number of WBCs Counted in Chamber 0 1–5 6–10 11–20 21

Number of Cells on Cytocentrifuge Slide 0–40 20–100 60–150 150–250 251

Figure 10–7 Normal lymphocytes. Some cytocentrifuge distortion of cytoplasm (×1000).

In operation

This cutaway drawing illustrates, at left, the in-load position which shows the sample chamber assembly in a tilted-back position, so that the sample is not absorbed by the filter card. During spinning, centrifugal force tilts the assembly upright and forces the sample to flow toward the microscope slide.

Figure 10–6 Cytospin 3 cytocentrifuge specimen processing assembly (Courtesy of Shandon, Inc., Pittsburgh, PA).

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Figure 10–8 Normal lymphocytes and monocytes (×500).

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CSF.11 The presence of increased numbers of these normal cells (termed pleocytosis) is considered abnormal, as is the finding of immature leukocytes, eosinophils, plasma cells, macrophages, increased tissue cells, and malignant cells. When pleocytosis involving neutrophils, lymphocytes, or monocytes is present, the CSF differential count is associated most frequently with its role in providing diagnostic information about the type of microorganism that is causing an infection of the meninges (meningitis). A high CSF WBC count of which the majority of the cells are neutrophils is considered indicative of bacterial meningitis. Likewise, a CSF WBC count with a high percentage of lymphocytes and monocytes that is moderately elevated suggests meningitis of viral, tubercular, fungal, or parasitic origin. As seen in Table 10-3, many pathological conditions other than meningitis can be associated with abnormal cells in the CSF. Cell forms differing from those found in blood include macrophages, choroid plexus and ependymal cells, spindleshaped cells, and malignant cells.

Technical Tip 10-5. Because laboratory personnel become so accustomed to finding neutrophils, lymphocytes, and monocytes, they should be careful not to overlook other types of cells in the CSF.

are lost more rapidly in CSF. Neutrophils associated with bacterial meningitis may contain phagocytized bacteria (Figs. 10-10 and 10-11). Although of little clinical significance, neutrophils may be increased after hemorrhage in the CNS, repeated lumbar punctures, and injection of medications or radiographic dye. Neutrophils with pyknotic nuclei indicate degenerating cells. They may resemble nucleated red blood cells (NRBCs) but usually have multiple nuclei. When a single nucleus is present, these neutrophils can appear similar to NRBCs (Fig. 10-12). NRBCs are seen as a result of contamination from bone marrow during the spinal tap (Figs. 10-13 and 10-14). This is found in approximately 1% of specimens.12 Capillary structures and endothelial cells may be seen after a traumatic tap (Fig. 10-15).

Lymphocytes and Monocytes A mixture of lymphocytes and monocytes is common in cases of viral, tubercular, and fungal meningitis. Reactive lymphocytes containing increased dark blue cytoplasm and clumped chromatin frequently are present in conjunction with normal cells during viral infections (Fig. 10-16). Increased lymphocytes are seen in cases of both asymptomatic HIV infection and AIDS. A moderately elevated WBC count (less than 50 WBCs/µL) with increased normal and reactive lymphocytes and plasma cells may indicate multiple sclerosis or other degenerative neurological disorders.13

Neutrophils

Eosinophils

In addition to bacterial meningitis, increased neutrophils are seen in the early stages (1 to 2 days) of viral, fungal, tubercular, and parasitic meningitis. Neutrophils also may contain cytoplasmic vacuoles after cytocentrifugation (Fig. 10-9). Granules also

Increased eosinophils are seen in the CSF in association with parasitic infections, fungal infections (primarily Coccidioides immitis), and introduction of foreign material, including medications and shunts, into the CNS (Fig. 10-17).

Table 10–3

Predominant Cells Seen in CSF

Type of Cell

Major Clinical Significance

Microscopic Findings

Lymphocytes

Normal Viral, tubercular, and fungal meningitis HIV infection and AIDS Multiple sclerosis Degenerative disorders

All stages of development may be found

Neutrophils

Parasitic infections Bacterial meningitis Early cases of viral, tubercular, and fungal meningitis Cerebral hemorrhage Cerebral abscess CNS infarction Injection of medications or radiographic dye into the subarachnoid space Metastatic tumors Repeated lumbar punctures

Granules may be less prominent than in blood Cells disintegrate rapidly

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Predominant Cells Seen in CSF—cont’d

Type of Cell

Major Clinical Significance

Microscopic Findings

Monocytes

Found mixed with lymphocytes

Macrophages

Normal Viral, tubercular, and fungal meningitis Multiple sclerosis Parasitic infections Fungal infections Coccidioidal meningitis Introducing medications and shunts in the CNS RBCs in spinal fluid

Blast forms

Contrast media Acute leukemia

Eosinophils

Lymphoma cells Plasma cells

Ependymal, choroidal, and spindleshaped cells Malignant cells

May contain phagocytized RBCs appearing as empty vacuoles or ghost cells, hemosiderin granules, and hematoidin crystals Lymphoblasts, myeloblasts, or monoblasts Resemble lymphocytes with cleft nuclei Traditional and classic forms seen

Disseminated lymphomas Multiple sclerosis Guillain-Barre syndrome Sarcoidosis Parasitic infections Syphilitic meningitis Tuberculous meningitis Lymphocyte reactions Diagnostic procedures

Reactive lymphocytes Seen in clusters with distinct nuclei and distinct cell walls Seen in clusters with fusing of cell borders and nuclei

Metastatic carcinomas Primary central nervous system carcinoma

Figure 10–9 Neutrophils with cytoplasmic vacuoles resulting from cytocentrifugation (×500).

Figure 10–10 Neutrophils with intracellular bacteria (×1000).

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Figure 10–11 Neutrophils with intracellular and extracellular bacteria (×1000).

Figure 10–12 Neutrophils with pyknotic nuclei. Notice the cell with a single nucleus in the center (×1000).

Figure 10–13 Nucleated RBCs seen with bone marrow contamination (×1000).

Figure 10–14 Bone marrow contamination (×1000). Notice the immature RBCs and granulocytes.

Figure 10–15 Capillary and tissue fragments from a traumatic tap (×100).

Figure 10–16 Broad spectrum of lymphocytes and monocytes in viral meningitis (×1000).

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Figure 10–17 Eosinophils (×1000). Notice cytocentrifuge distortion.

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Figure 10–19 Macrophages showing erythrophagocytosis (×500).

Macrophages The purpose of macrophages in the CSF is to remove cellular debris and foreign objects, such as RBCs. Macrophages appear within 2 to 4 hours after RBCs enter the CSF and frequently are seen after repeated taps. They tend to have more cytoplasm than monocytes in the peripheral blood (PB) (Fig. 10-18). The finding of increased macrophages indicates a previous hemorrhage (Fig. 10-19). Further degradation of the phagocytized RBCs results in the appearance of dark blue or black iron-containing hemosiderin granules (Figs. 10-20 through 10-23). Yellow hematoidin crystals represent further degeneration. They are iron-free, consisting of hemoglobin and unconjugated bilirubin (Figs. 10-24 and 10-25).

Nonpathologically Significant Cells

Figure 10–20 Macrophage with RBC remnants (×500).

Nonpathologically significant cells are seen most frequently after diagnostic procedures, such as pneumoencephalography, and in fluid obtained from ventricular taps or during neurosurgery. The cells often appear in clusters and can be distinguished from malignant cells by their uniform appearance. Choroidal cells are from the epithelial lining of the choroid plexus. They are seen singularly and in clumps. Usually nucleoli are absent, and nuclei have a uniform appearance (Fig. 10-26).

Figure 10–21 Macrophage with aggregated hemosiderin granules (×500).

Figure 10–18 Macrophages. Notice the large amount of cytoplasm and vacuoles (×500).

Ependymal cells are from the lining of the ventricles and neural canal. They have less defined cell membranes and frequently are seen in clusters. Often nucleoli are present (Fig. 10-27). Spindle-shaped cells represent lining cells from the arachnoid. Usually they are seen in clusters and may be seen with systemic malignancies (Fig. 10-28).

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Figure 10–22 Macrophage containing hemosiderin stained with Prussian blue (×250).

Figure 10–23 Macrophage with coarse hemosiderin granules (×500).

Figure 10–24 Macrophage containing hemosiderin and hematoidin crystals (500).

Figure 10–25 Macrophages with hemosiderin and hematoidin (×250). Notice the bright yellow color.

Figure 10–26 Choroidal cells showing distinct cell borders and nuclear uniformity (×500).

Figure 10–27 Ependymal cells. Notice the nucleoli and less distinct cell borders (×1000).

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Figure 10–28 Cluster of spindle-shaped cells (×500).

Malignant Cells of Hematologic Origin Lymphoblasts, myeloblasts, and monoblasts (Figs. 10-29 to 10-31) in the CSF are seen frequently as a serious complication of acute leukemias. Often nucleoli are more prominent than in blood smears.

Figure 10–29 Lymphoblasts from acute lymphocytic leukemia (×500).

Figure 10–30 Myeloblasts from acute myelocytic leukemia (×500).

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Figure 10–31 Monoblasts and two lymphocytes (×1000). Notice the prominent nucleoli.

Lymphoma cells are seen in the CSF also and indicate dissemination from the lymphoid tissue. They resemble large and small lymphocytes and usually appear in clusters of large, small, or mixed cells based on the classification of the lymphoma. Nuclei may appear cleaved, and prominent nucleoli are present (Figs. 10-32 to 10-34).

Figure 10–32 Cleaved and noncleaved lymphoma cells (×1000).

Figure 10–33 Lymphoma cells with nucleoli (×500).

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Part Three | Other Body Fluids chemical composition is controlled by the blood–brain barrier, reference values for CSF chemicals are not the same as the plasma values. Abnormal values result from alterations in the permeability of the blood–brain barrier or increased production or metabolism by the neural cells in response to a pathological condition. They seldom have the same diagnostic significance as plasma abnormalities. The clinically important CSF chemicals are few; under certain conditions, it may be necessary to measure a larger variety.

Cerebrospinal Protein

Figure 10–34 Burkitt lymphoma. Notice characteristic vacuoles (×500).

Figure 10–35 Medulloblastoma (×1000). Notice cellular clustering, nuclear irregularities, and rosette formation.

Malignant Cells of Nonhematologic Origin Metastatic carcinoma cells of nonhematologic origin are primarily from malignancies in the lung, breast, renal system, and gastrointestinal system. Cells from primary CNS tumors include astrocytomas, retinoblastomas, and medulloblastomas (Fig. 10-35). They usually appear in clusters and must be distinguished from normal clusters of ependymal, choroid plexus, lymphoma, and leukemia cells. Fusing of cell walls and nuclear irregularities and hyperchromatic nucleoli are seen in clusters of malignant cells.

Technical Tip 10-6.

Slides containing abnormal cells must be referred to pathology following facility protocol.

Chemistry Tests Because CSF is formed by filtration of the plasma, one would expect to find the same low-molecular-weight chemicals in the CSF that are found in the plasma. This is essentially true; however, because the filtration process is selective and the

The chemical test performed most frequently on CSF is the protein determination. Normal CSF contains a very small amount of protein. Reference values for total CSF protein usually are listed as 15 to 45 mg/dL but are somewhat method dependent, and higher values are found in infants and people over age 40.14 This value is reported in milligrams per deciliter and not grams per deciliter, as are plasma protein concentrations. In general, the CSF contains protein fractions similar to those found in serum; however, the ratio of CSF proteins to serum proteins varies among the fractions. As in serum, albumin makes up most of the CSF protein. But in contrast to serum, transthyretin (previously called prealbumin) is the second most prevalent fraction in CSF. The alpha globulins include primarily haptoglobin and ceruloplasmin. Transferrin is the major beta globulin present; also, a separate carbohydrate-deficient transferrin fraction, referred to as “tau,” is seen in CSF but not in serum. CSF gamma globulin is primarily immunoglobulin G (IgG), with only a small amount of immunoglobulin A (IgA). Immunoglobulin M (IgM), fibrinogen, and beta lipoprotein are not found in normal CSF.15

Clinical Significance of Elevated Protein Values Elevated total protein values are seen most frequently in pathological conditions. Abnormally low values are present when fluid is leaking from the CNS. The causes of elevated CSF protein include damage to the blood–brain barrier, immunoglobulin production within the CNS, decreased normal protein clearance from the fluid, and neural tissue degeneration. Meningitis and hemorrhage conditions that damage the blood–brain barrier are the most common causes of elevated CSF protein. Many other neurological disorders can elevate the CSF protein, and finding an abnormal result on clear fluid with a low cell count is not unusual (Box 10-1).

Methodology The two techniques used most routinely for measuring total CSF protein use the principles of turbidity production and dyebinding ability. The turbidity method has been adapted to automated instrumentation in the form of nephelometry. Methods for measuring CSF protein are available for most automated chemistry analyzers.

Protein Fractions Routine CSF protein procedures are designed to measure total protein concentration. However, diagnosis of neurological

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Chapter 10 | Cerebrospinal Fluid Box 10–1

Clinical Causes of Abnormal CSF Protein Values*

Elevated Results • Meningitis

• Myxedema

• Hemorrhage

• Cushing disease

• Primary CNS tumors

• Connective tissue disease

• Multiple sclerosis

• Polyneuritis

• Guillain-Barré syndrome

• Diabetes

• Neurosyphilis

• Uremia

• Polyneuritis Decreased Results • CSF leakage/trauma

• Rapid CSF production

• Recent puncture

• Water intoxication

*Reference values for protein are usually 15 to 45 mg/dL, but are method dependent, and higher values are found in infants and people older than 40 years of age.

disorders associated with abnormal CSF protein often requires measurement of the individual protein fractions. Protein that appears in the CSF as a result of damage to the integrity of the blood–brain barrier contains fractions proportional to those in plasma, with albumin present in the highest concentration. Diseases, including multiple sclerosis, that stimulate the immunocompetent cells in the CNS show a higher proportion of IgG. To determine accurately whether IgG is increased because it is being produced within the CNS or is elevated as the result of a defect in the blood–brain barrier, comparisons must be made between serum and CSF levels of albumin and IgG. Methods include the CSF/serum albumin index to evaluate the integrity of the blood–brain barrier and the CSF IgG index to measure IgG synthesis within the CNS. The CSF/serum albumin index is calculated after determining the concentration of CSF albumin in milligrams per deciliter and the serum concentration in grams per deciliter. The formula used is as follows: CSF/serum albumin index =

Normal IgG index values vary slightly among laboratories; however, in general, values greater than 0.70 indicate IgG production within the CNS. Techniques for measuring CSF albumin and globulin have been adapted to automated instrumentation.

Electrophoresis and Immunophoretic Techniques The primary purpose for performing CSF protein electrophoresis is to detect oligoclonal bands, which represent inflammation within the CNS. The bands are located in the gamma region of the protein electrophoresis, indicating immunoglobulin production. To ensure that the oligoclonal bands are present as the result of neurological inflammation, simultaneous serum electrophoresis must be performed. Disorders, such as leukemia, lymphoma, and viral infections, may produce serum banding, which can appear in the CSF as a result of leakage at the blood–brain barrier or traumatic introduction of blood into the CSF specimen. Banding representing both systemic and neurological involvement is seen in the serum and CSF with HIV infection.17 The presence of two or more oligoclonal bands in the CSF that are not present in the serum can be a valuable tool in diagnosing multiple sclerosis, particularly when accompanied by an increased IgG index (Fig. 10-36). Other neurological disorders, including encephalitis, neurosyphilis, Guillain-Barré syndrome, and neoplastic disorders, also produce oligoclonal banding that may not be present in the serum. Therefore, the presence of oligoclonal banding must be considered in conjunction with clinical symptoms. Oligoclonal banding remains positive during remission of multiple sclerosis but disappears in other disorders.13

CSF albumin (mg/dL) Serum albumin (g/dL)

An index value less than 9 represents an intact blood– brain barrier. The index increases relative to the amount of damage to the barrier. Calculation of an IgG index, which is actually a comparison of the CSF/serum albumin index with the CSF/serum IgG index, compensates for any IgG entering the CSF via the blood–brain barrier.16 It is performed by dividing the CSF/serum IgG index by the CSF/serum albumin index as follows: IgG index =

267

CSF IgG (mg/dL)/serum IgG (g/dL) CSF albumin (mg/dL)/serum albumin (g/dL)

Figure 10–36 Normal and abnormal oligoclonal banding. (Photograph courtesy of the University of Pittsburgh Department of Pathology, Case Index files, Case 059; http://path.upmc.edu/cases/case59. html, with permission.)

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Low protein levels in the CSF make concentration of the fluid before performing electrophoresis essential for most electrophoretic techniques. Better resolution can be obtained using CSF immunofixation electrophoresis (IFE) and isoelectric focusing (IEF) followed by silver staining. Specimen concentration is not required by the more sensitive IEF procedure. These techniques are also the method of choice when determining whether a fluid is actually CSF. CSF can be identified based on the appearance of the previously mentioned extra isoform of tau transferrin that is found only in CSF.18

Myelin Basic Protein Myelin basic protein (MBP) is a major component of the myelin nerve sheath surrounding axons of nerves in the nervous system. The presence of MBP in the CSF indicates recent destruction of the myelin sheath that protects the axons of the neurons (demyelination). The course of multiple sclerosis can be monitored by measuring the amount of MBP in the CSF.19 MBP also may provide a valuable measure of the effectiveness of current and future treatments. Other causes of increased MBP include CNS trauma, encephalopathies, Guillain-Barre syndrome, lupus, and brain tumors.20 Immunoassay techniques are used for measurement.21

CSF Glucose Glucose enters the CSF by selective transport across the blood– brain barrier, which results in a reference value that is approximately 60% to 70% that of the plasma glucose. If the plasma glucose is 100 mg/dL, then a reference CSF glucose would be approximately 65 mg/dL. For an accurate evaluation of CSF glucose, a blood glucose test must be run for comparison. The blood glucose should be drawn about 2 hours before the spinal tap to allow time for equilibration between the blood and fluid. CSF glucose is analyzed using the same procedures employed for blood glucose. Specimens should be tested immediately because glycolysis occurs rapidly in CSF. The diagnostic significance of CSF glucose is confined to finding values that are decreased relative to plasma values. Elevated CSF glucose values are always a result of plasma elevations. Low CSF glucose values can be of considerable diagnostic value in determining the causative agents in meningitis. A markedly decreased level of CSF glucose accompanied by an increased WBC count and a large percentage of neutrophils indicates bacterial meningitis. If the WBCs are lymphocytes instead of neutrophils, tubercular meningitis is suspected. Likewise, if a normal value for CSF glucose is found with an increased number of lymphocytes, the diagnosis would favor viral meningitis. Classic laboratory patterns such as those just described may not be found in all cases of meningitis, but they can be helpful when they appear. Decreased values for CSF glucose are caused primarily by alterations in the mechanisms of glucose transport across the blood–brain barrier and by increased use of glucose by the brain cells. The common tendency to associate decreased glucose totally with its use by microorganisms and leukocytes cannot account for the variations in glucose concentrations seen in different types of meningitis and the decreased levels seen in other disorders producing damage to the CNS.22

CSF Lactate CSF lactate levels can be a valuable aid in diagnosing and managing meningitis cases. In bacterial, tubercular, and fungal meningitis, CSF lactate levels greater than 25 mg/dL occur much more consistently than do decreased glucose and provide more reliable information when the initial diagnosis is difficult. Levels greater than 35 mg/dL are seen frequently with bacterial meningitis, whereas in viral meningitis, lactate levels remain lower than 25 mg/dL. CSF lactate levels remain elevated during initial treatment but fall rapidly when treatment is successful, thus offering a sensitive method for evaluating the effectiveness of antibiotic therapy. Tissue destruction within the CNS due to oxygen deprivation (hypoxia) increases levels of lactic acid in the CSF. Therefore, elevated CSF lactate is not limited to meningitis and can result from any condition that decreases oxygen flow to the tissues. Frequently CSF lactate levels are used to monitor patients with severe head injuries. RBCs contain high concentrations of lactate, and results that are falsely elevated may be obtained on xanthochromic or hemolyzed fluid.11

CSF Glutamine Glutamine is produced from ammonia and ␣-ketoglutarate by the brain cells. This process serves to remove the toxic metabolic waste product ammonia from the CNS. The normal concentration of glutamine in the CSF is 8 to 18 mg/dL.23 Elevated levels are associated with liver disorders that result in increased blood and CSF ammonia. Excess ammonia in the CNS increases glutamine synthesis; therefore, determining CSF glutamine provides an indirect test for the presence of excess ammonia in the CSF. Several methods of assaying glutamine are available and are based on the measurement of ammonia liberated from the glutamine. This is preferred over the direct measurement of CSF ammonia because the glutamine concentration remains more stable than the volatile ammonia concentration in the collected specimen. The CSF glutamine level also correlates with clinical symptoms much better than does the blood ammonia.23 As the concentration of ammonia in the CSF increases, the supply of ␣-ketoglutarate becomes depleted; glutamine can no longer be produced to remove the toxic ammonia, and coma ensues. Some disturbance of consciousness is almost always seen when glutamine levels are more than 35 mg/dL.15 Therefore, the CSF glutamine test is requested frequently for patients with coma of unknown origin. In addition, approximately 75% of children with Reye syndrome have elevated levels of CSF glutamine.24 A summary of CSF chemistry tests is presented in Table 10-4.

Microbiology Tests The role of the microbiology laboratory in analyzing CSF is to identify the causative agent in meningitis. For positive identification, the microorganism must be recovered from the fluid by growing it on the appropriate culture medium. This can take anywhere from 24 hours in cases of bacterial meningitis to 6 weeks for tubercular meningitis. Consequently, in many

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Chapter 10 | Cerebrospinal Fluid Table 10–4

CSF Chemistry Tests

Chemical Substance

Reference Concentration Value, Normal CSF

Significance of Increased Concentration

Significance of Decreased Concentration

Protein

15–45 mg/dL

CSF leakage

Glucose

60%–70% of plasma concentration 10–24 mg/dL 8–18 mg/dL

Meningitis Hemorrhage Multiple sclerosis None

Lactate Glutamine

>35 mg/dL: Bacterial meningitis >35 mg/dL: Some disturbance of consciousness

instances, the CSF culture is a procedure that is actually confirmatory rather than diagnostic. However, the microbiology laboratory does have several methods available to provide information for a preliminary diagnosis. These methods include the Gram stain, acid-fast stain, India ink preparation, immunologic assays (latex agglutination, coagglutination, immunoassay, and counterimmunoelectrophroesis), and molecular diagnostic nucleic acid amplification tests, such as polymerase chain reaction (PCR) assay. In Table 10-5, laboratory tests used in the differential diagnosis of meningitis are compared.

Gram Stain The Gram stain is performed routinely on CSF from all suspected cases of meningitis, although its value lies in detecting bacterial and fungal organisms. All smears and cultures should be performed on concentrated specimens because, often, only a few organisms are present at the onset of the disease. The CSF should be centrifuged at 1500 g for 15 minutes, and slides Table 10–5

269

Bacterial, tubercular, and fungal meningitis None None

and cultures should be prepared from the sediment.25 Use of the cytocentrifuge provides a highly concentrated specimen for Gram stains. Even when concentrated specimens are used, at least a 10% chance exists that Gram stains and cultures will be negative. Thus, blood cultures should be taken because often the causative organism is present in both the CSF and the blood.11 A CSF Gram stain is one of the most difficult slides to interpret because the number of organisms present is usually small, and they can be overlooked easily, resulting in a false-negative report. Also, false-positive reports can occur if precipitated stain or debris is mistaken for microorganisms. Therefore, considerable care should be taken when interpreting a Gram stain. Organisms encountered most frequently include Streptococcus pneumoniae (gram-positive cocci), Haemophilus influenzae (pleomorphic gram-negative rods), Escherichia coli (gram-negative rods), and Neisseria meningitidis (gram-negative cocci). The gram-positive cocci Streptococcus agalactiae and the gram-positive rods Listeria monocytogenes may be encountered in newborns.

Major Laboratory Results for Differential Diagnosis of Meningitis

Bacterial

Viral

Tubercular

Fungal

Elevated WBC count Neutrophils present

Elevated WBC count Lymphocytes present

Marked protein elevation

Moderate protein elevation Normal glucose level Normal lactate level

Elevated WBC count Lymphocytes and monocytes present Moderate to marked protein elevation Decreased glucose level Lactate level >25 mg/dL

Elevated WBC count Lymphocytes and monocytes present Moderate to marked protein elevation Normal to decreased glucose level Lactate level >25 mg/dL Positive India ink with Cryptococcus neoformans Positive immunological test for C. neoformans PCR positive for Cryptococcus neoformans

Markedly decreased glucose level Lactate level >35 mg/dL

Positive Gram stain and bacterial antigen tests PCR positive for Streptococcus pneumonia, Streptococcus agalactiae, Neisseria meningitidis, Haemophilus influenzae, Listeria monocytogenes, Escherichia coli K1

Pellicle formation PCR positive for enterovirus, herpes simplex virus, parechovirus

PCR positive for Mycobacterium tuberculosis

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270 Part Three | Other Body Fluids Acid-fast or fluorescent antibody stains are not performed routinely on specimens unless tubercular meningitis is suspected. Considering the length of time required to culture Mycobacteria, a positive report from this smear is extremely valuable. Specimens from possible cases of fungal meningitis are Gram stained and often have an India ink preparation performed on them to detect the presence of thickly encapsulated Cryptococcus neoformans (Fig. 10-37). As one of the complications of AIDS that occurs more frequently, cryptococcal meningitis is now encountered commonly in the clinical laboratory. Particular attention should be paid to the Gram stain for the classic starburst pattern produced by Cryptococcus, as this may be seen more often than a positive India ink (Fig. 10-38).26

Immunologic Assays Latex agglutination tests to detect the presence of C. neoformans antigen in serum and CSF provide a more sensitive method than the India ink preparation. However, immunologic testing results should be confirmed by culture and demonstration of the organisms by India ink because false-positive reactions do

occur. Interference by rheumatoid factor is the most common cause of false-positive reactions.26 Several commercial kits with pretreatment techniques are available and include incubation with dithiothreitol or pronase and boiling with ethylenediaminetetra-acetic acid.27,28 An enzyme immunoassay technique has been shown to produce fewer false-positive results. The lateral flow assay (LFA) can provide a rapid method for detecting C. neoformans with a high sensitivity and specificity. The procedure utilizes a reagent strip coated with monoclonal antibodies that react with the cryptococcal polysaccharide capsule.29 Latex agglutination and enzyme-linked immunosorbent assay (ELISA) methods provide a rapid means for detecting and identifying microorganisms in CSF. Test kits are available to detect Streptococcus group B; H. influenzae type B; S. pneumoniae; N. meningitidis A, B, C, Y, and W135; Mycobacterium tuberculosis; C. immitis; and E. coli K1 antigens. The bacterial antigen test (BAT) does not appear to be as sensitive to N. meningitidis as it is to the other organisms. The BAT should be used in combination with results from the hematology and clinical chemistry laboratories for diagnosing meningitis.30 The Gram stain is still the recommended method for detecting organisms.31 The amoeba Naegleria fowleri is an opportunistic parasite found in ponds, small lakes, and even chlorinated swimming pools. The Naegleria enters the nasal passages and migrates along the olfactory nerves to invade the brain. Motile trophozoites can be observed microscopically by examining a wet preparation of CSF. Nonmotile trophozoites may be seen on cytocentrifuged stained smears accompanied by increased WBCs and no bacteria. Figure 10-39 shows a Naegleria trophozoite. Notice the elongated form with a tapered posterior.32

PCR Molecular Diagnostic Testing

Figure 10–37 India ink preparation of C. neoformans capsular halo (×400). Notice budding yeast form. (Courtesy of Ann K. Fulenwider, MD.)

Figure 10–38 Gram stain of C. neoformans showing starburst pattern (×1000). (Courtesy of Ann K. Fulenwider, MD.)

Gram staining is a specific method for detecting bacteria in CSF, but that method lacks sensitivity. Culture is more sensitive, but detection rates depend on the number of viable bacteria. Nucleic acid amplification tests can detect the cause of meningitis with a small amount of the pathogen’s DNA. Universal PCR detects pathogens with increased sensitivity and specificity. PCR assays are based on the amplification of regions of ribosomal RNA (rRNA) genes to detect and differentiate causative pathogens of meningitis.33

Figure 10–39 N. fowleri trophozoite. (From Leventhal & Cheadle, Medical Parasitology, 6th ed., 2012, FA Davis Company, Philadelphia, with permission.)

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Serological Testing In addition to the serological procedures performed for identification of microorganisms, serological testing of the CSF is performed to detect the presence of neurosyphilis. The use of penicillin in the early stages of syphilis has greatly reduced the number of neurosyphilis cases. Consequently, the number of requests for serological tests for syphilis on CSF is currently low. However, detecting the antibodies associated with syphilis in the CSF still remains a necessary diagnostic procedure. Although many different serological tests for syphilis are available when testing blood, the procedure recommended by the CDC to diagnose neurosyphilis is the Venereal Disease Research Laboratory (VDRL), even though it is not as sensitive as the fluorescent treponemal antibody-absorption (FTA-ABS) test for syphilis. The rapid plasma reagin (RPR) test is not recommended because it is less sensitive than the VDRL. If the FTA-ABS is used, care must be taken to prevent contamination with blood because the FTA-ABS remains positive in the serum of treated cases of syphilis.34

Technical Tip 10-7.

The methodology for preparing a simulated CSF for student laboratory testing is included in the Instructor Resources located at DavisPlus.35,36

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

For additional resources please visit www.fadavis.com

22. 23.

References 1. Scanlon, VC and Sanders, T: Essentials of Anatomy and Physiology, 7th ed. FA Davis Company, Philadelphia, 2015. 2. Edlow, JA, and Caplan, LR: Avoiding pitfalls in the diagnosis of subarachnoid hemorrhage. N Engl J Med 342:29–36, 2000. 3. Nagda, KK: Procoagulant activity of cerebrospinal fluid in health and disease. Indian J Med Res 74:107–110, 1981. 4. Chow, G, and Schmidley, JW: Lysis of erythrocytes and leukocytes in traumatic lumbar punctures. Arch Neurol 41:1084–1085, 1984. 5. Seehusen, DA, Reeves, MM, and Fomin, DA: Cerebrospinal fluid analysis. Am Fam Physician 68(6):1103–1108, 2003. 6. Glasser, L: Tapping the wealth of information in CSF. Diagn Med 4(1):23–33, 1981. 7. Tanada H, Ikemoto T. Masutani, R, Tanaka H, and Takubo T: Evaluation of the automated hematology analyzer ADVIA®120 for cerebrospinal fluid analysis and usage of unique hemolysis reagent. International Journal of Laboratory Hematology. Int. J. Lab Hematol. 2014 Feb;36(1):83-91. Doi: 10.1111/ijlh.12130. Epub August 13, 2013. Web site: https://www.ncbi.nlm.nih. gov/pubmed/23937551 Accessed June 27, 2019. 8. CAP TODAY 2019. Beckman Coulter, Unicel DxH900 Product Guide. Web site: https://captodayonline.com/productguides/ beckman-coulter-unicel-dxh-900-hematology-analyzersnovember-2018.html. Accessed June 27, 2019. 9. Hod EA, Brugnara C, Pilichowska, M, et al: Automated cell counts on CSF samples: A multicenter performance evaluation of the GloCyte system. International Journal of Laboratory Hematology. Int J Lab Hematol. 2018 Feb;40(1):56-65. Doi: 10.1111/ijlh.

24. 25. 26. 27. 28. 29. 30. 31.

32. 33.

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12728. Epub September 7, 2017. Web site: https://www.ncbi. nlm.nih.gov/pubmed/28880455 Accessed June 27, 2019. University of Virginia Health Sciences Center: Clinical Laboratory Procedure Manual. Charlottesville, VA, 1993. Kjeldsberg, CR, and Knight, JA: Body Fluids: Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous and Synovial Fluids: A textbook Atlas. ASCP, Chicago, 1993. Abrams, J, and Schumacher, HR: Bone marrow in cerebrospinal fluid and possible confusion with malignancy. Arch Pathol Lab Med 110:366–369, 1986. Bentz, JS: Laboratory investigation of multiple sclerosis. Lab Med 26(6):393–399, 1995. Biou, D, et al: Cerebrospinal fluid protein concentrations in children: Age-related values in patients without disorders of the central nervous system. Clin Chem 46(3):399–403, 2000. Fishman, RA: Cerebrospinal Fluid in Diseases of the Nervous System, ed 2. WB Saunders, Philadelphia, 1992. Hershey, LA, and Trotter, JL: The use and abuse of the cerebrospinal fluid IgG profile in the adult: A practical evaluation. Ann Neurol 8(4):426–434, 1980. Grimaldi, LME, et al: Oligoclonal IgG bands in cerebrospinal fluid and serum during asymptomatic human immunodeficiency virus infection. Ann Neurol 24:277–279, 1988. Rouah, E, Rogers, BB, and Buffone, GJ: Transferrin analysis by immunofixation as an aid in the diagnosis of cerebrospinal fluid otorrhea. Arch Pathol Lab Med 111:756–757, 1987. Whitaker, JN: Myelin basic protein in cerebrospinal fluid and other body fluids. Multiple Sclerosis 4(1):16–21, 1998. Karcher, DS, McPherson, RA: Cerebrospinal, synovial, serous body fluids and alternative specimens. In: McPherson, RA, Pincus, MR, eds. Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd ed. Philadelphia: Elsevier Saunders: 480–490, 2011. Okta, M, et al: Evaluation of an enzyme immunoassay for myelin basic protein in CSF. Clin Chem 46:1326–1330, 2000. Menkes, J: The causes of low spinal fluid sugar in bacterial meningitis: Another look. Pediatrics 44(1):1–3, 1969. Hourani, BT, Hamlin, EM, and Reynolds, TB: Cerebrospinal fluid glutamine as a measure of hepatic encephalopathy. Arch Intern Med 127:1033–1036, 1971. Glasgow, AM, and Dhiensiri, K: Improved assay for spinal fluid glutamine and values for children with Reye’s syndrome. Clin Chem 20(6):642–644, 1974. Murray, PR, and Hampton, CM: Recovery of pathogenic bacteria from cerebrospinal fluid. J Clin Microbiol 12:554–557, 1980. Sato, Y, et al: Rapid diagnosis of cryptococcal meningitis by microscopic examination of centrifuged cerebrospinal fluid sediment. J Neurol Sci 164(1):72–75, 1999. Eng, RHK, and Person, A: Serum cryptococcal antigen determination in the presence of rheumatoid factor. J Clin Microbiol 14:700–702, 1981. Stockman, L, and Roberts, GD: Specificity of the latex test for cryptococcal antigen: A rapid simple method for eliminating interference. J Clin Microbiol 17(5):945–947, 1983. Knight, FR: New enzyme immunoassay for detecting cryptococcal antigen. J Clin Pathol 45(9):836–837, 1992. Klausner, JD, Vijayan, T, Chiller, T: Sensitivity and specificity of a new cryptococcal antigen lateral flow assay in serum and cerebrospinal fluid. MLO 45(3):16,18,20, 2013. Wojewoda, C: Bacterial Antigen Testing, The Good, the Not So Bad and the Ugly. NewsPath. http://www.cap.org/apps/docs/ newspath/1102/bacterial_antigen_testing.pdf. Accessed March 31, 2020. Leventhal, R and Cheadle, RF: Medical Parasitology, ed.6, FA Davis Company, Philadelphia, 2012. Meyer, T, Franke, G, Polywka, KA, Lutgehetmann, M, Gbadamosi, J, Magnus, T, Aepfelbacher, M: Patel, R, Editor:

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272 Part Three | Other Body Fluids Improved Detection of Bacterial Central Nervous System Infections by Use of Broad-Range PCR Assay. J Clin Microbiol 52(5):1751–1753, 2014. DOI: 10.1128/JCM.00469-14. https:// jcm.asm.org/content/52/5/1751. Accessed June 29, 2019. 34. Davis, LE, and Schmitt, JW: Clinical significance of cerebrospinal fluid tests for neurosyphilis. Ann Neurol 25:50–53, 1989.

35. Albright, RE, et al: Issues in cerebrospinal fluid management. Am J Clin Pathol 95(3):397–401, 1991. 36. Lofsness, KG, and Jensen, TL: The preparation of simulated spinal fluid for teaching purposes. Am J Med Technology 49(7):493–496, 1983.

Study Questions 1. CSF is produced mainly in the: A. Bone marrow B. Peripheral blood C. Choroid plexuses D. Subarachnoid space 2. The functions of the CSF include all of the following except: A. Removing metabolic wastes B. Producing an ultrafiltrate of plasma C. Supplying nutrients to the CNS D. Protecting the brain and spinal cord 3. The CSF flows through the: A. Choroid plexus B. Pia mater C. Subarachnoid space D. Dura mater 4. Substances present in the CSF are controlled by the: A. Arachnoid granulations B. Blood–brain barrier C. Presence of one-way valves D. Blood–CSF barrier

7. Place the appropriate letter in front of the statement that best describes CSF specimens in these two conditions: A. Traumatic tap B. Intracranial hemorrhage Even distribution of blood in all tubes Xanthochromic supernatant Concentration of blood in Tube 1 is greater than in Tube 3 Specimen contains clots 8. The presence of xanthochromia can be caused by all of the following except: A. Immature liver function B. RBC degradation C. A recent hemorrhage D. Elevated CSF protein 9. A web-like pellicle in a refrigerated CSF specimen indicates: A. Tubercular meningitis B. Multiple sclerosis C. Primary CNS malignancy D. Viral meningitis

5. What department is the CSF tube labeled 3 routinely sent to? A. Hematology B. Chemistry C. Microbiology D. Serology

10. Given the following information, calculate the CSF WBC count: cells counted, 80; dilution, 1:10; large Neubauer squares counted, 10. A. 8 B. 80 C. 800 D. 8000

6. The CSF tube that should be kept at room temperature is: A. Tube 1 B. Tube 2 C. Tube 3 D. Tube 4

11. A CSF WBC count is diluted with: A. Distilled water B. Normal saline C. Acetic acid D. Hypotonic saline

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273

12. A total CSF cell count on a clear fluid should be: A. Reported as normal B. Not reported C. Diluted with normal saline D. Counted undiluted

20. Hemosiderin granules and hematoidin crystals are seen in: A. Lymphocytes

13. The purpose of adding albumin to CSF before cytocentrifugation is to: A. Increase the cell yield B. Decrease the cellular distortion C. Improve the cellular staining D. Both A and B

21. Myeloblasts are seen in the CSF: A. In bacterial infections B. In conjunction with CNS malignancy C. After cerebral hemorrhage D. As a complication of acute leukemia

14. The primary concern when pleocytosis of neutrophils and lymphocytes is found in the CSF is: A. Meningitis B. CNS malignancy C. Multiple sclerosis D. Hemorrhage 15. Neutrophils with pyknotic nuclei may be mistaken for: A. Lymphocytes B. Nucleated RBCs C. Malignant cells D. Spindle-shaped cells 16. The presence of which of the following cells is increased in a parasitic infection? A. Neutrophils B. Macrophages C. Eosinophils D. Lymphocytes 17. Macrophages appear in the CSF after: A. Hemorrhage B. Repeated spinal taps C. Diagnostic procedures D. All of the above

B. Macrophages C. Ependymal cells D. Neutrophils

22. Cells resembling large and small lymphocytes with cleaved nuclei represent: A. Lymphoma cells B. Choroid cells C. Melanoma cells D. Medulloblastoma cells 23. The reference range for CSF protein is: A. 6 to 8 g/dL B. 15 to 45 g/dL C. 6 to 8 mg/dL D. 15 to 45 mg/dL 24. CSF can be differentiated from serum by the presence of: A. Albumin B. Globulin C. Transthyretin D. Tau transferrin 25. In serum, the second most prevalent protein is IgG; in CSF, the second most prevalent protein is: A. Transferrin B. Transthyretin C. Prealbumin D. Ceruloplasmin

18. Nucleated RBCs are seen in the CSF as a result of: A. Elevated blood RBCs B. Treatment of anemia C. Severe hemorrhage D. Bone marrow contamination

26. Elevated values for CSF protein can be caused by all of the following except: A. Meningitis B. Multiple sclerosis C. Fluid leakage D. CNS malignancy

19. After a CNS diagnostic procedure, which of the following might be seen in the CSF? A. Choroidal cells B. Ependymal cells C. Spindle-shaped cells D. All of the above

27. The integrity of the blood–brain barrier is measured using the: A. CSF/serum albumin index B. CSF/serum globulin ratio C. CSF albumin index D. CSF IgG index

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28. Given the following results, calculate the IgG index: CSF IgG, 50 mg/dL; serum IgG, 2 g/dL; CSF albumin, 70 mg/dL; serum albumin, 5 g/dL. A. 0.6 B. 6.0 C. 1.8 D. 2.8

34. Measurement of which of the following can be replaced by CSF glutamine analysis in children with Reye syndrome? A. Ammonia B. Lactate C. Glucose D. ␣-Ketoglutarate

29. The CSF IgG index calculated in Study Question 28 indicates: A. Synthesis of IgG in the CNS B. Damage to the blood–brain barrier C. Cerebral hemorrhage D. Lymphoma infiltration

35. Before performing a Gram stain on CSF, the specimen must be: A. Filtered B. Warmed to 37°C C. Centrifuged D. Mixed

30. The finding of oligoclonal bands in the CSF and not in the serum is seen with: A. Multiple myeloma B. CNS malignancy C. Multiple sclerosis D. Viral infections

36. All of the following statements are true about cryptococcal meningitis except: A. An India ink preparation is positive B. A starburst pattern is seen on Gram stain C. The WBC count is over 2000 D. A confirmatory immunology test is available

31. Which condition is suggested by the following results: a CSF glucose of 15 mg/dL, WBC count of 5000, 90% neutrophils, and protein of 80 mg/dL? A. Fungal meningitis B. Viral meningitis C. Tubercular meningitis D. Bacterial meningitis

37. The most sensitive and specific method to detect the causative organism in meningitis is: A. Gram stain B. Culture and sensitivity C. India ink stain D. PCR assay

32. A patient with a blood glucose of 120 mg/dL would have a normal CSF glucose of: A. 20 mg/dL B. 60 mg/dL C. 80 mg/dL D. 120 mg/dL 33. CSF lactate will be more consistently decreased in: A. Bacterial meningitis B. Viral meningitis C. Fungal meningitis D. Tubercular meningitis

38. The test of choice to detect neurosyphilis is the: A. RPR B. VDRL C. FAB D. FTA-ABS

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275

Case Studies and Clinical Situations 1. Three tubes of CSF containing evenly distributed visible blood are drawn from a 75-year-old disoriented patient and delivered to the laboratory. Initial test results are as follows: WBC count: 250 µL Protein: 150 mg/dL Glucose: 70 mg/dL Gram stain: No organisms seen Differential: Neutrophils, 68%; monocytes, 3%; lymphocytes, 28%; eosinophils, 1% Many macrophages containing ingested RBCs a. What is the most probable condition indicated by these results? State two reasons for your answer. b. Are the elevated WBC count and protein significant? Explain your answer. c. Are the percentages of the cells in the differential of any significance? Explain your answer. d. What two other structures besides RBCs might be contained in the macrophages? e. If the blood was distributed unevenly and nucleated RBCs and capillary structures were seen instead of macrophages, what would this indicate? 2. A patient with AIDS is hospitalized with symptoms of high fever and rigidity of the neck. Routine laboratory tests on the CSF show a WBC count of 100/µL with a predominance of lymphocytes and monocytes, glucose of 55 mg/dL (plasma: 85 mg/dL), and a protein of 70 mg/dL. The Gram stain shows a questionable starburst pattern. a. What additional microscopic examination should be performed? b. If the test is positive, what is the patient’s diagnosis? c. If the results of the test are questionable, what additional testing can be performed? d. What could cause a false-positive reaction in this test? e. If the tests named in a and c are negative, the glucose level is 35 mg/dL, and a pellicle is observed in the fluid, what additional testing should be performed? f. If CSF and serum IEF was performed on this patient, what unusual findings might be present? 3. A 35-year-old woman is admitted to the hospital with symptoms of intermittent blurred vision, weakness, and loss of sensation in her legs. A lumbar puncture is performed with the following results: Appearance: Colorless, clear WBC count: 35 cells/µL (90% lymphocytes) Glucose: 60 mg/dL (plasma: 100 mg/dL) Protein: 60 mg/dL (serum: 8 g/dL) Albumin: 40 mg/dL (serum: 6 g/dL) IgG globulin: 20 mg/dL (serum: 2 g/dL)

a. Name and perform the calculation to determine the integrity of the patient’s blood–brain barrier. b. Does the patient have an intact barrier? c. Name and perform the calculation used to determine whether IgG is being synthesized within the CNS. d. What does this result indicate? e. Considering the patient’s clinical symptoms and the calculation results, what diagnosis is suggested? f. If immunofixation electrophoresis is performed on the patient’s serum and CSF, what findings would be expected? g. What substance in the CSF can be measured to monitor this patient? 4. A 5-year-old female patient is admitted to the pediatric ward with a temperature of 105°F, lethargy, and cervical rigidity. A lumbar spinal tap is performed, and three tubes of cloudy CSF are delivered to the laboratory. Preliminary test results are as follows: Appearance: Cloudy WBC count: 800 cells/␮L Differential: 80% lymphocytes, 15% monocytes, 5% neutrophils Protein: 65 mg/dL Glucose: 70 mg/dL Gram stain: No organisms seen a. From these results, what preliminary diagnosis could the physician consider? b. Is the Gram stain result of particular significance? Why or why not? c. Are the lymphocytes significant? Why or why not? d. Would a CSF lactate test be of any value for the diagnosis? Why or why not? 5. State possible technical errors that could result in the following discrepancies: a. An unusual number of Gram stains reported as grampositive cocci fail to be confirmed by positive cultures. b. A physician complains that CSF differentials are being reported only as polynuclear and mononuclear cells. c. Bacteria observed on the cytospin differential cannot be confirmed by Gram stain or culture. d. The majority of CSF specimens sent to the laboratory from the neurology clinic have glucose readings of less than 50% of the corresponding blood glucose results performed in the clinic.

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CHAPTER

11

Semen LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 11-1 State the structures involved in sperm production and their function.

11-10 Describe the appearance of normal sperm, including structures and their functions.

11-2 Describe the four components of semen with regard to source and function.

11-11 Differentiate between routine and strict criteria for evaluating sperm morphology.

11-3 Explain the procedures for collecting and handling semen specimens.

11-12 Given an abnormal result in a routine semen analysis, determine additional tests that might be performed.

11-4 Describe the normal appearance of semen and three abnormalities in appearance.

11-13 Describe the two methods routinely used to detect antisperm antibodies.

11-5 State two possible causes of low semen volume.

11-14 List two methods for identifying a questionable fluid as semen.

11-6 Discuss the significance of semen liquefaction and viscosity. 11-7 Calculate a sperm concentration and count when provided with the number of sperm counted, the dilution, the area of the counting chamber used, and the ejaculate volume. 11-8 Define round cells, and explain their significance.

11-15 State the World Health Organization reference values for routine and follow-up semen analysis. 11-16 Discuss the types and significance of sperm function tests. 11-17 Describe methods of quality control appropriate for semen analysis.

11-9 State the two parameters to consider when evaluating sperm motility.

KEY TERMS Acrosomal cap

Prostate gland

Spermatozoa

Andrology

Semen

Testes

Bulbourethral gland

Seminal vesicles

Vasectomy

Epididymis

Seminiferous tubules

Viscosity

Liquefaction

Spermatids

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Introduction Advances in the field of andrology and assisted reproductive technology (ART), as well as increased concern over fertility, particularly by couples choosing to have children later in life, have resulted in increased emphasis on semen analysis. Patients with abnormal results on the routine semen analysis performed in the clinical laboratory often are referred to specialized andrology laboratories for further testing to determine the need for in vitro fertilization (IVF). Clinical laboratory personnel also may be employed in andrology laboratories and perform both routine and specialized testing. In addition to fertility testing, the clinical laboratory performs postvasectomy semen analysis and forensic analyses to determine the presence of semen.

Physiology Semen is composed of four fractions that are contributed by the testes, epididymis, seminal vesicles, prostate gland, and bulbourethral glands (Fig. 11-1). Each fraction differs in its composition, and the mixing of all four fractions during ejaculation is essential for the production of a normal semen specimen (Table 11-1). The testes are paired glands in the scrotum that contain the seminiferous tubules for the secretion of sperm. The external location of the scrotum contributes to a lower scrotum temperature that is optimal for sperm development. Germ cells for the production of spermatozoa are located in the epithelial cells of the seminiferous tubules. Specialized Sertoli cells provide support and nutrients for the germ cells as they undergo mitosis and meiosis (spermatogenesis). When spermatogenesis is complete, the immature sperm (nonmotile) enter the epididymis. In the epididymis, the sperm mature and develop flagella. The entire process takes approximately 90 days. The sperm remain stored in the epididymis until ejaculation, at which time they are propelled through the ductus deferens (vas deferens) to the ejaculatory ducts. The ejaculatory ducts receive both the sperm from the ductus deferens and fluid from the seminal vesicles. The seminal vesicles produce most of the fluid present in semen (60% to 70%), and this fluid is the transport medium for the sperm. The fluid contains a high concentration of fructose and flavin. Spermatozoa metabolize the fructose for the energy needed for the flagella to propel them through the female reproductive tract. In the absence of fructose, sperm do not display motility in the semen analysis. Flavin is responsible for the gray appearance of semen, as well as the blue to yellow fluorescence when semen is visualized under ultraviolet light (Wood’s lamp).1 Various proteins secreted by the seminal vesicles are involved in the coagulation of the ejaculate. The muscular prostate gland, located just below the bladder, surrounds the upper urethra and aids in propelling the sperm through the urethra by contractions during ejaculation. Approximately 20% to 30% of the semen volume is acidic fluid produced by the prostate gland. The milky acidic fluid contains high concentrations of acid phosphatase, citric acid, zinc, and

proteolytic enzymes responsible for both the coagulation and liquefaction of the semen after ejaculation. The bulbourethral glands, located below the prostate, contribute about 5% of the fluid volume in the form of a thick, alkaline mucus that helps neutralize acidity from the prostate secretions and the vagina. It is important for semen to be alkaline to neutralize the vaginal acidity present as a result of normal bacterial vaginal flora. Without this neutralization, sperm motility would be diminished.

Specimen Collection The variety in the composition of the semen fractions makes proper collection of a complete specimen essential for accurate evaluation of male fertility. Most of the sperm are contained in the first portion of the ejaculate, making complete collection essential for accurate testing of both fertility and postvasectomy specimens. When a part of the first portion of the ejaculate is missing, the sperm count will be decreased, the pH will be falsely increased, and the specimen will not liquefy. Likewise, when part of the last portion of ejaculate is missing, the semen volume is decreased, the sperm count is falsely increased, the pH is falsely decreased, and the specimen will not clot. Patients should receive detailed written instructions for specimen collection. Specimens are collected after a period of sexual abstinence of at least 2 days to not more than 7 days.2 Specimens collected after prolonged abstinence tend to have higher volumes and decreased motility.3 When performing fertility testing, the World Health Organization (WHO) recommends that two or three specimens be collected not less than 7 days or more than 3 weeks apart, with two abnormal specimens considered significant. The laboratory should provide the patient with warm sterile glass or plastic containers. Whenever possible, the specimen is collected in a room provided by the laboratory. However, if this is not appropriate, the specimen should be

SUMMARY 111

Semen Production

Structure

Function

Seminiferous tubules of testes Epididymis Ductus deferens

Spermatogenesis

Seminal vesicles Prostate gland

Bulbourethral glands

Sperm maturation Propel sperm to ejaculatory ducts Provide nutrients for sperm and fluid Provides enzymes and proteins for coagulation and liquefaction Add alkaline mucus to neutralize prostatic acid and vaginal acidity

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Chapter 11 | Semen 279

Seminal vesicle Urinary bladder

Rectum Ejaculatory duct

Vas deferens

Prostate gland

Urethra

Penis

Epididymis

Glans penis

Anus Testis Scrotum

Bulbourethral gland

Ureter Urinary bladder Vas deferens Prostate gland

Seminal vesicle Bulbourethral gland

Epididymis Testis Urethra Penis

Figure 11–1 The male genitalia. Top, sagittal view; bottom, anterior view.

Table 11–1

Semen Composition

Spermatozoa Seminal fluid Prostate fluid Bulbourethral glands

5% 60%–70% 20%–30% 5%

kept at room temperature and delivered to the laboratory within 1 hour of collection. Laboratory personnel must record the patient’s name and birth date, the period of sexual abstinence, the completeness of the specimen, any difficulties with collection, and the times of specimen collection and specimen receipt. Specimens awaiting analysis should be kept at 37°C. Specimens should be collected by masturbation into the container. If this is not possible, only nonspermicidial, nonlubricant-containing rubber or polyurethane condoms should be used. Ordinary condoms are not acceptable because they contain spermicides.

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280 Part Three | Other Body Fluids

Technical Tip 11-1. Coitus interruptus is not a reliable means of semen collection because the first portion of the ejaculate, which contains the highest number of spermatozoa, may be lost and the low pH of the vaginal fluid may affect sperm motility.2

Table 11–2

Reference Values for Semen Analysis2,6

Parameter Volume Viscosity

Specimen Handling All semen specimens are potential reservoirs for HIV, herpes viruses, and hepatitis viruses, and standard precautions must be observed at all times during analysis. Specimens are discarded as biohazardous waste. Sterile materials and techniques must be used when semen culture is to be performed or when the specimen is to be processed for bioassay, intrauterine insemination (IUI), or IVF.2

pH Sperm concentration Sperm count Motility

Semen Analysis Semen analysis for fertility evaluation consists of both macroscopic and microscopic examination. Parameters reported include appearance, volume, viscosity, pH, sperm concentration and count, motility, and morphology. Reference values are shown in Table 11-2.

Quality Vitality (% alive) Morphology

Appearance Normal semen has a gray-white color, appears translucent, and has a characteristic musty odor. When the sperm concentration is very low, the specimen may appear almost clear.2 Increased white turbidity indicates the presence of white blood cells (WBCs) and infection within the reproductive tract. If required, specimen culturing is performed before continuing with the semen analysis. During microscopic examination, WBCs must be differentiated from immature sperm (spermatids). The leukocyte esterase reagent strip test may be useful to screen for the presence of WBCs.4 Varying amounts of red coloration are associated with the presence of red blood cells (RBCs) and are abnormal. Yellow coloration may be caused by urine contamination, specimen collection after prolonged abstinence, and medications. Urine is toxic to sperm, thereby affecting the evaluation of motility.

Liquefaction A fresh semen specimen is clotted and should liquefy within 30 to 60 minutes after collection; therefore, recording the time of collection is essential for evaluating semen liquefaction. Failure of liquefaction to occur within 60 minutes may be caused by a deficiency in prostatic enzymes and should be reported. Analysis of the specimen cannot begin until liquefaction has occurred. If after 2 hours the specimen has not liquified, an equal volume of Dulbecco’s phosphate-buffered saline (DPBS) (Procedure 11-1) or proteolytic enzymes, such as alpha-chymotrypsin or bromelain (Procedure 11-2), may be added to induce liquefaction and allow the rest of the analysis to be performed. These treatments may affect biochemical tests, sperm motility, and sperm morphology, so their use must be documented. The dilution of semen with

Round cells

Reference Values

Lower Reference Limit1

2–5 mL Pours in droplets within 60 minutes 7.2– 8.0 >20 million/mL

1.5 mL

>40 million/ ejaculate >50% within 1 hr >2.0 or a, b, c in Table 11-3 >75% >14% normal forms (strict criteria) >30% normal forms (routine criteria) 7.2 15 million/mL

58% 4% normal forms

1Lower

reference limits recommended by the WHO (2010) for semen characteristics.

bromelain must be accounted for when calculating sperm concentration.2 Jelly-like granules (gelatinous bodies) may be present in liquefied semen specimens and have no clinical significance. Mucous strands, if present, may interfere with semen analysis.2

Technical Tip 11-2. Delivery of the semen specimen to the laboratory within 1 hour of collection and documentation of the collection time are critical to evaluate liquefaction.

Volume Normal semen volume ranges between 2 and 5 mL. It can be measured by pouring the specimen into a clean graduated cylinder calibrated in 0.1-mL increments. Increased volume may be seen after periods of extended abstinence. Decreased volume is associated more frequently with infertility and may indicate improper functioning of one of the semen-producing organs, primarily the seminal vesicles. Also, incomplete specimen collection must be considered.

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PROCEDURE 11-1 Semen Dilution With Dulbecco’s Phosphate-Buffered Saline2 Prepare an equal volume of diluent and semen (one part diluent and one part semen) using Dulbecco’s phosphatebuffered saline. Repeated pipetting of the prepared dilution will induce liquefaction. Preparation of Dulbecco’s Phosphate-Buffered Saline2 1. Using a 1-L volumetric flask, add the following: a. 750 mL of purified water b. 0.20 g of potassium chloride (KCL) c. 0.20 g of potassium dihydrogen phosphate (KH2PO4) d. 0.10 g of magnesium chloride hexahydrate (MgCl2.6H2O)

2.

3.

4. 5.

e. 8.0 g of sodium chloride (NaCl) f. 2.16 g of disodium hydrogen phosphate heptahydrate (Na2HPO4.7H2O) g. 1.00 g of D-glucose In a 10-mL volumetric flask, dissolve 0.132 g of calcium chloride dehydrate (CaCl2.2H2O) in 10 mL of purified water. To prevent precipitation, add calcium chloride dehydrated solution to the 1-L flask slowly, stirring continuously. Adjust the pH to 7.4 with 1 mol/L sodium hydroxide (NaOH). Make up to 1000 mL with purified water.

Technical Tip 11-3. Gently mixing the specimen container during liquefaction can help produce a homogeneous specimen.2

PROCEDURE 11-2 Digestion with Bromelain2 1. Prepare 10 IU/mL bromelain in Dulbecco’s phosphate-buffered saline. a. Into a 100-mL volumetric flask, add 1000 IU of bromelain. b. Add 60 mL Dulbecco’s phosphate-buffered saline. c. Mix to dissolve. It takes about 15 to 20 minutes. Bring the volume to the calibration mark using buffered saline. 2. Dilute one part semen 1:2 with the 10 IU/mL bromelain (one part semen + one part bromelain solution). 3. Stir with a pipette tip. 4. Incubate at 37°C for 10 minutes. 5. Mix the sample well before analysis.

pH The pH of semen indicates the balance between the pH values from the acidic prostatic secretion and the alkaline seminal vesicles secretion. The pH should be measured within 1 hour of ejaculation due to the loss of CO2 that occurs.2 The normal pH of semen is alkaline with a range of 7.2 to 8.0. Increased pH indicates infection within the reproductive tract. A decreased pH may be associated with increased prostatic fluid, obstruction of the ejaculatory duct, or poorly developed seminal vesicles.2 Semen for pH testing can be applied to the pH pad of a urinalysis reagent strip and the color compared with the manufacturer’s chart. Dedicated pH testing paper with a range of 4.0 to 10.0 also can be used.The results should be recorded to the nearest 0.1 pH unit. Also, a pH meter device that provides a numerical value can be used.

Viscosity

Sperm Concentration and Sperm Count

Specimen viscosity refers to the consistency of the fluid and may be related to specimen liquefaction. Specimens that are liquefied incompletely are clumped and highly viscous. The normal semen specimen should be drawn into a pipette easily and form small discrete droplets that do not appear clumped or stringy when falling by gravity from the pipette. Droplets that form threads longer than 2 cm are considered highly viscous and are recorded as abnormal. Ratings of 0 (watery) to 4 (gel-like) can be assigned to the viscosity report.5 Viscosity also can be reported as low, normal, or high. Increased viscosity and incomplete liquefaction impede testing for sperm motility, sperm concentration, antisperm antibody detection, and measurement of biochemical markers.2

Even though fertilization is accomplished by one spermatozoon, the actual number of sperm present in a semen specimen is a valid measurement of fertility. Various factors can affect sperm concentration, such as the days of sexual abstinence before the collection, infection, or stress; therefore, more than one semen specimen should be evaluated for infertility studies. Reference values for sperm concentration are commonly listed as greater than 20 to 250 million sperm per milliliter; concentrations between 10 and 20 million per milliliter are considered borderline. The total sperm count for the ejaculate can be calculated by multiplying the sperm concentration by the specimen volume. Total sperm counts greater than 40 million per ejaculate are considered normal (20 million per milliliter × 2 mL).

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282 Part Three | Other Body Fluids In the clinical laboratory, sperm concentration is usually performed using the Neubauer counting chamber. The sperm are counted in the same manner as cells in the cerebrospinal fluid cell count, that is, by diluting the specimen and counting the cells in the Neubauer chamber. The amount of the dilution and the number of squares counted vary among laboratories. The dilution used most commonly is 1:20 prepared using a mechanical (positive-displacement) pipette.6 Dilution of the semen is essential because it immobilizes the sperm before counting. The traditional diluting fluid contains sodium bicarbonate and formalin, which immobilize and preserve the cells; however, good results also can be achieved using saline and distilled water. Using the Neubauer hemocytometer, sperm usually are counted in the four corner and center squares of the large center square, similar to a manual RBC count (Fig. 11-2). Both sides of the hemocytometer are loaded and allowed to settle for 3 to 5 minutes; then they are counted, and the counts should agree within 10%. An average of the two counts is used in the calculation. If the counts do not agree, both the dilution and the counts are repeated. Counts are performed using either phase or bright-field microscopy. The addition of a stain, such as crystal violet, to the diluting fluid aids in visualization when using bright-field microscopy. Only fully developed sperm should be counted. Immature sperm and WBCs, often referred to as “round” cells, must not be included. However, their presence can be significant, and they may need to be identified and counted separately. Stain included in the diluting fluid aids in differentiating between immature sperm cells (spermatids) and leukocytes, and they can be counted in the same manner as mature sperm. A count

greater than 1 million leukocytes per milliliter is associated with inflammation or infection of the reproductive organs that can lead to infertility. The presence of more than 1 million spermatids per milliliter indicates disruption of spermatogenesis. This may be caused by viral infections, exposure to toxic chemicals, and genetic disorders.

Calculating Sperm Concentration and Sperm Count Calculation of sperm concentration depends on the dilution used and the size and number of squares counted. When using the 1:20 dilution and counting the five squares (RBCs) in the large center square, as described previously, the number of sperm can be multiplied by 1,000,000 (add 6 zeros) to equal the sperm concentration per milliliter. Notice that, unlike blood cell counts, the sperm concentration is reported in millions per milliliter rather than microliters. Sperm concentration also can be calculated using the basic formula for cell counts covered in Chapter 10. Because this formula provides the number of cells per microliter, the figure that is calculated must be multiplied by 1000 to calculate the number of sperm per milliliter. The total sperm count is calculated by multiplying the number of sperm per milliliter by the specimen volume. EXAMPLES 1. Using a 1:20 dilution, an average of 60 sperm are counted in the five RBC counting squares on both sides of the hemocytometer. Calculate the sperm concentration per milliliter and the total sperm count in a specimen with a volume of 4 mL. 60 sperm counted × 1,000,000 = 60,000,000 sperm/mL 60,000,000 sperm/mL × 4 mL = 240,000,000 sperm/ ejaculate

W

W

R

600 sperm counted × 20 (dilution) 60,000 sperm/µL = 2 sq mm × (squares counted) × (volume counted) 0.1 mm (depth) 60,000 sperm/µL × 1000 = 60,000,000 sperm/mL 60,000,000/mL × 2 mL = 120,000,000 sperm/ejaculate

R

R

R

W

2. In a 1:20 dilution, 600 sperm are counted in two WBC counting squares. Calculate the sperm concentration per milliliter and the total sperm count in a specimen with a volume of 2 µL.

R

W

Figure 11–2 Areas of the Neubauer counting chamber used for red and white blood cell counts. W, typical WBC counting area; R, typical RBC counting area.

Several methods have been developed using specially designed and disposable counting chambers that do not require specimen dilution. Comparison of these methods and the standard Neubauer counting chamber method showed poor correlation with the Neubauer method and also among themselves. The WHO states that the “validity of these alternative counting chambers must be established by checking chamber dimensions, comparing results with the improved Neubauer hemocytometer method, and obtaining satisfactory performance as shown by an external quality control program.”2

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Chapter 11 | Semen

Sperm Motility

Table 11–4

The presence of sperm capable of forward, progressive movement is critical for fertility because once presented to the cervix, the sperm must propel themselves through the cervical mucosa to the uterus, fallopian tubes, and ovum. Traditionally, clinical laboratory reporting of sperm motility has been a subjective evaluation performed by examining an undiluted specimen and determining the percentage of motile sperm and the quality of the motility. Sperm motility should be assessed using a well-mixed, liquefied semen specimen within 1 hour of specimen collection. The practice of examining sperm motility at timed intervals over an extended period has been shown to serve no useful purpose.7 To provide continuity in reporting, laboratories should place a consistent amount of semen on a slide under the same size cover slip, such as 10 µL under a 22 × 22 mm cover slip using a calibrated positive-displacement pipette, and allow it to settle for 1 minute. This procedure should be done in duplicate for accuracy. Then the percentage of sperm showing actual forward movement can be estimated after evaluating approximately 20 high-power fields. An alternative procedure is to examine 200 sperm per slide and count the percentages of the different motile categories using a manual cell counter.2 Motility is evaluated by both speed and direction. Grading can be done using a scale of 0 to 4, with 4 indicating rapid, straightline movement and 0 indicating no movement (Table 11-3). A minimum motility of 50% with a rating of 2.0 after 1 hour is considered normal.3 The WHO uses a rating scale of a, b, c, and d (see Table 11-3). Interpretation states that within 1 hour, 50% or more of the sperm should be motile in categories a, b, and c, or 25% or more should show progressive motility (a and b).6 The WHO Laboratory Manual for the Examination and Processing of Human Semen (2010)2 currently recommends a simpler system for grading motility that does not include speed because of the difficulty in standardized reporting. Motility is graded as progressive motility (PM), nonprogressive motility (NP), and immotility (IM). Motility must be specified as total motility (PM and NP) or progressive motility (PM).2 (See Table 11-4.)

Sperm Motility Grading

Table 11–3 Grade

WHO Criteria

4.0

a

3.0

b

2.0

b

1.0 0

c d

Sperm Motility Action Motile with rapid, straight-line motility Motile with slower speed, some lateral movement Motile with slow forward progression, noticeable lateral movement Motile without forward progression No movement

Progressive motility (PM) Nonprogressive motility (NP) Immotility (IM)

283

Alternative Sperm Motility Grading Criteria2 Sperm moving linearly or in a large circle Sperm moving with an absence of progression No movement

The presence of a high percentage of immobile sperm and clumps of sperm requires further evaluation to determine sperm vitality or the presence of sperm agglutinins.

Automated Semen Analysis Systems In recent years, instrumentation capable of performing computer-assisted semen analysis (CASA) has been developed. CASA provides objective determination of both sperm velocity and trajectory (direction of motion). Also, sperm concentration and morphology are included in the analysis. Currently, CASA instrumentation is found primarily in laboratories that specialize in andrology and perform a high volume of semen analysis. Studies comparing several automated systems with manual methods demonstrate a good correlation. Automated systems, however, provide faster results with higher precision and standardization, as well as reduced potential for human error.8 Three different automated systems are the Sperm Class Analyzer (SCA, Microptic, Spain),9 the CEROS CASA systems (Hamilton Thorne, Beverly, MA), and the Automated Sperm Quality Analyzers (SQAVision, Medical Electronic Systems, Los Angeles, CA). CASA systems capture and process microscopic images by computer software to detect sperm motility. The SCA consists of a microscope equipped with a digital camera, motorized heating stage, and image analysis software with an analytical filter that eliminates other bodies that could cause inconsistent analysis, such as cell debris, isolated sperm heads, or flagella. The analyzer measures sperm concentration, motility, vitality, DNA fragmentation, acrosome reaction, morphology, and leukocytes.9 The SQA measures sperm concentration, percent motility, percent normal morphology, concentration of motile and functional sperm, concentration of progressively motile sperm, velocity, and sperm motility index using electro-optics combined with spectrophotometry and computer algorithms.

Sperm Morphology Just as the presence of a normal number of sperm that are nonmotile produces infertility, the presence of sperm that are morphologically incapable of fertilization also results in infertility. Sperm morphology is evaluated with respect to the structure of the head, neckpiece, midpiece, and tail. Abnormalities in head morphology are associated with poor ovum penetration, whereas abnormalities in the neckpiece, midpiece, and tail affect motility. The normal sperm has an oval-shaped head approximately 5 µm long and 3 µm wide with a long, flagellar tail approximately 45 µm long (Fig. 11-3). Critical to ovum penetration is the

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284 Part Three | Other Body Fluids Acrosome Head

Cell membrane Nucleus

(Figs. 11-4 and 11-5). Abnormal sperm tails are frequently doubled, coiled, or bent (Figs. 11-6 and 11-7). A neckpiece that is abnormally long may cause the sperm head to bend backward and interfere with motility (Fig. 11-8).

Midpiece Mitochondria

Tail (flagellum)

Figure 11–4 Spermatozoon with double head, hematoxylin–eosin (×1000). Figure 11–3 Normal spermatozoon structure.

enzyme-containing acrosomal cap located at the tip of the head. The acrosomal cap should encompass approximately half of the head and cover approximately two thirds of the sperm nucleus.6 The neckpiece attaches the head to the tail and the midpiece. The midpiece is approximately 7 µm long and is the thickest part of the tail because it is surrounded by a mitochondrial sheath that produces the energy required by the tail for motility.

Technical Tip 11-4. Sperm motility can be evaluated at room temperature or at 37°C. When assessing motility at 37°C, the specimen should be incubated at this temperature and the preparation made with prewarmed slides and cover slips.2

Sperm morphology is evaluated from a thinly smeared, stained slide under oil immersion. Smears are made by placing approximately 10 µL of semen near the frosted end of a clean microscope slide. Place a second slide with a clean, smooth edge in front of the semen drop at a 45-degree angle and draw the slide back to the edge of the drop of semen, allowing the semen to spread across the end. When the semen is evenly distributed across the spreader slide, lightly pull the spreader slide forward with a continuous movement across the first slide to produce a smear. Staining can be performed using Wright’s, Giemsa, Shorr, or Papanicolaou stain; the stain used is a matter of laboratory preference. Air-dried slides are stable for 24 hours. At least 200 sperm should be evaluated and the percentage of abnormal sperm reported. Abnormalities in head structure that are identified routinely include double heads, giant and amorphous heads, pinheads, tapered heads, and constricted heads

Figure 11–5 Spermatozoon with amorphous head, hematoxylin– eosin (×1000).

Figure 11–6 Spermatozoon with double tail, hematoxylin–eosin (×1000).

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Figure 11–7 Common abnormalities of sperm heads and tails.

Normal

Double head

Giant head

Tapered head

Constricted head

Amorphous head

Double tail

Coiled tail

Pinhead

Spermatid

Calculating Round Cells Differentiation and enumeration of round cells (immature sperm and leukocytes) also can be made during the morphology examination (Fig. 11-9). Peroxidase-positive granulocytes are the predominant form of leukocyte in semen and can be differentiated further from spermatogenic cells and lymphocytes using a peroxidase stain. By counting the number of spermatids or leukocytes seen in conjunction with 100 mature sperm, the amount per milliliter can be calculated using the following formula, where N is the number of spermatids or neutrophils counted per 100 mature sperm, and S is the sperm concentration in millions per milliliter: Figure 11–8 Spermatozoon with bent neck and spermatid, hematoxylin–eosin (×1000).

Additional parameters in evaluating sperm morphology include measuring head, neck, and tail size; measuring acrosome size; and evaluating for the presence of vacuoles. Inclusion of these parameters is referred to as Kruger’s strict criteria.10 Strict criteria evaluation requires the use of a stage micrometer or morphometry.11 At present, evaluation of sperm morphology using strict criteria is not performed routinely in the clinical laboratory but is recommended by the WHO.6 Strict criteria evaluation is an integral part of evaluations in assisted reproduction. Normal values for sperm morphology depend on the evaluation method used and vary from greater than 30% normal forms when using routine criteria to greater than 14% normal forms when using strict criteria.6

C =

N × S 100

This method can be used when counting but cannot be performed during the hemocytometer count and to verify counts performed by hemocytometer. More than 1 million WBCs per milliliter per ejaculate indicates an inflammatory condition associated with infection and poor sperm quality; it may impair sperm motility and DNA integrity.2

Additional Testing Should abnormalities be discovered in any of these routine parameters, additional tests may be requested (Table 11-5). The most common are tests for sperm vitality, seminal fluid fructose level, sperm agglutinins, and microbial infection.

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Figure 11–9 Immature spermatozoa, hematoxylin–eosin (×1000).

Table 11–5

Additional Testing for Abnormal Semen Analysis

Abnormal Result

Possible Abnormality

Test

Decreased motility with normal count Decreased count

Vitality

Eosin–nigrosin stain

Decreased motility with clumping

Normal analysis with continued infertility

Lack of seminal vesicle support medium Male antisperm antibodies

Female antisperm antibodies

Fructose level

Mixed agglutination reaction and immunobead tests Sperm agglutination with male serum Sperm agglutination with female serum

Sperm Vitality Decreased sperm vitality may be suspected when a specimen has a normal sperm concentration with markedly decreased motility. Sperm vitality should be assessed within 1 hour of ejaculation. Vitality is evaluated by mixing the specimen with an eosin–nigrosin stain, preparing a smear, and counting the number of dead cells in 100 sperm using a bright-field or phase-contrast microscope. Living cells are not infiltrated by the dye and remain bluish white, whereas dead cells stain red against the purple background (Fig. 11-10). Normal vitality requires 50% or more living cells and should correspond to

Figure 11–10 Nonviable spermatozoa demonstrated by the eosin– nigrosin stain (×1000).

the motility evaluated previously. The presence of a large proportion of vital but immobile cells may indicate a defective flagellum, whereas a high number of immotile and nonviable cells may indicate epididymal pathology.2

Seminal Fluid Fructose Low sperm concentration may be caused by lack of the support medium produced in the seminal vesicles, which can be indicated by a fructose level that is low to absent in the semen. Low fructose levels are caused by abnormalities of the seminal vesicles, bilateral congenital absence of the vas deferens, obstruction of the ejaculatory duct, partial retrograde ejaculation, and androgen deficiency.2 Specimens can be screened for the presence of fructose using the resorcinol test that produces an orange color when fructose is present (Procedure 11-3). A normal quantitative level of fructose is equal to or greater than 13 µmol per ejaculate. This can be determined using spectrophotometric methods. Specimens for fructose levels should be tested within 2 hours of collection or frozen to prevent fructolysis.

Antisperm Antibodies Antisperm antibodies can be present in both men and women. They may be detected in semen, cervical mucosa, or serum and are considered a possible cause of infertility. It is not unusual for both partners to demonstrate antibodies, although male antisperm antibodies are encountered more frequently.

PROCEDURE 11-3 Seminal Fructose Screening Test7 1. Prepare reagent (50 mg resorcinol in 33 mL concentrated HCl diluted to 100 mL with water). 2. Mix 1 mL of semen with 9 mL of reagent. 3. Boil. 4. Observe for orange-red color.

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Chapter 11 | Semen 287 Under normal conditions, the blood–testes barrier separates sperm from the male immune system. When this barrier is disrupted, as can occur after surgery, vasectomy reversal (vasovasostomy), trauma, and infection, the antigens on the sperm produce an immune response that damages the sperm. The damaged sperm may cause the production of antibodies in the female partner.12 The presence of antibodies in a male subject can be suspected when clumps of sperm are observed during a routine semen analysis. Sperm-agglutinating antibodies cause sperm to stick to each other in a head-to-head, head-to-tail, or tailto-tail pattern.2 The agglutination is graded as “few,” “moderate,” or “many” on microscopic examination. The presence of antisperm antibodies in a female subject results in a normal semen analysis accompanied by continued infertility. The presence of antisperm antibodies in women may be demonstrated by mixing the semen with the female cervical mucosa or serum and observing for agglutination. A variety of immunoassay kits are available for both semen and serum testing. Two tests used frequently to detect the presence of antibody-coated sperm are the mixed agglutination reaction (MAR) test and the immunobead test. The MAR test is a screening procedure used primarily to detect the presence of immunoglobulin G (IgG) antibodies. The semen sample containing motile sperm is incubated with IgG antihuman globulin (AHG) and a suspension of latex particles or treated RBCs coated with IgG. The bivalent AHG binds simultaneously to both the antibody on the sperm and the antibody on the latex particles or RBCs, forming microscopically visible clumps of sperm and particles or cells. A finding of less than 10% of the motile sperm attached to the particles is considered normal. The immunobead test is a more specific procedure in that it can be used to detect the presence of IgG, IgM, and IgA antibodies and demonstrates the area of the sperm (head, neckpiece, midpiece, or tail) being affected by the autoantibodies. Head-directed antibodies can interfere with penetration into the cervical mucosa or ovum, whereas tail-directed antibodies affect movement through the cervical mucosa.13 In the immunobead test, sperm are mixed with polyacrylamide beads known to be coated with either anti-IgG, anti-IgM, or anti-IgA. Microscopic examination of the sperm shows the beads attached to sperm at particular areas. Depending on the type of beads used, the test could be reported as “IgM tail antibodies,” “IgG head antibodies,” and so forth. The presence of beads on less than 50% of the sperm is considered normal as defined by the WHO.2

Microbial and Chemical Testing The presence of more than 1 million leukocytes per millimeter indicates infection within the reproductive system, frequently the prostate. Routine aerobic and anaerobic cultures and tests for Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum are performed most frequently. Additional chemical testing performed on semen may include determining the levels of neutral α-glucosidase, free L-carnitine, glycerophosphocholine, zinc, citric acid, glutamyl

transpeptidase, and prostatic acid phosphatase. Just as decreased fructose levels are associated with a lack of seminal fluid, decreased neutral α-glucosidase, glycerophosphocholine, and L-carnitine suggest a disorder of the epididymis. Decreased zinc, citric acid, glutamyl transpeptidase, and acid phosphatase indicate a lack of prostatic fluid (Table 11-6). Spectrophotometric methods are used to quantitate citric acid and zinc. On certain occasions, the laboratory may be called on to determine whether semen is actually present in a specimen. A primary example is in cases of alleged rape or sexual assault. Microscopically examining the vaginal fluid specimen for the presence of sperm may be possible, with the best results being obtained by enhancing the specimen with xylene and examining under phase microscopy.14 Motile sperm can be detected for up to 24 hours after intercourse, whereas nonmotile sperm can persist for 3 days. As the sperm die off, only the heads remain and may be present for 7 days after intercourse. Seminal fluid contains a high concentration of prostatic acid phosphatase, so detecting this enzyme can aid in determining the presence of semen in a specimen. A more specific method is the detection of seminal glycoprotein p30 (prostatic specific antigen [PSA]), which is present even in the absence of sperm.15 Often, further information can be obtained by performing ABO blood grouping and DNA analysis on the specimen.

Postvasectomy Semen Analysis Postvasectomy semen analysis is a much less involved procedure compared with infertility analysis because the only concern is the presence or absence of spermatozoa. The length of time required for complete sterilization can vary greatly among patients and depends on both time and number of ejaculations. Therefore, finding viable sperm in a postvasectomy patient is not uncommon, and care should be taken not to overlook even a single sperm. Specimens are tested routinely at monthly intervals, beginning at 2 months postvasectomy and continuing until two consecutive monthly specimens show no spermatozoa. Recommended testing includes examining a wet preparation using phase microscopy for the presence of motile and nonmotile sperm. A negative wet preparation is followed by specimen centrifugation for 10 minutes and examination of the sediment.7

Technical Tip 11-5. A single “motile” sperm on a wet preparation is an indication of an unsuccessful vasectomy.

Table 11–6

Reference Semen Chemical Values2

Neutral α-glucosidase Zinc Citric acid Acid phosphatase

≥20 mU/ejaculate ≥2.4 µmol/ejaculate ≥52 µmol/ejaculate ≥200 units/ejaculate

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Sperm Function Tests Advances in assisted reproduction and IVF have resulted in a need for more sophisticated semen analysis to assess not only the characteristics of sperm but also the functional ability. The tests are performed most commonly in specialized andrology laboratories and include the hamster egg penetration assay, cervical mucus penetration test, hypo-osmotic swelling test, and the in vitro acrosome reaction (Table 11-7).16

Semen Analysis Quality Control Traditionally, routine semen analysis has been subject to very little quality control,17 a situation that has resulted from a lack of appropriate control materials and the subjectivity of the motility and morphology analyses. The analysis is rated as a high complexity test under the Clinical Laboratory Improvement Amendments, and standards for testing personnel must be observed. Increased interest in fertility testing has promoted the development of quality control materials and in-depth training programs. The standardized procedures developed by the WHO have provided a basis for laboratory testing and reporting. The use of CASA has aided in reducing the subjectivity of the analysis. However, even computerized analysis has been shown to vary among operators.18 Now laboratories can participate in proficiency testing programs offered by the College of American Pathologists and the American Association of Bioanalysts that include sperm concentration, vitality, and morphology. Commercial qualitycontrol materials and training aids are available and should be incorporated into laboratory protocols.

Table 11–7

Sperm Function Tests

Test

Description

Hamster egg penetration

Sperm are incubated with species-nonspecific hamster eggs, and penetration is observed microscopically Observation of sperm’s ability to penetrate partner’s midcycle cervical mucus Sperm exposed to lowsodium concentrations are evaluated for membrane integrity and sperm viability Evaluation of the acrosome to produce enzymes essential for ovum penetration

Cervical mucus penetration Hypo-osmotic swelling

In vitro acrosome reaction

For additional resources please visit www.fadavis.com

References 1. Noureddine, M: Forensic Tests for Semen: What you should know. Bodily Fluids and Forensics. Web site: https://ncforensics. wordpress.com/2011/10/19 forensic-tests-for-semen-what-youshould-know/. Accessed July 1, 2019. 2. WHO Laboratory Manual for the Examination and Processing of Human Semen. World Health Organization. WHO Press, Geneva, Switzerland, 2010. 3. Sarhar, S, and Henry, JB: Andrology laboratory and fertility assessment. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods. WB Saunders, Philadelphia, 1996. 4. Lopez, A, et al: Suitability of solid-phase chemistry for quantification of leukocytes in cerebrospinal, seminal and peritoneal fluid. Clin Chem 33(8):1475–1476, 1987. 5. Overstreet, JW, and Katz, DF: Semen analysis. Urol Clin North Am 14(3):441–449, 1987. 6. World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Interaction. Cambridge University Press, London, 1999. 7. Sampson, JH, and Alexander, NJ: Semen analysis: A laboratory approach. Lab Med 13(4):218–223, 1982. 8. Lammers, J, Splingart, C, Barriere, P, Jean, M, and Freour, T: Double-blind prospective study comparing two automated sperm analyzers versus manual semen assessment. J Assist Reprod Genet 31:35, 2014. doi: 10.1007/s10815-013-0139-2. Web site: https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC3909144/. Accessed July 1, 2019. 9. Sperm Class Analyzer® CASA System Product Brochure. Microptic Automatic Diagnostic Systems.https://www. micropticsl.com/products/sperm-class-analyzer-casa-system/. Accessed March 31, 2020. 10. Kruger, T, et al: Predictive value of sperm morphology in IVF. FertilSteril 112–117, 1988. 11. Harr, R: Characterization of spermatozoa by planar morphometry. Clin Lab Sci 10(4):190–196, 1997. 12. Cearlock, DM: Autoimmune antispermatozoa antibodies in men: Clinical detection and role in infertility. Clin Lab Sci 2(3): 165–168, 1989. 13. Marshburn, PB, and Kutteh, WH: The role of antisperm antibodies in infertility. Fertil Steril 61:799–811, 1994. 14. Fraysier, HD: A rapid screening technique for the detection of spermatozoa. J Forensic Sci 32(2):527–528, 1987. 15. Graves, HC, Sensabaugh, CF, and Blake, ET: Postcoital detection of a male-specific semen protein, application to the investigation of rape. N Engl J Med 312(6):338–343, 1985. 16. Yablonsky, T: Male fertility testing. Lab Med 27(6):378–383, 1996. 17. Baker, DJ, et al: Semen evaluations in the clinical laboratory. Lab Med 25(8):509–514, 1994. 18. Krause, W, and Viethen, G: Quality assessment of computerassisted semen analysis (CASA) in the andrology laboratory. Andrologia 31(3):125–129, 1999.

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Study Questions 1. Maturation of spermatozoa takes place in the: A. Sertoli cells B. Seminiferous tubules C. Epididymis D. Seminal vesicles

8. An increased semen pH may be caused by: A. Poorly developed seminal vesicles B. Increased prostatic secretions C. Obstruction of the ejaculation duct D. Prostatic infection

2. Enzymes for the coagulation and liquefaction of semen are produced by the: A. Seminal vesicles B. Bulbourethral glands C. Ductus deferens D. Prostate gland

9. Proteolytic enzymes may be added to semen specimens to: A. Increase the viscosity B. Dilute the specimen C. Decrease the viscosity D. Neutralize the specimen

3. The major component of seminal fluid is: A. Glucose B. Fructose C. Acid phosphatase D. Citric acid 4. If the first portion of a semen specimen is not collected, the semen analysis will have which of the following? A. Decreased pH B. Increased viscosity C. Decreased sperm count D. Decreased sperm motility 5. Failure of laboratory personnel to document the time a semen specimen is collected primarily affects the interpretation of semen: A. Appearance B. Volume C. pH D. Viscosity 6. Liquefaction of a semen specimen should take place within: A. 1 hour B. 2 hours C. 3 hours D. 4 hours 7. A semen specimen delivered to the laboratory in a condom has a normal sperm count and markedly decreased sperm motility. This indicates: A. Decreased fructose B. Antispermicide in the condom C. Increased semen viscosity D. Increased semen alkalinity

10. The normal sperm concentration is: A. Less than 20 million/µL B. More than 20 million/mL C. Less than 20 million/mL D. More than 20 million/µL 11. Given the following information, calculate the sperm concentration: dilution, 1:20; sperm counted in five RBC squares on each side of the hemocytometer, 80 and 86; volume, 3 mL. A. 80 million/mL B. 83 million/mL C. 86 million/mL D. 169 million/µL 12. Using the information from question 11, calculate the sperm concentration when 80 sperm are counted in 1 WBC square and 86 sperm are counted in another WBC square. A. 83 million/mL B. 166 million per ejaculate C. 16.6 million/mL D. 50 million per ejaculate 13. The primary reason to dilute a semen specimen before performing a sperm concentration is to: A. Immobilize the sperm B. Facilitate the chamber count C. Decrease the viscosity D. Stain the sperm

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290 Part Three | Other Body Fluids 14. When performing a sperm concentration, 60 sperm are counted in the RBC squares on one side of the hemocytometer and 90 sperm are counted in the RBC squares on the other side. The specimen is diluted 1:20. The: A. Specimen should be rediluted and counted B. Sperm count is 75 million/mL C. Sperm count is greater than 5 million/mL D. Sperm concentration is abnormal 15. Sperm motility evaluations are performed: A. Immediately after the specimen is collected B. Within 1 hour of collection C. After 3 hours of incubation D. At 6-hour intervals for 1 day 16. The percentage of sperm showing average motility that is considered normal is: A. 25% B. 50% C. 60% D. 75% 17. The purpose of the acrosomal cap is to: A. Penetrate the ovum B. Protect the nucleus C. Create energy for tail movement D. Protect the neckpiece 18. The sperm part containing a mitochondrial sheath is the: A. Head B. Neckpiece C. Midpiece D. Tail 19. All of the following are associated with sperm motility except the: A. Head B. Neckpiece C. Midpiece D. Tail 20. The morphological shape of a normal sperm head is: A. Round B. Tapered C. Oval D. Amorphous

21. Normal sperm morphology when using the WHO criteria is: A. >30% normal forms B. 15% abnormal forms D. 40 U/L

Elevated

100,000 RBCs/µL indicates blunt trauma injury

WBC count 500 cells/µL Differential Other Tests

Normal Bacterial peritonitis, cirrhosis Bacterial peritonitis, malignancy

Carcinoembryonic antigen CA 125 Glucose

Malignancy of gastrointestinal origin Malignancy of ovarian origin Decreased in tubercular peritonitis, malignancy Increased in pancreatitis, gastrointestinal perforation Increased in gastrointestinal perforation Ruptured or punctured bladder

Amylase Alkaline phosphatase Blood urea nitrogen/ creatinine Gram stain and culture Acid-fast stain Adenosine deaminase

Bacterial peritonitis Tubercular peritonitis Tubercular peritonitis

of bile, which can be confirmed using standard chemical tests for bilirubin. Blood-streaked fluid is seen after trauma and with cases of tuberculosis, intestinal disorders, and malignancy. Chylous or pseudochylous material may be present with cases of trauma or blockage of lymphatic vessels.

Laboratory Tests Normal WBC counts are less than 500 cells/µL, and the count increases with bacterial peritonitis and cirrhosis.4 To distinguish between those two conditions, an absolute neutrophil count should be performed. An absolute neutrophil count >250 cells/µL or >50% of the total WBC count indicates infection. Lymphocytes are the predominant cell in tuberculosis and peritoneal carcinomatosis.

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Cellular Examination Examination of ascitic exudates for the presence of malignant cells is important for detecting tumors of primary and metastatic origin. Malignancies are most frequently of gastrointestinal, prostate, or ovarian origin. Other cells present in ascitic fluid include leukocytes, abundant mesothelial cells, and macrophages, including lipophages (Fig. 13-17). Microorganisms, including bacteria, yeast, and Toxoplasma gondii, may also be present (Fig. 13-18). Malignant cells of ovarian, prostatic, and colonic origin, often containing mucin-filled vacuoles, are seen frequently (Figs. 13-19 to 13-22). Psammoma bodies containing concentric striations of collagen-like material can be seen in benign conditions and also are associated with ovarian and thyroid malignancies (Fig. 13-23).

Chemical Testing

Figure 13–19 Ovarian carcinoma showing community borders, nuclear irregularity, and hyperchromatic nucleoli (×500).

Chemical examination of ascitic fluid consists primarily of glucose, amylase, and alkaline phosphatase determinations. Glucose is decreased below serum levels in bacterial and tubercular peritonitis and malignancy. Amylase is determined on ascitic fluid to ascertain cases of pancreatitis, and it may be elevated

Figure 13–20 Ovarian carcinoma cells with large mucin-containing vacuoles (×500).

Figure 13–17 Lipophages (macrophages containing fat droplets) in peritoneal fluid (×500).

Figure 13–18 Budding yeast in peritoneal fluid (×400).

Figure 13–21 Adenocarcinoma of the prostate showing cytoplasmic vacuoles, community borders, and hyperchromatic nucleoli (×500).

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316 Part Three | Other Body Fluids sensitive and specific method for the diagnosis of bacterial infections, including M. tuberculosis.15

Serological Tests Measurement of the tumor markers CEA and CA 125 is a valuable procedure for identifying the primary source of tumors producing ascitic exudates. The presence of CA 125 antigen with a negative CEA suggests the source is from the ovaries, fallopian tubes, or endometrium.9

For additional resources please visit www.fadavis.com Figure 13–22 Colon carcinoma cells containing mucin vacuoles and nuclear irregularities (×400).

Figure 13–23 Psammoma bodies exhibiting concentric striations (×500).

in patients with gastrointestinal perforations. An elevated alkaline phosphatase level is also highly diagnostic of intestinal perforation. Measurements of blood urea nitrogen and creatinine in the fluid are requested when a ruptured bladder or accidental puncture of the bladder during the paracentesis is of concern. Bilirubin is measured when leakage of bile into the peritoneum is suspected after trauma or surgery. Bile contains primarily conjugated bilirubin; therefore, a test for total bilirubin is acceptable.10 Amylase or lipase can be measured to determine whether the pancreatitis or damage to the pancreas is accounting for the accumulation of these pancreatic enzymes in the ascitic fluid.10

Microbiology Tests Gram stains and bacterial cultures for both aerobes and anaerobes are performed when bacterial peritonitis is suspected. Inoculation of fluid into blood culture bottles immediately at the bedside increases the recovery of anaerobic organisms. Acidfast stains, adenosine deaminase, and cultures for tuberculosis may also be requested. Molecular testing using PCR is a more

References 1. Clinical and Laboratory Standards Institute: Body Fluid Analysis for Cellular Composition, Approved Guideline H56-A, Wayne, PA, 2006, CLSI. 2. Jay, SJ: Pleural effusions: Definitive evaluation of the exudate. Postgrad Med 80(5):180–188, 1986. 3. Light, RW: Clinical practice: Pleural effusion. N Engl J Med 346:1971–1977, 2002. 4. Cooper, A: Blood Sciences Test. Transudate or Exudate. Exeter Clinical Laboratory International. Web site: www.exeterlaboratory.com/test/transudate-or-exudate/. Published March 19, 2019. Accessed July 10, 2019. 5. Kjeldsberg, CR, and Knight, JA: Body Fluids: Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous and Synovial Fluids. A Textbook Atlas. ASCP, Chicago, 1993. 6. Valdez, L, et al: Cholesterol: A useful parameter for distinguishing between pleural exudates and transudates. Chest 99(5): 1097–1102, 1991. 7. Porcel, JM, and Light, RW: Diagnostic approach to pleural fluid effusion in adults. Am Fam Physician 73:1211–1220, 2006. 8. Boka, K: Pleural Effusion Workup. Medscape. Web site: https://emedicine.medscape.com/article/299959-workup#c9. Published December 28, 2018. Accessed July 10, 2019. 9. Colice, GL, et al: Medical and surgical treatment of parapneumonic effusions: An evidence-based guideline. Chest 118: 1158–1171, 2000. 10. Clinical and Laboratory Science Institute: Analysis of Body Fluids in Clinical Chemistry, Approved Guideline, C49-A, Wayne, PA, 2007, CLSI. 11. Na, MJ: Diagnostic Tools of Pleural Effusion. NCBI. 76(5): 199–210, 2014 DOI: 10.4046/trd.2014.76.5.199. Web site: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050067/. Published May 29, 2014. Accessed July 10, 2019. 12. Porcel, JM, et al: Use of a panel of tumor markers (carcinoembryonic antigen, cancer antigen 125, carbohydrate antigen 15-3 and cytokeratin 19 fragments) in pleural fluid for differential diagnosis of benign and malignant effusions. Chest 126: 1757–1763, 2004. 13. Jagminas, L: Diagnostic Peritoneal Lavage. Medscape. Web site: https:/emedicine.medscape.com/article/82888-overview#a1. Published October 20, 2017. Accessed July 10, 2019. 14. Runyan, BA, et al: The serum-ascites albumin gradient is superior to the exudate transudate concept in the differential diagnosis of ascites. Ann Intern Med 117:215–218, 1992. 15. Suceveanu, AI, Todescu, D, Mazilu, L, Manousos, FG, Julea, R, Voinea, F, Dumitru, E, and Suceveanu, AP: Modern Tools for Diagnosis in Tuberculous Ascites. DOI: 10.5772/intechopen. 70417. Web site: https://www.intechopen.com/books/ascitesphysiopathology-treatment-complications-and-prognosis/ modern-tools-for-diagnosis-in-tuberculous-ascites. Published November, 29, 2017. Accessed July 10, 2019.

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Study Questions 1. The primary purpose of serous fluid is to: A. Remove waste products B. Lower capillary pressure C. Lubricate serous membranes D. Nourish serous membranes 2. The membrane that lines the wall of a cavity is the: A. Visceral B. Peritoneal C. Pleural D. Parietal 3. During normal production of serous fluid, the slight excess of fluid is: A. Absorbed by the lymphatic system B. Absorbed through the visceral capillaries C. Stored in the mesothelial cells D. Metabolized by the mesothelial cells 4. Production of serous fluid is controlled by: A. Capillary oncotic pressure B. Capillary hydrostatic pressure C. Capillary permeability D. All of the above 5. An increase in the amount of serous fluid is called a/an: A. Exudate B. Transudate C. Effusion D. Malignancy 6. Pleural fluid is collected by: A. Pleurocentesis B. Paracentesis C. Pericentesis D. Thoracentesis 7. Place the appropriate letter in front of the following statements describing transudates and exudates. A. Transudate B. Exudate Caused by increased hydrostatic pressure Caused by increased capillary permeability Caused by decreased oncotic pressure Caused by congestive heart failure Malignancy related Tuberculosis related Endocarditis related Clear appearance

8. Fluid: serum protein and lactic dehydrogenase ratios are performed on serous fluids: A. When malignancy is suspected B. To classify transudates and exudates C. To determine the type of serous fluid D. When a traumatic tap has occurred 9. Which of the following requires the most additional testing? A. Transudate B. Exudate 10. An additional test performed on pleural fluid to classify the fluid as a transudate or exudate is the: A. WBC count B. RBC count C. Fluid:cholesterol ratio D. Fluid-to-serum protein gradient 11. A milky-appearing pleural fluid indicates: A. Thoracic duct leakage B. Chronic inflammation C. Microbial infection D. Both A and B 12. Which of the following best represents a hemothorax? A. Blood HCT: 42 Fluid HCT: 15 B. Blood HCT: 42 Fluid HCT: 10 C. Blood HCT: 30 Fluid HCT: 10 D. Blood HCT: 30 Fluid HCT: 20 13. All of the following are normal cells seen in pleural fluid except: A. Mesothelial cells B. Neutrophils C. Lymphocytes D. Mesothelioma cells 14. A differential observation of pleural fluid associated with tuberculosis is: A. Increased neutrophils B. Decreased lymphocytes C. Decreased mesothelial cells D. Increased mesothelial cells

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15. All of the following are characteristics of malignant cells except: A. Cytoplasmic molding B. Absence of nucleoli C. Mucin-containing vacuoles D. Increased nucleus:cytoplasm ratio

21. The recommended test for determining whether peritoneal fluid is a transudate or an exudate is the: A. Fluid:serum albumin ratio B. Serum ascites albumin gradient C. Fluid:serum lactic dehydrogenase ratio D. Absolute neutrophil count

16. A pleural fluid pH of 6.0 indicates: A. Esophageal rupture B. Mesothelioma C. Malignancy D. Rheumatoid effusion

22. Given the following results, classify this peritoneal fluid: serum albumin, 2.2 g/dL; serum protein, 6.0 g/dL; fluid albumin, 1.6 g/dL. A. Transudate B. Exudate

17. Plasma cells seen in pleural fluid indicate: A. Bacterial endocarditis B. Primary malignancy C. Metastatic lung malignancy D. Tuberculosis infection

23. Differentiation between bacterial peritonitis and cirrhosis is done by performing a/an: A. WBC count B. Differential C. Absolute neutrophil count D. Absolute lymphocyte count

18. A significant cell found in pericardial or pleural fluid that should be referred to cytology is a: A. Reactive lymphocyte B. Mesothelioma cell C. Monocyte D. Mesothelial cell 19. Another name for a peritoneal effusion is: A. Peritonitis B. Lavage C. Ascites D. Cirrhosis 20. A test performed primarily on peritoneal lavage fluid is a/an: A. WBC count B. RBC count C. Absolute neutrophil count D. Amylase

24. Detection of the CA 125 tumor marker in peritoneal fluid indicates: A. Colon cancer B. Ovarian cancer C. Gastric malignancy D. Prostate cancer 25. Chemical tests primarily performed on peritoneal fluid include all of the following except: A. Amylase B. Glucose C. Alkaline phosphatase D. Calcium 26. Cultures of peritoneal fluid are incubated: A. Aerobically B. Anaerobically C. At 37°C and 42°C D. Both A and B

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Case Studies and Clinical Situations 1. Fluid from a patient with congestive heart failure is collected by thoracentesis and sent to the laboratory for testing. It appears clear and pale yellow and has a WBC count of 450/µL, fluid:serum protein ratio of 0.35, and fluid:serum LD ratio of 0.46. a. What type of fluid was collected? b. Based on the laboratory results, would this fluid be considered a transudate or an exudate? Why? c. List two other tests that could be performed to aid in classifying this fluid. 2. A cloudy pleural fluid has a glucose level of 30 mg/dL (serum glucose level is 100 mg/dL) and a pH of 6.8. a. What condition do these results indicate? b. What additional treatment might the patient receive, based on these results? 3. The following results were obtained on a peritoneal fluid: serum albumin, 2.8 g/dL; fluid albumin, 1.2 g/dL. a. Calculate the SAAG. b. Is this a transudate or an exudate? Why? c. What is the most probable cause of the effusion?

4. Paracentesis is performed on a patient with ascites. The fluid appears turbid and has an elevated WBC count. Additional tests ordered include an absolute granulocyte count, amylase, creatinine, CEA, and CA 125. a. What is the purpose for the absolute granulocyte count? If it is less than 250 cells/µL, what condition is indicated? b. If the amylase level is elevated, what is its significance? State an additional test that might be ordered. c. Explain the significance of an elevated creatinine level. d. What is the purpose of the CEA and CA 125 tests? 5. Describe a situation in which paracentesis might be performed on a patient who does not have ascites. If the RBC count is 300,000/µL, what does this indicate? 6. Microscopic examination of an ascitic fluid shows many cells with nuclear and cytoplasmic irregularities containing psammoma bodies. The CEA test result is normal. What additional test would be helpful?

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CHAPTER

14

Bronchoalveolar Lavage Fluid LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 14-1 State the indications for performing a bronchoalveolar lavage (BAL).

14-4 Describe the appearance of BAL fluid in normal and abnormal conditions.

14-2 Describe the procedure for performing a BAL.

14-5 Discuss the normal and abnormal cellular composition of BAL fluid.

14-3 Explain the procedures for the collecting, handling, and transport of specimens for BAL.

KEY TERMS Bronchoalveolar lavage (BAL)

Bronchoscopy

Flow cytometry

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Clinical Significance Analyzing specimens obtained by bronchoalveolar lavage (BAL) is a method for obtaining cellular, immunologic, and microbiological information from the lining fluid of the lower respiratory tract. BAL is particularly useful in evaluating patients who are immunocompromised or patients with any number of possible diagnoses, including interstitial lung disease (infectious, noninfectious, immunologic, or malignant), airway diseases, suspected alveolar hemorrhage, pulmonary alveolar proteinosis, and Langerhans cell histiocytosis, as well as exposure to dust and asbestos. Often, BAL is used in conjunction with high-resolution computerized tomography (HRCT), medical history, and physical examination to determine the need for a surgical biopsy.

Specimen Collection During bronchoscopy, a fiber-optic bronchoscope is guided into a selected bronchopulmonary segment, usually the right middle or lingular lobe of the lung; however, target areas are better defined using HRCT before the procedure. Optimal targets are areas of alveolar ground-glass opacity, more prominent nodular profusion, or fine reticulation.1 The segment to be lavaged should be recorded on the requisition form. Aliquots of sterile normal saline are instilled into the alveolar spaces through the bronchoscope to mix with the bronchial contents and are aspirated for cellular examination and culture. In this manner, cells and organisms in the alveoli distant to the bronchoscope can be sampled.2 The instillation volume is between 100 and 300 mL of sterile saline in 20- to 50-mL aliquots.2 The first aliquot is discarded; the remaining aliquots are sent either individually or pooled for further analysis.2 The desired fluid volume for analysis is 10 to 20 mL (minimal volume is 5 mL). Optimal sampling retrieves greater than 30% with a typical recovery range of 50% to 70%. Low-volume recovery (less than 25%) caused by fluid retention in the lung may appear in cases of chronic obstructive lung diseases and should be noted on the requisition form.

Specimen Handling and Transport Specimens should be kept at room temperature during transport to the laboratory and processed immediately. When delivery to the laboratory is delayed for longer than 30 minutes, specimens should be transported on ice (4°C).1 Specimens that will not be analyzed immediately should be centrifuged, the cells resuspended in a nutrient-supplemented medium, and refrigerated at 4°C for up to 24 hours.1 Cell counts should be performed within 1 hour, but are stable for up to 3 hours if the fluid is in a nutrient-supplemented medium.2 Specimens can be filtered through loose gauze (50- to 70-µm nylon filter) to remove mucus, phlegm, and dust.

Color and Clarity Diagnostic tests on BAL fluid include a cell count with differential, microbiological studies, and cytopathology. A macroscopic observation is recorded describing the color and clarity

of the specimen. The appearance of the BAL fluid can provide valuable diagnostic information. The color of BAL fluid can be described as: • Colorless • Milky white • Light brown-beige • Gray-beige (typical for smokers) • Red The clarity refers to the transparency or turbidity of the BAL fluid. It is described as: • Clear • Hazy • Cloudy • Turbid A bloody BAL fluid with increasing intensities during the sequential aliquots indicates acute diffuse alveolar hemorrhage, whereas orange-red BAL fluid is the result of an older hemorrhagic syndrome and would be evaluated for intracellular iron content by cytochemistry.2 A milky or light brown-beige–colored BAL fluid indicates an accumulation of phospholipid–protein complexes derived from pulmonary surfactant in the lung alveoli and strongly suggests pulmonary alveolar proteinosis. The BAL fluid should be centrifuged if it looks milky. If typical lipid–protein complexes exist, a creamy layer forms on top of the rest of the BAL after centrifugation.2 The presence of clots should be noted. The fluid volume is measured, and cell counts and differential counts are performed.

Cell Counts Counts for white blood cells (WBCs) and red blood cells (RBCs) are performed on BAL and may be diluted to facilitate counting using a hemocytometer. Cell viability can be determined by adding Trypan blue. Counts also can be performed with certain automated cell counters as designated by the manufacturer. When cell concentration is less than the automated instrument’s linearity specifications, hemocytometry should be used.2 If a hemocytometer is used, WBC counts may be diluted using the BMP LeukoChek system to facilitate counting. A BMP LeukoChek system is available with a 1 to 100 dilution of ammonium oxalate to lyse the RBCs. When the RBCs have lysed and the solution is clear, plate the fluid on a hemocytometer and allow the cells to settle for 5 minutes. Count all cells in the 18 squares on both sides of the hemocytometer and calculate the average of the two sides. Using the following formula, calculate the WBC count:3 WBC/µL =

average number of cells × dilution factor × 10 9 squares

RBC counts may be diluted with isotonic saline using an MLA pipette. Plate the fluid on a hemocytometer and allow it to settle for 5 minutes. Count both sides of the

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Chapter 14 | Bronchoalveolar Lavage Fluid 323 hemocytometer and use the following formula to calculate the RBC/µL3: RBC/µL =

number of cells × dilution factor × 10 Number of squares counted

Cells must be distributed evenly over the hemocytometer surface. For a leukocyte count within the reference range, there should be no more than a 15-cell difference between the highest and lowest total number of cells found among the squares counted. For a RBC count, there should be no more than a 30-cell difference between the highest and lowest total number of cells found among the squares counted. Total cells counted on each side of the counting chamber should agree within 10% of each other. Counts that do not meet this standard should not be reported. Mix the specimen well and repeat the count. If clumps of cells or clots are present, note on the requisition form that the “cell count may be inaccurate due to clumps of cells and/or clots.”

Technical Tip 14-1. BAL body fluids may not be counted on the Sysmex instrumentation because of the varied types of cells present.3

Figure 14–1 Bronchoalveolar lavage: Normal macrophages and lymphocytes (×1000).

A lymphocyte differential count equal to or greater than 25% suggests granulomatous lung disease, whereas a lymphocyte differential count greater than 50% suggests hypersensitivity pneumonitis or nonspecific interstitial pneumonia.1 The ratio of CD4 to CD8 lymphocytes further defines the disease process (Table 14-1). Immunologic analysis is performed by flow cytometry.

Leukocytes

Neutrophils

Evaluating the predominant inflammatory cellular pattern provides valuable information to the clinician in determining a differential diagnosis. The morphological appearance of cells and particles, such as the morphology of macrophages in extrinsic allergic alveolitis and sarcoidosis, or the detection of dust particles in occupational exposure conditions provides diagnostic information.2

Neutrophils are the primary granulocyte seen, with a normal value of less than 3%. They are elevated in patients who are cigarette smokers and in cases of bronchopneumonia, toxin exposure, and diffuse alveolar damage. A neutrophil count equal to or greater than 50% strongly suggests acute lung injury, aspiration pneumonia, or suppurative infection.1

Visit www.fadavis.com for Video 14-1 (Preparation of a cytocentrifuge slide).

Differential slides are prepared by cytocentrifugation using routine procedures with staining (Wright-Giemsa or May Grunwald–Giemsa), and at least 300 cells, but often 500 to 1000 cells, are counted and classified.4 Cells seen in BAL fluid include macrophages, lymphocytes, ratio of CD4+ and CD8+ lymphocytes (CD4/CD8 ratio), neutrophils, eosinophils, ciliated columnar bronchial epithelial cells, and squamous epithelial cells.

Eosinophils Eosinophils, usually less than 1% to 2% of the total cells, are elevated in cases of asthma, drug-induced lung disease, infections (parasitic, mycobacterial, or fungal), hypersensitivity, pneumonitis, and eosinophilic pneumonia. An eosinophil differential

CD4/CD8 T Cell Findings Associated With Disease Conditions

Macrophages

Table 14–1

Macrophages, often containing a variety of phagocytized material, are the cells seen most frequently, in numbers ranging from 56% to 80% (Fig. 14-1). Phagocytized material includes hemosiderin; golden, brown, or black pigment inclusions; or foamy cells. A predominance of macrophages containing smokingrelated inclusions suggests smoking-related interstitial lung disease or pulmonary Langerhans cell histiocytosis.1

CD4/CD8 Ratio

Conditions

Low

Hypersensitivity pneumonitis Silicosis Drug-induced disease HIV infection Tuberculosis Malignancies Sarcoidosis Connective tissue disorder

Lymphocytes Lymphocytes, normally constituting 1% to 15% of the cell population, are increased in cases of interstitial lung disease, drug reactions, pulmonary lymphoma, and nonbacterial infections.

Normal High

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count greater than or equal to 25% diagnoses eosinophilic lung disease.1

Box 14–1

Mast Cells

Bacteria

A mast cell differential count greater than 1% combined with a lymphocyte count greater than 50% and a neutrophil count greater than 3% strongly suggests hypersensitivity pneumonitis.1

Staphylococcus aureus (methicillin-sensitive S. aureus [MSSA]) and methicillin-resistant S. aureus (MRSA)

Erythrocytes

Mycoplasma pneumoniae

The presence of erythrocytes indicates an acute alveolar hemorrhage. Phagocytosed erythrocytes suggest that an alveolar hemorrhage has occurred within the past 48 hours, whereas hemosiderin-laden macrophages indicate an alveolar hemorrhage older than 48 hours.

Legionella pneumophila

Epithelial Cells Ciliated columnar bronchial epithelial cells are more numerous in bronchial wash specimens than in bronchial lavage specimens because of the more vigorous washing technique. In a lavage specimen, the presence of these cells normally ranges from 4% to 17% (Fig. 14-2).

Microbiological Tests Fungal elements, bacteria, and viral inclusions also may be observed in respiratory specimens. Box 14-1 lists the organisms that may be identified. Quantitative or semiquantitative cultures are useful for diagnosis in cases of ventilator-associated pneumonia and can diagnose the infection if the organism is identified. Newer molecular techniques have allowed for a rapid diagnosis of organisms in BAL fluid.5 With the increasing concern about health care–acquired infections and antibiotic-resistant microorganisms, BAL is performed more frequently on patients using ventilators to detect infection and monitor antibiotic therapy. BAL is becoming an important diagnostic test for Pneumocystis jirovecii5 (previously Pneumocystis carinii) in patients who are immunocompromised. With P. jirovecii, characteristic

Microorganisms Isolated From Bronchoalveolar Lavage Fluid

Streptococcus pneumoniae Chlamydia Strongyloides stercoralis Actinomyces Mycobacterium tuberculosis Klebsiella pneumoniae Escherichia coli Viruses Cytomegalovirus (CMV) Herpes simplex virus (HSV) Varicella-zoster virus (VZV) Adenoviruses Measles virus Influenza A and B viruses Respiratory syncytial virus Fungal Aspergillus Pneumocystis jirovecii (formerly P. carinii) Histoplasma capsulatum Blastomyces dermatitidis Coccidioides immitis Cryptococcus neoformans Mucor Candida

amorphous material is seen microscopically under low power, and organisms are visible under high power (Figs. 14-3 and 14-4).6 Cryptococcus neoformans has become a significant opportunistic pathogen in patients with AIDS. A diagnosis of pulmonary cryptococcosis can be made by demonstrating a positive cryptococcal antigen in respiratory specimens exhibiting yeast cells that morphologically resemble C. neoformans. The extent of the cryptococcal infection correlates with the antigen titer.

Cytology Figure 14–2 Bronchoalveolar lavage: Ciliated bronchial epithelial cells; notice the eosinophilic bar (×1000).

Cytological studies include observing sulfur granules (actinomycetes), hemosiderin-laden macrophages, Langerhans cells, cytomegalic cells, fat droplets seen in fat embolism with an Oil

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Chapter 14 | Bronchoalveolar Lavage Fluid 325 Red O stain, and lipid-laden alveolar macrophages using a Sudan III stain. Periodic acid–Schiff staining or Oil Red O staining may be useful in diagnosing cases of pulmonary alveolar proteinosis or aspiration.1 Dust particle inclusions indicate pneumoconioses or asbestos exposure. Specimens are evaluated by a pathologist in cytology whenever malignancy is suspected.

For additional resources please visit www.fadavis.com

References Figure 14–3 Bronchoalveolar lavage: Amorphous material associated with P. carinii when examined under low power (×100).

Figure 14–4 Bronchoalveolar lavage: Characteristic cup-shaped organisms indicating P. carinii (×1000).

1. Meyer, KC, Raghu, G, Baughman, RP, et al: An official American Thoracic Society clinical practice guideline: The clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease. Am J Respir Crit Care Med 185(9):1004–1014. DOI:10.1164/ rccm.201202-0320ST. https://www.atsjournals.org/doi/full/ 10.1164/rccm.201202-0320ST. Published May 1, 2012. Accessed: October 29, 2019. 2. Clinical and Laboratory Standards Institute: Body Fluid Analysis for Cellular Composition; Approved Guideline. CLSI document H56-A. Clinical and Laboratory Standards Institute. Wayne, PA 2006, CLSI. 3. Bronchoalveolar Lavage, BAL Cell Count and Differential. Methodist Hospital: Clinical Laboratory Procedure Manual. Omaha, NE, January 5, 2012. 4. Jacobs, JA, DeBrauwer, EI, et al: Accuracy and precision of quantitative calibrated loops in transfer of bronchoalveolar lavage fluid. J Clin Micro 38(6):2117–2121, 2000. 5. Baldassarri, RJ, Rebecca, Kumar, D, Baldassarri, S, and Cai, G: Diagnosis of Infectious Diseases in the Lower Respiratory Tract. A Cytopathologist’s Perspective. Arch Pathol Lab Med. 2019;143: 683–694; DOI: 10.5858/ arpa.2017-0573-RA. Web site: https:// www.archivesofpathology.org/doi/pdf/10.5858/arpa.2017-0573RA. Accessed September 10, 2019. 6. Linder, J: Bronchoalveolar Lavage. ASCP, Chicago, 1988.

Study Questions 1. All of the following could be diagnosed by collecting and analyzing a BAL except: A. Asbestos-related pulmonary disease (dust particles) B. Interstitial lung disease C. Alveolar hemorrhage D. Meningitis 2. What procedure is used for bronchoalveolar lavage? A. Bronchoscopy B. Arthrocentesis C. Colonoscopy D. Thoracentesis

3. In bronchoalveolar lavage, the targeted area of the lung is: A. Flushed with antibiotics B. Rinsed with sterile saline C. Rinsed with water D. Flushed with a fluorometric stain 4. A BAL fluid that appears orange-red is an indication of which of the following: A. Acute diffuse alveolar hemorrhage B. Alveolar proteinosis C. Patient who is a heavy smoker D. Older hemorrhage syndrome

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5. Cell counts from a BAL fluid must be performed within: A. 1 hour B. 3 hours C. 24 hours D. 36 hours 6. An elevated CD4/CD8 lymphocyte ratio indicates: A. Sarcoidosis B. Tuberculosis C. HIV infection D. Silicosis 7. Immunological study of cells is typically performed by: A. Cytocentrifugation B. Flow cytometry C. Differential count D. Hemocytometer cell count

9. What is an opportunistic pathogen in patients with AIDS that can be recovered in BAL fluid? A. Toxoplasma gondii B. Legionella pneumophila C. Cryptococcus neoformans D. Mycobacterium tuberculosis 10. The stain used in cytology for the diagnosis of lipidladen alveolar macrophages is: A. Periodic acid stain B. Oil Red O stain C. Sudan III stain D. Iron stain

8. The cell in a BAL fluid seen most frequently is: A. Eosinophil B. Neutrophil C. Lymphocyte D. Macrophage

Case Studies and Clinical Situations 1. A patient presented in the emergency department with a cough, shortness of breath, and hemoptysis (coughing up blood). A bronchoscopy with bronchoalveolar lavage was performed. a. What would the specimen possibly look like macroscopically? b. How would an acute diffuse alveolar hemorrhage appear? c. What cellular element would suggest that the alveolar hemorrhage occurred within the past 48 hours? d. How would an older hemorrhagic syndrome appear? e. What intracellular content would be present in the case of an older hemorrhagic syndrome? f. What procedure would be used to identify the intracellular content?

2. A 35-year-old male with a history of HIV infection is admitted to the hospital with fever, dyspnea, and a cough. BAL is performed. a. Which of the following findings from the BAL may be indicative of his condition? 1. Presence of lymphocytes with increased ratio of CD4 and CD8 2. Presence of lymphocytes with decreased ratio of CD4 and CD8 3. Increase in eosinophils 4. >5% of epithelial cell counts b. What opportunistic organism is often seen in HIV/AIDS patients? c. What is a confirmatory test?

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CHAPTER

15

Amniotic Fluid LEARNING OUTCOMES Upon completing this chapter, the reader will be able to: 15-1 State the functions of amniotic fluid.

15-7 Interpret a Liley graph.

15-2 Describe the formation and composition of amniotic fluid.

15-8 Describe the analysis of amniotic fluid for the detection of neural tube disorders.

15-3 Differentiate maternal urine from amniotic fluid.

15-9 Explain the physiological significance of the lecithinsphingomyelin (L/S) ratio.

15-4 State indications for performing an amniocentesis.

15-10 State the relationship of phosphatidyl glycerol to FLM.

15-5 Describe the specimen-handling and processing procedures for testing amniotic fluid for bilirubin, fetal lung maturity (FLM), and cytogenetic analysis.

15-11 Define lamellar bodies and describe their significance to FLM.

15-6 Discuss the principle of the spectrophotometric analysis for evaluation of hemolytic disease of the fetus and newborn.

15-12 Discuss the principle of and sources of error for the L/S ratio, Amniostat-FLM, lamellar body count, and Foam Stability Index for FLM.

KEY TERMS Amniocentesis Amnion Amniotic fluid Cytogenetic analysis Fetal lung maturity (FLM)

Hemolytic disease of the fetus and newborn (HDFN)

Meconium

Lamellar bodies

Polyhydramnios

Lecithin-sphingomyelin ratio (L/S ratio)

Oligohydramnios Respiratory distress syndrome (RDS) Surfactants

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328 Part Three | Other Body Fluids

Introduction

Umbilical cord

Although the testing of amniotic fluid is associated frequently with cytogenetic analysis, the clinical laboratory also performs several significant tests on amniotic fluid. Because amniotic fluid is a product of fetal metabolism, the constituents that are present in the fluid provide information about the metabolic processes taking place during—as well as the progress of—fetal maturation. When conditions arise that adversely affect the fetus, the danger to the fetus must be measured against the ability of the fetus to survive an early delivery. The tests covered in this chapter are used to determine the extent of fetal distress and fetal maturity (Table 15-1). Advances in prenatal screening using a blood specimen from a pregnant woman for fragments of cell-free DNA produced by the placenta to determine chromosomal abnormalities have led to a decrease in testing amniotic fluid obtained by amniocentesis. However, often confirmatory testing is necessary after a positive prenatal blood screening test or to test for conditions not covered by the screening test.

Physiology Function Amniotic fluid is present in the amnion, a membranous sac that surrounds the fetus (Fig. 15-1). The amnion, a membrane composed of cuboidal cells, is metabolically active and is

Table 15–1

Tests for Fetal Well-Being and Maturity

Test

Reference Values at Term14

Bilirubin scan

ΔA450 >0.025

Alphafetoprotein Lecithinsphingomyelin ratio Amniostat-FLM

75 Osm/kg 250 mg/d Iron supplements containing vitamin C Failure to wait the specified time after sample is applied to add the developer reagent

been converted to porphyrins. As hemoglobin progresses through the intestinal tract, bacterial actions degrade it to porphyrin that the gFOBT cannot detect, thereby making the HemoQuant test more sensitive to upper GI bleeding. Falsenegative results from upper GI bleeding can be seen with the gFOBT. Additionally, the porphyrin-based test is not affected by the presence of reducing or oxidizing substances or the water content of the fecal specimen. False-positive results can occur with the porphyrin-based test when nonhuman sources of blood (red meat) are present; therefore, patients should be instructed to avoid red meat for 3 days before the test. This test is performed primarily by reference laboratories and not performed routinely in hospital laboratories.

Quantitative Fecal Fat Testing Quantitative fecal fat analysis is used as a confirmatory test for steatorrhea. As discussed, quantitative fecal analysis requires the collection of at least a 3-day specimen. The patient must maintain a regulated intake of fat (50 to 150 g/d) for 2 to 3 days before and during the stool collection period. The specimen is collected in a large, preweighed container. Before analysis, the specimen is weighed and homogenized. Refrigerating the specimen prevents any bacterial degradation. The method used routinely for fecal fat measurement is the Van de Kamer titration, although gravimetric, near-infrared reflectance spectroscopy (NIRS), and nuclear magnetic resonance spectroscopy methods are available.12 In the titration method, fecal lipids are converted to fatty acids and titrated to a neutral endpoint with sodium hydroxide. Approximately 80% of the total fat content is measured by titration, whereas the gravimetric method measures all fecal fat. A drawback of the titration/gravimetric method is that it is time consuming and uses solvents that are corrosive and flammable. A rapid (5 minutes) and safe procedure for analyzing quantitative fecal fat is the hydrogen nuclear magnetic resonance spectroscopy (1H NMR) method. In this method, the homogenized specimen is microwaved-dried and analyzed. The results correlate well with the gravimetric method, and it is widely used in reference laboratories.18

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348 Part Three | Other Body Fluids The fat content is reported as grams of fat or the coefficient of fat retention per 24 hours. Reference values based on a 100 g/d intake are 1 to 6 g/d, or a coefficient of fat retention of at least 95%. The coefficient of fat retention is calculated as follows: (dietary fat – fecal fat) × 100 (dietary fat)

Although the Van de Kamer titration is the gold standard for fecal fat, the acid steatocrit (see Procedure 16-6) is a rapid test to estimate the amount of fat excretion.19 It is similar to the

PROCEDURE 16-6 Acid Steatocrit 1. Dilute 0.5 g of feces from a spot collection 1 to 4 with deionized water. 2. Vortex for 2 minutes to homogenize the specimen. 3. Add a volume of 5 N perchloric acid equal to 20% of the homogenate, and then vortex the mixture for 30 seconds. Confirm the pH to be 4.5 2+ to 4+ Absent or present Absent or present

Appearance

Thin, homogeneous, white-togray vaginal discharge

White, curd-like vaginal discharge

pH WBCs Lactobacilli Clue cells Other cells

>4.5 Rare or absent Rare or absent >20%

3.8–4.5 3+ to 4+ Present Absent Large clumps of epithelial cells

Other organism

Increase in small curved bacilli, coccobacilli, and pleomorphic bacilli Positive

Budding yeast cells and pseudohyphae

Trichomonas frequently associated with other organisms

Negative

Positive

Confirmatory tests: DNA probe, proline amino peptidase, OSOM BVBLUE Rapid Test

Confirmatory tests: DNA probe, OSOM BVBLUE Rapid Test

Confirmatory tests: DNA probe or culture, OSOM Trichomonas Rapid Test

Amine (whiff) test Other tests

Desquamative Inflammatory Vaginitis

Atrophic Vaginitis

Excessive purulent vaginal discharge, vaginal erythema

Excessive purulent vaginal discharge, vaginal erythema

>4.5 3+ to 4+ Absent or reduced

>4.5 3+ to 4+ Decreased

Occasional parabasal or basal cells >1+ RBCs 2+ gram-positive cocci

Occasional parabasal or basal cells >1+ RBCs Increased grampositive cocci and gram-negative rods; decreased large rods Negative

Negative

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Chapter 17 | Vaginal Secretions In addition to evaluating vaginal secretions for infections, tests are performed on vaginal secretions to detect the placental α1-microglobulin (PAMG-1) protein, insulin-like growth factor binding protein-1 (IGFBP-1) to diagnose ruptured fetal membranes, or fetal fibronectin enzyme to assess the risk of preterm delivery. The fern test (see Chapter 15) is used to identify amniotic fluid that may be present when the amniotic sac has ruptured.2

Specimen Collection and Handling The health-care provider collects vaginal secretions during a pelvic examination. Detailed instructions and the specific manufacturer’s collection and transport devices must be provided and are specific to the organism sought. Correct specimen handling and timely transport to the laboratory are important for optimal detection of the responsible pathogen. A speculum moistened with warm water is used to visualize the vaginal fornices. Lubricants may contain antibacterial agents and must not be used. The specimen is collected by swabbing the vaginal walls and vaginal pool to collect epithelial cells along with the vaginal secretions using one or more sterile, polyester-tipped swabs on a plastic shaft or swabs specifically designated by the manufacturer.3 Cotton swabs should not be used because cotton is toxic to Neisseria gonorrhoeae, the wood in a wooden shaft may be toxic to Chlamydia trachomatis, and calcium alginate can inactivate herpes simplex virus (HSV) for viral cultures.4 The health-care provider performs a gross examination of the vaginal secretions and then places the swab in a tube containing 0.5 to 1.0 mL of sterile physiological saline. The tube is sealed for transport to the laboratory, where the specimen is processed for microscopic analysis. The swab should be twirled in the saline vigorously to dislodge particulates from the swab. Failure to dislodge particles may lead to erroneous results. Specimens should be tested with pH paper before being placed in saline.2 An alternative method of specimen preparation is to dilute a sample of vaginal discharge in one to two drops of normal saline solution directly on a microscope slide. Then a second sample is placed in 10% KOH solution in the same manner. Cover slips are placed over both slides for microscopic examination.5 Properly labeled specimens should be placed in a biohazard bag with the requisition form and transported to the laboratory as soon as possible. The requisition form must include the patient’s name and unique identifier, as well as a patient medical history that should include menstrual status; use of vaginal creams, lubricants, and douches; and recent exposure to sexually transmitted diseases.3 Specimens should be analyzed immediately, but if a delay in transport or analysis is necessary, specimen handling is based on the suspected pathogen. Specimens must be kept at room temperature to preserve the motility of Trichomonas vaginalis and the recovery of N. gonorrhoeae, whereas specimens for C. trachomatis and HSV must be refrigerated to prevent overgrowth of normal flora.4 Specimens for T. vaginalis should be examined within 2 hours of collection.2

357

Technical Tip 17-2. Keep specimens for suspected T. vaginalis at room temperature and examine within 2 hours of collection to visualize movement of the flagella or undulating membrane on a wet prep. When not moving, Trichomonas may resemble a white blood cell (WBC), transitional, or renal tubular epithelial (RTE) cell.

Color and Appearance Normal vaginal fluid appears white with a flocculent discharge. Microscopically, normal vaginal flora includes a predominance of large, rod-shaped, gram-positive lactobacilli and squamous epithelial cells. WBCs may be present, and red blood cells (RBCs) will be present if the patient is menstruating3 (Table 17-2). Abnormal vaginal secretions may appear as an increased thin, homogeneous, white-to-gray discharge often seen in BV; as a white “cottage cheese–like” discharge particular for Candida infections; or as an increased yellow-green, frothy, adherent discharge associated with T. vaginalis.3 C. trachomatis may present with a yellow, opaque cervical discharge.6

Diagnostic Tests pH The health-care provider can perform a vaginal pH test when performing a pelvic examination. The test should be performed before placing the swab into saline or KOH solutions. Commercial pH test paper with a narrow pH range is recommended to evaluate pH values in the 4.5 range more accurately. The test paper is placed in the pooled vaginal secretion, and the color change is compared with a chart with corresponding pH values. Factors that can interfere with the pH test include contamination of the vaginal secretions with cervical mucus, semen, and blood.6 The pH test helps to differentiate the causes of vaginitis, as shown in Table 17-1. The vaginal pH is usually about 4.5 in women with vulvovaginal candidiasis but is above 4.5 in women with BV, trichomoniasis, desquamative inflammatory vaginitis (DIV), and atrophic vaginitis.1,2,4 Table 17–2

Normal Findings in Vaginal Secretions

Appearance pH Amine (whiff) test WBCs Lactobacilli Clue cells Other cells Other organisms

White, flocculent discharge 3.8–4.2 Negative Rare to 2+ Predominant Absent Absent (except RBCs during menses) Other lactobacilli subgroups, occasional yeast

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358 Part Three | Other Body Fluids As described previously, normal vaginal flora includes a predominance of the bacteria lactobacilli, which produce the end-product lactic acid from glycogen metabolism. Lactic acid provides an acidic vaginal environment with a pH value between 3.8 and 4.5. This acidity suppresses the overgrowth of infectious organisms such as Mobiluncus, Prevotella, and Gardnerella vaginalis, and therefore maintains the balance of normal vaginal bacteria flora.7 Some lactobacilli subgroups also produce hydrogen peroxide, which is toxic to pathogens and helps keep the vaginal pH acidic to provide protection from urogenital infections. BV has been associated with the absence of hydrogen peroxide–producing lactobacilli.3 Estrogen production also is necessary to preserve an acidic vaginal environment.6

Microscopic Procedures Usually vaginal infections are diagnosed from microscopic examination. Saline wet mounts and KOH mounts are the initial screening tests, and the Gram stain is used as a confirmatory examination for yeast or BV.2 Slides are prepared from the saline specimen solution that was made from the vaginal swab immediately after collection. Three clean glass slides (if a Gram stain is requested) are labeled with the patient’s name and a unique identifier. A drop of specimen is placed on each slide using a disposable transfer pipette. An alternative method is to press the swab against the slide and then roll the swab over the slide. The slide for Gram stain is allowed to dry and then is heat-fixed for the Gram stain procedure and examination performed in the microbiology section of the laboratory. For wet mount examinations, cells and organisms are quantified per high power field (hpf) (40×); for Gram stains, cells and organisms are reported per oil immersion field (100×).

Technical Tip 17-3. The Seattle STD/HIV Prevention Training Center has developed the YouTube video titled Examination of Vaginal Wet Preps, an excellent online overview of performing the microscopic examination of vaginal secretion. It is available at https://www.youtube.com/watch?v=8dgeOPGx6YI.8 Wet Mount Examination For the saline wet mount examination, a cover slip is placed on the specimen carefully to exclude air bubbles. The slide is examined microscopically using the low power (10×) and high

dry power (40×) objective with a bright-field microscope. Using the low power objective (100× magnification), the slide is scanned for an even distribution of cellular components, types and numbers of epithelial cells, clumping of epithelial cells, and the presence of budding yeast or pseudohyphae. Then the slide is examined using the high power objective (400× magnification), and the organisms and cells are counted and reported per hpf using the criteria in Table 17-3. Typical constituents found in vaginal fluid wet mounts include squamous epithelial cells, WBCs, RBCs, clue cells, parabasal cells, basal cells, bacteria, motile T. vaginalis, yeast, and hyphae/ pseudohyphae. Intravaginal medications might leave oil droplets that can interfere with the interpretation of wet mounts. In this case, a Gram stain is useful to detect yeast or BV.2 Squamous Epithelial Cells Squamous epithelial cells measure 25 to 70 µm in diameter and exhibit a polygonal “flagstone” appearance. They contain a prominent nucleus that is centrally located and about the size of a RBC, as well as a large amount of irregular cytoplasm, lacking granularity, with distinct cell margins (Fig. 17-1). These large, flat cells originate from the linings of the vagina and female urethra and are present in significant numbers in the vaginal secretions of a healthy female. Clumps of epithelial cells, as observed in Figure 17-2, are an indication of the presence of increased numbers of yeast.3 Clue Cells Clue cells are an abnormal variation of the squamous epithelial cell and are distinguished by coccobacillus bacteria attached in clusters on the cell surface, spreading past the edges of the cell and making the border appear indistinct or stippled. Bacteria should cover at least 75% of the epithelial cell. This gives the cell a granular, irregular appearance sometimes described as “shaggy.” Clue cells are diagnostic of BV caused by G. vaginalis (Fig. 17-3). The presence of clue cells also can be found in urine sediment and should be confirmed by the procedures already described. White Blood Cells WBCs measure 14 to 16 µm in diameter and exhibit a granular cytoplasm. Often they are described as polymorphonuclear (PMN) WBCs because of their characteristic multilobed nucleus (Fig. 17-4). Normally, WBCs are present in rare to scanty numbers in vaginal secretions. More than 3+ WBCs in vaginal

PROCEDURE 17-1 pH Test 1. Using a circular motion, gently apply the vaginal secretion over the surface of the pH test paper. 2. Immediately observe the color reaction on the paper, and compare it to a color comparison chart to determine the pH of the sample. 3. Record the results.

Table 17–3 Rare 1+ 2+ 3+ 4+

Quantitation Scheme for Microscopic Examinations2

Fewer than 10 organisms or cells/slide Fewer than 1 organism or cell/hpf 1–5 organisms or cells/hpf 6–30 organisms or cells/hpf >30 organisms or cells/hpf

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A Figure 17–1 Squamous epithelial cells identifiable under low power (×100).

B

Figure 17–2 Clump of squamous epithelial cells (×400).

Figure 17–4 A. White blood cells. Notice the multilobed nucleoli (×400). B. Stained smear.

Figure 17–5 Normal red blood cells (×400). Figure 17–3 Clue cells (×400).

secretions suggest vaginal candidiasis; atrophic vaginitis; or infections with Trichomonas, Chlamydia, N. gonorrhoeae, or HSV.3 Red Blood Cells RBCs appear as smooth, nonnucleated, biconcave disks measuring approximately 7 to 8 µm in diameter (Fig. 17-5). RBCs

can be somewhat distorted in vaginal specimens. Usually RBCs are not seen in vaginal secretions, but they might be present during menstruation or due to a desquamative inflammatory process.2 RBCs can be confused with yeast cells and are distinguished from yeast cells by KOH, which will lyse the RBCs but allow the yeast cells to remain intact.

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360 Part Three | Other Body Fluids Parabasal Cells Parabasal cells are round to oval-shaped and measure 16 to 40 µm in diameter. The ratio of nucleus to cytoplasm is 1:1 to 1:2, with marked basophilic granulation or amorphic basophilic structures (“blue blobs”) in the surrounding cytoplasm. They are located in the luminal squamous epithelium of the vaginal mucosa. Although it is rare to find parabasal cells in vaginal secretions, less mature cells may be found if the patient is menstruating or if the patient is postmenopausal.2 Increased numbers of parabasal cells, if present with large numbers of WBCs, can indicate DIV2 (Fig. 17-6). Basal Cells Basal cells are located deep in the basal layer of the vaginal stratified epithelium. These cells are round and measure 10 to 16 µm in diameter. They have a ratio of nucleus to cytoplasm of 1:2. Basal cells are distinguished from WBCs, which are similar in size, by their round rather than lobed nucleus. They are not normally seen in vaginal fluid and, if present and accompanied by large numbers of WBCs and altered vaginal flora, can suggest DIV.2

suggests an alteration in the normal flora. Often the lactobacilli are replaced by increased numbers of any of the following: • Mobiluncus spp. (thin, curved, gram-negative, motile rods) • Prevotella spp. • Porphyromonas spp. • Bacteroides spp. (anaerobic gram-negative rods) • Gardnerella vaginalis (short, gram-variable coccobacilli) • Peptostreptococcus spp. (gram-positive cocci) • Enterococcus spp. (gram-negative cocci) • Mycoplasma hominis • Ureaplasma urealyticum Trichomonas vaginalis T. vaginalis is an atrial flagellated protozoan that can cause vaginal inflammation and infection in women. The organism is oval shaped, measures 5 to 18 µm in diameter, and has four anterior flagella and an undulating membrane that extends half the length of the body.9 An axostyle bisects the trophozoite longitudinally

Bacteria The vagina is a nonsterile environment with complex endogenous bacterial flora that vary with the age and hormonal status of the patient. Lactobacillus spp. normally comprise the largest portion of vaginal bacteria.7 They appear as large, gram-positive, nonmotile rods and produce lactic acid, which maintains the vaginal pH at 3.8 to 4.5 (Fig. 17-7 A and B). Hydrogen peroxide, produced by lactobacilli subgroups, also can help to suppress the overgrowth of other organisms. Other bacteria commonly present include anaerobic streptococci, diphtheroids, coagulasenegative staphylococci, and α-hemolytic streptococci. When conditions are present that cause an imbalance in the normal flora, vaginitis can occur. Absent or decreased numbers of lactobacilli relative to the number of squamous epithelial cells

A

B

Figure 17–6 Parabasal cell surrounded by epithelial cells (×400).

Figure 17–7 Bacteria. A. Large rods characteristic of Lactobacilli, the predominant bacteria in normal vaginal secretions (×400). B. Bacteria with white blood cells (×400).

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Chapter 17 | Vaginal Secretions and protrudes from the posterior end, which enables the organism to attach to the vaginal mucosal and cause tissue damage (Fig. 17-8). The “jerky” motion of the flagella and undulating membrane characteristic of T. vaginalis can be observed in a wet mount. Care must be taken not to confuse T. vaginalis with sperm, which have only a single tail, a much smaller head (approximately one-half the diameter of a RBC), and no axostyle. In addition, nonmotile trichomonads can be mistaken for WBCs (Fig. 17-9). T. vaginalis organisms quickly lose their viability after collection. Specimens must be examined as soon as possible or, if necessary, maintained at room temperature for a maximum of 2 hours before preparing the wet mount to observe the organism’s motility. Trichomonas also can be seen in a urinary microscopic sample but cannot be reported unless motility is observed, either in movement across the slide or just in the tail. A dead trichomonad tends to appear oval and slightly larger than a WBC. Yeast Cells Candida albicans and non-Candida spp. cause most fungal infections, but an occasional yeast in vaginal secretions is considered part of the normal flora. Yeast cells appear on a wet mount as both budding yeast cells (blastospores) (Fig. 17-10) or as hyphae, which are long filaments that grow and form a mycelium (Fig. 17-11). Pseudohyphae—multiple buds that do not detach and form chains—also can be seen. It can be

PROCEDURE 17-2 Saline Wet Mount2 1. Prepare a clean glass slide labeled with the patient’s name and unique identifier. 2. Place one drop of vaginal specimen on the slide. 3. Cover the slide with a cover slip, removing any air bubbles. 4. Examine the slide with the 10× objective for epithelial cells and any budding yeast cells or pseudohyphae. 5. Examine the slide with the 40× objective, and quantify organisms and cells per hpf. 6. Record the results.

difficult to distinguish yeast cells from RBCs on a wet mount because both measure about 7 to 8 µm in diameter; however, differentiation can be made using the KOH test. Yeast cells stain gram positive.

KOH Preparation and Amine Test The KOH slide is prepared and the amine (whiff) test is performed by placing a drop of the saline specimen prepared from the collection swab onto a clean slide that is labeled properly

N U

C

P

Figure 17–8 Trichomonas vaginalis. (From Leventhal and Cheadle, Medical Parasitology, 6th edition, 2012, F.A. Davis Company, Philadelphia, with permission.)

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A

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Part Three | Other Body Fluids and adding one drop of 10% KOH solution. The slide is checked immediately for a “fishy” amine odor. The result is reported as positive (presence of fishy odor) or negative (absence of fishy odor). Increased numbers of anaerobic bacteria in the vagina produce polyamines that are released into the vaginal fluid. The odor results from trimethylamine, a volatilization product of amines, when the KOH is added. Because volatile amines are not present in normal vaginal secretion, a positive test result suggests BV caused by increased numbers of G. vaginalis in conjunction with Mobiluncus spp. and with T. vaginalis. After the amine test has been performed, place a cover slip over the specimen, taking care to exclude air bubbles. Allow the KOH preparation to rest for 5 minutes to dissolve epithelial and blood cells. Heat may be applied to speed the dissolving process. One drop of 10% glycerin may be added after

Figure 17–9 Trichomonas vaginalis in wet mount. (Courtesy of the U.S. Department of Health and Human Services, via the CDC.)

PROCEDURE 17-3 KOH Preparation2 1. Prepare a clean glass slide labeled with the patient’s name and unique identifier. 2. Place one drop of vaginal specimen on the slide. 3. Add one drop of 10% KOH to the slide. 4. Allow the KOH slide preparation to rest for up to 5 minutes to allow cellular tissue and other debris to dissolve. Gentle heating may speed the dissolving process. 5. Cover the specimen with a cover slip, removing any air bubbles. 6. Examine the slide under the 10× objective for overall assessment and for yeast pseudohyphae. 7. Switch to the 40× objective to examine for budding yeast cells (smaller blastopore blastospore). 8. Record the results.

Figure 17–10 Budding yeast cells (×400).

PROCEDURE 17-4 Amine (Whiff) Test

Figure 17–11 Yeast cells showing mycelial forms (×400).

1. Apply one drop of the saline vaginal fluid suspension to the surface of a clean glass slide. 2. Add one drop of 10% KOH directly to the vaginal sample. 3. Holding the slide in one hand, gently fan above the surface of the slide with the other hand and assess for the presence of a fishy amine odor. 4. Report as positive or negative. Positive: The presence of a fishy odor after adding KOH. Negative: The absence of a fishy odor after adding KOH.

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Chapter 17 | Vaginal Secretions the KOH to prevent specimen deterioration.2 The slide is examined under low power (100× magnification) for the presence of yeast pseudohyphae and under high dry power (400× magnification) to identify smaller blastospores (yeast cells).2

Other Diagnostic Tests Although the wet mount and KOH slide examinations and the amine test are used commonly to diagnose BV, other tests may be required for a confirmatory diagnosis. These include Gram stain, specimen culture, DNA probe testing, and point-of-care test kits.

Gram Stain The Gram stain is considered the gold standard in identifying the causative organisms for BV. It also provides a permanent record of the patient specimen. A scored Gram stain system is a weighted combination of the following morphotypes: Lactobacillus acidophilus (large gram-positive rods), G. vaginalis and Bacteroides spp. (small gram-variable or gram-negative rods), and Mobiluncus spp. (curved gram-variable rods). The types of bacterial morphophytes are evaluated and scored. For example, Lactobacillus morphophytes are the predominant bacteria in normal vaginal flora; therefore, if 4+ Lactobacillus morphophytes are present on Gram stain, and Gardnerella and Bacteroides spp. morphophytes and curved gram-variable rods are absent, the score is 0. As indicated in Table 17-4, a Nugent score of 0 to 3 is considered normal vaginal flora, whereas a score of 4 to 6 is reported as intermediate, and a score of 7 or more is diagnostic of BV.

Culture Culture, using various types of media, is the gold standard test for detecting yeast and Trichomonas; however, it is more time consuming and requires up to 2 days for a result. Culture for G. vaginalis is not diagnostic for BV because it is part of the normal flora in 50% of healthy women.

Table 17–4

Nugent Gram Stain Criteria to Diagnose Bacterial Vaginosis

Lactobacillus

Gardnerella and Bacteroides spp. Morphophytes

4+ 3+ 2+ 1+ 0

0 1+ 2+ 3+ 4+

Curved GramVariable Rods 0 1+ or 2+ 3+ or 4+

Points 0 1 2 3 4

Note: Points are added according to the morphotypes seen. Add the points for all three columns for a final sum. A score of 7 or higher indicates bacterial vaginosis.6

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Special media called Diamond medium is required for determining the presence of T. vaginalis. A commercial transport and culture pouch system for the detection of Trichomonas is now available (InPouch TV, Biomed Diagnostics, White City, OR). The specimen must be inoculated into the pouch within 30 minutes of collection and then is incubated for 5 days at 37°C in a CO2 atmosphere. The pouch is examined microscopically daily for motile trichomonads.

DNA Testing DNA hybridization probe methods have been developed to identify the specific causative pathogen for vaginitis. A DNA probe testing system, Affirm VPIII (Becton, Dickinson, Franklin Lakes, NJ), is available for differential diagnosis of G. vaginalis, Candida spp., and T. vaginalis. It is easy to perform, and results are available in 1 hour, with a sensitivity of 95%. This test is significantly more sensitive than wet mount microscopy and is less subjective to personal bias compared with traditional microscopic tests. New molecular assays are being developed continually for the diagnosis of vaginitis. Trichomonas also can be detected by DNA probes amplified by polymerase chain reaction (PCR). Enzymes are added to the specimen that amplifies specific regions of the DNA of T. vaginalis by PCR. Then the number of DNA fragments is calculated. This is the most accurate diagnostic method, and it has the advantage of detecting nonviable organisms.10

Point-of-Care Tests Various rapid diagnostic tests are available to quickly screen for the causative agents of vaginitis, and they provide a higher sensitivity and specificity for the organism sought. For example, proline aminopeptidase activity in vaginal secretions can be detected by rapid antigen tests to identify G. vaginalis.6 The OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, MA) is an immunochromatographic strip test that detects T. vaginalis antigen from vaginal swabs in 10 minutes. The test is performed by placing the vaginal swab in the kit’s sample buffer. The Trichomonas proteins are solubilized into the buffer. The test stick coated with antiTrichomonas antibodies is placed into the sample mixture. The solution migrates up the stick and, if Trichomonas antigens are present, they will react with the antibodies on the stick. A visible blue line and a red internal control line indicate a positive result. The OSOM BVBLUE test (Genzyme Diagnostics, Cambridge, MA) detects vaginal fluid sialidase, an enzyme produced by the bacterial pathogens associated with BV, such as Gardnerella, Bacteroides, Prevotella, and Mobiluncus. The test takes 1 minute to perform and is read by examining the change in the color of the solution: blue or green is positive, yellow is negative. Commercial tests to measure an elevated vaginal pH (VSSense Pro Swab) and the presence of amines (FemExam pH and Amines TestCard, Litmus Concepts, Santa Clara, CA) use pH indicators and an amine test system that is read visually to identify BV and Trichomonas.

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Vaginal Disorders

Trichomoniasis

Bacterial Vaginosis

Trichomoniasis is caused by the parasitic protozoon T. vaginalis. The infection is transmitted by sexual intercourse, and it causes vaginitis in women and, sometimes, urethritis in men. The infection is classified as an STI, frequently occurs with infections of gonorrhea or Chlamydia, and has been associated with enhanced transmission rates of HIV.5,7,11 In pregnant women, a correlation has been found between a T. vaginalis infection and low birth weight, premature rupture of membranes, and preterm delivery.11 Trichomoniasis is characterized by a green-to-yellow frothy vaginal discharge, malodor, pruritus, irritation, dysuria, dyspareunia, and vaginal mucosa erythema, although some patients are asymptomatic. Patients may present with a “strawberry cervix” because of punctuate hemorrhages.11 Usually males are asymptomatic or may present with urethritis. Trichomoniasis can be diagnosed with the wet mount and microscopic examinations, visualizing the motile trichomonads in a fresh specimen; however, this method has a sensitivity of only 60% to 70%.5 The test must be performed within 2 hours of specimen collection to preserve the viability of the organism. WBCs and lactobacilli bacteria also are present with a T. vaginalis infection. The vaginal pH is greater than 4.5, and the amine test from the KOH preparation will be positive. If the wet mount is negative for motile trichomonads, a culture using Diamond medium or the commercially available pouch system (InPouch TV, Biomed Diagnostics, White City, OR) is recommended for detection of T. vaginalis. The DNA probe test system, Affirm VPIII (Becton, Dickinson, Franklin Lakes, NJ), and the point-of-care rapid antigen detection test, OSOM Trichomonas Rapid Test (Genzyme Diagnostics, Cambridge, MA), are available with increased sensitivity and specificity for T. vaginalis. The recommended treatment for trichomoniasis is metronidazole. For patients who develop an allergy to metronidazole or for whom treatment is not effective, a newer drug, tinidazole, is available. All sexual partners of patients, even if asymptomatic, should be treated to avoid reinfection.

BV is the most common cause of vaginitis, affecting 40% to 50% of women of childbearing age.1 It occurs when there is an imbalance in the ratio of normal vaginal bacterial flora. The predominant organism in the vaginal flora is lactobacilli, which produce lactic acid that maintains the vaginal pH between 3.8 and 4.5. Additionally, certain subsets of lactobacilli produce hydrogen peroxide, which prevents the overgrowth of normal vaginal flora. As the vaginal pH becomes alkaline, lactobacilli are replaced by an overgrowth of G. vaginalis, Mobiluncus spp., Prevotella spp., Porphyromonas, Peptostreptococcus, Mycoplasma hominis, and Ureaplasma spp. The malodor and increased abnormal vaginal discharge result from this mix of organisms and is more apparent after intercourse.11 BV is associated with new or multiple sex partners, frequent douching, use of intrauterine devices, pregnancy, and a lack of the protective lactobacilli.1 There is evidence that BV is a risk factor for the premature rupture of membranes and preterm labor for pregnant women. Additional complications include pelvic inflammatory disease and endometritis, as well as an increased risk for acquiring some sexually transmitted infections (STIs) such as HIV, N. gonorrhoeae, C. trachomatis, and HSV-2.1,5 BV is diagnosed by examining the vaginal secretions for abnormal appearance or quantity, performing the pH and amine tests, and microscopically observing the wet mount for the presence of clue cells and the absence of WBCs and lactobacilli morphotypes. According to Amsel’s Diagnostic Criteria, three of the following four features must be present for the diagnosis of BV: 1. Thin, white, homogeneous discharge 2. Vaginal fluid pH greater than 4.5 3. A positive amine (whiff) test 4. Presence of clue cells on microscopic examination5 The Gram stain is the gold standard for determining the ratio of each bacterial morphotype and offers a definitive diagnosis. Other tests used to diagnose BV include the DNA hybridization probe test; Affirm VPIII (Becton, Dickinson), which detects G. vaginalis; a proline aminopeptidase test (Pip Activity TestCard, Quidel, San Diego, CA); and the OSOM BVBLUE test (Genzyme Diagnostics, Cambridge, MA). Treatment is recommended for women to relieve vaginal symptoms and reduce the risk of infection. In addition, benefits of treatment include reducing the risk of acquiring an STI and reducing the risks associated with pregnancy. The recommended treatments are metronidazole (Flagyl), metronidazole gel, or clindamycin cream.5

Technical Tip 17-4. The detection of clue cells in the wet mount examination of vaginal discharge is the most useful single indicator for the diagnosis of BV.

Candidiasis Vulvovaginal candidiasis is caused by an infection with the yeast Candida. It is a common cause of vaginitis, and nearly 75% of adult women have at least one yeast infection in their lifetime.11 Most yeast infections are caused by C. albicans, but other nonalbican species, such as C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei, have been isolated as the cause. Candida is part of the normal vaginal flora, but an infection occurs when there is a change in the vaginal environment that permits the overgrowth of Candida and symptoms of the infection to occur. Conditions that can cause a change in the vaginal environment include the use of broad-spectrum antibiotics, oral contraceptives, or estrogen replacement therapy; hormonal changes that occur with pregnancy; ovulation; and menopause. Increased infection rates occur in patients who are immunocompromised or those with conditions such as diabetes mellitus, iron deficiency, and HIV infection. The infection is found

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Chapter 17 | Vaginal Secretions predominantly in women of childbearing age, who are producing large amounts of estrogen. Estrogen causes the vagina to mature and produce glycogen, which facilitates the growth and adherence of C. albicans.11 Typical clinical symptoms of vulvovaginal candidiasis include genital itching or burning, dyspareunia; dysuria; and the presence of an abnormal thick, white, curd-like vaginal discharge. The pH of the vaginal fluid remains normal (3.8 to 4.5), and the amine test is negative. In vulvovaginal candidiasis, the microscopic examination of the saline and KOH wet prep and Gram stain will reveal budding yeast and pseudohyphae forms, large numbers of WBCs, lactobacilli, and large clumps of epithelial cells. A culture and DNA hybridization probe (Affirm VPIII Microbial Identification System; Becton, Dickinson, Franklin Lakes, NJ) analysis can be performed to confirm the clinical diagnosis and to identify the species of Candida. Yeast infections are treated with over-the-counter or prescription azole antifungal agents. They may be intravaginal, suppository, or oral agents, and the regimen depends on the medication. Over-the-counter intravaginal medications include butoconazole, clotrimazole, tioconazole, and miconazole. For patients with recurrent infections (four or more episodes per year), a prescription medication using a longer treatment regimen with either oral fluconazole or intravaginal butoconazole, nystatin, and terconazole may be more effective.5 Vulvovaginal candidiasis is not acquired through sexual intercourse, so treatment of sexual partners is not indicated.

Desquamative Inflammatory Vaginitis DIV is a syndrome characterized by profuse purulent vaginal discharge, vaginal erythema, and dyspareunia.3 There is a heterogeneous group of causes of DIV; however, β-hemolytic gram-positive streptococci can be cultured from most patients.3 The syndrome also can occur secondary to atrophic vaginitis in postmenopausal women as a result of decreased estrogen. The vaginal secretion pH is greater than 4.5, and the amine test is negative. Wet mount and Gram stain microscopic examination of the vaginal secretions reveal large numbers of WBCs, RBCs, occasional parabasal and basal cells, squamous epithelial cells, and reduced or absent lactobacilli that have been replaced by gram-positive cocci (refer back to Table 17-1). DIV is treated with 2% clindamycin.3 Hormone replacement therapy is effective for patients with DIV secondary to atrophic vaginitis.

Atrophic Vaginitis Atrophic vaginitis is a syndrome found in postmenopausal women. This syndrome is caused by thinning of the vaginal mucosa because of reduced production of both estrogen and glycogen. As a result, the vaginal environment changes, and the balance of normal flora is altered. Clinical symptoms include vaginal dryness and soreness, dyspareunia, inflamed vaginal mucosa, and purulent discharge. The vaginal secretion pH is greater than 4.5, and the amine test is negative. Microscopic evaluation is similar to that for DIV and includes large numbers of WBCs and the presence of RBCs, occasional

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parabasal and basal cells, squamous epithelial cells, and decreased numbers of lactobacilli that have been replaced by gram-positive cocci and gram-negative rods.3 Treatment of atrophic vaginitis is estrogen replacement. Topical vaginal ointments are used initially; however, for frequent, recurrent episodes of atrophic vaginitis, oral or transcutaneous (patch) modes are more effective.

Additional Procedures for Vaginal Secretion Associated With Pregnancy As noted previously in this chapter, complications from vaginitis syndromes can include premature rupture of fetal membranes and a high risk of preterm labor. The following tests are used to evaluate these conditions: • Fetal fibronectin (fFN) enzyme test • AmniSure (AmniSure International) test to detect the amniotic fluid protein PAMG-1 • Actim PROM (Cooper Surgical Medical Devices) test to detect the amniotic fluid protein IGFBP-1, also referred to as placental protein 12 (PP12) • ROM Plus (Clinical Innovations) to detect the amniotic fluid proteins alpha-fetoprotein (AFP) and IGFBP-1 The fern test, described in Chapter 15, is another test that determines the presence of amniotic fluid in vaginal secretions. Patient history, amniotic pooling in the fornix of the vagina, a vaginal pH greater than 7.0, and a positive fern test are strong indicators of amniotic sac rupture.

Fetal Fibronectin Test Preterm delivery, defined as delivery before the completion of 37 weeks’ gestation, is the leading cause of neonatal mortality and morbidity in the United States.12 fFN is an adhesive glycoprotein in the extracellular matrix at the maternal and fetal interface within the uterus. It is elevated during the first 24 weeks of pregnancy but then diminishes. The presence of fFN in vaginal secretions between 24 and 34 weeks’ gestation is associated with preterm delivery. The test can be used by health-care providers as a means to better manage patient care, and it can be performed routinely as part of a prenatal visit for asymptomatic women between 22 and 30 weeks’ gestation or in symptomatic pregnant women between 24 and 34 weeks. Symptoms of preterm delivery include a change in vaginal secretions, vaginal bleeding, uterine contractions, abdominal or back discomfort, pelvic pressure, and cramping. The specimen is obtained by rotating the swab provided in the specimen collection kit across the posterior fornix of the vagina for 10 seconds to absorb the vaginal secretions. The swab must not be contaminated with lubricants, creams, soaps, or disinfectants that may interfere with the antibody–antigen reaction in the test system. The methods for detection of the fFN enzyme immunoassay are solid-phase enzyme-linked immunosorbent assay (ELISA) or lateral flow, solid-phase immunochromatographic

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assay using the Rapid fFN cassette. In the fFN enzyme immunoassay, the vaginal sample is incubated with FDC-6, a monoclonal antibody specific for fFN, and the presence or absence of the fFN is determined spectrophotometrically at a wavelength of 550 nm. The rapid fFN assay is a qualitative test for the detection of fFN that uses a Rapid fFN cassette kit and a TLiIQ analyzer. The specimen swab is placed into an extraction buffer and filtered with a plunger filter. The filtered sample is dispensed onto the sample application well of the Rapid fFN cassette. The sample flows from an absorbent pad across a nitrocellulose membrane by capillary action through a reaction zone containing murine monoclonal anti-fFN antibody conjugated to blue microspheres. The monoclonal antibody FDC-6 is specific for fFN. The conjugate, embedded in the membrane, is mobilized by the flow of the sample. Then the sample flows through a zone containing goat polyclonal antihuman fibronectin antibody that captures the fibronectin–conjugate complexes. The remaining sample flows through a zone containing goat polyclonal antimouse IgG antibodies, which captures unbound conjugate, resulting in a control line. After 20 minutes, the intensities of the test line and control line are interpreted with the TLiIQ analyzer. The results are reported as positive or negative.13 Symptomatic pregnant women with a positive fFN test are at increased risk for delivery in less than or equal to 7 to 14 days from specimen collection, and asymptomatic pregnant women are at increased risk for delivery in less than or equal to 34 weeks and 6 days of gestation.13

AmniSure Test The risk of premature delivery also may be caused by the premature rupture of fetal membranes (PROM). In addition, rupture of fetal membranes can cause infection, fetal distress, prolapse of the umbilical cord, postnatal endometritis, and placental abruption. A symptom of fetal membrane rupture is leakage of amniotic fluid. PAMG-1 is present in high levels in amniotic fluid and low levels in blood; therefore, it is a reliable marker of fetal membrane rupture. A normal level of PAMG-1 in pregnant women ranges from 0.05 to 0.22 mg/mL, which might increase to 3 mg/mL when vaginitis is present. Fetal membrane rupture causes increased concentrations of amniotic fluid in the vaginal secretions and can raise the PAMG-1 levels to 2,000 to 25,000 mg/mL.14 The AmniSure test quickly identifies patients with PROM, and appropriate intervention can take place. The AmniSure ROM test (AmniSure International) is a qualitative rapid test that uses an immunochromatographic device. A sample of vaginal secretions collected with a swab is placed into a vial with solvent. The swab is rotated for 1 minute to enable the solvent to extract the sample from the swab, and then the swab is discarded. The AmniSure test strip is placed into the vial. Monoclonal antibodies with colloidal gold particles attached are located on the pad region of the test strip. The antibodies attach to the PAMG-1 in the sample and transport it to the test region. The solution flows from the pad region of the strip to the test region. The test region of the test strip has antibodies immobilized on it. If PAMG-1 is present in the

patient sample, it will bind with antibodies in the test region and produce a line. This line is produced by gold dye attached to conjugated antibodies and indicates a rupture of fetal membranes. The second control line is designed to indicate that the test is functioning well. The test is read immediately or within 10 minutes. A positive test result indicates a membrane rupture and is indicated by the presence of two lines. When only the control line is present, it is reported as negative for membrane rupture. The test should be performed immediately after collection, but if there is a delay in testing, the specimen can be maintained in a closed sample vial and refrigerated for 6 hours. False positives can be caused by the presence of large amounts of blood in the vaginal sample.

Actim PROM The Actim PROM (CooperSurgical, Trumbull, CT) is a 5-minute test that can be used bedside to diagnose patients with PROM.15 It is a rapid immunoassay test that specifically detects IGFBP-1 in the vaginal fluid. IGFBP-1 accumulates at high concentrations in amniotic fluid. When the membranes are ruptured, IGFBP-1 is detectable in a vaginal swab sample. The swab is held in the vaginal fornix for 10 to 15 seconds. Then the swab is placed in the specimen extraction solution and swirled vigorously for 10 to 15 seconds. The yellow area of the dipstick is placed into the extraction solution and held there until the liquid front reaches the result area, at which point the dipstick is removed. The test is interpreted in 5 minutes. A positive result will show two lines on the dipstick, indicating that the membranes have ruptured. A negative result will show only one control line, indicating that the membranes are intact. Blood, urine, semen, bath and odor products, common vaginal infections, and medications will not interfere with the testing. The test has been designed to minimize interference from bleeding, but in cases of heavy bleeding, the blood present may have a higher concentration of IGFBP-1 protein.16

ROM Plus The ROM Plus fetal membranes rupture test is a qualitative immunochromatographic test for the detection of amniotic fluid in vaginal secretions of pregnant women. This test is unique in that it detects both AFP and IGFBP-1 protein, also known as PP12, from amniotic fluid in vaginal secretions. The ROM Plus test strip is a lateral flow device. The sample is collected by placing the swab on the vaginal mucosal lining for 15 seconds. Then the swab is mixed into a vial containing 400 µL of buffer solution, and the diluted sample is applied to the sample pad of the test strip via the sample well on the cassette. The liquid moves chromatographically and unidirectionally toward the absorbent pad. During migration, the sample reacts with monoclonal and polyclonal antibodies bound to the test strip membrane. These antibodies are immunoreactive to a combination of AFP and IGFBP-1. If the sample contains AFP and IGFBP-1, it binds to the antibody test line, causing the test line to appear and indicate a positive result. Two lines will be visible. If the sample does not contain the amniotic markers, only the control line will be visible, indicating a negative result.17

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Chapter 17 | Vaginal Secretions IGFBP-1 concentration in amniotic fluid is between 10,500 and 350,000 ng/mL. AFP concentration in amniotic fluid is from 2,800 to 26,000 ng/mL. Serum IGFBP-1 concentration is from 55 to 242 U/mL (equivalent to 33 to 290 ng/mL). Concentrations of IGFBP-1 in amniotic fluid can be 100 to 1,000 times higher than that in maternal serum.17

8. 9.

10.

For additional resources please visit www.fadavis.com

References 1. Egan, MA, and Lipsky, MS: Diagnosis of vaginitis, Am Fam Physician 62(5):1095–1104, 2000. Web site: http://www.aafp. org/afp/2000/0901/p1095.html. Accessed October 4, 2019. 2. Clinical and Laboratory Standards Institute. Provider-Performed Microscopy Testing: Approved Guideline, ed. 2. CLSI document POCT10-A2. CLSI, Wayne, PA, 2011, CLSI. 3. Metzger, GD: Laboratory diagnosis of vaginal infections. Clin Lab Sci 11:47–52, 1998. 4. Woods, GL, and Croft, AC: Specimen collection and handling for diagnosis of infectious diseases. In Henry, JB (ed): Clinical Diagnosis and Management by Laboratory Methods, ed 22. Elsevier Saunders, Philadelphia, 2011. 5. Centers for Disease Control and Prevention: Diseases characterized by vaginal discharge. Sexually Transmitted Diseases Treatment Guidelines, 2010. Web site: http://www.cdc.gov/std/ treatment/2010/vaginal-discharge.htm. Accessed April 14, 2020. 6. French, L, Horton, J, and Matousek, M: Abnormal vaginal discharge: Using office diagnostic testing more effectively, J Fam Practice 53(10):805–814, 2004. Web site: https://www. mdedge.com/familymedicine/article/60266/womens-health/ abnormal-vaginal-discharge-using-office-diagnostic. Accessed July 23, 2019. 7. Fader, RC: Anaerobes of clinical importance. In Mahon, CR, Lehman, DC, and Maneselis, G: Textbook of Diagnostic

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16. 17.

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Microbiology, ed 4. Saunders Elsevier, Maryland Heights, MO, 2011. Seattle STD/HIV Prevention Training Center. Examination of vaginal wet preps (video). Web site: https://www.youtube.com/ watch?v=8dgeOPGx6YI. Accessed July 27, 2019. Smith, LA: Diagnostic Parasitology. In Mahon, CR, Lehman, DC, Maneselis, and Maneselis, G: Textbook of Diagnostic Microbiology, ed 4. Saunders Elsevier, Maryland Heights, MO, 2011. Patil, MJ, Magamoti, JM, and Metgud, SC: Diagnosis of Trichomonas vaginalis from vaginal specimens by wet mount microscopy, in pouch TV culture system, and PCR. J. Global Infect Dis [serial online] [cited 2012 Jul 6] 4:22–25, 2012. Web site: http://www.jgid.org/text.asp?2012/4/1/22/ 93756. Accessed July 23, 2019. Keen, EF, and Aldous, WK: Genial infections and sexually transmitted diseases. In Mahon, CR, Lehman, DC, Maneselis, G: Textbook of Diagnostic Microbiology, ed 4, Saunders Elsevier, Maryland Heights, MO, 2011. Lockwood, CJ, Senyei, AE, Dische, MR, Casal, DC, et al: Fetal fibronectin in cervical and vaginal secretions as a predictor of preterm delivery. New Engl J Med 325:669–674, 1991. Fetal Fibronectin Enzyme Immunoassay and Rapid fFN for the TLiIQ System. AW-04196-002 Rev.002, Hologic, Inc. Web site: http://www.ffntest.com/;hcp/science_fetal.html. Accessed July 23, 2019. Cousins, LM, et al: AmniSure Placental Alpha Microglobulin-1 Rapid Immunoassay versus standard diagnostic methods for detection of rupture of membranes. Am J Perinatol 22(6): 317–320, Aug 2005. Abdelazim, IA: Insulin-like growth factor binding protein-1 (Actim PROM test) for detection of premature rupture of fetal membranes. J. Obstet Gynaecol Res. 2014 Apr;40(4):961–967. Doi: 10.1111/jog.12296. Epub 2014 Feb 26. https://www. ncbi.nlm.nih.gov/pubmed/24612210. Accessed July 23, 2019. Actim PROM brochure. Cooper Surgical. www.coopersurgical. com. 2014. Accessed July 23, 2019. ROM Plus Fetal Membranes Rupture Test Instructions for Use brochure. (Package insert). Clinical Innovations. www. clinicalinnovations.com. Accessed July 23, 2019.

Study Questions 1. Which of the following would not be a reason to collect a vaginal fluid for analysis? A. Vaginitis B. Complications of pregnancy resulting in preterm delivery C. Forensic testing in a sexual assault D. Pregnancy testing 2. Which of the following organisms might not be detected if the specimen for vaginal secretion analysis had been refrigerated? A. Prevotella bivia B. Lactobacillus acidophilus C. Trichomonas vaginalis D. Candida albicans

3. The appearance of the vaginal discharge in vulvovaginal candidiasis is described as: A. Clear and colorless B. Thin, homogeneous, white-to-gray discharge C. White, curd-like D. Yellow-green and frothy 4. A normal range for a vaginal pH is: A. 3.8 to 4.5 B. 5.0 to 6.0 C. 6.0 to 7.0 D. 7.0 to 7.4

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5. Which of the following tests differentiates budding yeast cells from RBCs? A. pH B. Saline wet mount C. KOH prep D. Whiff test 6. Which of the following constituents is normal in healthy vaginal fluid secretions? A. Lactobacilli B. Basal cells C. Trichomonas vaginalis D. Pseudohyphae 7. Vaginal specimens collected for a saline wet prep should be: A. Refrigerated to preserve motility B. Prepared as soon as possible C. Mailed to a reference laboratory D. Preserved with potassium hydroxide 8. A positive amine (whiff) test is observed in which of the following syndromes? A. Bacterial vaginosis B. Vulvovaginal candidiasis C. Atrophic vaginitis D. Desquamative inflammatory vaginitis 9. A squamous epithelial cell covered with coccobacilli that extends beyond the cytoplasm margin is a: A. Basal cell B. Parabasal cell C. Clue cell D. Blastospore 10. All of the following are diagnostic of bacterial vaginosis except: A. Vaginal pH of 3.8 B. Presence of clue cells C. Positive amine (whiff) test D. Thin, homogeneous, white-to-gray vaginal discharge

11. Which of the following organisms produces lactic acid and hydrogen peroxide to maintain an acidic vaginal environment? A. Gardnerella vaginalis B. Mobiluncus spp. C. Lactobacilli spp. D. β-Hemolytic streptococci 12. All of the following are diagnostic of vulvovaginal candidiasis except: A. Large numbers of WBCs B. Presence of clue cells C. Positive KOH test D. Vaginal pH of 4.0 13. All of the following are diagnostic of trichomoniasis except: A. Vaginal pH of 6.0 B. Positive amine test C. Positive KOH test D. Motile trichomonads present 14. The bacteria associated with desquamative inflammatory vaginitis is: A. β-Hemolytic streptococci B. Trichomonas vaginalis C. Gardnerella vaginalis D. Mycoplasma hominis 15. The protein present in vaginal secretions that can identify patients who are at risk for preterm delivery is: A. Human chorionic gonadotropin B. Estrogen C. PAMG-1 D. Fetal fibronectin 16. Which of the following immunochromatographic tests detects both AFP and IGFBP-1 proteins to diagnose PROM? A. AmniSure ROM test B. Actim PROM C. ROM Plus D. Fetal fibronectin

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Case Studies and Clinical Situations 1. A 30-year-old woman has symptoms of dysuria, vaginal itching, and a white, curd-like discharge. During her visit at the women’s clinic, the patient revealed that she had recently completed a regimen of broad-spectrum antibiotics as treatment for a urinary tract infection. Her health-care provider takes a swab of the vaginal secretions for analysis. a. What tests will be performed on the vaginal specimen? b. Based on the patient history and observation of the vaginal secretion, which test will be diagnostic for the probable diagnosis? c. What confirmatory test can be performed? d. What is the probable diagnosis? e. What is the first choice of treatment? 2. A sexually active teenager visited the women’s clinic complaining of vaginal itching and soreness. She indicated that she was experiencing increased vaginal secretions that were frothy and yellow to green. Upon examination, the health-care provider noted a strawberry-like cervix and performed a pH test on the secretions. The pH was 5.5, and the wet prep demonstrated “swimming” organisms. a. What is the probable diagnosis? b. What other tests can be performed to confirm this diagnosis?

c. What is the best course of treatment? d. Should her sexual partner be treated? e. List three complications that can occur with this disorder. 3. During a routine visit with the gynecologist, a 60-year-old woman complained of vaginal dryness and soreness. During the examination, the health-care provider noted erythema of the vaginal mucosa. The pH of the vaginal secretions was 6.0. The KOH and amine (whiff) tests were negative. The microscopic examination revealed epithelial cells, basal cells, decreased lactobacilli, and increased gram-positive cocci and gram-negative rods. a. What is the name of this condition? b. Explain how this condition can occur. c. What is the treatment for this condition?

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Answers to Study Questions, Case Studies, and Clinical Situations Chapter 1 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

C C C A D A B A C D D C B

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

A B D A B C C A B D B B A

27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

C D D D B 2, 1, 2, 3, 2, 1 D C D C D

Case Studies and Clinical Situations 1. a. Review of the procedure by a designated authority has not been documented. b. Instructions and training are not being provided to personnel performing collections. c. A safety statement about the heat produced by the reaction is not in the procedure manual. d. The bottles have not been dated and initialed. 2. a. Corrective action; proficiency survey tests should be rotated among personnel performing the tests. b. Accept; quality control (QC) on the Clinitest tablets must be performed only when they are used to perform a test. c. Corrective action; documentation of technical competency should be performed on all personnel working in the section and educational qualifications assessed. 3. a. The procedure was being performed incorrectly. The correct timing of the glucose reaction was not being done. b. The technologist performing the test. QC ensures that the reagents and instrument are working properly and that the technologist is performing the test correctly.

c. At the beginning of each shift, when a new bottle of reagent is opened, or as stated in the procedure manual. d. Refer to the procedure manual. Check expiration dates of controls and reagents. Open and test a new bottle of control or reagents. e. When the controls are within range. 4. a. Delay in testing the specimen. b. Procedure manual. c. Delta check. d. Incident report. e. Treatment of the patient will be delayed because the specimen will need to be recollected and tested. Extra expense incurred. f. Errors should be corrected as soon as possible following the facility’s policy. The original result must not be erased.

Chapter 2 Study Questions 1. 2. 3. 4.

A C B D

5. 6. 7. 8.

D C A C

9. B 10. C

Case Studies and Clinical Situations 1. a. No. The large amount of blood and leukocytes on the chemical reagent strip does not correlate with 0 to 2 RBCs/hpf and the 0 to 2 WBCs/hpf for the microscopic result. b. The MLS student may not have mixed the specimen before pouring the urine into the aliquot tube, causing the cellular constituents to reside at the bottom of the original urinalysis container. c. The quality control specimens should be tested for precision and accuracy before patient specimens are tested and the results reported. 2. a. Sysmex XN, Glocyte, iQ200, and Advia 2120i. b. Advia 2120i. c. Up to 4 hours.

371

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Chapter 3 Study Questions 1. 2. 3. 4. 5. 6. 7.

C B C A D C C

8. 9. 10. 11. 12. 13. 14.

A C A B D D A

15. 16. 17. 18. 19. 20.

C C A D B B

Case Studies and Clinical Situations 1. a. It could have increased pH, nitrite, and bacteria and decreased clarity, glucose, ketones, bilirubin, urobilinogen and WBCs, RBCs, and casts. b. Decreased glucose and decreased ketones. c. Reject the specimen and recollect. d. The specimen was refrigerated and was brought immediately to the laboratory. 2. a. It would be less clear. b. Additional epithelial cells and bacteria (making it not acceptable for a culture). 3. a. The results would be decreased falsely. b. The patient needs to collect another 24-hour specimen. 4. a. Yes. b. The correct ratio of additive to urine must be maintained. The excess preservative can be toxic to bacteria, causing false-negative results. 5. a. Specimen temperature. b. The temperature would be lower than body temperature. c. The specimen tested was not from the defendant. d. A chain-of-custody form (COC) that is filled out accurately. The specimen could be collected as a “witnessed” collection.

21. 22. 23. 24. 25.

D D B D D

26. 27. 28. 29. 30.

Study Questions

Study Questions

9. 10. 11. 12. 13. 14. 15. 16.

B A C D D B B D

17. B 18. B: Beta2microglobulin; B: Creatinine; B: Cystatin C; A: 125Iiodothalamate 19. B 20. 69 mL/min

D 600 mL/min C B

1. a. 160 mg/dL to 180 mg/dL. b. Renal tubular reabsorption is impaired. 2. a. Juxtaglomerular apparatus → Angiotensinogen → Renin → Angiotensin I → Angiotensin II. b. Vasoconstriction, increased sodium reabsorption, and increased aldosterone to retain sodium. c. Production of renin decreases and, therefore, the actions of the RAAS. 3. a. The physician can calculate the approximate creatinine clearance using the MDRD-IDMS-traceable formula. b. The cystatin C test and the beta2-microglobulin test serum tests. c. No. The beta2-microglobulin test requires a normal immune system, and malignancies can affect the immune system; therefore, the test cannot be reliable in patients with immunologic disorders and malignancies. 4. a. Yes. Serum from the midnight specimen is not being separated from the clot and refrigerated in a timely manner. b. Lactic acid will be present in serum that is not separated from the clot and will affect the freezingpoint osmolarity readings. c. If the laboratory is using a freezing-point osmometer, results will be affected by alcohol ingestion; vapor pressure results would not be affected and could be used as a comparison. 5. a. Diabetes insipidus. b. Neurogenic diabetes insipidus. c. Nephrogenic diabetes insipidus.

Chapter 5

B D C D A B C D

31. 32. 33. 34.

Case Studies and Clinical Situations

Chapter 4 1. 2. 3. 4. 5. 6. 7. 8.

C C B A +0.5

1. 2. 3. 4. 5. 6. 7. 8.

A D A D C A C C

9. 10. 11. 12. 13. 14. 15. 16.

D A C B D A C B

17. 18. 19. 20. 21. 22. 23. 24.

D D C B B A B D

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Case Studies and Clinical Situations 1. a. An elevated pH and a positive reagent strip reaction for nitrite. b. The reagent strip specific gravity would be much lower if the patient had been given radiographic dye. c. The reagent strip test for bilirubin would be positive. d. The reagent strip reaction for blood would be positive, and RBCs would be seen in the microscopic examination. 2. a. 1.018. b. Yes. c. It would agree with the reagent strip reading because, like the osmometer, the reagent strip is not affected by high-molecular-weight substances. 3. Hemoglobin and myoglobin. a. Examine the patient’s plasma/serum. The breakdown of red blood cells to hemoglobin produces a red serum/plasma. Myoglobin is produced from skeletal muscle and is rapidly cleared from the plasma/serum. The serum/plasma color would not be affected. 4. a. The woman has been eating fresh beets. b. Yes. The pH of the woman’s urine is acidic or she has not recently consumed fresh beets. 5. No. The urine can contain increased pH, glucose, ketones, bilirubin, urobilinogen, nitrite, and small amounts of cellular structures.

Chapter 6 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

A D A C D A D B D 2, 1, 2, 3, 1, 2, 3 B A A D B A

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

C A A C A B C A C B A D A C 1, 2, 1, 2, 1, 2 32. B

33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

A 1, 3, 4, 2 A D C A C D A B D C B C C A C

Case Studies and Clinical Situations 1. a. The blood glucose is elevated and has exceeded the renal tubular maximum (Tm) for glucose. b. Diabetes mellitus. c. It indicates diabetes mellitus–related renal disease. d. Renal tubular reabsorption disorders. 2. a. Yellow foam. b. Possible biliary duct obstruction preventing bilirubin from entering the intestine. c. Icteric. d. Protection from light. 3. a. Hemoglobinuria. b. Increased hemoglobin presented to the liver results in increased bilirubin entering the intestine for conversion to urobilinogen. c. The circulating bilirubin is unconjugated. d. It would if a Multistix reagent strip is used and would not if a Chemstrip is used. A Watson-Schwartz test is more specific for porphobilinogen. 4. a. Negative chemical reactions for blood and nitrite. Ascorbic acid interference for both reactions. A random specimen or further reduction of nitrite could cause the negative nitrite. b. Glucose, bilirubin, LE. Ascorbic acid is a strong reducing agent that interferes with the oxidation reaction in the glucose test. Ascorbic acid combines with the diazo reagent in the bilirubin and LE tests, lowering the sensitivity. c. The dark yellow color may be caused by betacarotene and vitamin A, and some B vitamins also produce yellow urine. d. Nonnitrite–reducing microorganisms; lack of dietary nitrate; antibiotic administration. 5. a. To check for possible exercise-induced abnormal results. b. Negative protein and blood, possible changes in color and specific gravity. c. Renal. 6. a. No, the specimen is clear. b. Myoglobinuria. c. Muscle damage from the accident (rhabdomyolysis). d. Yes. Myoglobin is toxic to the renal tubules. 7. a. Laboratory personnel are not capping the reagent strip containers tightly in a timely manner. b. Personnel performing the CLIA-waived reagent strip test are not waiting 2 minutes to read the LE reaction. c. The student is not mixing the specimen. d. The reagent strips have deteriorated, and the quality control on the strips was not performed before reporting the results.

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Chapter 7 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

C D D C A B C D C D D A C B C A D D

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

B C A B C D D B D A B C C D D D A C

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

C D A C D A A C D C 4, 3, 5, 1 3, 5, 2, 6, 4 4, 8, 7, 6, 1, 5, 3 50. 3, 5, 2, 1, 7, 4

Case Studies and Clinical Situations 1. a. Yeast grows best at a low pH with an increased concentration of glucose. b. Yes, this exceeds the renal threshold. c. No, yeast is not capable of reducing nitrate to nitrite. d. Moderate blood with no RBCs. e. Myoglobin is the cause of the positive chemical test result for blood. The patient has been bedridden for an extended period, causing muscle destruction. 2. a. The large objects are in a different plane from that of the urinary constituents. b. Contamination by artifacts. c. No, because they are in a different plane. d. Polarizing microscopy. 3. a. Renal tubules. b. Yes, viral infections can cause tubular damage. c. RTE cells absorb the bilirubin-containing urinary filtrate. d. Liver damage inhibits processing of reabsorbed urobilinogen. e. Hemolytic anemia. 4. a. The patient is taking a pigmented medication, such as phenazopyridine. b. Yes. c. Ask what medications the patient is taking. d. Ampicillin.

5. a. b. c. d. 6. a. b.

c. d.

7. a. b.

Calcium oxalate. Monohydrate and dihydrate calcium oxalate. Oval: monohydrate; envelope: dihydrate. Monohydrate. Microscopic results do not match the chemical tests for blood, nitrite, and leukocyte esterase. The specimen has been unpreserved at room temperature for too long, the cells have disintegrated, and the bacteria have converted the nitrite to nitrogen. The pH. Ask the clinic personnel to instruct the patient to collect a midstream clean-catch specimen and have the specimen delivered to the laboratory immediately. No, because they are associated with strenuous exercise. The positive blood reaction is from hemoglobinuria or myoglobinuria resulting from participating in a contact sport. The protein is orthostatic.

c. Increased excretion of RTE cell lysosomes in the presence of dehydration. 8. a. Yes, the waxy casts are probably an artifact, such as a diaper fiber. Waxy casts are not associated with negative urine protein. b. No, this is normal after an invasive procedure. c. Yes, tyrosine crystals are seen in patients with severe liver disease; therefore, the bilirubin should be positive. The crystals may be an artifact or from a medication. d. Yes, uric acid crystals may be mistaken for cystine crystals. e. Yes, radiographic dye crystals associated with a high specific gravity resemble cholesterol crystals. f. No, Trichomonas is carried asymptomatically by men. g. No, calcium carbonate crystals are found in alkaline urine; therefore, clumps of amorphous phosphates may be present.

Chapter 8 Study Questions 1. 2. 3. 4. 5. 6. 7.

B C B C B A C

8. 9. 10. 11. 12. 13. 14.

D A C C C B D

15. 16. 17. 18. 19. 20.

A C A A A D

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Case Studies and Clinical Situations 1. a. Acute glomerulonephritis. b. M protein in the cell wall of the group A streptococcus. c. Glomerular bleeding. d. No, they are also passing through the damaged glomerulus. e. Good prognosis with appropriate management of secondary complications. f. Henoch-Schönlein purpura. 2. a. IgA nephropathy. b. Serum IgA level. c. Chronic glomerulonephritis/end-stage renal disease. d. Impaired renal tubular reabsorption associated with end-stage renal disease. e. The specific gravity is the same as that of the ultrafiltrate, indicating a lack of tubular concentration. f. The presence of extreme urinary stasis. 3. a. Nephrotic syndrome. b. Nephrotic syndrome may be caused by sudden, severe hypotension. c. Changes in the electrical charges of the shield of negativity produce increased membrane permeability. d. Decreased plasma albumin lowers the capillary oncotic pressure, causing fluid to enter the interstitial tissue. e. Reabsorption of filtered lipids by the RTE cells. 4. a. Minimal change disease. b. Nephrotic syndrome, focal segmental glomerulosclerosis. c. Good prognosis with complete remission. 5. a. Goodpasture syndrome. b. The autoantibody attaches to the glomerular capillaries, causing complement activation and destruction of the capillaries. c. Granulomatosis with polyangiitis. d. Antineutrophilic cytoplasmic antibody. e. Granuloma formation resulting from autoantibodies binding to neutrophils in the vascular walls and initiating an immune response. 6. a. Cystitis, UTI. b. The specimen is very dilute. c. Irritation of the urinary tract will cause a small amount of bleeding. The cells and bacteria may cause a trace protein, or it may be a false-positive due to the high pH. d. Yes, glitter cells are seen in hypotonic urine.

e. f. 7. a. b. c. d. e. f. g. 8. a. b. c. d. 9. a. b. c. d. e.

10. a.

Female children. Pyelonephritis. Intravenous pyelogram. Chronic pyelonephritis. WBC cast. Reflux nephropathy. Performing a Gram stain. Radiographic dye. Permanent tubular damage and progression to chronic, end-stage renal disease. Abnormal. Acute interstitial nephritis. This disorder is an inflammation, not an infection. Discontinue the medication because it is causing the allergic reaction. Acute renal failure. The prerenal sudden decrease in blood flow to the kidneys. Lack of renal concentrating ability. Tubular damage. The increased diameter of the damaged distal convoluted tubule and extreme urinary stasis, allowing casts to form in the collecting ducts. Renal lithiasis.

b. The high specific gravity. c. Yes, the dark yellow color and high specific gravity indicate a concentrated urine, which induces the formation of renal calculi. d. Calcium oxalate. e. Increased hydration and dietary changes.

Chapter 9 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

C A C B A A C B D D

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

D B C A D B, A, B, B, A D B B B

21. 22. 23. 24. 25. 26. 27.

D D D C B D D, F, A, E, C, B

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Case Studies and Clinical Situations 1. a. b. c. d. 2. a. b. c. 3. a. b. c. d. e. 4. a. b. c. d. e. 5. a. b.

c. 6. a. b. c. 7. a. b. c. d. e.

Underdevelopment of the liver. Yes, with severe acquired liver disease. Tyrosine crystals; leucine crystals, bilirubin crystals. Protect the specimen from light. Isovaleric acidemia. Maple syrup urine disease. Yes, the MS/MS screen would be positive. Renal lithiasis. Impaired renal tubular reabsorption of cystine. Lysine, arginine, ornithine. They are more soluble than is cystine. The disorder is inherited cystinuria. Yes. Yes, uric acid crystals accumulating on the surface of the diaper could have an orange color. Lesch-Nyhan disease. Yes, the disease is inherited as sex-linked recessive. Hypoxanthine guanine phosphoribosyltransferase. Yes. The urine may contain melanin or homogentisic acid. Yes. Melanin will react with sodium nitroprusside, the reagent used on reagent strips for the detection of ketones. Yes. Homogentisic acid turns black in alkaline urine. Yes, the blue color could indicate the presence of indican in the urine. Hartnup disease. Good with proper dietary supplements. The Ehrlich reaction. Acetylacetone. Porphobilinogen. Blood. Free erythrocyte protoporphyrin (FEP).

Chapter 10 Study Questions 1. 2. 3. 4. 5. 6. 7. 8.

C B C B A B B, B, A, A C

9. 10. 11. 12. 13. 14. 15. 16.

A C C D D A B C

17. 18. 19. 20. 21. 22. 23. 24.

D D D B D A B D

25. 26. 27. 28. 29.

C C A C A

30. 31. 32. 33. 34.

C D C B A

35. 36. 37. 38.

C C D B

Case Studies and Clinical Situations 1. a. Cerebral hemorrhage because of the presence of erythrophagocytosis, even distribution of blood, and patient’s history. b. No, they would be consistent with peripheral blood entering the CSF. c. No, they are consistent with the percentages seen in peripheral blood. d. Hemosiderin granules and hematoidin crystals. e. A traumatic tap. 2. a. An India ink preparation. b. Cryptococcus meningitis. c. Immunologic testing for Cryptococcus. d. Rheumatoid factor. e. Acid-fast staining and culture. f. Noticeable oligoclonal bands in both the CSF and serum. 3. a. CSF/serum albumin index = 6.7. b. Yes. c. IgG index = 1.5. d. Immunoglobulin synthesis within the CNS. e. Multiple sclerosis. f. Oligoclonal banding only in the CSF. g. Myelin basic protein. 4. a. Viral, tubercular, or fungal meningitis. b. No, the Gram stain would be negative in viral and tubercular and not always positive in fungal meningitis. c. Yes. Lymphocytes are very predominant in viral meningitis. d. Yes, a CSF lactate level of 25 mg/dL or less would aid in confirming viral meningitis. The lactate level would be higher in cases of bacterial, tubercular, and fungal meningitis. 5. a. Stain precipitate is being confused with gram-positive cocci. b. Differentials are being reported from the counting chamber. c. The albumin is contaminated. d. The specimens are not being delivered promptly to the laboratory.

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Chapter 11 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

C D B C D A B D C B B

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

C A A B B A C A C A A

23. 24. 25. 26. 27. 28. 29. 30. 31.

D B D B D B C A B

Case Studies and Clinical Situations 1. a. b. c. d.

2.

3. 4. 5.

6.

Sperm concentration, motility, and morphology. 21,000,000; no. 1,800,000; no. Yes. The normal sperm concentration is 20 to 60 million/mL. Spermatid counts over 1 million are considered abnormal. Both of these abnormal results, as well as the abnormal motility, are related to defects in sperm maturation. a. Male antisperm antibodies may form after vasovasostomy procedures. b. The MAR test and the immunobead test. c. The MAR test detects the presence of IgG male sperm antibodies. The immunobead test delineates the areas of the sperm (head, tail, neck) that are affected by the antibodies. d. Clumping, ovum penetration, and motility. The specimen contains urine, which is toxic to sperm, therefore decreasing viability. The specimen was collected improperly, and the first part of the ejaculation was lost. a. Yes, insufficient prostatic fluid is present. b. Zinc, citrate, and acid phosphatase. c. Sperm motility is affected severely. a. Acid phosphatase and seminal glycoprotein p30 tests. b. Microscopic examination for the presence of sperm.

Chapter 12

B C A A C A

15. 16. 17. 18. 19. 20.

B B A D B C

21. 22. 23. 24. 25.

C A D C A

Case Studies and Clinical Situations 1. a. Sterile, heparinized tube, liquid EDTA tube, nonanticoagulated tube. b. MSU crystals are seen in gout. c. Highly birefringent, needle-shaped crystals under polarized light that turn yellow when aligned with the slow vibration of red compensated polarized light. d. Infection is frequently a complication of severe inflammation. 2. a. WBC diluting fluid containing acetic acid was used. b. Normal, hypotonic, or saponin-containing saline should be used. c. Crystal-induced inflammatory and septic. d. Gram stain and culture, crystal examination. 3. a. Noninflammatory. b. Hydroxyapatite crystals. c. Glucose. A normal result is consistent with noninflammatory arthritis. 4. a. Fibrinogen. b. EDTA or heparinized tube. c. No, the bacteria will be trapped in the clot.

Chapter 13 Study Questions 1. 2. 3. 4. 5. 6. 7.

C D A D C D B, A, A, A, B, B, B, A 8. B

9. 10. 11. 12. 13. 14. 15. 16. 17.

B C D D D C B A D

18. 19. 20. 21. 22. 23. 24. 25. 26.

B C B B B C B D D

Case Studies and Clinical Situations

Study Questions 1. B 2. A 3. A

9. 10. 11. 12. 13. 14.

4. B 5. B, C, B, A, D, B, D

6. A 7. B 8. B

1. a. Pleural fluid. b. Transudate because all the test results are consistent with those of a transudate. c. Pleural fluid-to-serum ratios of cholesterol and bilirubin.

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Answers to Study Questions, Case Studies, and Clinical Situations

2. a. b. 3. a. b. c. 4. a.

Pneumonia. Chest tube drainage. 1.6. Transudate. The SAAG is above 1.1. Hepatic disorder. To differentiate between cirrhosis and peritonitis; cirrhosis. b. Pancreatitis or gastrointestinal perforation; alkaline phosphatase. c. Rupture or accidental puncture of the bladder. d. To detect the presence of gastrointestinal (CEA) and ovarian (CA 125) cancers. 5. The patient has been a victim of blunt trauma, and the physician wants to determine whether abdominal bleeding is occurring; abdominal bleeding. 6. Thyroid profile; CA 125.

Chapter 14 Study Questions 1. 2. 3. 4.

D A B D

5. 6. 7. 8.

A A B D

9. C 10. C

Case Studies and Clinical Situations 1. a. Red and cloudy. b. Bloody fluid with increasing intensities during sequential aliquots. c. Phagocytosed erythrocytes. d. Orange-red fluid. e. Hemosiderin-laden macrophages. f. Prussian blue for hemosiderin-laden macrophages; Sudan III stain for lipid-laden alveolar macrophages. 2. a. Presence of lymphocytes with decreased ratio of CD4 and CD8. b. Cryptococcus neoformans. c. Demonstration of a positive cryptococcal antigen in the BAL specimen. The extent of the cryptococcal infection correlates with the antigen titer.

Chapter 15 Study Questions 1. 2. 3. 4.

B C A C

5. 6. 7. 8.

B A D 2, 4, 1, 3

9. 10. 11. 12.

A C B True

13. A 14. True 15. C

16. True 17. C 18. B

19. B 20. D

Case Studies and Clinical Situations 1. a. Yes. b. FLM. c. The level of phosphatidylglycerol present in the fetal lungs. d. Phosphatidylglycerol is essential for FLM, and levels do not always parallel lecithin levels in fetuses of diabetic mothers. 2. a. A neural tube disorder, such as spina bifida or anencephaly. b. An acetylcholinesterase level. c. The amniotic fluid specimen contains blood. 3. a. False-positive result. b. False-positive result. c. No effect. d. False-positive result. 4. a. False-positive result. b. False-positive result. c. False-positive or test interference. d. No effect. 5. Both a and c are correct. The incorrect specimen was sent, or the specimen was exposed to light. 6. a. Premature rupture of the membranes. b. Fern test, pH test, nitrazine test, AmniSure ROM test, Actim PROM test, and ROM+ test. c. PAMG-1, IGFBP-1, AFP. d. Actim PROM test.

Chapter 16 Study Questions 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

C A C B D D D C D C

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

A C D B B False C C C D

21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

B C B A B B A C C B

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Case Studies and Clinical Situations 1. a. Secretory diarrhea. b. Stool culture. c. Probable: Salmonella, Shigella, Campylobacter, Yersinia, Escherichia coli; Improbable: Staphylococcus, Vibrio. d. Osmotic diarrhea. 2. a. Microscopic examination for fecal fats. b. Neutral fats stain directly and appear as large, orangered droplets; soaps and fatty acids appear as smaller orange-red droplets after pretreatment of the specimen with heat and acetic acid. c. Quantitative fecal fat test. d. Bulky and frothy. e. Muscle fiber screening and the gelatin test for trypsin. f. Muscle fiber: failure to include red meat in the diet. g. Chymotrypsin or elastase I. 3. a. Patient #1: gastric reflux medication containing bismuth may produce black stools. Patient #2: medications, such as aspirin and other NSAIDs, may cause gastric bleeding. Patient #3: red meat was not avoided for 3 days before specimen collection. b. Provide dietary and medication instructions to patients. c. The Hemoccult ICT immunochemical test. 4. a. The APT test cannot be performed because the hemoglobin is already denatured. It must be red blood. b. The pH will be low because increased carbohydrates are available for bacterial metabolism. c. The infant had ingested maternal blood. d. Yes, adequate carbohydrates are not present, and fats are being metabolized for energy.

Chapter 17 Study Questions 1. 2. 3. 4. 5. 6.

D C C A C A

7. 8. 9. 10. 11. 12.

B A C A C B

13. 14. 15. 16.

C A D C

Case Studies and Clinical Situations 1. a. Vaginal pH, saline and KOH wet preps, Gram stain. b. KOH will reveal budding yeast. c. Culture and DNA direct hybridization probe (Affirm VPIII). d. Vulvovaginal candidiasis caused by Candida albicans. e. Antifungal agents. 2. a. Trichomoniasis caused by Trichomonas vaginalis. b. Wet mount, vaginal pH, amine test from KOH prep, DNA probe (Affirm VPIII), OSOM Trichomonas Rapid Test. c. Metronidazole. d. Yes. e. Complications include low birth rate, premature rupture of membranes, and preterm delivery during pregnancy. 3. a. Desquamative inflammatory vaginitis secondary to atrophic vaginitis. b. Reduced estrogen production in postmenopausal women. c. Hormone replacement therapy (estrogen).

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Abbreviations A2LA AABB A:C ACE AChE ADA ADH AER AFP AGN AHG AIDS AIN ALA ANA ANCA AOA APR ARF ART ASHI ASO ATN BAL BAT B2M BCAAs BSI BUN BV CAP CASA CDC CEA CGN CHP CKD CLIA CLRW CLSI CNS COC COLA CPPD CPR CR ch CSF CT

American Association for Laboratory Accreditation American Association of Blood Banks albumin:creatinine [ratio] angiotensin-converting enzyme acetylcholinesterase adenosine deaminase antidiuretic hormone albumin excretion rate alpha-fetoprotein acute glomerulonephritis antihuman globulin acquired immunodeficiency syndrome acute interstitial nephritis α-aminolevulinic acid antinuclear antibody antineutrophilic cytoplasmic antibody American Osteopathic Association auto particle recognition acute renal failure assisted reproductive technology American Society of Histocompatibility and Immunogenetics antistreptolysin O acute tubular necrosis bronchoalveolar lavage bacterial antigen test beta2 microglobulin branched-chain amino acids body substance isolation blood urea nitrogen bacterial vaginosis College of American Pathologists computer-assisted semen analysis Centers for Disease Control and Prevention carcinoembryonic antigen chronic glomerulonephritis chemical hygiene plan chronic kidney disease Clinical Laboratory Improvement Amendments clinical laboratory reagent water Clinical and Laboratory Standards Institute central nervous system chain of custody Commission on Laboratory Assessment calcium pyrophosphate dihydrate cardiopulmonary resuscitation core channel cerebrospinal fluid computed tomography

CTAB Cu2O CuSO4 CV DBDH DCT DI DIDNTB

DIV DNPH DOT EDS EDTA eGFR ELISA EPS EQA ESRD EU FAH FAST FDA FEP fFN FISH FLM FOBT FSGS FTA-ABS GAGs GALE GALK GALT GC-MS gFOBT GFR GHS GI GPA 1H NMR H+ H2PO4 Hb HbA HbF HBV hCG

cetyltrimethylammonium bromide cuprous oxide copper sulfate coefficient of variation diisopropyl benzene dihydroperoxide distal convoluted tubule diabetes insipidus (3⬘,3⬙, diodo 4⬘,4⬙-dihydroxy-5⬘,5⬙dinitrophenyl)-3,4,5,6-tetra-bromosulphonphtalein desquamative inflammatory vaginitis 2,4-dinitrophenylhydrazine Department of Transportation early dumping syndrome ethylenediaminetetraacetic acid estimated glomerular filtration rate enzyme-linked immunosorbent assay expressed prostatic specimen external quality assessment end-stage renal disease Ehrlich unit fumarylacetoacetate hydrolase focused assessment with sonography for trauma Food and Drug Administration free erythrocyte protoporphyrin fetal fibronectin fluorescence in situ hybridization fetal lung maturity fecal occult blood testing focal segmental glomerulosclerosis fluorescent treponemal antibody-absorption glycosaminoglycans galactose-4-epimerase galactokinase galactose-1-phosphate uridyl transferase gas chromatography–mass spectrometry guaiac-based fecal occult blood test glomerular filtration rate Globally Harmonized System gastrointestinal granulomatosis with polyangiitis hydrogen nuclear magnetic resonance spectroscopy titratable acid/hydrogen ion hydrogen phosphate ion hemoglobin hemoglobin A hemoglobin F hepatitis B virus human chorionic gonadotropin 381

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382

Abbreviations

HCV HCO3 HDFN HF-BF HICPAC 5-HIAA HIV HLA-B12 hpf HRCT HSV IBS IDMS IEF IEM IFE iFOBT IgA IgG IgM IGFBP-1 IQCP ISO IUI IVF KDIGO KOH LBC LC-MS/MS LD LE LED LFA LIS lpf L/S MAR MBP MCD MDRD MESNA MGN MoM mOsm MPGN MPSs MSAFP MS/MS MSU MSUD NaCl NaOH NFPA NH4+ NIRS

hepatitis C virus bicarbonate ion hemolytic disease of the fetus and newborn high-fluorescing body fluid Healthcare Infection Control Practices Advisory Committee 5-hydroxyindoleacetic acid human immunodeficiency virus human leukocyte antigen-B12 high-power field high-resolution computed tomography herpes simplex virus irritable bowel syndrome isotope dilution mass spectrophotometry isoelectric focusing inborn error of metabolism immunofixation electrophoresis immunochemical fecal occult blood test immunoglobulin A immunoglobulin G immunoglobulin M insulin-like growth factor binding protein-1 individualized quality control plan International Organization for Standardization intrauterine insemination in vitro fertilization Kidney Disease: Improving Global Outcomes potassium hydroxide lamellar body count liquid chromatography–tandem mass spectrometry lactic dehydrogenase leukocyte esterase light-emitting diode lateral flow assay laboratory information system low-power field lecithin-sphingomyelin ratio mixed agglutination reaction myelin basic protein minimal change disease modification of diet in renal disease mercaptoethane sulfonate sodium membranous glomerulonephritis multiples of the median milliosmole membranoproliferative glomerulonephritis mucopolysaccharidoses maternal serum alpha-fetoprotein tandem mass spectrophotometry monosodium urate (uric acid) maple syrup urine disease sodium chloride sodium hydroxide National Fire Protection Association ammonium ion near-infrared spectroscopy

NKDEP NRBC NS NTD OD OGTT OSHA PAA PAH PAMG-1 PB PCR PCT PEP PG pKa PKU PM PMNs POC PP12 PPE PPMT PROM PSA PSP PT QA QC QM QMS RA RAAS RBC RCA RCF RDS RF RGE RNA RPGN RPM RPR RTE SAAG SDS SD SF ch SG SKY SLE SP SPS SSA STI

National Kidney Disease Education Program nucleated red blood cell nephrotic syndrome neural tube defect optical density oral glucose tolerance test Occupational Safety and Health Administration plasma amino acids p-aminohippuric acid placental alpha microglobulin peripheral blood polymerase chain reaction proximal convoluted tubule postexposure prophylaxis phosphatidyl glycerol dissociation constant phenylketonuria preventive maintenance polymorphonuclear white blood cells point of care placental protein 12 personal protective equipment pre- and postmassage test premature rupture of membranes prostate-specific antigen phenolsulfonphthalein proficiency testing quality assessment quality control quality management quality management system rheumatoid arthritis renin–angiotensin–aldosterone system red blood cell root cause analysis relative centrifugal force respiratory distress syndrome rheumatoid factor rapid gastric emptying ribonucleic acid rapidly progressive (or crescentic) glomerulonephritis revolutions per minute rapid plasma reagin renal tubular epithelial (cells) serum-ascites albumin gradient Safety Data Sheet standard deviation surface channel specific gravity fluorescent mapping spectral karyotyping systemic lupus erythematosus Standard Precautions sodium polyanethol sulfonate sulfosalicylic acid sexually transmitted infection

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Abbreviations TAT TC-BF THP TJC TLC Tm TMB TMG

turnaround time total nucleated body fluid Tamm-Horsfall protein The Joint Commission thin-layer chromatography maximal reabsorptive capacity/tubular reabsorptive maximum 3,3⬘,5,5⬘-tetramethylbenzidine maximal tubular reabsorption capacity for glucose

UP UTI VB1 VB2 VB3 VDRL VUR WBC WBC-BF WHO

Universal Precautions urinary tract infection voided bladder 1 voided bladder 2 voided bladder 3 Venereal Disease Research Laboratory vesicoureteral reflux white blood cell white blood cell body fluid World Health Organization

383

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Glossary accreditation The process by which a program or institution documents meeting established guidelines accuracy Closeness of the measured result to the true value acholic stools Pale-colored stools acrosomal cap Tip of a spermatozoa head, which contains enzymes for entry into an ovum active transport Movement of a substance across cell membranes into the bloodstream by electrochemical energy acute glomerulonephritis (AGN) Disease marked by the sudden onset of symptoms consistent with damage to the glomerular membrane acute interstitial nephritis (AIN) Inflammation of the renal interstitium and tubules, frequently caused by a reaction to a medication acute-phase reactants Low-molecular-weight plasma proteins associated with infection and inflammation acute tubular necrosis (ATN) Disorder affecting the renal tubular cells caused by decreased renal blood flow or toxic substances aerosol Fine suspension of particles in air afferent arteriole A small branch of the renal artery through which blood flows to the glomerulus of the kidney albinism An inherited condition marked by decreased production of melanin albuminuria Protein (albumin) in the urine aldosterone A hormone that regulates reabsorption of sodium in the distal convoluted tubule alimentary tract The digestive tract, including structures between the mouth and the anus alkaptonuria Homogentisic acid in the urine caused by a failure to inherit the gene responsible for the production of homogentisic acid oxidase aminoaciduria Disorder in which increased amino acids are present in the urine amniocentesis Transabdominal puncture of the uterus and amnion to obtain amniotic fluid amnion The membranous sac that contains the fetus and amniotic fluid amniotic fluid Liquid that surrounds the fetus during gestation amyloid material A starch-like protein–carbohydrate complex that is deposited abnormally in tissue in some patients in chronic disease states anaphylaxis Severe reaction caused by an autoimmune reaction to certain antigenic compounds andrology The study of diseases of the male reproductive organs antidiuretic hormone (ADH) Hormone produced by the hypothalamus to regulate water reabsorption in the collecting duct

antiglomerular basement membrane antibody Autoantibody against alveolar and glomerular capillary basement membranes found in patients with Goodpasture syndrome antineutrophilic cytoplasmic antibody (ANCA) Autoantibody that reacts with neutrophils in the renal tubules; provides a laboratory test for granulomatosis with polyangiitis anuria Complete stoppage of urine flow arachnoid granulations Projections on the arachnoid membrane of the brain through which cerebrospinal fluid is reabsorbed arthritis Inflammation of the synovial joints arthrocentesis The puncture of a joint to obtain synovial fluid ascites Abnormal accumulation of peritoneal fluid ascitic fluid Watery fluid that accumulates in the peritoneal cavity in certain disease conditions ascorbic acid A strong reducing agent that prevents oxidation of the chromogen and may produce false-negative results on some tests astrocytomas Tumors of the brain and spinal cord atrophic vaginitis Syndrome in postmenopausal women caused by reduced estrogen production auto-checks Automatic checks performed by semiautomated analyzers to identify strip type and humidity exposure auto particle recognition (APR) Capability of some equipment to categorize and count urine particles automatically in uncentrifuged urine based on size, shape, texture, and contrast autovalidated Automatic release of test results into the patient record autoverification An automated comparison of patient results within laboratory analyzers with predetermined, preapproved criteria programmed into them azotemia Increased nitrogenous waste products in the blood bacterial endocarditis Inflammation of the endocardial membrane of the heart caused by bacterial infection bacterial vaginosis (BV) Inflammation of the vagina most often caused by Gardnerella vaginalis bacteriuria Bacteria in the urine basal cells Cells located in the basal layer of the vaginal stratified epithelium beta2 microglobulin A subunit of the class I major compatibility antigens that enters the blood at a constant rate bilirubin A bright yellow pigment produced in the degradation of heme biliverdin Green pigment formed by the oxidation of bilirubin biohazardous Pertaining to a hazard caused by infectious organisms birefringent The ability to refract light in two directions 385

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386 Glossary blood–brain barrier The barrier between the brain tissue and capillary blood that controls the passage of substances in the blood to the brain and cerebrospinal fluid body substance isolation A guideline stating that all moist body substances are capable of transmitting disease Bowman capsule Part of the nephron that contains the glomerulus bright-field microscopy A procedure by which magnified images appear dark against a bright background bronchoalveolar lavage (BAL) Diagnostic procedure by which cells and other components from bronchial and alveolar spaces are obtained for various studies bronchoscopy Procedure during which a fiber-optic bronchoscope is guided into a selected bronchopulmonary segment, usually the right middle or lingular lobe of the lung; however, target areas are better defined using highresolution computed tomography (HRCT) before the procedure bulbourethral glands Two small glands located on each side of the prostate gland carcinogenic Capable of causing cancer casts Elements excreted in the urine in the shape of renal tubules catheterized specimen A urine specimen collected by passing a sterile tube into the bladder chain of custody (COC) Step-by-step documentation of the handling and testing of legal specimens chain of infection A continuous link in the transmission of harmful microorganisms between a source and a susceptible host chemical hygiene plan (CHP) Protocol established for the identification, handling, storage, and disposal of all hazardous chemicals choroid plexuses A network of capillaries in the ventricle of the brain that produces cerebrospinal fluid chronic glomerulonephritis (CGN) Kidney disorder caused by slow, cumulative damage and scarring of the glomeruli; may lead to kidney failure and/or end-stage renal disease chylous effusion Occurs when chylous material accumulates in the pleural space, usually secondary to disruption of thoracic lymphatics chylous material A milky lymphatic fluid that contains triglycerides and chylomicrons cirrhosis Chronic liver disease that results in loss of liver cell function clarity Transparency of urine, ranging from clear to turbid clearance tests Standard tests used to measure the filtering capacity of the glomeruli Clinical Laboratory Improvement Amendments (CLIA) Governmental regulatory agency that establishes quality standards for laboratory testing Clinical and Laboratory Standards Institute (CLSI) Nonprofit organization that publishes recommendations for laboratory tests clue cells Squamous epithelial cells covered with the gramnegative bacteria Gardnerella vaginalis

coefficient of variation (CV) Standard deviation expressed as a percentage of the mean collecting duct Part of the nephron where the final concentration of urine takes place through the reabsorption of water colligative property Freezing point, boiling point, vapor pressure, or osmotic pressure property that is mathematically proportional to the concentration of a solution concentration tests Tests to determine the ability of the tubules to reabsorb the essential salts and water that have been nonselectively filtered by the glomerulus constipation Infrequent production of feces that results in small, hard stools control mean Average of all data points control range Limit within which expected control values lie, usually plus or minus two standard deviations from the mean countercurrent mechanism A selective urine concentration process in the ascending and descending loops of Henle creatinine A substance formed by the breakdown of creatine during muscle metabolism creatinine clearance A test used to measure the glomerular filtration rate crenated Shrunken and irregularly shaped or notched cylindroids Formation of casts at the junction of the ascending loop of Henle and the distal convoluted tubule may produce structures with a tapered end cylindruria The presence of urinary casts cystatin C Small protein produced at a constant rate by all nucleated cells cystinosis An inherited recessive disorder that disrupts the metabolism of cystine cystinuria Cystine in the urine that occurs as a result of a defect in the renal tubular reabsorption of amino acids cystitis An inflammation of the bladder cytogenetic analysis An analysis of cellular chromosomes dark-field microscopy Microscopic technique by which magnified images appear bright against a dark background delta check A quality management (QM) procedure that compares a patient’s test results with the previous results demyelination The destruction of the myelin sheath that protects a nerve density Concentration of solutes present per volume of solution desquamative inflammatory vaginitis (DIV) Syndrome characterized by purulent vaginal discharge, vaginal erythema, and dyspareunia diarrhea Watery stools diarthroses Freely movable joints Diazo reaction A reaction in various diseases consisting of a red discoloration of the urine on addition of diazobenzenesulfonic acid digital imaging Capability of some equipment to capture detailed images of urine particles in urine samples that require further review disinfectant A substance that destroys microorganisms that is used on surfaces rather than the skin

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Glossary distal convoluted tubule Part of the nephron between the ascending loop of Henle and the collecting duct where the final concentration of urinary filtrate begins dysentery An inflammation of the intestines that is caused by microorganisms and results in diarrhea dysmorphic Irregularly shaped dyspareunia Abnormal pain during sexual intercourse dyspnea Difficulty breathing dysuria Painful urination edema An accumulation of fluid in the tissues efferent arteriole The small renal artery branch through which blood flows away from the glomerulus effusion An accumulation of fluid between the serous membranes Ehrlich reaction Urobilinogen reacts with p-dimethylaminobenzaldehyde (Ehrlich reagent) to produce colors ranging from light to dark pink electronic quality control Mechanical or electrical sample used in place of a liquid control to verify reliability of test results endogenous procedure A test that uses a substance originating within the body epididymis Small structure that forms the first part of the secretory duct of the testes erythrophagocytosis Engulfment of red blood cells by macrophages examination variable Processes that occur during testing of a sample exogenous procedure A test that requires a substance to be infused into the body external quality assessment (EQA) Testing of unknown samples received from an outside accrediting agency to provide validation of the quality of patient test results external quality control Commercial controls used to verify accuracy and reliability of patient test results exudate Serous fluid effusion caused by conditions producing damage to the serous membranes Fanconi syndrome A group of disorders marked by renal tubular dysfunction associated with some inherited and acquired conditions fenestrated endothelium The endothelial cells of the capillary wall that differ from those in other capillaries by containing pores ferritin A major storage form of iron found in the liver, spleen, and bone marrow fetal lung maturity (FLM) The presence of a sufficient amount of surfactant lipoproteins to maintain alveolar stability first morning specimen The first voided urine specimen collected immediately upon arising; recommended screening specimen flatus Gas expelled from the anus flow cytometry Used to measure the forward scattered, side scattered, and fluorescence light characteristics of particles present in urine fluorescence microscopy Microscopic technique by which naturally fluorescent substances or those that have been

387

stained by a fluorescent dye produce an image when illuminated with a light of a specific wavelength focal segmental glomerulosclerosis (FSGS) Disorder that affects only certain areas of the glomerulus and produces heavy proteinuria fomite Inanimate object that can serve as a reservoir for pathogenic organisms free water clearance A test to determine the ability of the kidney to respond to the state of body hydration fructosuria The presence of fructose in the urine galactosuria The presence of galactose in the urine Gardnerella vaginalis Rod-shaped bacteria that causes bacterial vaginitis gastrocolic fistula Abnormal passageway between the stomach and the colon gestational diabetes Diabetes that some women get during pregnancy in which the body has too much glucose in the blood; glucose crosses the placenta but insulin does not, so the baby develops high glucose levels ghost cells Red blood cells that have lost their hemoglobin, leaving only the cell membrane; appear in hyposthenuric urine Globally Harmonized System (GHS) An international effort to standardize both the classification of hazardous chemicals and the symbols used to communicate these hazards on labels and in Safety Data Sheet (SDS) documentation glomerular filtration barrier Structure of the walls of the glomerular capillaries that prevents the filtration of large molecules from the blood into the urine filtrate glomerular filtration rate (GFR) The volume of plasma that is filtered by the glomerulus in a specified time glomerulonephritis An inflammation of the glomerulus that results in impaired glomerular filtration glomerulus Tuft of capillary blood vessels located in Bowman capsule where filtration occurs glucosuria The amount of glucose in the glomerular filtrate exceeds the capacity of the renal tubule to reabsorb it glycogenesis The conversion of glucose to glycogen glycogenolysis The conversion of glycogen to glucose glycosaminoglycans A group of large compounds located primarily in the connective tissue; also known as mucopolysaccharides glycosuria Glucose in the urine (glucosuria) Goodpasture syndrome An autoimmune disorder characterized by morphological changes to the glomeruli resembling those in rapidly progressive glomerular nephritis gout Disorder related to elevated serum uric acid that results in the accumulation of uric acid crystals in a moveable joint granuloma Modular accumulation of inflammatory cells granulomatosis with polyangiitis (GPA) Disorder that causes a granuloma-producing inflammation of the small blood vessels primarily of the kidney and respiratory system; formerly called Wegener granulomatosis Greiss reaction Nitrite at an acidic pH reacts with an aromatic amine (para-arsanilic acid or sulfanilamide) to form

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388 Glossary a diazonium compound that then reacts with tetrahydrobenzoquinolin compounds to produce a pink-colored azo dye Guillain-Barré syndrome Autoimmune disorder that causes destruction of the myelin sheath that surrounds the peripheral nerves, resulting in loss of motor function Hartnup disease A recessive inherited disorder marked by intestinal absorption abnormalities and renal aminoaciduria hematoidin Yellow, crystalline substance that results from the destruction of red blood cells hematuria Blood in the urine hemoglobinuria Hemoglobin in the urine hemolytic disease of the fetus and newborn (HDFN) Rh incompatibility between mother and fetus that can cause hemolysis of the fetal red blood cells hemoptysis Blood in the sputum hemosiderin An insoluble form of storage iron; a product of red blood cell hemolysis hemothorax The accumulation of blood in the pleural cavity Henoch-Schönlein purpura Disease involving inflammation of small blood vessels, causing them to start leaking; occurs primarily in children after upper respiratory infections histogram A graphical display of data using bars of different heights homocystinuria The presence of homocysteine in the urine caused by an inherited autosomal recessive disorder hyaluronic acid Glycosaminoglycan found in synovial fluid that provides lubrication to the joints hydrostatic pressure Pressure exerted by a liquid hyperglycemia Elevated glucose levels in the blood hypernatremia Elevated blood sodium levels hypersthenuric Pertaining to urine specific gravity greater than the 1.010 of the glomerular filtrate hyponatremia Decreased blood sodium levels hyposthenuric Pertaining to urine specific gravity lower than the 1.010 of the glomerular filtrate hypoxia Lack of oxygen iatrogenic Pertaining to a condition caused by treatment, medications, or diagnostic procedures IgA nephropathy Damage to the glomerular membrane caused the deposition of IgA immune complexes on the membrane immune complexes Antigen–antibody combinations in vitro fertilization (IVF) Fertilization between an ovum and a sperm performed in the laboratory inborn error of metabolism (IEM) Failure to inherit the gene to produce a particular enzyme indicanuria The presence of indican in the urine infection control Procedures to control and monitor infections within an institution infertility The inability to conceive interference-contrast microscopy A procedure by which three-dimensional images of a specimen are obtained internal quality control Electronic, internal, and procedural controls contained within the test system that ensure its reliability

interstitial Pertaining to spaces between tissue cells ischemia Deficiency of blood to a body area isosthenuric Pertaining to urine specific gravity the same as the 1.010 of the glomerular filtrate jaundice Yellow appearance of skin, mucous membranes, and eye sclera due to increased amounts of bilirubin in the blood juxtaglomerular apparatus Specialized cells located on the afferent arteriole that regulate secretion of renin ketonuria Ketones in the urine Köhler illumination Adjustments made to the microscope condenser when objectives are changed labia The outer folds of the vagina lactobacilli A group of gram-positive rod-shaped bacteria lactosuria The presence of lactose in the urine lamellar bodies Organelles produced by type II pneumonocytes in the fetal lung that contain lung surfactants Langerhans cells Pancreatic cells lecithin Phospholipid that forms part of the cell wall used to determine fetal lung maturity lecithin-sphingomyelin ratio (L/S ratio) A comparison of lung surfactants that is performed to determine fetal lung maturity Lesch-Nyhan disease An inherited sex-linked recessive purine metabolism disorder marked by excess uric acid crystals in the urine leukocyturia Leukocytes (white blood cells) in the urine light-emitting diode (LED) A semiconductor diode that emits light when conducting current lipiduria The presence of lipids in the urine lipophages Macrophages that have ingested fat globules seen in peritoneal fluid liquefaction The conversion of solid or coagulated material to a liquid form lithiasis The formation of renal calculi (kidney stones) lithotripsy A procedure that uses ultrasonic waves to crush renal calculi loops of Henle The U-shaped part of the renal tubule that consists of a thin descending limb and a thick ascending limb lysosomes Cellular organelles that contain digestive enzymes macula densa Specialized cells located on the distal convoluted tubule that interact with the juxtaglomerular cells malabsorption Impaired absorption of nutrients by the intestine maldigestion Impaired digestion of foodstuffs Maltese cross formation Cross-shaped inclusions in the urinary sediment of patients with nephrotic syndrome seen under polarized light due to the birefringence of lipid droplets maple syrup urine disease (MSUD) An autosomal recessive trait that causes increased levels of the branched-chain amino acids leucine, isoleucine, and valine and their ketone acids in the urine maximal reabsorptive capacity (Tm) The maximum reabsorption ability for a solute by renal tubules meconium The dark-green mucus-containing stool formed by a fetus

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Glossary medullary interstitium Spaces between the cells in the medulla of the kidney that contain highly concentrated fluid medulloblastomas Malignant tumor of the fourth ventricle and cerebellum melanoma A tumor of the melanogen-producing cells, which frequently is malignant melanuria Increased melanin in the urine melituria Increased urinary sugar membranous glomerulonephritis (MGN) Type of glomerulonephritis that develops when inflammation of kidney structures causes problems with kidney function membranoproliferative glomerulonephritis (MPGN) Specific type of glomerular disease that is characterized by immune complex deposits in the kidneys glomerular mesangium and a thickening of the basement membrane; occurs when the body’s immune system functions abnormally meninges Protective membranes around the brain and spinal cord meningitis Inflammation of the meninges, frequently caused by microbial infection mesothelial cells Cells that line the serous membranes metabolic acidosis A decrease in the blood pH caused by a metabolic increase in acidic elements microalbuminuria Low levels of urine protein that are not detected by routine reagent strips midstream clean-catch specimen Specimen collected in a sterile container after cleansing the glans penis or urinary meatus; the first portion of urine is voided into the toilet, the midportion is collected, and the remaining portion is voided into the toilet minimal change disease (MCD) Disease that produces little cellular change in the glomerulus, except for some damage to the podocytes and the shield of negativity, allowing for increased protein filtration; also known as lipid nephrosis or nil disease Mobiluncus spp. Curved, gram-variable, rod-shaped bacteria mucin Glycoprotein found in mucus and in the skin, connective tissues, tendons, and cartilage mucopolysaccharides (MPS) Glycosaminoglycans that consist of a protein core with polysaccharide branches mucopolysaccharidoses (MPSs) A group of genetic disorders marked by excess mucopolysaccharides in blood and urine multiple myeloma Malignant disorder that results in infiltration of the bone marrow by plasma cells myoglobin Iron-containing protein found in muscle tissue myoglobinuria Myoglobin in the urine nephron A functional unit of the kidney that forms urine nephropathy Disease of the kidneys nephrotic syndrome (NS) A renal disorder marked by massive proteinuria, lipiduria, and edema caused by disruption of the glomerular membrane neutrophages Vacuolated macrophages containing phagocytized neutrophils; formerly called Reiter cells nocturia Excessive urination during the night

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occult blood Blood that is not visible to the naked eye Occupational Safety and Health Administration (OSHA) The government agency created to protect employees from potential health hazards in the workplace through the development and monitoring of regulations ochronosis Deposit of brown pigment in the body tissues, particularly noticeable in the ears oligoclonal bands Electrophoretic bands migrating in the gamma region that are present in cerebrospinal fluid and serum oligohydramnios Decreased amniotic fluid oliguria A marked decrease in urine flow oncotic pressure The osmotic pressure of a substance in solution caused by the presence of colloids organic acidemias The accumulation of organic acids in the blood, mainly isovaleric, propionic, and methylmalonic acids orthostatic proteinuria Increased protein in urine only when an individual is in an upright position osmolality The osmotic pressure of a solution expressed in milliosmoles per liter; it is affected only by the number of particles present osmolar clearance The amount of plasma filtered each minute to produce a urine with the same osmolarity as plasma osmolarity The osmotic pressure of a solution expressed in milliosmoles per kilogram; it is affected only by the number of particles present osmotic diarrhea An increased retention of water and solutes in the large intestine associated with malabsorption and maldigestion osmotic gradient The difference in the concentration of substances on either side of a membrane oval fat bodies Lipid-containing renal tubular epithelial (RTE) cells pancreatic insufficiency The decreased ability of the pancreas to secrete digestive enzymes parabasal cell Cells located in the luminal squamous epithelium of the vaginal mucosa paracentesis Surgical puncture into the abdominal cavity to obtain peritoneal fluid parietal membrane Serous membrane that lines the walls of the pleural, pericardial, and peritoneal cavities passive transport Movement of molecules across a membrane by diffusion because of a physical gradient pentosuria The presence of pentose sugars in the urine pericardiocentesis Surgical puncture into the pericardial cavity to obtain pericardial fluid pericarditis An inflammation of the membranes enclosing the heart peritoneal lavage Introduction and subsequent removal of fluid into the peritoneal cavity to detect the presence of abnormal substances peritonitis An inflammation of the membranes that line the peritoneal cavity peritubular capillaries The capillaries that surround the renal tubules

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390 Glossary personal protective equipment (PPE) Items used to protect the body from infectious agents phase-contrast microscopy Procedure in which magnified images show varied intensities of light and dark and are surrounded by halos phenazopyridine Medication for urinary tract infection that produces a thick, orange urine specimen phenylketonuria (PKU) The presence of abnormal phenylalanine metabolites in the urine phosphatidyl glycerol Phospholipid found in amniotic fluid that is used to confirm fetal lung maturity pigmented villonodular synovitis Proliferation of synovial cells forming brown nodules, resulting in inflammation, pain, and hemorrhagic effusions pleocytosis Increased numbers of normal cells in the cerebrospinal fluid podocytes Epithelial cells of the inner lining of Bowman capsule that contain foot-like processes polarizing microscopy A procedure in which magnified birefringent images appear bright or colored against a black background polydipsia Excessive thirst polyhydramnios Excessive amniotic fluid polyuria Marked increase in urine flow porphobilinogen Immediate precursor of the porphyrins involved in the synthesis of heme porphyrias Disorders of porphyrin metabolism that are inherited or acquired porphyrins Intermediate compounds in the synthesis of heme porphyrinuria The presence of porphyrins in the urine postexamination variable Process that affects the reporting and interpretation of test results postexposure prophylaxis (PEP) Preventative treatment provided after exposure to a potentially harmful agent postrenal proteinuria Increased protein in the urine caused by infections/inflammation that add protein to the urine after its formation precision Reproducibility of a test result preexamination variable Process that occurs before collection of a sample prerenal proteinuria Increased protein in the urine caused by factors affecting the plasma before it reaches the kidney preventive maintenance (PM) Checks on instruments and equipment on a regular schedule proficiency testing (PT) Performance of tests on specimens provided by an external monitoring agency prostate gland Muscular gland surrounding the male urethra protein error of indicators Indicators change color in the presence of protein at a constant pH proteinuria Protein in the urine (albuminuria) proximal convoluted tubule The nearest tubule to the glomerulus where reabsorption of essential substances begins pruritus Symptom of itching

pseudochylous effusion Cloudy to opalescent effusions that are not of primary lymphatic origin but arise from the breakdown products of any chronic effusion pseudochylous material Milky effusion that does not contain chylomicrons pulmonary infarction Blockage of the pulmonary artery resulting in destruction of lung tissue pseudogout Arthritic disorder caused by the accumulation of calcium pyrophosphate crystals in a moveable joint purpura Small capillary hemorrhages pyelonephritis Infection of the renal tubules pyknotic Referring to a dense, round nucleus pyuria The presence of white blood cells (pus) in the urine quality assessment (QA) Methods used to measure quality patient care quality control (QC) Methods used to monitor the accuracy of procedures quality management (QM) The overall process of guaranteeing quality patient care, regulated throughout the total testing system quality management system (QMS) All of the laboratory’s policies, processes, procedures, and resources needed to achieve quality testing quality system The overall laboratory policies, procedures, processes, and resources to achieve quality test results radioisotope A substance that emits radiant energy ragocytes Neutrophils that contain ingested clumps of IgG random specimen Urine collected at any time without prior patient preparation rapidly progressive (or crescentic) glomerulonephritis (RPGN) A more serious form of acute glomerular disease that often terminates in renal failure reflectance photometry A lamp and monochromator provide an incident light beam that is directed to a colored reaction product refractive index A comparison of the velocity of light in the air with the velocity of light in a solution refractometry Measurement of the light-bending capability of solutions Reiter cells Vacuolated macrophages containing ingested neutrophils associated with nonspecific arthritic inflammation; see also neutrophages reliability The ability to maintain both precision and accuracy renal plasma flow The volume of plasma passing through the kidneys per minute renal proteinuria Protein in the urine caused by impaired renal function renal threshold Plasma concentration of a substance at which active transport stops and increased amounts are excreted in the urine renal tubular acidosis The inability to produce an acidic urine in the presence of metabolic acidosis renin Proteolytic enzyme produced by the kidney that reacts with angiotensinogen to produce angiotensin to increase blood pressure

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Glossary renin–angiotensin–aldosterone system (RAAS) Regulates flow of blood to and within the kidneys by responding to changes in blood pressure and plasma sodium content resolution The ability to separate fine structures for visualization of detail respiratory distress syndrome (RDS) Disease caused by lack of lung surfactant retinoblastomas Malignant glioma of the retina seen in young children rhabdomyolysis Muscle destruction rheumatoid factor Immunoglobulin associated with rheumatoid arthritis root cause analysis (RCA) A systematic process for identifying “root causes” of problems or events and an approach for responding to them sarcoidosis Multisystem disease caused by infiltration of the organs by T lymphocytes and phagocytes that form granulomas in the tissues Safety Data Sheet (SDS) A document provided by the vendor or manufacturer of a chemical substance that describes the chemical’s characteristics scattergram A graphic representation of points referencing two variables secretory diarrhea The increased secretion of water and electrolytes into the large intestine caused by bacterial enterotoxins semen Fluid containing spermatozoa seminal vesicles Two sac-like structures close to the prostate that produce the majority of the seminal fluid seminiferous tubules Tubules that produce or conduct semen sensitivity The lowest level of an analyte that a test can detect serous fluid Fluid formed as a plasma ultrafiltrate that provides lubrication between the parietal and visceral serous membranes serum-ascites albumin gradient (SAAG) Calculation used to distinguish if a peritoneal effusion is a transudate or an exudate shield of negativity Negative ions in the glomerular filtration barrier that prevent small proteins, such as albumin, from entering the urine filtrate shift Abrupt change in the mean of a series of results Sjögren syndrome An autoimmune disorder in which autoimmune white blood cells (WBCs) attack the moistureproducing glands specific gravity The density of a solution compared with that of a similar volume of distilled water, influenced by both the number and size of the particles present specificity The likelihood of measuring the analyte desired spermatids Immature spermatozoa spermatozoa Sperm cells sphingomyelin Phospholipid found in amniotic fluid used to determine fetal lung maturity standard deviation (SD) Measurement statistic that indicates the average distance each data point is from the mean

391

Standard Precautions (SP) Guidelines recommended by the Centers for Disease Control (CDC) describing personnel protective practices steatorrhea Excess fat in the feces stercobilinogen Substance derived from urobilinogen that is found in the feces and is oxidized to form urobilin, forming the brown color of feces stools Fecal material discharged from the large intestine subarachnoid space The area between the arachnoid and pia mater membranes suprapubic aspiration The technique used to obtain sterile urine specimens for bacterial culture or cytological examination in which a sterile needle is introduced through the abdomen into the bladder surfactants Phospholipids secreted by type II pneumocytes to maintain alveolar integrity syncytia A group of cells with continuous adjoining cell walls synovial fluid Plasma ultrafiltrate that contains hyaluronic acid and provides lubrication of the joints synoviocytes Cells in the synovial membrane that secrete hyaluronic acid systemic lupus erythematosus (SLE) Autoimmune disorder that affects the connective tissue and results in damage to organs, particularly the kidneys and joints Tamm-Horsfall protein (THP) Mucoprotein found in the matrix of renal tubular casts (see uromodulin) tamponade Buildup of pericardial fluid affecting the heart test utilization Correct procedures for ordering a test thoracentesis Surgical puncture into the thoracic cavity to collect pleural fluid thrombosis Formation of a blood clot timed specimen Urine specimen collected over an interval of time for a quantitative analysis of a urine chemical, usually a 24-hour collection titratable acidity Hydrogen ions in the urine that can be quantitated by titration with a base to a pH of 7.4 transudate Serous effusion produced as a result of disruption of fluid production and regulation between the serous membranes traumatic tap Surgical puncture contaminated with capillary blood trend Gradual change in one direction of the mean of a control substance Trichomonas vaginalis Flagellated protozoan that infects the vagina trichomoniasis Vaginal infection caused by Trichomonas vaginalis tubular reabsorption Substances moved from the tubular filtrate into the blood by active or passive transport tubular secretion The passage of substances from the blood in the peritubular capillaries to the tubular filtrate tubulointerstitial disease Renal disease that affects both the renal tubules and renal interstitium turnaround time (TAT) Time from ordering a test through analysis in the laboratory to the charting of the report

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392 Glossary tyrosyluria The presence of tyrosine in the urine Universal Precautions (UP) Centers for Disease Control (CDC) guideline stating that all patients are capable of transmitting bloodborne disease urobilin The oxidized form of urobilinogen that provides the brown color to feces urobilinogen A compound formed in the intestines by the bacterial reduction of bilirubin urochrome Yellow pigment produced by endogenous metabolism that imparts the yellow color to urine uroerythrin Pink pigment in urine derived from melanin metabolism that attaches to urates in the sediment uromodulin Glycoprotein that is the only protein produced by the kidney tubules; forms the matrix of casts (formerly called Tamm-Horsfall protein) uromodulin-associated kidney disease Autosomal mutation of the gene producing uromodulin that results in the destruction of the renal tubular cells vaginal pool Mucus and cells in the posterior fornix of the vagina

vaginitis Inflammation of the vagina vasa recta A network of capillaries that surrounds the loop of Henle vasectomy Surgical removal of all or part of the vas deferens for the purpose of male sterilization vasopressin Antidiuretic hormone that regulates reabsorption of water by the collecting ducts vasovasostomy Repair of a severed vas deferens to restore fertility vesicoureteral reflux (VUR) Urine in the bladder passing back into the ureters visceral membrane The serous membrane covering the organs contained within a cavity viscosity The amount of resistance to flow in a liquid vulvovaginal candidiasis Inflammation of the vulva, vagina, or vulvovaginal glands caused by Candida albicans xanthochromia Yellowish discoloration of the cerebrospinal fluid yeast Fungus that reproduces by budding

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Index Page numbers follow by f indicate figures; page numbers followed by t indicate tables.

A

Abnormal urine color, 127–129, 128f, 129f Abnormal urine crystals, 34–37, 206–209, 207t, 208–209f Accreditation agencies, 62 Accuracy, 66 ACE. see angiotensin-converting enzyme (ACE) Acetest tablets, 152 Acetic acid stain, 171, 171t Acetylcholinesterase (AChE), 331 Acholic stools, 343 Acid-albumin test, 247 Acid-base balance, 111–113 Acidic urine, crystals in, 26–29, 201–204, 202–204f Acidosis metabolic, 113 renal tubular, 113 Acrosomal cap, 284 Actim PROM test, 366 Active transport, 110, 110t Acute glomerulonephritis (AGN), 220, 223t, 224t Acute intermittent porphyria, 245t Acute interstitial nephritis (AIN), 228, 228t, 229b Acute-phase reactants, 144 Acute pyelonephritis, 226–227, 228t, 229b Acute renal failure (ARF), 225 Acute tubular necrosis (ATN), 222, 227t Addis, Thomas, 92 Addis count, 170 ADH. see antidiuretic hormone (ADH) ADVIA2120i with Body Fluids Software, 77t, 87 Afferent arteriole, 106, 109f AGN. see acute glomerulonephritis (AGN) AHG. see antihuman globulin (AHG) AIN. see acute interstitial nephritis (AIN) Air bubble artifacts in urinary sediment, 40, 210 Albinism, 239 Albumin, reagent strips reaction to, 147 Albumin:creatinine ratio, 147 Albumin/protein:creatinine ratio, 148 Albuminuria, 92 Alcohol-based cleansers, 54 Aldosterone, 109 Alimentary tract, 340 Alkaline urine, crystals in, 30–33, 204–205, 204–206f Alkaptonuria, 129, 236, 239 Alpha-fetoprotein (AFP), 328t, 329, 332 Alport syndrome, 224t, 225, 225t Altered motility, 341–342 Amber urine, 127–128 American Association for Laboratory Accreditation (A2LA), 62 American Association of Blood Banks (AABB), 62 American Osteopathic Association (AOA), 62

American Society of Histocompatibility and Immunogenetics (ASHI), 62 Amino acid disorders, 236–243 branched-chain, 239–240, 240f phenylalanine-tyrosine disorders, 236–239, 238f Ammonia, 113 measurement, 119 Ammonium, 94t Ammonium biurate, 33, 202t, 205, 206f Amniocentesis, 329–330, 330b Amnion, 328, 328f Amniostat-FLM, 328t Amniotic fluid, 328–335 chemical composition, 329 color and appearance, 331, 331t cytogenetic analysis of, 328, 328t differentiating maternal urine from, 329 function, 328, 328f physiology, 328–329, 328f specimen collection, 329–330, 330b specimen handling and processing, 330–331 tests for fetal distress, 331–332, 332–333f tests for fetal maturity, 333–335 volume, 328–329 AmniSure test, 366 Amorphous phosphates, 30, 201t, 204, 204f Amorphous urates, 27, 201–202, 201t, 202f Ampicillin crystals, 37, 207t, 209, 210f Amyloid material, 145 Anaphylaxis, 222 ANCA. see antineutrophilic cytoplasmic antibody (ANCA) Andrology, 278 Angiotensin-converting enzyme (ACE), 109 Antidiuretic hormone (ADH), 93, 118 Antiglomerular basement membrane antibody, 221 Antihuman globulin (AHG), 287 Antineutrophilic cytoplasmic antibody (ANCA), 221 Antinuclear antibody (ANA), 312 Antisperm antibodies, 286–287 Anuria, 93 APT test (fetal hemoglobin), 348–349 Arachnoid, 254, 254f Arachnoid granulations, 254 ARF. see acute renal failure (ARF) ART. see assisted reproductive technology (ART) Arthritis. see synovial fluid Arthrocentesis, 294 Artifacts, urinary sediment, 38–40, 209–211, 210–211f Artificial imaging identification (AII) software, 86 Ascites, 314 Ascitic fluid, 314 Ascorbic acid, 150, 154 Assisted reproductive technology (ART), 278, 288 Astrocytomas, 266 ATN. See acute tubular necrosis (ATN) Atrophic vaginitis, 356t, 357, 365

AUTION Eleven AE-4022, 77t, 78f AUTION HYBRID AU-4050, 77t AUTION MAX AX-4030, 77t, 80f Auto image evaluation module (AIEM) software, 85 Automated body fluid analysis, 77t, 87–88, 88t semen analysis, 283 Automated microscopy, 77t, 79–86, 80–85f Automated urinalysis, 76–88 automated microscopy, 77t, 79–86, 80–85f combination systems, 86–87, 86f fully automated chemistry analyzers, 77t, 78–79 reflectance photometry, 76, 76t semi-automated urine chemistry analyzers, 76–78, 77t, 78–79f Autovalidated results, 81 Autoverification, 69

B

Bacteria, 11, 42, 171t, 187–188, 188f bronchoalveolar lavage (BAL) fluid, 324, 324b casts, 20, 195 diarrhea and, 341–342 fecal, 343–344 pleural fluid testing for, 312 vaginal secretions, 360–361, 360–362f Bacterial endocarditis, 312 Bacterial vaginosis (BV), 356, 356t, 364 Bacteroides, 360, 363 BAL. see bronchoalveolar lavage (BAL) Basal cells, 358, 360 Bence Jones protein, 144 Berger disease, 221–222 Beta2-microglobulin (B2M), 113, 116 Bilirubin, 127–128 amniotic fluid, 331, 332f in amniotic fluid, 328t clinical significance of, 155, 155t crystals, 36, 207t, 208, 209f in jaundice, 155t in peritoneal fluid, 316 production of, 154–155, 155f reagent strip reactions, 155–156 interference, 156 Biliverdin, 128 Biohazardous material, 51, 52f decontamination, 54–56 symbol for, 56f Biological hazards, 50–56, 50t, 52f infectious agent, 50 reservoir, 50–51 Standard Precautions, 51–54 Birefringent, 176 Black urine, 129 Blast forms in cerebrospinal fluid (CSF), 261t Blood casts, 172t in cerebrospinal fluid (CSF), 256, 258, 262f, 263

393

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394

Index

in feces, 345–347, 346f traumatic tap, 256–257 in urine, 128, 128f, 129f, 152–154 clinical significance, 153 reagent strip reactions, 153–154 interference, 154 Blood-brain barrier, 254 Blood flow, renal, 106, 108b tests, 119 Blood urea nitrogen (BUN), 220 Blue urine, 129 Body fluid analyzers, 77t, 87–88, 88t Body fluids, 41–46 cerebrospinal fluid, 41–42 peritoneal fluid, 43 pleural fluid, 44–45 synovial fluid, 46 Body fluid white blood cell count (WBC-BF), 88 Body surface area, 115, 115f Bowman capsule, 107, 221 Brain, 254–255f. see also cerebrospinal fluid (CSF) Branched-chain amino acids disorders, 239–240, 240f Bright, Richard, 92 Bright-field microscopy, 174f, 176 Broad casts, 25, 198–199, 199f Bronchoalveolar lavage (BAL) fluid, 322–325 cell counts, 322–323 clinical significance, 322 color and clarity, 322 cytology studies, 324–325 eosinophils in, 323–324 epithelial cells in, 324, 324f erythrocytes in, 324 leukocytes in, 323 lymphocytes in, 323, 323t macrophages in, 323, 323f mast cells in, 324 microbiological tests, 324, 324b, 325f neutrophils in, 323 specimen collection, 322 specimen handling and transport, 322 Bronchoscopy, 322 Brown urine, 128–129 Bryant, Thomas, 92 Bulbourethral glands, 278 Burkitt lymphoma, 266f

C

Calcium, 94t Calcium carbonate, 32, 202t, 205, 205f Calcium oxalate, 28–29, 201t, 203–204, 204f Calcium phosphate, 32, 202t, 205 Calibration/calibration verification, 62 Campylobacter, 341, 343 Candidiasis, 364–365 Candida albicans, 12 Carbohydrate disorders, 247 Carbohydrates in feces, 349 Carcinoembryonic antigen (CEA), 312 Cardiac tamponade, 312 Cardiopulmonary resuscitation (CPR), 60 CASA. See Computer-assisted semen analysis (CASA) Casts, 17–25, 168, 171t bacterial, 20, 195 blood (hemoglobin), 172t

broad, 25, 198–199, 199f coarse granular inclusion, 172t composition and formation, 191–192 detection of, 169–170 epithelial cell, 20, 195, 195–196f fatty, 23, 172t, 177, 196, 196f, 210 gram stain, 173 granular, 21–22, 197–198, 197–198f hyaline, 17–18, 171, 172t, 178–179, 190, 192, 192f lipid stains, 171 mixed cellular, 23, 196–197 Prussian blue stain, 173 RBC, 18, 180, 192–194, 192–194f red cell inclusion, 172t renal tubular epithelial cells, 185 waxy, 24, 172t, 198, 198f WBC, 19, 194–195, 194–195f Catheterized specimen, 97t, 98 CD4/CD8 T cells, 323, 323t Cell counts bronchoalveolar lavage (BAL) fluid, 322–323 cerebrospinal fluid (CSF), 257–258, 257f synovial fluid, 296 Cells, urinalysis, 2–10 epithelial cells, 5–6 oval fat bodies, 10 red blood cells, 2–3 renal tubular epithelial cells, 8–9 transitional epithelial (urothelial) cells, 7–8 white blood cells, 3–5 Cellular structure of glomerulus, 106–107, 109f Centers for Disease Control and Prevention (CDC), 50 on bloodborne pathogens exposure, 53 on hand washing, 54 on porphyrin disorders, 243 Central nervous system (CNS), 254 tumors of, 266 Centrifugation, macroscopic screening, 168–169 Cerebrospinal fluid (CSF), 41–42, 254–271 appearance of, 255–256, 256f, 256t cell count, 257–258, 257f chemistry tests, 266–268, 267b, 267f, 269t differential count, 258–266 cellular constituents, 259–266, 260–261t, 261–266f cytocentrifugation, 259, 259f, 259t neutrophils, 260, 261–262f formation and physiology, 254, 254–255f glucose, 268 glutamine, 268 lactate, 268 in malignancies, 265, 265–266f microbiology tests, 268–270, 270f gram stain, 269–270 immunologic assays, 270 PCR molecular diagnostic testing, 270 nonpathologically significant cells in, 263, 264–265f serological testing, 271 specimen collection and handling, 254–255, 255f traumatic collection (tap), 256–257 Cerebrospinal protein, 266–268, 267b, 267f Cetyltrimethylammonium bromide (CTAB) turbidity test, 247 Chain of custody (COC), 100

Chain of infection, 50–51, 52f Chemical hazards, 50t, 56–58, 58f Globally Harmonized System (GHS), 58 handling, 57 hygiene plan, 57 labeling, 57 Safety Data Sheets, 57–58 spills and exposure, 56–57 symbols, 59f waste disposal, 58 Chemical hygiene plan (CHP), 57, 58f Chemistry tests cerebrospinal fluid (CSF), 266–268, 267b, 267f, 269t feces, 345–349, 350t peritoneal fluid, 315–316, 315–316f pleural fluid, 310–312, 312t semen, 287, 287t synovial fluid, 300–301 Chlamydia trachomatis, 357, 364 Chloride, 94t Cholesterol crystals, 35, 206, 207t, 208f Choroidal cells, 261t Choroid plexuses, 254 Chronic glomerulonephritis (CGN), 221, 223t, 224t Chronic pyelonephritis, 227, 228t, 229b Chylous material, 308–309, 309t Clarity and appearance bronchoalveolar lavage fluid, 322 cerebrospinal fluid (CSF), 255–256, 256t feces, 343 pericardial fluid, 312 peritoneal fluid, 314 pleural fluid, 308–309, 308t semen, 280 synovial fluid, 295–296 urine, 129–131, 129t, 130b vaginal secretions, 357 Clearance tests, 113–115 Clinical and Laboratory Standards Institute (CLSI), 50, 93 Clinical Laboratory Improvement Amendments (CLIA), 67, 76 Clinical laboratory reagent water (CLRW), 64 Clinitek Advantus, 77t, 79f Clinitek Atlas, 77t, 80f, 86, 86f CLINITEK AUWi Pro System, 77t Clinitek Microalbumin reagent strips, 147, 148 Clinitek Novus, 77t Clinitek Status, 77t, 79f Clinitest, 150–151 Clostridium, 341 Clots, blood, 257 Clue cells, 5, 358 Coarse granular inclusion casts, 172t Cobas 6500 Urine Analyzer, 77t, 87 Cobas u 411 Urine Analyzer, 77t, 78f Cobas u 601 Urine Analyzer, 77t Cobus u 701 Microscopy Analyzer, 77t Coefficient of variation (CV), 67 Collecting duct, 111 Collecting duct cells, 8 College of American Pathologists (CAP), 62 Colligative properties, 133, 134t Color amniotic fluid, 331, 331t bronchoalveolar lavage fluid, 322 feces, 343

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Index peritoneal fluid, 314 pleural fluid, 308–309 semen, 280 synovial fluid, 295–296 urine, 126–127t, 126–129 vaginal secretions, 357 Commercial systems, macroscopic screening, 169 Commission on Laboratory Assessment (COLA), 62 Composition, urine, 93, 94t Computed tomography (CT), 314 Computer-assisted semen analysis (CASA), 283 Concentration tests, 117 Confirmatory tests, reagent strip, 142 Congenital erythropoietic porphyria, 245t Constipation, 341 Containers, specimen, 94 Copper reduction test, 150 Core channel (CR ch) stains, 81 Cough etiquette, 53 Countercurrent mechanism, 111 CPR. see Cardiopulmonary resuscitation (CPR) Creatinine, 94t in amniotic fluid versus maternal urine, 329 clearance tests, 113, 114–115, 115f reagent strips reaction to, 147–148 Crenated cells, 179, 179f Critical results, 69, 70f Cryptosporidium, 341 Crystals, synovial fluid, 296 types of, 297, 298t Crystals, urinary, 171t abnormal, 34–37, 206–209, 207t, 208–209f formation, 199 general identification techniques, 201, 201–202t normal acidic, 26–29, 201–204, 202–204f alkaline, 30–33, 204–205, 204–206f CSF. see Cerebrospinal fluid (CSF) Culture, vaginal secretions, 363 Cryptococcus neoformans, 270, 270f Cystatin C, 113, 116 Cystine crystals, 34, 206, 207t, 208f disorders, 242–243 Cystinosis, 34, 236, 242–243 Cystinuria, 34, 236, 242 Cystitis, 226, 228t, 229b Cytocentrifugation, 259, 259f, 259t Cytodiagnostic urine testing, 173 Cytology studies bronchoalveolar lavage (BAL) fluid, 324–325 pericardial fluid, 312–314, 313f

D

Dark-field microscopy, 174f, 177–178, 178f Dark yellow urine, 127–128 D-dimer test, 257 Dekkers, Frederik, 92 Delta check, 69 Density, 117 Depolarized side scatter (DSS) detector, 81 Desquamative inflammatory vaginitis (DIV), 356t, 357, 365 Diabetes insipidus, 94, 94t nephrogenic, 226, 227t osmolality in, 117, 118f

Diabetes mellitus, 94, 94t Diabetic nephropathy, 145, 224t, 226 Diarrhea, 341–342, 341–342t Differential count cerebrospinal fluid (CSF), 258–266 synovial fluid, 296–297 Differential interference-contrast microscopy, 177–178, 178f Digital imaging, 82 Dirui FUS – 100/200 with H-800, 77t Disinfectants, 55 Disposal of biological waste, 54, 56–57 Distal convoluted tubule, 106, 109f Distal tubular cells, 8 DNA testing, vaginal secretions, 363 Documentation on bloodborne pathogens, 53 Drug specimen collection, 100–101 Dura mater, 254, 254f DxH 900 Hematology Analyzer, 77t Dysentery, 343 Dysmorphic red blood cells, 2, 179, 180f Dyspareunia, 356 Dysuria, 356

E

Early dumping syndrome (EDS), 342 Education and competency assessment of personnel, 62 Efferent arteriole, 106 Effusions, 306, 307b chylous, 309, 309t pseudochylous, 309, 309t synovial fluid, 296 Ehrlich test, 243 Electrical hazards, 50t, 59–60 Electronic quality control, 62, 67 Emergency shower, 58f Endogenous procedures, 114 End-stage renal disease (ESRD), 221 Engineering controls for bloodborne pathogens, 53 Enterococcus, 360 Environmental control, 53 Environmental Protection Agency (EPA), 58 Enzyme-linked immunosorbent assay (ELISA), 270, 365–366 Enzymes in feces, 349 in synovial fluid, 301 Eosinophils, 3, 44, 46, 181, 182f in bronchoalveolar lavage (BAL) fluid, 323–324 in cerebrospinal fluid (CSF), 260, 261t, 263f in pleural fluid, 309 Ependymal cells, 261t Epithelial cells, 5–6, 171t, 182–187, 183f in bronchoalveolar lavage (BAL) fluid, 324, 324f casts, 20, 195, 195–196f oval fat bodies, 186–187, 186–187f renal tubular, 8–9, 184–186, 185–186f, 222 squamous, 182–184, 183–184f transitional, 7–8, 184, 184f Equipment maintenance, 62 Erythrocytes in bronchoalveolar lavage (BAL) fluid, 324 Erythropoietic protoporphyria, 245t Escherichia coli, 341, 343 Ethylenediaminetetraacetic acid (EDTA), 295

395

Examination variables, 61, 64–67, 66–68f Exogenous procedures, 114 External quality assessment (EQA), 62, 67 External quality control, 62, 66–67, 68f Exudates, 307, 308t, 314 Eye wash station, 58f

F

Face shields, 54 Fanconi syndrome, 145, 225, 227t Fat droplets in feces, 344–345, 344–345f, 348 in synovial fluid, 296 Fatty casts, 23, 172t, 177, 196, 196f, 210 Fecal analysis, 340–350 chemical testing, 345–349, 350t diarrhea and steatorrhea, 341–342, 341–342t macroscopic screening, 342–343, 343t microscopic examination, 343–345, 344–345f physiology of, 340–341, 340f specimen collection, 342 usefulness of, 340 Fecal enzymes, 349 Fecal material artifacts, 210, 210f Fecal occult blood testing (FOBT), 345–347, 346f Fecal trypsin, 349 Fenestrated endothelium, 107 Fern test, 329 Fetal fibronectin test (fFN), 365–366 Fetal lung maturity (FLM), 330, 333 Fetus in amniotic sac, 328, 328f AmniSure test and, 366 cytogenetic testing of, 328, 328t fetal fibronectin test (fFN) and, 365–366 hemolytic disease of the fetus and newborn (HDFN), 330, 331–332, 332–333f lung maturity, 330, 333 neural tube defects (NTDs) in, 329, 332 ROM/PROM tests, 366–367 Fibers, 39–40 Fibrin, 296 Fire/explosive hazards, 50t, 60–61, 61t First morning specimen, 96–97 5-hydroyindoleacetic acid, 241–242 Flatus, 340 Flow cytometry, 80, 323 Fluorescence in situ hybridization (FISH), 330 Fluorescence microscopy, 174f, 178, 178f Fluorescent mapping spectral karyotyping (SKY), 330 Fluorescent treponemal antibody-absorption (FTA-ABS), 271 Foam Stability Index, 328t, 334 Focal segmental glomerulosclerosis (FSGS), 222, 224t, 225t Focused assessment with sonography for trauma (FAST), 314 Food and Drug Administration (FDA), 67 Formation, urine, 93 Fomites, 50 Four-glass collection, 97t Free erythrocyte protoporphyrin (FEP), 243 Free water clearance, 118–119 Freezing-point osmometers, 117–118 Fructosuria, 247 Fully automated chemistry analyzers, 77t, 78–79

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396

Index

Fungi bronchoalveolar lavage (BAL) fluid, 322, 324b FUS-100 system, 77t, 86 FUS-200 system, 77t, 86

G

Galactokinase (GALK), 247 Galactose, 151 Galactose-1-phosphate uridyl transferase (GALT), 247 Galactosuria, 247 Gardnerella vaginalis, 358, 360, 363, 364 Gas chromatography-mass spectrometry (GC-MS), 239 Gastrocolic fistulas, 344 Ghost cells, 179 Globally Harmonized System (GHS), 58, 60f, 61b GloCyte Automated Cell Counter, 88 Glomerular disorders, 220–222 clinical information associated with, 224–225t glomerulonephritis, 220–222 laboratory testing in, 223–224t Glomerular filtration, 106–109, 109f renin-angiotensin-aldosterone system (RAAS), 106, 108–109, 110b, 110f tests, 113–116 Glomerular filtration barrier, 106, 109f Glomerular filtration rate (GFR), 106 estimated, 115–116 Glomerular pressure, 107–108 Glomerular proteinuria, 144–145 Glomerulonephritis, 220–222 acute glomerulonephritis (AGN), 220, 223t, 224t Alport syndrome, 224t, 225, 225t chronic glomerulonephritis (CGN), 221, 223t, 224t diabetic nephropathy, 145, 224t, 226 Fanconi syndrome, 225 focal segmental glomerulosclerosis (FSGS), 222, 224t, 225t Goodpasture syndrome, 220–221, 223t, 224t granulomatosis with polyangiitis (GPA), 221, 223t, 224t Henoch-Schönlein purpura, 221, 223t, 224t immunoglobulin A nephropathy, 221–222, 223t, 225t membranoproliferative glomerulonephritis (MPGN), 221, 223t, 224t membranous glomerulonephritis (MGN), 221, 223t, 224t minimal change disease (MCD), 222, 223t, 225t nephrogenic diabetes insipidus, 226 nephrotic syndrome (NS), 220, 222, 223t, 225t rapidly progressive (crescentic) glomerulonephritis (RPGN), 220, 223t, 224t renal glycosuria, 226 uromodulin-associated kidney disease, 226 Glomerulus, 106 Gloves, 51–52 starch granules from, 210 Glucose, 148–151 in amniotic fluid versus maternal urine, 329 cerebrospinal fluid (CSF), 268

clinical significance, 148–149, 149f Clinitest, 151 copper reduction test, 150 reaction interference, 150 reagent strip (glucose oxidase) reaction, 149 in synovial fluid, 300 Glucose tolerance specimens, 97 Glucosuria, 149 Glutamine, cerebrospinal fluid (CSF), 268 Glycosaminoglycans (GAGs), 246 Glycosuria, 149 Goggles, 54 Gold and mercury treatments, 221 Goodpasture syndrome, 220–221, 223t, 224t Gout, 297 Gowns, 52 Gram stain, 171t, 173 cerebrospinal fluid (CSF), 269–270 feces, 343 vaginal secretions, 356, 363, 363t Granular casts, 21–22, 197–198, 197–198f Granulomatosis with polyangiitis (GPA), 221, 223t, 224t Green urine, 129 Group A streptococcal enzyme tests, 220 Guaiac-based fecal occult blood tests (gFOBT), 345–346, 346f

H

Hair and fiber artifacts, 210, 211f Hand hygiene, 51, 52f, 54, 55–56 Hansel stain, 171t, 173 Harmonic oscillation densitometry, 133 Healthcare Infection Control Practices Advisory Committee (HICPAC), 51 Hematoidin crystals, 263 Hematuria, 152, 153 Heme formation, 244f Hemocytometer, 322 Hemoglobinuria, 152, 153, 154 Hemolytic disease of the fetus and newborn (HDFN), 330, 331–332, 332–333f Hemothorax, 308 Henoch-Schönlein purpura, 221, 223t, 224t Hepatic disease, 36 Hepatitis B, 221 Hereditary and metabolic tubular disorders, 225–226, 227t Herpes simplex virus (HSV), 357, 364 High-fluorescing body fluid cells (HF-BF), 88 Hippocrates, 92 Histograms, 80, 81 History of urinalysis, 92–93, 92–93f Hoesch test, 243, 246 Hoffman microscopy, 177 Homocystinuria, 243 Human gonadotropin (hCG), 66 Human immunodeficiency virus (HIV), 364 Human leukocyte antigen B12 (HLA-B12), 222 Hunter syndrome, 246 Hurler syndrome, 246 Hyaline casts, 17–18, 171, 172t, 178–179, 190, 192, 192f Hydrogen nuclear magnetic resonance spectroscopy (H NMR) method, 347 Hydrostatic pressure, 106, 306

Hyperglycemia, 149 Hypernatremia, 118 Hypersthenuric urine, 131 Hypertonic urine, 2, 3 Hyponatremia, 118 Hyposthenuric urine, 131 Hypoxia, 268

I

IChemVELOCITY, 77t, 80f Ictotest tablets, 156 Immunochemical fecal occult blood test (iFOBT), 346–347 ImmunoDip reagent strips, 147 Immunofixation electrophoresis (IFE), 268 Immunoglobulin A (IgA), 220 nephropathy, 221–222, 223t, 225t Immunoglobulin G (IgG) antihuman globulin (AHG), 287 cerebrospinal fluid (CSF), 266 Immunoglobulin M (IgM), 266 Inborn error of metabolism (IEM), 236 Indicanuria, 236, 241 Individualized quality control plan (IQCP), 68–69, 69f, 70f Infection control, 50, 52f Infectious agents, 50, 52f reservoir, 50–51 Instrumentation and equipment, 64, 66, 66f Insulin-like growth factor binding protein-1 (IGFBP-1), 329, 357 Integrity, specimen, 95–96, 95t Interference-contrast microscopy, 174f, 177 Internal quality control, 62, 67 International Organization for Standardization (ISO), 61 Interpreting results, 69 Interstitial disorders, 226–228, 228t, 229b Inulin clearance, 114 In vitro fertilization (IVF), 278, 288 IQ 200 Automated Urine Microscopy, 77t, 83, 85, 85f IQ200 body fluid analysis, 88 IQ 200 Sprint, 77t IQ 200 using Body Fluids Software, 77t IRICELL Urinalysis – IQ Series Urinalysis Workcell, 77t, 86, 86f Irritable bowel syndrome (IBS), 342 Isoelectric focusing (IEF), 268 Isosthenuric urine, 131 Isotope dilution mass spectrophotometry (IDMS) reference method, 115–116 IVF. see in vitro fertilization (IVF)

J

Jaundice, 154 The Joint Commission, 60, 62 Joint disorders. see synovial fluid Juxtaglomerular apparatus, 108, 109f

K

Ketones clinical significance, 151 reagent strip reactions, 152 interference, 152

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Index Ketonuria, 151 Köhler illumination, 175–176

L

Labeling chemical, 57, 59f specimen, 64, 64b, 95 Laboratory coats, 54 Laboratory information system (LIS), 76 LabUMat 2 system, 77t, 87 Lactate cerebrospinal fluid (CSF), 268 synovial fluid, 301 Lactobacilli, 360, 360f, 363 Lactoferrin latex agglutination test, 344 Lactosuria, 247 Lamellar body count, 328t, 334–335 density procedures, 334 Latex agglutination tests, 270 Lead poisoning, 245t Lecithin-sphingomyelin (L/S) ratio, 328t, 287–334 Lesch-Nyhan disease, 247 Leucine crystals, 34, 207t, 208, 209f Leukocyte esterase, 11, 23 clinical significance, 159 reagent strip reaction, 160 interference, 160 Leukocytes in bronchoalveolar lavage (BAL) fluid, 323 fecal, 343–344 Levy-Jennings control charts, 67, 67f, 81 Light-emitting diode (LED), 76 Liley graph, 331, 333f Linens, handling of, 53 Lipid stains, 171, 171t, 173 Lipiduria, 187 Lipophages, 315, 315f Liquid chromatography-tandem mass spectrometry (LC-MS/MS), 247 Liquefaction, semen, 278, 280 Lithotripsy, 229 Liver disease, 34, 35 Liver disorders, crystals associated with, 207–208, 208f Loops of Henle, 106 Lymphocytes, 3 in bronchoalveolar lavage (BAL) fluid, 323, 323t in cerebrospinal fluid (CSF), 41, 260, 260t, 262f in peritoneal fluid, 43 in pleural fluid, 44–45, 309 Lymphoma cells, 261t, 265–266f

M

Macrophages in bronchoalveolar lavage (BAL) fluid, 323, 323f in cerebrospinal fluid (CSF), 261t, 263, 263f in pleural fluid, 309 Macroscopic screening, 168t centrifugation, 168–169 commercial systems, 169 correlating results, 170, 171t fecal analysis, 342–343, 343t

reporting microscopic examination, 170 sediment examination, 169–170 sediment preparation, 169 specimen preparation, 168 specimen volume, 168 volume of sediment, 169 Macula densa, 108, 109f Malabsorption, 341 Maldigestion, 341 Malignancies cerebrospinal fluid (CSF) in, 261t, 265, 265–266f feces appearance with, 343 peritoneal fluid in, 315, 315–316f pleural fluid in, 309, 310, 310f, 311f, 312b Malignant melanoma, 129 Maltese cross formation, 176, 210 Maple syrup urine disease (MSUD), 236, 240 Masks, goggles, and face shields, 54 Mass spectrophotometry, tandem (MS/MS), 236, 237f, 243 Mast cells in bronchoalveolar lavage (BAL) fluid, 324 Maximal reabsorptive capacity, 111 Means of transmission, 51, 52f Meat fibers in feces, 344, 344f Meconium, 331 Medical controls for bloodborne pathogens, 53 Medullary interstitium, 106 Medulloblastomas, 266, 266f Melanuria, 236, 239 Melituria, 247 Membranoproliferative glomerulonephritis (MPGN), 221, 223t, 224t Membranous glomerulonephritis (MGN), 221, 223t, 224t Meninges, 254, 254f Meningitis, 254, 260, 269t Mercaptoethane sulfonate sodium (MESNA), 152 Mesothelial cells, 43, 44, 306 in pleural fluid, 309, 310f Metabolic acidosis, 113 Metabolic disorders, urine screening for, 236t Micral-Test reagent strips, 147 Microalbuminuria, 145 testing for, 146–147 Microbiological tests bronchoalveolar lavage (BAL) fluid, 324, 324b, 325f cerebrospinal fluid (CSF), 268–270, 270f peritoneal fluid, 316 pleural fluid, 312 semen, 287, 287t synovial fluid, 301 Microorganisms, 11–13 Microscopes, 173–176, 174f Microscopy, 168–211 automated, 77t, 79–86, 80–85f bacteria, 187–188, 188f cytodiagnostic urine testing, 173 differential interference-contrast, 177–178, 178f epithelial cells, 182–187, 183–187f expected staining reactions of, 172t feces, 343–345, 344–345f fluorescence, 174f, 178, 178f Köhler illumination, 175–176 macroscopic screening, 168–170

397

microscope, 173–176, 174f modulation-contrast, 177 red blood cells, 179–180f, 179–181 sediment examination techniques, 170–178 sediment stains, 171–172t, 171–173 types of, 174f, 176–177 vaginal secretions, 358–363, 358t, 359–362f white blood cells, 181–182, 181–182f Midstream clean-catch specimen, 97t, 98 Minimal change disease (MCD), 222, 223t, 225t Mixed agglutination reaction (MAR), 287 Mixed cellular casts, 23, 196–197 Mobiluncus, 358, 360, 363, 364 Modification of Diet in Renal Disease (MDRD) study, 115–116 Modulation-contrast microscopy, 177 Monocytes in cerebrospinal fluid (CSF), 41, 260, 261t, 262f in peritoneal fluid, 43 in pleural fluid, 44 Mononuclear cells, 182, 182f Morphology, sperm, 283–285, 284–285f Motility, sperm, 283, 283t Mouth, nose, and eye protection, 52 MSUD. see maple syrup urine disease (MSUD) Mucin clot, 296 Mucopolysaccharide disorders, 246–247 Mucopolysaccharidoses (MPSs), 246 Mucus, 16, 190, 191f Multiple myeloma, 144 Multiple sclerosis, 254 Multiples of the median (MoM), 332 Muscle fibers in feces, 344, 344f Mycoplasma hominis, 360, 364 Myelin basic protein (MBP), 268 Myoglobinuria, 153–154

N

N95 respirators, 52 Naegleria fowleri, 270, 270f National Kidney Disease Education Program (NKDEP), 115–116 Near-infrared reflectance spectroscopy (NIRS), 347 Neisseria gonorrhoeae, 357, 364 Nephrogenic diabetes insipidus, 226, 227t Nephrons, 106, 107f, 108f Nephrotic syndrome (NS), 220, 222, 223t, 225t Neubauer hemocytometer, 257, 257f, 282, 282f Neural tube defects (NTDs), 329, 332 Neutrophages, 296–297 Neutrophils in bronchoalveolar lavage (BAL) fluid, 323 in cerebrospinal fluid (CSF), 41–42, 260, 260t, 261–262f in pleural fluid, 44–45, 309 in synovial fluid, 46 Newborn urine screening. see urine screening Nitrite clinical significance, 157–158 reagent strip reactions, 158 interference, 158–159 Nocturia, 93 Nomarski microscopy, 177–178, 178f Nonpathologically significant cells in cerebrospinal fluid (CSF), 263, 264–265f

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398

Index

Nonpathological turbidity of urine, 129, 130b Normal urine color, 126–127, 128f Normal urine crystals acidic, 201–204, 202–204f alkaline, 204–205, 204–206f Nucleated red blood cells (NRBCs), 260

O

Occult blood in feces, 345–347, 346f Occupational Exposure to Bloodborne Pathogens Standard, 53 Occupational health and bloodborne pathogens, 53 Occupational Safety and Health Administration (OSHA), 50 Occupational Exposure to Bloodborne Pathogens Standard, 53 Ochronosis, 239 Odor semen, 280 urine, 134, 134t Oil droplet artifacts, 40, 210, 210f Oligoclonal bands, 267, 267f Oligohydramnios, 328 Oliguria, 93 Oncotic pressure, 106, 306 Optical density, 328t, 331 Oral glucose tolerance test (OGTT), 97 Orange urine, 127–128 Organic acidemias, 236, 240 Orthostatic (postural) proteinuria, 145 Osmolality, 117, 118f, 132–133, 134t Osmolar clearance, 118–119 Osmolarity, 118 Osmotic diarrhea, 341 Osmotic gradient, 106 OSOM BVBLUE test, 363 OSOM Trichomonas Rapid Test, 363 “Out-of-control” procedures, 67, 68f Oval fat bodies, 10, 186–187, 186–187f, 210 Overflow disorders, 236, 237t

P

P-aminohippuric acid (PAH) test, 119 Pancreatic insufficiency, 344 Pancreatitis, 309 Parabasal cells, 358, 360, 360f Parasites, 14–15, 189, 189–190f, 341 Parietal membrane, 306 Passive transport, 110, 110t Pathogens, 50 Pathological turbidity of urine, 130–131, 130b Patient care equipment, 53 Patient placement, 53 Pediatric specimens, 100 Pentosuria, 247 Peptostreptococcus, 360, 364 Pericardial fluid, 312–314, 313f, 313t Pericarditis, 312 Peripheral blood (PB), 263 Peritoneal fluid, 43, 314–316, 314t, 315–316f Peritoneal lavage, 314 Peritonitis, 314 Peritubular capillaries, 106 Personal protective equipment (PPE), 51, 52f, 54 for bloodborne pathogens, 53 Personnel assessment, 68

PH semen, 281 urine, 142–143, 143t vaginal secretions, 357–358 Phase-contrast microscopy, 174f, 176 Phenazopyridine, 128 Phenolsulfonphthalein test, 120 Phenylalanine-tyrosine disorders, 236–239, 238f Phenylketonuria (PKU), 236, 237 Phosphate, 30–31, 94t amorphous, 30 calcium, 32 Phosphatidyl glycerol (PG), 334 Physical hazards, 50t, 61 Physiology amniotic sac, 328, 328f cerebrospinal fluid (CSF), 254, 254–255f fecal analysis, 340–341, 340f renal, 106–113 semen, 278, 279f, 279t synovial fluid, 294, 294f, 294t Pia mater, 254, 254f Pigmented villonodular synovitis, 297 Pink urine, 128–129 Pinworm, 14 PKU. see phenylketonuria (PKU) Placental alpha macroglobulin (PAMG-1), 329, 357 Placental protein 12 (PP12), 329 Plasma cells in cerebrospinal fluid (CSF), 261t in pleural fluid, 309 Pleocytosis, 260 Pleural fluid, 44–45, 308–312 appearance, 308–309, 308t chemistry tests, 310–312, 312t hematology tests, 309–310, 309–311f, 309t microbiological and serological tests, 312 Podocytes, 107 Point-of-care (POC) instrument, 67 Point-of-care tests, vaginal secretions, 363 Polarization, synovial fluid crystal, 298–300, 299–300f Polarizing microscopy, 171, 174f, 176–177, 177f Pollen grain artifacts, 210, 210f Polydipsia, 94 Polyhydramnios, 328 Polymerase chain reaction (PCR), 269, 270, 312 Polymorphonuclear (PMN) white blood cells, 358 Polyuria, 93, 94, 94t Porphobilinogen, 128 Porphyria cutanea tarda, 245t Porphyrin-based fecal occult blood test, 347 Porphyrin disorders, 243–246, 244f, 245t Porphyrins, 128 Porphyrinuria, 243 Porphyromonas, 360, 364 Portal of entry, 51, 52f Portal of exit, 51, 52f Postexamination variables, 61, 68–69 Postexposure prophylaxis (PEP), 53 Postrenal proteinuria, 145 Postvasectomy semen analysis, 287 Potassium, 94t Potassium hydroxide (KOH) examination, 356, 357, 361–362 Pre- and postmassage test (PPMT), 99

Precision, 66 Preexamination variables, 61, 62–64, 63f, 64b, 65f Pregnancy-associated vaginal secretions, 365–367 Premature rupture of membranes (PROM), 329, 330t, 366–367 Prerenal proteinuria, 144 Preservation, specimen, 96, 96t Preventive maintenance (PM), 64 Prevotella, 358, 360, 364 Proficiency testing (PT), 62, 67 Prostate gland, 278 Prostatitis specimen, 97t, 98–100 Protein, 23, 25, 143–148 in amniotic fluid versus maternal urine, 329 cerebrospinal fluid (CSF), 266–268, 267b, 267f reagent strip reactions, 146–148, 146t in synovial fluid, 300 Protein error of indicators, 146 Proteinuria, 143–148 clinical significance of, 144 postrenal, 145 prerenal, 144 renal, 144–145 Protozoa, 341 Provider-performed microscopy procedures (PPMP) tests, 68 Proximal convoluted tubule, 106 Proximal tubular cells, 8 Pruritus, vaginal, 356 Prussian blue stain, 171t, 173 Pseudochylous material, 308–309, 309t Pseudogout, 297 Pulmonary infarction, 309 Purine disorders, 247 Pyelonephritis acute, 226–227, 228t, 229b chronic, 227, 228t, 229b

Q

Qualitative fecal fats, 344–345, 344–345f Quality assessment (QA), 68 Quality control (QC), 61, 66–67 cerebrospinal fluid (CSF) cell counts, 258 individualized plan, 68–69, 69f, 70f reagent strips, 142 semen analysis, 288 Quality control plan (QCP), 68 Quality management (QM), 61–62 errors in, 70 Quality management system (QMS), 61 Quantitative fecal fat testing, 347–348 Queenan curve, 331, 333f

R

RAAS. see renin-angiotensin-aldosterone system (RAAS) Radioactive hazards, 50t, 58–59 Radiographic dye crystals, 38, 207, 207t Radioisotopes, 58 Radionucleotides, 116 Ragocytes, 297 Random specimen, 96 Rapid gastric emptying (RGE), 342 Rapidly progressive (crescentic) glomerulonephritis (RPGN), 220, 223t, 224t

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Index Rapid plasma reagin (RPR) test, 271 Reagents, 64 Reagent strips, 140–141f, 140–142 confirmatory testing, 142 handling and storing, 141 quality control of, 142 reactions, 143, 146–148, 146t albumin, 147 blood, 153–154 glucose oxidase, 149 ketones, 152 leukocyte esterase, 160 nitrite, 158–159 specific gravity, 160–161, 160f urinary bilirubin, 155–156 urobilinogen, 157 technique, 140–141 Reagent strip specific gravity, 133 Record-keeping, 62 Red blood cells (RBCs), 2–3, 81, 168, 171t in acute glomerulonephritis (AGN), 220 bronchoalveolar lavage (BAL) fluid, 322–323 casts, 18, 180, 192–194, 192–194f in cerebrospinal fluid (CSF), 256, 258, 262f, 263 in pleural fluid, 44–45 in semen, 280 serous fluid, 307 in synovial fluid, 296 in urine, 128–129, 152, 179–180f, 179–181 vaginal secretions, 359, 359f in vaginal secretions, 357 Red cell inclusion casts, 172t Red O stain, 171, 171t Red urine, 128–129 Reflectance photometry, 76, 76t Refractometry, 131–132, 132f Reiter cells, 296–297 Rejection, specimen, 64, 64b, 95 Relative centrifugal force (RCF), 168–169 Reliability, 66 Renal blood flow, 106, 108b tests, 119 Renal disease, 220–230 glomerular disorders, 220–222, 223–224t interstitial disorders, 226–228, 228t laboratory testing in, 223–224t renal failure, 228–229, 229b renal lithiasis, 229–230 tubular disorders, 222, 225–226 urine screening for, 236, 237t Renal failure, 228–229, 229b Renal function, 105–120 renal disease and, 220–230 renal physiology and, 106–113 tests, 113–119 glomerular filtration, 113–116 tubular reabsorption, 116–119, 117–118f tubular secretion and renal blood flow, 119 Renal glycosuria, 226, 227t Renal lithiasis, 229–230 Renal physiology, 106–113, 107f blood flow, 106, 108b glomerular filtration, 106–109, 109f renin-angiotensin-aldosterone system (RAAS), 106, 108–109, 110b, 110f

tubular reabsorption, 106, 110–111, 110t tubular secretion, 106, 111–113, 112–113f Renal plasma flow, 106 Renal proteinuria, 144–145 Renal threshold, 111 Renal tubular acidosis, 113 Renal tubular epithelial (RTE) cells, 8–9, 184–186, 185–186f, 222 Renin, 109 Renin-angiotensin-aldosterone system (RAAS), 106, 108–109, 110b, 110f Requisition form, specimen, 63, 95 Reservoir, 50–51, 52f Resolution, 174 Respiratory distress syndrome (RDS), 333 Respiratory hygiene, 53 Respiratory tract amniotic fluid from, 329 bronchoalveolar lavage of, 322–325 Results critical, 69, 70f errors in, 69 interpreting, 69 Retinoblastomas, 266 Revolutions per minute (RPM), 169 Rhabdomyolysis, 153 Rheumatoid factor (RF), 270, 312 Ribonucleic acid (RNA), 80 Risk assessment (RA), 68 ROM Plus test, 366–367 Root cause analysis (RCA), 64 Ropes test, 296 Round renal tubular epithelial (RTE) cells, 182 RPGN. see rapidly progressive (crescentic) glomerulonephritis (RPGN) RTE. see renal tubular epithelial (RTE) cells Rupture of membranes (ROM), 330t, 366–367

S

Safety Data Sheets (SDS), 57–58, 61b Safety hazards, 50–62 biological, 50–56, 50t, 52f chemical, 50t, 56–58, 58f electrical, 50t, 59–60 fire/explosive, 50t, 60–61, 61t physical, 50t, 61 quality management (QM) for, 61–62 radioactive, 50t, 58–59 sharps, 50t, 53, 56 Standard Precautions (SP) against, 51–54 types of, 50, 50t Safety programs, 62 Salmonella, 341, 343 Scattergrams, 80, 81, 82f Schistosoma haematoblum, 15 Secretory diarrhea, 341 Sediment constituents, 178–179 artifacts, 209–211, 210–211f bacteria, 187–188, 188f casts, 168–170, 171–172t, 191–199, 192–199f crystals, 171t, 199, 201–202t, 201–209, 202–209f epithelial cells, 182–187, 183–187f expected staining reactions of, 172t mucus, 190, 191f parasites, 189, 189–190f red blood cells, 179–180f, 179–181

399

spermatozoa, 189–190, 190f white blood cells, 181–182, 181–182f yeast, 168, 179, 179f, 188, 189f Sediment examination techniques, 169–178 preparation, 169 volume, 169 Sediment stains, 171–173 characteristics of, 171t expected reactions of sediment constituents, 172t supravital stains, 171, 172t Semen, 278–288. See also spermatozoa analysis, 280–285 additional testing for abnormalities, 285–288, 286–288t, 286f appearance, 280 automated, 283 liquefaction, 280 microbial and chemical testing, 287, 287t pH, 281 postvasectomy, 287 quality control, 288 reference values for, 280t sperm concentration and count, 281–282, 282f sperm morphology, 283–285, 284–285f sperm motility, 283, 283t viscosity, 281 volume, 280 composition, 279t physiology, 278, 279f, 279t specimen collection, 278–280 specimen handling, 280 Semi-automated urine chemistry analyzers, 76–78, 77t, 78–79f Seminal fluid fructose, 286 Seminal vesicles, 278 Seminiferous tubules, 278 Sensitivity of testing, 69 Serological tests cerebrospinal fluid (CSF), 271 peritoneal fluid, 316 pleural fluid, 312 synovial fluid, 301 Serous fluid, 306–316, 306f formation, 306, 307f general laboratory procedures, 307–308 pericardial fluid, 312–314, 313f, 313t peritoneal fluid, 314–316, 314t, 315–316f pleural fluid, 308–309t, 308–312, 309–311f, 312b, 312t specimen collection and handling, 306–307 transudates and exudates, 307, 308t Sexually transmitted infections (STIs), 364 Sharps hazards, 50t, 53, 56 Shield of negativity, 107, 109f Shigella, 341, 343 Siemens Multistix Pro 10 reagent strips, 148 Sjögren syndrome, 221 SLE. see systemic lupus erythematosus (SLE) Sodium, 94t Sodium urates, 29, 201t Specific gravity, 131–133, 131t osmolality, 132–133, 134t reagent strip reaction, 133, 160–161, 160f interference, 160–161 refractometry, 131–132, 132f tubular reabsorption tests, 117, 117f

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400

Index

Specificity of testing, 69 Specimen collection amniotic fluid, 329–330, 330b bronchoalveolar lavage fluid, 322 cerebrospinal fluid (CSF), 254–255, 255f fecal, 342 semen, 278–280 serous fluid, 306–307 urine containers, 94 labels, 64, 64b, 95 requisition form, 63, 95 vaginal secretions, 357 Specimen handling amniotic fluid, 330–331 bronchoalveolar lavage fluid, 322 cerebrospinal fluid (CSF), 254–255, 255f semen, 280 synovial fluid, 294–295, 295t urine integrity, 95–96, 95t preservation, 96, 96t vaginal secretions, 357 Specimen rejection, urine, 64, 64b, 95 Specimens, urine collection and handling, 63–64, 64b, 94–96 criteria for rejection of, 64, 64b policy for handling mislabeled, 64, 64b preservation of, 96, 96t rejection of, 64, 64b types of, 96–100, 97t 24-hour, 97–98, 97t, 98b catheterized, 97t, 98 drug specimen collection, 100–101 first morning, 96–97, 97t four-glass collection, 97t midstream clean-catch, 97t, 98 pediatric, 100 pre- and postmassage test (PPMT), 99 prostatitis specimen, 97t, 98–100 random, 96, 97t suprapubic aspiration, 97t, 98 three-glass collection, 97t, 98–99 Sperm, 16 Spermatids, 280 Spermatozoa. see also semen antisperm antibodies, 286–287 concentration and count, 281–282, 282f function tests, 288, 288t morphology, 283–285, 284–285f motility, 283, 283t production of, 278 seminal fluid fructose, 286 in urine, 189–190, 190f vitality, 286 Spills, 54–57 Spinal cord, 254–255f. see also cerebrospinal fluid (CSF) Spindle-shaped cells, 261t Squamous epithelial cells, 182–184, 183–184f vaginal secretions, 358, 359f Stamey-Meares test for prostatitis, 99–100 Standardization, 62 Standard Precautions (SP), 51–54, 52f, 94 Staphylococcus, 341, 343 Starch granules artifacts, 38, 210, 210f Steatorrhea, 342 Stercobilinogen, 156

Sternheimer-Malbin stain, 171, 171t Subarachnoid space, 254 Sudan III stain, 171, 171t Sulfonamide crystals, 37, 207t, 208–209, 209f Sulfosalicylic acid precipitation (SSA) test, 146, 146f Suprapubic aspiration, 97t, 98 Supravital stains, 171, 172t Surface channel (SF ch) stains, 81 Surfactants, 333 Susceptible host, 51, 52f Syncytia, 184 Synovial fluid, 46, 294–301 cell counts, 296 chemistry tests, 300–301 color and clarity, 295–296 crystals, 296 polarization, 298–300, 299–300f slide preparation, 297–298 types of, 297, 298t differential count, 296–297, 297t microbiological tests, 301 physiology, 294, 294f, 294t serological tests, 301 specimen collection and handling, 294–295, 295t viscosity, 296 Synoviocytes, 296 Syphilis cerebrospinal fluid (CSF) testing for, 271 secondary, 221 Sysmex UD-10 Fully Automated Urine Particle Digital Imaging Device, 81, 82, 83f Sysmex UF 1000i system, 77t, 80–82, 80–82f Sysmex UF-5000 Fully Automated Urine Particle Analyzer, 81, 82–85f Sysmex UN-2000 system, 77t, 81, 82, 82f Sysmex XN-Series using Body Fluids mode, 77t, 87–88, 88t Systemic lupus erythematosus (SLE), 220, 221, 309f

T

Tamm-Horsfall protein (THP), 144 Testes, 278 Testing procedure, 66 Test utilization, 62 Thin-layer chromatography (TLC), 330 Three-glass collection, 97t, 98–99 Thrombosis, 221 Timed specimen, 97–98, 97t, 98b Titratable acidity, 119 Toluidine blue stain, 171t Total nucleated body fluid cell count (TC-BF), 88 Training, 62, 68 Transitional epithelial (urothelial) cells, 7–8, 184, 184f Transudates, 307, 308t, 314 Traumatic collection of cerebrospinal fluid (CSF), 256–257 Trichomonads, 14 Trichomonas vaginalis, 14, 357, 360–361, 361–362f, 363 Trichomoniasis, 356, 356t, 357, 360–361, 315–362f, 363, 364 Triple phosphate, 202t, 204–205, 205f Tryptophan disorders, 241–242, 241f

Tubular concentration, 111 Tubular disorders, 222, 225–226 hereditary and metabolic, 225–226, 227t Tubular proteinuria, 145 Tubular reabsorption, 106, 110–111, 110t renal glycosuria and, 226 tests, 116–119, 117–118f Tubular secretion, 106, 111–113, 112–113f tests, 119 Tubulointerstitial disease, 226 Turnaround time (TAT), 62–63 24-hour (or timed) specimen, 97–98, 97t, 98b 2% acetic acid stain, 171, 171t Tyrosine crystals, 34, 35, 207t, 208f metabolism, 237–239, 238f Tyrosyluria, 236, 237–239

U

UDP-galactose-4-epimerase (GALE), 247 Universal Precautions (UP), 51 Urea, 93, 94t clearance, 113 Ureaplasma urealyticum, 360, 364 Uric acid, 26–27, 94t, 201t, 202–203, 203f in synovial fluid, 301 Urinalysis, 2–40, 76–88 artifacts, 38–40 automation, 76–88 casts, 17–25 cause-and-effect diagram for, 63f cells, 2–10 chemical examination, 140–161 crystals, 26–37 history and importance, 92–93, 92–93f microorganisms, 11–13 microscopic examination, 168–211 miscellaneous structures, 16 parasites, 14–15 physical examination, 126–134 Urinalysis Data Manager (UDM), 82 Urinalysis procedure manual, 62–70 components of, 62 critical results, 69, 70f electronic controls, 67 examination variables, 64–67, 66–68f example of, 63f external quality control, 66–67, 67f, 68f individualized quality control plan, 68–69, 69f, 70f instrumentation and equipment, 64, 66, 66f internal quality control, 67 interpreting results, 69 personnel and facilities, 68 postexamination variables, 68–69, 69f preexamination variables, 62–64, 63f, 64b, 65f proficiency testing, 67 quality control, 66 reagents, 64 result errors, 69 specimen collection and handling, 63–64, 64b testing procedure, 66 Urinary ammonia, 119 Urinary tract infection (UTI), 11, 19, 20, 36, 188, 226 Urine acidic, 201–204, 202–204f

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Index alkaline, 204–205, 204–206f bilirubin in, 154–156, 155f, 155t blood in, 128, 128f, 129f, 152–154 chemical examination of, 140–161 clarity of, 129–131, 129t, 130b color of, 126–127t, 126–129 abnormal, 127–129, 128f, 129f normal, 126–127, 128f composition of, 93, 94t crystals, 171t, 199, 201–202t, 201–209, 202–209f differentiation of amniotic fluid from maternal, 329 disposal of, 54, 56–57 formation of, 93 glucose in, 148–151 ketones in, 151–152 leukocyte esterase in, 159–160 nitrite in, 157–159 odor of, 134, 134t pH of, 142–143, 143t physical examination of, 126–134 protein in, 143–148 sediment constituents (see sediment constituents, urine) specific gravity of, 117, 117f, 131–133, 131t specimen collection and handling, 63–64, 64b, 65f, 94–96, 95t urobilinogen in, 155f, 155t, 156–157 Urine chemistry analyzers, 76–88 fully automated, 77t, 78–79 semi-automated, 76–78, 77t, 78–79f Urine screening, 236–247 alkaptonuria, 129, 236, 239 amino acid disorders, 236–243, 238f, 240–241f branched-chain, 239–240, 240f carbohydrate disorders, 247 cystine disorders, 236, 242–243 melanuria, 236, 239 metabolic disorders, 236t mucopolysaccharide disorders, 246–247 newborn, 236, 237f organic acidemias, 236, 240 overflow versus renal disorders, 236, 237t phenylketonuria (PKU), 236, 237 porphyrin disorders, 243–246, 244f, 245t purine disorders, 247 tryptophan disorders, 241–242, 241f tyrosyluria, 236, 237–239 Urine sediment constituents. see sediment constituents, urine Urine specimens. see specimens, urine Urine volume, 93–94 Urinometry, 133

UriSed 2 system, 77t, 85–86, 87 UriSed 3 system, 77t, 85–86, 87 Urisys 1100 system, 78f Urisys 1800 system, 77t Urisys 2400 system, 77t, 79f Urobilin, 127 Urobilinogen, 156–157 clinical significance, 157 in jaundice, 155t production of, 155f reaction strip reactions, 157 interference, 157 Urochrome, 126–127 Uroerythrin, 127 Uromodulin, 144, 190 Uromodulin-associated kidney disease, 226, 227t UTI. see urinary tract infection (UTI) UX-2000 system, 77t, 87

V

Vaginal disorders, 364–365 atrophic vaginitis, 356t, 357, 365 bacterial vaginosis (BV), 356, 356t, 364 candidiasis, 364–365 desquamative inflammatory vaginitis (DIV), 356t, 357, 365 trichomoniasis, 356, 356t, 357, 360–361, 361–362f, 363, 364 Vaginal pool, 357 Vaginal secretions, 356–367 color and appearance, 357, 357t diagnostic tests, 357–363 microscopic procedures, 358–363, 358t, 359–362f pH, 357–358 disorders and laboratory findings associated with, 356–357, 356t pregnancy-associated, 365–367 specimen collection and handling, 357 Vaginitis atrophic, 356t, 357, 365 desquamative inflammatory vaginitis (DIV), 356t, 357, 365 pH and, 357 Van de Kamer titration, 347–348 Vapor pressure osmometers, 118 Variegate porphyria, 245t Vasa recta, 106 Vasectomy, 287 Vasopressin (antidiuretic hormone [ADH]), 111 Vasovasostomy, 287 Venereal Disease Research Laboratory (VDRL), 271

401

Vesicoureteral reflux (VUR), 226 Vibrio cholerae, 341, 343 Viruses bronchoalveolar lavage (BAL) fluid, 324, 324b Visceral membrane, 306 Viscosity semen, 281 synovial fluid, 296 Volume analysis semen, 280 urine, 93–94 Volume of sediment, macroscopic screening, 169 Vulvovaginal candidiasis, 356, 356t

W

Waste disposal biological, 54, 56–57 chemical, 58 Watson-Schwartz test, 243, 245–246 Waxy casts, 24, 172t, 198, 198f Wet mount examination, vaginal secretions, 358, 358t White blood cells (WBCs), 3–5, 81, 168, 171t in acute glomerulonephritis (AGN), 220 body fluid count, 88 bronchoalveolar lavage (BAL) fluid, 322–323 casts, 19, 194–195, 194–195f cerebrospinal fluid (CSF), 255f in cerebrospinal fluid (CSF), 257–258, 257f esterase in, 159 peritoneal fluid, 314–315, 315–316f in semen, 280 serous fluid, 307 in synovial fluid, 296 in urine, 181–182, 181–182f vaginal secretions, 358–359, 359f Work practice controls for bloodborne pathogens, 53 World Health Organization (WHO), 278, 283

X

Xanthrochromia, 256 Xanthrochromic supernatant, 257 X-ray procedures, 38

Y

Yeast, 12–13, 168, 179, 179f, 188, 189f vaginal secretions, 358, 361, 362f Yersinia, 343