Essentials of Clinical Pathology [1 ed.]
 2031708910, 9789380704197

Table of contents :
Prelims
Chapter-01_Examination of Urine
Chapter-02_Renal Function Tests
Chapter-03_Diabetes Mellitus
Chapter-04_Liver Function Tests
Chapter-05_Disorders of Lipids and Biochemical Cardiac Markers
Chapter-06_Examination of Cerebrospinal Fluid
Chapter-07_Examination of Pleural and Peritoneal Fluids
Chapter-08_Examination of Sputum
Chapter-09_Examination of Feces
Chapter-10_Gastric Analysis
Chapter-11_Tests for Malabsorption and Pancreatic Function
Chapter-12_Thyroid Function Tests
Chapter-13_Pregnancy Tests
Chapter-14_Infertility
Chapter-15_Semen Analysis
Chapter-16_Hematopoiesis
Chapter-17_Collection of Blood
Chapter-18_Estimation of Hemoglobin
Chapter-19_Packed Cell Volume
Chapter-20_Total Leukocyte Count
Chapter-21_Reticulocyte Count
Chapter-22_Blood Smear
Chapter-23_Red Cell Indices
Chapter-24_Erythrocyte Sedimentation Rate
Chapter-25_Examination of Bone Marrow
Chapter-26_Diagnosis of Malaria and Other Parasites in Blood
Chapter-27_Laboratory Tests in Anemia
Chapter-28_Laboratory Tests in Hematological Malignancies
Chapter-29_Laboratory Tests in Bleeding Disorders
Chapter-30_Laboratory Tests in Thrombophilia
Chapter-31_Laboratory Tests in Porphyrias
Chapter-32_Automation in Hematology
Chapter-33_Blood Group Systems
Chapter-34_Blood Grouping
Chapter-35_Collection of Donor Blood, Processing and Storage
Chapter-36_Screening Tests for Infections Transmissible by Transfusion
Chapter-37_Compatibility Test (Cross-match)
Chapter-38_Adverse Effects of Transfusion
Chapter-39_Blood Components
General References
Index

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Essentials of

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Clinical Pathology

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Essentials of

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Clinical Pathology

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Associate Professor Department of Pathology Government Medical College Nagpur, Maharashtra, India

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Shirish M Kawthalkar

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JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD Nagpur • St Louis (USA) • Panama City (Panama) • London (UK) • New Delhi • Ahmedabad Bengaluru • Chennai • Hyderabad • Kochi • Kolkata • Lucknow • Mumbai

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Published by Jitendar P Vij Jaypee Brothers Medical Publishers (P) Ltd

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Corporate Office 4838/24 Ansari Road, Daryaganj, New Delhi - 110002, India, Phone: +91-11-43574357, Fax: +91-11-43574314

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Ahmedabad, Phone: Rel: +91-79-32988717, e-mail: [email protected] Bengaluru, Phone: Rel: +91-80-32714073, e-mail: [email protected] Chennai, Phone: Rel: +91-44-32972089, e-mail: [email protected] Hyderabad, Phone: Rel:+91-40-32940929, e-mail: [email protected] Kochi, Phone: +91-484-2395740, e-mail: [email protected] Kolkata, Phone: +91-33-22276415, e-mail: [email protected] Lucknow, Phone: +91-522-3040554, e-mail: [email protected] Mumbai, Phone: Rel: +91-22-32926896, e-mail: [email protected] Nagpur, Phone: Rel: +91-712-3245220, e-mail: [email protected]

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Offices in India

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Registered Office B-3 EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi - 110 002, India Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021 +91-11-23245672, Rel: +91-11-32558559, Fax: +91-11-23276490, +91-11-23245683 e-mail: [email protected], Website: www.jaypeebrothers.com

Essentials of Clinical Pathology © 2010, Shirish M Kawthalkar

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Overseas Offices • North America Office, USA, Ph: 001-636-6279734, e-mail: [email protected], [email protected] • Central America Office, Panama City, Panama, Ph: 001-507-317-0160, e-mail: [email protected], Website: www.jphmedical.com • Europe Office, UK, Ph: +44 (0) 2031708910, e-mail: [email protected]

All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author and the publisher. This book has been published in good faith that the material provided by author is original. Every effort is made to ensure accuracy of material, but the publisher, printer and author will not be held responsible for any inadvertent error (s). In case of any dispute, all legal matters are to be settled under Delhi jurisdiction only. First Edition: 2010 ISBN 978-93-80704-19-7 Typeset at JPBMP typesetting unit Printed at

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Preface

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The major aims of this book are discussion of (i) use of laboratory tests in the investigation and management of common diseases, and (ii) basic biochemical and pathological principles underlying the application of laboratory tests. The book has been written keeping in mind mainly the curricula of undergraduate students of pathology. It should also prove to be appropriate for postgraduate residents and students of medical laboratory technology. The laboratory tests that are demonstrated to and performed by medical students in pathology practical class and during university examination are given in more detail. To keep pace with new knowledge and advances, principles of currently performed techniques in clinical laboratory practice have also been outlined. Most of the chapters are followed by reference ranges and critical values for ready access. Critical values or action values are those laboratory results that require immediate attention of the treating clinician. While interpreting results of laboratory tests, it is necessary to follow two fundamental rules of laboratory medicine: (i) diagnosis should never be made from a single abnormal test result (since it is affected by a number of preanalytical and analytical factors), and (ii) try to arrive at a single diagnosis (rather than multiple diagnoses) from all the abnormal test results obtained. Clinical pathology is the second major subdivision of the discipline of pathology after anatomic pathology. It is concerned with laboratory investigations for screening, diagnosis, and overall management of diseases by analysis of blood, urine, body fluids, and other specimens. The specialties included under the discipline of clinical pathology are clinical chemistry, hematology, blood banking, medical microbiology, cytogenetics, and molecular genetics. However, scope of this book does not allow microbiology and genetics to be included in this book. I must appreciate and recognize the unstinting support of my parents, my beloved wife Dr Anjali, and my two children, Ameya and Ashish during preparation of this book. I am thankful to Dr HT Kanade, Dean, Government Medical College, Akola, Dr Smt Deepti Dongaonkar, Dean, Government Medical College, Nagpur, Dr BB Sonawane, Professor and Head, Department of Pathology, Government Medical College, Akola, and Dr WK Raut, Professor and Head, Department of Pathology, Government Medical College, Nagpur, for encouraging me in undertaking this project for the benefit of medical students. I express my thanks to Mr JP Vij and his outstanding team of M/s Jaypee Brothers Medical Publishers for undertaking to publish this book, being patient with me during the preparation of the manuscript, and bringing it out in an easy-to-read and reader-friendly format. Although I have made every effort to avoid any mistakes and errors, some may persist and feedback in this regard will be highly appreciated. Shirish M Kawthalkar

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Contents Section 1 Chemical Pathology and Related Studies 1. Examination of Urine ................................................................................................................................................. 3 2. Renal Function Tests ................................................................................................................................................ 30 3. Diabetes Mellitus ..................................................................................................................................................... 39 4. Liver Function Tests ................................................................................................................................................. 52 5. Disorders of Lipids and Biochemical Cardiac Markers .................................................................................... 69 6. Examination of Cerebrospinal Fluid .................................................................................................................... 80 7. Examination of Pleural and Peritoneal Fluids .................................................................................................... 91 8. Examination of Sputum........................................................................................................................................... 99 9. Examination of Feces ............................................................................................................................................. 104 10. Gastric Analysis ...................................................................................................................................................... 121 11. Tests for Malabsorption and Pancreatic Function ........................................................................................... 127 12. Thyroid Function Tests ......................................................................................................................................... 137 13. Pregnancy Tests ...................................................................................................................................................... 146 14. Infertility .................................................................................................................................................................. 150 15. Semen Analysis ....................................................................................................................................................... 159

Section 2 Laboratory Hematology 16. Hematopoiesis ......................................................................................................................................................... 169 17. Collection of Blood ................................................................................................................................................. 179 18. Estimation of Hemoglobin ................................................................................................................................... 183 19. Packed Cell Volume ............................................................................................................................................... 188 20. Total Leukocyte Count .......................................................................................................................................... 192 21. Reticulocyte Count ................................................................................................................................................. 196 22. Blood Smear ............................................................................................................................................................. 200 23. Red Cell Indices ...................................................................................................................................................... 213 24. Erythrocyte Sedimentation Rate .......................................................................................................................... 215 25. Examination of Bone Marrow .............................................................................................................................. 220 26. Diagnosis of Malaria and Other Parasites in Blood ........................................................................................ 229 27. Laboratory Tests in Anemia ................................................................................................................................. 244 28. Laboratory Tests in Hematological Malignancies ........................................................................................... 273 29. Laboratory Tests in Bleeding Disorders ............................................................................................................ 288 30. Laboratory Tests in Thrombophilia .................................................................................................................... 311 31. Laboratory Tests in Porphyrias ............................................................................................................................ 314 32. Automation in Hematology .................................................................................................................................. 319

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Essentials of Clinical Pathology

Section 3 Practical Blood Transfusion 33. 34. 35. 36. 37. 38. 39.

Blood Group Systems ............................................................................................................................................ Blood Grouping ...................................................................................................................................................... Collection of Donor Blood, Processing and Storage ........................................................................................ Screening Tests for Infections Transmissible by Transfusion ...................................................................... Compatibility Test (Cross-match) ....................................................................................................................... Adverse Effects of Transfusion ............................................................................................................................ Blood Components .................................................................................................................................................

329 336 341 347 352 354 359

General References ...................................................................................................................................................... 365 Index ........................................................................................................................................................................... 367

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Chemical Pathology and Related Studies

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1

Examination of Urine

COMPOSITION OF NORMAL URINE

COLLECTION OF URINE

Urinalysis is one of the most commonly performed laboratory tests in clinical practice. Composition of normal urine is shown in Table 1.1.

There are various methods for collection of urine. Method of collection to be used depends on the nature of investigation (Boxes 1.1 and 1.2).

INDICATIONS FOR URINALYSIS 1. Suspected renal diseases like glomerulonephritis nephrotic syndrome, pyelonephritis, and renal failure 2. Detection of urinary tract infection 3. Detection and management of metabolic disorders like diabetes mellitus 4. Differential diagnosis of jaundice 5. Detection and management of plasma cell dyscrasias 6. Diagnosis of pregnancy.

Time of Collection 1. A single specimen: This may be a first morning voiding, a random specimen, or a post-prandial specimen. The first voided specimen in the morning is the most concentrated and has acidic pH in which formed elements (cells and casts) are well preserved. This specimen is used for routine examination, fasting glucose, proteins, nitrite, microscopic analysis for cellular elements, pregnancy test, orthostatic proteinuria, and bacteriological analysis.

Table 1.1: Composition of normal urine (24 hour) in adults

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Parameters

Values

Volume Specific gravity Osmolality pH Glucose Proteins Urobilinogen Porphobilinogen Creatinine Urea nitrogen Uric acid Sodium Potassium Chloride Calcium (low calcium diet) Formiminoglutamic acid (FIGlu) Red cells, epithelial cells, and white blood cells

600-2000 ml 1.003-1.030 300-900 mOsm/kg 4.6-8.0 3.5 gm/24 hr) Hypoalbuminemia (350 mg/dl) Lipiduria

ria correlates with severity of disease and prognosis. Serial estimations of urinary protein are also helpful in monitoring response to treatment. Most severe degree of proteinuria occurs in nephrotic syndrome (Box 1.6). 2. Tubular proteinuria: Normally, glomerular membrane, although impermeable to high molecular weight proteins, allows ready passage to low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal. Urine electrophoresis shows prominent α- and βbands (where low molecular weight proteins migrate) and a faint albumin band (Fig. 1.3). Tubular type of proteinuria is commonly seen in acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of kidney, interstitial nephritis, cystinosis, Fanconi syndrome and rejection of kidney transplant. Purely tubular proteinuria cannot be detected by reagent strip test (which is sensitive to albumin), but heat and acetic acid test and sulphosalicylic acid test are positive. 3. Overflow proteinuria: When concentration of a low molecular weight protein rises in plasma, it “overflows” from plasma into the urine. Such proteins are immunoglobulin light chains or Bence Jones proteins (plasma cell dyscrasias), hemoglobin (intravascular hemolysis), myoglobin (skeletal muscle trauma), and lysozyme (acute myeloid leukemia type M4 or M5). 4. Hemodynamic proteinuria: Alteration of blood flow through the glomeruli causes increased filtration of proteins. Protein excretion, however, is transient. It is seen in high fever, hypertension, heavy exercise, congestive cardiac failure, seizures, and exposure to cold. Postural (orthostatic) proteinuria occurs when the subject is standing or ambulatory, but is absent in recumbent position. It is common in adolescents (3-5%)

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and is probably due to lordotic posture that causes inferior venacaval compression between the liver and vertebral column. The condition disappears in adulthood. Amount of proteinuria is 300

< 20 20-200 >200

< 20 20-300 >300

< 20 20-200 >200

< 30 30-300 >300

< 2.5 2.5-25 >25

• Detection of microalbuminuria or early diabetic nephropathy • To follow response to therapy in renal disease Proteinuria >1500 mg/ 24 hours indicates glomerular disease; proteinuria >3500 mg/24 hours is called as nephrotic range proteinuria; in tubular, hemodynamic and post renal diseases, proteinuria is usually < 1500 mg/ 24 hours. Grading of albuminuria is shown in Table 1.5. There are two methods for quantitation of proteins: (1) Estimation of proteins in a 24-hour urine sample, and (2) Estimation of protein/creatinine ratio in a random urine sample. 1. Quantitative estimation of proteins in a 24-hour urine sample: Collection of a 24-hour sample is given earlier. Adequacy of sample is confirmed by calculating expected 24-hour urine creatinine excretion. Daily urinary creatinine excretion depends on muscle mass and remains relatively constant in an individual patient. In adult males creatinine excretion is 14-26 mg/kg/24 hours, while in women it is 11-20 mg/kg/24 hours. Various methods are available for quantitative estimation of proteins: Esbach’s albuminometer method, turbidimetric methods, biuret reaction, and immunologic methods. 2. Estimation of protein/creatinine ratio in a random urine sample: Because of the problem of incomplete collection of a 24-hour urine sample, many laboratories measure protein/creatinine ratio in a random urine sample. Normal protein/creatinine ratio is < 0.2. In low-grade proteinuria it is 0.2-1.0; in moderate, it is 1.0-3.5; and in nephrotic- range proteinuria it is > 3.5. Microalbuminuria This is defined as urinary excretion of 30 to 300 mg/24 hours (or 2-20 mg/dl) of albumin in urine. Significance of microalbuminuria 1. Microalbuminuria is considered as the earliest sign of renal damage in diabetes mellitus (diabetic nephropathy). It indicates increase in capillary

permeability to albumin and denotes microvascular disease. Microalbuminuria precedes the development of diabetic nephropathy by a few years. If blood glucose level and hypertension are tightly controlled at this stage by aggressive treatment then progression to irreversible renal disease and subsequent renal failure can be delayed or prevented. 2. Microalbuminuria is an independent risk factor for cardiovascular disease in diabetes mellitus. Detection of microalbuminuria: Microalbuminuria cannot be detected by routine tests for proteinuria. Methods for detection include: • Measurement of albumin-creatinine ratio in a random urine sample • Measurement of albumin in an early morning or random urine sample • Measurement of albumin in a 24 hr sample Test strips that screen for microalbuminuria are available commercially. Exact quantitation can be done by immunologic assays like radioimmunoassay or enzyme linked immunosorbent assay. Bence Jones Proteinuria Bence Jones proteins are monoclonal immunoglobulin light chains (either κ or λ) that are synthesized by neoplastic plasma cells. Excess production of these light chains occurs in plasma cell dyscrasias like multiple myeloma and primary amyloidosis. Because of their low molecular weight and high concentration they are excreted in urine (overflow proteinuria). Bence Jones proteins have a characteristic thermal behaviour. When heated, Bence Jones proteins precipitate at temperatures between 40°C to 60°C (other proteins precipitate between 60-70°C), and precipitate disappears on further heating at 85-100°C (while precipitate of other proteins does not). When cooled (60-85°C), there is reappearance of precipitate of Bence Jones proteins. This test, however, is not specific for Bence Jones proteins and both false-positive and -negative results can occur. This test has been replaced by protein electrophoresis of concentrated urine sample (Fig. 1.7).

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Essentials of Clinical Pathology glucosuria or glycosuria (Box 1.7). Glycosuria results if the filtered glucose load exceeds the capacity of renal tubular reabsorption. Most common cause is hyperglycemia from diabetes mellitus. Causes of Glycosuria

Fig. 1.7: Urine protein electrophoresis showing heavy Bence Jones proteinuria (red arrow) along with loss of albumin and other low molecular weight proteins in urine

Further evaluation of persistent overt proteinuria is shown in Figure 1.8. Glucose The main indication for testing for glucose in urine is detection of unsuspected diabetes mellitus or follow-up of known diabetic patients. Practically all of the glucose filtered by the glomeruli is reabsorbed by the proximal renal tubules and returned to circulation. Normally a very small amount of glucose is excreted in urine (< 500 mg/24 hours or 2.0 grams/dl): Brick- red precipitate.

Fig. 1.9: Principle of Benedict’s qualitative test for sugar in urine. Sensitivity is 200 mg of glucose/dl

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Essentials of Clinical Pathology Sensitivity of the test is about 100 mg glucose/dl of urine. False positive test occurs in the presence of oxidizing agent (bleach or hypochlorite used to clean urine containers), which oxidizes the chromogen directly. False-negative test occurs in the presence of large amounts of ketones, salicylates, ascorbic acid, and severe Escherichia coli infection (catalase produced by organisms in urine inactivates hydrogen peroxide). Ketones

Fig. 1.10: Grading of Benedict’s test (above) and reagent strip test (below) for glucose

B. Clinitest tablet method (Copper reduction tablet test): This is a modified form of Benedict’s test in which the reagents are present in a tablet form (copper sulphate, citric acid, sodium carbonate, and anhydrous sodium hydroxide). Sensitivity is 200 mgs/dl of glucose. 2. Reagent strip method This test is specific for glucose and is therefore preferred over Benedict’s and Clinitest methods. It is based on glucose oxidase-peroxidase reaction. Reagent area of the strips is impregnated with two enzymes (glucose oxidase and peroxidase) and a chromogen. Glucose is oxidized by glucose oxidase with the resultant formation of hydrogen peroxide and gluconic acid. Oxidation of chromogen occurs in the presence of hydrogen peroxide and the enzyme peroxidase with resultant color change (Fig. 1.11). Nature of chromogen and buffer system differ in different strips. The strip is dipped into the urine sample and color is observed after a specified time and compared with the color chart provided (Fig. 1.10). This test is more sensitive than Benedict’s qualitative test and specific only for glucose. Other reducing agents give negative reaction.

Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric acid, and acetone) in urine is called as ketonuria. Ketones are breakdown products of fatty acids and their presence in urine is indicative of excessive fatty acid metabolism to provide energy. Causes of Ketonuria Normally ketone bodies are not detectable in the urine of healthy persons. If energy requirements cannot be met by metabolism of glucose (due to defective carbohydrate metabolism, low carbohydrate intake, or increased metabolic needs), then energy is derived from breakdown of fats. This leads to the formation of ketone bodies (Fig. 1.12). 1. Decreased utilization of carbohydrates a. Uncontrolled diabetes mellitus with ketoacidosis: In diabetes, because of poor glucose utilization, there is compensatory increased lipolysis. This causes increase in the level of free fatty acids in plasma. Degradation of free fatty acids in the liver leads to the formation of acetoacetyl CoA which then forms ketone bodies. Ketone bodies are strong acids and produce H+ ions, which are neutralized by bicarbonate ions; fall in bicarbonate (i.e. alkali) level produces ketoacidosis. Ketone bodies also increase the plasma osmolality and cause cellular dehydration. Children and young adults with type 1 diabetes are

Fig. 1.11: Principle of reagent strip test for glucose in urine. Each mole of glucose produces one mole of peroxide, and each mole of peroxide reduces one mole of oxygen. Sensitivity is 100 mg glucose/100 ml

Examination of Urine

Fig. 1.12: Formation of ketone bodies. A small part of acetoacetate is spontaneously and irreversibly converted to acetone. Most is converted reversibly to β-hydroxybutyrate

especially prone to ketoacidosis during acute illness and stress. If glycosuria is present, then test for ketone bodies must be done. If both glucose and ketone bodies are present in urine, then it indicates presence of diabetes mellitus with ketoacidosis (Box 1.8). In some cases of diabetes, ketone bodies are increased in blood but do not appear in urine. Presence of ketone bodies in urine may be a warning of impending ketoacidotic coma. b. Glycogen storage disease (von Gierke’s disease) 2. Decreased availability of carbohydrates in the diet: a. Starvation b. Persistent vomiting in children c. Weight reduction program (severe carbohydrate restriction with normal fat intake) 3. Increased metabolic needs: a. Fever in children b. Severe thyrotoxicosis c. Pregnancy d. Protein calorie malnutrition Tests for Detection of Ketones in Urine The proportion of ketone bodies in urine in ketosis is variable: β-hydroxybutyric acid 78%, acetoacetic acid 20%, and acetone 2%.

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No method for detection of ketonuria reacts with all the three ketone bodies. Rothera’s nitroprusside method and methods based on it detect acetoacetic acid and acetone (the test is 10-20 times more sensitive to acetoacetic acid than acetone). Ferric chloride test detects acetoacetic acid only. β-hydroxybutyric acid is not detected by any of the screening tests. Methods for detection of ketone bodies in urine are Rothera’s test, Acetest tablet method, ferric chloride test, and reagent strip test. 1. Rothera’s’ test (Classic nitroprusside reaction) Acetoacetic acid or acetone reacts with nitroprusside in alkaline solution to form a purple-colored complex (Fig. 1.13). Rothera’s test is sensitive to 1-5 mg/dl of acetoacetate and to 10-25 mg/dl of acetone. Method 1. Take 5 ml of urine in a test tube and saturate it with ammonium sulphate. 2. Add a small crystal of sodium nitroprusside. Mix well. 3. Slowly run along the side of the test tube liquor ammonia to form a layer. 4. Immediate formation of a purple permanganate colored ring at the junction of the two fluids indicates a positive test (Fig. 1.14). False-positive test can occur in the presence of L-dopa in urine and in phenylketonuria. 2. Acetest tablet test This is Rothera’s test in the form of a tablet. The Acetest tablet consists of sodium nitroprusside, glycine, and an alkaline buffer. A purplelavender discoloration of the tablet indicates the presence of acetoacetate or acetone (≥ 5 mg/dl). A rough estimate of the amount of ketone bodies can be obtained by comparison with the color chart provided by the manufacturer.The test is more sensitive than reagent strip test for ketones.

Box 1.8: Urine ketones in diabetes Indications for testing • At diagnosis of diabetes mellitus • At regular intervals in all known cases of diabetes, and in gestational diabetes • In known diabetic patients during acute illness, persistent hyperglycemia (>300 mg/dl), pregnancy, clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain)

Fig. 1.13: Principles of Rothera’s test and reagent strip test for ketone bodies in urine. Ketones are detected as acetoacetic acid and acetone but not β-hydroxybutyric acid

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Essentials of Clinical Pathology Table 1.6: Urine bilirubin and urobilinogen in jaundice Urine test

Hemolytic jaundice

Hepatocellular Obstructive jaundice jaundice

1. Bilirubin

Absent

Present

Present

Increased

Absent

2. Urobilinogen Increased

In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically apparent. In a fever of unknown origin bilirubinuria suggests hepatitis.

Fig. 1.14: Rothera’s tube test and reagent strip test for ketone bodies in urine

3. Ferric chloride test (Gerhardt’s): Addition of 10% ferric chloride solution to urine causes solution to become reddish or purplish if acetoacetic acid is present. The test is not specific since certain drugs (salicylate and L-dopa) give similar reaction. Sensitivity of the test is 25-50 mg/ dl. 4. Reagent strip test: Reagent strips tests are modifications of nitroprusside test (Figs 1.13 and 1.14). Their sensitivity is 5-10 mg/dl of acetoacetate. If exposed to moisture, reagent strips often give false-negative result. Ketone pad on the strip test is especially vulnerable to improper storage and easily gets damaged. Bile Pigment (Bilirubin) Bilirubin (a breakdown product of hemoglobin) is undetectable in the urine of normal persons. Presence of bilirubin in urine is called as bilirubinuria. There are two forms of bilirubin: conjugated and unconjugated. After its formation from hemoglobin in reticuloendothelial system, bilirubin circulates in blood bound to albumin. This is called as unconjugated bilirubin. Unconjugated bilirubin is not water-soluble, is bound to albumin, and cannot pass through the glomeruli; therefore it does not appear in urine. The liver takes up unconjugated bilirubin where it combines with glucuronic acid to form bilirubin diglucuronide (conjugated bilirubiun). Conjugated bilirubin is watersoluble, is filtered by the glomeruli, and therefore appears in urine. Detection of bilirubin in urine (along with urobilinogen) is helpful in the differential diagnosis of jaundice (Table 1.6).

Presence of bilirubin in urine indicates conjugated hyperbilirubinemia (obstructive or hepatocellular jaundice). This is because only conjugated bilirubin is water-soluble. Bilirubin in urine is absent in hemolytic jaundice; this is because unconjugated bilirubin is water-insoluble. Tests for Detection of Bilirubin in Urine Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or fluorescent light) and on standing at room temperature. Biliverdin cannot be detected by tests that detect bilirubin. Therefore fresh sample that is kept protected from light is required. Findings associated with bilirubinuria are shown in Box 1.9. Methods for detection of bilirubin in urine are foam test, Gmelin’s test, Lugol iodine test, Fouchet’s test, Ictotest tablet test, and reagent strip test. 1. Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white. 2. Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Fig. 1.15). 3. Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test. Box 1.9: Clinical and laboratory findings in bilirubinuria • Jaundice • Urine color: Dark yellow with yellow foam • Elevated serum conjugated bilirubin

Examination of Urine

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Fig. 1.16: Positive Fouchet’s test for bilirubin in urine

Fig. 1.15: Positive Gmelin’s test for bilirubin showing play of colors

4. Fouchet’s test: This is a simple and sensitive test. i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex). ii. Filter to obtain the precipitate on a filter paper. iii. To the precipitate on the filter paper, add 1drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml). iv. Immediate development of blue-green color around the drop indicates presence of bilirubin (Fig. 1.16). 5. Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl). Bile Salts Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation). Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice,

bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay’s surface tension test. The property of bile salts to lower the surface tension is utilized in this test. Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent. Thymol (used as a preservative) gives false positive test. Urobilinogen Conjugated bilirubin excreted into the duodenum through bile is converted by bacterial action to urobilinogen in the intestine. Major part is eliminated in the feces. A portion of urobilinogen is absorbed in blood, which undergoes recycling (enterohepatic circulation); a small amount, which is not taken up by the liver, is excreted in urine. Urobilinogen is colorless; upon oxidation it is converted to urobilin, which is orange-yellow in color. Normally about 0.5-4 mg of urobilinogen is excreted in urine in 24 hours. Therefore, a small amount of urobilinogen is normally detectable in urine. Urinary excretion of urobilinogen shows diurnal variation with highest levels in afternoon. Therefore, a 2-hour post-meal sample is preferred. Causes of Increased Urobilinogen in Urine 1. Hemolysis: Excessive destruction of red cells leads to hyperbilirubinemia and therefore increased formation of urobilinogen in the gut. Bilirubin, being of unconjugated type, does not appear in urine. Increased urobilinogen in urine without bilirubin is

18

Essentials of Clinical Pathology

typical of hemolytic anemia. This also occurs in megaloblastic anemia due to premature destruction of erythroid precursors in bone marrow (ineffective erythropoiesis). 2. Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells. Causes of Reduced Urobilinogen in Urine 1. Obstructive jaundice: In biliary tract obstruction, delivery of bilirubin to the intestine is restricted and very little or no urobilinogen is formed. This causes stools to become clay-colored. 2. Reduction of intestinal bacterial flora: This prevents conversion of bilirubin to urobilinogen in the intestine. It is observed in neonates and following antibiotic treatment. Testing of urine for both bilirubin and urobilinogen can provide helpful information in a case of jaundice (Table 1.6). Tests for Detection of Urobilinogen in Urine Fresh urine sample should be used because on standing urobilinogen is converted to urobilin, which cannot be detected by routine tests. A timed (2-hour postprandial) sample can also be used for testing urobilinogen. Methods for detection of increased amounts of urobilinogen in urine are Ehrlich’s aldehyde test and reagent strip test. 1. Ehrlich’s aldehyde test: Ehrlich’s reagent (pdimethylaminobenzaldehyde) reacts with urobilinogen in urine to produce a pink color. Intensity of color developed depends on the amount of urobilinogen present. Presence of bilirubin interferes with the reaction, and therefore if present, should be removed. For this, equal volumes of urine and 10% barium chloride are mixed and then filtered. Test for urobilinogen is carried out on the filtrate. However, similar reaction is produced by porphobilinogen (a substance excreted in urine in patients of porphyria). Method: Take 5 ml of fresh urine in a test tube. Add 0.5 ml of Ehrlich’s aldehyde reagent (which consists of hydrochloric acid 20 ml, distilled water 80 ml, and paradimethylaminobenzaldehyde 2 gm). Allow to stand at room temperature for 5 minutes. Development of pink color indicates normal amount of urobilinogen. Dark red color means increased amount of urobilinogen (Fig. 1.17). Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. For this, Watson-Schwartz

Fig. 1.17: Ehrlich’s aldehyde test for urobilinogen

test is used. Add 1-2 ml of chloroform, shake for 2 minutes and allow to stand. Pink color in the chloroform layer indicates presence of urobilinogen, while pink coloration of aqueous portion indicates presence of porphobilinogen. Pink layer is then decanted and shaken with butanol. A pink color in the aqueous layer indicates porphobilinogen (Fig. 1.18). False-negative reaction can occur in the presence of (i) urinary tract infection (nitrites oxidize urobilinogen to urobilin), and (ii) antibiotic therapy (gut bacteria which produce urobilinogen are destroyed). 2. Reagent strip method: This method is specific for urobilinogen. Test area is impregnated with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate. Blood The presence of abnormal number of intact red blood cells in urine is called as hematuria. It implies presence of a bleeding lesion in the urinary tract. Bleeding in urine may be noted macroscopically or with naked eye (gross hematuria). If bleeding is noted only by microscopic examination or by chemical tests, then it is called as occult, microscopic or hidden hematuria. Causes of Hematuria 1. Diseases of urinary tract • Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus nephritis, Henoch-Schonlein purpura

Examination of Urine

19

Fig. 1.18: Interpretation of Watson-Schwartz test

• Nonglomerular diseases: Calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, after strenuous physical exercise, diseases of prostate (benign hyperplasia of prostate, carcinoma of prostate). 2. Hematological conditions: Coagulation disorders, sickle cell disease Presence of red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria. Tests for Detection of Blood in Urine 1. Microscopic examination of urinary sediment: Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test. 2. Chemical tests: These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red

cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Fig. 1.19). Intensity of color produced is proportional to the amount of hemoglobin present. Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria. • Benzidine test: Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive. • Orthotoluidine test: In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test. • Reagent strip test: Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).

Fig. 1.19: Principle of chemical test for red cells, hemoglobin, or myoglobin in urine

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Essentials of Clinical Pathology

Fig. 1.20: Evaluation of positive chemical test for blood in urine

Causes of false-positive tests: • Contamination of urine by menstrual blood in females • Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection. Causes of false-negative tests: • Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative. • Use of formalin as a preservative for urine Evaluation of positive chemical test for blood is shown in Figure 1.20. Hemoglobin Presence of free hemoglobin in urine is called as hemoglobinuria. Causes of Hemoglobinuria 1. Hematuria with subsequent lysis of red blood cells in urine of low specific gravity. 2. Intravascular hemolysis: Hemoglobin will appear in urine when haptoglobin (to which hemoglobin binds in plasma) is completely saturated with hemoglobin. Intravascular hemolysis occurs in infections (severe falciparum malaria, clostridial infection, E. coli septicemia), trauma to red cells (march hemoglobinuria, extensive burns, prosthetic heart valves),

glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation. Tests for Detection of Hemoglobinuria Tests for detection of hemoglobinuria are benzidine test, orthotoluidine test, and reagent strip test. Hemosiderin Hemosiderin in urine (hemosiderinuria) indicates presence of free hemoglobin in plasma. Hemosiderin appears as blue granules when urine sediment is stained with Prussian blue stain (Fig. 1.21). Granules are located inside tubular epithelial cells or may be free if cells have disintegrated. Hemosiderinuria is seen in intravascular hemolysis. Myoglobin Myoglobin is a protein present in striated muscle (skeletal and cardiac) which binds oxygen. Causes of myoglobinuria include injury to skeletal or cardiac muscle, e.g. crush injury, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.

Examination of Urine

Fig. 1.21: Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue)

Chemical tests used for detection of blood or hemoglobin also give positive reaction with myoglobin (as both hemoglobin and myoglobin have peroxidase activity). Ammonium sulfate solubility test is used as a screening test for myoglobinuria (Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and is precipitated. A positive chemical test for blood done on supernatant indicates myoglobinuria). Distinction between hematuria, hemoglobinuria, and myoglobinuria is shown in Table 1.7. Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection) In addition to direct microscopic examination of urine sample, chemical tests are commercially available in a

21

reagent strip format that can detect significant bacteriuria: nitrite test and leucocyte esterase test. These tests are helpful at places where urine microscopy is not available. If these tests are positive, urine culture is indicated. 1. Nitrite test: Nitrites are not present in normal urine; ingested nitrites are converted to nitrate and excreted in urine. If gram-negative bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests. As E. coli is the commonest organism causing urinary tract infection, this test is helpful as a screening test for urinary tract infection. Some organisms like Staphylococci or Pseudomonas do not reduce nitrate to nitrite and therefore in such infections nitrite test is negative. Also, urine must be retained in the bladder for minimum of 4 hours for conversion of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred. Sufficient dietary intake of nitrate is necessary. Therefore a negative nitrite test does not necessarily indicate absence of urinary tract infection. The test detects about 70% cases of urinary tract infections. 2. Leucocyte esterase test: It detects esterase enzyme released in urine from granules of leucocytes. Thus the test is positive in pyuria. If this test is positive, urine culture should be done. The test is not sensitive to leucocytes < 5/HPF.

MICROSCOPIC EXAMINATION Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”. Urine consists of various microscopic, insoluble, solid elements in suspension. These elements are classified as

Table 1.7: Differentiation between hematuria, hemoglobinuria, and myoglobinuria Parameter

Hematuria

Hemoglobinuria

Myoglobinuria

1. Urine color

Normal, smoky, red, or brown

Pink, red, or brown

Red or brown

2. Plasma color

Normal

Pink

Normal

3. Urine test based on

Positive

Positive

Positive

peroxidase activity 4. Urine microscopy

Many red cells

Occasional red cell

Occasional red cell

5. Serum haptoglobin

Normal

Low

Normal

6. Serum creatine kinase

Normal

Normal

Markedly increased

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Essentials of Clinical Pathology

organized or unorganized. Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 1.8. Different types of urinary sediments are shown in Figure 1.22. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance. Specimen: The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be

examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine. Method: A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Fig. 1.23). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination. Cells Cellular elements in urine are shown in Figure 1.24.

Table 1.8: Urinary findings in renal diseases Condition

Albumin

RBCs/HPF

WBCs/HPF

Casts/LPF

Others

1. Normal

0-trace

0-2

0-2

Occasional (Hyaline)



2. Acute glomerulonephritis

1-2+

Numerous; dysmorphic

0-few

Red cell, granular

Smoky urine or hematuria

3. Nephrotic syndrome

>4+

0-few

0-few

Fatty, hyaline, Waxy, epithelial

Oval fat bodies, lipiduria

4. Acute pyelonephritis

0-1+

0-few

Numerous

WBC, granular

WBC clumps, bacteria, nitrite test

HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells.

Fig. 1.22: Different types of urinary sediment

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Examination of Urine

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Red Blood Cells

Fig. 1.23: Preparation of urine sediment for microscopic examination

Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology. The quantity of red cells can be reported as number of red cells per high power field. Causes of hematuria have been listed earlier.

Fig. 1.24: Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa

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Essentials of Clinical Pathology

White Blood Cells (Pus Cells) White blood cells are spherical, 10-15 μ in size, granular in appearance in which nuclei may be visible. Degenerated white cells are distorted, smaller, and have fewer granules. Clumps of numerous white cells are seen in infections. Presence of many white cells in urine is called as pyuria. In hypotonic urine white cells are swollen and the granules are highly refractile and show Brownian movement; such cells are called as glitter cells; large numbers are indicative of injury to urinary tract. Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of clumps is suggestive of urinary tract infection. Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection. In urinary tract infection, following are usually seen in combination: • Clumps of pus cells or pus cells >10/HPF • Bacteria • Albuminuria • Positive nitrite test Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelonephritis). Eosinophils (>1% of urinary leucocytes) are a characteristic feature of acute interstitial nephritis due to drug reaction (better appreciated with a Wright’s stain). Renal Tubular Epithelial Cells Presence of renal tubular epithelial cells is a significant finding. Increased numbers are found in conditions causing tubular damage like acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy metal poisoning. These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen. Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations.

diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma. Oval Fat Bodies These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria. Spermatozoa They may sometimes be seen in urine of men. Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and all types of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis. Organisms Organisms detectable in urine are shown in Figure 1.25. Bacteria Bacteria in urine can be detected by microscopic examination, reagent strip tests for significant bacteriuria (nitrite test, leucocyte esterase test), and culture. Method of collection for bacteriologic examination is given earlier in Box 1.2. Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >10 3 colonyforming units/ml of urine in a suprapubic aspiration sample.

Squamous Epithelial Cells Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus. Transitional Epithelial Cells Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and

Fig. 1.25: Organisms in urine: (A) Bacteria, (B) Yeasts, (C) Trichomonas, and (D) Egg of Schistosoma haematobium tahir99 - UnitedVRG vip.persianss.ir

Examination of Urine 1. Microscopic examination: In a wet preparation, presence of bacteria should be reported only when urine is fresh. Bacteria occur in combination with pus cells. Gram’s-stained smear of uncentrifuged urine showing 1 or more bacteria per oil-immersion field suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, then urine is probably contaminated with vaginal flora. Also, presence of only bacteria without pus cells indicates contamination with vaginal or skin flora. 2. Chemical or reagent strip tests for significant bacteriuria: These are given earlier. 3. Culture: On culture, a colony count of >105/ml is strongly suggestive of urinary tract infection, even in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gramnegative enteric bacteria, particularly Escherichia coli. If three or more species of bacteria are identified on culture, it almost always indicates contamination by vaginal flora. Negative culture in the presence of pyuria (‘sterile’ pyuria) occurs with prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (irrespective of cause), female genital tract infection, and non-specific urethritis in males. Yeast Cells (Candida) These are round or oval structures of approximately the same size as red blood cells. In contrast to red cells, they show budding, are oval and more refractile, and are not soluble in 2% acetic acid. Presence of Candida in urine may suggest immunocompromised state, vaginal candidiasis, or diabetes

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mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state. Trichomonas vaginalis These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility. Eggs of Schistosoma haematobium Infection by this organism is prevalent in Egypt. Microfilariae They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel. Casts Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Fig. 1.26). Casts are best seen under low power

Fig. 1.26: Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts tahir99 - UnitedVRG vip.persianss.ir

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Essentials of Clinical Pathology

objective (×10) with condenser lowered down to reduce the illumination. Casts are the only elements in the urinary sediment that are specifically of renal origin. Casts (Fig. 1.27) are of two main types: • Noncellular: Hyaline, granular, waxy, fatty • Cellular: Red blood cell, white blood cell, renal tubular epithelial cell. Hyaline and granular casts may appear in normal or diseased states. All other casts are found in kidney diseases. Non-cellular Casts Hyaline casts: These are the most common type of casts in urine and are homogenous, colorless, transparent, and refractile. They are cylindrical with parallel sides and blunt, rounded ends and low refractive index. Presence of occasional hyaline cast is considered as normal. Their presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of TammHorsfall protein. They occur transiently after strenuous

muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria. Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis. Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure. Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol

Fig. 1.27: Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast, (F) White cell cast, and (G) Epithelial cast

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Examination of Urine esters) in Tamm-Horsfall protein matrix. They are seen in nephrotic syndrome. Broad casts: Broad casts form in dilated distal tubules and are seen in chronic renal failure and severe renal tubular obstruction. Both waxy and broad casts are associated with poor prognosis. Cellular Casts To be called as cellular, casts should contain at least three cells in the matrix. Cellular casts are named according to the type of cells entrapped in the matrix. Red cell casts: These are cylindrical structures with red cells in Tamm-Horsfall protein matrix. They may appear brown in color due to hemoglobin pigmentation. These have greater diagnostic importance than any other cast. If present, they help to differentiate hematuria due to glomerular disease from hematuria due to other causes. RBC casts usually denote glomerular pathology e.g. acute glomerulonephritis. White cell casts: These are cylindrical structures with white blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the interstitium and therefore presence of leucocyte casts indicates tubulointerstitial disease like pyelonephritis. Renal tubular epithelial cell casts: These are composed of renal tubular epithelial cells that have been sloughed off. They are seen in acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even an occasional renal tubular cast is a significant finding. Crystals Crystals are refractile structures with a definite geometric shape due to orderly 3-dimensional arrangement of its atoms and molecules. Amorphous material (or deposit) has no definite shape and is commonly seen in the form of granular aggregates or clumps. Crystals in urine (Fig. 1.28) can be divided into two main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states). However, crystals found in normal urine can also be seen in some diseases in increased numbers. Most crystals have no clinical importance (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, before reporting presence of any abnormal crystals, it is necessary to confirm them by chemical tests.

27

Normal Crystals Crystals present in acid urine a. Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine. b. Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning. c. Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C. Crystals present in alkaline urine: a. Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve. b. Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits. • Phosphate crystals Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffinlids”), or may have a feathery fern-like appearance. Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (starshaped, plates or prisms). • Amorphous phosphates: These occur as colorless small granules, often dispersed. All phosphates are soluble in dilute acetic acid. 



c. Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C. Abnormal Crystals They are rare, but result from a pathological process. These occur in acid pH, often in large amounts. Abnormal

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Essentials of Clinical Pathology

Fig. 1.28: Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin, (4) Tyrosine, (5) Sulfonamide, and (6) Leucine

crystals should not be reported on microscopy alone; additional chemical tests are done for confirmation. 1. Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones. 2. Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners,

and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope. 3. Bilirubin crystals: These are small (5 μ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease. tahir99 - UnitedVRG vip.persianss.ir

Examination of Urine 4. Leucine crystals: These are refractile, yellow or brown, spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along with tyrosine in severe liver disease (cirrhosis). 5. Tyrosine crystals: They appear as clusters of fine, delicate, colorless or yellow needles and are seen in liver disease and tyrosinemia (an inborn error of metabolism). They dissolve in alkali. 6. Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.

REFERENCE RANGES Volume in 24 hours: Adults: 600-2000 ml Color: Pale yellow to colorless Appearance: Clear Odor: Aromatic Specific gravity: 1.003-1.030 Osmolality: 300-900 mOsm/kg of water pH: 4.6-8.0 (Average: 6.0) Proteins: Qualitative test: Negative Quantitative test: < 150 mg/24 hours Albumin: < 30 mg/24 hours Glucose: Qualitative test: Negative Quantitative test: < 500 mg/24 hours (< 15 mg/dl) Ketones: Qualitative test: Negative Bilirubin: Negative Bile salts: Negative Occult blood: Negative Urobilinogen: 0.5-4.0 mg/24 hours Myoglobin (Ammonium sulphate solubility test): Negative Microscopy: 1-2 red cells, pus cells, or epithelial cells/ HPF; occasional hyaline cast/LPF; Phosphate, oxalate, or urate crystals depending on urine pH.

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CRITICAL FINDINGS • • • •

Strongly positive test for glucose and ketone bodies Positive test for reducing sugar in an infant Hemoglobinuria Red cell casts or >50% dysmorphic red cells on microscopic examination • Abnormal crystals like cysteine, leucine, or tyrosine.

BIBLIOGRAPHY 1. Burtis CA, Ashwood ER (Eds). Tietz fundamentals of clinical chemistry (5th Ed). Philadelphia; WB Saunders Company, 2001. 2. Carroll MF, Temte JL. Proteinuria in adults: A diagnostic approach. Am Fam Physician 2000;62:1333-40. 3. Cheesbrough M. District laboratory practice in tropical countries. Part 1 and Part 2. Cambridge; Cambridge University Press, 1998. 4. Grossfeld GD, Wolf JS, Litwin MS, et al. Asymptomatic microscopic hematuria in adults: Summary of the AUA best policy recommendations. Am Fam Physician 2001; 63:1145-54. 5. Henry JB (Ed): Clinical diagnosis and management by laboratory methods. (20th Ed). Philadelphia; WB Saunders Company, 2001. 6. King M. A medical laboratory for developing countries. London. Oxford University Press, 1973. 7. Mathieson PW. The cellular basis of albuminuria. Clinical Science 2004;107:533-8. 8. Simerville JA, Maxted WC, Pahira JJ. Urinalysis: A comprehensive review. Am Fam Physician 2005;71: 1153-62. 9. Wallach J. Interpretation of diagnostic tests. (7th Ed). Philadelphia. Lippincott Williams and Wilkins, 2000. 10. World Health Organization. Manual of basic techniques for a health laboratory (2nd Ed). Geneva; World Health Organization, 2003.

tahir99 - UnitedVRG vip.persianss.ir

2

Renal Function Tests

Kidney is a highly specialized organ that performs following functions: • Maintenance of extracellular fluid volume and composition: Kidney regulates water and electrolyte balance, acid-base balance, and fluid osmotic pressure. • Excretion of metabolic waste products (blood urea, creatinine, uric acid) and drugs, but retention of essential substances (like glucose and amino acids). • Regulation of blood pressure by renin-angiotensin mechanism • Synthesis of erythropoietin, a hormone which stimulates erythropoiesis • Production of vit. D3 (active form of vit. D) from vit. D2, which stimulates absorption of calcium from gastrointestinal tract.

FACTORS AFFECTING RENAL FUNCTION Kidney function is affected by following factors: • Diffuse renal disease. • Pre-renal conditions—Decreased renal blood flow as in dehydration, congestive cardiac failure and shock. • Post-renal conditions—Obstruction to urinary outflow.

INDICATIONS FOR RENAL FUNCTION TESTS 1. Early identification of impairment of renal function in patients with increased risk of chronic renal disease: Early detection and treatment of renal impairment in chronic renal disease prevent complications of chronic renal failure and is associated with improved prognosis. Laboratory tests can be applied in individuals who are at increased risk of developing chronic renal disease (Box 2.1) to detect renal functional impairment at an early stage and to detect degree of kidney damage. 2. Diagnosis of renal disease 3. Follow the course of renal disease and assess response to treatment.

Box 2.1: Conditions with increased risk of chronic renal disease • • • • • • • •

Diabetes mellitus Hypertension Autoimmune diseases like systemic lupus erythematosus Older age (GFR declines with age) Family history of renal disease Systemic infection Urinary tract infection Lower urinary tract obstruction

4. Plan renal replacement therapy (dialysis or renal transplantation) in advanced renal disease. 5. Adjust dosage of certain drugs (e.g. chemotherapy) according to renal function.

CLASSIFICATION OF RENAL FUNCTION TESTS Renal function tests can be classified as shown in Table 2.1. In practice, the commonly performed renal function tests are routine urinalysis, estimation of serum creatinine, blood urea nitrogen (BUN), BUN/Serum creatinine ratio, creatinine clearance test (or estimation of GFR from serum creatinine value by a prediction equation), and estimation of urine concentrating ability (water deprivation test). Urine examination is the first test performed in patients suspected of having renal disease. It is the simplest and the least expensive renal function test. In urine examination parameters that can assess renal function are urine volume in 24 hours, specific gravity, osmolality, proteinuria, and microscopic examination of urinary sediment. Tests to Evaluate Glomerular Function The best test to assess overall kidney function is estimation of glomerular filtration rate or GFR (Box 2.2). GFR varies according to age, sex, and body surface area. tahir99 - UnitedVRG vip.persianss.ir

Renal Function Tests

31

Table 2.1: Classification of renal function tests Tests to evaluate glomerular function

Tests to evaluate tubular function

1. Clearance tests to measure glomerular 1. Tests to assess proximal tubular function: filtration rate: Inulin clearance, 125I-iothalamate clearance, 51Cr-EDTA clearance, Cystatin C • Glycosuria, phosphaturia, uricosuria clearance, Creatinine clearance, and Urea • Generalized aminoaciduria clearance • Tubular proteinuria 2. Calculation of creatinine clearance from • Fractional sodium excretion prediction equations 2. Tests to assess distal tubular function: 3. Blood biochemistry: Serum creatinine, • Specific gravity and osmolality of urine Blood urea nitrogen (BUN), and BUN/serum creatinine ratio • Water-deprivation test and water-loading test 4. Microalbuminuria and albuminuria • Ammonium chloride loading test

Box 2.2: Glomerular filtration rate (GFR) • Best test for assessment of excretory renal function • Varies according to age, sex, and body weight of an individual; a normal GFR also depends on normal renal blood flow and pressure. • Normal GFR in young adults is 120-130 ml/min per 1.73 m2. • Creatinine clearance is commonly used as a measure of GFR. Equations can be used to estimate GFR from serum creatinine value. • GFR declines with age (due to glomerular arteriolosclerosis) • GFR 800 mOsm/kg of water or specific gravity is ≥1.025 following dehydration, concentrating ability of renal tubules is normal. However, normal result does not rule out presence of renal disease. False result will be obtained if the patient is on low-salt, low-protein diet or is suffering from major electrolyte and water disturbance. 4. Water loading antidiuretic hormone suppression test This test assesses the capacity of the kidney to make urine dilute after water loading. After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in 15-30 minutes. The urine is

Renal Function Tests collected at hourly intervals for the next 4 hours for measurements of urine volume, specific gravity, and osmolality. Plasma levels of antidiuretic hormone and serum osmolality should be measured at hourly intervals. Normally, more than 90% of water should be excreted in 4 hours. The specific gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg. Plasma level of antidiuretic hormone should be appropriate for serum osmolality. In renal function impairment, urine volume is reduced (60 years: 8-21 mg/dl Serum creatinine: Adult males: 0.7-1.3 mg/dl Adult females: 0.6-1.1 mg/dl Children ( 5 mg/dl BUN: > 80 mg/dl

BIBLIOGRAPHY 1. Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart MJ, Shepherd J. Clinical Biochemistry: An Illustrated Colour Text (3rd Ed). Edinburgh: Churchill Livingstone 2004. 2. Johnson CA, Levey AS, Coresh J, Levin A, Lau J, Eknoyan G. Clinical practice guidelines for chronic kidney disease in adults: Part II. Glomerular filtration rate, proteinuria, and other markers Am Fam Physician 2004;70:1091-7. 3. Stevens LA, Coresh J, Green T, Levey AS. Assessing kidney function-measured and estimated glomerular filtration rate. N Engl J Med 2006;354:2473-83. 4. Stevens LA, Levey AS. Measurement of kidney function. Med Clin N Am 2005;89:457-73.

3

Diabetes Mellitus

Diabetes mellitus (DM) is a metabolic group of disorders characterized by persistent hyperglycemia due to deficiency and/or diminished effectiveness of insulin. There are derangements of carbohydrate, protein, and fat metabolism due to failure of insulin action on target cells. Typical features of DM are as follows: • Fasting hyperglycemia • Glycosuria • Symptoms due to marked hyperglycemia: polyuria, polydipsia, weight loss, weakness, polyphagia, and blurred vision • Long-term complications like atherosclerosis (leading to ischemic heart disease, cerebrovascular disease, and peripheral vascular disease) and microangiopathy (which can cause nephropathy with risk of renal failure; retinopathy with potential loss of vision; and peripheral neuropathy with risk of foot ulcers, amputations, or Charcot joints). • Acute metabolic complications (hyperosmolar hyperglycemic state, diabetic ketoacidosis). • Susceptibility to infections especially of skin, respiratory tract, and urinary tract.

METABOLIC ACTIONS OF INSULIN Insulin is the major hormone regulating blood glucose level. Insulin is synthesized by β cells of pancreas as preproinsulin, which is rapidly converted to proinsulin. Proinsulin is a single chain polypeptide. In the Golgi apparatus, proinsulin is broken down into 2 units- insulin (51 amino acids) and C (connecting)-polypeptide (31 amino acids) (Fig. 3.1). Both insulin and C peptide are stored in membrane-bound granules in the cytoplasm of β cells. Upon stimulation (mainly by blood glucose), both insulin and C peptide are released in circulation. C peptide is often measured as a marker of activity of β cells. C peptide has no known function. Insulin acts on various cells (especially those of liver, muscle, and adipose tissue) through receptors. Important actions of insulin are shown in Box 3.1.

Fig. 3.1: Proinsulin, insulin, and C-peptide. The biochemical cleavage of proinsulin to insulin and C-peptide occurs in Golgi apparatus of β cell. Secretion of insulin is stimulated by glucose, mannose, amino acids, and sulfonylureas

“Stress hormones” like glucagons, glucocorticoids, growth hormone, and adrenaline oppose action of insulin.

CLASSIFICATION OF DIABETES MELLITUS According to American Diabetes Association (1997), DM is classified into following types: • Type 1 (Absolute deficiency of insulin due to destruction of β cells of pancreas) – Immune mediated – Idiopathic

40

Essentials of Clinical Pathology Box 3.1: Major actions of insulin

• Increases: – Uptake of glucose in skeletal muscle and adipose tissue – Glycogenesis in liver and muscle – Fatty acid and triglycerides in liver and adipose tissue – Protein synthesis in liver and muscle • Decreases: – Gluconeogenesis in liver – Glycogenolysis in liver and muscle – Lipolysis in adipose tissue – Ketogenesis in liver

• Type 2 (Insulin resistance along with relative deficiency of insulin secretion) • Other specific types • Gestational DM (onset or first recognition of glucose intolerance during pregnancy).

Fig. 3.2: Pathogenesis of type I diabetes mellitus

Type 1 Diabetes Mellitus It accounts for 5-10% of all cases of DM. This was previously called as insulin-dependent DM or IDDM (because insulin therapy is essential to prevent ketosis), juvenile-onset DM (because it commonly presents during childhood or adolescence), brittle DM, or ketosis-prone DM. It is characterized by absolute deficiency of insulin secretion. Cell-mediated autoimmune destruction of β cells of pancreas is responsible for majority of cases of type 1 DM (immune-mediated type 1 DM), leading to inability of pancreas to synthesize insulin. There is

infiltration by cytotoxic CD8+ T lymphocytes in and around islets. It is thought that many cases follow a viral infection that has damaged the islet cells of pancreas (Fig. 3.2). Markers of immune destruction of β cells, which can be detected in peripheral blood, are islet cell antibodies, autoantibodies to insulin, autoantibodies to glutamic acid decarboxylase (GAD65), and autoantibodies to tyrosine phosphatases (IA-2 and IA-2b). The disease has strong association with HLA DR3 and HLA DR4 haplotypes (Fig. 3.3). This type occurs mainly in

Fig. 3.3: HLA-linked genetic predisposition to type 1 diabetes mellitus

Diabetes Mellitus children and adolescents, but can occur at any age. These patients are also at risk of other autoimmune disorders like Graves’ disease, Hashimoto’s thyroiditis, vitiligo, Addison’s disease, pernicious anemia, etc. Some cases of type 1 DM do not have any known etiologies or evidence of autoimmunity. These individuals are of Asian or African origin and their disease is strongly inherited. This form of type 1 DM is called as idiopathic DM. Type 2 Diabetes Mellitus This is the most common form of DM comprising about 90-95% of all patients of DM. This was previously called as non-insulin-dependent DM (NIDDM), maturity-onset DM (because onset usually occurs during adult life), stable DM, or ketosis-resistant DM. It is characterized by insulin resistance along with relative deficiency of insulin secretion (i.e. inadequate insulin secretory response to overcome peripheral insulin resistance). (Fig. 3.4). Type 2 DM is not HLA-linked and there is no role of autoimmunity in its pathogenesis. It has a strong genetic predisposition. Type 2 DM occurs more frequently in individuals with positive family history (parents or siblings with DM), obesity (≥ 20% over ideal body weight or body mass index ≥ 25 kg/m2), hypertension (>140/90 mm Hg in adults), dyslipidemia, lack of physical activity, pre-diabetes (impaired fasting glucose or impaired glucose tolerance), and prior gestational DM. Type 2 diabetes is more common in certain racial groups like South Asians and Africans. Rising trend of type 2 DM is due to increasing tendency towards obesity in urban populations coupled with high-calorie diet. Differences between type 1 and type 2 DM are listed in Table 3.1.

41

Other Specific Types There are several forms of DM associated with underlying conditions: • Genetic defects of β cell function: In these disorders, insulin secretion from β cells is impaired. These are called as maturity onset diabetes of the young (MODY). They are inherited in an autosomal dominant manner and they are caused by mutations in genetic loci such as hepatic nuclear factor, glucokinase, etc. • Genetic defects in insulin action: These result from mutations in insulin receptor gene. • Diseases of exocrine pancreas: Diseases causing generalized pancreatic damage can result in DM. These include cystic fibrosis, hemochromatosis, chronic pancreatitis, trauma, pancreatectomy, and pancreatic cancer. • Endocrine disorders: Several hormones inhibit the action of insulin. Excessive secretion of these hormones will cause DM. Hyperglycemia is corrected following resolution of the primary endocrinopathy. Endocrine disorders associated with hyperglycemia are: – Acromegaly: Excess growth hormone. – Cushing’s syndrome: Excess cortisol. – Glucagonoma: Excess glucagon – Pheochromocytoma: Excess epinephrine. – Hyperthyroidism: Excess thyroxine • Drug- or chemical-induced DM: Drugs or chemicals can impair insulin secretion or insulin action. Destruction of β cells and formation of islet cell antibodies have also been reported with some drugs. Examples include thiazide diuretics, α-interferon, and glucocorticoids. • Infections: Certain viral infections (such as Coxsackie virus B, congenital rubella, cytomegalovirus) can cause destruction of β cells. • Other genetic syndromes sometimes associated with DM: Many genetic syndromes (e.g. Down’s syndrome, Klinefelter’s syndrome, Turner’s syndrome) are associated with increased risk of developing DM. Gestational Diabetes Mellitus (GDM)

Fig. 3.4: Pathogenesis of type 2 diabetes mellitus

This refers to the onset or first recognition of glucose intolerance during pregnancy. GDM occurs in 2-3% of all pregnant women. It is associated with increased risk of high birth weight of the newborn, cardiac defects, polyhydramnios, intrauterine fetal loss (due to placental insufficiency), premature birth, hypertension during pregnancy, pre-eclampsia, and alteration in duration of pregnancy. Early diagnosis and treatment

42

Essentials of Clinical Pathology Table 3.1: Differences between type 1 and type 2 diabetes mellitus

Parameter 1. Previous names 2. 3. 4. 5.

% of all DM cases Age of onset Obesity Presentation

6. 7. 8. 9. 10. 11. 12. 13.

Genetic predisposition Glucose intolerance Ketoacidosis Hyperosmolar hyperglycemic state Serum insulin Autoimmunity (Islet cell antibodies) HLA-linkage Pathogenesis

14. Serum C-peptide level 15. Predisposing factors 16. Insulin requirement for treatment

Type 1 DM

Type 2 DM

Type I, IDDM, Juvenile-onset, Ketosis-prone, Brittle 5-10% < 35 years No (weight loss common) Acute; symptoms (classical) since few weeks Low Marked Common Uncommon Undetectable Majority of cases Yes (DR3, DR4) Absolute insulin deficiency

Type II, NIDDM, maturity-onset, Ketosis-resistant, Stable 90-95% > 35 years Common Insidious; symptoms since few months or years Strong Mild Uncommon Common Normal or high No No Insulin resistance with relative insulin deficiency Normal or high Obesity, lack of physical activity In some situations

Very low to absent Viral infections, toxins Always

of GDM are essential to prevent perinatal morbidity and mortality. After delivery, GDM can have following course: 1. Return to normal glucose tolerance (however, many of these patients are likely to develop DM during subsequent years), or 2. Persistence of DM or impaired glucose tolerance. Patient should be reassessed 6 weeks or later following delivery. Risk factors for GDM are shown in Box 3.2.

acute complications of DM and microangiopathy, but are at increased risk of cardiovascular disease (1.5 times normal individuals). Development of DM can be delayed or prevented through modest amount of weight loss, diet modification, and regular moderate exercise in patients with prediabetes.

PREDIABETES

The metabolic syndrome refers to a constellation of lipid and non-lipid risk factors that are of metabolic origin and associated with risk of cardiovascular disease. The Adult Treatment Panel III (ATP III) 1 of the National Cholesterol Education Program in 2001 proposed criteria for diagnosing the metabolic syndrome. The metabolic syndrome is diagnosed when three or more out of following five criteria are present. • Abdominal obesity – Men: Waist circumference > 40 inches (102 cm) – Women: Waist circumference > 35 inches (88 cm) • Fasting glucose ≥ 110 to < 126 mg/dl (As these criteria were proposed in 2001, the fasting plasma glucose value should be reduced to 100 mg/dl according to revised criteria proposed by American Diabetes Association in 2004). • Blood pressure ≥ 130/> 85 mm Hg • Triglycerides ≥ 150 mg/dl (> 1.7 mmol/L)

Prediabetes is a state in which plasma glucose level is higher than normal but not high enough for diagnosis of DM. It is also referred to as impaired fasting glucose (IFG) or impaired glucose tolerance (IGT), depending on which test is used for its detection. Studies have shown that majority of individuals with prediabetes develop type 2 DM within 10 years. Prediabetic persons do not develop

Box 3.2: Risk factors for gestational DM • • • • •

Past history of GDM Previous high-birth-weight baby Obesity Family history of diabetes mellitus High-risk ethnic group: South Asian or African.

METABOLIC SYNDROME (INSULIN RESISTANCE SYNDROME, REAVEN’S SYNDROME, SYNDROME X)

Diabetes Mellitus • Plasma high density lipoprotein (HDL)-cholesterol – Men: < 40 mg/dl (< 1 mmol/L) – Women: < 50 mg/dl (< 1.3 mmol/L) Metabolic syndrome is common in Asian Indians in Britain and in Africa. It is said that insulin resistance is common to all above risk factors and plays an etiological role. These persons have increased risk of developing Type 2 DM.

METABOLIC ALTERATIONS IN DIABETES MELLITUS In DM, there are abnormalities of carbohydrate metabolism (hyperglycemia, glycosuria, impaired glucose tolerance), protein metabolism (increased protein catabolism with muscle wasting, gluconeogenesis), and fat metabolism (increased fatty acid synthesis, ketosis) (Fig. 3.5). Metabolic alterations in type 2 DM are less severe than in type 1 DM. • Hyperglycemia: Hyperglycemia is due to deficient uptake of glucose by muscle and fat cells (due to

43

insulin deficiency). In reaction to this, there are compensatory glycogenolysis (breakdown of glycogen to glucose) and gluconeogenesis (formation of glucose from non-carbohydrates like proteins), which contribute to hyperglycemia. Typical clinical features of hyperglycemia are polyuria, polydipsia, polyphagia, weakness, weight loss, and blurring of vision. • Glycosuria: Glycosuria results when blood glucose level exceeds renal threshold (180 mg/dl or 10 mmol/ L in most individuals). Excess glucose increases osmolality of glomerular filtrate with resultant osmotic diuresis and polyuria. This causes depletion of water and electrolytes, cellular dehydration, and intense thirst (polydipsia). Insulin deficiency leads to catabolism of proteins, and released amino acids are used for formation of glucose (gluconeogenesis). Breakdown of lipids also occurs and coupled with proteolysis, lead to negative energy balance and weight loss. This in turn induces polyphagia (increased appetite).

Fig. 3.5: Metabolic alterations in diabetes mellitus

44

Essentials of Clinical Pathology

• Ketosis: Insulin deficiency leads to increased degradation of lipids (lipolysis), resulting in increased levels of free fatty acids in circulation. These are converted to acetoacetyl CoA in the liver. Acetoacetyl CoA, in turn, is converted to ketone bodies. If muscles or other tissues do not utilize ketone bodies equal to the rate of their formation, they accumulate in blood. With the rise in blood levels of ketone bodies (ketonemia), they are excreted in urine (ketonuria). Ketone bodies are strong acids, which dissociate and release H+ ions. Bicarbonate removes these H+ ions in plasma and the level of bicarbonate falls. This produces metabolic acidosis. Symptoms of metabolic acidosis are nausea, vomiting, and hyperpnea (air hunger). Diabetic ketoacidosis (DKA) is a typical feature of type 1 DM. • Hyperosmolar hyperglycemic state (HHS): This occurs in type 2 patients due to combination of severe dehydration (secondary to sustained osmotic diuresis coupled with inadequate fluid intake) and hyperglycemia. It occurs usually in elderly with unrecognized DM or DM of recent onset.

LONG-TERM COMPLICATIONS OF DIABETES MELLITUS In long standing DM of both types, a wide variety of lesions develop in many organs, which are important causes of morbidity and mortality. Macroangiopathy (Macrovascular disease): In DM, atherosclerosis of aorta and of medium size arteries (like coronary, cerebral, and peripheral) occurs earlier in life and is more extensive than in non-diabetic patients. It can cause myocardial infarction, cerebrovascular accident, and gangrene of lower extremities. Pathogenesis of atherosclerosis in DM is related to hyperinsulinemia with peripheral insulin resistance and dyslipidemia (raised triglycerides, low high density lipoprotein or HDL, and raised low density lipoprotein or LDL). Microangiopathy (Microvascular disease): Microangiopathy is due to poor diabetes control (Box 3.3). Microangiopathy (thickening of walls of small blood vessels with narrowing of lumina) is common in kidneys (leading to renal insufficiency), retina (visual impairment), and peripheral nerves (sensory, motor or autonomic neuropathy). Infections: Diabetic patients are susceptible to infections of skin, respiratory tract (pneumonia, tuberculosis), and kidneys (pyelonephritis).

Box 3.3: Microangiopathy in diabetes mellitus • Risk is directly related to presence of high glucose level for long duration • Improved glycemic control significantly reduces the risk • Consists of: – Retinopathy: Visual loss can occur due to vitreous hemorrhage (from proliferating retinal vessels) and maculopathy – Nephropathy: Early stage is characterized by increased glomerular filtration rate and microalbuminuria; with progressive renal damage, overt proteinuria and renal failure develop. – Neuropathy: Postural hypotension, impotence, sensory and motor neuropathy, foot ulcer.

Average life expectancy of DM patients is reduced. Usual causes of death in DM include myocardial infarction, stroke, renal failure, infections, and ketoacidotic or hyperosmolar hyperglycemic coma.

ROLE OF LABORATORY TESTS IN DIABETES MELLITUS In DM, applications of laboratory tests are as follows: • Diagnosis of DM • Screening of DM • Assessment of glycemic control • Assessment of associated long-term risks • Management of acute metabolic complications. Laboratory Tests for Diagnosis of Diabetes Mellitus Diagnosis of DM is based exclusively on demonstration of raised blood glucose level (hyperglycemia). The current criteria (American Diabetes Association, 2004) for diagnosis of DM are as follows: Typical symptoms of DM (polyuria, polydipsia, weight loss) and random plasma glucose ≥ 200 mg/dl (≥ 11.1 mmol/L) Or Fasting plasma glucose ≥ 126 mg/dl (≥ 7.0 mmol/L) Or 2-hour post glucose load (75 g) value during oral glucose tolerance test ≥ 200 mg/dl (≥ 11.1 mmol/L). If any one of the above three criteria is present, confirmation by repeat testing on a subsequent day is necessary for establishing the diagnosis of DM. However, such confirmation by repeat testing is not required if

Diabetes Mellitus

45

patient presents with (a) hyperglycemia and ketoacidosis, or (b) hyperosmolar hyperglycemia. The tests used for laboratory diagnosis of DM are (1) estimation of blood glucose and (2) oral glucose tolerance test.

Addition of sodium fluoride is not necessary if plasma is separated from whole blood within 1 hour of blood collection. Plasma is preferred for estimation of glucose since whole blood glucose is affected also by concentration of proteins (especially hemoglobin).

Estimation of Blood Glucose

There are various methods for estimation of blood glucose: • Chemical methods: – Orthotoluidine method – Blood glucose reduction methods using neocuproine, ferricyanide, or copper.

Measurement of blood glucose level is a simple test to assess carbohydrate metabolism in DM (Fig. 3.6). Since glucose is rapidly metabolized in the body, measurement of blood glucose is indicative of current state of carbohydrate metabolism. Glucose concentration can be estimated in whole blood (capillary or venous blood), plasma or serum. However, concentration of blood glucose differs according to nature of the blood specimen. Plasma glucose is about 15% higher than whole blood glucose (the figure is variable with hematocrit). During fasting state, glucose levels in both capillary and venous blood are about the same. However, postprandial or post glucose load values are higher by 20-70 mg/dl in capillary blood than venous blood. This is because venous blood is on a return trip after delivering blood to the tissues. When whole blood is left at room temperature after collection, glycolysis reduces glucose level at the rate of about 7 mg/dl/hour. Glycolysis is further increased in the presence of bacterial contamination or leucocytosis. Addition of sodium fluoride (2.5 mg/ml of blood) maintains stable glucose level by inhibiting glycolysis. Sodium fluoride is commonly used along with an anticoagulant such as potassium oxalate or EDTA.

Chemical methods are less specific but are cheaper as compared to enzymatic methods. • Enzymatic methods: These are specific for glucose. – Glucose oxidase-peroxidase – Hexokinase – Glucose dehydrogenase Chemical methods have now been replaced by enzymatic methods. Terms used for blood glucose specimens: Depending on the time of collection, different terms are used for blood glucose specimens. • Fasting blood glucose: Sample for blood glucose is withdrawn after an overnight fast (no caloric intake for at least 8 hours). • Post meal or postprandial blood glucose: Blood sample for glucose estimation is collected 2 hours after the subject has taken a normal meal. • Random blood glucose: Blood sample is collected at any time of the day, without attention to the time of last food intake. Oral Glucose Tolerance Test (OGTT)

Fig. 3.6: Blood glucose values in normal individuals, prediabetes, and diabetes mellitus

Glucose tolerance refers to the ability of the body to metabolize glucose. In DM, this ability is impaired or lost and glucose intolerance represents the fundamental pathophysiological defect in DM. OGTT is a provocative test to assess response to glucose challenge in an individual (Fig. 3.7). American Diabetes Association does not recommend OGTT for routine diagnosis of type 1 or type 2 DM. This is because fasting plasma glucose cutoff value of 126 mg/dl identifies the same prevalence of abnormal glucose metabolism in the population as OGTT. World Health Organization (WHO) recommends OGTT in those cases in which fasting plasma glucose is in the range of impaired fasting glucose (i.e. 100-125 mg/ dl). Both ADA and WHO recommend OGTT for diagnosis of gestational diabetes mellitus.

46

Essentials of Clinical Pathology 3. A single venous blood sample is collected 2 hours after the glucose load. (Previously, blood samples were collected at ½, 1, 1½, and 2 hours, which is no longer recommended). 4. Plasma glucose is estimated in fasting and 2-hour venous blood samples. Interpretation of blood glucose levels is given in Table 3.2.

Fig. 3.7: Oral glucose tolerance curve

Preparation of the Patient • Patient should be put on a carbohydrate-rich, unrestricted diet for 3 days. This is because carbohydrate-restricted diet reduces glucose tolerance. • Patient should be ambulatory with normal physical activity. Absolute bed rest for a few days impairs glucose tolerance. • Medications should be discontinued on the day of testing. • Exercise, smoking, and tea or coffee are not allowed during the test period. Patient should remain seated. • OGTT is carried out in the morning after patient has fasted overnight for 8-14 hours. Test 1. A fasting venous blood sample is collected in the morning. 2. Patient ingests 75 g of anhydrous glucose in 250-300 ml of water over 5 minutes. (For children, the dose is 1.75 g of glucose per kg of body weight up to maximum 75 g of glucose). Time of starting glucose drink is taken as 0 hour.

OGTT in gestational diabetes mellitus: Impairment of glucose tolerance develops normally during pregnancy, particularly in 2nd and 3rd trimesters. Following are the recent guidelines of ADA for laboratory diagnosis of GDM: • Low-risk pregnant women need not be tested if all of the following criteria are met, i.e. age below 25 years, normal body weight (before pregnancy), absence of diabetes in first-degree relatives, member of an ethnic group with low prevalence of DM, no history of poor obstetric outcome, and no history of abnormal glucose tolerance. • Average-risk pregnant women (i.e. who are in between low and high risk) should be tested at 24-28 weeks of gestation. • High-risk pregnant women i.e. those who meet any one of the following criteria should be tested immediately: marked obesity, strong family history of DM, glycosuria, or personal history of GDM. Initially, fasting plasma glucose or random plasma glucose should be obtained. If fasting plasma glucose is ≥ 126 mg/dl or random plasma glucose is ≥ 200 mg/dl, repeat testing should be carried out on a subsequent day for confirmation of DM. If both the tests are normal, then OGTT is indicated in average-risk and high-risk pregnant women. There are two approaches for laboratory diagnosis of GDM • One step approach • Two step approach In one step approach, 100 gm of glucose is administered to the patient and a 3-hour OGTT is performed. This test may be cost-effective in high-risk pregnant women.

Table 3.2: Interpretation of oral glucose tolerance test Parameter

Normal

Impaired fasting glucose

Impaired glucose tolerance

Diabetes mellitus

1. Fasting (8 hr)

< 100

100-125



≥ 126

2. 2 hr OGTT

< 140

300 mg/24 hours). Significance of microalbuminuria in DM is as follows: • It is the earliest marker of diabetic nephropathy. Early diabetic nephropathy is reversible. • It is a risk factor for cardiovascular disease in both type 1 and type 2 patients. • It is associated with higher blood pressure and poor glycemic control. Specific therapeutic interventions such as tight glycemic control, administration of ACE (angiotensinconverting enzyme) inhibitors, and aggressive treatment of hypertension significantly slow down the progression of diabetic nephropathy. In type 2 DM, screening for microalbuminuria should begin at the time of diagnosis, whereas in type 1 DM, it should begin 5 years after diagnosis. At this time, a routine reagent strip test for proteinuria is carried out; if negative, testing for microalbuminuria is done. Thereafter, in all patients who test negative, screening for microalbuminuria should be repeated every year. Screening tests for microalbuminuria include: • Albumin to creatinine ratio in a random urine sample • Urinary albumin excretion in a 24-hour urine sample. Reagent strip tests to detect microalbuminuria are available. Positive results should be confirmed by more

Fig. 3.8: Evolution of diabetic nephropathy. In 80% of patients with type 1 DM, microalbuminuria progresses in 10-15 years to overt nephropathy that is then followed in majority of cases by progressive fall in GFR and ultimately end-stage renal disease. Amongst patients with type 2 DM and microalbuminuria, 20-40% of patients progress to overt nephropathy, and about 20% of patients with overt nephropathy develop end-stage renal disease. Abbreviation: GFR: Glomerular filtration rate

50

Essentials of Clinical Pathology

specific quantitative tests like radioimmunoassay and enzyme immunoassay. For diagnosis of microalbuminuria, tests should be positive in at least two out of three different samples over a 3 to 6 month period. Lipid Profile Abnormalities of lipids are associated with increased risk of coronary artery disease (CAD) in patients with DM. This risk can be reduced by intensive treatment of lipid abnormalities. Lipid parameters which should be measured include: • Total cholesterol • Triglycerides • Low-density lipoprotein (LDL) cholesterol • High-density lipoprotein (HDL) cholesterol The usual pattern of lipid abnormalities in type 2 DM is elevated triglycerides, decreased HDL cholesterol and higher proportion of small, dense LDL particles. Patients with DM are categorized into high, intermediate and low-risk categories depending on lipid levels in blood (Table 3.3). Annual lipid profile is indicated in all adult patients with DM.

Laboratory Tests in the Management of Acute Metabolic Complications of Diabetes Mellitus The three most serious acute metabolic complications of DM are: • Diabetic ketoacidosis (DKA) • Hyperosmolar hyperglycemic state (HHS) • Hypoglycemia The typical features of DKA are hyperglycemia, ketosis, and acidosis. The common causes of DKA are infection, noncompliance with insulin therapy, alcohol abuse and myocardial infarction. Patients with DKA present with rapid onset of polyuria, polydipsia, polyphagia, weakness, vomiting, and sometimes abdominal pain. Signs include Kussmaul’s respiration, odour of acetone on breath (fruity), mental clouding, and dehydration. Classically, DKA occurs in type 1, while HHS is more typical of type 2 DM. However, both complications can occur in either types. If untreated, both events can lead to coma and death. Hyperosmolar hyperglycemic state is characterized by very high blood glucose level (> 600 mg/dl), hyperosmolality (>320 mOsmol/kg of water), dehydration, lack of ketoacidosis, and altered mental status. It usually occurs in elderly type 2 diabetics. Insulin secretion is adequate to prevent ketosis but not hyperglycemia. Causes of HHS are illness, dehydration, surgery, and glucocorticoid therapy. Differences between DKA and HHS are presented in Table 3.4.

Table 3.3: Categorization of cardiovascular risk in diabetes mellitus according to lipid levels (American Diabetes Association) Category

Low density lipoproteins

High density lipoproteins

Triglycerides

High-risk

≥130

≥ 400

Intermediate risk Low-risk

100-129 < 100

< 35 (men) < 45 (women) 35-45 > 45 (men) > 55 (women)

200-399 < 200

Table 3.4: Comparison of diabetic ketoacidosis and hyperosmolar hyperglycemic state

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Parameter

Diabetic ketoacidosis

Hyperosmolar hyperglycemic state

Type of DM in which more common Age Prodromal clinical features Abdominal pain, Kussmaul’s respiration Acidosis Plasma glucose Serum bicarbonate Blood/urine ketones β-hydroxybutyrate Arterial blood pH Effective serum osmolality* Anion gap**

Type 1 Younger age < 24 hrs Yes Moderate/Severe > 250 mg/dl 600 mg/dl) >15 mEq/L ± Normal or raised Normal (>7.30) Increased (>320) Variable

*Osmolality: Number of dissolved (solute) particles in solution; normal: 275-295 mOsmol/kg ** Anion gap: Difference between sodium and sum of chloride and bicarbonate in plasma; normal average value is 12

Diabetes Mellitus Laboratory evaluation consists of following investigations: • Blood and urine glucose • Blood and urine ketone • Arterial pH, Blood gases • Serum electrolytes (sodium, potassium, chloride, bicarbonate) • Blood osmolality • Serum creatinine and blood urea. Testing for ketone bodies: Ketone bodies are formed from metabolism of free fatty acids and include acetoacetic acid, acetone and β-hydroxybutyric acid. Indications for testing for ketone bodies in DM include: • At diagnosis of diabetes mellitus • At regular intervals in all known cases of diabetes, during pregnancy with pre-existing diabetes, and in gestational diabetes • In known diabetic patients: during acute illness, persistent hyperglycemia (> 300 mgs/dl), pregnancy, and clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain). An increased amount of ketone bodies in patients with DM indicate impending or established diabetic ketoacidosis and is a medical emergency. Method based on colorimetric reaction between ketone bodies and nitroprusside (by dipstick or tablet) is used for detection of both blood and urine ketones. Test for urine ketones alone should not be used for diagnosis and monitoring of diabetic ketoacidosis. It is recommended to measure β-hydroxybutyric acid (which accounts for 75% of all ketones in ketoacidosis) for diagnosis and monitoring DKA.

REFERENCE RANGES • Venous plasma glucose: Fasting: 60-100 mg/dl At 2 hours in OGTT (75 gm glucose): 6.2 mEq/L • Serum bicarbonate: < 10 mEq/L or > 40 mEq/L • Serum chloride: < 80 mEq/L or > 115 mEq/L

BIBLIOGRAPHY 1. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004; 24:S5-S10. 2. American Diabetes Association. Gestational diabetes mellitus. Diabetes Care 2004;27:S88-S90. 3. American Diabetes Association. Hyperglycemic crises in diabetes. Diabetes Care 2004;27:S94-S102. 4. American Diabetes Association. Screening for type 2 diabetes. Diabetes Care 2004;27:S11-S14. 5. American Diabetes Association. Tests of glycemia in diabetes. Diabetes Care 2004;27:S91-S93. 6. Lernmark A. Type 1 diabetes. Clin Chem 1999;45: 1331-38. 7. Lebovitz HE. Type 2 diabetes: An overview. Clin Chem 1999;45:1339-45. 8. Reaven GM. The metabolic syndrome: Requiescat in pace. Clin Chem 2005;51:931-8. 9. Reinauer H, Home PD, Kanagasabapathy AS, Heuck C. Laboratory diagnosis and monitoring of diabetes mellitus. Geneva. World Health Organization, 2002. 10. Sacks DB, Bruns DE, Goldstein DE, Maclaren NK, McDonald JM, Parrott M. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem 2002; 48:436-72. 11. Trachtenbarg DE. Diabetic ketoacidosis. Am Fam Physician 2005;71:1705-14.

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4

Liver Function Tests

FUNCTIONS OF LIVER 1. Excretory function: Liver cells metabolize and excrete endogenous as well as exogenous substances. Liver regulates bilirubin metabolism by secretion and excretion of bilirubin. 2. Synthetic function: Synthesis of proteins like albumin, α- and β-globulins, transport proteins and many coagulation proteins occurs in the liver. Liver also produces triglycerides, cholesterol, lipoproteins, and primary bile acids. Albumin maintains osmotic pressure of plasma, transports various compounds, and acts as a protein reserve. Liver does not synthesize immunoglobulins and complement. 3. General metabolic functions: Liver regulates carbohydrate, lipid, and protein metabolism. Glycogen, derived from monosaccharides, is stored in the liver. When carbohydrate intake is reduced, blood glucose level is maintained by breakdown of stored glycogen (glycogenolysis). If needed, amino acids and fat are converted to glucose by the liver (gluconeogenesis). Synthesis of triglycerides, phospholipids, cholesterol, and lipoproteins occurs in the liver. Liver also esterifies cholesterol, and forms bile acids from cholesterol (Fig. 4.1). Bile acids are essential for fat absorption from the intestine. Lipoproteins help in transport of fats. Besides synthesis of various proteins and enzymes, liver is the site for deamination and transamination of amino acids. Ammonia is converted to urea in the urea cycle and detoxified in the liver. 4. Liver is the storage site for iron, glycogen, and vitamins. 5. During fetal life, hematopoiesis occurs in the liver. It is also a site for destruction of damaged red cells (immune hemolysis). 6. Liver is the major organ for catabolism of steroid hormones.

Fig. 4.1: Formation of bile acids and bile salts

NORMAL BILIRUBIN METABOLISM Bilirubin is mostly (85%) produced from breakdown of hemoglobin of old red cells in reticuloendothelial cells (macrophages), mainly in spleen. A smaller amount is derived from premature destruction of red cell precursors in bone marrow, and from myoglobin, cytochromes, and peroxidases. Steps in metabolism of bilirubin are outlined below (Fig. 4.2). 1. Hemoglobin is degraded within macrophages to form heme and globin; globin consists of amino acids

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53

Fig. 4.2: Normal bilirubin metabolism

2.

3.

4.

5.

which are recycled. Heme (iron + protoporphyrin) releases iron, which is stored as ferritin. Protoporphyrin is first converted to biliverdin, which is then reduced to bilirubin. Bilirubin is released from macrophages into the circulation where it binds with albumin. This is called as unconjugated bilirubin. It is soluble in lipid but insoluble in water. Bilirubin-albumin complex reaches the liver where it is taken up by the hepatocytes. Bilirubin is set free in the cytoplasm, while albumin is released back into the circulation. Bilirubin is conjugated with glucuronic acid to form bilirubin monoglucuronide and diglucuronide (conjugated bilirubin); this process is mediated by the enzyme glucuronyl transferase. Conjugated bilirubin is more soluble in water. Conjugated bilirubin is secreted from the hepatocyte into the biliary canaliculi, from where it passes into the bile duct and gallbladder along with bile (bilirubin monoglucuronide 25% and bilirubin diglucuronide 75%). Bilirubin reaches the small intestine via the common bile duct. Bile also contains bile salts, which

are necessary for digestion and absorption of fat from the small intestine. 6. When bilirubin reaches the large intestine, it is converted by bacterial action to a group of compounds known as urobilinogen by the action of bacterial enzymes. 7. Most of the urobilinogen is excreted in feces as urobilin and is responsible for brown coloration of feces. A part of urobilinogen is absorbed into the circulation from where it reaches the liver, is taken by the hepatocytes, and is again re-excreted in bile (enterohepatic circulation). A small amount of urobilinogen in circulation escapes clearance by the liver and is excreted in urine.

INDICATIONS AND LIMITATIONS OF LIVER FUNCTION TESTS Liver function tests (LFT) are the various laboratory tests that are used to: • Screen for liver disease; • Identify the nature of liver disease (hepatocellular, cholestatic, or infiltrative);

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• Assess severity and prognosis of liver disease; and • Follow up the course of liver disease Box 4.1: Limitations of liver function tests • Do not necessarily assess liver function • Lack sensitivity (i.e. may be normal in some liver diseases like cirrhosis) • Lack specificity (i.e. may be abnormal in non-liver disorders e.g. serum albumin is low in nephrotic syndrome and in cirrhosis)

The term ‘liver function tests’ is a misnomer since most of these tests are used for identification of liver disease, its severity, and its type and do not necessarily assess liver function. Generally these tests, which are performed in combination, are abnormal in liver disease, and the pattern of abnormality is indicative of the nature of liver disease. Since liver has a large amount of anatomical and functional reserve and capacity for rapid regeneration, functional deficiency becomes apparent if there is an extensive liver damage. Also, these tests are not specific for liver disease (except serum bile acids) and are abnormal in various non-hepatic conditions (Boxes 4.1 and 4.2). Therefore, laboratory tests should be selected and interpreted in the context of clinical features and other investigations. An isolated abnormality of a single liver function test usually means a non-hepatic cause. If several liver function tests are simultaneously abnormal, then hepatic etiology is likely.

Box 4.2: Non-hepatic causes of abnormal liver function tests • Increased serum bilirubin: – Hemolysis – Ineffective erythropoiesis – Resorption of a large hematoma • Increased aminotransferases: – Muscle injury – Alcohol abuse – Myocardial infarction • Increased serum alkaline phosphatase: – Pregnancy – Bone disease • Low serum albumin: – Poor nutritional status – Proteinuria – Malabsorption – Severe illness causing protein catabolism

Table 4.1: Commonly performed liver function tests Test

Hepatic cause of abnormality

1. Serum alanine aminotransferase

Hepatocellular injury

2. Serum aspartate aminotransferase

Hepatocellular injury

3. Serum alkaline phosphatase

Cholestasis

4. Serum bilirubin 5. Serum albumin

Defective conjugation or excretion Decreased synthesis

CLASSIFICATION OF LIVER FUNCTION TESTS Liver function tests can be classified as follows: 1. Tests that assess excretory function of the liver: Bilirubin in serum and urine, and urobilinogen in urine and feces. 2. Tests that assess synthetic and metabolic functions of the liver: Serum proteins, serum albumin, serum albumin/globulin (A/G) ratio, prothrombin time (PT), and blood ammonia level. 3. Tests that assess hepatic injury (liver enzyme studies): Serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase, serum γ-glutamyl transferase (GGT), and 5’-nucleotidase (5’-NT). 4. Tests that assess clearance of exogenous substances by the liver: Bromosulphthalein excretion test. Commonly performed liver function tests are listed in Table 4.1. Tests that Assess Excretory Function of the Liver Jaundice Jaundice (from French jaune, meaning yellow) or icterus refers to yellow discoloration of skin, sclera, and mucous membranes due to increased level of serum bilirubin. Jaundice becomes clinically evident when serum bilirubin level exceeds 2.0 mg/dl. There are various methods for classification of jaundice as follows: 1. According to the main type of bilirubin increased in plasma: • Predominantly unconjugated hyperbilirubinemia: Indirect or unconjugated bilirubin is > 85% of total; causes are hemolysis, resorption of a large hematoma, ineffective erythropoiesis, Gilbert’s syndrome, physiologic jaundice of newborn, and Crigler-Najjar syndrome.

Liver Function Tests • Predominantly conjugated hyperbilirubinemia: Direct or conjugated bilirubin is >50% of total; causes are hepatitis, cirrhosis, cholestasis, drugs (anabolic steroids, oral contraceptives), toxins, Dubin-Johnson syndrome, and Rotor syndrome. • Mixed (conjugated + unconjugated) hyperbilirubinemia: Conjugated bilirubin is 20-50% of total; it results from viral or alcoholic hepatitis. 2. According to etiology: • Hemolytic: The increased rate of red cell destruction causes increased haemoglobin breakdown to bilirubin in reticuloendothelial cells; this exceeds the capacity of conjugation in liver. • Hepatocellular: Inability of hepatocytes to conjugate and/or excrete bilirubin. • Obstructive: Failure of excretion of conjugated bilirubin into the intestine, causing its regurgitation in circulation. 3. According to site of disease: • Prehepatic • Hepatic • Posthepatic A simple classification is division of jaundice into three main types: prehepatic, hepatic, and posthepatic. This classification is the basis for identifying the cause of jaundice (Table 4.2). 1. Prehepatic jaundice: There is excessive formation of bilirubin exceeding the capacity of the liver to conjugate it for excretion. The type of bilirubin increased in serum is of unconjugated type. Bilirubin is absent in urine since unconjugated bilirubin is water-insoluble. Urobilinogen is increased in urine

55

and feces. Jaundice is usually mild (serum bilirubin 50%) Present Decreased Common duct stone Abnormal that is corrected with vit K Marked rise of serum ALP (>3 times normal)

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase

Fig. 4.4: Investigation of jaundice. ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase

unconjugated bilirubin is 90% or more. This is because conjugated bilirubin is rapidly secreted into the bile after its formation and removed through the gut. Conjugated bilirubin is composed of blirubin glucuronide, bilirubin diglucuronide, and delta (δ) bilirubin. Delta bilirubin represents bilirubin covalently bound to albumin in circulation. Normally, δ bilirubin is absent or present in

very small amount. In cholestasis, proportion of δ-bilirubin increases. Owing to its longer half-life, it is cleared slowly from circulation. Conjugated bilirubin is weakly bound to albumin, is water-soluble, and can be excreted in urine. Unconjugated bilirubin is tightly bound to albumin and is water-insoluble. As bilirubin is altered by exposure to light, sample should be kept in the dark.

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Fig. 4.5: Estimation of serum bilirubin by diazo method

There are two methods for estimation of serum bilirubin: diazo method and spectrophotometry. In diazo method, total and direct-reacting bilirubin are measured, and indirect bilirubin is obtained by subtracting direct from total bilirubin (Fig. 4.5). Estimation of both types of bilirubin is helpful in the differential diagnosis of jaundice. In post-hepatic type of jaundice, direct bilirubin is the predominant form (> 50% of the total). In hepatocellular jaundice, direct bilirubin is usually between 20-50% of total. Indirect bilirubin predominates in hemolysis, Gilbert’s syndrome, and Crigler-Najjar syndrome (direct bilirubin is < 15% of total). Box 4.3: Serum bilirubin • Direct bilirubin (Conjugated bilirubin): It reacts directly with diazo reagent. It consists of monoconjugated bilirubin, diconjugated bilirubin, and bilirubin tightly bound to albumin (delta bilirubin). • Indirect bilirubin (Unconjugated bilirubin): It reacts with diazo reagent in the presence of alcohol. It consists of bilirubin bound to albumin. It is calculated as ‘total bilirubin minus direct bilirubin’.

Direct spectroscopic estimation is used for measurement of total serum bilirubin in newborns and infants (1.5), and serum protein electrophoresis. 1. Total serum proteins: These can be estimated by refractometer method or by biuret method. In refractometer method, refractive index of the solution (which depends on solute concentration) is measured. Refractive index varies mainly with concentration of proteins and is affected very little by electrolytes

Liver Function Tests and other molecules. Protein concentration is read directly from the scale on the refractometer. In biuret method, copper ions react with peptide bonds of proteins and form a violet-colored compound. Intensity of this color (measured colorimetrically) is proportional to the concentration of proteins. Total serum protein level is affected by both albumin and gamma globulins. In cirrhosis, decrease in albumin level is often compensated by increase in the level of gamma globulins; therefore, estimation of total serum proteins is of limited value in cirrhosis. Estimation of serum albumin and serum protein electrophoresis are more helpful. 2. Serum albumin: Albumin is synthesized exclusively in liver and constitutes about 60% of total proteins in serum; therefore its estimation is an important investigation in liver disease. Half-life of albumin is about 20 days and therefore fall in its level in response to decreased synthesis is not immediately apparent. Therefore, in acute liver disease (e.g. viral hepatitis), there is little change in albumin level. Serum albumin level is low in chronic liver disease (cirrhosis) and correlates with synthetic capacity of hepatocytes; therefore, it is helpful in following progression of cirrhosis. Also, fall in serum albumin level correlates with severity of ascites. In cirrhosis and in chronic active hepatitis, serum gamma globulins are increased due to inflammation. Low albumin and raised gamma globulins in serum cause reversal of albumin/globulin ratio. Serum albumin is estimated by bromocresol green method. Bromocresol green is an indicator dye, which when added to serum, binds selectively and tightly to albumin and becomes blue in color. Absorbance (in a spectrophotometer at 632 nm) is directly proportional to concentration of albumin. Causes of decreased serum albumin: • Decreased intake: malnutrition. • Decreased absorption: malabsorption syndromes. • Decreased synthesis: liver disease, chronic infections. • Increased catabolism: thyrotoxicosis, fever, malignancy, infections. • Increased loss: nephrotic syndrome, severe burns, protein-losing enteropathies, ascites • Increased blood volume: pregnancy, congestive cardiac failure. As low serum albumin occurs in diseases other than those of liver, serum albumin is a sensitive but nonspecific test for liver disease. 3. Serum protein electrophoresis: Details of serum protein electrophoresis are given in Chapter 28 “Laboratory Tests in Hematological Malignancies”. In liver disease, following changes may be seen on protein electrophoresis (Fig. 4.6):

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1. In cirrhosis, albumin may be reduced and there may be polyclonal increase of IgG and IgA, with β-γ bridging. (IgA migrates between β and γ regions which obscures the demarcation between β and γ peaks). 2. In primary biliary cirrhosis, there is polyclonal increase of IgM. 3. In α1-antitrypsin deficiency (associated with cirrhosis) α1- globulin band is reduced. 4. In chronic active hepatitis, IgG is elevated. Prothrombin Time (PT) Most of the coagulation proteins are synthesized in the liver. Vitamin K is required for the synthesis of factors II, VII, IX, and X by the hepatocytes; therefore these factors are called as vitamin K-dependent factors. Synthesis of these factors is deficient in hepatocellular disease. In obstructive jaundice, vitamin K (a fat-soluble vitamin) cannot be absorbed due to the absence of bile in the intestine. PT measures three out of four vitamin K-dependent factors (II, VII, and X) and is prolonged in hepatocellular disease and in obstructive jaundice. Intramuscular injection of vitamin K corrects prolonged PT in obstructive jaundice but not in hepatocellular jaundice. In acute fulminant liver failure, marked prolongation of PT is an unfavourable prognostic sign. To distinguish between a prolonged PT due to hepatocellular disease from that due to cholestasis with fat malabsorption, PT is repeated after administration of vitamin K. Reduction of prolonged PT occurs in cholestatic liver disease, but not in hepatocellular disease. Blood Ammonia Blood ammonia is mainly derived from gastrointestinal tract. In the intestine, bacterial enzymes act on nitrogencontaining foods to produce ammonia, which is carried to the liver via portal vein. In the liver, ammonia is converted to non-toxic urea in the urea cycle. Increased blood ammonia levels are seen in: • Fulminant hepatic failure • Cirrhosis • Reye’s syndrome • “Shunting” of portal blood to systemic circulation • Gastrointestinal hemorrhage (there is increased production of ammonia from blood proteins by bacterial enzymes). In hepatic disease, gastrointestinal hemorrhage is associated with increased risk of hepatic encephalopathy. • Inherited deficiencies of urea cycle enzymes. If raised, estimation of blood ammonia is likely to be helpful in patients with coma of unknown origin, since it is indicative of hepatic encephalopathy.

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Fig. 4.6: Serum protein electrophoresis patterns and densitometer scans in normal individuals and in cirrhosis of liver and α1-antitrypsin deficiency

Tests which Assess Hepatic Injury (Liver Enzyme Studies) Serum enzyme changes in liver disease result from hepatocyte damage and do not indicate hepatic functional capacity. In the investigation of liver disease, following serum enzymes are measured: • Serum aspartate aminotransferase or AST (formerly called serum glutamic-oxaloacetic transaminase or SGOT) • Serum alanine aminotransferase or ALT (formerly called serum glutamic-pyruvic transaminase or SGPT) • Serum alkaline phosphatase or ALP • γ-Glutamyl transferase or GGT (also called as γ-glutamyl transpeptidase)

• 5’-nucleotidase (5’-NT) Locations of enzymes in liver cell are shown in Figure 4.7. Degree of elevation of a particular enzyme depends on pattern of hepatic damage. Serum Aminotransferases Serum aminotransferases are the sensitive markers of acute hepatocellular injury. ALT is a cytosolic enzyme while AST is both cytosolic and mitochondrial. Normally, aminotransferases are present in serum at a low level. When necrosis or death of cells containing these enzymes occurs, aminotransferases are released into the blood and their concentration in blood increases. This level correlates with extent of tissue damage.

Liver Function Tests Most marked elevations of ALT and AST (>15 times normal) are seen in acute viral hepatitis, toxin-induced hepatocellular damage (e.g. carbon tetrachloride), and centrilobular necrosis due to ischemia (congestive cardiac failure). Moderate elevations (5-15 times) occur in chronic hepatitis, autoimmune hepatitis, alcoholic hepatitis, acute biliary tract obstruction, and drug-induced hepatitis. Mild elevations (1-3 times) are seen in cirrhosis, nonalcoholic steatosis, and cholestasis. Determinations of these enzymes are helpful in the differential diagnosis of hepatocellular from cholestatic jaundice. Increase of AST and ALT is much more in hepatocellular jaundice (>500 units/ml) than in cholestatic jaundice (2.0) is highly suggestive of alcoholic hepatitis, while ratio 500 IU) • Mild increase of ALP (3 times normal) Elevation of GGT and 5’-NT Mild or no increase of ALT and AST (usually 2.0, ↑ GGT Liver biopsy Raised immunoglobulins, low albumin, antinuclear antibody+, anti-smooth muscle antibody+, liver kidney microsomal antibody+ Transferrin saturation >45%, genetic testing Low ALP, high serum bilirubin, low serum ceruloplasmin

Fig. 4.9: Evaluation of acute hepatocellular injury

• In patients with cholestatic or infiltrative pattern of injury, imaging studies should be done for diagnosis of obstruction. If there is no evidence of obstruction, liver biopsy is usually done (Table 4.5 and Fig. 4.10). Severity of liver disease is assessed by serum bilirubin, serum albumin, and prothrombin time. Child-Turcotte-Pugh classification is commonly used to assess severity of cirrhosis and is based on both clinical and laboratory parameters (Table 4.6).

LIVER BIOPSY Liver biopsy is a procedure in which a small piece of liver tissue is removed and examined microscopically to determine the cause and severity of liver disease. Paul Ehrlich performed the first percutaneous liver biopsy in 1883 in Germany. Since then the technique has been modified and now a variety of approaches can be used to obtain a liver biopsy sample. Before proceeding with

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Table 4.5: Differentiation of causes of raised serum alkaline phosphatase in jaundice Cause

Clinical features

Investigations

1. Primary biliary cirrhosis

Middle-aged female; pruritus; xanthelasma

Antimitochondrial antibody; raised serum cholesterol

2. Primary sclerosing cholangitis

Adult male; associated inflammatory bowel disease

Antinuclear cytoplasmic antibody, endoscopic retrograde cholangiopancreatography

3. Extrahepatic biliary obstruction Dark urine; clay-colored stools; palpable gallbladder ±

Ultrasonography for dilated bile ducts; endoscopic retrograde cholangiopancreatography

Fig. 4.10: Evaluation of raised serum alkaline phosphatase in cholestatic injury

the biopsy, potential risks of the procedure and benefits of histological examination should be assessed and the procedure should be performed only when benefits out weigh the risks.

TYPES OF LIVER BIOPSY Currently, several methods are available for obtaining liver tissue specimen. Choice of the method depends on its availability, clinical situation, and preference of the

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Essentials of Clinical Pathology Table 4.6: Child-Turcotte-Pugh grading system to assess severity of cirrhosis

Parameter

Score 1

Score 2

Score 3

Serum bilirubin (mg/dl)

3

Serum albumin (g/dl)

≥ 3.5

2.8-3.5

3-5 seconds above control value, platelet count < 50,000/ml, or bleeding time ≥10 minutes are considered as contraindications to liver biopsy. If liver biopsy is essential, then biopsy can be performed after administration of vitamin K, fresh frozen plasma, or platelet concentrate. Alternatively, transjugular approach can be used. In patients with hemophilia who are infected with hepatitis C or B virus through blood transfusion, liver biopsy can be carried out to assess liver damage after correcting coagulation deficiency.

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Liver Function Tests • Tense ascites: Tense ascites is associated with failure to obtain liver tissue (due to increased distance between abdominal wall and liver) and risk of bleeding in ascites. Biopsy can be performed after removal of ascitic fluid or through transjugular approach. • Hydatid cyst of liver: Puncture of hydatid cyst will lead to spread of cysts throughout the abdomen and sometimes, anaphylactic reaction. • Suspected hemangioma or other highly vascular tumor • Amyloidosis is associated with increased risk of bleeding. Patients with encephalopathy, hepatic failure with severe jaundice, severe congestive cardiac failure, and advanced age are at increased risk of complications following liver biopsy. Pre-requisites: • Prior imaging of liver should be carried out to identify any abnormality, to define borders, and to determine relative positions of gallbladder, lungs, and kidneys. • Routine hemogram • Coagulation profile • Informed consent • Blood grouping and cross matching Method: 1. Patient lies in supine position. 2. After selecting the site for liver biopsy, a local anesthetic is injected. 3. A small incision is made and the biopsy needle is passed into the liver. Patient is asked to hold his/her breath in expiration. Method of obtaining liver tissue depends on the type of needle used. 4. After needle is removed, patient lies supine or on the right side and is closely monitored especially during first 6 hours for early detection of complications. Complications: 1. Pain 2. Intraperitoneal hemorrhage: Patients at particular risk of bleeding are those with cirrhosis and malignancy in liver. These patients should not be biopsied on outpatient basis. 3. Biliary peritonitis due to puncture of gallbladder. 4. Puncture of other organs like kidney, lung, and colon. Overall mortality following liver biopsy is reported to be 0.1%. Percutaneous Guided Liver Biopsy In percutaneous guided liver biopsy, needle is inserted into the liver through the abdomen or lower chest during

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real time imaging of the liver (ultrasonography, computed tomography, or magnetic resonance imaging). This approach is suitable for biopsy of focal lesions. Percutaneous ‘Plugged’ Liver Biopsy In this method, after taking the biopsy sample, the outer sheath of the needle is left in place and the obturator holding the liver biopsy tissue is removed. A cannula is then introduced through the outer sheath and gel or gel foam is injected to seal or ‘plug’ the needle track in the liver. This method is said to reduce the risk of bleeding in individuals with impaired coagulation. Transvenous (Transjugular) Liver Biopsy Liver biopsy is obtained from within the vascular system of liver. A small catheter is inserted through the jugular vein in the neck and radiologically guided, via right atrium and inferior vena cava, into the hepatic vein. The biopsy needle is then inserted through the catheter, advanced into the liver, and biopsy is taken. This method is suitable in severe coagulation disorders and in massive ascites. Due to the risk of cardiac arrhythmias (as the catheter passes through the atrium), close cardiac monitoring is required during this procedure. Laparoscopic Liver Biopsy A laparoscope is introduced through an incision in the abdominal wall, and liver biopsy is obtained under direct visualization. Usually, such a biopsy is taken when a focal lesion is incidentally detected on diagnostic laparoscopy of abdomen.

REFERENCE RANGES Serum alanine aminotransferase (ALT, SGPT): 5-42 U/L Serum aspartate aminotransferase (AST, SGOT): 5-40 U/L Serum alkaline phosphatase (ALP): • Children: 25-350 U/L • Adult males: 25-120 U/L • Adult females: 25-90 U/L AST/ALT ratio: 0.7-1.4 Serum bilirubin: • Total: 0.3-1.0 mg/dl • Direct (Conjugated): 0-0.2 mg/dl Serum proteins, total: 5.5-8.0 gm/dl Serum albumin: 3.5-5.0 gm/dl Serum globulins: 1.8-3.5 gm/dl Albumin/Globulin (A/G) ratio: >1.5

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Serum protein electrophoresis: Albumin 52-65% α1 globulin: 2.5-5% α2 globulin: 7-13% β globulin: 8-14% γ globulin: 12-22% Prothrombin time: 11-15 seconds Plasma ammonia: 9-33 μmol/L Serum gammaglutamyl transferase: • Males: Up to 40 U/L • Females: Up to 25 U/L

CRITICAL VALUES Plasma ammonia: >40 μmol/L Serum bilirubin: >15 mg/dl in newborns

BIBLIOGRAPHY 1. American Gastroenterological Association Clinical Practice Committee: AGA technical review on liver chemistry tests. Gastroenterology 2002;123:1367-84. 2. American Gastroenterological Association Medical Position Statement: Evaluation of liver chemistry tests. Gastroenterology 2002;123:1364-6 3. Beckingham IJ, Ryder SD. Investigation of liver and biliary disease. BMJ 2001;322:33-6. 4. Black ER. Diagnostic strategies and test algorithms in liver disease. Clin Chem 1997;43:1555-60. 5. Burke MD. Liver function: test selection and interpretation of results. Clin Lab Med 2002;22:377-90.

6. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of hepatic injury. II. Recommendations for use of laboratory tests in screening, diagnosis, and monitoring. Clin Chem 2000; 46:2050-68. 7. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB. Diagnosis and monitoring of hepatic injury. I. Performance characteristics of laboratory tests. Clin Chem 2000;46:2027-49. 8. Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart MJ, Shepherd J. Clinical Biochemistry. An Illustrated Colour Text, 3rd Ed. Edinburgh. Churchill Livingstone. 2004. 9. Grant A, Neuberger J. Guidelines on the use of liver biopsy in clinical practice. Gut 1999; 45 (Suppl IV): IV1-IV11. 10. Johnston DE. Special considerations in interpreting liver function tests. Am Fam Physician 1999;59:2223-30. 11. Limdi JK, Hyde GM. Evaluation of abnormal liver function tests. Postgrad Med J 2003;79;307-12. 12. Lucey MR, Brown KA, Everson GT, et al. Minimal criteria for placement of adults on the liver transplant waiting list; a report of a national conference organized by the American Society of Transplant Physicians and the American Association for the Study of Liver Disease. Liver Transpl Surg 1997;3:628-37. 13. Pugh RN, Murray-Lyon IM, Dawson JL, et al. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973;60:646-9. 14. Roche SP, Kobos R. Jaundice in the adult patient. Am Fam Physician 2003;69:299-304. 15. Thapa BR, Walia A. Liver function tests and their interpretation. Indian J Pediatr 2007;74:663-71.

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Disorders of Lipids and Biochemical Cardiac Markers DISORDERS OF LIPIDS The major lipids present in blood are cholesterol, fatty acids, and triglycerides. Lipid disorders are common in clinical practice, and some of them are associated with an increased risk of atherosclerotic cardiovascular disease. Cardiovascular disease is a major cause of mortality in persons under the age of 60, and proper management of lipid abnormalities significantly reduces this risk. Lipids are insoluble in plasma and are therefore transported in circulation in association with proteins. These complexes of lipids and proteins are known as lipoproteins. Dyslipidemias are disorders of lipoprotein metabolism.

PHYSIOLOGY Cholesterol and Triglycerides The two major lipids in blood are cholesterol and triglycerides. Since they are insoluble in water, they are carried by lipoproteins. Cholesterol is a lipid found in all cell membranes and in blood plasma. Cholesterol is an essential component of the cell membranes, and is necessary for synthesis of steroid hormones, and for the formation of bile acids. Cholesterol is synthesized by liver and many other organs, and is also ingested in the diet. Triglycerides are lipids in which three long-chain fatty acids are attached to glycerol. Triglycerides serve as a source of energy. They are present in dietary fat and also synthesized by liver and adipose tissue. Lipoproteins Cholesterol and triglycerides are not soluble in water and are transported in blood incorporated into lipoproteins. Lipoproteins are spherical macromolecular complexes consisting of (i) a central core of lipids (cholesterol ester,

triglycerides, fat-soluble vitamins) that is surrounded by (ii) a surface monolayer composed of phospholipids, free cholesterol, and apoproteins (Fig. 5.1). The surface of lipoproteins is water-soluble or polar while core is hydrophobic or non-polar. There are five major lipoprotein classes: chylomicrons, very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) (Fig. 5.2 and Table 5.1). Triglycerides are carried mainly by chylomicrons, VLDL, and LDL, while major lipoproteins for cholesterol transport are LDL and HDL. Chylomicrons: These are the largest and the least dense of the lipoproteins, and transport exogenous lipids to various cells. Dietary fat is incorporated into chylomicrons by intestinal epithelial cells. The lipid core of

Fig. 5.1: Basic structure of a lipoprotein molecule. Lipoproteins are spherical aggregates of lipids and apolipoproteins. They consist of a core of triglycerides and cholesterol esters surrounded by a shell of phospholipids and cholesterol. Apolipoproteins are embedded in the shell. The larger the lipid core, the lower is its density. Lipoproteins are classified into 5 types: chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL)

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Fig. 5.2: Lipoproteins and their composition

Table 5.1: Summary of major lipoproteins Lipoprotein

Major lipid carried

Formation

Function

1. Chylomicron

Triglyceride

Secreted by intestinal epithelial cells

Transport of exogenous triglycerides, cholesterol, and fat-soluble vitamins absorbed from food to the peripheral tissues

2. Very low density lipoprotein 3. Low density lipoprotein

Triglycerides, Cholesterol Cholesterol

Secreted by liver

Transport of endogenous triglycerides to adipose tissue and muscle from liver Transport of cholesterol from liver to peripheral tissues

4. High density lipoprotein

Cholesterol

Formed from modification of VLDL by lipoprotein lipase in peripheral tissues Secreted from liver

chylomicrons consists mainly of triglycerides, some cholesterol and fat-soluble vitamins. Intestinal cells secrete chylomicrons into the lymphatics, which then enter the bloodstream via the thoracic duct. In circulation, chylomicrons are acted upon by lipoprotein lipase to release triglycerides; further hydrolysis of triglycerides by lipoprotein lipase causes release of free fatty acids that are then taken up by adipose tissue and muscle. Liver takes up the cholesterol-rich chylomicron remnant particle and cholesterol enters the metabolic pathway. Very low-density lipoproteins (VLDL): VLDL particle is synthesized by the liver. It transports triglycerides and

“Reverse cholesterol transport”, i.e. from peripheral tissues to liver

cholesterol. It carries most of the endogenous triglyceride from liver to adipose tissue and muscle. Triglyceride is removed by the action of lipoprotein lipase in the circulation and VLDL particle becomes smaller, when it is called as intermediate density lipoprotein (IDL). Further processing of IDL leads to the formation of low-density lipoprotein (LDL), which is the major carrier for cholesterol. Intermediate density lipoprotein (IDL): This is the remnant of VLDL formed when triglycerides are removed from VLDL by lipoprotein lipase in circulation.

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Disorders of Lipids and Biochemical Cardiac Markers About half of IDL is cleared from blood by the liver and the remaining half is further processed to form LDL. Normally, IDL is not detected in plasma as it is formed transiently. Low-density lipoprotein (LDL): LDL is the major carrier lipoprotein for cholesterol from liver to the peripheral tissues. It is formed from VLDL. LDL plays a major role in the genesis of atherosclerosis. LDL is taken up by the cells through the LDL receptor, a glycoprotein. The LDL receptor is present on the surface of all cells and recognizes apolipoprotein B on the surface of LDL particle. After internalization of LDL particle, the lipoprotein is catabolized and the receptor is recycled back to the cell surface. Intracellularly, LDL is degraded to free cholesterol that is needed for cellular needs. The level of LDL in circulation is determined by number and function of LDL receptors. Joseph Goldstein and Michael Brown were awarded the Nobel Prize for Physiology or Medicine in 1985 for characterizing the LDL receptor. Genetic absence of LDL receptors leads to familial hypercholesterolemia. High-density lipoprotein (HDL): HDL binds to peripheral tissues that have apolipoprotein A receptors and takes up cholesterol. HDL cholesterol is either taken by the liver or is incorporated into IDL to form LDL. Lipoprotein (a) or Lp (a): Attachment of apolipoprotein (a) molecule to apolipoprotein B molecule on the surface of LDL particle leads to the formation of a new particle called as lipoprotein (a). Excess Lp (a) is associated with risk of atherosclerosis. Lipoprotein Metabolism There are two pathways of lipoprotein metabolism: exogenous and endogenous. Exogenous pathway: Small intestinal cells absorb fatty acids and cholesterol, esterify them into triglycerides and

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cholesterol esters and incorporate them into the core of the chylomicrons. Chylomicrons are secreted into the lymphatics from where they reach the bloodstream via thoracic duct. In the circulation, endothelial-bound lipoprotein lipase hydrolyzes triglycerides in the chylomicron core and releases free fatty acids that are then taken up by the adipose tissue and muscle; in these the free fatty acids are converted to triglycerides and stored. The remaining smaller chylomicron is taken by the liver, degraded, and cholesterol contained therein is then used for the formation of bile acids, incorporated into cell membranes, secreted in blood as lipoprotein cholesterol, or excreted in bile (Fig. 5.3). Endogenous pathway: This pathway is divided into: • Apo B-100 lipoprotein system • Apo A-I lipoprotein system Apo B-100 lipoprotein system: In the liver, triglycerides and cholesterol are assembled with apo B-100 and phospholipids to produce VLDL. VLDL represents the major export pathway for cholesterol from liver. After its secretion from the liver, lipoprotein lipase on capillary endothelium hydrolyzes triglyceride in the core of the VLDL particles resulting in the formation of (cholesterol ester-rich) intermediate density lipoproteins (IDL). Further degradation results in the formation of LDL particles that are rich in cholesterol ester. LDL particles are taken up by all nucleated cells through LDL receptors (receptor- mediated endocytosis) or by other scavenger routes (e.g. monocytes or foam cell in atheromatous plaques). Apo A-I lipoprotein system: High-density lipoprotein (HDL) particles are synthesized by liver and intestine and participate in reverse cholesterol transport. HDL, rich in apo A-I, acquires free cholesterol from peripheral tissues, esterifies it, and either transfers it directly to the liver or to other lipoproteins (IDL and LDL), which then transport it to liver (Fig. 5.4).

Fig. 5.3: Exogenous lipid pathway showing route of metabolism of lipid absorbed from the intestine

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Fig. 5.4: Endogenous lipid pathway showing route of metabolism of endogenously synthesized lipid Abbreviations: VLDL: very low density lipoprotein; IDL: intermediate density lipoprotein; LDL: Low density lipoprotein; HDL: high density lipoprotein; LCAT: lecithin-cholesterol acyltransferase

Apolipoproteins These are proteins located on the surface of lipoproteins. They aid in lipid transport and delivery in three ways: (i) stabilizing structure of lipoproteins, (ii) serving as regulatory cofactors for enzymes that act on lipoproteins, and (iii) acting as ligands for binding to lipoprotein receptors. Important apolipoproteins are: • A-I: Activates lecithin-cholesterol acyl transferase (LCAT) • B-100: Ligand for LDL receptors • C-II: Activates lipoprotein lipase • E: Ligand for chylomicron remnant receptor in liver

CLASSIFICATION OF LIPOPROTEIN DISORDERS In clinical practice, lipoprotein disorders are classified as being primary (inherited disorders) or secondary (acquired disorders). Primary lipoprotein disorders are

commonly classified according to the Fredrickson or World Health Organization classification (Table 5.2). This classification is based on the particular pattern of lipid and lipoprotein abnormality. On electrophorsis, four principal bands are observed from cathode (–) to anode (+): chylomicrons, β (LDL), pre β (VLDL), and α (HDL) (Fig. 5.5). Fredrickson classification of hyperlipidemia is as follows: • Type I: Increased chylomicrons • Type IIA: Increased β lipoproteins (LDL) • Type IIB: Increased β and pre-β lipoproteins (LDL, VLDL) • Type III: Broad β lipoproteins (IDL) • Type IV: Increased pre-β lipoproteins (VLDL) • Type V: Increased chylomicrons and pre-β lipoproteins (chylomicrons, VLDL) Secondary lipoprotein diseases arise from an underlying cause such as diabetes mellitus, alcohol abuse, hypothyroidism, nephrotic syndrome, renal failure, and biliary cirrhosis of liver.

Table 5.2: Frederickson’s classification of primary lipoprotein disorders Types Prevalence

Appearance of plasma (fasting)

Increased particles

Blood lipids

I

Rarest

Creamy layer at the top

Chylomicron

Marked elevation of triglycerides

IIA

Common; 1:500

Clear (orange-yellow tint)

Low density lipoprotein

Increased total cholesterol

IIB

Common; 1:300

Clear to slightly turbid

Low density lipoprotein, Very low density lipoprotein

Increased triglycerides and total cholesterol

III

Uncommon

Thin creamy layer at the top; turbid to opaque plasma

Intermediate density lipoprotein

Increased triglycerides and total cholesterol

IV

Common; 1:500

Turbid to opaque

Very low density lipoprotein

Increased triglycerides

V

Uncommon

Creamy layer at the top; plasma turbid to opaque

Chylomicron, Very low density lipoprotein

Marked elevation of triglycerides

In routine clinical practice lipid abnormalities are identified by standard lipid assays. tahir99 - UnitedVRG vip.persianss.ir

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Fig. 5.5: Classification of lipoproteins by agarose gel electrophoresis

The main risk factors for coronary artery disease are age (men > 45 years, women > 55 years), dyslipidemia, family history of premature coronary heart disease, hypertension, and cigarette smoking. Lipid abnormalities associated with atherosclerosis and increased risks of coronary artery disease are (i) elevated LDL cholesterol, (ii) low HDL-cholesterol, and (iii) elevated triglycerides.

LABORATORY TESTS FOR LIPOPROTEIN DISORDERS Collection of sample: Sample for lipid analysis should be collected in dry EDTA anticoagulant (1 mg/ml). For estimation of triglycerides and lipoproteins, a 12-hour fasting sample is necessary. Triglycerides and LDL are affected by recent ingestion of food. Patient should be on the usual diet for 2-3 weeks before analysis. Lipid analysis should not be performed during acute illness and should be deferred for 3 months after major illness. Drugs affecting lipid levels should be avoided like steroids, oral contraceptives, etc. Approach to the evaluation of lipid disorder is shown in Figure 5.6.

It is recommended by National Cholesterol Education Program that a fasting lipid profile (consisting of triglycerides, total cholesterol, HDL, and LDL) be carried out every 5 years beginning at the age of 20 years. Desirable and high risk levels of serum lipids are shown in Table 5.3.

Identification of a lipid disorder: Following investigations on a fasting blood sample are usually adequate for identifying lipid abnormalities in majority of cases: • Total cholesterol • Triglycerides • HDL-cholesterol • LDL-cholesterol

(2) Serum triglycerides: Hypertriglyceridemia is a risk factor for coronary heart disease, but less significant than total cholesterol and lipoproteins. Patients with serum triglycerides >200 mg/dl have risk of atherosclerosis, and >1000 mg/dl are at increased risk of acute pancreatitis. Increase in triglyceride is often associated with low HDL. Causes of hypertriglyceridemia are listed in Table 5.5.

Fig. 5.6: Approach to the evaluation of a lipid disorder

(1) Total cholesterol: Causes of elevated serum cholesterol are listed in Table 5.4.

Table 5.3: Guidelines of National Cholesterol Education Program Adult treatment Panel III (All values in mg/dl) • • • •

Total cholesterol: Desirable: 150 mg/ dl). It occurs in tuberculous meningitis (fine cobweblike clot after 12-24 hours), purulent meningitis (pellicle forms early followed by a large clot), spinal block (complete clotting of CSF), and traumatic LP. Clot formation does not occur in subarachnoid hemorrhage. • Thick viscous CSF: This is seen cryptococcal meningitis, meningeal metastatic mucinous adenocarcinoma, severe meningitis, and release of nucleus pulposus fluid in CSF due to needle injury to the intervertebral disk.

3. Cell Counts in Cerebrospinal Fluid (1) Total leukocyte count: Cell count on CSF is done manually on undiluted sample in a counting chamber. Total leukocyte count increases in various disorders and along with differential count provides important diagnostic information. An increase in cell count in CSF is called as pleocytosis. It is essential to do microscopic examination of all CSF samples since white blood cell (WBC) count upto 200/cmm and red cell count upto 400/cmm are associated with clear appearance of CSF. For correct results: • Cell count should be done as soon as possible after collection of CSF since cellular disintegration occurs rapidly. Cells also adhere to the walls of the glass tubes. • CSF specimen collected in tube 3 should be used. • No dilution of CSF is usually required. A diluent should be used only if CSF is cloudy and likely to contain increased leukocytes. Method: i. CSF sample should be properly mixed. If CSF is clear, it is not diluted. If CSF appears cloudy or turbid, 1:20 dilution is made using 0.05 ml of CSF and 0.95 ml of Turk solution. (Composition of Turk solution: Glacial acetic acid 4 ml, methylene blue solution 10 drops, and distilled water to make 200 ml). ii. The counting chamber is covered with the coverslip provided. iii. The counting chamber is filled with the fluid and allowed to stand for 2 minutes for cells to settle. iv. For counting cells in CSF, Fuchs-Rosenthal counting chamber is preferred because its depth is twice that of improved Neubauer chamber. In this, cells are counted in 5 large squares (4 corner squares and one central square). v. If undiluted CSF is used, total number of cells counted in 5 squares represents total count per cmm of CSF. If CSF is diluted, the number of cells counted is multiplied by the dilution factor (i.e. 20). Causes of increased cell count in CSF: • Meningitis and other infections of CNS • Intracranial hemorrhage • Meningeal infiltration by malignancy • Repeated lumbar punctures • Injection of foreign substances (e.g. radiographic contrast media, drugs) in subarachnoid space. • Multiple sclerosis Presence of blood in CSF due to traumatic tap or subarachnoid hemorrhage artefactually raises the

Examination of Cerebrospinal Fluid

Fig. 6.6: Cells in CSF: (1) Many neutrophils in bacterial meningitis, (2) lymphocytes seen in normal CSF in adults, (3) Many red cells and a small lymphocyte (traumatic tap), (4) Increased monocytes in CSF, (5) Neutrophils, lymphocytes, and red cells in CSF, (6) Lining cells of pia and arachnoid in CSF, (7) Blast cells in CSF, (8) a malignant cell in CSF

leucocyte count by 1 WBC per 1000 red cells. This correction factor should be used if patient's hemogram is normal. If significant anemia or leukocytosis is present, then leukocyte count in CSF should be corrected as follows: Corrected WBC count in CSF = WBC count in CSF (cells/cmm) –

WBC count in blood × Red cell count in CSF

Red cell count in blood

(2) Differential leukocyte count: This provides information about relative proportion of various leukocytes.

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If CSF contains only a few cells, then it is centrifuged at high speed (3000 g) for 10 minutes and a smear is made from the sediment. If CSF contains many cells, then a smear is made directly from the uncentrifuged sample. After staining with a Romanowsky stain, smear is examined under the microscope (Fig. 6.6). Simple centrifugation of CSF often causes cell breakage and distortion. Cytospin preparation with cytocentrifuge (high speed centrifugation to concentrate cells on a slide in a uniform monolayer) has been recommended as it improves the cell yield and preserves the cell morphology well. In normal adults, differential count shows 70% lymphocytes and 30% monocytes. In young children, a higher proportion of monocytes (up to 70%) are present. Table 6.2 shows causes of increase in different types of leukocytes in CSF. (3) Other cells: Apart from mature blood cells, CSF may contain immature hematopoietic cells, tissue cells (ependymal cells, pia arachnoid mesothelial cells) and malignant cells. CSF examination is commonly carried out in acute lymphoblastic leukemia to detect involvement of CNS. Increased WBC count (>5/μl) with lymphoblasts is evidence of CNS involvement. 4. Chemical Examination of Cerebrospinal Fluid Routine chemical examination of CSF consists of estimation of proteins and glucose. CSF from tube 1 is used for chemical examination. (1) Estimation of proteins in CSF: Normal CSF protein level in adults is 15-45 mg/dl. An increase in CSF protein is a sensitive but non-specific indicator of CNS disease. CSF proteins may be normal during early stages of meningitis. Significant elevation (>150 mg/dl) occurs in bacterial meningitis.

Table 6.2: Differential cell count in CSF Predominant neutrophils

Predominant lymphocytes

Mixed cell pattern (neutrophils, lymphocytes, monocytes)

Predominant eosinophils

1. Meningitis: bacterial, early viral, fungal, early tuberculous 2. Subarachnoid hemorrhage 3. Repeated lumbar punctures 4. Introduction of anticancer drugs or contrast media in subarachnoid space 5. Meningeal metastasis

1. Meningitis: viral, tuberculous 2. Incompletely treated bacterial meningitis 3. Cysticercosis, toxoplasmosis 4. Multiple sclerosis 5. Subacute sclerosing panencephalitis

1. Tuberculous meningitis 2. Fungal meningitis 3. Chronic bacterial meningitis

1. Parasitic and fungal infections 2. Reaction to foreign material (e.g. shunts)

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There are various methods for estimation of CSF proteins. Turbidimetric method using trichloroacetic acid for precipitation of proteins is commonly used. In principle, trichloroacetic acid, when added to CSF, causes precipitation of proteins and a turbid solution is obtained. Amount of turbidity is compared with the turbidity of a known (standard) concentration of protein in a photoelectric colorimeter. If a sample is contaminated with blood while doing the lumbar puncture (traumatic tap), false elevation of proteins will occur. This can be corrected by deducting 1 mg/dl of protein for every 1000 red cells per cmm. For this correction to be accurate, red cell count and proteins should be estimated in the sample from the same tube of CSF. If facilities for estimation of CSF proteins are not available in the laboratory, then Pandy's test for globulins may be performed. In this test, CSF is added to saturated solution of phenol. If cloudiness develops immediately, it indicates presence of increased globulins and the test is reported as positive. If no cloudiness develops, the test is reported as negative (Fig. 6.7). Along with CSF proteins, it is necessary to simultaneously measure serum proteins for proper interpretation of results. CSF proteins are elevated in following conditions: • Increased capillary permeability of blood-brain barrier: Meningitis • Mechanical obstruction to circulation of CSF (causing increased fluid reabsorption due to stasis): Spinal cord tumor • Increased local (intrathecal) immunoglobulin (IgG) production: Multiple sclerosis, neurosyphilis, subacute sclerosing panencephalitis • Both increased capillary permeability and increased local immunoglobulin (IgG) production: GuillainBarré syndrome • Hemorrhage in CSF: Traumatic tap, subarachnoid hemorrhage. Marked elevation (>500 mg/dl) is noted in complete spinal block by a tumor, bacterial meningitis, and bloody CSF. Differential diagnosis of elevated proteins in CSF (increased capillary permeability of blood-brain barrier

Fig. 6.7: Pandy's test for globulins

vs. increased intrathecal synthesis of immunoglobulin G) can be made from parameters shown in Table 6.3. Albumin is neither synthesized nor metabolized in CNS. Therefore, increased CSF albumin/serum albumin ratio indicates increased permeability of blood-brain barrier. Immunoglobulin G (IgG) can be synthesized in CNS. Therefore, increased CSF IgG/serum IgG ratio indicates either increased permeability of blood-brain barrier or increased intrathecal synthesis of IgG. Increased CSF IgG/albumin index indicates local IgG production.

(2) Estimation of glucose in CSF: Normal CSF glucose is 2/3rds of blood glucose (CSF to blood glucose ratio is 0.6). A sample for blood glucose should be drawn 1 hour before LP for comparison with CSF glucose. After collection, CSF sample should be immediately processed for glucose estimation because falsely low result due to glycolysis may occur.

Table 6.3: Differentiation of causes of elevated proteins in cerebrospinal fluid CSF/Serum albumin ratio

CSF/Serum IgG ratio

CSF IgG/Albumin index

1. Increased 2. Normal

Increased Increased

Normal Increased

Causes

Increased permeability of blood-brain barrier Increased intrathecal synthesis of proteins

Examination of Cerebrospinal Fluid CSF glucose is measured by glucose oxidase method. Normal range is 45-80 mg/dl. CSF glucose 5% of neutrophils have 5 or more lobes. They are large in size and are also called as macropolycytes. They are seen in folate or vitamin B12 deficiency and represent one of the earliest signs. vi. Pelger-Huet cells: In Pelger-Huet anomaly (a benign autosomal dominant condition), there is failure of nuclear segmentation of granulocytes so that nuclei are rod-like, round, or have two segments. Such granulocytes are also observed in myeloproliferative disorders (pseudo-Pelger-Huet cells). vii. Atypical lymphocytes: These are seen in viral infections, especially infectious mononucleosis. Atypical lymphocytes are large, irregularly shaped lymphocytes with abundant cytoplasm and irregular nuclei. Cytoplasm shows deep basophilia at the edges and scalloping of borders. Nuclear chromatin is less dense and occasional nucleolus may be present. viii. Blast cells: These are most premature of the leukocytes. They are large (15-25 µm), round to oval cells, with high nuclear cytoplasmic ratio. Nucleus shows one or more nucleoli and nuclear chromatin is immature. These cells are seen in severe infections, infiltrative disorders, and leukemia. In leukemia and lymphoma, blood smear suggests the diagnosis or differential diagnosis and helps in ordering further tests (Fig. 22.13 and Box 22.3). 3. Differential leukocyte count (DLC): DLC refers to relative proportion of different leukocytes expressed as a percentage.

Fig. 22.13: Comparison of blood smears in (A) acute myeloid leukemia and (B) acute lymphoblastic leukemia

Blood Smear

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Box 22.3: Role of blood smear in leukemia • It suggests the diagnosis or differential diagnosis • It suggests further investigations to be performed for definitive diagnosis.

Principle: Leukocytes are counted on a blood smear and percentage of each type of leukocyte is recorded. Uses of DLC 1. To support the diagnosis of infectious, inflammatory, or allergic disorders. 2. Diagnosis of malignant blood disorders. Using a high power objective, leukocytes should be classified and counted moving from one field to another as shown in Figure 22.4. DLC should not be carried out on the edges and in the tail of the film. In a badly spread smear, polymorphonuclear neutrophils, monocytes, and abnormal cells tend to accumulate in 'feather edge' (tail) and lateral edges, while lymphocytes accumulate in the body of the smear. Also, cells in the body of the smear are shrunken and darkly stained. DLC on such a smear will not be representative. If count is high and abnormal cells are present, DLC should be done in the area just before the tail of the film. This is because morphology is better appreciated in this area. 4. Numerical abnormalities of leukocytes (Fig. 22.14): For meaningful interpretation, absolute count of leukocytes should be reported. These are obtained as follows:

Fig. 22.14: Numerical abnormalities of white blood cells: (A) Neutrophilia; (B) Eosinophilia; (C) Monocytosis; (D) Lymphocytosis

8. Malignant tumors 9. Physiologic causes: Exercise, labor, pregnancy, emotional stress. Leukemoid reaction: This refers to the presence of markedly increased total leukocyte count (>50,000/cmm) with immature cells in peripheral blood resembling leukaemia but occurring in non-leukemic disorders (Fig. 22.15). Its causes are: • Severe bacterial infections, e.g. septicemia, pneumonia • Severe hemorrhage • Severe acute hemolysis

Absolute leukocyte count = Percentage of leukocyte × Total leukocyte count/ml

Neutrophilia An absolute neutrophil count greater than 7500/µl is termed as neutrophilia or neutrophilic leukocytosis. Causes 1. Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection. 2. Tissue necrosis: Burns, injury, myocardial infarction. 3. Acute blood loss 4. Acute hemorrhage 5. Myeloproliferative disorders 6. Metabolic disorders: Uremia, acidosis, gout 7. Poisoning

Fig. 22.15: Leukemoid reaction in blood smear

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Essentials of Clinical Pathology Table 22.1: Differences between leukemoid reaction and leukemia

Parameter

Leukemoid reaction

Leukemia

1. Clinical presentation

Features of underlying disease; fever common

Splenomegaly

< 50000/µl Disappears with resolution of underlying disease Toxic granules, Döhle inclusion bodies Absent Few; cells up to myelocyte stage

Variable, usually > 1 lac/µl Progressive increase

2. Examination of blood a. Total leukocyte count b. Course of neutrophilia c. Evidence of infection d. Basophilia e. Immature cells

Absent Present Many; cells up to blasts

3. Examination of marrow

Myeloid hyperplasia

4. Clonality

Polyclonal

Monoclonal

5. Karyotype

Normal

Abnormal

• Poisoning • Burns • Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 22.1). Neutropenia: Absolute neutrophil count less than 2000/ µl is neutropenia. It is graded as mild (2000-1000/µl), moderate (1000-500/µl), and severe (< 500/µl). Causes I. Decreased or ineffective production in bone marrow: 1. Infections a. Bacterial: typhoid, paratyphoid, miliary tuberculosis, septicemia b. Viral: influenza, measles, rubella, infectious mononucleosis, infective hepatitis. c. Protozoal: malaria, kala azar d. Overwhelming infection by any organism 2. Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis. 3. Drugs: a. Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants. b. Dose-related: Anticancer drugs 4. Ionizing radiation 5. Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.

Increased blasts and immature cells of neutrophil series; Suppression of other cell lines

II. Increased destruction in peripheral blood: 1. Neonatal isoimmune neutropaenia 2. Systemic lupus erythematosus 3. Felty's syndrome III. Increased sequestration in spleen: 1. Hypersplenism. Eosinophilia This refers to absolute eosinophil count greater than 600/ µl. Causes 1. Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs. 2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis. 3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis. 4. Hematologic disorders: Chronic myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma. 5. Carcinoma with necrosis. 6. Radiation therapy. 7. Lung diseases: Loeffler's syndrome, tropical eosinophilia 8. Hypereosinophilic syndrome. Basophilia: Increased numbers of basophils in blood (>100/μl) occurs in chronic myeloid leukemia, polycythemia vera, idiopathic myelofibrosis, basophilic leukemia, myxedema, and hypersensitivity to food or drugs.

Blood Smear Monocytosis

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Box 22.4: Differential diagnosis of lymphocytosis

This is an increase in the absolute monocyte count above 1000/µl. Causes 1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar. 2. Recovery from neutropenia. 3. Autoimmune disorders. 4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease. 5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis. Lymphocytosis This is an increase in absolute lymphocyte count above upper limit of normal for age (4000/µl in adults, >7200/ µl in adolescents, >9000/µl in children and infants) (Box 22.4). Causes 1. Infections: • Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella • Bacterial: Pertussis, tuberculosis • Protozoal: Toxoplasmosis 2. Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma. 3. Other: Serum sickness, post-vaccination, drug reactions. Platelets Platelets are small, 1-3 µm in diameter, purple structures with tiny irregular projections on surface. In blood films prepared from non-anticoagulated blood (i.e. direct

• Mature lymphocytosis: Viral infections, whooping cough, tuberculosis, infectious lymphocytosis, chronic lymphocytic leukemia • Atypical lymphocytosis: Infectious mononucleosis, cytomegalovirus, toxoplasmosis, infectious hepatitis • Lymphoblasts: Acute lymphoblastic leukemia

fingerstick), they occur in clumps. If platelet count is done on automated blood cell counters using EDTA-anticoagulated blood sample, about 1% of persons show falsely low count due to the presence in them of EDTAdependent antiplatelet antibody. Examination of a parallel blood film is useful in avoiding the false diagnosis of thrombocytopenia in such cases. Occasionally, platelets show rosetting around neutrophils (platelet satellitism) (Fig. 22.16). This is seen in patients with platelet antibodies and in apparently normal persons. Blood smear examination can be helpful in determining underlying cause of thrombocytopenia such as leukemia, lymphoma, or microangiopathic hemolytic anemia (Box 22.5). Organisms Common parasites seen in blood are malaria parasites and microfilaria (See Chapter 26: Diagnosis of Malaria and Other Parasites in Blood). Other parasites that can Box 22.5: Role of blood smear in thrombocytopenia It suggests probable cause, e.g. leukemia, microangiopathic process like thrombotic thrombocytopenic purpura or disseminated intravascular coagulation, aplastic anemia, May-Hegglin anomaly.

Fig. 22.16: Causes of false thrombocytopenia on automated hematology analyzer: (A) Clumps of platelets; (B) Platelet satellitism

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be seen in blood smear are Leishmania donovani, and trypanosomes. Sometimes bacteria such as meningococci and fungi (e.g. Histoplasma capsulatum) can be observed within phagocytic leukocytes.

REFERENCE RANGES Normal Blood Smear Red blood cells: Normocytic and normochromic White blood cells: Total and differential leucocyte counts within normal limits Platelets: Adequate Parasites: Not seen. Differential Leukocyte Count • Neutrophils: 40-75% • Lymphocytes: 20-40% (in children between 4 months to 4 years of age, percentage of lymphocytes is more than neutrophils; this is called as inverted differential in children) • Monocytes: 2-10%

• Eosinophils: 1-6% • Basophils: 0-1% Critical Values • • • • • •

Blood smear showing sickle cells Blood smear showing blast cells Leukemoid reaction Suspected aplastic anemia Malarial parasites Absolute neutrophil count 1 year of age) as well as in adults is posterior iliac crest (posterior superior iliac spine). This site has a large reservoir of marrow, is located just beneath the skin and therefore easily accessible. Also, there are no large blood vessels or nerves close to this area, and as the patient’s back is towards the physician, patient’s apprehension is less. In obese patients, anterior superior iliac spine, being more easily localized, can be used. • Sternum: Previously, sternum was commonly used for aspiration of bone marrow in adults (at the level of second intercostal space in midline). However, it is associated with the risk of perforation of posterior sternal plate and puncturing of underlying large blood vessels and right atrium with serious consequences. It also causes greatest patient anxiety. This site should not be used in children as the bone is thin and marrow cavity is small. • Spinous processes of lumbar vertebra: This is an additional site for aspiration in adults. • Tibia: In infants under 1 year of age, marrow can be aspirated from the medial aspect of upper end of tibia just beneath tibial tuberosity. In older children, the tibial cortical bone becomes hard and cellularity of marrow decreases, and therefore this site is not used. Sites for bone marrow aspiration are shown in Figure 25.1.

METHOD Bone Marrow Aspiration 1. An informed consent should be obtained before the procedure. Bone marrow aspiration or biopsy should be performed by the physician. An assistant is required for preparation of smears. 2. A sterile tray should be prepared containing autoclaved bone marrow aspiration needle, sterile disposable syringes with needles, local anesthetic

Fig. 25.1: Sites of bone marrow aspiration (shaded areas)

Fig. 25.2: Klima and Salah bone marrow aspiration needles. Adjustable guard prevents damage to deeper structures. Stylet keeps needle patent during introduction

solution, clean and dry glass slides, spreader slide, gloves, drapes, gauze, and a skin antiseptic solution. All aseptic precautions should be observed during the procedure. Various bone marrow aspiration needles are available. Salah and Klima needles are commonly used (Fig. 25.2). Salah needle has a guard with a side screw, while Klima needle has a guard which screws along the length of the needle. Guards on these needles are adjustable to control the depth of penetration. The guard may slip from the Salah needle during the procedure. For aspiration from iliac crest and tibia, if required, guard may be removed to increase the length of the needle. Guard is essential during sternal aspiration to prevent penetration

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Fig. 25.4: (A) Bone marrow smear and (B) Bone marrow biopsy sample

Fig. 25.3: Jamshidi bone marrow biopsy needle

deeper than necessary and avoid damage to heart or large vessels. Jamshidi needle, which is longer, can be used for both aspiration and biopsy from iliac crest (Fig. 25.3). 3. For aspiration from posterior superior iliac spine, patient should lie on one side with back towards the physician, knees and hips flexed, and the knees drawn towards the chest. The site for aspiration should be selected and scrubbed with soap and water. After wearing sterile gloves, antiseptic solution is applied in a circular fashion, moving from center towards periphery. A sterile drape is placed over the area with its central opening over the aspiration site. 4. Skin and periosteum are infiltrated with a local anesthetic. First inject beneath the skin surface and advancing the needle further, a larger amount is injected into the periosteal surface. 5. After waiting for 5 minutes for anesthesia to take effect, bone marrow aspiration needle is inserted along with the fitted stylet. (Stylet prevents blockage of lumen of needle by tissues through which needle passes). When the bone is reached, the needle is rotated clockwise and anticlockwise and slowly advanced into the bone, maintaining steady and firm pressure. When the marrow is reached, a slight “give” (decrease in resistance) will be noted. The needle is advanced for 1-2 mm into the marrow and the stylet is removed. If the needle is placed correctly, it will be fixed by the surrounding bone and will remain rigid and unmoving.

6. A 5 or 10 ml syringe is attached to the needle and a small amount of marrow is aspirated (till the first drop of blood appears i.e. 0.25-0.50 ml) by quickly pulling the plunger of the syringe. Aspiration is associated with sharp pain (suction pain). Aspiration of larger amount of blood causes dilution of marrow sample by peripheral blood with subsequent difficulties in interpretation of smears. If no material is aspirated, stylet is replaced, needle is redirected, and aspiration attempted again. 7. The syringe should be handed over to the assistant for preparation of smears on glass slides. The smears should be made promptly, before clotting occurs, by putting one drop of the aspirated material near one end of a glass slide and spreading it similar to a blood film (Fig. 25.4). Before making smears, any excess blood on the slide should be sucked away by Pasteur pipette, leaving behind marrow particles. If immunophenotyping or cytogenetic analysis is to be carried out, further marrow sample should be aspirated in a second syringe and dispensed in a tube containing heparin anticoagulant. 8. After completion of aspiration, the stylet should be reinserted into the needle and the needle is removed. Sterile gauze is placed over the site and light pressure is applied till bleeding ceases. A larger dressing is then applied. Bone Marrow Trephine Biopsy Preparation of the patient and local anesthesia are similar to aspiration. A short acting intravenous sedative is preferable in adults. In children, general anesthesia may be necessary. Percutaneous trephine biopsy of bone marrow is commonly obtained from posterior superior iliac spine.

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Jamshidi or Islam needles are commonly used. Often, bone marrow aspiration and biopsy are combined together; aspiration is carried out first followed by biopsy. If both aspiration and biopsy are combined, then, after aspiration, either (i) the needle is advanced a little further (1-3 cm) into the bone, or (ii) the needle is withdrawn and reinserted through the same skin incision but placed at a different site in the bone (about 1 cm away). For biopsy, the needle should be advanced through the bone rotating it clockwise 10 times. The needle should be removed by anticlockwise rotation. The biopsy should be removed gently from the hub end of the needle by inserting the stylet through the point of the needle. For adequate assessment, biopsy should measure at least 1.6 cm in length (Fig. 25.4). Biopsy should be placed in a fixative solution (either 10% formalin or preferably Helly’s fluid). Dressing should be applied to the site similar to aspiration. Along with bone marrow aspiration/biopsy, peripheral blood smears should be prepared from finger prick and venous blood should be collected in EDTA anticoagulant for cell counts.

COMPLICATIONS OF BONE MARROW ASPIRATION AND/OR BIOPSY 1. Local infection: This complication, which is more likely to occur in neutropenic patients, can be prevented if strict aseptic precautions are observed. 2. Hemorrhage: Serious hemorrhage can occur if (i) marrow biopsy is done without adequate replacement cover in coagulation disorders, and (ii) great vessels or heart is injured during sternal aspiration. 3. Cardiac tamponade or mediastinitis: This is likely if posterior sternal plate is penetrated during sternal aspiration.

PROCESSING OF MARROW SPECIMENS Bone Marrow Aspiration A drop of aspirated marrow sample is put on each of the several glass slides, excess blood is removed by sucking with a Pasteur pipette, and smears are prepared with a ‘spreader’. The marrow particles are carried just behind the spreader and cellular trails are produced while spreading. The smears are allowed to dry in the air and are labeled. They are stained with one of the Romanowsky stains. The staining time for marrow films

is longer than that for blood films. On smears, marrow particles are dragged towards the tail end of the smear and after staining appear as dark blue-violet irregular granules. Rest of the film stains even pink. Routinely, one marrow smear (containing one or more marrow particles) should also be stained with Perl’s stain for assessment of bone marrow storage iron. Bone marrow aspiration provides following information: • Assessment of morphology of bone marrow cells. • Assessment of nature of hematopoiesis (normal, dyshematopoiesis) • Cytogenetic analysis • Immunophenotyping of abnormal cells in leukemias. • Cytochemistry for typing of leukemia • Iron stain for assessing iron stores and sideroblasts • Microbial culture, e.g. for tuberculosis With marrow aspiration, marrow architecture and cellular relations cannot be studied. Bone Marrow Trephine Biopsy If prior marrow aspiration is not satisfactory, imprint smears should be prepared by gently rolling the marrow biopsy specimen on a glass slide. These smears are stained with one of the Romanowsky stains for cytological evaluation. Biopsy specimen is then fixed in either 10% formalin or Helly’s fluid. (Helly’s fluid consists of potassium dichromate 2.5 gm; mercuric chloride 5 gm; 40% formalin 5 ml; and water 100 ml). The biopsy is processed (by decalcification, dehydration, clearing, and embedding) to obtain paraffin wax blocks. Less than 4 μm thick sections are cut and stained with hematoxylin and eosin stain and reticulin. It is recommended to mount 5 stepwise serial sections on one slide to increase the chance of detecting small focal lesions. In addition, Giemsa stain is also recommended for easier differentiation between blood and marrow cells and mast cells. Iron stain is less informative on biopsy since iron is usually lost during decalcification. Bone marrow biopsy provides following information: • Cellularity of bone marrow • Bone marrow architecture • Bone structure • Marrow fibrosis • Focal lesions (granulomas, metastatic deposits, infiltration by lymphoma).

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Table 25.1: Comparison of bone marrow aspiration and biopsy Parameter

Bone marrow aspiration

Bone marrow biopsy

1. Site

Iliac spine, sternum, tibia, spinous process of vertebra Morphology, cytochemistry, iron stain, immunophenotyping, culture Unexplained cytopenia, suspected hematological malignancy

Iliac spine (posterior superior)

2. Information obtained 3. Main indications

4. Needle used commonly 5. Studies done

6. Time for routine examination report

Salah, Klima Romanowsky stain, Iron stain, cytochemistry, cytogenetic or molecular genetic analysis, immunophenotyping, culture Same day

With marrow biopsy, detailed morphologic assessment of cells is often not satisfactory. Because of decalcification, paraffin wax- embedded sections show cellular distortion and shrinkage. Plastic embedding of marrow biopsy is advocated to obtain superior cytologic details (as decalcification step is not required). Comparison of bone marrow aspiration and biopsy is presented in Table 25.1.

EXAMINATION OF MARROW SPECIMENS Bone Marrow Aspiration Smears Peripheral blood smear in conjunction with routine hemogram (hemoglobin and cell counts) should be examined first before assessing bone marrow smears. Findings should be interpreted in the light of clinical features and relevant laboratory data. Examination of marrow smear consists of assessment of following features: • Cellularity • Differential count • Myeloid:erythroid ratio • Erythroid series: maturation sequence, type of maturation (normoblastic, micronormoblastic, megaloblastic), cytologic abnormalities. • Myeloid series: maturation sequence, cytologic abnormalities. • Megakaryocyte series: number, abnormal forms. • Lymphocyte series

Cellularity, architecture, fibrosis, focal lesions, bone structure Repeated dry tap, aplastic anemia, myelofibrosis, focal lesions, hairy cell leukemia, staging of lymphoma Jamshidi H and E stain, reticulin stain, immunohistochemistry

Up to 7 days

• Plasma cell series • Abnormal cells: blasts, carcinoma cells, and necrotic cells. • Parasites: malaria parasites, microfilaria, Leishmania donovani, and Histoplasma. • Iron content of marrow (on iron stain). Romanowsky-stained smears are first examined under low power objective (×10) to assess: • Cellularity of marrow particles • Number of megakaryocytes • Focal metastatic deposits • Cell distribution and selection of suitable area for detailed cytologic examination. Assessment of cellularity is based on examination of several marrow particles. Cellularity refers to the proportion of hematopoietic cells as compared to the fat cells in a marrow particle. Cellularity can also be assessed by examining the density of hematopoietic cells in cellular trails behind the marrow particles. Cellularity can be expressed either in percentage or stating whether marrow particles are normocellular, hypercellular, or hypocellular for age. Megakaryocytes are found in the tail of the smear or near the marrow particles. About 1-3 megakaryocytes are seen normally per low power field. Cellular trails are examined under high power (×40) and oil immersion objective (×100) for differential count and assessing erythroid and myeloid maturation. For differential count, at least 500 cells should be counted in

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cellular trails of particles. Normal differential count in bone marrow in adults is shown at the end of this chapter under “Reference Ranges”. Usually a 5-finger differential count is sufficient which consists of counting relative percentages of erythroid cells, myeloid cells, lymphoid cells, plasma cells, and blast cells. In acute leukemia, myelodysplastic syndrome, lymphoma infiltration, and plasma cell dyscrasias, a more detailed differential count is essential. Myeloid:erythroid (M:E) ratio is the ratio of all granulocytes and their precursors to all erythroid precursor cells. Normal M:E ratio ranges from 2:1 to 4:1. On an average, there are 3 myeloid precursors for every erythroid precursor. Since M:E ratio is relative it should be interpreted carefully. Increased M:E ratio is observed when myeloid series is hyperplastic as in infections and myeloproliferative disorders (chronic or acute myeloid leukemia) or when erythroid series is suppressed as in aplastic crisis of hemolytic anemia. Reduced M:E ratio is observed when myeloid series is depressed and in erythroid hyperplasia (e.g. hemolytic anemia). Maturation of erythroid and myeloid series should be assessed. Nature of erythroid maturation may be normoblastic, micronormoblastic, or megaloblastic. Dyshematopoietic features may be suggestive of myelodysplastic syndrome or congenital dyserythropoietic anemia. Detailed cytological evaluation of abnormal cells like blast cells, lymphoma cells, myeloma cells, storage cells,

necrotic cells, or metastatic cells should be carried out. Parasites like Leishmania donovani and Histoplasma capsulatum are detected in macrophages. Routinely, Perl’s stain for iron is done on a marrow smear (containing at least one marrow particle) to assess storage iron and number and types of sideroblasts. In acute leukemia, cytochemical stains like myeloperoxidase, nonspecific esterase, and periodic acid shiff are done for typing of acute leukemia. Iron Staining of Bone Marrow Aspiration Smears Presence of iron in marrow aspiration smears can be demonstrated by Perls’ Prussian blue reaction. In this method, ionic iron reacts with acid ferrrocyanide solution to form blue-colored ferric ferrocyanide. Iron appears as bright blue granular aggregates. Perls’ stain does not detect heme iron of hemoglobin. Iron staining is a valuable test for detection of iron deficiency and must be carried out as a routine on all aspiration smears (Fig. 25.5). Bone marrow aspiration smears containing at least one marrow particle are required for demonstration of storage iron in macrophages. Bone marrow biopsy sections are not suitable for evaluation of iron stores since some iron is lost during processing (decalcification step) of tissue. On marrow smears, stainable iron can be demonstrated in siderocytes, sideroblasts, and macrophages of reticuloendothelial system. Siderocytes are immature, non-nucleated red cells, which contain 1-2 granules of

Fig. 25.5: Iron stain on bone marrow aspiration smear. Left part of the figure shows blue stained iron granules. Right part of the figure shows no stainable iron

Examination of Bone Marrow non-heme iron. On Romanowsky-stained smears, these are called as Pappenheimer bodies. Erythroblasts containing aggregates of iron are called as sideroblasts. They are of three types—I, II, and III. In type I sideroblasts (which are seen normally), iron granules are small and few (1-4) in number. Normally, about 25-50% of erythroblasts contain 1-4, small iron granules. Types II and III sideroblasts are abnormal. In type II sideroblasts, iron granules are large and numerous. Type II sideroblasts are seen in hemolytic anemia and iron overload. In type III sideroblasts, iron granules are distributed in the form of a ring around the nucleus. The ‘ringed’ sideroblasts are seen in sideroblastic anemias. Iron granules in macrophages of reticuloendothelial system represent storage form of iron. Normally, a few, small granules are seen. In iron deficiency anemia, there is complete lack of stainable iron in the erythroblasts and macrophages. Lack of stainable iron in erythroblasts and increased amount of iron in macrophages is a feature of anemia of chronic disease. Marked increase of storage iron in macrophages occurs in thalassemias and iron overload. Bone Marrow Trephine Biopsy Hematoxylin and eosin-stained sections of marrow trephine biopsy are initially examined under low power objective to assess cellularity, to identify focal lesions like granuloma, lymphoma infiltrates and clumps of metastatic malignancy, and to assess bone structure and number of megakaryocytes. Because of variable cellularity between particles and dilution with peripheral blood in marrow aspiration smears, assessment of cellularity is more reliably performed on biopsy sections.

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Cellularity (Fig. 25.6) varies with the age of the patient. There is a gradual reduction in red marrow (hematopoietic tissue) and proportionate increase in fat cells as age advances. A rough estimate of normal cellularity is obtained by deducting 1 for each year of age from 100 and expressing it as a percentage; expected normal cellularity is ±10% of this value. For example, if age of the patient is 25 years, then expected normal cellularity is 65% to 85%. The cellularity in the first decade of life is about 80% while in adults it is about 50%. In older age (>70 years), cellularity decreases to about 30%. Also, normally, the cellularity immediately beneath the subcortical area is low as compared to the deeper medullary areas. Normally bone marrow biopsy shows bony trabecule (lamellar bone) separated by interconnecting spaces containing bone marrow. The bony trabecule are thin, irregular, and show osteocytes within lacune. The trabecule show lining of endosteum (osteoblasts and osteocytes). In a disease called osteopetrosis, bony trabecule are markedly thick with severe reduction in marrow space; there are cartilaginous plates in trabecular bone; and numerous osteoclasts are present along the endosteal surface. High power examination is carried out to assess relative proportion of myeloid and erythroid cells, location of hematopoietic precursors, pattern of infiltration of lymphoma cells, morphology of abnormal cells, and parasites. Normally, erythroid precursors are located in clusters in the center of marrow spaces, while granulocyte

Fig. 25.6: Cellularity of bone marrow on biopsy: (A) Normocellular; (B) Hypercellular; and (C) Hypocellular

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precursors are present close to the trabecule. In myelodysplastic syndrome, clusters of myeloblasts and promyelocytes are located in central marrow cavity; this is called as abnormal localization of immature precursors or ALIP. In infiltration of bone marrow by non-Hodgkin’s lymphoma, pattern of infiltration may be paratrabecular, focal non-paratrabecular, interstitial, and diffuse. Similarly, distinct patterns of infiltration are found in chronic lymphocytic leukemia like diffuse, interstitial, nodular, or mixed (nodular and interstitial) which has prognostic significance. In idiopathic myelofibrosis, collagen deposition can be made out readily on H and E stained sections. For demonstration of reticulin fibers, a silver impregnation technique is required. Reticulin fibers are increased in acute leukemias, chronic myeloid leukemia, polycythemia vera, myelofibrosis, hairy cell leukemia, metastatic carcinoma, and certain inflammatory conditions. If necessary, special stains like Ziehl-Neelsen stain, Giemsa stain, Congo red stain, stains for fungi, and immunoperoxidase stains can be applied. During decalcification of bone marrow biopsy specimen for paraffin wax embedding, many cellular enzymes are destroyed and therefore cytochemical stains (for typing of leukemias), which depend on enzymatic reactions within cells, cannot be applied. For plastic embedding of tissues, decalcification is not required and therefore cytochemistry is possible.

REFERENCE RANGES Differential Count in Bone Marrow in Adults • • • • • • • • •

Myeloblasts: 0-3% Promelocytes: 2-5% Neutrophil myelocytes: 8-15% Metamyelocytes: 9-24% Neutrophils (including band forms): 14-26% Erythroblasts: 15-36% Lymphocytes: 5-20% Plasma cells: 0-3% Myeloid:erythroid (M:E) ratio: 2:1 to 4:1.

Iron Staining of Bone Marrow Smears • Sideroblasts 30-50% of all erythroblasts • No ringed sideroblasts

CRITICAL VALUES • New diagnosis of a hematological malignancy or aplastic anemia or severe thrombocytopenia • Parasites.

BIBLIOGRAPHY 1. Bain BJ. Bone marrow aspiration. J Clin Pathol 2001;54:657-63. 2. Bain BJ. Bone marrow trephine biopsy. J Clin Pathol 2001;54:737-42. 3. Hoffbrand AV, Moss PAH, Pettit JE (Eds). Essential Hematology. (5th Ed). Blackwell Publishing Ltd, 2006. 4. Hyun BH, Gulati GL, Ashton JK. Bone marrow examination: techniques and interpretation. Hematology/ Oncology Clinics of North America 1988;4:513-52. 5. Knowles S and Hoff brand AV. Bone marrow aspiration and trephine biopsy. (1) BMJ 1980;281:204-5. (2) BMJ 1981;281:280-1.

26

Diagnosis of Malaria and Other Parasites in Blood MALARIA

LIFE CYCLE OF MALARIA PARASITES

Malaria is endemic in tropical and subtropical developing countries where it is a major public health problem. Every year there are about 300-500 million clinical cases of malaria with 1-2 million deaths, most of them in Africa. Inadequate administration and implementation of malaria control measures, failure of insecticides, and emergence of drug-resistance strains of the parasite are responsible for resurgence of malaria. There are four species of malaria parasites that infect humans: Plasmodium vivax, P. falciparum, P. malariae, and P. ovale. P. vivax is the most widely distributed species in the world. In India, the prevalent forms of infections are: P. vivax (70%), P. falciparum (25-30%), mixed P. vivax and P. falciparum (4-8%), and P. malariae (< 1%). P. malariae is localised to certain foci, especially in some areas of Karnataka. P. ovale is mainly restricted to West Africa.

Life history of malaria parasite consists of two cycles of development: asexual cycle or schizogony that occurs in humans and sexual cycle or sporogony that occurs in mosquitoes (Fig. 26.1). 1. Asexual cycle (human cycle, schizogony): This occurs in the liver cells and red blood cells of infected humans, and therefore humans are the intermediate hosts of the malaria parasite (Schizogony refers to the process of reproduction in protozoa in which there is production of daughter cells by fission). The human cycle begins when infected female Anopheles mosquito bites a person and sporozoites are injected into the circulation. There are four stages of human cycle.

Fig. 26.1: Life cycle of malaria parasite

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a. Pre-erythrocytic schizogony (Hepatic schizogony): Inoculated sporozoites rapidly leave the circulation to enter the liver cells where they develop into hepatic (pre-erythrocytic) schizonts (Schizonts are cells undergoing schizogony). One sporozoite produces one tissue form. Hepatic schizonts rupture to release numerous merozoites in circulation (Merozoites are daughter cells produced after schizogony). Up to 40,000 merozoites are produced in the hepatic schizont. In P. falciparum infection, all of the hepatic schizonts mature and rupture simultaneously; dormant forms do not persist in hepatocytes. In contrast, some of the sporozoites of P. vivax and P. ovale remain dormant after entering liver cells and develop into schizonts after some delay. Such persistent forms are called as hypnozoites; they develop into schizonts at a later date and are a cause of relapse. b. Erythrocytic schizogony: Merozoites released from rupture of hepatic schizonts enter the red blood cells via specific surface receptors. These merozoites become trophozoites that utilize red cell contents for their metabolism. A brown-black granular pigment (malaria pigment or hemozoin) is produced due to breakdown of hemoglobin by malaria parasites. The fully formed trophozoite develops into a schizont by multiple nuclear and cytoplasmic divisions. Mature schizonts rupture to release merozoites, red cell contents, malarial toxins, and malarial pigment. (This pigment is taken up by monocytes in peripheral blood and by macrophages of reticulo-endothelial system. In severe cases, organs which are rich in macrophages like spleen, liver, lymph nodes, and bone marrow become slate-gray or black in color due to hemozoin pigment). Rupture of red cell schizonts corresponds with clinical attack of malaria. Released merozoites infect new red cells and enter another erythrocytic schizogony cycle. This leads to rapid amplification of plasmodia in the red cells of the human host. In P. falciparum, P. vivax, and P. ovale infections, cycle of schizogony lasts for 48 hours, while in P. malarie infection it lasts for 72 hours. Merozoites of P. vivax and P. ovale preferentially invade young red cells or reticulocytes while those of P. falciparum infect red cells of all ages. Senescent red cells are preferred by P. malariae.

P. vivax, P. ovale, and P. malariae complete the erythrocyte schizogony in general circulation. Schizonts of P. falciparum induce membrane changes in red cells, which causes them to adhere to the capillary endothelial cells (cytoadherence). Therefore, in P. falciparum infection, erythrocyte schizogony is completed in capillaries of internal organs and usually only ring forms are seen in circulation. c. Gametogony: After several cycles of erythrocytic schizogony, some merozoites, instead of developing into trophozoites and schizonts, transform into male and female gametocytes. These sexual forms are infective to mosquito and the person harboring them is called as a “carrier”. Gametocytes are not pathogenic for humans. d. Exoerythrocytic schizogony: In P. vivax and P. ovale infections, some of the sporozoites in liver cells persist and remain dormant. These dormant forms in liver cells are called as hypnozoites. They become active and develop into schizonts a few days, months, or even years later. These schizonts rupture, release merozoites, and cause relapse. Exoerythrocytic schizogony is absent in P. falciparum infection and therefore relapse does not occur. Hence, P. vivax and P. ovale are called as relapsing plasmodia while P. falciparum and P. malariae are known as non-relapsing plasmodia. 2. Sexual cycle (mosquito cycle, sporogony): The sexual cycle begins when a female Anopheles mosquito ingests mature male and female gametocytes during a blood meal. First, 4-8 microgametes are produced from one male gametocyte (microgametocyte) in the stomach of the mosquito; this is called as exflagellation. The female gametocyte (macrogametocyte) undergoes maturation to produce one macrogamete. By chemotaxis, microgametes are attracted toward the macrogamete; one of the microgametes fertilizes the macrogamete to produce a zygote. The zygote becomes motile and is called as ookinete. Ookinete penetrates the lining of the stomach and comes to lie on the outer surface of the stomach where it develops into an oocyst. On further growth and maturation, multiple sporozoites are formed within the oocyst. After complete maturation, oocyst ruptures to release sporozoites into the body cavity of the mosquito. Most of the sporozoites migrate to the salivary glands. Infection is transmitted to the humans by the bite of the mosquito through saliva when it takes a blood meal.

Diagnosis of Malaria and Other Parasites in Blood

CLINICAL FEATURES Clinically malaria is characterized by paroxysms of highgrade fever along with anemia and splenomegaly. There are three stages of a typical malarial paroxysm: (i) cold stage (lasts for 20 min to 1 hr): There is sudden onset of fever, shivering, and extreme cold; (ii) hot stage (1-4 hr): Fever rises to its maximum and there are associated severe headache and body ache; (iii) sweating stage (2-3 hrs.): Patient perspires profusely and temperature falls. The duration of each paroxysm is about 6-10 hours and coincides with the rupture of erythrocytes and release of showers of merozoites. Febrile paroxysms recur every 48 hours (in P. vivax, P. falciparum, and P. ovale infections) that is called as tertian malaria or every 72 hours (in P.malariae infection) called as quartan malaria. Anemia usually results from destruction of parasitized red cells; other causes are suppression of erythropoiesis in bone marrow and immune hemolysis. Splenomegaly is an important feature and with repeated paroxysms there is a significant enlargement of spleen. Splenomegaly is secondary to activation and hyperplasia of mononuclear phagocytic system. Other clinical features include myalgia, joint pains, thrombocytopenia, and hypoglycemia. Relapse is a feature of malaria caused by P. vivax and P. ovale and is due to persistence of hypnozoites in liver cells. It may occur months or even years after the initial attack. Relapse does not occur with P. falciparum infection. Untreated P. falciparum infection can be severe and fatal. This is because of high degree of parasitemia and sequestration of parasitized red cells in capillaries of internal organs. The red cells containing trophozoites and schizonts of P. falciparum express, on their surface, positively charged parasite proteins and become ‘sticky’. These red cells adhere to each other and to endothelial cells of capillaries of internal organs (cytoadhesion). This leads to clogging of microcirculation by parasitized red cells with resultant rupture of capillaries. Two serious complications of falciparum malaria are cerebral malaria and blackwater fever. Cerebral malaria clinically manifests as hyperpyrexia, convulsions, coma, and sometimes death. In blackwater fever, there is sudden onset of massive intravascular hemolysis (which causes hemoglobinemia, hemoglobinuria, and hyperbilirubinemia) and high-grade fever. Renal failure is common. Parasites are not detectable in blood. Urine

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appears dark brown or black in color due to hemoglobinuria. Infection by P. malariae can cause nephrotic syndrome.

MODES OF TRANSMISSION Malaria is transmitted by the bite of certain species of infected female Anopheles mosquitoes. Other rare modes of transmission are blood transfusion, use of contaminated syringes or needles, and congenital (from mother to the fetus). Hepatic schizogony does not occur when malaria is transmitted through transfusion, contaminated syringes, or congenital route; therefore, in case of P. vivax infection, relapse will not occur.

ROLE OF GENETIC FACTORS IN MALARIA 1. Sickle cell trait and β -thalassemia trait: Young children with sickle cell trait (heterozygotes) develop comparatively mild form of falciparum malaria. However, persons with sickle cell anemia (homozygous for sickle cell gene) are not protected. Theory of balanced polymorphism proposes that selective advantage gained by sickle heterozygotes is balanced by disadvantage of homozygous state. β-thalassemia trait is also protective against severe falciparum malaria. 2. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency: Protection against severe falciparum malaria is afforded to G-6-PD-deficient female heterozygotes. 3. Newborns: For the first few months of life, newborn infant has high levels of hemoglobin F in red cells that suppresses the growth of malaria parasite. 4. Duffy antigen: The Fy (a-b-) phenotype (i.e. Duffy antigen-negative blood group) in blacks confers resistance against P. vivax infection. This is because P. vivax parasite enters the red cells at glycophorin receptors present on Duffy antigen site. P. vivax infection is very rare in West Africa due to the lack of Duffy antigen on red cells of the population. Methods of Diagnosis Malaria can be diagnosed by following methods: • Clinical diagnosis • Microscopic examination of blood smears • Rapid diagnostic tests (Detection of malaria parasite antigen or enzyme)

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• Fluorescence microscopy • Serologic techniques • Detection of nucleic acid of parasite by polymerase chain reaction. 1. Clinical diagnosis: This is the most common method for diagnosis of malaria in endemic countries. However, this method is unreliable since symptoms of malaria overlap with those of many other febrile illnesses like viral fever, viral encephalitis, and typhoid fever. 2. Microscopic examination of thick and thin smears: The most commonly used, fairly rapid, and accepted method for diagnosis of malaria is microscopic examination of stained blood smears. It remains the gold standard for diagnosis of malaria. Blood smear should be obtained before the next expected febrile paroxysm or at the onset of fever and chills. Collection of blood immediately following a paroxysm of fever is not likely to show intrerythrocytic parasites because of lysis of parasitized red cells. Blood sample should be taken before antimalarial drugs are given. After administration of antimalarial drugs, parasites become scarce in blood and alter in morphology. Ideally, blood should be collected by skin puncture and smears made immediately. Adhesion of blood to the slide and subsequent staining are affected if anticoagulated blood sample is used. If it is necessary to use anticoagulated blood, blood should be collected in EDTA and smear made as early as possible; delay causes morphologic changes in parasites, which may make diagnosis difficult. One thick smear (for detection of parasites) and one thin smear (for definitive identification of species) should be prepared (Fig. 26.2).

Fig. 26.2: Thick and thin blood smears (unstained)

After complete drying, thick smear is stained with a Giemsa stain or a Field’s stain. Red cells are lysed during staining and parasites are better visualised against a clear background. Thick smear (since it allows examination of a relatively large amount of blood) is more sensitive than thin smear for detection of malaria parasites, particularly if they are few in number. For thick smear, a large drop of blood is collected in the center of glass slide. It is spread with the corner of a spreader slide or a stick in such a manner that an evenly spread circular or rectangular smear of size 15 × 15 mm is obtained. After labeling, smears are allowed to dry in the air. For better results, thick smear can be dried in an incubator at 37°C for 15 min. Thick smear should not be fixed since it is to be dehemoglobinised. Thin smear is prepared as outlined in Chapter 22 (Blood Smear). It is air-dried, fixed with methanol, and stained with a Giemsa stain or Leishman stain. Thin smear is used for definitive identification of parasite species, determination of parasite density (if parasitemia is very high), and for detection of any associated morphologic abnormalities of blood cells. It is fixed with methanol for 1-2 minutes before staining. Thick and thin smears can also be prepared on the same slide. It is recommended to examine at least 100 oil immersion fields in thick smear and 200 oil immersion fields in thin smear before reporting the smear as negative for malaria parasite. If blood smear is negative for malaria parasite despite strong clinical suspicion, repeat smears should be taken. A buffy coat preparation can also be helpful in such a case. EDTA-anticoagulated blood is centrifuged and a smear is prepared from buffy coat layer and from red cells just below it. Parasitized red cells get concentrated just beneath the buffy coat layer. In P. vivax infection, almost all red cell stages of parasite are represented in the blood smear. In P. falciparum infection, usually only ring forms or gametocytes are seen. Presence of mature trophozoites and schizonts of P. falciparum in blood smear are indicative of a serious infection. If only gametocytes of P. falciparum are seen in untreated subjects, it denotes active, suppressed infection; if seen in treated subjects, it carries no significance. Identification of malaria parasites: Different stages of malaria parasites are found in peripheral blood. These are trophozoites, schizonts, and gametocytes. The four

Diagnosis of Malaria and Other Parasites in Blood species of malaria parasites can be distinguished morphologically by microscopic examination of thin blood smear. Microscopic examination of blood smears for diagnosis of malaria is a sensitive test; it can detect about 50 parasites/μl in experienced hands. Morphological features of malaria parasites are summarized and compared in Table 26.1. Stages of P. vivax and P.falciparum are shown in Figures 26.3 and 26.5. Blood smears in P. vivax and P. falciparum are shown in Figures 26.4 and 26.6 respectively. As compared to the recently available rapid immunochromatographic tests (see below), microscopic examination of the blood smear is relatively inexpensive. However, it needs good quality control, is laborintensive, and takes more time than immunochromato-

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graphic tests. Also, in some cases of falciparum malaria, parasites get sequestered in the capillaries of internal organs and are not detectable on blood smears (but can be detected by immunochromatographic tests). If P. falciparum is identified, it is necessary to estimate parasite density. Since gametocytes are non-pathogenic, they should be excluded while counting parasites. Two methods for estimation of parasite density are given below. 1. In a thick smear, number of parasites is counted till 200 white blood cells are observed. Number of parasites present per μl of blood is calculated from the following formula: Number of parasites Total leukocyte count/µl × ——————————— 200

Table 26.1: Morphological comparison of Plasmodia Plasmodium falciparum Often only ring forms and gametocytes are seen; multiple rings in a single cell common; red cell size normal; high parasite density. Early trophozoite Late trophozoite A delicate, small Compact blue ring with uniformly fine cytoplasmic 1-2 red chromatin dots ring with 1-2 small chromatin dots; ring may be attached to the red cell margin (accole form).

Schizont Very rarely seen except in cerebral malaria; contains 18-32 merozoites which fill 2/3rds of a red cell; a single block of dark brown pigment

Gametocyte Crescentic or bananashaped; larger than a red cell

Plasmodium vivax Often all stages are seen; single parasite in a red cell; red cell enlarged and shows Schuffner’s dots (fine, pink granules); medium parasite density. Blue cytoplasmic ring 1/3rd the diameter of the red cell; one side of ring thicker; red chromatin at thinner part of ring

Irregularly thick cytoplasmic ring (ameboid); large red chromatin dot

12-24 merozoites arranged rosette-like granular yellow-brown pigment in center

Large and spherical

Plasmodium ovale Often all stages are seen; single parasite in a red cell; red cell slightly enlarged, oval, and with fimbriated margins; prominent Schuffner’s dots. Thick, dense blue ring Compact ring; slightly 8-12 irregularly placed Oval; prominent with a large red amoeboid merozoites Schuffner’s dots chromatin dot Plasmodium malariae Often all stages are seen; single parasite in a red cell; red cell normal in size; no Schuffner’s dots; low parasite density. Similar to P. vivax Compact band-like ring 6-12 merozoites in a Round or oval across the red cell “daisy-head” pattern

•••••••••••••••••

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Fig. 26.3: Stages of P. vivax

2. In a thin smear, number of parasites amongst 1000 red cells is counted and reported as a percentage. Number of parasites in 1 μl of blood can be calculated if red cell count in millions/μl is known; if it is not known, then it can be arbitrarily taken as 5 million/ μl. Number of parasites in 1 μl of blood = Red cell count in million/cmm × parasite percentage

Estimation of degree of parasitemia is of significance in P. falciparum infection. Percentage parasitemia exceeding 10% is an indication for exchange transfusion. In patients taking antimalarial treatment, percent parasitemia should be obtained daily till no more parasites (excluding gametocytes) are detectable.

Fig. 26.4: P. vivax infection is associated with simultaneous presence of multiple stages

Usually, total leukocyte count is taken as 8000/ μl and the number of parasites is multiplied by 40 to get the result. However, if accurate leukocyte count is known, then a better estimate of parasite density is obtained. To obtain percent parasitemia, figure of number of parasites amongst 200 white blood cells is divided by 1250.

Reporting the result: In the presence of malaria parasite, blood smear should be reported as follows: • Blood smear is positive for malaria parasite • Name of species • Red cell stages seen • Parasite density (in case of P. falciparum) Comparison of morphology of P. vivax and P. falciparum is shown in Figure 26.7. 3. Detection of malaria parasite antigen by rapid immunochromatographic tests: In recent years, commercial manufacturers have introduced simple non-microscopic rapid diagnostic tests. These tests

Fig. 26.5: Stages of P. falciparum

Diagnosis of Malaria and Other Parasites in Blood

Fig. 26.6: Blood smear in P. falciparum infection showing ring forms.Usually a single stage is seen in P. falciparum infection

detect antigens of malaria parasites by immunochromatographic method. The antigens against which the commercial test kits are currently available are: • Histidine rich protein-2 (HRP-2) synthesized by asexual blood stages and young gametocytes of P. falciparum and expressed on red cell surface. • Parasite lactate dehydrogenase (pLDH), an enzyme of glycolytic pathway found in all four human malaria species; there are different forms of pLDH for each species. Level of pLDH correlates with parasite density.

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Many commercial test kits are available with differing test procedures. General principle of these tests is as follows. Blood sample is collected by skin puncture. It is then mixed with a buffer solution provided with the kit; this solution causes lysis of red cells and also contains a specific antibody labeled with a dye. If the antigen under investigation is present in blood, formation of antigenantibody complex occurs. The labeled antigen-antibody complex is then allowed to migrate along a nitrocellulose test strip by capillary action (The manufacturer has impregnated this test strip with a monoclonal antibody against the antigen across the strip. The strip also contains a line of positive control to check whether the test has been performed correctly and whether the reagents are functional). In the last step, a washing solution is added to remove hemoglobin from lysed red cells and to visualise the reaction. If the blood sample contains the antigen under investigation, the labeled antigenantibody complex will form, it will migrate up the strip, and will be captured by the pre-deposited monoclonal antibody; this results in the appearance of a colored line across the strip. Tests that detect P. falciparum, P. vivax, and other species are available. Tests which detect HRP-2 antigen of P. falciparum have sensitivity and specificity of >90% (if parasite density is greater than 100/μl). Commercial test kits can be helpful in following situations: • Confirmation of P. falciparum infection if there is uncertainty about the identity of malaria parasite on blood smear. • Confirmation of P. falciparum in mixed infections.

Fig. 26.7: Comparison of P. falciparum and P. vivax morphology

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• Diagnosis of P. falciparum in endemic remote peripheral areas where properly trained technicians and facilities for microscopic diagnosis are not available. Local health workers with little training can perform rapid diagnostic tests. • Rapid, self-diagnosis of malaria by tourists traveling to endemic countries. Some manufacturers have developed self-help diagnostic kits for travelers. Positive rapid diagnostic tests (for HRP-2) persist for several days following successful treatment of P. falciparum infection (due to persistence of antigens in blood). Therefore these tests are not useful in following response to treatment. Also these tests are not able to estimate parasite density. Distinction between nonpathogenic gametocyte stage and other pathogenic stages is not possible. Comparison between blood smear and rapid diagnostic tests is given in Table 26.2. 4. Fluorescence microscopy: There are two fluorescence microscopy techniques employed for diagnosis of malaria: quantitative buffy coat (QBC) system (Becton Dickinson), and Kawamoto technique. a. Quantitative Buffy Coat (QBC) system (Becton Dickinson): Blood is centrifuged in a special capillary tube which contains a float and which is coated with acridine orange (a fluorescent dye) and an anticoagulant. Following centrifugation, malaria parasites get concentrated in the upper layer of red cells just below the buffy coat and are stained with the fluorescent dye. When the capillary tube is viewed using a special objective (paralens) attached to the fluorescence microscope, malaria parasites fluoresce green yellow against a dark red-black background. Nuclei of trophozoites fluoresce bright green.

Acridine orange stains all the cells that contain nucleic acids. Therefore, considerable experience is required to distinguish fluorescing parasites from other cells containing nucleic acids. This method also needs expensive apparatus and materials. Howell-Jolly bodies (nuclear remnants in red cells in certain anemias) give a false positive reaction. Schizonts and gametocytes get localized in the buffy layer and are missed. Specific identification of species is difficult and it is necessary to use Romanowsky-stained smears for this purpose. Despite these disadvantages, this technique is a more rapid alternative to blood smears. b. Kawamoto technique: Blood smears are prepared, stained with a fluorescent dye (acridine orange), and examined by fluorescence microscopy. An interference filter designed for acridine orange is placed in the pathway of transmitted light beam and a barrier filter is placed in the eyepiece. Nuclei of malaria parasites fluoresce bright green and cytoplasm red. Nuclei of white cells also fluoresce bright green. Definitive species identification is difficult; therefore Romanowsky-stained smears are required for species diagnosis. Kawamoto technique is less expensive than QBC system. 5. Detection of nucleic acid sequences of malaria parasites: Malaria parasites can be detected by identification of specific nucleic acid sequences in their DNA. Methods based on polymerase chain reaction (PCR) have been developed for identification of DNA of malaria parasite. Species diagnosis is also possible. PCR-based methods can detect very low levels of parasites in blood (< 5 parasites/μl of blood) with very high sensitivity and specificity.

Table 26.2: Comparison of blood smear and commercial rapid diagnostic tests for malaria diagnosis Parameter

Blood smear

Rapid diagnostic tests

1. Sensitivity 2. Species identified 3. Parasite density 4. Performance 5. Time required 6. Detection of sequestered P. falciparum 7.Distinction between gametocytes and other stages 8. Cost

5-10 parasites/μl All Can be estimated Laborious 1 hour No Yes Low

40-100 parasites/μl Depends on test kit Cannot be estimated Easy 15-20 minutes Yes No High

Diagnosis of Malaria and Other Parasites in Blood Molecular methods can be useful in the diagnosis of malaria, in following response to treatment, in epidemiological surveys, and for screening of blood donors. They can also be used as a standard to judge other methods of malaria diagnosis. However, these methods cannot be routinely applied because of the high cost, need for special equipments and materials, and lengthy procedure (24 hours). Presently they can be used as a research tool in malaria control programs, and to carry out quality control checks on microscopic diagnosis. 6. Serologic methods: Indirect immunofluorescence or hemagglutination tests are available which detect antibodies against malaria parasites in patient’s serum. These tests are helpful for: (i) retrospective epidemiological surveys in endemic areas, (ii) screening of blood donors for asymptomatic infection, and (iii) retrospective diagnosis. However, these tests are not helpful for routine diagnosis of active infection as they cannot distinguish between current and past infection. Thus they cannot guide treatment.

LYMPHATIC FILARIASIS Lymphatic filariasis is a parasitic infection caused by nematode worms Wuchereria bancrofti, Brugia malayi, and Brugia timori. Lymphatic filariasis affects more than 120 million people in the tropical and subtropical countries. In India, lymphatic filariasis is caused by Wuchereria bancrofti (majority of cases) and Brugia malayi (mainly in southwest India). (Brugia timori is restricted to Indonesia). According to World Health Organization, one-third of the people affected by lymphatic filariasis are in India. The prevalence of this disease appears to be on the rise due mainly to proliferation of slums and expansion of breeding sites for mosquitoes. Life Cycle Filarial worms pass their life cycle in two hosts: humans (definitive host) and mosquitoes (intermediate host) (Fig. 26.8). The disease is transmitted to the humans by the bite of infected female mosquitoes of the genera Culex, Anopheles, or Aedes. The infective larvae are deposited on the human skin when the mosquito takes a blood meal. The larvae then enter the host through the puncture wound. The larvae reach the lymphatic channels and mature into adult male and female worms (5-18 months).

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Fig. 26.8: Life cycle of Wuchereria bancrofti

After fertilization, female worms produce numerous microfilariae, which migrate into venous blood (via large lymphatic ducts) and reach arterial circulation through pulmonary capillaries. The adult worms inhabit the lymphatics and live for many (5-10) years. The microfilariae are ingested by the mosquitoe when it takes a blood meal. Microfilariae cast off their sheaths in the stomach of the mosquitoe, penetrate the gut wall, and migrate into the thoracic muscles where they reside and grow into infective larvae. After about 2 weeks of development, the mature infective forms migrate to the mouthparts of the mosquitoe and are transmitted to the definitive host through the bite.

CLINICAL FEATURES Clinical features are variable. Also, after acquisition, infection may take years to manifest clinically. Many infected individuals do not exhibit any clinical manifestations even though they have numerous circulating microfilariae and harbor adult worms in lymphatics. In acute disease, repeated attacks of fever are associated with lymphangitis (filarial fever). Inflammation occurs in lymphatics of limbs, genitals (epididymis, spermatic cord, and vulva), and breasts. Lymphangitis is often associated with enlargement of regional lymph nodes (inguinal or axillary). Repeated episodes of lymphangitis cause fibrosis and obstruction of lymphatics with accumulation of lymph fluid in surrounding tissues (lymphedema). The late manifestations of filariasis are hydrocele and elephantiasis. In hydrocele, sac surrounding the testis is filled with fluid. In elephantiasis, there is extreme edematous enlargement of the affected part (limbs,

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external genitals, or breasts) with coarse thickening and fissuring of skin. Local bacterial and fungal infections are common. Microfilariae are usually not found in blood of patients with hydrocele and elephantiasis. In some cases, there is passage of milky-white chyle in urine (chyluria). This is due to rupture of distended urogenital lymphatics, which are connected to the intestinal lymphatic vessels carrying chyle. Microscopic examination of such urine shows microfilariae.

LABORATORY DIAGNOSIS Diagnostic methods in lymphatic filariasis include: • Microscopic examination of blood for demonstration of microfilaria • Detection of filarial antigen in blood by rapid immunochromatographic test • Demonstration of microfilariae in hydrocele fluid or chylous urine • Demonstration of adult worms in lymph node biopsy • Detection of parasite DNA by polymerase chain reaction. Microscopic Examination of Blood for Demonstration of Microfilaria Since some species exhibit periodicity (i.e. circulation of microfilariae in increased numbers at certain times of the day), blood should be collected at the correct time to improve the chances of detection. For Wuchereria bancrofti and Brugia malayi showing nocturnal periodicity, blood should be collected at night between 10 p.m. to 4 a.m. Microfilariae are present in greater numbers in capillary blood than in venous blood; therefore, skin puncture is preferred. Usually microfilariae are scanty in peripheral blood so that concentration techniques may be necessary for their demonstration. Following microscopic methods can be used for detection of microfilariae in peripheral blood: • Thick blood smear • Concentration techniques: membrane filtration, microhematocrit centrifugation, lysed venous blood technique, lysed capillary blood technique.

prepared from capillary blood collected at the appropriate time, and if clinical suspicion is strong, concentration techniques are employed. This is because circulating microfilariae are often scanty and sensitivity of microfilarial detection increases when volume of blood sampled is increased. Concentration techniques Membrane filtration: This is a sensitive method but is expensive for routine use in endemic areas. Anticoagulated venous blood (10 ml) is passed through a polycarbonate membrane filter of 3 μm or 5 μm pore size. Following this 10 ml of methylene blue saline solution is passed through the filter for staining the microfilariae. Microfilariae are trapped and retained on the filter, which is placed on a glass slide and examined under the microscope. Microhematocrit tube or capillary tube method: Two heparinised capillary tubes are filled with blood from skin punctures (or two plain capillary tubes are filled with anticoagulated venous blood). After sealing the dry ends with a suitable sealant, tubes are centrifuged in a microhematocrit centrifuge for about 5 minutes. The capillary tubes are placed on a glass slide and fixed with adhesive tape. Plasma just above the buffy coat layer is examined for motile microfilariae under the microscope (Fig. 26.9). Lysed venous blood method: 10 ml of venous blood is lysed by saponin-saline solution. The hemolysate is centrifuged, supernatant is discarded, and the sediment is placed on glass slide. After adding a drop of methylene blue solution, a coverslip is placed, and the preparation is examined under the microscope for microfilariae. Lysed capillary blood method: 0.1 ml of blood obtained by skin puncture is added to 1 ml of saponin-saline solution to cause lysis of red cells. After centrifugation, supernatant is discarded and sediment is placed on a glass slide. A drop of methylene blue solution is added and a coverslip is placed over it. The entire preparation is examined under the microscope for motile microfilariae.

Thick Blood Smear

Morphology of Microfilariae on Romanowsky-stained Blood Smears

A thick blood smear is spread from 20 μl of capillary blood on a glass slide, air-dried, and stained with a Romanowsky stain. If microfilariae are not detected in thick smears

Wuchereria bancrofti: Microfilariae measure about 300 μ in length and 8 μ in breadth. They have a hyaline sheath, which stains pink. There are distinct nuclei in the central

Diagnosis of Malaria and Other Parasites in Blood

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Fig. 26.9: Microhematocrit tube concentration technique for demonstration of microfilaria.

shows two distinct nuclei. Cephalic space is twice as long as it is broad. Instead of smooth curves to the body, there are kinks. Detection of Filarial Antigen in Blood by Rapid Immunochromatographic Test

Fig. 26.10: Microfilaria of W. bancrofti in blood smear

axis of the body. Nuclei are not present in the tip of the tail. Cephalic space (present at the anterior end) is as long as it is broad. Tip of the tail is bent backwards and body curves are few (Fig. 26.10). Brugia malayi: These measure about 230 μ × 6 μ in size. Sheath stains dark pink in color. The nuclei are crowded in the body, are blurred in outline, and the tip of the tail

A highly sensitive and specific test for diagnosis of active infection by Wuchereria bancrofti is ICT Filariasis Card Test (ICT Diagnostics), which is available commercially. This test detects circulating antigens of this organism in a fingrprick blood sample of infected individuals. In contrast to the microscopic tests, blood sample can be collected at any time of the day. There is no crossreactivity with other filarial organisms like Brugia malayi. Positive result is obtained even if microfilariae are not circulating and live adult worms are present in the lymphatics. The test remains positive for up to 18 months following successful therapy of filariasis. Apart from diagnosis of individual patients, this test can be applied to assess the prevalence of filarial infection in a population. Time required for the test is about 10-15 minutes. Other tests have been developed for detection of circulating filarial antigens like immunoradiometric assay and enzyme-linked immunosorbent assay (ELISA). However, these tests are expensive and more laborintensive for routine use. Also, they cannot be applied in field conditions.

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Detection of Filarial DNA by Polymerase Chain Reaction Filarial DNA can be detected in circulating blood by polymerase chain reaction. Although this test is highly sensitive and species-specific for diagnosis, it is laborious and expensive for routine use in developing countries.

VISCERAL LEISHMANIASIS Leishmaniases are a group of parasitic diseases caused by obligate intracellular protozoa of the genus Leishmania and transmitted by the bite of an infected female sandfly of the genus Phlebotomus (Africa, Asia, and Europe) or Lutzomyia (South and Central America). Leishmaniases are endemic in tropical and subtropical regions of 88 countries on five continents. There are about 12 million cases of leishmaniases worldwide and 1.5 to 2 million cases occur annually. Rising prevalence of leishmaniases is attributed to massive rural to urban migration, deforestation with development of newer dwelling sites, and newer irrigation projects. In addition, leishmania/human immunodeficiency virus coinfection is rapidly emerging as a new, severe disease. Leishmaniases occur in three different clinical forms: visceral, cutaneous, and mucocutaneous. Only visceral leishmaniasis is considered below. In Asia, visceral leishmaniasis (VL) is also called as kala azar or black sickness. This is the most severe form of leishmaniases and is caused by Leishmania donovani complex (which includes L. donovani, L. infantum, and L. chagasi species). Majority of cases of VL occur in Bangladesh, northeastern Brazil, northeast India, Nepal, and Sudan. In India, the causative organism is L. donovani and infection is anthroponotic (i.e. transmitted from one human to another through sandfly vector Phlebotomus). In India, leishmaniasis is prevalent in Assam and Bengal

along Brahmaputra and Ganges, Bihar, Orissa, and Andhra Pradesh. L. infantum is endemic in the Mediterranean basin and infection is zoonotic (i.e. transmitted to humans from animals like dogs through sandfly vector). L. chagasi occurs in Latin America with domestic dog as the primary reservoir of infection. Typical manifestations of VL include fever, enlargement of spleen, enlargement of liver, severe cachexia, pancytopenia (anemia, leukopenia, and thrombocytopenia), and hypergammaglobulinemia. Secondary infections are frequent. The name “ Kala-azar” is derived from the grayish coloration of skin that often develops during the course of disease. If untreated, death often follows. Post kala azar dermal leishmaniasis (PKDL) is a cutaneous form of leishmaniasis occurring after resolution of visceral leishmaniasis. It is observed in India and East Africa. It manifests as hypopigmented and raised erythematous lesions most prominently on the face. The lesions contain amastigote forms of L. donovani. Since Leishmania are present in large numbers in peripheral blood, they can be transmitted via sharing of needles among intravenous drug abusers. The typical clinical features of visceral leishmaniasis are not always seen and atypical presentations make diagnosis difficult. Response to treatment is poor and relapses are common.

Life Cycle of Leishmania donovani Leishmania donovani (LD) exists in two stages: promastigote (flagellated form found in sandfly vector) and amastigote (non-flagellated tissue-form found in mammalian host) (Fig. 26.11). Infection is transmitted to man when the infected female sandfly takes a blood meal

Fig. 26.11: Life cycle of Leishmania donovani

Diagnosis of Malaria and Other Parasites in Blood and promastigote forms are inoculated. These promastigotes are taken up by the macrophages of the reticuloendothelial system and are transformed into amastigote forms. The amastigote forms multiply by binary fission inside the macrophages, are released in circulation when the host cell ruptures, and are again taken up by new macrophages. In VL, whole of the reticuloendothelial system is progressively infected (including bone marrow, spleen, liver, lymph nodes, and blood monocytes). When the female sandfly takes a blood meal, it ingests intracellular and free amastigote forms present in blood. These then develop into promastigote forms in the midgut of the sandfly. After multiplication, promastigote forms fill the lumen of the midgut and spread forwards to the pharynx and buccal cavity. These are transmitted to man when the infected female sandfly takes a blood meal.

LABORATORY DIAGNOSIS OF VISCERAL LEISHMANIASIS Laboratory studies for diagnosis of VL include: • Examination of splenic aspirate, bone marrow aspirate, or buffy coat preparation of peripheral blood for amastigote forms • Detection of anti-Leishmania antibodies • Intradermal skin test • Culture of parasite • Animal inoculation studies • DNA studies Examination of Splenic Aspirate, Bone Marrow Aspirate, or Buffy Coat Preparation of Peripheral Blood for Amastigote Forms The most commonly used method for demonstration of the parasite is the examination of Giemsa- or Leishmanstained smears from relevant tissues. Usually, smear is prepared from splenic aspirate (sensitivity 95-98%), bone marrow aspirate (sensitivity 60-85%), or buffy coat (sensitivity 67-99%). After staining, smears are examined under oil-immersion lens of the light microscope for amastigote forms (also called as LD bodies). Amastigote forms are small (2-4 μ in diameter), round to oval, and have nucleus and a rod-shaped kinetoplast. Cytoplasm is pale blue while nucleus and kinetoplast stain pinkish red. Nucleus is relatively large and kinetoplast lies at right angles to it. In aspirate smears, amastigote forms are seen in groups inside macrophages or lying free between cells

241

Fig. 26.12: Leishmania donovani bodies in bone marrow (within a macrophage as well as lying free)

(Fig. 26.12). In buffy coat smears, they are seen inside monocytes or less commonly inside neutrophils. Amastigote forms should be distinguished from yeast forms of Histoplasma capsulatum (which show budding and do not show kinetoplast). Splenic aspirate has the highest sensitivity for detection of tissue forms of Leishmania donovani. Since splenic aspiration is associated with a risk of fatal hemorrhage, platelet count and prothrombin time should be obtained before the procedure. Splenic aspirate is contra-indicated if platelet count is 5 seconds. Tests for Detection of Anti-Leishmania Antibodies Tests are available for detection of raised non-specific immunoglobulins and specific anti-Leishmania antibodies. a. Tests that detect raised non-specific immunoglobulins: In VL, increased amounts of nonspecific polyclonal IgG and IgM antibodies are produced. Tests, which detect these, are formol gel (aldehyde) test and antimony test. However, these tests are nonspecific and therefore unreliable for diagnosis of VL. Formol gel (aldehyde) test can be used to support the diagnosis of VL. In this test, 1-2 drops of 40% formalin solution are added to 1 ml of patient’s serum in a test tube. The mixture is allowed to stand for 2 hr. In a positive test, serum becomes milky white and gels (usually within 2-20 min) (Fig. 26.13). This test becomes positive only when the disease is of greater than 3 months duration.

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Essentials of Clinical Pathology positive in persons who have recovered from kala azar. It is negative in active infection. Parasite Culture

Fig. 26.13: Formol gel test for visceral leishmaniasis

This test is also positive in other conditions with hypergammaglobulinemia like multiple myeloma and chronic liver disease. b. Tests that detect specific anti-Leishmania antibodies in serum: Various tests have been developed for detection of anti-Leishmania antibodies like complement fixation test, indirect immunofluorescence test, countercurrent immunoelectrophoresis, enzyme-linked immunosorbent assay, direct agglutination test, and latex agglutination test. In direct agglutination test (DAT), the antigen used is trypsin-treated promastigotes, which are fixed in formalin and stained with Coomasie brilliant blue. Patient’s serum is incubated with this antigen and agglutination is noted. Due to technical difficulties, the test is not widely employed in field conditions. A rapid and simple immunochromatographic strip test has been developed for diagnosis of VL. This test detects specific IgG anti-Leishmania antibodies against rk39 antigen. This test has been evaluated in field trials in India and is reported to have high sensitivity and specificity. Skin Test The Montenegro (or Leishmanin) skin test is based on delayed type of hypersensitivity reaction to Leishmaniasis. In this test, 0.5 ml of killed promastigotes are injected intradermally and the test is read after 72 hr. The test is

Cultures are usually made on Novy-McNeal Nicolle (NNN) medium. After inoculation with 1-2 drops of splenic or bone marrow aspirate, the culture is incubated at 22 to 28°C, and examined weekly for promastigote forms for upto 4 weeks. In vitro culture of infected tissue is not required for diagnosis in clinical practice. Parasite culture is necessary if other methods of diagnosis are negative in the presence of strong clinical suspicion, and to obtain sufficient quantity of antigen for direct agglutination test. Animal Inoculation Studies Laboratory animals (especially golden hamsters) are inoculated with infected tissue sample via intraperitoneal, intrasplenic, or intradermal route. In positive cases, parasite can be demonstrated in cutaneous lesions, liver, or spleen. The test becomes positive after several weeks or several months and therefore this test is not used for routine diagnosis. DNA Diagnosis Molecular methods based on polymerase chain reaction have been described for diagnosis of VL. In this test, DNA specific for Leishmania is identified. This test can be used for diagnosis, species identification, and in assessing response to treatment. Currently this test is largely a research tool.

BIBLIOGRAPHY 1. British Committee for Standards in Hematology. The laboratory diagnosis of malaria. Clin Lab Haem 1997; 19:165-70. 2. Cheesbrough M. District Laboratory Practice in Tropical Countries. 1998, Cambridge University Press. 3. Chatterjee KD. Parasitology (Protozoology and Helminthology). 9th ed. Calcutta. Published by the author, 1973. 4. Herwaldt BL. Leishmaniasis. Lancet 1999;354:1191-9. 5. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Hematology. (9th ed). London: Churchill Livingstone, 2001.

Diagnosis of Malaria and Other Parasites in Blood 6. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Reviews 2002;15:66-78. 7. Palmer CJ, et al. Evaluation of the OptiMal test for rapid diagnosis of P. vivax and P. falciparum malaria. J Clin microbial 1998;36:203-6. 8. Sundar S, Rai M. Laboratory diagnosis of visceral Leishmaniasis. Clinical and Diagnostic Laboratory Immunology 2002; 9:951-8. 9. Warhurst DC, Williams JE. Laboratory diagnosis of malaria. J Clin Pathol 1996;49:533-98. 10. Warrell DA, Gilles HM (Eds): Essential Malariology. (4th Ed). 2002. London. Arnold.

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11. Weil GJ, Lammie PJ, and Weiss N. The ICT Filariasis Test: A rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitology Today 1997;13: 401-4. 12. WHO information Fact sheets: The Leishmaniases and Leishmania/HIV co-infections. Fact sheet no. 116. Revised May 2000. Geneva. World Health Organization. 13. World Health Organization. New perspectives in malaria diagnosis. World Health Organization. 2000. Geneva, Switzerland. WHO/MAL/2000.1091. 14. World health Organization. Basic laboratory methods in medical parasitology. 1991. Geneva. World Health Organization.

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Laboratory Tests in Anemia

Anemia is defined as a reduction in hemoglobin concentration below the level, which is expected for healthy persons of same age and sex, and in the same environment. Adequate oxygen cannot be delivered to various organs and tissues due to low oxygen carrying capacity of blood. The normal hemoglobin ranges, as proposed by World Health Organization (WHO), are given at the end of this chapter under “Reference Ranges”. Anemia may occur without symptoms and may be detected incidentally during medical examination. When severe enough, clinical features due to anemia result from hypoxia such as fatigue, weakness, dizziness, fainting, and mental confusion. Pallor of skin, mucous membranes, and conjunctiva is present. Hyperdynamic

circulation causes palpitations and heart murmurs, and in severe cases congestive cardiac failure can develop, particularly in elderly. Anginal pain can result from myocardial hypoxia. Anemia is an objective sign of disease and needs further evaluation to determine the underlying cause and appropriate treatment.

CLASSIFICATION OF ANEMIAS There are two ways of classifying anemia: • Etiological classification • Morphological classification 1. Etiological classification: Anemia can result from a variety of causes (Table 27.1).

Table 27.1: Etiological classification of anemia I. Anemia due to decreased production of red blood cells • Nutritional deficiencies: Iron deficiency anemia, megaloblastic anemia due to deficiency of folate or vitamin B12 • Anemia of chronic disease • Sideroblastic anemia • Aplastic anemia • Anemia due to infiltration of bone marrow by malignant cells • Anemia of chronic renal failure II. Anemia due to increased destruction of red blood cells (Hemolytic anemia) A. Hereditary: • Defect in red cell membrane: Hereditary spherocytosis • Defect in hemoglobin: Sickle cell disease, thalassemia, hemoglobin E disease • Defect in red cell enzymes: Glucose-6-phosphate dehydrogenase deficiency B. Acquired: • Autoimmune hemolytic anemia • Paroxysmal nocturnal hemoglobinuria • Hemolytic transfusion reaction • Hemolytic disease of newborn • Mechanical hemolytic anemia • Hypersplenism • Malaria III. Anemia due to acute blood loss: Hemorrhage due to trauma, massive gastrointestinal bleeding, or child delivery.

Laboratory Tests in Anemia 2. Morphological classification: This classification is based on red cell size and hemoglobin content (red cell indices) in a case of anemia. This classification is given later in Table 27.16.

ANEMIAS DUE TO DECREASED PRODUCTION OF RED BLOOD CELLS Iron Deficiency Anemia Iron deficiency is the most common cause of anemia worldwide, and is a public health problem in developing countries. Pregnant women, women in reproductive age group, and children under 5 years of age are particularly susceptible for nutritional deficiency. In men and postmenopausal women, blood loss (especially from the gastrointestinal tract) is the main cause. Common causes are listed in Table 27.2. Table 27.2: Causes of iron deficiency anemia • Nutritional deficiency due to poor diet or increased requirements: infants and children (6 months-2 years), women in reproductive age group, pregnancy • Blood loss: a. Gastrointestinal: Esophageal varices, peptic ulcer, carcinoma of stomach, hookworm infestation, colorectal carcinoma, hemorrhoids b. Genitourinary tract: Menorrhagia, hematuria • Malabsorption: Celiac disease

Clinical Features

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Laboratory Features 1. Low hemoglobin and low packed cell volume 2. Low mean cell volume, mean cell hemoglobin, and mean cell hemoglobin concentration; red cell distribution width is increased. 3. Blood smear shows microcytic, hypochromic red cells and pencil cells (Fig. 27.1). 4. Serum ferritin is less than 15 μg/dl. The best determinant of storage iron is serum ferritin. Serum ferritin >100 μg/dl rules out diagnosis of iron deficiency in most cases. Serum ferritin is a storage form of iron and is reduced only in iron deficiency anemia. However, it is also an acute phase reactant. If iron deficiency anemia is associated with inflammatory, neoplastic, or liver disease, its concentration is raised; therefore, in these conditions, estimation of serum ferritin will not be helpful for diagnosis of iron deficiency. In the presence of an inflammatory disorder, assay for soluble transferrin receptor is preferable for diagnosis as it is not affected by inflammation. 5. Serum iron, total iron binding capacity (TIBC), and transferrin saturation: Serum iron is a measure of iron bound to transferrin (transport protein for iron). Serum iron level is affected by diurnal variation, ironcontaining medications, hemolysis, etc. Total iron binding capacity refers to the iron-binding sites of all the circulating transferrin. Transferrin saturation is the ratio of serum iron to transferrin. Typically, serum iron is low, TIBC is increased, and transferrin

Apart from nonspecific clinical manifestations of anemia, iron deficiency can cause koilonychia (flattened or spoonshaped nails), angular stomatitis, and glossitis. Stages of Iron Deficiency There are three stages of iron deficiency as shown in Box 27.1. Box 27.1: Stages of iron deficiency • Stage 1 (Iron depletion): Depletion of storage iron (low serum ferritin), normal transport iron (serum iron, total iron binding capacity), normal hemoglobin • Stage 2 (Low transport iron): Depletion of storage iron, low transport iron, normal hemoglobin • Stage 3: (Low hemoglobin production): Depletion of storage iron, low transport iron, low hemoglobin

Fig. 27.1: Blood smear in iron deficiency anemia showing microcytic hypochromic red cells and “pencil” cells

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saturation is 100 fl in adults). Elevation of mean cell volume is an early sign and precedes the onset of anemia. Pancytopenia is common. • Blood smear shows oval macrocytosis, basophilic stippling, Howell-Jolly bodies, and hypersegmentation of neutrophils (>5% of neutrophils showing 5 or more lobes) (Fig. 27.2). • Reticulocyte count is normal or low. • Bone marrow shows megaloblasts, erythroid hyperplasia, and giant metamyelocytes and bands (Fig. 27.3). • Vitamin assays: See Table 27.5. Identification of cause It is necessary to distinguish between folate and vitamin B12 deficiency and to determine the cause for appropriate treatment

Fig. 27.2: Blood smear in megaloblastic anemia showing oval macrocytes and a hypersegmented neutrophil

Fig. 27.3: Bone marrow smear showing megaloblasts and a giant metamyelocyte

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Fig. 27.4 Evaluation of suspected megaloblastic anemia. (*: Measurement of serum methylmalonic acid is said to be more sensitive than measurement of vitamin B12 and an earlier marker for detection of vitamin B12 deficiency)

Table 27.5: Differences between folate and vitamin B12 deficiency Parameter 1. 2. 3. 4. 5. 6. 7. 8.

Usual mechanism of deficiency Neurologic features Serum vitamin B12 Serum folate Red cell folate Serum homocysteine Serum methylmalonic acid Therapeutic trial

Folate deficiency

Vitamin B12 deficiency

Inadequate intake Absent Normal Low Low Increased Normal Optimal response to folate

Inadequate absorption May be present Low Normal or increased Low Increased Increased Optimal response to vitamin B12

(Table 27.5, and Fig. 27.4). Treatment only with folate in vitamin B12 deficiency can worsen the neurological abnormalities. In vitamin B 12 deficiency, test for vitamin B 12 absorption (Schilling test) can be carried out. In this test, urinary excretion of oral dose of radio-labeled vitamin B 12 is compared with the excretion of oral dose of radiolabeled vitamin B12 bound to intrinsic factor. In pernicious anemia, deficient absorption is corrected with addition of intrinsic factor.

Anemia of Chronic Disease Anemia of chronic disease is the most common form of anemia amongst hospitalized patients. The three disease categories associated with anemia of chronic disease are chronic infection, inflammation, and malignancy (Table 27.6). There is a block in release of storage iron from macrophages for erythropoiesis that is mediated by inflammatory cytokines. Anemia is mild (9-10 gm/dl) and non-progressive. Clinical features reflect underlying disease. The main differential diagnosis is iron deficiency anemia.

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Table 27.6: Diseases associated with anemia of chronic disease 1.

Chronic infections: Tuberculosis, urinary tract infection, bronchiectasis, osteomyelitis, subacute bacterial endocarditis

2.

Chronic inflammation: Rheumatoid arthritis, systemic lupus erythematosus.

3.

Malignancy

Laboratory Features • Normocytic normochromic anemia (70% cases) or microcytic hypochromic anemia (30% cases). • Decreased serum iron, decreased total iron binding capacity, and normal or raised serum ferritin. Serum transferrin receptor level is normal. • Increased marrow storage iron • Erythrocyte sedimentation rate is increased out of proportion to the degree of anemia. Sideroblastic Anemia In sideroblastic anemia, heme synthesis is deficient. There is a mitochondrial defect that leads to the failure of incorporation of iron into heme. Iron accumulates in mitochondria that surround the nucleus of erythroblasts forming ringed sideroblasts. Sideroblastic anemia is characterized by: • Dimorphic anemia (i.e. blood smear shows dual population of cells: normocytic normochromic, and microcytic hypochromic) (Fig. 27.5). • Ringed sideroblasts in bone marrow (see Fig. 27.5).

Sideroblastic anemia may be hereditary (X-linked) or acquired. Most cases are acquired and causes include: (i) drugs: isoniazid, chloramphenicol, cytotoxic drugs, (ii) alcoholism, (iii) lead poisoning, (iv) myelodysplastic syndrome, and (v) acute myeloid leukemia. Aplastic Anemia Aplastic anemia is characterized by pancytopenia in peripheral blood (reduction of red cells, leukocytes, and platelets in peripheral blood) and decreased cellularity in bone marrow. Aplastic anemia can occur at any age and clinical presentation is related to pancytopenia (anemia, risk of infections, and bleeding manifestations). Organomegaly is typically absent. Causes of aplastic anemia are listed in Table 27.7. Investigations in a Suspected Case of Aplastic Anemia • Tests for confirmation of aplastic anemia: Complete blood count (to demonstrate pancytopenia, low hemoglobin, and low reticulocyte count) and bone marrow examination (to demonstrate depletion of hematopoietic precursors and hypocellularity). Table 27.7: Causes of aplastic anemia Acquired Idiopathic Drugs: Idiosyncratic: antibacterials (chloramphenicol, sulfonamides), nonsteroidal anti-inflammatory drugs (phenylbutazone, indomethacin, piroxicam, diclofenac), antithyroid drugs, furosemide, phenothiazines, allopurinol, oral antidiabetics, Dose-related: Cytotoxic drugs Chemicals (e.g. benzene) or radiation Infections: Hepatitis; Epstain Barr virus, Mycobacteria Paroxysmal nocturnal hemoglobinuria Systemic lupus erythematosus Graft vs. host disease

Fig. 27.5: Blood smear (on left) in sideroblastic anemia showing dimorphic red cells, basophilic stippling, and a polychromatic red cell. Bone marrow smear stained with iron stain (on right) shows ringed sideroblasts

Inherited Fanconi’s anemia, Dyskeratosis congenita

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• Tests for diagnosis of underlying cause: Viral studies (hepatitis A antibody, hepatitis B surface antigen, hepatitis C antibody), Ham’s test and/or flow cytometry for lack of CD55 and CD59 (for paroxysmal nocturnal hemoglobinuria), detailed drug history, antinuclear antibody test (for systemic lupus erythematosus), and cytogenetic analysis (for Fanconi anemia). Etiology remains unknown in 50% cases of aplastic anemia. Severity of Aplastic Anemia 1. Severe aplastic anemia: Aplastic anemia is considered to be severe when following features are present (Camitta et al, 1976): • Bone marrow cellularity is either < 25% of normal or is 25-50% of normal with 400) biochemical variants of G6PD enzyme have been reported due to point mutations or deletions in the gene. In India, G6PD Mediterranean, G6PD KeralaKalyan, and G6PD Orissa variants are more common. G6PD deficiency leads to inability of red cells to remove toxic hydrogen peroxide (H2O2), an oxidative metabolite. Accumulated H2 O2 leads to oxidation of hemoglobin with subsequent denaturation and precipitation of globin chains. Precipitated globin form inclusions attached to red cell membrane (Heinz bodies). Red cells containing Heinz bodies are destroyed in spleen (extravascular hemolysis). Oxidant damage also causes peroxidation of membrane lipids of red cells leading to intravascular hemolysis. G6PD deficiency is usually asymptomatic. It can cause (i) neonatal jaundice, (ii) acute hemolytic anemia on exposure to oxidant stress (Table 27.12), or (iii) chronic hemolytic anemia. Examination of blood smear during hemolytic episode shows polychromasia, fragmented red cells, spherocytes, bite cells (red cells having bitten out margins) and half-ghost cells (one half of the red cell is empty and the other half is filled with hemoglobin) (Fig. 27.11). Biochemical abnormalities include increased serum bilirubin, hemoglobinemia, and hemoglobinuria

Table 27.12: Causes of hemolysis in glucose-6phosphate dehydrogenase deficiency 1. Bacterial and viral infections 2. Drugs: • Antimalarials: Primaquine, pamaquine • Antibacterials: Sulfonamides, nalidixic acid, nitrofurantoin, dapsone • Antipyretics and analgesics 3. Chemicals: Naphthalene balls

Fig. 27.11: Blood smear during acute hemolysis in glucose 6 phosphate dehydrogenase deficiency showing a bite cell and hemi-ghost cells

(due to intravascular hemolysis). Heinz bodies can be demonstrated by methyl violet staining. The screening tests used for G6PD deficiency are fluorescent spot test, methemoglobin reduction test, and dye decolorization test.

IMMUNE HEMOLYTIC ANEMIAS Immunological destruction of red cells occurs when antibody and/or complement bind to red cell membrane. Hemolysis may be extra- or intravascular. Classification of immune hemolytic anemias is shown in Table 27.13. Warm antibody type autoimmune hemolytic anemia occurs mainly in persons over 50 years of age. Mild jaundice and splenomegaly are common. Red cells coated with IgG are recognized by Fc receptors on macrophages and phagocytosed in spleen. In many cases, phagocytosis is incomplete with formation of spherocytes (Fig. 27.12). Red cells coated with IgG are detected by direct antiglobulin test (Fig. 27.34, later). Cold agglutinin disease is characterized by acrocyanosis (cyanosis of fingers, toes, nose, and ears) due to the presence of cold agglutinins that cause agglutination of erythrocytes on exposure to cold. The antibody is IgM, cold-reactive, and after binding to red cells activates complement. The disease usually occurs in older individuals. Blood smear shows autoagglutination of red cells (Fig. 27.13); preparation of blood smear after

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Essentials of Clinical Pathology Table 27.13: Classification of immune hemolytic anemias

1. Autoimmune hemolytic anemia • Warm antibody type – Primary or idiopathic – Secondary: infections, autoimmune disorders, lymphoma, chronic lymphocytic leukemia • Cold antibody type – Cold agglutinin disease – Paroxysmal cold hemoglobinuria 2. Alloimmune hemolytic anemia • Hemolytic disease of newborn • Hemolytic transfusion reaction 3. Drug-induced hemolytic anemia

Fig. 27.12: Blood smear in warm antibody type autoimmune hemolytic anemia showing spherocytes, polychromasia, and late normoblast

Fig. 27.13: Blood smear in cold agglutinin disease showing large clusters of red cells (autoagglutination)

warming of blood sample causes disappearance of autoagglutination. Direct antiglobulin test is positive due to the presence of complement on red cells.

case of anti-D, mother is Rh D-negative and father is Rh D-positive. Rh HDN develops during second or subsequent pregnancies if the fetus is Rh D-positive. Clinical presentation is variable. Rh HDN may manifest with (i) mild anemia and jaundice, (ii) icterus gravis neonatorum with kernicterus, or (iii) hydrops fetalis with intrauterine fetal death. The usual clinical presentation of ABO HDN is mild anemia and jaundice.

HEMOLYTIC DISEASE OF NEWBORN Hemolytic disease of newborn (HDN) is characterized by destruction of red cells of the fetus or neonate due to antibodies produced by the mother. Allo-antibodies develop in the mother against foreign red cell antigens of the fetus inherited from the father. Leakage of fetal red cells during pregnancy or delivery into the maternal circulation stimulates formation of antibodies. Passage of IgG maternal antibodies across placenta into the fetal circulation causes hemolysis of fetal red cells. The two main red cell antigens responsible for HDN are Rh and ABO. In Rh HDN, antibodies develop against anti-D and less commonly against anti-C or anti-E. In

Antenatal investigations consist of: 1. Maternal: These are ABO and Rh grouping, antibody screening, antibody identification, and antibody titration: If antibody is clinically significant, titer is determined. Titer >1:32 or a rising titer showing increase of 2 dilutions or more is significant and amniocentesis should be performed to determine severity of disease (Fig. 27.14).

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Fig. 27.14: Approach to the management of hemolytic disease of newborn

2. Fetal: This is amniocentesis or cordocentesis for assessment of severity of hemolysis by determining bilirubin level. Liley’s graph is used for predicting severity of HDN and management of the fetus. Liley’s graph plots degree of absorption at 450 nm versus gestational age in weeks on a semilogarithmic graph paper. After delivery, Kleihauer-Betke acid elution test (Fig. 27.15) is used to identify number of fetal red cells in maternal circulation, and calculate the number of vials of Rh immune globulin to be infused to prevent maternal immunization (Fig. 27.16). Investigations in newborn consist of: 1. Blood grouping 2. Blood smear (Fig. 27.17) 3. Direct antiglobulin test (DAT) on cord red cells: Positive DAT (Fig. 27.34, later) can result from coating of red cells of the newborn with immune anti-A or anti-B from group O mother, or immune anti-D from Rh-negative mother. 4. Estimation of bilirubin and hemoglobin Differences between Rh and ABO HDN are presented in Table 27.14.

Fig. 27.15: Acid elution or Kleihauer-Betke test. Maternal blood sample is withdrawn within 2 hours of birth, a blood smear is prepared and flooded with acid. Hemoglobin in adult red cells (HbA) is washed out by the acid solution, while hemoglobin in fetal red cells (HbF) is not. After counterstaining with safranin, red cells containing HbA appear pale, while cells containing HbF appear dark. Acid elution test is performed to assess amount of fetomaternal hemorrhage and to calculate the dose of Rh immune globulin

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Fig. 27.16: Use of Kleihauer-Betke test to determine the dose of anti-D following delivery by a Rh D-negative woman of a Rh D-positive baby. The maternal sample should be taken within 2 hours of delivery or any other sensitizing event

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired clonal stem cell disorder in which red cells are abnormally sensitive to the hemolytic action of the complement. PNH red cells are deficient in several cell membrane proteins including CD 55 (decay accelerating factor) and CD 59 (membrane inhibitor of reactive lysis or MIRL). Normally, small amount of complement is being continuously generated via alternate complement pathway. CD 55 inactivates C3 convertase while CD 59 inhibits the formation of membrane attack complex and protects the red cells against complement-mediated attack. In a typical case of PNH, red cell destruction occurs at night so that hemoglobinuria (passage of reddishbrown urine) is noticed after getting up in the morning. However, presentation is often variable and may be in the form of pancytopenia, aplastic anemia, chronic intravascular hemolysis, or recurrent venous thromboses. Some cases of PNH evolve to acute myeloid leukemia. Laboratory diagnosis of PNH is based on: • Evidence of intravascular hemolysis: Hemoglobinuria, hemosiderinuria, methemalbuminemia, and increased indirect serum bilirubin.

Fig. 27.17: Blood smear in Rh hemolytic disease of newborn showing erythroblasts and polychromatic cells

• Demonstration of increased sensitivity of red cells to complement: Sucrose hemolysis test, Ham’s test. • Flow cytometric analysis of blood cells for lack of CD55 or CD59.

MICROANGIOPATHIC HEMOLYTIC ANEMIA This is a form of mechanical hemolytic anemia resulting from intravascular fragmentation and lysis of red cells

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Table 27.14: Comparison of Rh and ABO hemolytic disease of newborn Parameter

Rh HDN

ABO HDN

1. 2. 3. 4. 5. 6. 7. 8. 9.

D-negative D-positive Second or subsequent Common Common Usual Erythroblastosis Strongly positive Rh immune globulin

O A or B First Uncommon Uncommon Rare Spherocytes Weakly positive or negative None

Blood group of mother Blood group of fetus Pregnancy usually affected Hydrops fetalis Stillbirth Severe anemia Blood smear Direct antiglobulin test Prevention

diagnosis of anemia are estimation of hemoglobin and packed cell volume. Estimation of Hemoglobin

Fig. 27.18: Blood smear in microangiopathic hemolytic anemia showing many fragmented red cells

due to vascular endothelial abnormalities. Common causes include thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, disseminated intravascular coagulation, eclampsia, disseminated malignancy, and generalized vasculitis. Blood film shows numerous fragmented red cells (schistocytes) (Fig. 27.18).

APPROACH TO DIAGNOSIS OF ANEMIA Evaluation of a case of anemia consists of: • Establishing the presence and severity of anemia, and • Determining the cause of anemia. Establishing the Presence and Severity of Anemia Clinical features of anemia are non-specific and their assessment is often subjective. Laboratory methods for

Cyanmethemoglobin method is the method of choice. Recently introduced WHO hemoglobin color scale is a simple and inexpensive method and well suited for under-resourced laboratories (see Chapter 18: Estimation of Hemoglobin). Depending on hemoglobin concentration, anemia is graded as mild (hemoglobin less than lower limit of normal but above 10.0 gm/dl), moderate (hemoglobin 7-10 gm/dl), and severe (hemoglobin < 7 gm/dl). Measured hemoglobin level depends on amount of hemoglobin in red blood cells and blood volume. Pseudoanemia or spurious anemia results from relative increase in plasma volume in pregnancy, splenomegaly, congestive cardiac failure, and paraproteinemias. Therefore, hemoglobin level should be interpreted in the light of clinical features. Estimation of Packed Cell Volume (PCV) PCV is the volume of packed red cells obtained after centrifugation of a sample of anticoagulated blood in a Wintrobe tube or a capillary tube (see Chapter 19 : Packed Cell Volume). Hematocrit is the equivalent measurement derived on automated blood cell analyzer from the red cell indices. Although not entirely synonymous, the terms PCV and hematocrit are used interchangeably in clinical practice. PCV is about three times the value of hemoglobin concentration and thus can be used to cross-check the hemoglobin value. Sometimes, additional information can be gained from the observation of color of plasma and thickness of

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buffy coat layer. Normal plasma is straw-colored; it is colorless in iron deficiency anemia, pink in hemoglobinemia, and yellow in hemolysis. A thick buffy coat indicates leukocytosis and/or thrombocytosis. Determining the Cause of Anemia Determining the cause of anemia depends on evaluation of clinical and laboratory studies (Fig. 27.19). Evaluation of Clinical Features in Anemia Clinical features in a case of anemia depend on (i) severity of anemia, (ii) cause of anemia, and (iii) rapidity of development of anemia. Anemia may be acute or chronic. Acute anemia may be due to either acute blood loss or acute hemolysis, and symptoms are often due to loss of circulatory volume. Chronic anemia is tolerated well due to compensatory mechanisms. Chronic anemia may be symptomatic or may be detected incidentally during the course of investigations for some other disease. Clinical features, which are related to the cause of anemia, are shown in Table 27.15.

Fig. 27.19: Approach to diagnosis of anemia

Basic Laboratory Studies in Anemia Peripheral blood smear: In majority of cases, if correlated with clinical features, blood smear findings suggest the

Table 27.15: Clinical features and the cause of anemia Clinical feature

Probable cause of anemia

1. Pregnancy, females of reproductive age group, growing children 2. Chronic blood loss 3. Chronic alcoholism

Nutritional deficiency

4. Ingestion of drugs: • Prolonged intake of aspirin • Cytotoxic drugs • Alpha methyl dopa, rifampicin 5. Family history of anemia, jaundice, gallstones, or splenectomy 6. Organomegaly (hepatomegaly, splenomegaly, lymphadenopathy) 7. Community: • Sindhis, Lohanas • Parsees, Vataliya Prajapatis • Tribal groups 8. Deformities of skull and facial bones like malar prominence, frontal bossing, depressed bridge of nose

Iron deficiency Folate deficiency, gastrointestinal blood loss, sideroblastic anemia Iron deficiency from gastric bleeding Aplastic anemia Autoimmune hemolytic anemia Inherited hemolytic anemia Hematologic malignancy

Thalassemia Glucose 6 phosphate dehydrogenase deficiency Sickle cell disease Thalassemia

Laboratory Tests in Anemia probable cause of anemia. Apart from morphological type of anemia and abnormality of red cells, blood smear also provides information regarding disorders of white cells (e.g. leukemias), and platelets (thrombocytopenia). (See Chapter 22: Blood Smear). Reticulocyte count: It is a measure of the capacity of the bone marrow to produce red cells. Reticulocyte count is mainly useful for diagnosis of anemia due to decreased red cell production or ineffective erythropoiesis (in which reticulocyte count is low). Increased reticulocyte count appropriate for degree of anemia indicates effective red cell production. Causes of low reticulocyte count are megaloblastic anemia, aplastic anemia, bone marrow infiltration, and alcoholism. Causes of increased reticulocyte count are response to hematinic therapy in nutritional anemia, hemolysis, and bleeding (see Chapter 21: Reticulocyte Count). If reticulocyte count is increased, then tests for hypoproliferative anemias are generally not required (such as iron studies, vitamin B12 and folate assays, bone marrow examination).

261

Red cell indices: Among the red cell indices, mean cell volume is the most useful for classification of anemia into normocytic, microcytic, and macrocytic types (Table 27.16). Red cell distribution width is used to distinguish between iron deficiency anemia and thalassemia (see Chapter 23: Red Cell Indices). Microcytic Hypochromic Anemia An approach for evaluation for microcytic hypochromic anemia is shown in Figure 27.20. The distinction between different causes of microcytic anemia is shown in Table 27.3 earlier. Macrocytic Anemias Evaluation of macrocytic anemia is presented in Figure 27.21. Normocytic Normochromic Anemia Evaluation of normocytic anemia is shown in Figure 27.22.

Table 27.16: Morphological classification of anemia Microcytic hypochromic anemia (MCV < 80 fl; MCHC < 30 gm/dl) • • • •

Iron deficiency anemia Thalassemia Anemia of chronic disease Sideroblastic anemia Macrocytic anemia* (MCV > 100 fl; MCHC 30-35 gm/dl)

• Megaloblastic – Folate or vitamin B12 deficiency

• Non-megaloblastic – Alcoholism – Liver disease – Hemolytic anemia – Myelodysplastic syndrome – Hypothyroidism

Normocytic and normochromic anemia (MCV 80-100 fl; MCHC 30-35 gm/dl) • Reticulocyte count increased – Recent blood loss – Hemolysis

• Reticulocyte count low – Aplastic anemia – Chronic renal failure – Anemia of chronic disease – Anemia due to infiltration of marrow

*Another way of classification of macrocytic anemia: (1) Round macrocytosis: alcoholism, liver disease, hypothyroidism; and (2) oval macrocytosis: folate or vitamin B12 deficiency, myelodysplastic syndrome. Abbreviations: MCV: mean cell volume; MCHC: mean cell hemoglobin concentration

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Fig. 27.20: Evaluation of microcytic hypochromic anemia

Fig. 27.21: Evaluation of macrocytic anemia

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263

Fig. 27.22: Evaluation of normocytic normochromic anemia

Hemolytic Anemia Increased rate of red cell destruction which cannot be compensated by increased red cell production by the bone marrow leads to hemolytic anemia. Normally, red cells have a life span of about 120 days after which they are destroyed. In hemolytic anemia, red cell life-span is shortened. Causes of hemolytic anemia are listed earlier in Table 27.1. Apart from general features of anemia, hemolytic anemia may manifest clinically with jaundice, and in chronic cases with gallstones and splenomegaly. Family history may be positive in hereditary hemolytic anemias, and occurrence in a particular ethnic group may be suggestive of a particular disorder (e.g. thalassemia, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency). In acquired hemolytic anemias, evidence of an underlying disorder may be present, e.g. malaria, septicemia, drug ingestion, lymphoproliferative disorder, connective tissue disease, disseminated cancer, etc.

Approach to the diagnosis of hemolytic anemia consists of determining (i) presence of hemolysis, (ii) whether hemolysis is intravascular or extravascular, and (iii) cause of hemolysis. 1. Establishing the presence of hemolysis: Laboratory features of excessive red cell destruction are: • Increased indirect serum bilirubin • Increased urobilinogen in urine • Decreased or absent plasma haptoglobin (protein that binds free hemoglobin in plasma) • Appearance of free hemoglobin in plasma or urine • Increased serum lactate dehydrogenase Laboratory features of increased red cell production by the bone marrow (in response to hemolysis) are: • Increased reticulocyte count • Polychromatic cells and nucleated red cells on blood smear • Erythroid hyperplasia in bone marrow.

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The three typical laboratory features indicative of hemolytic anemia are low hemoglobin, increased reticulocyte count, and raised indirect serum bilirubin. 2. Whether hemolysis is intravascular or extravascular: In intravascular hemolysis, red cell destruction occurs within circulation (Fig. 27.23), while in extravascular hemolysis, red cells are destroyed by macrophages of the reticuloendothelial system in spleen and liver (Fig. 27.24). Main causes of intravascular hemolysis are hemolytic transfusion reaction, glucose-6-phosphate dehydrogenase deficiency, blackwater fever in falciparum malaria, septicemia, autoimmune hemolytic anemia (some types), and paroxysmal nocturnal hemoglobinuria. Laboratory features of intravascular hemolysis are: • Hemoglobinemia • Decreased or absent plasma haptoglobin • Hemosiderinuria • Methemalbuminemia: Methemalbumin has a characteristic absorption band at 558 nm (Schumm’s test).

In extravascular hemolysis, unconjugated bilirubin in serum and urobilinogen in urine are increased. However, hemoglobinemia, hemoglobinuria, hemosiderinuria, and methemalbuminemia are absent. 3. Defining cause of hemolysis: Usually, clinical features and morphology of red cells on blood smear suggest the probable cause of hemolytic anemia (Fig. 27.25). Specific laboratory tests are then carried out for definitive diagnosis. Specific laboratory studies to establish the cause of hemolytic anemia are: a. Tests in hereditary hemolytic anemias: • Hemoglobin disorders: sickling test, hemoglobin electrophoresis, high performance liquid chromatography, estimation of hemoglobin A2, estimation of hemoglobin F, red cell inclusions. • Red cell enzyme defects: test for glucose-6phosphate dehydrogenase deficiency • Red cell membrane disorders: osmotic fragility test, autohemolysis test

Fig. 27.23: Catabolism of hemoglobin following intravascular hemolysis.

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265

b. Tests in acquired hemolytic anemias: • Immune hemolytic anemias: antiglobulin (Coombs’) test • Paroxysmal nocturnal hemoglobinuria: sucrose hemolysis test, Ham’s acidified serum lysis test, flow cytometric analysis. Specific Laboratory Studies in Anemia Vitamin B12 and Folate Vitamin B12 and folate are measured in patients with (i) MCV>100 fl (macrocytosis), (ii) macrocytic anemia, or (iii) neurological and psychiatric abnormalities. Some investigators also recommend measurement of serum methylmalonic acid and serum homocysteine due to interpretive difficulties associated with vitamin assays. Iron Studies

Fig. 27.24: Catabolism of hemoglobin following extravascular hemolysis

Iron studies for diagnosis of iron deficiency anemia include serum iron, total iron binding capacity, transferrin saturation, and serum ferritin. Before testing, causes of iron deficiency and presence of any chronic disease should be assessed. Serum ferritin is superior to other tests for diagnosis of iron deficiency (< 15 μg/dl is diagnostic of iron deficiency, while >100 μg/dl excludes iron deficiency).

Fig. 27.25: Evaluation of hemolytic anemia

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Bone Marrow Examination If facilities for iron studies and vitamin assays are not available, examination of bone marrow is indicated to (i) distinguish between causes of microcytic hypochromic anemia by assessing storage iron and ringed sideroblasts, and (ii) distinguish between megaloblastic and nonmegaloblastic macrocytosis. Bone marrow biopsy is essential for diagnosis of aplastic anemia. Tests for Hemoglobin S Two tests are available for detection of Hb S: • Sickle cell slide test • Solubility test for hemoglobin S 1. Sickle cell slide test: When red cells containing Hb S are deprived of oxygen, they become sickle-shaped. Reducing agent that is used to remove oxygen from red cells is 2% sodium metabisulphite. A drop of capillary or anticoagulated venous blood is mixed on a glass slide with a drop of 2% sodium metabisulphite. A coverslip is placed over the mixture and sealed with petroleum jelly-paraffin wax. The preparation is examined under the microscope after 30 minutes. If sickle cells are not seen, examine the slide again after 2 hours, and 24 hours. The test is reported as negative if the red cells remain round, and as positive if red cells become sickle shaped (crescentshaped with pointed ends) or holly-leaf shaped (Fig. 27.26).

Fig. 27.26: Sickle cell slide test

A positive test indicates presence of Hb S. The test cannot differentiate between sickle cell trait and sickle cell disease. However, if the test is positive, then on the basis of blood smear examination, a probable diagnosis of sickle cell trait (target cells) or sickle cell disease (presence of sickle cells and target cells) may be suggested. It is necessary to perform hemoglobin electrophoresis for confirmation of Hb S and to differentiate between various sickle cell disorders (sickle cell trait, sickle cell anemia, sickle cell-β thalassemia, sickle cell-DPunjab disease, etc.). False-negative test can occur if the reagent is outdated or not freshly prepared, concentration of Hb S is low (e.g. in infants below 6 months, following recent blood transfusion), or if there is severe anemia. False-positive test can occur if the concentration of sodium metabisulphite is excessive or if there is drying of the wet preparation. 2. Solubility test for hemoglobin S: In this test, anticoagulated blood is added to the reagent solution consisting of phosphate buffer, saponin, and sodium dithionite. Red cells are hemolyzed and Hb S, if present, is reduced by dithionite. Hb S forms tactoids which refract light and the solution appears turbid (Fig. 27.27). The solution remains clear with other hemoglobins like Hb A, Hb F, Hb C, Hb D, Hb G, and Hb O-Arab. The test cannot differentiate between various sickle cell disorders. Causes of false-positive test include polycythemia, marked leukocytosis, paraproteinemias, and hyperlipidemia.

Fig. 27.27: Solubility test for sickle hemoglobin. In a negative test, black lines kept behind the test tube are visible. In a positive test, black lines are not visible because the solution becomes turbid

Laboratory Tests in Anemia Causes of false-negative test are old or outdated reagent, low concentration of Hb S (e.g. infants 80% cells monocytic

NSE+

7. AML M6

>20% of nonerythroid cells are myeloblasts; >50 of all nucleated cells are erythroblasts; marked dyserythropoiesis >20% of blasts that may be small, lymphoblast-like or large with fine chromatin and prominent nucleoli and cytoplasmic blebs

PAS+

8. AML M7

Platelet peroxidase+ (EM)

t (9;22)

CD41+, CD61+, HLADR+

MPO: Myeloperoxidase; EM: Electron microscopy; Type I blasts: No azurophil granules in cytoplasm; Type II blasts: Few azurophil granules; NSE: Non-specific esterase; PAS: Periodic Acid Schiff; TLC: Total leucocyte count

and immunophenotyping. The important cytochemical studies in acute leukemias are (Fig. 28.3): • Myeloperoxidase (identifies an enzyme present in primary granules of granulocytic cells) • Non-specific esterase (identifies an enzyme present in large amounts in monocytic cells) • Periodic acid Schiff (identifies intracellular glycogen). Myeloperoxidase (MPO) • MPO enzyme is located in primary (azurophil) and secondary granules in all stages of neutrophil series (Fig. 28.4).

• MPO stain is positive in AML M1, AML M2, AML M3, and AML M4 (only in myeloblasts). In AML M0, MPO activity is visible only by electron microscopy. • Lymphoblasts are negative for MPO. Nonspecific Esterase (NSE) NSE activity is strongly positive in monocytic series (monoblasts, promonocytes, and monocytes). It allows diagnosis of AML M4 and AML M5 (Fig. 28.5). Sodium fluoride may be added that renders monocytic cells negative.

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277

Fig. 28.1: (A) AML M0; (B) AML M1; (C) AML M2; (D) AML M3; (E) AML M4; (F) AML M5; (G) AML M6; (H) AML M7

Fig. 28.2: (A) ALL L1; (B) ALL L2; (C) ALL L3

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Essentials of Clinical Pathology Table 28.5: Differences between acute lymphoblastic leukemia and acute myeloid leukemia

Parameter

Acute lymphoblastic leukemia

Acute myeloid leukemia

1. Predominant age

Children (peak 3-4 years)

Adults

2. Morphology of blasts • Size of blasts • Cytoplasm • Granules in cytoplasm • Nuclear chromatin • Nucleoli • Auer rods

Small Scanty Absent Coarse 0-2 Absent

Large Moderate May be present Fine >3 Pathognomonic, if present

3. Cytochemistry • Myeloperoxidase • Periodic acid Schiff

Negative Block-like

Positive Diffuse

4. Immunophenotyping

B or T lymphoid markers

Myeloid markers

5. Prognosis

Curable in majority of children

Curable in minority of adults

Fig. 28.3: Principle cytochemical reactions in acute leukemias

Periodic Acid Schiff (PAS) • PAS reagent stains glycogen in the cytoplasm. • Lymphoblasts show block-like positivity (Fig. 28.6). • PAS is positive in 70% of ALL L1 and ALL L2 cases. Lymphoblasts in ALL L3 are negative. Another stain that may be used is Sudan black B that dentifies phospholipids and other neutral fats in membranes of both primary and specific granules in granulocytes.

3. Immunophenotyping: This technique consists of identification of antigens present on leukemic cells in blood or bone marrow with fluorescently-labeled monoclonal antibodies. As blood and bone marrow cells are in fluid suspension, flow cytometery is the method of choice. Cell surface antigens are named according to the cluster of differentiation (CD) system. Specific antigens are expressed on cells of different lineages at different stages of development. A panel

Laboratory Tests in Hematological Malignancies

Fig. 28.4: Myeloperoxidase (MPO) stain. MPO is a marker for primary azurophil granules and is the routine initial cytochemical stain in all acute leukemias. Dark brown granules in the cytoplasm of blasts differentiate AML from ALL. However, negative MPO reaction does not exclude AML

Fig. 28.5: Nonspecific esterase reaction. This stain is used for demonstration of monocytic differentiation in acute myeloid leukemia

consisting of a combination of specific antibodies is employed to determine the immunophenotype and diagnostic classification of leukemias. It is especially

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Fig. 28.6: Periodic acid Schiff reaction. Coarsely granular (‘block-like’) perinuclear staining pattern is observed in some cases of acute lymphoblastic leukemia. Negative PAS staining is not helpful for differentiating AML from ALL

helpful if morphology and cytochemistry are ambiguous. A large number of antigens can be assessed simultaneously on a single specimen. The immunologic profiles of cell lineages are follows: • Primitive stem cell markers: CD34, CD117, TdT • Myeloid lineage: CD13, CD33, cytoplasmic myeloperoxidase • Monocytic lineage: CD14, CD64 • Erythroid lineage: Glycophorin A • Megakaryocytic lineage: CD41, CD42, CD61 • B-ALL: CD19, CD20, CD21, CD22, CD79a, CD10 (common ALL antigen or CALLA), cytoplasmic μ chain, surface immunoglobulin • T-ALL: CD2, CD3, CD5, CD7, cytoplasmic CD3. For further subclassification of B-ALL (into pro-B ALL, common ALL, Pre-B ALL, mature B ALL) or TALL (into pro-T ALL, pre-T ALL, cortical T ALL, mature T ALL), another set of antibodies is needed. Terminal deoxynucleotidyl transferase (TdT) is positive in all forms of ALL except L3 type. Immunophenotyping is essential for diagnosis of AML M0 in which myeloperoxidase is negative on light microscopy, myeloid-specific antigens (CD13, CD33, cytoplasmic myeloperoxidase, CD117) are positive, and T or B lymphoid markers are absent. CD14 and antilysozyme are strongly expressed in AML with significant

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monocytic component (AML M4 and M5). In acute erythroleukemia (AML M6), glycophorin A and in acute megakaryocytic leukemia (AML M7), platelet glycoprotein antigens (CD41, CD42, and CD61) are demonstrated. Immunophenotyping is also used for detection of minimal residual disease. Detection of minimal residual disease (MRD) consists of identification of a small population of leukemic cells among a large population of normal cells after therapy to determine whether residual disease is present that may later cause a relapse. MRD can also be detected by morphology, cytogenetic analysis, and molecular analysis. 4. Cytogenetic analysis: Structural or numerical abnormalities of chromosomes are detected by cytogenetic analysis or karyotyping (Table 28.6). With cytogenetic analysis, a variety of gross alterations can be detected such as translocations, deletions, and duplications. In contrast to molecular methods, cytogenetic analysis is more widely available. In acute leukemias, cytogenetic abnormalities are linked to the pathogenesis of the disease. Applications of cytogenetic analysis in acute leukemia are • Diagnosis of specific types of AML: WHO classification of AML recognizes distinct entities, which have clonal, recurrent cytogenetic abnormalities. Their identification is necessary as they have a correlation with response to therapy. • Prediction of prognosis: Certain cytogenetic abnormalities are associated with poor prognosis and their detection may affect treatment strategies. Table 28.6: Chromosomal abnormalities in acute leukemias Chromosomal abnormality

Prognosis

Acute lymphoblastic leukemia 1. 2. 3. 4. 5. 6.

t(9;22)(q34;q11.2) t(4;11)(q21;q23) t(1;19)(q23;p13.3) t(12;21)(p13;q22) Hyperdiploidy>50 Hypodiploidy

Poor Poor Average Good Good Poor

Acute myeloid leukemia 1. 2. 3. 4.

t(8;21)(q22;q22) t(15;17)(q22;q12) inv(16) Monosomy 7

Good Good Good Poor

• Detection of minimal residual disease: Relapse following therapy is due to the persistence of viable leukemic cells following cytotoxic chemotherapy. Leukemic cells on the background of normal cells can be detected by morphology, cytogenetics, immunophenotyping, and molecular methods. • To establish clonality i.e. determining whether a cell population is derived from a single cell (monoclonal) or from multiple cells (polyclonal); it is helpful mainly in B or T cell lymphomas. • To detect chromosomal disorders which predispose to acute leukemia, e.g. trisomy 21. 5. Molecular genetic analysis: This is used for: • Detection of clonality by gene rearrangement studies (by polymerase chain reaction or Southern blot analysis). • Detection of minimal residual disease: Molecular methods like polymerase chain reaction can be helpful in detecting a small submicroscopic population of residual leukemic cells when peripheral blood and bone marrow examinations appear normal. • Diagnosis of specific types of acute leukemias by specific molecular probes: Molecular methods are used for detection of chromosomal translocations that generate fusion transcripts and chimeric proteins. The commonly used methods are reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent in situ hybridization (FISH). The technique detects t(15;17) in acute promyelocytic leukemia that generates PML/RARα fusion gene, and t(9;22) in B-ALL that generates bcr/abl fusion gene. Detection of these translocations is also helpful for determining prognosis and response to treatment. • Detection of opportunistic pathogens in immunocompromised patients.

CHRONIC LEUKEMIAS Chronic leukemias are heterogeneous disorders characterized by neoplastic proliferation of maturelooking cells of myeloid or lymphoid lineage. General differences between acute and chronic leukemias are outlined in Table 28.7. Chronic leukemias are of two main types: • Chronic myeloid leukemia • Chronic lymphoid leukemias (Table 28.8).

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Table 28.7: General differences between acute and chronic leukemias Parameter

Acute leukemias

Chronic leukemias

Clinical presentation Hematological features in marrow and blood Course

Usually sudden and fulminant Immature blast cells

Usually incidental or insidious onset Mature and differentiated cells

Aggressive

Indolent

Table 28.8: Chronic lymphoid leukemias B cell type

T cell type

1. 2. 3. 4. 5. 6.

1. 2. 3. 4.

Chronic lymphocytic leukemia Prolymphocytic leukemia Waldenström macroglobulinemia Hairy cell leukemia Plasma cell leukemia Leukemic phase of non-Hodgkin’s lymphoma

Chronic Myeloid Leukemia Chronic myeloid leukemia (CML) is a chronic myeloproliferative neoplasm originating from a pleuripotent hematopoietic stem cell and characterized by predominant proliferation of granulocytic cells. There are three phases of CML: chronic phase (3-5 years), accelerated phase (6-12 months), and blast crisis (2-4 months). CML is associated with a characteristic cytogenetic abnormality called Philadelphia chromosome that results from t(9;22)(q34;q11) (Fig. 28.7). This leads to juxtaposition of c-abl gene from chromosome 9 to bcr gene on chromosome

Fig. 28.7: Translocation between chromosomes 9 and 22 causing formation of Philadelphia chromosome

Prolymphocytic leukemia Large granular lymphocytic leukemia Adult T cell leukemia/lymphoma Sezary syndrome

22. The chimeric gene bcr-abl codes a tyrosine kinase that affects cell proliferation. Chronic phase: The average age at diagnosis is 45 years. In chronic phase, the usual presenting features include weakness, weight loss, abdominal fullness, easy bruisability, and splenomegaly. Blood smear in chronic phase of CML shows marked leukocytosis, immature white blood cells, basophilia, eosinophilia, anemia, and thrombocytosis (Fig. 28.8).

Fig. 28.8: Blood smear in chronic phase of chronic myeloid leukemia showing all forms of immature white cells and a basophil

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Essentials of Clinical Pathology Box 28.1: Fluorescence in situ hybridization (FISH)

Fig. 28.9: Diagrammatic representation of fluorescence in situ hybridization (FISH) in interphase nuclei. (A) Normal interphase nucleus showing two red dots (located on abl genes on two chromosomes number 9) and two green dots (located on bcr genes on two chromosomes number 22). (B) Interphase nucleus of a leukemic cell in chronic myeloid leukemia showing one red dot (abl on normal chromosome 9), one green dot (bcr on normal chromosome 22), and a combined dot on fused bcr-abl gene on Philadelphia chromosome. This fusion causes a fluorescent color change (red+green = yellow) which is not shown in the figure

Bone marrow shows markedly increased myeloid to erythroid ratio, < 10% blasts, and increased reticulin or fibrosis. Diagnostic feature is Philadelphia chromosome on cytogenetic analysis, and presence of a bcr-abl fusion gene on fluorescence in situ hybridization (FISH) (Fig. 28.9). Principle of FISH is shown in Box 28.1. Leukocyte alkaline phosphatase (LAP) score: This cytochemical stain is used to demonstrate presence and amount of the enzyme alkaline phosphatase within neutrophils. Blood smear is prepared from finger stick, air-dried, fixed, and stained. Enzyme activity is indicated by the presence of bright blue granules in neutrophils; nuclei are stained red (Fig. 28.10). In chronic myeloid leukemia, LAP is either absent or low. In leukemoid reaction and in other myeloproliferative neoplasms (polycythemia vera, essential thrombocythemia, and myelofibrosis), LAP score is increased. Therefore, LAP score is useful in differentiating CML from these disorders. Depending on the intensity of staining, LAP score is graded in each neutrophil as follows: • 0: Negative; No granules • 1: Positive with very few granules • 2: Positive with few to moderate number of granules • 3: Strongly positive with numerous granules • 4: Very strongly positive with cytoplasm crowded with granules.

This is a combination of cytogenetic and molecular techniques used to identify and localize the presence or absence of specific chromosomes or chromosomal regions through hybridization of fluorescent probes that bind specifically to its complementary target sequence. Fluorescent microscope is used to detect the presence or absence of fluorescent signals. FISH can be used in metaphase chromosomes (FISH-metaphase) or in interphase cells (FISH-interphase), and is a very versatile procedure. The technique consists of (1) Preparation of fluorescent probes: Probes are short sequences of single-stranded DNA that are complementary to the portion of gene of interest; probes are labeled with a fluorescent dye, (2) Preparation of metaphase chromosomes or interphase cells that are fixed on a microscope slide, (3) Hybridization: Fluorescentlylabeled probe is applied to the denatured chromosomal DNA and incubated. Attachment or hybridization occurs between the probe and the complementary DNA sequence. Nonhybridized probes are removed by washing, (4) Detection: This consists of observation of hybridization under fluorescent lighting. Fluorescent signals indicate hybridization or presence of complementary sequence of DNA, while absence of fluorescent signals indicates absence of complementary sequence of DNA. Each probe can be labeled with a different coloured fluorescent dye, so that a number of different probes can be simultaneously detected in the same preparation. In interphase FISH, probes are directly introduced into the cell. In contrast to conventional cytogenetic anlysis that requires 7-10 days for detection of chromosomal abnormalities, interphase FISH provides rapid diagnosis within 1-2 days. Unlike, metaphase FISH, actual chromosomes cannot be visualized with interphase FISH and the location of abnormality on the chromosome cannot be seen.

Fig. 28.10: Leukocyte alkaline phosphatase reaction: three neutrophils with score: 0, two neutrophils with a score of 2, and one neutrophil with score of 3

Laboratory Tests in Hematological Malignancies LAP score in an individual smear is the sum of the scores of 100 consecutive neutrophils. Normal range is 40-100. Chronic phase of CML should be differentiated from leukemoid reaction in which myeloid cells increase in number in peripheral blood secondary to infections, inflammation, and other disorders (See Table 22.1 in Chapter 22: Blood Smear). Accelerated phase: During accelerated phase, basophils and blast cells increase in number and patient increasingly becomes resistant to therapy. Blast crisis: Blast crisis results when blast cells become > 20% in blood or bone marrow. Additional cytogenetic abnormalities develop in accelerated and blast phases. Chronic Lymphocytic Leukemia Chronic lymphocytic leukemia (CLL) is a neoplastic disorder characterized by monoclonal proliferation of immunologically incompetent, slowly dividing, mature B lymphocytes. Most patients are adults >60 years. Clinical features include insidious onset of weakness, weight loss, susceptibility to infections, lymphadenopathy, and splenomegaly. About 25% of patients are asymptomatic. Laboratory features include absolute lymphocytosis (> 5000/cmm) consisting of small mature lymphocytes, smudge cells, and typical immunophenotype (weak

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surface membrane immunoglobulin, single light chain, CD19, CD20, and T-associated antigen CD5). The neoplastic lymphocytes are more fragile than normal cells and are often disrupted while spreading blood smear producing smudge (or basket) cells (Fig. 28.11). Complications include infections, autoimmune hemolytic anemia, and transformation to prolymphocytic leukemia or large cell lymphoma (Richter’s syndrome).

PLASMA CELL DYSCRASIAS Plasma cell dyscrasias are a group of disorders (Table 28.9) characterized by clonal proliferation of plasma cells or plasmacytoid lymphocytes and monoclonal production of immunoglobulins. Features of plasma cell dyscrasias are summarized in Table 28.10. Laboratory investigations in plasma cell dyscrasias are shown in Box 28.2. The laboratory findings that are suspicious of a plasma cell dyscrasia are raised erythrocyte sedimentation rate, rouleaux formation on blood smear, increased plasma cells in bone marrow (Fig. 28.12), renal impairment with bland urinary sediment, unexplained lytic bone lesions, anemia associated with renal failure and bone Table 28.9: Plasma cell dyscrasias 1. 2. 3. 4. 5. 6.

Multiple myeloma Plasmacytoma Waldenström macroglobulinemia Amyloidosis Heavy chain disease Monoclonal gammopathy of undetermined significance (MGUS)

Table 28.10: Characteristics of plasma cell dyscrasias Multiple myeloma: Osteolytic lesions, bone pain, pathological fractures, raised erythrocyte sedimentation rate, hypercalcemia, anemia, renal disease, serum M protein >3 g/dl, urinary M protein, plasma cells >10% in bone marrow Solitary plasmacytoma: Single tumor in bone, no urine or serum protein abnormalities, no myeloma cells in marrow Waldenström macroglobulinemia: Organomegaly, hyperviscocity, Serum IgM >3 g/dl, infiltration of bone marrow with plasmacytoid lymphocytes

Fig. 28.11: Blood smear in chronic lymphocytic leukemia showing small mature-looking lymphocytes and a smudge cell

Monoclonal gammopathy of undetermined significance: No osteolytic lesions/hypercalcemia/anemia/renal disease, serum M protein 4,00,000/μl) in chronic myeloproliferative disorders is sometimes associated with thrombosis and bleeding manifestations. Causes of thrombocytopenia: Thrombocytopenia is defined as platelet count below 1,50,000/μl. Causes of thrombocytopenia are listed earlier in Table 29.2. Evaluation of thrombocytopenia is shown in Figure 29.10. Causes of thrombocytosis: Thrombocytosis is defined as platelet count greater than 4,00,000/μl. Its causes are (i) primary: chronic myeloproliferative disorders like chronic myeloid leukemia, essential thrombocythemia, idiopathic myelofibrosis, and polycythemia vera; (ii) secondary (reactive): disseminated malignancy, hemorrhage, splenectomy, chronic inflammation, and iron deficiency anemia with bleeding.

Fig. 29.10: Evaluation of thrombocytopenia

Laboratory Tests in Bleeding Disorders Automated Method Automated hematology analyzers more precisely count platelets. However, these are expensive and have high running costs. Platelet counting is usually based on the principle of aperture impedance. A smaller aperture tube is required for platelets and a threshold level needs to be set to exclude larger red cells and smaller debris. False elevation of platelet count by this method can result from the presence of fragments of red or white cells, microspherocytes, and elevated cryoglobulins. Causes of pseudothrombocytopenia (falsely low platelet count) are: clumping of platelets in EDTA-anticoagulated venous blood sample (due to the presence of EDTA-dependent platelet antibody in some patients which is active only in vitro), platelet satellitism (adherence of platelets to neutrophils in EDTA-anticoagulated sample), platelet clumping due to the presence of platelet cold agglutinins in blood, and presence of giant platelets (which are not counted as platelets by electronic cell analyzers). Some analyzers can measure platelet distribution width (PDW), which is a measure of degree of variation of platelet size present in a blood sample. High PDW is seen in myeloproliferative disorders. In secondary thrombocytosis, PDW is normal. Mean platelet volume (MPV) is increased when thrombocytopenia is due to peripheral platelet destruction and is normal or low when thrombocytopenia is due to defective platelet production. Young platelets with residual RNA are called as reticulated platelets. These can be identified by some analyzers and flow cytometers. Increased numbers are seen in idiopathic thrombocytopenic purpura and lower numbers in aplastic anemia. Bleeding Time The bleeding time test assesses primary hemostasis (vascular and platelet components) and is dependent on adequate functioning of platelets and blood vessels. In this test, a superficial skin puncture or incision is made and the time required for bleeding to stop is measured. Three methods are commonly used: Duke’s, Ivy’s, and template. Duke’s method, which measures bleeding time following ear lobe puncture, is not advocated since it cannot be standardized and can cause a large local hematoma. In Ivy’s method, 3 punctures are made on the volar surface of the forearm with a lancet (cutting depth 2-2.5 mm) under standardized venous

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pressure (40 mm Hg). A disadvantage with this method is closure of puncture wound before cessation of bleeding. Template method uses a special surgical blade, which makes a larger cut (5 mm long and 1 mm deep). Although template method is better, it can produce a large scar and even a keloid in predisposed individuals. Ivy’s method is described below. Ivy’s method Principle: Three standard punctures are made with a lancet on the volar surface of the forearm under standard pressure, and the average time required for bleeding to cease from the puncture sites is measured. Equipment 1. Sphygmomanometer 2. Sterile disposable lancets (2-2.5 mm blade with shoulder, which limits the depth of penetration). 3. Stopwatch 4. Filter paper Method 1. A sphygmomanometer cuff is wrapped around the upper arm and inflated to 40 mm of Hg. 2. The dorsal surface of the forearm is cleansed with 70% ethanol and allowed to dry. 3. Three punctures are made (about 5 cm apart) in quick succession with a lancet (Superficial veins, and scars or bruises should be avoided). 4. A stopwatch is started as soon as a puncture is made. One stopwatch is needed for each puncture. 5. Blood oozing from the puncture wound is gently blotted with a filter paper at 15 seconds intervals. Avoid directly touching the edges of the wound. 6. The timer is stopped when blood no longer stains the filter paper. 7. Time required for bleeding to cease from all the three puncture wounds is noted. The average time is reported as the bleeding time. 8. Sterile adhesive strip is applied over the puncture. Reference range: 2-7 minutes. Majority of individuals have bleeding time less than 4 minutes. It should be reported in minutes or nearest half minute. If bleeding continues beyond 20 minutes, BT should be reported as >20 minutes and the test is discontinued. Causes of prolongation of bleeding time 1. Thrombocytopenia: If platelet count is less than 1,00,000/ml, bleeding time should not be performed, as it will be prolonged. With a very low platelet count, bleeding may be difficult to control.

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Fig. 29.11: Evaluation of prolonged bleeding time

2. Disorders of platelet function 3. von Willebrand disease 4. Disorders of blood vessels Evaluation of prolonged bleeding time is shown in Figure 29.11. Platelet Function Analyzer (PFA-100) This is a newly introduced screening test for platelet function that assesses both platelet adhesion and aggregation. This method uses an instrument called as PFA-100 in which anticoagulated whole blood is passed at a high shear rate through small membranes that have been coated with either collagen and epinephrine or collagen and ADP. Platelets adhere to each membrane and gradually occlude an aperture at the centre of the membrane. The time required for complete occlusion of the aperture is called as closure time. Normal closure time is 1-3 minutes. The PFA-100 test is performed initially with the collagen/epinephrine membrane; if closure time is normal, there is no significant platelet function defect. If closure time with collagen/epinephrine is prolonged, test with collagen/ADP is carried out; if normal, aspirin-induced platelet dysfunction is the probable cause; if prolonged, other platelet function defect (acquired or inherited) is likely. This test is more sensitive than bleeding time to assess primary hemostasis, sensitive for detection of von Willebrand disease and easy to perform. However, in the presence of thrombocytopenia and anemia, closure

time is prolonged. Also, in the presence of a strong clinical suspicion of a platelet function defect and normal PFA100 result, further testing is still necessary. Clotting Time This is a crude test and is now replaced by activated partial thromboplastin time. Clotting time measures the time required for the blood to clot in a glass test tube kept at 37°C. Prolongation of clotting time only occurs in severe deficiency of a clotting factor and is normal in mild or moderate deficiency. Prothrombin Time (PT) PT assesses coagulation factors in extrinsic pathway (F VII) and common pathway (F X, F V, prothrombin, and fibrinogen) (Fig. 29.12). Principle: Tissue thromboplastin and calcium are added to plasma and clotting time is determined. The test determines the overall efficiency of extrinsic and common pathways. Equipment 1. Water bath at 37°C 2. Test tubes (75 × 12 mm) 3. Stopwatch Reagents 1. Thromboplastin reagent: This contains tissue factor and phospholipids and is available commercially. 2. Calcium chloride 0.025 mol/liter. Specimen: Platelet-poor citrated plasma (Box 29.2).

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Laboratory Tests in Bleeding Disorders

Fig. 29.12: Principle of prothrombin time

Box 29.2: Collection of blood for coagulation studies Venous blood is collected from antecubital fossa with a plastic, siliconized glass, or polypropylene syringe and a large bore needle (20 or 21 G in adults, 22 or 23 G in infants). Blood should never be collected from indwelling intravenous lines, as these often contain heparin. Glass syringe should not be used for collection since it activates coagulation. The blood is drawn gently but quickly after a single, smooth venepuncture. The needle is detached from the syringe, and the sample is passed gently into the plastic container. After capping the container, the blood and the anticoagulant are mixed immediately by gentle inversion 5 times. The anticoagulant used for coagulation studies is trisodium citrate (3.2%), with anticoagulant to blood proportion being 1:9. Most coagulation studies require platelet poor plasma (PPP). To obtain PPP, blood sample is centrifuged at 3000-4000 revolutions per minute for 15-30 minutes. Coagulation studies are carried out within 2 hours of collection of sample.

Method 1. Deliver 0.1 ml of plasma in a glass test tube kept in water bath at 37°C. 2. Add 0.1 ml of thromboplastin reagent and mix. 3. After 1 minute, add 0.1 ml of calcium chloride solution. Immediately start the stopwatch and record the time required for clot formation. Normal range: 11-16 seconds. Causes of prolongation of PT 1. Treatment with oral anticoagulants 2. Liver disease 3. Vitamin K deficiency 4. Disseminated intravascular coagulation 5. Inherited deficiency of factors in extrinsic and common pathways.

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Uses of PT 1. To monitor patients who are on oral anticoagulant therapy: PT is the standard test for monitoring treatment with oral anticoagulants. Oral anticoagulants inhibit carboxylation of vitamin K-dependent factors (Factors II, VII, IX, and X) and make these factors inactive. In patients receiving oral anticoagulants, PT should be reported as a ratio of PT of patient to PT of control; it should not be reported as percentage. Various types of thromboplastin reagents obtained from different sources (like ox brain, rabbit brain, or rabbit lung) are available for PT test. These differ in their responsiveness to deficiency of vit. K-dependent factors. Technique of PT is also different in different laboratories. For standardization and to obtain comparable results, it is recommended to report PT (in persons on oral anticoagulants) in the form of an International Normalized Ratio (INR). INR =

PT of patient PT of control

ISI

International Sensitivity Index (ISI) of a particular tissue thromboplastin is derived (by its manufacturer) by comparing it with a reference thromboplastin of known ISI. INR should be maintained in the therapeutic range for the particular indication (INR of 2.0-3.0 for prophylaxis and treatment of deep venous thrombosis; INR of 2.5-3.5 for mechanical heart valves). Therapeutic range provides adequate anticoagulation for prevention of thrombosis and also checks excess dosage, which will cause bleeding. 2. To assess liver function: Liver is the site of synthesis of various coagulation factors, including vitamin Kdependent proteins. Therefore PT is a sensitive test for assessment of liver function. 3. Detection of vitamin K deficiency: PT measures three of the four vitamin K-dependent factors (i.e. II, VII, and X). 4. To screen for hereditary deficiency of coagulation factors VII, X, V, prothrombin, and fibrinogen. Activated Partial Thromboplastin Time (APTT) APTT is a measure of coagulation factors in intrinsic pathway (F XII, F XI, high molecular weight kininogen, prekallikrein, F IX, and F VIII) and common pathway (F X, F V, prothrombin, and fibrinogen) (Fig. 29.13). tahir99 - UnitedVRG vip.persianss.ir

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Fig. 29.13: Principle of activated partial thromboplastin time

Principle: Plasma is incubated with an activator (which initiates intrinsic pathway of coagulation by contact activation). Phospholipid (also called as partial thromboplastin) and calcium are then added and clotting time is measured. Equipment: This is same as for PT. Reagents 1. Kaolin 5 gm/liter: This is a contact activator. 2. Phospholipid: Various APTT reagents are available commercially, which contain phospholipids. 3. Calcium chloride 0.025 mol/liter. Specimen: Citrated platelet poor plasma (Box 29.2). Method 1. Mix equal volumes of phospholipid reagent and calcium chloride solution in a glass test tube and keep in a waterbath at 37°C. 2. Deliver 0.1 ml of plasma in another test tube and add 0.1 ml of kaolin solution. Incubate at 37°C in the waterbath for 10 minutes. 3. After exactly 10 minutes, add 0.2 ml of phospholipidcalcium chloride mixture, start the stopwatch, and note the clotting time.

Normal range: 30-40 seconds. Causes of prolongation of APTT 1. Hemophilia A or B. 2. Deficiencies of other coagulation factors in intrinsic and common pathways. 3. Presence of coagulation inhibitors 4. Heparin therapy 5. Disseminated intravascular coagulation 6. Liver disease Uses of APTT 1. Screening for hereditary disorders of coagulation: Since deficiencies of F VIII (hemophilia A) and F IX (hemophilia B) are relatively common, APTT is the most important screening test for inherited coagulation disorders. APTT detects deficiencies of all coagulation factors except F VII and F XIII. PT is also performed along with APTT. Prolongation of both PT and APTT is indicative of deficiency of coagulation factors in common pathway. Normal PT with prolongation of APTT is indicative of intrinsic pathway deficiency (particularly of F VIII or IX). 2. To monitor heparin therapy: Heparin potentiates the action of natural anticoagulant antithrombin III which

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Laboratory Tests in Bleeding Disorders is an inhibitor of thrombin and activated factors IX, X, and XI. Full dose heparin therapy needs monitoring by APTT to maintain the dose in the therapeutic range (1.5 to 2.5 times the upper reference limit of APTT). 3. Screening for circulating inhibitors of coagulation: APTT is prolonged in the presence of specific inhibitors (which are directed against specific coagulation factors) and non-specific inhibitors (which interfere with certain coagulation reactions). Mixing experiment for detection of inhibitors: Mixing studies are used to distinguish between factor deficiencies and factor inhibitors (specific coagulation factor inhibitor or non-specific inhibitor such as lupus anticoagulant). If APTT is prolonged, patient’s plasma is mixed with an equal volume of normal plasma (called as a 50:50 mix) and APTT is repeated. In coagulation factor deficiency, prolongation of APTT gets corrected by more than 50% of the difference between the clotting times of control and test plasma. In the presence of lupus anticoagulant, there is no such correction. With lupus anticoagulant, APTT remains prolonged after mixing and for 2 hours following incubation. With F VIII inhibitor (which is timeand temperature-dependent), prolonged APTT gets immediately corrected after mixing, but becomes prolonged after incubation (Fig. 29.14).

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Thrombin Time (TT) Thrombin time assesses the final step of coagulation i.e. conversion of fibrinogen to fibrin by thrombin (Fig. 29.15). Principle: Thrombin is added to patient’s plasma and time required for clot formation is noted. Equipment: Same as for PT. Reagent: Thrombin solution. Specimen: Citrated platelet poor plasma (Box 29.2). Method: Take 0.1 ml of buffered saline in a test tube and add 0.1 ml of plasma. Note clotting time after addition of 0.1 ml of thrombin solution. Normal range: ± 3 seconds of control.

Fig. 29.15: Principle of thrombin time

Fig. 29.14: Interpretation of mixing experiment. Lupus inhibitor is an immediate-acting inhibitor, while F VIII inhibitor is a time-dependent inhibitor

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Causes of Prolongation of TT 1. Disorders of fibrinogen: Prolongation of TT occurs in afibrinogenemia (virtual absence of fibrinogen), hypofibrinogenemia (fibrinogen less than 100 mgs/dl), and dysfibrinogenemia (dysfunctional fibrinogen). 2. Heparin therapy: Heparin inhibits action of thrombin. 3. Presence of fibrin degradation products (FDPs): These interfere with fibrin monomer polymerization. TT is repeated using a mixture of normal plasma and patient’s plasma. If TT remains prolonged, then FDPs are present (provided patient is not receiving heparin). Interpretation of Screening Tests In a patient with a bleeding disorder, results of all the screening tests should be interpreted together (Fig. 29.16). 1. Selective thrombocytopenia: If low platelet count is the only abnormality on hemostasis screen, blood smear and bone marrow examinations are required mainly to exclude underlying hematologic disease like aplastic anemia, leukemia, lymphoma, or myelodysplastic syndrome. In the absence of a hematologic disorder, normal or increased numbers of megakaryocytes in bone marrow is indicative of peripheral destruction of platelets (e.g. idiopathic thrombocytopenic purpura, drugs, infections, collagen disorders).

2. Selective prolongation of bleeding time: This occurs in disorders of platelet function, von Willebrand disease, and vascular disorders. For definitive diagnosis of platelet function defects, platelet aggregation studies are required. In vascular disorders, associated clinical and laboratory features are often suggestive of diagnosis. 3. Selective prolongation of APTT: Isolated prolongation of APTT suggests deficiency of coagulation factors in intrinsic pathway, especially that of F VIII (hemophilia A) or F IX (hemophilia B). Definitive diagnosis is based on specific coagulation factor assay. Deficiency of F XII, high molecular weight kininogen, or prekallikrein is not associated with clinical bleeding. Acquired causes of prolongation of APTT are heparin therapy and circulating inhibitors of coagulation. 4. Prolongation of APTT and bleeding time: This combination is observed in von Willebrand disease. Platelet aggregation studies are required for definitive diagnosis. 5. Selective prolongation of PT: Isolated prolongation of PT is suggestive of F VII deficiency. Acquired causes, which cause prolongation of only PT, are early vitamin K deficiency and start of oral anticoagulant therapy. 6. Prolongation of both PT and APTT: This indicates deficiency of one or more factors in common pathway

Fig. 29.16: Evaluation of a suspected bleeding disorder. BT: bleeding time; PC: platelet count; PT: prothrombin time; APTT: activated partial thromboplastin time

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Laboratory Tests in Bleeding Disorders (i.e. F X, F V, prothrombin, or fibrinogen), or dysfibrinogenemia. Other causes are heparin therapy, liver disease, vit K deficiency (in which prolongation of PT is more as compared to APTT), and oral anticoagulant therapy. 7. All screening tests abnormal: Combination of low platelet count and prolongation of both PT and APTT is seen in acute disseminated intravascular coagulation (DIC) and liver disease. DIC should be suspected when a patient with some underlying condition (like sepsis, major trauma, malignancy, snake bite, obstetric problem, etc.) develops acute bleeding manifestations. Other laboratory abnormalities in DIC are fragmented red cells on blood smear, and raised levels of FDPs, and D-dimer. In liver disease, in contrast to DIC, fragmented red cells are absent, D-dimer test is negative, liver function tests are abnormal, and F VIII level is normal. 8. All screening tests normal: If, in a patient suspected of having a bleeding disorder, all the screening tests are normal, possibilities include mild forms of von Willebrand disease, some platelet function defects, vascular disorder, mild coagulation factor deficiency, or F XIII deficiency. Investigations in such cases include blood smear, platelet function studies, workup for von Willebrand disease, and clot solubility test for F XIII deficiency. Specific Tests for Hemostasis 1. Platelet aggregation studies: Platelet aggregation tests are carried out in specialized hematology

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laboratories if platelet dysfunction is suspected. These tests are usually indicated in patients presenting with mucocutaneous type of bleeding and in whom screening tests reveal normal platelet count, prolonged bleeding time, normal prothrombin time, and normal activated partial thromboplastin time. Platelet aggregation studies are carried out on platelet-rich plasma using aggregometer. When a platelet aggregating agent is added to platelet-rich plasma, platelets form aggregates and optical density falls (or light transmission increases); this is recorded by a chart recorder on a strip chart. Commonly used platelet aggregating agents are ADP (adenosine 5diphosphate), epinephrine (adrenaline), collagen, arachidonic acid, and ristocetin. ADP (low dose) and epinephrine induce primary and secondary waves of aggregation (biphasic curve). Primary wave is due to the direct action of aggregating agent on platelets. Secondary wave is due to thromboxane A2 synthesis and secretion from platelets. Collagen, arachidonic acid and ristocetin induce a single wave of aggregation (monophasic curve) Normal aggregation curve is shown in Figure 29.17. Aggregation patterns in various platelet function defects are shown in Figures 29.18 to 29.20, and in Table 29.8. 2. Factor VIII assay: One stage F VIII assay based on APTT is usually performed for diagnosis of F VIII deficiency. Coagulation factor assays are based on the ability of patient’s plasma to correct specific factordeficient plasma. The abilities of the dilutions of standard plasma (normal plasma) and patient’s plasma to correct the APTT of the plasma known to

Fig. 29.17: Normal platelet aggregation curves tahir99 - UnitedVRG vip.persianss.ir

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Essentials of Clinical Pathology

Fig. 29.18: Platelet aggregation curves in von Willebrand disease and Bernard-Soulier syndrome (absent aggregation with ristocetin, normal aggregation with ADP, epinephrine, and arachidonic acid)

Fig. 29.19: Platelet aggregation curves in storage pool defect (absent second wave of aggregation with ADP and epinephrine, absent or greatly diminished aggregation with collagen, and normal ristocetin aggregation)

Fig. 29.20: Platelet aggregation curves in Glanzmann’s thrombasthenia (absent aggregation with all agonists except ristocetin)

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Table 29.8: Laboratory features of platelet function disorders Disorder

Platelet aggregation pattern

Other features

Bernard Soulier syndrome

Normal with ADP, epinephrine, collagen, and arachidonic acid; deficient with ristocetin Deficient with ADP, epinephrine, collagen, and arachidonic acid; normal with ristocetin Primary wave with ADP and epinephrine, normal with arachidonic acid, deficient with collagen, normal with ristocetin Primary wave with ADP and epinephrine, deficient with arachidonic acid, deficient with collagen, normal with ristocetin Normal with ADP, epinephrine, collagen, and arachidonic acid; deficient with ristocetin

Autosomal recessive; severe bleeding; giant platelets

Glanzmann’s thrombasthenia

Storage pool defect

Aspirin-like defect

von Willebrand disease

Autosomal recessive; severe bleeding; small and discrete platelets; defective clot retraction Defects of platelet granules; platelet dense granules are decreased with deficient release of ADP, ATP, calcium, and serotonin

Autosomal dominant/recessive; abnormality in aggregation corrected with cryoprecipitate

be completely deficient in F VIII are compared. The results are plotted (clotting times in seconds vs. percent factor activity) on a log/log graph paper. The 1:10 dilution is considered as 100% activity. F VIII activity of 50-150% is considered as normal. 3. Detection of fibrinogen/fibrin degradation products (FDPs): FDPs are fragments produced by proteolytic digestion of fibrinogen or fibrin by plasmin. For determination of FDPs, blood is collected in a tube containing thrombin (to remove all fibrinogen by converting it into a clot) and soybean trypsin inhibitor (to inhibit plasmin and thus prevent in vitro breakdown of fibrin). A suspension of latex particles linked to antifibrinogen antibodies (or fragments D and E) is mixed with dilutions of patient’s serum on a glass slide. If FDPs are present, agglutination of latex particles occurs (Fig. 29.21). The highest dilution of serum at which agglutination is detected is used to determine concentration of FDPs. Increased levels of FDPs occur in fibrinogenolysis or fibrinolysis. This occurs in disseminated intravascular coagulation, deep venous thrombosis, severe pneumonia, and recent myocardial infarction. 4. Detection of D-dimers: D-dimer is derived from the breakdown of fibrin by plasmin and D-dimer test is used to evaluate fibrin degradation. Blood sample can be either serum or plasma. Latex or polystyrene

Fig. 29.21: Principle of latex agglutination test for fibrinogen/fibrin degradation products

microparticles coated with monoclonal antibody to Ddimer are mixed with patient’s sample and observed for microparticle agglutination. As the particle is small, turbidometric endpoint can be determined in automated instruments. D-dimer and FDPs are raised in disseminated intravascular coagulation, intravascular thrombosis (myocardial infarction, stroke, venous thrombosis, pulmonary embolism), and during postoperative

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period or following trauma. D-dimer test is commonly used for exclusion of thrombosis and thrombotic tendencies. 5. Estimation of fibrinogen: Fibrinogen is commonly measured by Clauss method that consists of modification of thrombin time by diluting plasma; thrombin time of diluted plasma is inversely proportional to concentration of fibrinogen. Fibrinogen can also be estimated by immunological method; in dysfibrinogenemia, fibrinogen estimated by functional assay (Clauss method) is abnormal while immunological assay is normal. 6. Platelet glycoprotein analysis: This is done by flow cytometric analysis for detection of lack of GpIb/IX in Bernanrd Soulier syndrome (deficiency of CD42), and lack of GpIIb/IIIa in Glanzmann’s thrombasthenia (deficiency of CD41, CD61).

REFERENCE RANGES • Bleeding time: Ivy method: 2-7 minutes; Template method: 2.5-9.5 minutes • Prothrombin time: 11-16 seconds • Activate partial thromboplastin time: 30-40 seconds • Thrombint time: ±3 seconds of control • Plasma fibrinogen: 200-400 mg/dl • Fibrinogen/fibrin degradation products: < 10 μg/ml • D-dimer: Qualitiative: Negative; Quantitative: < 200 mg/L

• Factors II, V, VII, VIII, IX, X, XI, XII: 50-150% • vWF:Ag: 50-150%

CRITICAL VALUES • Prothrombin time: > 30 seconds or > 3 times control value • Activated partial thromboplastin time: ≥ 75 seconds • Platelet count < 20,000/cmm or > 1 million/cmm • D-dimer: Positive • Plasma fibrinogen: < 100 mg/dl

BIBLIOGRAPHY 1. Evatt BL, Gibbs WN, Lewis SM, McArthur JR. Fundamental Diagnostic Hematology: The Bleeding and Clotting Disorders (2nd ed), 1992. US Dept. of health and Human Services, Atlanta, Georgia and World Health Organization, Geneva, Switzerland. 2. Kawthalkar SM. Essentials of Hematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006. 3. Lewis SM, Bain BJ, bates I (Eds). Dacie and Lewis Practical Hematology (9th ed). London: Churchill Livingstone, 2002. 4. Provan D, Krentz A. Oxford Handbook of Clinical and Laboratory Investigations (2002). Oxford university Press. Oxford. 5. Shrikhande AV, Warhadpande MS, Kawthalkar SM. A laboratory manual of coagulation (1994). Dept. of Pathology. Govt. Medical College, Nagpur. 6. Wallach J. Interpretation of Diagnostic Tests (7th ed). Philadelphia: Lippincott Williams and Wilkins, 2000.

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Laboratory Tests in Thrombophilia Thrombophilia is a hemostatic disorder in which there is a predisposition to thrombosis due to an inherited or acquired condition (Table 30.1). It results from alteration in hemostatic constituents of blood.

INHERITED THROMBOPHILIA 1. Factor V Leiden (Activated protein C resistance): Factor V Leiden (so-called because of its discovery in Leiden, Netherlands) is the most common inherited cause of thrombophilia. Mutation in FV gene (transmitted through autosomal dominant inheritance) leads to replacement of arginine by glutamine at position 506. This substitution renders activated form of FV resistant to degradation by activated protein C. More than 95% cases of activated protein C resistance are due to FV Leiden (other causes of activated protein C resistance being pregnancy, oral contraceptive use, malignancy, and other FV mutations). The terms FV Leiden and activated protein C resistance are not synonymous. Failure of

removal of F Va leads to increased prothrombinase complex (F Xa-Va-PL-Ca) activity, and greater thrombin generation. It is primarily associated with increased risk of venous thrombosis. Heterozygotes have 2-6% increased risk, while homozygotes have 50-100 times risk. Risk of thrombosis is greatly increased during pregnancy and following oral contraceptive use. Laboratory tests for FV Leiden include activated protein C resistance assay and genetic analysis. In activated protein C resistance assay, activated partial thromboplastin time (APTT) on the patient’s sample is compared with APTT done after addition of activated protein C to plasma sample. The ratio is called as activated protein C sensitivity ratio. In normal subjects, addition of activated protein C to the plasma sample will prolong APTT because activated protein C inhibits F V and F VIII. In F V Leiden, APTT shows only slight prolongation. Low activated protein C ratio indicates more resistance to

Table 30.1: Conditions associated with increased risk of thrombosis Inherited (Primary) 1. 2. 3. 4. 5. 6. 7.

Factor V Leiden (Activated protein C resistance), i.e. Abnormal F V that resists degradation by protein C Prothrombin gene mutation Hyperhomocysteinemia Deficiency or decreased activity of protein C Deficiency or decreased activity of protein S Deficiency or decreased activity of antithrombin III Dysfibrinogenemia

Acquired (Secondary) 1. Oral contraceptive therapy 2. 3. 4. 5. 6. 7. 8. 9. 10.

Pregnancy Antiphospholipid syndrome Paroxysmal nocturnal hemoglobinuria Malignancy Disseminated intravascular coagulation Heparin-induced thrombocytopenia Thrombotic thrombocytopenic purpura Nephrotic syndrome Myeloproliferative disorders

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2.

3.

4.

5.

Essentials of Clinical Pathology

activated protein C. If the screening test is positive for activated protein C resistance, genetic testing is performed. Genetic analysis by polymerase chain reaction can detect heterozygous and homozygous states. Prothrombin gene G20210A mutation: A G→A mutation at position 20210 of prothrombin gene is the second most common cause of inherited thrombophilia. This mutation, by some unknown mechanism, leads to a rise in the concentration of prothrombin, thus making available large amounts of prothrombin for conversion to thrombin. It is implicated in causation of both arterial and venous thrombosis and pregnancy-associated thrombosis. Diagnosis is based on genetic analysis. Deficiency of protein C and S: Protein C, when activated by thrombin, inactivates factors V and VIII. Protein S serves as a cofactor in this reaction. In protein C and S deficiency, thrombin generation is increased, producing hypercoagulability. Both protein C and protein S deficiencies primarily cause venous thrombosis. Diagnosis of protein C deficiency requires quantification of protein C concentration. Distinction between deficiency and dysfunction of protein C is based on functional assay for protein C. Protein S deficiency is detected by quantification of protein S (both free and bound forms). Deficiency of antithrombin III: Antithrombin III is the natural inhibitor of thrombin, F Xa, IXa, XIa, and XIIa. Deficiency state is associated with venous thrombosis. Homozygous state is incompatible with life. Hyperhomocysteinemia: Increased levels of the amino acid homocysteine can occur in various inherited disorders like homocystinuria, cystathione synthase deficiency, and C677T gene polymorphism in the methyl tetrahydrofolate reductase (MTHFR) gene. Acquired causes of hyperhomocysteinemia are vitamin B12 deficiency, folate deficiency, and vitamin B6 deficiency. Hyperhomocysteinemia is associated with increased risk of both arterial and venous thrombosis. Laboratory tests are assay for homocyseine or genetic analysis of MTHFR gene.

ACQUIRED THROMBOPHILIA 1. Oral contraceptive therapy and pregnancy: Increased predisposition to thrombosis results from

increased synthesis of coagulation factors in liver and decreased synthesis of antithrombin III. 2. Antiphospholipid syndrome: Antiphospholipid antibodies are autoantibodies directed against antigens composed of phospholipids. They are of two types: lupus anticoagulant (so named because it was first detected in a patient with systemic lupus erythematosus) and anticardiolipin antibodies. Lupus anticoagulant is detected by prolongation of phospholipid-dependent coagulation tests such as activated partial thromboplastin time (APTT) and dilute Russell’s viper venom time. If APTT is prolonged, the test is repeated after mixing the sample 50:50 with normal plasma. If not corrected, it suggest presence of lupus anticoagulant (Box 30.1). Anticardiolipin antibodies are detected by enzyme linked immunosorbent assay (ELISA). Antiphospholipid antibodies are associated with arterial and venous thrombosis, spontaneous, recurrent abortions, and thrombocytopenia. Antiphospholipid syndrome is present if antiphospholipid antibodies (lupus anticoagulant or anticardiolipin antibodies or both) are present along with an episode of arterial or venous thrombosis, thrombocytopenia, or frequent second trimester abortions. Antiphospholipid antibodies may occur without any underlying disorder (primary) or in association with systemic lupus erythematosus, Sjogren’s syndrome, rheumatoid arthritis, human immunodeficiency virus infection or malignancy (secondary). 3. Heparin-induced thrombocytopenia: This complication occurs in 1-3% of patients receiving any type of heparin. Platelet count should be checked every alternate day in patients receiving heparin. The condition should be suspected when patient is not

Box 30.1: Criteria for diagnosis of lupus anticoagulant (International Society of Thrombosis and Hemostasis) • Prolongation of a phospholipid-dependent screening test for coagulation like activated partial thromboplastin time (APTT) or dilute Russell’s viper venom time • Mixing study with APTT using 50:50 mixture of patient’s and normal plasma shows no correction, indicating that prolongation is due to an inhibitor. • Demonstration of antiphospholipid nature of antibodies by neutralizing them with high concentration of platelets (platelet neutralization test) • No other cause for thrombosis

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Laboratory Tests in Thrombophilia responding to heparin, and develops thrombocytopenia (50% below baseline level) and thrombosis. These patients are at risk of developing acute myocardial infarction, stroke, peripheral arterial thrombosis and deep venous thrombosis. The pathogenesis consists of binding of IgG-heparinplatelet factor 4 complexes to Fc receptors on platelets that causes activation and aggregation of platelets as well as clearance of coated platelets by macrophages. This leads to thrombosis and thrombocytopenia.

LABORATORY TESTING IN THROMBOPHILIA Laboratory studies will identify the underlying cause in majority of patient presenting with thrombosis. The screening tests for thrombophilia are not readily available. Also, testing for thrombophilia is expensive, rarely helps in acute patient management, and results may not be interpreted correctly, leading to improper treatment. Concentration solely on thrombophilia detection can overlook a serious underlying disorder. Testing for thrombophilia should be ordered in selected patients in whom such complex and costly testing will be of significant benefit for management. Indications for Testing for Thrombophilia 1. 2. 3. 4.

Thrombosis at young age with no obvious risk factors. Unexplained recurrent thrombosis. Thrombosis at unusual locations. Strong family history of thrombosis (in first degree relative).

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5. Recurrent abortions (≥ 3) 6. Pregnancy-associated thrombosis. Laboratory Tests The tests should be performed atleast 4-6 weeks after the acute thrombotic event and discontinuation of warfarin, as acute states cause elevation of acute phase reactants (that interfere with testing and cause false positive results). 1. For inherited thrombophilia: • DNA analysis for F V Leiden • DNA analysis for prothrombin gene mutation • Test for homocysteine level. • Testing for protein C, protein S, and antithrombin III deficiency or dysfunction 2. For acquired thrombophilia: • Exclusion of diabetes mellitus, hyperlipidemia, myeloproliferative disorders, paroxysmal nocturnal hemoglobinuria • Test for lupus anticoagulant and anticardiolipin antibodies.

BIBLIOGRAPHY 1. Brandt JT, Triplett DA, Alving B, et al. Criteria for the diagnosis of lupus anticoagulants: an update. Thromb Hemost 1995;74:1185-90. 2. Van Cott EM, Laposata M. Laboratory evaluation of hypercoagulable states. Hematology/Oncology Clinics of North America 1998;12:1141-66. 3. Whiteman T, Hassouna HI. Hypercoagulable states. Hematology/Oncology Clinics of North America 2000; 14:355-77.

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Laboratory Tests in Porphyrias Porphyrias (from Greek porphura meaning purple pigment; the name is probably derived from purple discoloration of some body fluids during the attack) are a heterogeneous group of rare disorders resulting from disturbance in the heme biosynthetic pathway leading to the abnormal accumulations of red and purple pigments called as porphyrins in the body. Heme, a component of hemoglobin, is synthesized through

various steps as shown in Figure 31.1. Each of the steps is catalyzed by a separate enzyme; if any of these steps fails (due to hereditary or acquired cause), precursors of heme (porphyrin intermediates) accumulate in blood, get deposited in skin and other organs, and excreted in urine and feces. Depending on the site of defect, different types of porphyrias are described with varying clinical features, severity, and the nature of accumulated porphyrin.

Fig. 31.1: Figure on left shows steps in the biosynthesis of heme. Some steps (first and last three) occur in mitochondria, while some occur in cytosol. Figure on right shows deficiency state associated with each enzyme. Deficiency of ALA synthase is associated with sideroblastic anemia, and deficiencies of other enzymes cause porphyria

Laboratory Tests in Porphyrias Porphyria has been offered as a possible explanation for the medieval tales of vampires and werewolves; this is because of the number of similarities between the behavior of persons suffering from porphyria and the folklore (avoiding sunlight, mutilation of skin on exposure to sunlight, red teeth, psychiatric disturbance, and drinking of blood to obtain heme). Porphyrias are often missed or wrongly diagnosed as many of them are not associated with definite physical findings, screening tests may yield false-negative results, diagnostic criteria are poorly defined and mild disorders produce an enzyme assay result within ‘normal’ range. Heme is mainly required in bone marrow (for hemoglobin synthesis) and in liver (for cytochromes). Therefore, porphyrias are divided into erythropoietic and hepatic types, depending on the site of expression of disease. Hepatic porphyrias mainly affect the nervous system, while erythropoietic porphyrias primarily affect the skin. Porphyrias are also classified into acute and nonacute (or cutaneous) types depending on clinical presentation (Table 31.1). Inheritance of porphyrias may be autosomal dominant or recessive. Most acute porphyrias are inherited in an autosomal dominant manner (i.e. inheritance of one abnormal copy of gene). Therefore, the activity of the deficient enzyme is 50%. When the level of heme falls in the liver due to some cause, activity of ALA synthase is stimulated leading to increase in the levels of heme

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precursors up to the point of enzyme defect. Increased levels of heme precursors cause symptoms of acute porphyria. When the heme level returns back to normal, symptoms subside. Accumulation of porphyrin precursors can occur in lead poisoning due to inhibition of enzyme aminolevulinic acid dehydratase in heme biosynthetic pathway. This can mimick acute intermittent porphyria.

CLINICAL FEATURES Clinical features of porphyrias are variable and depend on type. Acute porphyrias present with symptoms like acute and severe abdominal pain/vomiting/constipation, chest pain, emotional and mental disorders, seizures, hypertension, tachycardia, sensory loss, and muscle weakness. Cutaneous porphyrias present with photosensitivity (redness and blistering of skin on exposure to sunlight), itching, necrosis of skin and gums, and increased hair growth over the temples (Table 31.2). Symptoms can be triggered by drugs (barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines), emotional or physical stress, infection, dieting, fasting, substance abuse, premenstrual period, smoking, and alcohol. Autosomal dominant porphyrias include acute intermittent porphyria, variegate porphyria, porphyria

Table 31.1: Various classification schemes for porphyrias Classification based on predominant clinical manifestations

Classification based on site of expression of disease

Classification based on mode of clinical presentation

Neuropsychiatric 1. Acute intermittent porphyria

Hepatic 1. ALA-dehydratase porphyria

2. ALA-dehydratase porphyria (Plumboporphyria)

2. Acute intermittent porphyria

Acute 1. ALA-dehydratase porphyria (Plumboporphyria) 2. Acute intermittent porphyria

Cutaneous (Photosensitivity) 1. Congenital erythropoietic porphyria 2. Porphyria cutanea tarda 3. Erythropoietic protoporphyria

3. Hereditary coproporphyria 4. Variegate porphyria

3. Hereditary coproporphyria 4. Variegate porphyria Non-acute (cutaneous) 1. Porphyria cutanea tarda

Mixed (Neuropsychiatric and cutaneous)

Erythropoietic porphyria 1. Congenital erythropoietic porphyria 2. Erythropoietic protoporphyria

1. Hereditary coproporphyria 2. Variegate porphyria

Hepatic/Erythropoietic 1. Porphyria cutanea tarda

2. Congenital erythropoietic porphyria 3. Erythropoietic protoporphyria

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Essentials of Clinical Pathology Table 31.2: Clinical characteristics of porphyrias

Porphyria

Deficient enzyme

Clinical features

Inheritance

Initial test

1. Acute intermittent porphyria (AIP)*

PBG deaminase

Autosomal dominant

2. Variegate porphyria

Protoporphyrinogen oxidase

Urinary PBG; urine becomes brown, red, or black on standing Urinary PBG

3. Hereditary coproporphyria

Coproporphyrinogen oxidase

4. Congenital erythropoietic porphyria

Uroporphyrinogen cosynthase

5. Porphyria cutanea tarda*

Uroporphyrinogen decarboxylase

Acute neurovisceral attacks; triggering factors+ (e.g. drugs, diet restriction) Acute neurovisceral attacks + skin fragility, bullae Acute neurovisceral attacks + skin fragility, bullae Onset in infancy; skin fragility, bullae; extreme photosensitivity with mutilation; red teeth and urine (pink red urinestaining of diapers) Skin fragility, bullae

6. Erythropoietic protoporphyria*

Ferrochelatase

Acute photosensitivity

Autosomal dominant

Autosomal dominant

Urinary PBG

Autosomal recessive

Urinary/fecal total porphyrins; ultraviolet fluorescence of urine, feces, and bones

Autosomal dominant (some cases) Autosomal dominant

Urinary/fecal total porphyrins Free erythrocyte protoporphyrin

Disorders marked with * are the three most common porphyrias. PBG: Porphobilinogen

cutanea tarda, erythropoietic protoporphyria (most cases), and hereditary coproporphyria. Autosomal recessive porphyrias include: congenital erythropoietic porphyria, erythropoietic protoporphyria (few cases), and ALAdehydratase porphyria (plumboporphyria).

LABORATORY DIAGNOSIS Porphyria can be diagnosed through tests done on blood, urine, and feces during symptomatic period. Timely and accurate diagnosis is required for effective management of porphyrias. Due to the variability and a broad range of clinical features, porphyrias are included under differential diagnosis of many conditions. All routine hospital laboratories usually have facilities for initial investigations in suspected cases of porphyrias; laboratory tests for identification of specific type of porphyrias are available in specialized laboratories. Initial Studies In suspected acute porphyrias (acute neurovisceral attack), a fresh randomly collected urine sample (10-20 ml) should

be submitted for detection of excessive urinary excretion of porphobilinogen (PBG) (Fig. 31.2). In AIP, urine becomes red or brown on standing (Fig. 31.3). In suspected cases of cutaneous porphyrias (acute photosensitivity without skin fragility), free erythrocyte protporphyrin or FEP in EDTA blood (for diagnosis of erythrocytic protoporphyria) and for all other cutaneous porphyrias (skin fragility and bullae), examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma for excess porphyrins are necessary (Fig. 31.4 and Table 31.2). Apart from diagnosis, the detection of excretion of a particular heme intermediate in urine or feces can help in detecting site of defect in porphyria. Heme precursors up to coproporphyrinogen III are water-soluble and thus can be detected in urine. Protoporphyrinogen and Protoporphyrin are insoluble in water and are excreted in bile and can be detected in feces. All samples should be protected from light. Samples required are (i) 10-20 ml of fresh random urine sample without any preservative; (ii) 5-10 g wet weight of fecal sample, and (iii) blood anticoagulated with EDTA.

Laboratory Tests in Porphyrias

Fig. 31.2: Evaluation of acute neurovisceral porphyria

Fig. 31.3: Red coloration of urine on standing in acute intermittent porphyria

Fig. 31.4: Evaluation of cutaneous porphyrias

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Essentials of Clinical Pathology Table 31.3: Diagnostic patterns of concentrations of heme precursors in acute porphyrias

Porphyria

Urine

Feces

Acute intermittent porphyria

PBG, Copro III



Variegate porphyria

PBG, Copro III

Proto IX

Hereditary coproporphyria

PBG, Copro III

Copro III

PBG: Porphobilinogen; Copro III: Coproporphyrinogen III; Proto IX: Protoporphyrin IX

Table 31.4: Diagnostic patterns of concentrations of heme precursors in cutaneous porphyrias Porphyria

Urine

Feces

Erythrocytes

Congenital erythropoietic porphyria

Uro I, Copro I

Copro I



Porphyria cutanea tarda

Uroporphyrin

Isocopro



Erythropoietic protoporphyria





Protoporphyrin

Uro I: Uroporphyrinogen I; Copro I: Coproporphyrinogen I; Isocopro: Isocoproporphyrinogen

Test for Porphobilinogen in Urine

Tests for Porphyrins in Erythrocytes and Plasma

Ehrlich’s aldehyde test is done for detection of PBG. Ehrlich’s reagent (p-dimethylaminobenzaldehyde) reacts with PBG in urine to produce a red color. The red product has an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. Since both urobilinogen and porphobilinogen produce similar reaction, further testing is required to distinguish between the two. Urobilinogen can be removed by solvent extraction. (See WatsonSchwartz test in Chapter 1: Examination of Urine). Levels of PBG may be normal or near normal in between attacks. Therefore, samples should be tested during an attack to avoid false-negative results.

Visual examination for porphyrin fluorescence, and solvent fractionation and spectrophotometry have now been replaced by fluorometric methods.

Test for Total Porphyrins in Urine Total porphyrins can be detected in acidified urine sample by spectrophotometry (Porphyrins have an intense absorbance peak around 400 nm). Semiquantitative estimation of porphyrins is possible. Test for Total Porphyrins in Feces Total porphyrins in feces can be determined in acidic extract of fecal sample by spectrophotometry; it is necessary to first remove dietary chlorophyll (that also absorbs light around 400 nm) by diethyl ether extraction.

Further Testing If the initial testing for porphyria is positive, then concentrations of porphyrins should be estimated in urine, feces, and blood to arrive at specific diagnosis (Tables 31.3 and 31.4) In latent porphyrias and in patients during remission, porphyrin levels may be normal; in such cases, enzymatic and DNA testing is necessary for diagnosis. If porphyria is diagnosed, then it is necessary to investigate close family members for the disorder. Positive family members should be counseled regarding triggering factors.

BIBLIOGRAPHY 1. Deacon AC, Elder GH: Frontline tests for the investigation of suspected porphyria. J Clin Pathol 2001; 54; 500-7. 2. Crook MA: Clinical chemistry and metabolic medicine. Seventh edition. London. Edward Arnold, 2006. 3. Thadani H, Deacon A, Peters T: Diagnosis and management of porphyria. BMJ 2000; 320; 1647-51.

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Automation in Hematology

AUTOMATED HEMATOLOGY ANALYZER Automation is a process of replacement of tasks hitherto performed by humans by computerized methods. Until recently, hematological tests were performed only by manual methods. These methods, though still performed in many peripheral laboratories, are laborintensive, and involve use of hemocytometers (counting chambers), centrifuges, Wintrobe tubes, photometers, and stained blood smears. Hematology cell analyzers can generate the blood test results rapidly and also perform additional tests not possible by manual technology. Both manual and automated laboratory techniques have advantages and disadvantages, and it is unlikely that one will completely replace the other. Advantages of Automated Hematology Analyzer • Speed with efficient handling of a large number of samples • Accuracy and precision in quantitative blood tests • Ability to perform multiple tests on a single platform • Significant reduction of labor requirements • Invaluable for accurate determination of red cell indices. Disadvantages of Automated Hematology Analyzer • Flags: Flagging of a laboratory test result demands labour-intensive manual examination of a blood smear • Comments on red cell morphology cannot be generated. Abnormal red cell shapes (such as fragmented cells) cannot be recognized. • Erroneously increased or decreased results due to interfering factors • Expensive with high running costs.

Automated hematology analyzers are of two main types: • Semi-automated: Some steps like dilution of blood sample are performed by the technologist; can measure only a few parameters • Fully automated: Require only anticoagulated blood sample; measure multiple parameters.

PRINCIPLES OF WORKING Automated hematology analyzers work on different principles: • Electrical impedance • Light scatter • Fluorescence • Light absorption • Electrical conductivity. Most analyzers are based on a combination of different principles. 1. Electrical impedance: This is the classic and timetested technology for counting cellular elements of blood. As this method of cell counting was first developed by Coulter Electronics, it is also called as Coulter principle (Fig. 32.1). Two electrodes placed in isotonic solutions are separated by a glass tube having a small aperture. A vacuum is applied and as a cell passes through the aperture, flow of current is impeded and a voltage pulse is generated. The requisite condition for cell counting by this method is high dilution of sample so that minimal numbers of cells pass through the aperture at one point of time. There are two electrodes on either side of the aperture; as the solution in which the cells are suspended is an electrolyte solution, an electric current is generated between the two electrodes. When a cell passes through this narrow aperture across which a current is flowing, change in electrical

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Essentials of Clinical Pathology

2.

Fig. 32.1: Coulter principle of electrical impedance

3. resistance (i.e. momentary interruption of electrical current between the two electrodes) occurs. A small pulse is generated due to a temporary increase in impedance. This pulse is amplified, measured, and counted. The height of the pulse is proportional to cell volume. The width of the pulse corresponds with the time required for the cell to traverse the aperture. Cells that do not pass through the center of the aperture generate a distorted pulse that is not representative of the cell volume. Some analyzers use hydrodynamic focusing to force the cells through the central path so that all cells take the same path for volume measurement. An anticoagulated whole blood sample is aspirated into the system, divided into two portions, and mixed with a diluent. One dilution is passed to the red cell aperture bath (for red cell and platelet counting), and the other is delivered to the WBC aperture bath (where a reagent is added for lysis of red cells and release hemoglobin; this portion is used for leukocyte counting followed by estimation of hemoglobin). Particles between 2-20 fl are counted as platelets,

4.

5.

while those between 36-360 fl are counted as red cells. Hemoglobin is estimated by light transmission at 535 nm. Light scatter: Each cell flows in a single line through a flow cell. A laser device is focused on the flow cell; as the laser light beam strikes a cell it is scattered in various directions. One detector captures the forward scatter light (forward angle light scatter or FALS) that is proportional to cell size and a second detector captures side scatter (SS) light (90°) that corresponds to the nuclear complexity and granularity of cytoplasm. This simultaneous measurement of light scattered in two directions is used for distinguishing between granulocytes, lymphocytes, and monocytes. Fluorescence: Cellular fluorescence is used to measure RNA (reticulocytes), DNA (nucleated red cells), and cell surface antigens. Light absorption: Concentration of hemoglobin is measured by absorption spectrophotometry, after conversion of hemoglobin to cyanmethemoglobin or some other compound. In some analyzers, peroxidase cytochemistry is used to classify leukocytes; the peroxidase activity is determined by absorbance. Electrical conductivity: Some analyzers use conductivity of high frequency current to determine physical and chemical composition of leucocytes for their classification.

PARAMETERS MEASURED BY HEMATOLOGY ANALYZERS Parameters measured by hematology analyzers and their derivation are shown in Tables 32.1 and 32.2. Most automated hematology analyzers measure red cell count, red cell indices (mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration), hemoglobin, hematocrit, total leukocyte count, differential leukocyte count (three-part or five-part), and platelet count.

Table 32.1: Parameters measured by hematology analyzers Parameters measured by most analyzers

Parameters measured by some analyzers

• • • • • • • •

• • • • • •

RBC count Hemoglobin Mean cell volume Mean cell hemoglobin Mean cell hemoglobin concentration WBC count WBC differential Platelet count

Red cell distribution width Reticulocyte count Reticulocyte hemoglobin content Mean platelet volume Platelet distribution width Reticulated platelets

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Table 32.2: Parameters reported by hematology analyzers Parameters measured directly or derived through histogram

Parameters measured through calculation

• • • • • • • • •

• Hematocrit • Mean cell hemoglobin • Mean cell hemoglobin concentration

RBC count Mean cell volume (Derived from RBC histogram) Red cell distribution width (Derived from RBC histogram) Hemoglobin Reticulocyte count WBC count Differential WBC count (Derived through WBC histogram) Platelet count Mean platelet volume (Derived from platelet histogram)

Estimation of Hemoglobin Hemoglobin is measured directly by a modification of cyanmethemoglobin method (all hemoglobins are converted to cyanmethemoglobin by potassium ferricyanide; cyanmethemoglobin has a broad absorbance peak at 540 nm). Some analyzers use a nonhazardous reagent such as sodium lauryl sulphate. A non-ionic detergent is added for rapid red cell lysis and to minimize turbidity caused by cell membranes and plasma lipids. Estimation of Red Blood Cell Count and Mean Cell Volume (MCV) Red cell count and cell volume are directly measured by aperture impedance or light scatter analysis. In a red cell histogram, cell numbers are plotted on Y-axis, while cell volume is indicated on X-axis (Fig. 32.2). The analyzer counts those cells as red cells volume of which ranges between 36 fl and 360 fl. MCV is used for morphological classification of anemia into microcytic, macrocytic, and normocytic types.

Fig. 32.2: Diagrammatic representation of red cell histogram obtained by aperture impedance. The analyzer counts cells between 36 fl and 360 fl as red cells. Although leukocytes are present and counted along with red cells in the diluting fluid, their number is not statistically significant. Only if leukocyte count is markedly elevated (>50,000/μl), histogram and the red cell count will be affected. Area of the peak between 60 fl and 125 fl is used for calculation of mean cell volume and red cell distribution width. Abnormalities in red cell histogram include: (1) Left shift of the curve in microcytosis, (2) Right shift of the curve in macrocytosis, and (3) Bimodal peak of the curve in double (dimorphic) population of red cells

Estimation of MCH, MCHC, and Hematocrit These parameters are obtained indirectly through calculations.

Mean cell volume (fl) Hematocrit (%) = ——————————— Red cell count (106/μl)

Hemoglobin (g/l) Mean cell hemoglobin (pg) = ——————————— Red cell count (106/μl)

Estimation of Red Cell Distribution Width (RDW)

Hemoglobin (g/dl) Mean cell hemoglobin = ——————————— Hematocrit (%) concentration (g/dl)

RDW is a quantitative measure of variation in sizes of red cells and is expressed as coefficient of variation of red cell size distribution. It is equivalent to anisocytosis observed on blood smear. It is derived from red cell histogram in some analyzers. RDW is usually elevated

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in iron deficiency anemia, but not in β-thalassemia minor and anemia of chronic disease (other causes of microcytic anemia). However, this distinction is not absolute and there is a significant overlap between values among patients. Raised RDW requires examination of blood smear. Among the red cell values generated by the analyzer (red cell count, hemoglobin, hematocrit, MCV, MCH, MCHC, and RDW), most important for decisionmaking are hemoglobin, hematocrit, and MCV. WBC Differential Hematology analyzers can either generate a 3-part differential (differential count reported as lymphocytes, monocytes, and granulocytes) or a 5-part differential (lymphocytes, monocytes, neutrophils, eosinophils, and basophils). The 3-part differential counting is based on electrical impedance volume measurement of leukocytes. In volume histogram for WBCs, approximate numbers of cells are plotted on Y-axis and cell size on X-axis. Those cells with volume 35-90 fl are designated as lymphocytes, cells with volume 90-160 fl as mononuclear cells, and cells with volume 160-450 fl as neutrophils (Fig. 32.3). Any deviation from the expected histogram is flagged by the analyzer, mandating review of blood smear. A large proportion of 3-part differential counts are ‘flagged’ to avoid missing abnormal cells.

Instruments measuring a 5-part differential work on a combination of different principles, e.g. light scatter, impedance, and electrical conductivity, a combination of light scatter, peroxidase staining, and resistance of basophils to lysis in acid buffer, etc. Platelet Count Platelets are difficult to count because of their small size, marked variation in size, tendency to aggregation, and overlapping of size with microcytic red cells, cellular fragments, and other debris. In hematology analyzers, this difficulty is addressed by mathematical analysis of platelet volume distribution so that it corresponds to lognormal distribution. Platelets are counted by electrical impedance method in the RBC aperture, and a histogram is generated with platelet volume on X-axis and relative cell frequency on Y-axis (Fig. 32.4). Normal platelet histogram consists of a right-skewed single peak. Particles greater than 2 fl and less than 20 fl are classified as platelets by the analyzer. Two other platelet parameters can be obtained from platelet histogram using computer technology: mean platelet volume (MPV) and platelet distribution width (PDW). Some analyzers can generate another parameter called as reticulated platelets. MPV refers to the average size of platelets and is obtained from mathematical calculation. Normal MPV

Fig. 32.3: Diagrammatic representation of WBC histogram. WBC histogram analysis shows relative numbers of cells on Y-axis and cell size on X-axis. The lytic agent lyses the cytoplasm that collapses around the nucleus causing differential shrinkage. The analyzer sorts the WBCs according to the nuclear size into 3 main groups (3-part differential): Cells with 35-90 fl volume are designated as lymphocytes, cells with 90-160 fl volume are designated as monocytes, and cells with 160-450 fl volume are designated as neutrophils. Abnormalities in WBC histogram include: (1) Peak to the left of lymphocyte peak: Nucleated red cells, (2) Peak between lymphocytes and monocytes: Blast cells, eosinophilia, basophilia, plasma cells, and atypical lymphocytes, and (3) Peak between monocytes and neutrophils: Left shift

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graph are determined by the degree of side scatter, degree of forward scatter, light absorption by the cell, and cytochemical staining (if used). The forward angle light scatter (FALS) is represented on Y-axis, and the side scatter (SS) is represented on X-axis. Low FALS and low SS are indicative of lymphocytes; with subsequent increasing FALS and SS, monocytes, neutrophils, and lastly eosinophils are designated in the graph. Counting of basophils is based on a different technology. Fig. 32.4: Diagrammatic representation of normal platelet histogram: Counting and sizing of platelets by electrical impedance method occurs in the RBC aperture. The counter designates particles between sizes 2 fl and 20 fl as platelets. Abnormalities in platelet histogram result from interferences such as cytoplasmic fragments (peak at left end of histogram) or severely microcytic red cells and giant platelets (peak at right end of histogram)

is 7-10 fl. Increased MPV (> 10 fl) results from presence of immature platelets in circulation; peripheral destruction of platelets stimulates megakaryocytes to produce such platelets (e.g. in idiopathic thrombocytopenic purpura). Decreased MPV (< 7 fl) is due to presence of small platelets in circulation (in conditions associated with reduced production of platelets in bone marrow). PDW is analogous to RDW and is a measure of variation in size of platelets (normal 100/μl in blood.

BIBLIOGRAPHY

Fig. 36.7: Principle of Venereal Disease Research Laboratory (VDRL) slide test for syphilis

1. Chatterjee K, Sen A. Step by Step Blood Transfusion Services. A Practical Manual on the Technical and Clinical Aspects. New Delhi: Jaypee Brothers and Medical Publishers (P) Ltd, 2006. 2. Kawthalkar SM. Essentials of Haematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006.

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Compatibility Test (Cross-match) When the recipient’s ABO and Rh blood groups are determined, the donor blood unit that is ABO and Rh compatible is selected, and compatibility test is carried out. The purpose of compatibility test is to prevent the transfusion of incompatible red cell units and thus avoidance of hemolytic transfusion reaction in the recipient. Compatibility test detects (i) major ABO grouping error, and (ii) most clinically significant antibodies reactive against donor red cells. There are two types of cross-match: major cross-match (testing recipient’s serum against donor’s red cells) and minor cross-match (testing donor’s serum against recipient’s red cells). However, minor cross-match is considered as less important since antibodies in donor blood unit get diluted or neutralized in recipient’s plasma. Also, if antibody screening and identification is being carried out, minor cross-matching is not essential. Therefore, only the red cells from the donor unit are tested against the recipient’s serum and the name compatibility test has replaced the term cross-matching. For transfusion of platelets or fresh frozen plasma, cross-matching is not required. However, fresh frozen plasma should be ABO-compatible. A full cross-matching procedure consists of: • Immediate spin cross-match at room temperature, and • Indirect antiglobulin test at 37°C.

IMMEDIATE SPIN CROSS-MATCH • The purpose of this test is to detect ABO incompatibility. Equal volumes of 2% saline suspension of red cells of donor and recipient’s serum are mixed, incubated at room temperature for 5 minutes, and centrifuged. Agglutination or hemolysis indicates incompatibility (Fig. 37.1).

Fig 37.1: Immediate spin cross-match

Causes of False-negative Test 1. A2B donor red cells and group B recipient serum. 2. Rapid complement fixation of potent ABO antibodies with bound complement interfering with agglutination. Causes of False-positive Test 1. Rouleaux formation 2. Cold-reactive antibodies: If agglutination disappears by keeping the tube at 37°C for 10 minutes, presence of cold agglutinins is confirmed.

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recipient’s serum. Agglutination or hemolysis at any stage is indicative of incompatibility.

EMERGENCY CROSS-MATCH If blood is required urgently, ABO and Rh grouping are carried out by rapid slide test and immediate spin cross match (i.e. the first stage of cross match) is performed (to exclude ABO incompatibility). If the blood unit is compatible, then after issuing it, remaining stage of the cross-match is completed. If any incompatibility is detected, the concerned physician is immediately informed about the incompatibility detected.

ANTIBODY SCREENING AND IDENTIFICATION

Fig 37.2: Indirect antiglobulin test for detection of clinically significant IgG antibodies

INDIRECT ANTIGLOBULIN TEST Saline-suspended red cells of the donor after being incubated in patient’s serum are washed in saline and antiglobulin reagent is added. Following re-centrifugation, examine for agglutination or hemolysis (Fig. 37.2). This test detects most of the clinically significant IgG antibodies. If agglutination or hemolysis is not observed in any of the above stages, donor unit is compatible with

Screening for unexpected or irregular antibodies is done during pre-transfusion testing in recipient’s serum and in donor’s blood. In this test, serum of the recipient is tested against a set of three group O screening cells of known antigenic type. If unexpected antibodies are detected, then they are identified and blood unit that lacks the corresponding antigen is selected for compatibility test.

BIBLIOGRAPHY 1. Kawthalkar SM. Essentials of Hematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006. 2. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Hematology (9th Ed). London: Churchill Livingstone, 2001.

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Adverse Effects of Transfusion Blood transfusion is a life saving procedure in an appropriate setting and there are no side-effects in majority of cases. However, it is a potentially harmful procedure and every recipient of transfusion is at risk of an adverse reaction. It should be prescribed only if there is a definite clinical indication. This is because even with best possible blood banking standards, transmission of infections or other complications can occur. Adverse effects of transfusion are listed in Table 38.1. Main causes of transfusion-related deaths are: • Immediate acute hemolytic transfusion reaction (ABO incompatibility) • Pulmonary edema and congestive heart failure (circulatory overload) • Bacterial contamination of blood unit • Transfusion of physically damaged red cells (e.g. by heat, cold) • Transfusion-associated graft vs. host disease

the recipient. It results from transfusion of ABOmismatched blood to the recipient due most commonly to a clerical error. Most severe reaction occurs if group A blood is transfused to a group O recipient. Pathophysiology consists of antigen-antibody reaction that leads to complement activation and intravascular hemolysis. This causes hypotension, shock, acute renal failure, and disseminated intravascular coagulation (Fig. 38.1). Signs and symptoms (that appear within minutes of starting transfusion) include fever, pain at the infusion site, loin pain, tachycardia, hemoglobinuria, and hypotension. In anesthetized patients, bleeding and hypotension are the only indications.

ACUTE HEMOLYTIC TRANSFUSION REACTION This is a medical emergency and results from intravascular destruction of donor red cells by antibodies in Table 38.1: Adverse effects of transfusion Immediate

Delayed

1. Acute hemolytic transfusion reaction 2. Febrile non-hemolytic transfusion reaction 3. Allergic reactions 4. Anaphylactic reactions 5. Transfusion-associated lung injury 6. Volume overload 7. Bacterial contamination of donor unit

1. Delayed hemolytic transfusion reaction 2. Transmission of infections 3. Iron overload 4. Graft vs. host disease 5. Post-transfusion purpura

Fig. 38.1: Pathophysiology of acute hemolytic transfusion reaction

Adverse Effects of Transfusion Laboratory features are: • Hemoglobinemia (pink coloration of plasma after centrifugation of post-transfusion sample) • Positive direct antiglobulin test • Hemoglobinuria • Schistocytes (fragmented red cells) and spherocytes on blood smear • Elevated indirect serum bilirubin

FEBRILE NON-HEMOLYTIC TRANSFUSION REACTION This is the most common transfusion reaction. It occurs in about 1% of all transfusions and is defined as an unexplained rise of temperature of atleast 1°C during or shortly after transfusion. It is caused by the release of pyrogenic cytokines from white cells (during storage of blood unit or following transfusion due to reaction of alloantibodies with white cells of donor). This reaction is common in multiply-transfused patients. Signs and symptoms include fever, chills, and tachycardia. Diagnosis depends on exclusion of other causes of febrile transfusion reaction. Transfusion reactions presenting with fever are shown in Figure 38.2.

BACTERIAL CONTAMINATION OF DONOR UNIT Transfusion of an infected blood product is more common with platelet concentrates since platelets are stored at a higher temperature (20-24°C) that promotes multiplication of contaminating bacteria. Organisms depend on the nature of blood product. Platelets are usually contaminated with gram-positive cocci, while red cells are contaminated with Yersinia enterocolitica, Escherichia coli, or Pseudomonas species. Signs and symptoms include high grade fever with rigors, hypotension, and shock. Laboratory studies include inspection of blood bag for discoloration, and

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Gram staining and culture of blood from the blood bag and from the recipient. Direct antiglobuin test is negative.

TRANSFUSION-ASSOCIATED LUNG INJURY This is an acute respiratory disorder that manifests with fever, chills, dyspnea, and dry cough. X-ray shows diffuse pulmonary infiltrates. One probable mechanism is reaction of anti-HLA or anti-neutrophil antibodies in donor blood with leukocytes of the recipient leading to the formation of leukocyte aggregates; these aggregates deposit in pulmonary vasculature and cause increased vascular permeability and pulmonary edema.

DELAYED HEMOLYTIC TRANSFUSION REACTION This is a hemolytic transfusion reaction occurring several days or weeks after transfusion. This occurs in individuals who have been sensitized to a red cell antigen by a previous transfusion or pregnancy so that the antibody is present in a low titer. On re-exposure, there is a secondary IgG immune response and mainly exravascular hemolysis. This reaction is typically associated with Kidd antibodies. Signs and symptoms include fever, mild jaundice, and mild anemia. Laboratory features include raised indirect serum bilirubin, spherocytes on blood smear, anemia, and positive direct antiglobulin test. Acute and delayed hemolytic transfusion reactions are compared in Table 38.2.

ANAPHYLACTIC REACTION This rare reaction occurs in IgA-deficient recipients in whom anti-IgA antibodies react with IgA in donor plasma, leading to activation of complement and formation of anaphylatoxins (C3a and C5a). Signs and symptoms include development of acute hypotension, shock, and dyspnoea after transfusion of a few drops of blood.

Fig. 38.2: Transfusion reactions presenting with fever

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Essentials of Clinical Pathology Table 38.2: Comparison of acute and delayed hemolytic transfusion reactions

Parameter

Acute

Delayed

1. Type of antibody 2. Nature of antibody 3. Time of appearance of clinical features after transfusion 4. Site of hemolysis 5. Clinical features

Anti-ABO Naturally-occurring Within minutes

Anti-Kidd Immune After several days or weeks

Intravascular Fever, chills, back pain, acute renal failure, DIC

Extravascular Fever, mild jaundice, anemia

ALLERGIC REACTION This results from type I hypersensitivity reaction to some donor plasma proteins. It is the second most frequently reported transfusion reaction. Signs and symptoms include mild urticaria, rash, and pruritus.

VOLUME OVERLOAD This occurs if transfusion rate is too rapid, or excessive, or if cardiac or renal impairment is present. It causes cardiac failure and lung edema.

IRON OVERLOAD Each unit of blood contains 200 mg of iron. Patients receiving regular transfusion therapy such as those with thalassemia develop manifestations of parenchymal damage due to iron accumulation.

TRANSMISSION OF INFECTIONS Organisms transmissible by transfusion are listed in Table 36.1 (See Chapter 36: Screening Tests for Infections Transmissible by Transfusion). 1. Hepatitis A virus: Hepatitis A is rarely transmitted by transfusion, as the duration of viremia is short. Donors with hepatitis A or who are in close contact with a hepatitis A are deferred for 1 year. 2. Hepatitis B virus: In India, prevalence of hepatitis B virus is 1.5 to 4%, and there are 43 million carriers. Hepatitis B virus, a DNA virus, can be transmitted by both cellular and plasma components. Incubation period is 2-6 months. All blood donations are tested for HBsAg by a sensitive method that has greatly reduced the risk of transmission. During early period of infection, HBsAg may be undetectable; during this window period, antibodies to the hepatitis B core

antigen (anti-HBc) may be the only evidence of disease. HBsAg-positive donors are permanently excluded from donations. Liver diseases caused by hepatitis B virus include: subclinical hepatitis, acute icteric hepatitis, fulminant hepatitis (massive hepatic necrosis), chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 3. Hepatitis C virus: In India, prevalence of hepatitis C virus is reported to be 1.66% and there are 15 million carriers. This RNA virus is the most common cause of transfusion-transmitted hepatitis. It is transmitted by both cellular and plasma components. Incubation period is about 8 weeks. Persistent infection is the rule. Chronic hepatitis is very common (usually mild). Cirrhosis occurs in a minority of patients, and hepatocellular carcinoma occurs in about 10% with cirrhosis. The test used for donor screening is anti-HCV antibody test. From infection till the appearance of anti-HCV positivity (70-80 days), HCV RNA can be detected by PCR testing. 4. Human immunodeficiency virus (HIV): In India, adult prevalence is 0.7%. Two genetically different but closely related viruses are recognized: HIV-1 and HIV-2. Both are RNA retroviruses. Natural history of HIV infection consists of (i) acute, self-limited, “flulike” illness occurring 3-6 weeks after infection, (ii) chronic phase lasting for several years that may be asymptomatic or associated with persistent lymphadenopathy, and (iii) final, full-blown phase with opportunistic infections and malignancies. HIV can be transmitted by both cellular and plasma components. The test used for detection of infection is antiHIV-1/2 antibodies by enzyme immunoassay. To

Adverse Effects of Transfusion reduce the window period from infection to appearance of antibodies from about 22 days to 10 days, nucleic acid testing (NAT) for HIV RNA is recommended. 5. Treponema pallidum: Trasfusion-transmission of syphilis is rare since T. pallidum does not survive in refrigerated storage and is inactivated at 4°C after 4 days; however, fresh blood and platelet concentrates can transmit the organism. The main value of screening test is as a marker of high-risk behavior. 6. Malaria parasites: Malaria parasites are readily transmitted by transfusion. In endemic areas, it is not practical to reject all potential donors with history of malaria in the past. In endemic areas, the only safe prevention is administration of preventive antimalarial drugs to all recipients of transfusion.

COMPLICATIONS ASSOCIATED WITH MASSIVE TRANSFUSION Massive transfusion refers to transfusion of stored blood equivalent to patient’s blood volume in 24 hours. Morbidity and mortality is due to rapid blood loss coupled with transfusion of stored blood. Storage of blood is associated with loss of 2,3diphosphoglycerate, lowering of pH, loss of ATP, loss of platelet function, and depletion of coagulation factors. Microaggregates composed of leukocytes and platelets

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form gradually in stored blood. Rapid transfusion of large volumes of stored blood leads to: • Dilution of platelets and coagulation factors • Hyperkalemia (due to release of potassium from stored red cells) • Hypocalcemia (due to binding of calcium by citrate) • Hypothermia (due to rapid infusion of large amount of cold blood) • Adult respiratory distress syndrome due to migration of microaggregates to lungs.

RECOGNITION AND INVESTIGATION OF A TRANSFUSION REACTION All reactions following blood transfusion should be considered as hemolytic in nature and should be investigated accordingly (Fig. 38.3). 1. Transfusion should be immediately stopped, leaving open intravenous line with normal saline. 2. All paperwork and blood bag should be checked for clerical error. More than 90% of hemolytic transfusions result from a clerical error (i.e. a wrong unit of blood is given to the wrong recipient). 3. Blood bank is informed immediately and the blood bag, administration set, and post-transfusion blood and urine samples should be sent to the blood bank. 4. Evidence of hemolysis: Obtain a post-transfusion blood sample from the recipient, centrifuge, and observe for pink discoloration of overlying plasma (hemoglobinemia); this is the most rapid way of

Fig. 38.3: Immediate management of a suspected transfusion reaction. All reactions should be assumed to be hemolytic and investigated accordingly

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detecting intravascular hemolysis if pre-transfusion sample is normal. Similarly, visual examination of patient’s urine can be done for hemoglobinuria. Blood smear examination will show fragmented red cells and spherocytes. Indirect serum bilirubin is raised. 5. Evidence of blood group incompatibility: Perform a direct antiglobulin test (DAT) on post- and pretransfusion blood samples. Positive DAT on posttransfusion sample (with negative test on pretransfusion sample) is indicative of an immunological hemolytic transfusion reaction. Blood group incompatibility will also be revealed on (i) repeat ABO grouping on recipient’s pre- and post-transfusion samples and on donor unit, and (ii) repeat crossmatching of donor blood against recipient’s pre- and post-transfusion samples.

6. Investigations for detection of complications of hemolytic transfusion reaction: • Tests for disseminated intravascular coagulation: Blood smear, coagulation screen, and test for fibrin degradation products • Tests for acute renal failure: Blood urea, serum creatinine, and serum electrolytes 7. Bacteriological culture if the cause of the acute transfusion is still not clear.

BIBLIOGRAPHY 1. Kawthalkar SM. Essentials of Hematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006. 2. World Health Organization: Blood Transfusion Safety: The Clinical Use of Blood. Geneva: World Health Organization, 2002.

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Blood Components

A single whole blood donation can be separated into different components to provide treatment to more than one patient. One unit of whole blood can be broken down into one unit of packed red cells, one unit of platelets, and one unit of fresh frozen plasma/cryoprecipitate. This practice avoids wastage of collected whole blood (each component is stored at a temperature that is optimal for that component), allows administration of specific replacement therapy, and also avoids transfusion of unnecessary blood elements that are not required by the patient. Terms used in transfusion therapy are shown in Table 39.1. There are two methods for collection of blood for preparation of blood components: 1. Single whole blood donation: Preparation of blood components has been greatly facilitated by the introduction of double and triple bags having closed integral tubing. After collection of a unit of whole blood in the primary bag, blood components can be separated from one another by differential centrifu-

gation due to differences in their specific gravities. After their separation, various components can be transferred from one bag to another in a closed circuit thus avoiding exposure to external environment and maintaining the sterility. Blood should be processed for component separation within 6 hours of collection (Figs 39.1 and 39.2). 2. Apheresis: This is a procedure in which a suitable donor is connected to an automated cell separator machine (that is essentially designed as a centrifuge) through which whole blood is withdrawn, the desired blood component is retained, and the remainder of the blood is returned back to the donor. Depending on the component that is separated and removed, the procedure is called as plateletpheresis, leukapheresis, or plasmapheresis.

WHOLE BLOOD Whole blood is one unit of donor blood collected in a suitable anticoagulant-preservative solution (citrate phosphate dextrose adenine or CPDA-1). Its total volume

Table 39.1: Definitions used in transfusion therapy Blood product A therapeutic substance prepared from human blood Whole blood One unit of non-separated donor blood collected in an appropriate container containing anticoagulant-preservative solution Blood component A constituent separated from whole blood by differential centrifugation or that is obtained directly from donor by apheresis Plasma derivative Human plasma proteins obtained from multiple donor units of plasma under pharmaceutical manufacturing conditions. These products are heat-treated or chemical-treated to inactivate lipid-enveloped viruses. Plasma derivatives like factor concentrates and immunoglobulins can also be prepared by recombinant DNA technology

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Fig. 39.1: Blood components are prepared from a unit of whole blood within 6 hours of collection. Initial light centrifugation separates red cells from platelets and plasma. Heavy centrifugation of platelet-rich plasma separates platelets and plasma

Fig. 39.2: Principle of preparation of blood components from one unit of whole blood

is about 400 ml (350 ml of blood + 49 ml of anticoagulant). It consists of cellular elements and plasma. Whole blood is stored in an approved blood bank refrigerator at 4°-6°C. Shelf life of such blood (collected in CPDA anticoagulant) is 35 days. It does not contain functionally effective platelets and labile coagulation factors (F V and

F VIII). Transfusion of whole blood should commence within 30 minutes of removal from the refrigerator, and should be complete within 4 hours of starting. Transfusion of one unit raises hemoglobin by 1 gm/dl or hematocrit by 3%. Indications and contraindications for whole blood transfusion are given in Table 39.2.

Blood Components Table 39.2: Indications and contraindications for whole blood transfusion Indications • Acute blood loss with hypovolemia • Exchange transfusion in neonates • Non-availability of red cell concentrate or suspension

Table 39.4: Indications for packed red cell transfusion • Anemia: Chronic severe anemia, severe anemia with congestive cardiac failure, anemia in elderly • Acute blood loss (transfused along with a crystalloid or a colloid solution)

Contraindications • Chronic anemia with compromised cardiovascular function

BLOOD COMPONENTS Blood components are listed in Table 39.3.

RED CELL COMPONENTS 1. Packed red cells: Packed red cells are prepared by removing most of the plasma from one unit of whole blood (hematocrit 70-75%). Whole blood is either allowed to sediment overnight in a refrigerator at 26°C or is spun in a refrigerated centrifuge. Supernatant plasma is then separated from red cells in a closed system by transferring it to the attached empty satellite bag. Red cells and a small amount of plasma are left behind in the primary blood bag. Packed red cells have a high viscosity and therefore the rate of infusion is slow. Transfusion of one unit of red cells increases hemoglobin by 1 gm% (or increases hematocrit by 3%). Indications for packed red cells are shown in Table 39.4. 2. Red cells in additive solution (Red cell suspension): These are red cells with minimal residual plasma and an additive solution (SAG-M which contains saline, adenine, glucose, and mannitol). This increases shelf life from 35 days to 42 days. After collection of whole Table 39.3: Blood components Cellular components • Red cells: Packed red cells, red cells in additive solution, leukocyte-poor red cells, washed red cells, frozen red cells, irradiated red cells • Platelets: Platelet concentrate, apheresis platelets • Granulocytes: granulocyte concentrate Plasma components • Fresh frozen plasma • Cryoprecipitate

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3.

4.

5.

6.

blood in the primary collection bag (containing CPDA1), maximum amount of plasma is removed (after centrifugation) and transferred to one satellite bag. The additive solution from the second satellite bag is transferred into the primary collection bag (containing packed red cells) in a closed system. Indications for red cells in SAG-M are similar to those for packed red cells. Leukocyte-poor red cells: Leukocyte-poor red cells contain < 5 × 106 white cells per bag. Methods for leukocyte depletion are (i) leukocyte-reduction filters, and (ii) removal of buffy coat. Indications for leucocyte-poor red cells are: (i) prevention of HLA immunization in patients who are likely to receive allogeneic bone marrow transplantation, (ii) prevention of febrile nonhemolytic transfusion reactions in persons receiving multiple transfusions, and (iii) prevention of transmission of cytomegalovirus. Washed red cells: Red cells can be washed with normal saline to remove plasma proteins, white cells, and platelets. Such red cells are used for IgA-deficient individuals who have developed anti-IgA antibodies, as exposure will lead to anaphylaxis. Frozen red cells: If a cryoprotective agent such as glycerol is added, red cells can be stored frozen for upto 10 years. This method can be used for storage for donor red cells with rare blood groups, for future autologous transfusion, and for individuals who have repeated febrile nonhemolytic transfusion reactions. Irradiated red cells: Gamma-irradiation of red cells inactivates lymphocytes and prevents graft vs. host disease. Irradiated red cells are indicated for intrauterine or premature neonate transfusions, and in individuals with immunodeficiency, and in those receiving blood from first-degree relative donors.

PLATELETS Platelet concentrates can be obtained from single donor units or by plateletpheresis. 1. Platelet concentrate (Random donor platelets prepared from whole blood unit): One unit of whole blood is centrifuged (light spin) to obtain platelet-rich plasma

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(PRP). PRP is then transferred to the attached satellite bag and spun (high spin) to get platelets at the bottom and supernatant plasma. Most of the supernatant is returned back to the primary collection bag or to another satellite bag, leaving behind 50-60 ml of plasma with the platelets. Platelets are stored at 20°-24°C with continuous agitation (in a storage device called platelet agitator). Maximum period of storage is 5 days. One unit of platelet concentrate contains > 45 × 109 platelets. Transfusion of one unit will raise the platelet count in the recipient by about 5000/μl. The usual adult dose is 4-6 units of platelet concentrate (or 1 unit/10 kg of body weight). These units (which are from different donors) are pooled into one bag before transfusion. This dose will raise the platelet count by 20,000 to 40,000/μl. 2. Plateletpheresis (Single donor platelets): In platelet pheresis, a donor is connected to a blood cell separator machine in which whole blood is collected in an anticoagulant solution, platelets are separated and retained, and remaining components are returned back to the donor. With this method, a large number of platelets can be obtained from a single donor (equivalent to 6 units of platelet concentrate). This method is especially suitable if HLA-matched platelets are required (i.e. if patient has developed refractoriness to platelet transfusion due to the formation of alloantibodies against HLA antigens). The usual indications and contraindications for administering platelets are shown in Table 39.5.

PLASMA COMPONENTS The main plasma components are fresh frozen plasma and cryoprecipitate. 1. Fresh frozen plasma (FFP): FFP is prepared from whole blood within 6 hours of collection because after this time labile coagulation factors are lost. Plasma is Table 39.5: Indications and contraindications to platelet transfusions Indications • Bleeding due to decreased platelet production • Bleeding in hereditary disorders of platelet function • Massive blood transfusion Contraindications • Thrombotic thrombocytopenic purpura • Hemolytic uremic syndrome

Table 39.6: Indications for fresh frozen plasma • Multiple coagulation factor deficiencies: liver disease, warfarin overdose, massive blood transfusion • Disseminated intravascular coagulation • Inherited deficiency of a coagulation factor for which no specific replacement therapy is available • Thrombotic thrombocytopenic purpura

separated from whole blood by centrifugation, expressed into the attached satellite bag, and rapidly frozen at –20°C or at lower temperature. FFP contains all the coagulation factors. FFP can be stored for 1 year if temperature is maintained below –25°C. When required for transfusion, FFP is thawed between 30-37°C and then stored in the refrigerator at 2-6°C. Since labile coagulation factors rapidly deteriorate, FFP should be transfused within 2 hours of thawing. Indications for FFP are shown in Table 39.6. 2. Cryoprecipitate: Cryoprecipitate is prepared from plasma that has been freshly separated (within 6 hours of collection) by rapidly freezing it at -20°C or lower and thawing it slowly at 4-6°C. A white flocculent precipitate and plasma are obtained. The mixture is centrifuged and supernatant plasma is removed leaving behind sediment of cryoprecipitate suspended in 10-20 ml of plasma. The unit is then refrozen (-20°C or colder) and can be stored at this temperature for 1 year. When needed, cryoprecipitate is thawed at 30-37°C, required donations are pooled and transfused to the patient. Cryoprecipitate contains F VIII, von Willebrand factor, fibrinogen, F XIII, and fibronectin. Indications for cryoprecipitate are F VIII deficiency (if F VIII concentrate is not available), von Willebrand disease, and deficiency of fibrinogen.

PLASMA DERIVATIVES Plasma derivatives are manufactured by fractionation of large volumes of pooled human plasma. Important plasma derivatives are listed in Table 39.7. 1. Human albumin solutions: Albumin is prepared by cold ethanol fractionation of pooled plasma and is sterilized during manufacture to destroy viruses and bacteria. Albumin is used as a replacement fluid in therapeutic plasma exchange, and for treatment of diuretic-resistant edema of hypoproteinemia.

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Blood Components Table 39.7: Plasma derivatives 1. 2. 3. 4. 5.

Human albumin solutions F VIII concentrate F IX concentrate Prothrombin complex concentrate Immunoglobulins

2. F VIII concentrate: Freeze-dried F VIII concentrate is prepared by fractionation from large pools of fresh frozen plasma. To reduce the risk of transmission of viral infections, it is treated with heat or chemicals during manufacturing process. F VIII concentrate is the treatment of choice for treatment of hemophilia A and severe von Willebrand disease. 3. Prothrombin complex concentrate (PCC): PCC contains factors II, VII, IX, and X, and also protein C and S. Main uses of PCC are (i) deficiency of F IX, (ii) deficiency of F VIII with development of inhibitors against F VIII, and (iii) inherited deficiency of factors II, VII, and X. A serious risk of PCC is thrombotic complications due to the presence of small amounts of activated coagulation factors. 4. Immunoglobulins: Immunoglobulins are obtained by cold ethanol fractionation of large pools of human plasma. They are of two types: specific and nonspecific.

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a. Non-specific immunoglobulins: These are derived from the pooled plasma of non-selected donors. Some indications include (i) passive prophylaxis of viral infections like hepatitis, rubella, and measles, (ii) treatment of hypogammaglobulinaemia, (iii) autoimmune thrombocytopaenic purpura to induce a rise in platelet count, and (iv) neonatal sepsis. b. Specific immunoglobulins: They are obtained from donors who have selected high titer IgG antibodies. Anti-RhD immunoglobulin is prepared from plasma of Rh-negative donors who have produced anti-D following immunization; it is used for prevention of sensitization to RhD antigen in Rhnegative women giving birth to a Rh-positive baby. Other specific immunoglobulins include hepatitis B immune globulin, varicella-zoster immune globulin, and tetanus immune globulin that are used for passive prophylaxis of infections.

BIBLIOGRAPHY 1. Kawthalkar SM. Essentials of Hematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006. 2. World Health Organization. Blood Transfusion Safety: The Clinical Use of Blood. World Health Organization. Geneva, 2002.

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General References 1. Cheesbrough M. District laboratory practice in tropical countries. Part 1 and Part 2. Cambridge. Cambridge University Press, 1998. 2. Crook MA. Clinical chemistry and metabolic medicine (7th Ed). London: Edward Arnold (Publishers) Ltd, 2006. 3. Wallach J. Interpretation of diagnostic tests (7th Ed). Philadelphia: Lippincott Williams and Wilkins, 2000. 4. Mitchell RN, Kumar V, Abbas AK, Fausto N. Robbins and Cotran pathologic basis of disease (7th Ed). Philadelphia: Saunders, 2006. 5. Henry JB. Clinical diagnosis and management by laboratory methods (20th Ed). Philadelphia: WB Saunders Company, 2001. 6. Burtis CA, Ashwood ER. Tietz fundamentals of clinical chemistry (5th Ed). Philadelphia: WB Saunders Company,. 2001. 7. World Health Organization. Manual of basic techniques for a health laboratory (2nd Ed). Geneva: World Health Organization, 2003. 8. Provan D, Krentz A. Oxford Handbook of Clinical and Laboratory Investigation. Oxford. Oxford University Press, 2002. 9. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Haematology (9th Ed). London: Churchill Livingstone, 2001. 10. Provan D, Singer CRJ, Baglin T, Lilleyman J. Oxford Handbook of Clinical Haematology (2nd Ed). Oxford: Oxford University Press, 2004. 11. King M. A medical laboratory for developing countries. London: Oxford University Press, 1973. 12. Kawthalkar SM. Essentials of Haematology. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd, 2006. 13. Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart MJ, Shepherd J. Clinical biochemistry. An illustrated colour test. 3rd Ed. Edinburgh: Churchill Livingstone, 2004. 14. Hoffbrand AV, Pettit JE, Moss PAH. Essential Haematology (4th Ed). Oxford. Blackwell Science Ltd, 2001. 15. Chatterjee K, Sen A. Step by step blood transfusion services. A practical manual on the technical and clinical aspects. New Delhi: Jaypee Brothers and Medical Publishers (P) Ltd, 2006.

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Index A Abnormal coagulation profile 66 crystals 28 red cell arrangement 206 ABO grouping 336 system 329 Abortion 148 Acetest tablet method 15 test 15 Acid load test 37 phosphatase 166 Acquired disorders of coagulation 296 inhibitors of coagulation 296 Activated partial thromboplastin time 303 protein C resistance 311 Acute coronary syndrome 74 hemolytic transfusion reaction 354 hepatitis 66 leukemias 273 myeloid leukemia 9 phase reactants 218 Advantages of automated hematology analyzer 319 Adverse effects of transfusion 354 Albumin 96 Albuminuria 35 Alcoholic liver disease 66 Alicylates 13 Alimentary glycosuria 12 Alkaline picrate reaction 34 Allergic reaction 356 Ammonium chloride loading test 37 magnesium phosphate 27 urate crystals 27 Amorphous urates 27

Amylase 97, 135 Anaphylactic reaction 355 Anemia of chronic disease 248 Angina pectoris 76 Animal inoculation studies 242 Antibodies of ABO system 331 Antibody screening and identification 353 Antigens of ABO system 330 Rh system 332 Antiglobulin test 270 Antiphospholipid syndrome 312 Antisperm antibodies 164 Antithyroid antibodies 143 Anuria 5 Aplastic anemia 249 Apolipoproteins 71 Appearance of sputum 99 Approach to diagnosis of anemia 259 bleeding disorders 297 Apt test 118 Ascaris lumbricoides 112 Ascorbic acid 13 Asexual cycle 229 Assessment of severity of inflammatory disorders 218 Atypical lymphocytes 208 Autohemolysis test 270 Automated hematology analyzer 319 method 301 Automation in hematology 319

B B cell development 175 ontogeny 175 Bacteria 24, 350 Bacterial contamination of donor unit 355 culture 81 proliferation 4

Basal acid output 123, 125 body temperature 156 Basic laboratory studies in anemia 260 Basophilic stippling 205 Basophils 174, 203, 207 Bence Jones proteinuria 11 Benedict’s qualitative solution 13 test 13 Benedict’s test 14 Bentiromide test 134 Bernard Soulier syndrome 293 Bile pigment 16 salts 17 Biliary peritonitis 67 Bilirubin 16, 97 crystals 28 Bioassays 148 Biochemical analysis of semen 165 cardiac marker 74 studies 77 Biosynthesis of thyroid hormones 137 Blast cells 208 Bleeding diathesis 66, 82 disorders 291 Blood 18 ammonia 59 biochemistry 33 components 359, 361 group A 330 AB 330 B 330 O 330 systems 329, 335 mixed CSF 83 smear 200 transfusion 343 urea nitrogen 33 vessel wall 288

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Boiling test 9 Bone marrow aspiration 222, 224, 241 smears 225 examination 266 iron stain 246 trephine biopsy 223, 224, 227 Boric acid 5 Breath CO2 tests 131 hydrogen test 130, 131 tests 134 Broad casts 26 Bromosulphthalein excretion test 63 Bromothymol blue 7 Brugia malayi 239 Bulbourethral glands 159 Butter fat test 129

C Cabot’s rings 206 Calcium carbonate crystals 27 hydrogen phosphate 27 oxalate crystals 27 Capillary tube method 238 Cardiac tamponade 224 Cardiorespiratory compromise 82 Casts 25 Catabolism of steroid hormones 52 Catheter specimen 4 Causes of ascites 95 decreased CSF pressure 83 serum creatinine level 34 total T4 143 erroneous results 323 false negative test 20, 118, 352 positive test 19, 118, 352 female infertility 154 glycosuria 12 hematuria 18 hemoglobinuria 20 increased ALP 61 BUN 34

cell count in CSF 84 CK 78 MCV 213 serum creatinine level 34 total T4 142 urobilinogen in urine 17 ketonuria 14 low MCV 213 malabsorption 118, 127 male infertility 151 pleural effusion 91 positive test 271 prolongation of bleeding time 301 proteinuria 8 Cell 22 count 93, 98 ontogeny 175 Cellular casts 27 elements 3 Cephalic phase 121 Cephalosporins 13 Cerebrospinal fluid 80, 85 Cervical mucus penetration test 166 Charcot joints 39 Cholestasis 63 Cholesterol 69 crystals 28 Christmas disease 294 Chromosomal analysis 152 Chronic leukemias 280 liver disease 66 lymphocytic leukemia 283 myeloid leukemia 281 renal disease 30 Chylomicrons 69 Chylothorax 94 Classic nitroprusside reaction 15 Classical hemophilia 294 Classification of acute leukemias 273 anemias 244 diabetes mellitus 39 intestinal parasites of humans 107 lipoprotein disorders 72 liver function tests 54 renal function tests 30

Clean-catch specimen 4 Clearance of radiolabeled agents 32 Clearance tests 32 Clinitest tablet method 14 Clot formation 84 Clotting time 302 Coagulation system 289 Coccidia 111 Collection methods 4 Collection of blood 179 cerebrospinal fluid 80 donor blood 344 pleural fluid 91 sample 95, 123 semen for investigation of infertility 159 specimen for parasites 105 sputum 99 urine 3 Colorimetric methods 183 Combined acidity 124 Commercial automated culture systems 102 Compatibility test 352 Complete blood count including blood smear 298 Complications associated with massive transfusion 357 Complications of bone marrow aspiration 224 lumbar puncture 82 Composition of normal cerebrospinal fluid in adult 80 urine 3 Computer-assisted semen analysis 166 Concepts of universal donor and recipient 332 Congo red test 126 Conjugated bilirubin 56, 58 hyperbilirubinemia 56 Contraindications to gastric analysis 123 lumbar puncture 82 Coombs’ test 270 Copper reduction methods 13 tablet test 14

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Index Correction for abnormal solute concentration 7 dilution 7 temperature 7 Correction of TLC for nucleated red cells 194 Counting cells 194 Creatine kinase 78 Creatinine clearance 32 Crigler-Najjar syndrome 55 Criteria for selection of blood donors 342 Crohn’s disease 211 Cryptococcus neoformans 87 Cryptosporidium parvum 111 Crystals 27 present in acid urine 27 alkaline urine 27 CSF culture 88 protein electrophoresis 88 Culture 102 Cushing’s syndrome 12 Cyanmethemoglobin method 185 Cyclospora cayetanensis 111 Cystatin C clearance 32 Cysteine crystals 28 Cytogenetic analysis 280 Cytological examination of sputum 103 Cytoplasmic vacuoles 208

D D-dimer test 94 Decrease in glucose 4 Decreased utilization of carbohydrates 14 Deficiency of antithrombin III 312 protein C and S 312 Delayed hemolytic transfusion reaction 355 Demonstration of eggs of A. lumbricoides 112 hookworm eggs 113 trophozoites 110 Dental treatment 343

Detection of antigen in stool samples 111 filarial antigen 239 DNA 240 infection 218 microalbuminuria 11 nucleic acid sequences of malaria parasites 236 parasites 104 postoperative infection 219 Determination of blood group substances 166 Determining cause of anemia 260 Diabetes mellitus 4, 12, 39-41, 44 Diacetyl monoxime urea method 34 Diagnosis of malaria and other parasites in blood 229 renal disease 30 Diagnostic thoracentesis 91 Diazo method 58 Differential leukocyte count 85, 208 Diffusion of iodide 137 Dilution of blood 193 Direct antiglobulin test 270 bilirubin 58 fluorescent antibody assay 111 spectroscopic estimation 58 tests 133 wet mount of CSF 87 Directed donor 342 Disadvantages of automated hematology analyzer 319 Disintegration of cellular elements 4 Disorders of blood vessels 292 lipids 69 platelet 292, 293 thyroid 139 Disseminated intravascular coagulation (DIC) 296 Distal tubular function 36 DNA diagnosis 242 Döhle inclusion bodies 207 Donation interval 342 Donor reactions 345

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Double oxalate 182 Drugs 12 Dubin-Johnson syndrome 56 Duffy antigen 231 D-xylose absorption test 130

E Ectopic pregnancy 147 Eggs of schistosoma haematobium 25 Ehrlich’s aldehyde test 18 Electrical conductivity 320 impedance 319 Emergency cross-match 353 Endocrine component 133 diseases 12 Endocytosis of colloid droplets 138 Endogenous pathway 71 Endometrial biopsy 155 Endoscopic biopsy of ulcer in intestine 110 Entamoeba histolytica 108 Enterobius vermicularis 114 Enzymatic methods 35 Enzyme-linked immunosorbent assay 348 Eosinophil 174, 203, 207 Eosinophilia 210 Epididymis 159 Errors in blood collection 194 filling of chamber 194 pipetting 194 Erythrocyte sedimentation rate 215, 219 Erythrocytic schizogony 230 Erythropoiesis 171 Escherichia coli 6, 7 Esophageal disease 94 Esophagogastroduodenoscopy 126 Estimation of blood glucose 45 creatinine clearance from serum creatinine 33 fecal enzymes 134 fat 129, 135

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glucose in CSF 86 hemoglobin 183, 268, 321, 344 progesterone in mid-luteal phase 157 protein 11 red blood cell count 321 red cell distribution width 321 Ethylene diamine tetra-acetic acid (EDTA) 181 Evaluation of chronic diarrhea 104 clinical features in anemia 260 dysentery 104 malabsorption syndromes 127 Examination of ascitic fluid 96 blood smear 203 bone marrow 220 cerebrospinal fluid 80 feces 104 marrow specimens 225 pleural fluid 93 seminal fluid 160 splenic aspirate 241 sputum 99 smear 101 urine 3 Excretion of metabolic waste products 30 Excretory function 52 Exocrine component 131 Exoerythrocytic schizogony 230 Exogenous pathway 71 Extrahepatic biliary obstruction 66 Exudates 93

F Factor V Leiden 311 Factors affecting erythrocyte sedimentation rate 215 renal function 30 False reactions in ABO grouping 338 Fasting blood glucose 45 glucose 3 plasma glucose 47 Fatty casts 26 Febrile non-hemolytic transfusion reaction 355

Fecal fat 129 osmotic gap 119 pH 119 Female infertility 154 Fern test 156 Ferric chloride test 15, 16 Fibrinolytic system 290 Flagging 323 Floatation techniques 107 Florence test 166 Flow cytometric analysis 272 Fluorescence 320, 325 microscopy 101, 236 Foam test 16 Follicle stimulating hormone 150, 151 Follicular cell and proteolysis 138 Formalin 5 Formation of crystals 4 Fouchet’s test 17 Fractional excretion of sodium 35 test meal 126 Free acidity 124 thyroxine 143 Fresh frozen plasma (FFP) 362 Froin’s syndrome 84 Frozen red cells 361 Functions of cerebrospinal fluid 80 liver 52 Further testing 318

G Gametogony 230 Gasometric method 183 Gastric analysis 121 phase 121 Gastrin 132 Gaucher’s disease 221 General metabolic functions 52 Generalized aminoaciduria 35 Gestational diabetes mellitus 41 trophoblastic disease 148 Giardia intestinalis 110

Gilbert’s syndrome 55 Glanzmann’s thrombasthenia 294 Glitter cells 24 Glomerular proteinuria 8 Glomerulonephritis nephrotic syndrome 3 Glucose 12 6-phosphate dehydrogenase deficiency 231, 255 tolerance test 12 Glycated hemoglobin 47 Glycosuria 6, 35, 43, 48 without hyperglycemia 12 Gmelin’s test 16 Gram’s stained smear 25 smear 87 Granular casts 26 Graves’ disease 144 Gross appearance of cerebrospinal fluid 83

H Haemophilus influenzae 82, 88, 100 Ham’s acidified serum lysis test 271 Hamster egg penetration assay 166 Hay’s surface tension test 17 HDL-cholesterol 74 Heat and acetic acid test 9 Heinz bodies 199, 269 Helminths 112 Hematopoiesis 52, 169 Hemiglobincyanide method 185 Hemocytometer 192 Hemodynamic proteinuria 9 Hemoglobin 20, 172 content 204 D 254 electrophoresis 267 Hemolysis 17 Hemolytic anemia 261 disease of newborn 256 uremic syndrome 293 Hemophilia A 294 B 294 Hemorrhage 18, 224

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Index Hemorrhagic disease of newborn 296 fluid 93, 96 Hemosiderin 20 Heparin 182 induced thrombocytopenia 312 Hepatic injury 59 jaundice 55 schizogony 230 Hepatitis A virus 356 B virus 348, 356 C virus 349, 356 Hepatocellular injury 63 Hereditary disorders of hemoglobin 250 spherocytosis 254 Herniation of brain 82 High density lipoprotein 71 fever 9 performance liquid chromatography 268 Histoplasma capsulatum 212, 226 Histoplasmosis 221 Hodgkin’s disease 211 Hollander’s test 125 Hookworms 113 Hormonal studies 152 Howell-Jolly bodies 199, 205 Human albumin solutions 362 chorionic gonadotropin 146 cycle 229 immunodeficiency virus 343, 349, 356 leukocyte antigens 175 Hydatid cyst of liver 67 Hydrochloric acid 5, 122 Hyperglycemia 43 Hyperhomocysteinemia 312 Hyperosmolar hyperglycemic state (HHS) 44 Hypersegmented neutrophils 208 Hyperthyroidism 139 Hypertonic urine 22 Hypo-osmotic swelling of flagella 166

Hypothyroidism 140 Hysterosalpingo-contrast sonography 157 Hysterosalpingography (HSG) 157

I Identification of adult worms 112 cause of dyslipidemia 74 larvae of S. stercoralis 114 lipid disorder 73 malaria parasites 232 rotavirus 105 trophozoites and cysts on stool 108 Iliac spines 222 Illness 343 Immediate spin cross-match 352 Immune hemolytic anemias 255 thrombocytopenic purpura 292 Immunochemical tests 118 Immunoelectrophoresis 285 Immunofixation electrophoresis 286 Immunological assays 148 Immunophenotyping 278 Importance of HLA antigens 177 Increase in pH 4 Increased destruction in peripheral blood 210 sequestration in spleen 210 soluble transferrin receptor 246 testosterone 157 Indications and limitations of liver function test 53 Indications for abdominal paracentesis 95 bone marrow aspiration 221 biopsy 221 examination 221 gastric analysis 122 hemoglobin 183 lumbar puncture 81 measurement of erythrocyte sedimentation 216 renal biopsy 37 function tests 30

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semen analysis 159 testing for thrombophilia 313 urinalysis 3 Indirect antiglobulin test 271, 353 bilirubin 58 tests 134 urinary tract infection 21 Induction of sputum 99 Infants 4 Infection in spinal canal 82 Infectious disease 157, 343 Infertility 150 Infiltrative diseases 63 Inherited disorders of coagulation 294 thrombophilia 311 Insulin hypoglycemia test 125 resistance syndrome 42 Intermediate density lipoprotein (IDL) 70 Interpretation of liver function tests 63 screening tests 306 Intrahepatic cholestasis 56 Intraperitoneal hemorrhage 67 Intravascular hemolysis 9, 264 Inulin clearance 32 Investigation of male infertility 152 pyrexia of unknown origin 221 Iron deficiency anemia 245 overload 356 staining of bone marrow aspiration smears 226 studies 265 Irradiated red cells 361 Isomorphic red cells 23 Isoniazid 13 Isospora belli 111 Ivy’s method 301

J Jaffe’s reaction 34 Jaundice 54

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K Kala azar 221 Karyopyknotic index (KI) 156 Kawamoto technique 236 Ketones 14 Ketosis 43 Kidney function 30 Klebsiella pneumoniae 100

L Laboratory diagnosis of acute leukemias 275 visceral leishmaniasis 241 cerebrospinal fluid 82 Laboratory testing in thrombophilia 313 Laboratory tests for diagnosis of diabetes mellitus 44 human chorionic gonadotropin 148 lipoprotein disorders 73 screening of diabetes mellitus 47 Laboratory tests in anemia 244 bleeding disorders 288 hematological malignancies 273 management of acute metabolic complication of diabetes mellitus 50 porphyrias 314 thrombophilia 311 Laboratory tests to assess glycemic control 47 long-term risks 49 Lactate dehydrogenase 97 Lactation 343 Lactose tolerance test 130 Laparoscopic liver biopsy 67 Laparoscopy and dye hydrotubation test 157 Latex agglutination tests 87, 88 LDL-cholesterol 74 Leishman stain 202 Leishmania donovani 211, 226 Leucine crystals 29 Leucocyte esterase test 21 Leukemoid reaction 209 Leukocyte-poor red cells 361

Life cycle of Leishmania donovani 240 malaria parasites 229 Light absorption 320 scatter 325 Lipoproteins 69 Liquid biopsy of urinary tract 21 Litmus paper test 7 Liver biopsy 64 disease 296 function tests 52 transplantation 66 Loeffler’s syndrome 210 Loop of Henle 8 Loss of ketone bodies 4 Low-density lipoprotein (LDL) 71 Lugol iodine test 16 Lundh meal 133 Lung diseases 210 Lupus pleuritis 94 Luteinizing hormone 150 Lymphatic filariasis 237 Lymphocytes 175, 207 Lympocytotoxicity test 177 Lysed capillary blood method 238 venous blood method 238

M Macro method 188 Macroangiopathy 44 Macrocytic anemias 261 Macrophages 52 Macrovascular disease 44 Malabsorption syndromes 127 Malaria 229, 343 Male infertility 150 Manual method 192, 299 Mastalgia 155 Maximum acid output (MAO) 123 Mean cell hemoglobin 213 concentration 214 volume 213

Mediastinitis 224 Megaloblastic anemia 246 Membrane filtration 238 Metabolic actions of insulin 39 alterations in diabetes mellitus 43 syndrome 42 Metabolism of thyroid hormones 138 Method of gastric analysis 123 Methods for estimation of BUN 34 erythrocyte sedimentation 216 hemoglobin 183 serum creatinine 34 Methods for HLA antigen typing 177 Micro method 189 Microalbuminuria 11, 35, 49 Microangiopathic hemolytic anemia 258 Microangiopathy 44 Microcytic hypochromic anemia 221, 261 Microfilariae 25 Microhematocrit tube 238 Microplate method 338 Microscopic examination of blood for demonstration 238 urinary sediment 19 Microsporidia 111 Microvascular disease 44 Midstream specimen 4 Miliary tuberculosis 221 Mixed lymphocyte culture 177 reaction 177 Modes of transmission 231 Molecular genetic analysis 280 techniques 177 Molecular methods 102 Monocytes 174, 203, 207 Moraxella catarrhalis 100 Morphological classification of anemia 213 Morphology 275

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Index Morphology of abnormal leukocytes 207 microfilariae on Romanowsky stained 238 normal leukocytes 207 Mosquito cycle 230 Mycobacterium tuberculosis 87, 101 Myeloperoxidase (MPO) 276 Myocardial infarction 219 Myoglobin 20, 78

N Naegleria fowleri 87 Nalidixic acid 13 NBT-PABA test 134 Neimann-Pick disease 221 Neisseria meningitidis 82 Neonatal screening for hypothyroidism 144 Nephropathy 39 Nephrotic syndrome 6 Neurogenic phase 121 Neutropenia 210 Neutrophilia 209 Neutrophils 174, 203 Nitrite test 21 Nitroprusside test 16 Non-cellular casts 26 Non-endocrine diseases 12 Non-specific esterase (NSE) 276 Normal absorption of carbohydrates 127 bilirubin metabolism 52 bone marrow 220 crystals 27 gastric anatomy and physiology 121 pregnancy 146 Normocytic normochromic anemia 261 Numerical abnormalities of leukocytes 209

O Obstructive jaundice 18 OGTT in gestational diabetes mellitus 46 Oligoclonal bands 89 Oliguria 5

Oral contraceptive therapy and pregnancy 312 glucose tolerance test 45 Organ transplantation 177 Organisms 24, 211 Orthostatic proteinuria 3 Osmotic fragility test 269 Ounting chamber with cover glass 192 Oval fat bodies 24 Overflow proteinuria 9 Oxidation of bilirubin to biliverdin 4 urobilinogen to urobilin 4 Oxyhemoglobin method 186

P P. falciparum 231 P. ovale 231 P. vivax 231 Packed cell volume 188 red cells 361 Pancreas 131 Pancreatic disease 12, 94 function tests 131 Pancreolauryl test 134 Pappenheimer bodies 199, 205 Para-aminosalicylic acid 13 Parasite culture 242 Parasites 203, 351 Paroxysmal nocturnal hemoglobinuria 258 Peak acid output 123, 125 Pelger-Huet cells 208 Penicillins 13 Percutaneous blind liver biopsy 66 guided liver biopsy 67 plugged liver biopsy 67 Periodic acid Schiff (PAS) 278 Peripheral blood smear 260 neuropathy 39 Peritoneal fluid 95 pH indicator paper 7 meter 7

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Phosphates 27 Physiologic jaundice of newborn 56 Physiology of hemostasis 288 Plasma cell dyscrasias 9, 283 components 362 derivatives 362 proteins 289 Plasmodium species 351 Platelet 203, 211, 288, 361 concentrate 361 count 299, 322 function analyzer 302 glycoprotein analysis 310 pheresis 362 Pleural biopsy 94 effusion 91 fluid 91 Polychromatic cells 203 Polymerase chain reaction 87, 88, 177 Polymorphonuclear neutrophil 207 Polyuria 5 Postcoital test 165 Posthepatic jaundice 56 Postprandial blood glucose 45 Post-puncture headache 82 Post-renal proteinuria 9 Post-transfusion purpura 293 Postural proteinuria 9 Prediabetes 42 Pre-erythrocytic schizogony 230 Pregnancy 343 test 3, 146 Prehepatic jaundice 55 Preparation of blood smear 200 slides 106 Preservation of urine sample 4 Primary biliary cirrhosis 56, 66 sclerosing cholangitis 56, 66 Primed lymphocyte typing 177 Processing of donor blood 345 marrow specimens 224 Production of vit. D3 30 Professional donors 342

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Prostate 159 Proteins 8, 97 Proteinuria 6 Prothrombin complex concentrate 363 time 59, 302 Protozoa 107 Pseudochylous effusion 93 Pseudomonas aeruginosa 100 Pus cells 23 Pyelonephritis 3 Pyloric part 121 Pyrexia 66

Q Qualitative tests 147 Quantitative buffy coat 236 estimation of fecal fat 118 proteins 10 tests 147 Queckenstedt’s test 81

R Radioactive iodine uptake test 144 Random blood glucose 45 donor platelets 361 Reaction and pH 7 Reagent strip method 7, 14, 18 test 7, 10, 15, 16 Red blood cells 22, 171 Red cell 203 casts 27 components 361 distribution width 214 enzymes 172 inclusions 205, 268 indices 213, 261 membrane 172 suspension 361 Red cells in additive solution 361 Red cells with abnormal shape 205 size 204 staining 204

Reduction of intestinal bacterial flora 18 Refractometer method 7, 58 Regular cycles 155 Regulation of blood pressure 30 Renal biopsy 37 failure 3 function tests 30 transplantation 30 tubular epithelial cell 24, 27 Replacement blood donor 342 Response to oral iron therapy 246 Restriction fragment length polymorphism 177 Reticulocyte count 196261, 323 Retinopathy 39 Reverse passive hemagglutination assay 348 Reye’s syndrome 59 Rh antibodies 335 D grouping 338 system 332 Rheumatoid effusion 94 Role of genetic factors in malaria 231 laboratory tests in diabetes mellitus 44 Rothera’s nitroprusside method 15 test 15 Rouleaux formation 338 Round cells 163 Roundworm 112

S Sahli’s acid hematin method 184 Scattergram 323 Schizogony 229 Screening tests for hemostasis 298 infections transmissible by transfusion 347 Secondary lipoprotein diseases 72 Secretin-cerulin test 133 Sedimentation rate 215 techniques 107

Self-monitoring of blood glucose (SMBG) 48 Semen 159 analysis 152, 159 Seminal fluid 159 vesicles 159 Sensitivity of test 13 Sequence of filling of tubes 182 Serologic methods 237 tests 110, 87, 88 Serological testing 177 Serum albumin 59 alkaline phosphatase 61 aminotransferases 60 bilirubin 56 creatinine 34, 35 ferritin 245 iron 245 lipase 135 protein electrophoresis 59, 284 triglycerides 73 Sexual cycle 230 Sickle cell disorders 253 slide test 266 Sideroblastic anemia 249 Significance of erythrocyte sedimentation rate 215 microalbuminuria 11 Sims-Huhner test 165 Single donor platelets 362 radial immunodiffusion 286 Sites for bone marrow aspiration 222 Skeletal muscle trauma 9 Skin piercing 343 puncture 179 test 242 Slide test 336 Small lymphocytes 203 Solubility test for hemoglobin S 266 Sources of error in manual blood cell count 194 Specific gravity 6 method 186

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Index Specific tests for hemostasis 307 Sperm count 162 function tests 165 morphology 162 motility 161 viability 161 vitality 161 Spermatozoa 24, 159 Sperms 159 Spinnbarkeit test 156 Spinous processes of lumbar vertebra 222 Sporogony 230 Spot check of gastric pH 126 Squamous epithelial cells 24 Stages of erythrocyte sedimentation rate 215 iron deficiency 245 Staining of blood smear 202 Staphylococcus aureus 100 Statistical error 194 Stellar phosphate 27 Sterile’ pyuria 25 Sternum 222 Straw-colored transudative fluid 93 Streptococcus pneumoniae 100 pyogenes 100 Streptomycin 13 Strongyloides stercoralis 113 Subarachnoidal epidermal cyst 82 Subdural hematoma 82 Sucrose hemolysis test 271 Sulfonamide crystals 29 Sulphosalicylic acid test 10 Suspected acute leukemia 221 chronic lymphoid leukemias 221 infections 221 myelodysplastic syndrome 221 myeloproliferative disorders 221 plasma cell dyscrasia 221 storage disorder 221 Synthesis of erythropoietin 30 Synthetic function 52

T T cell development 175 ontogeny 175 Taenia saginata 116 solium 115 Tallqvist hemoglobin chart 184 Tamm-Horsfall protein 8, 25 Telescoped urinary sediment 24 Tense ascites 67 Terminology in flow cytometry 324 Test for fructose 165 malabsorption of fat 118 occult blood in stools 117 pancreatic arylesterase 134 porphobilinogen in urine 318 reducing sugars 119 total porphyrins in feces 318 urine 318 urobilinogen in feces 119 Testes 159 Testicular biopsy 152 Testing for ketone bodies 51 Tests for detection of anti-leishmania antibodies 241 bilirubin in urine 16 blood in urine 19 glucose in urine 13 hemoglobinuria 20 ketones in urine 15 occult blood in feces 117 urobilinogen in urine 18 Tests for evaluate glomerular function 30 tubular function 35 gastric analysis 125 glucose-6-phosphate dehydrogenase deficiency 270 hemoglobin S 266 malabsorption and pancreatic function 127 ovulation 155

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pancreatic function 133 paroxysmal nocturnal hemoglobinuria 271 porphyrins in erythrocytes and plasma 318 Thalassemias 250 Therapeutic thoracentesis 92 Thick blood smear 238 exudative fluid 93 viscous CSF 84 Thoracentesis 91 Thoracoscopy 95 Thrombin time (TT) 305 Thrombopoiesis 178 Thrombotic thrombocytopenic purpura 293 Thymol 5 Thyroid function tests 137, 142 hormones 137 scintiscanning 144 stimulating hormone 142 Thyrotropin releasing hormone stimulation test 143 Tibia 222 Time of collection 3 Titration 124 Toluene 5 Total acidity 124 cholesterol 73 iron binding capacity 245 leukocyte count 84, 192 serum proteins 58 thyroxine 142 Toxic granules 207 Transferrin saturation 245 Transfusion associated lung injury 355 Transitional epithelial cells 24 Transvaginal ultrasonography 157 Transvenous liver biopsy 67 Treponema pallidum 350, 357 Trichomonas vaginalis 25 Trichuris trichiura 113 Triglycerides 69

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Essentials of Clinical Pathology

Triple phosphates 27 Trisodium citrate 182 Troponins 78 True chylous effusion 93 Trypanosomes 87 Tube method 337 Tubeless gastric analysis 126 Tuberculous pleural effusion 94 Tubes for collection of blood 182 Tubular proteinuria 9, 35 Turk solution 84 Types of blood donors 341 liver biopsy 65 Tyrosine crystals 29

U Ultrasonography (USG) 156 Unconjugated bilirubin 58 Unexplained cytopenia 221 Unstable angina 76 Urea clearance 33 Urease-berthelot reaction 34 Uric acid crystals 27 Urinary albumin excretion 49 concentration of sodium 35 Urine bilirubin and urobilinogen 58 osmolality 36 specific gravity 36

Urinometer method 6 Urobilinogen 17 Uses of C-reactive protein 218 PCV 188 red cell indices 213

V Vagina 166 Vaginal cytology 156 Vas deferens 159 Vasectomy 166 Venous blood collection 179 Very low-density lipoproteins (VLDL) 70 Viral hepatitis 343 Viruses 348 Visceral leishmaniasis 240 Viscosity 161 Vitamin B12 and folate 265 K deficiency 296 Volume of donation 343 von Willebrand disease (vWD) 294

W Washed red cells 361 Water deprivation test 36 loading antidiuretic hormone suppression test 36

Waxy cast 26 WBC cytogram 323 Wedge method 200 Westergren method 216, 219 White blood cells 23, 174 cell 203, 206 casts 27 count 96 WHO hemoglobin color scale 185 Wilson’s disease 66 Wintrobe method 188, 218 mixture 182 Wuchereria bancrofti 238

X Xanthochromia 84

Y Yeast cells 25 Yersinia pestis 100

Z Zeta sedimentation ratio (ZSR) 218 Ziehl-Neelsen smear 87 technique 101 Zollinger-Ellison syndrome 122

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