DNA analysis for missing person identification in mass fatalities 9780429253614, 0429253613, 9781306410243, 130641024X, 9781466514287, 1466514280

Table of contents :
Content: Preface
Acknowledgments
About the Author
1 Human Identification through DNA Analysis
1.1 DNA
1.2 Types of DNA Analysis
1.3 A History of DNA Profiling
1.4 Using DNA for Identification of Human Remains
1.5 The DNA Analysis Process
1.6 Explaining the DNA Process to Non-Technical Personnel
Additional Resources
2 Mass Fatalities
2.1 Definition of a Mass Fatality
2.2 Jurisdictional Issues
2.3 Causes of Mass Fatality Incidents
2.4 Mass Fatality Response Overview
2.5 Managing the Mass Fatality Response Operations
2.6 Factors Impacting a Mass Fatality Response. 2.7 Finances and Politics2.8 Availability of Antemortem Records and DNAReference Samples
Additional Resources
3 Postmortem Functions-Body Recovery andMorgue Operations
3.1 Field Operations (Body Recovery)
3.2 Morgue Operations
Additional Resources
Attachment A
Attachment B
4 Antemortem Functions-Family AssistanceOperations
4.1 Function of Family Assistance Operations
4.2 Personnel
4.3 Creating a Reported Missing Case
4.4 Antemortem Information
4.5 Information Technology Support
4.6 Providing Information to the Public
4.7 Financial Assistance
4.8 Notification and Release. 4.9 Grief Support4.10 Family Assistance Centers (FACs)
4.11 Family Assistance Operations Relationship with theMorgue
Additional Resources
Attachment A
5 Identification of Remains
5.1 Identification
5.2 Types of Identification
5.3 Identification of Bodies
5.4 Presentation and Review of Proposed Identification
5.5 Acceptance/Authorization of Identification
5.6 Family Notification of Identification
5.7 Release of Remains and Personal Property
Additional Resources
Attachment A
Attachment B
6 Identification and Collection of BiologicalSamples from Human Remains. 6.1 Special Considerations for Sample Collections6.2 Determining the Best Sample to Collect
6.3 Collecting Multiple Samples
6.4 Establishing DNA Sample Protocol
Additional Resources
7 Identification and Collection of DNA ReferenceSamples
7.1 The Reported Missing
7.2 Chain of Custody
7.3 Reference Sample Types
7.4 Pedigree
7.5 Scheduling Collections
7.6 Collecting Kinship Samples
Additional Resources
8 Application of DNA Technology for HumanIdentification
8.1 DNA Profiling Process Overview
8.2 DNA Extract Assessment
8.3 Amplification Strategies and Considerations. 8.4 DNA Separation and DNA Profile Generation8.5 Emerging DNA Technologies
8.6 Duplicate Testing and Profile Verification
8.7 Options for Testing
Additional Resources
Attachment A
9 DNA Profile Analysis and Interpretation
9.1 Parameters for Acceptable DNA STR Profiles
9.2 Data Review
9.3 Documentation of Data Review
9.4 Case Evaluation, Kinship Screening, and KinshipCalculations
9.5 Kinship Analysis
9.6 Reporting Matches
Additional Resources
10 DNA Sample, Case, and Data Tracking UsingInformation Technology Tools
10.1 Laboratory Information Management System (LIMS).

Citation preview

DNA Analysis for

Missing Person Identification in Mass Fatalities

Amanda C. Sozer, PhD

DNA Analysis for

Missing Person Identification in Mass Fatalities

DNA Analysis for

Missing Person Identification in Mass Fatalities Amanda C. Sozer, PhD

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2014 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20131119 International Standard Book Number-13: 978-1-4665-1428-7 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents

Preface xv Acknowledgments xvii About the Author xix

1

Human Identification through DNA Analysis 1.1 DNA 1.2 Types of DNA Analysis 1.2.1 Autosomal Short Tandem Repeats 1.2.2 Y-STR Haplotype 1.2.3 Mitochondrial Polymorphism 1.3 A History of DNA Profiling 1.4 Using DNA for Identification of Human Remains 1.5 The DNA Analysis Process 1.5.1 Organizing and Planning 1.5.2 Sample Selection and Collection 1.5.3 Extraction/Purification 1.5.4 Amplification 1.5.5 Data Generation/Analysis 1.5.6 Data Interpretation 1.5.7 Profile Comparison 1.5.8 Kinship Analysis 1.5.9 Reporting the Profile Matches 1.6 Explaining the DNA Process to Non-Technical Personnel 1.6.1 Common Questions 1.6.1.1 Why Identify the Remains? 1.6.1.2 How Is the Testing Done? 1.6.1.3 How Long Will the Process Take? 1.6.1.4 How Can I Help Identify My Loved One? 1.6.1.5 What Are the Sources of DNA Samples that Can Be Used? 1.6.1.6 What Are Useful Sources of DNA from the Victim? 1.6.1.7 How Can DNA from Relatives Be Used? 1.6.1.8 Why Might DNA Analysis Not Work? 1.6.1.9 How Much Will Testing Cost? v

1 1 2 2 5 5 8 9 10 11 11 12 13 14 15 16 17 18 18 20 20 20 20 21 21 21 21 21 22

vi

Contents

1.6.1.10 Who Issues the DNA Identification Reports? 22 1.6.1.11 Are THere Any Guidelines for Using DNA? 22 1.6.1.12 Who Will Have Access to My DNA and Profile? 22 Additional Resources 22

2

Mass Fatalities

23

2.1 2.2 2.3 2.4

Definition of a Mass Fatality 23 Jurisdictional Issues 23 Causes of Mass Fatality Incidents 25 Mass Fatality Response Overview 27 2.4.1 Field Operations/Body Recovery 28 2.4.2 Morgue Operations 28 2.4.3 Reporting the Missing and Presumed Deceased 29 2.4.4 Collection of Antemortem Information about the Deceased 30 2.4.5 Comparison of Antemortem and Postmortem Information 31 2.5 Managing the Mass Fatality Response Operations 31 2.6 Factors Impacting a Mass Fatality Response 32 2.6.1 Closed versus Open Events 32 2.6.2 Number of Deceased 33 2.6.3 Rate of Recovery 34 2.6.4 Condition of Human Remains 35 2.6.5 Fragmentation 35 2.6.6 Decomposition 35 2.6.7 Commingling 36 2.7 Finances and Politics 36 2.8 Availability of Antemortem Records and DNA Reference Samples 37 Additional Resources 37

3

Postmortem Functions—Body Recovery and Morgue Operations 3.1

Field Operations (Body Recovery) 3.1.1 Locating Remains 3.1.2 Personnel 3.1.3 Health and Safety Concerns 3.1.3.1 Mass Graves 3.1.3.2 Environmental Hazards 3.1.3.3 Families On-Site

39 39 39 41 43 44 44 44

Contents

3.1.4 Assessing a Disaster Site 3.1.5 Documentation 3.1.5.1 Other Field Documentation 3.1.5.2 Associated and Unassociated Property 3.1.6 Removal of Remains 3.1.6.1 Mass Graves 3.1.6.2 Commingled Remains 3.1.6.3 Personal Items 3.1.6.4 Chain of Custody 3.1.7 Transportation and Storage 3.2 Morgue Operations 3.2.1 Personnel 3.2.2 Safety 3.2.3 Numbering the Human Remains 3.2.4 Morgue Examinations 3.2.4.1 Photography 3.2.4.2 Forensic Pathology 3.2.4.3 Body Radiography 3.2.4.4 Fingerprints 3.2.4.5 Dental 3.2.4.6 Forensic Anthropology 3.2.4.7 DNA 3.2.4.8 Administrative 3.2.5 Documentation 3.2.5.1 Data Management Additional Resources Attachment A Attachment B

4

vii

44 45 46 46 47 48 48 48 48 48 49 49 51 51 52 54 54 54 55 55 55 56 56 56 57 58 58 58

Antemortem Functions—Family Assistance Operations 73 4.1 Function of Family Assistance Operations 4.2 Personnel 4.3 Creating a Reported Missing Case 4.4 Antemortem Information 4.5 Information Technology Support 4.6 Providing Information to the Public 4.7 Financial Assistance 4.8 Notification and Release 4.9 Grief Support 4.10 Family Assistance Centers (FACs) 4.10.1 Reception

73 74 75 76 76 78 79 79 79 80 80

viii

Contents

4.10.2 Child Care/Play Area 80 4.10.3 Private Meeting Rooms 81 4.10.4 Office Space for FAC Staff 81 4.10.5 Nutritional Services 81 4.10.6 Quiet Room 81 4.10.7 Communications Center 81 4.10.8 Security 81 4.10.9 Medical Care 82 4.10.10 DNA Operations 82 4.11 Family Assistance Operations Relationship with the Morgue 82 Additional Resources 83 Attachment A 83

5

6

Identification of Remains

93

5.1 Identification 5.2 Types of Identification 5.2.1 Tentative Identification 5.2.2 Presumptive Identification 5.2.3 Positive Identification 5.3 Identification of Bodies 5.3.1 Appropriate Personnel 5.3.2 Identification Standards and Guidelines 5.4 Presentation and Review of Proposed Identification 5.5 Acceptance/Authorization of Identification 5.6 Family Notification of Identification 5.7 Release of Remains and Personal Property Additional Resources Attachment A Attachment B

93 94 94 94 95 96 96 97 97 98 98 99 99 100 101

Identification and Collection of Biological Samples from Human Remains 6.1 6.2

6.3 6.4

103

Special Considerations for Sample Collections Determining the Best Sample to Collect 6.2.1 Ease of Sample Collection 6.2.2 Ease of DNA Profiling 6.2.3 DNA Profiling Success Collecting Multiple Samples Establishing DNA Sample Protocol 6.4.1 Order of Sample Preference and Sample Quantity 6.4.2 Safety Precautions and Contamination Issues

103 104 105 106 107 108 108 110 110

Contents

6.4.3 Packaging, Labeling, and Storage Additional Resources

7

The Reported Missing Chain of Custody Reference Sample Types 7.3.1 Direct References 7.3.2 Personal Items 7.3.3 Family References/Kinship Samples 7.4 Pedigree 7.5 Scheduling Collections 7.6 Collecting Kinship Samples Additional Resources

9

111 113

Identification and Collection of DNA Reference Samples 115 7.1 7.2 7.3

8

ix

115 116 117 117 118 120 121 123 123 125

Application of DNA Technology for Human Identification 127 8.1

DNA Profiling Process Overview 8.1.1 Sample Receipt and Accessioning 8.1.2 Aliquotting and Sample Evaluation 8.1.3 Preparation of the Sample 8.1.4 DNA Extraction Methods 8.2 DNA Extract Assessment 8.3 Amplification Strategies and Considerations 8.3.1 General Considerations 8.4 DNA Separation and DNA Profile Generation 8.4.1 DNA Profile Generation 8.5 Emerging DNA Technologies 8.6 Duplicate Testing and Profile Verification 8.7 Options for Testing 8.7.1 Selecting a Laboratory Additional Resources Attachment A

127 128 129 130 130 130 131 131 133 133 133 134 134 134 136 137

DNA Profile Analysis and Interpretation

147

9.1  Parameters for Acceptable DNA STR Profiles 9.1.1 Allele Sizing 9.1.2 Peak Morphology 9.1.2.1 Spurious Peaks (Background, Stutter, Dye Blobs, Spikes, –A, Pull-Up)

147 148 150 150

x

Contents

9.2

Data Review 153 9.2.1 Evaluation of Controls 153 9.2.2 Evaluation of Allelic Ladders 157 9.2.3 Evaluation of Internal Lane Standard (ILS) 157 9.2.4 Evaluation of the DNA Profiles from Tested Samples 157 9.2.5 Degradation 158 9.2.6 Allelic Dropout 159 9.2.7 Tri-Allelic Patterns 159 9.2.8 Microvariants 159 9.3 Documentation of Data Review 160 9.4 Case Evaluation, Kinship Screening, and Kinship Calculations 162 9.4.1 Population Databases 162 9.4.2 Sample Checks 164 9.4.2.1 Elimination Sample Check 164 9.4.2.2 Duplicate Sample Verification 164 9.4.2.3 Amelogenin 164 9.4.3 Reference Sample Validation 165 9.4.3.1 Kinship Reference Samples 165 9.4.3.2 Relationship Verification 165 9.4.3.3 Kinship Simulation 165 9.4.3.4 Direct Reference Samples 166 9.4.3.5 Relationship Verification 166 9.4.4 Personal Item Samples 166 9.4.4.1 Profile Quality 166 9.4.4.2 Elimination Samples 166 9.4.4.3 Mixtures 166 9.4.4.4 Relationship Verification 167 9.4.5 Screening 167 9.4.6 Direct Matches 167 9.4.7 Calculations 167 9.4.8 Incomplete Loci 169 9.5 Kinship Analysis 169 9.5.1 Likelihood Ratio 169 9.5.2 Prior Odds 170 9.5.3 Mutations 172 9.5.4 Unexpected Non-Relatedness 173 9.6 Reporting Matches 174 9.6.1 Special Situations 174 Additional Resources 175

Contents

10

DNA Sample, Case, and Data Tracking Using Information Technology Tools

xi

177

10.1 Laboratory Information Management System (LIMS) 177 10.2 Assigning a Reported Missing Case 178 10.3 Collection of Samples from Unidentified Human Remains 178 10.4 Collection Reference Samples 179 10.5 Tracking the Sample during Testing and Data Analysis 181 10.6 DNA Profile Interpretation and Management 184 10.7 Report Writing 185 10.8 Communication Logs 186 10.9 Security 186 10.10 Quality Control 187 10.11 Work Lists 187 10.12 Maintaining Fiscal Responsibility 187 10.13 Acquiring the LIMS 189 Additional Resources 189 Appendix A 189

11

Implementing and Maintaining a Quality DNA Program 195 11.1 Accreditation and Its Role in International Recognition 195 11.1.1 Standards 196 11.1.2 Accreditation Bodies 197 11.1.3 Other Resources for Guidance 197 11.1.4 Quality System Elements 198 11.1.4.1 Organization and Management 198 11.1.5 Document Control 200 11.1.6 Review of Requests, Tenders, and Contracts 201 11.1.7 Subcontracting 201 11.1.8 Purchasing Services and Supplies 201 11.1.9 Customer Service and Complaints 202 11.1.10 Deviations and Corrective Action 202 11.1.11 Control of Records 203 11.1.12 Internal Audits 205 11.1.13 Management Reviews 205 11.1.14 Personnel 206 11.1.15 Accommodation and Environmental Conditions 206 11.1.16 Test and Calibration Methods and Method Validation 208 11.1.17 Validation of Methods 210

xii

Contents

11.1.18 Uncertainty of Measurement 11.1.19 Control of Data 11.1.20 Equipment 11.1.21 Sampling 11.1.22 Handling of Test and Calibration Items 11.1.23 Assuring the Quality of Test and Calibration Results (Quality Assurance, Quality Control, Proficiency Testing) 11.1.24 Reporting the Results Additional Resources Attachment A

12

Laboratory Development

210 210 211 211 211 212 213 214 215

217

12.1 Laboratory Operations Strategy 217 12.1.1 Mission, Vision, Goals, and Objectives of Laboratory 217 12.1.2 Needs of the Individuals and Organizations with Interest in the Laboratory Operations 220 12.2 Laboratory Functions 220 12.3 Sample Types and Number Estimates 222 12.4 Laboratory Design and Layout 224 12.5 Staffing and Training 224 12.6 Quality Assurance and Quality Control 224 12.7 Equipment and Supplies 226 12.8 Validation 226 12.8.1 Stages of Validation 227 12.8.2 Guidelines and Organizational Standards 227 12.8.3 The Validation Process 227 12.8.4 Testing Methods and Procedures 227 Additional Resources 231 Attachment A 231

13

Delivering Effective Training 13.1 Defining Stakeholder Learning Needs 13.2 Key Factors for Successful Training 13.2.1 Identify Participants and Schedule Training 13.3 Getting Ready for Training 13.3.1 Training Materials and Delivery Methods 13.3.2 Training Environment 13.3.3 Training Evaluation Methods Additional Resources Attachment A

253 253 254 254 255 255 259 259 259 260

Contents

Attachment B Attachment C Attachment D

xiii

261 262 263

Terminology 267

Preface

DNA Analysis for Missing Person Identification in Mass Fatalities was initially started in 2008 as short individual training chapters. As part of an initiative supported by a U.S. Department of State grant, the chapters were originally designed as background information to accompany a 20-day instruction for Iraqi scientists on mass fatality response operations and forensic DNA’s critical role. These university scientists from across Iraq were tasked with the responsibility of increasing awareness for the use of forensic DNA to identify victims of mass fatalities. Forensic DNA profiling is an important technology used in the identification of victims of mass fatalities and supports human rights and rule of law initiatives. In teaching these most amazing, dedicated, and inquisitive Iraqi scientists, it was quickly apparent that a subject as complex as mass fatality response operations and the role of forensic DNA was difficult to capture in 100 pages. As such, this document grew in length outside the scope of the original project. Additionally, new DNA technologies have posed an added challenge of keeping the document up to date scientifically. Regardless, this document became what I consider to be the most comprehensive general text on mass fatality response and the use of DNA for identifications written to date and holds tremendous value to organizations contemplating the use of DNA in human identification initiatives following mass fatalities. Amanda Sozer SNA International

xv

Acknowledgments

This book was written with the help and input of a number of individuals. I would especially like to thank Shelly Beckwith for her help with organizing the interpretation of DNA data (one of the most complex issues in the use of DNA following mass fatalities), Susan Peters for her expertise in de-convoluting complex issues, program development, and training, Arbie Goings for his input on mass fatalities, body recovery, and family assistance center operations, Julia Powers for her help in the identification of human remains, Dave Boyer for his proficiency in sample collection, Jamie Handelsman for her innovative approaches to training, William Watson for his creativity in generating diagrams to express complex DNA processes, the staff of Future Technologies Inc., for their help with datatracking, George Riley for his input on quality systems, and Brendan Sozer, Kaitlyn Andrews-Rice, Kaitlyn French, and Web Bist for their help in getting the document ready for publication. This book is in honor of the many scientists, educators, and human rights organizations that are dedicated to bringing truth and understanding to family and friends following a mass fatality.

xvii

About the Author

Amanda Sozer, Ph.D., president of SNA International, received her B.A. from Rutgers University and her Ph.D. from the University of Tennessee–Oak Ridge Graduate School of Biomedical Sciences at Oak Ridge National Laboratory. Sozer has worked in forensics for over 20 years directing forensic laboratories and programs. In addition to directing forensic DNA laboratories she served as a technical contractor to the U.S. National Institute of Justice (NIJ) and worked on the DNA backlog reduction programs for no-suspect forensic cases and convicted offender outsourcing programs, which resulted in the processing of millions of samples. Following 9/11 Sozer served on and facilitated the NIJ Kinship and Data Analysis Panel for the World Trade Center victim identification effort and was instrumental in writing NIJ’s Lessons Learned from 9/11: DNA Identification in Mass Fatality Incidents. Sozer managed the Hurricane Katrina victim DNA identification effort, overseeing the collection, testing, analysis, and matching of thousands of samples. She facilitated the writing of the AABB Guidelines for Mass Fatality DNA Identification Operations. Sozer has also written strategic plans and mass fatality response plans for a number of organizations in the United States, and recently led a subject matter expert group developing guidelines for scientists working on human rights projects for the American Academy for the Advancement of Science. Sozer was the technical lead on a U.S. State Department project to strengthen forensic DNA capabilities in Iraq and spearheaded the DNA technical portion of a State Department forensic DNA needs assessment in Afghanistan. She has worked on numerous local, state, and federal forensic projects in the United States and human identification forensic projects and human identification initiatives in Guatemala, Cyprus, Iraq, Afghanistan, Jordan, Dominican Republic, Colombia, Lebanon, the Phillipines, and Libya. xix

Chapter Human Identification through DNA Analysis

1

Following a mass fatality, human remains are identified for the purpose of repatriation, the certification of death, the issuance of legal documents, and for providing truth and understanding to families. The use of DNA in the identification process is becoming increasingly common. This chapter reviews the basics of DNA and provides an overview of the DNA profiling process.

1.1 DNA Deoxyribonucleic acid (DNA) contains the genes, or instructions, for cell growth and function. It is found in all nucleated cells in the body. DNA strands are quite long, approximately 3 billion base pairs. These long strands are packaged into bundles, called chromosomes, inside the nucleus, as shown in Figure 1.1. In addition, DNA is found in the mitochondria, organelles found in cells responsible for cellular energy production. Mitochondria, as seen in Figure 1.2, are present in high numbers (100–1,000) in each cell. The mitochondrial genome is a relatively small, single, circular DNA chromosome of only about 16,500 base pairs. However, within each mitochondrion there are multiple copies of its chromosome (2–10). Therefore each cell potentially contains hundreds to thousands of copies of the mitochondrial chromosome. Regardless of where the DNA is found in the cell, DNA has a double helix structure similar to a twisted ladder, with the legs of the ladder consisting of two antiparallel sugar phosphate backbones. The “rungs” of the ladder are composed of four different nucleotide bases (adenine, thymine, guanine, and cytosine) that hydrogen bond into set base pairs. Figure  1.3 illustrates the base pairing in DNA. Encoded within the sequence of the three billion base pairs that make up the DNA of the human genome are the individual genes or instructions necessary for producing and regulating the processes responsible for life. Since all humans are structurally and functionally similar (one head, two arms, one heart, etc.), the majority of their DNA is identical. However, there are areas in the DNA where genetic differences between individuals can be detected. 1

2

DNA Analysis for Missing Persons in Mass Fatalities

Figure 1.1  DNA structure. Cell membrane

Mitochondrions

Ribosomes

Nucleus

Cytoplasm

Figure 1.2  Human cell.

Differences in the length or sequence of the DNA, called polymorphisms, are responsible for the variations between individual genomes. This uniqueness combined with the underlying inheritance of these polymorphisms from an individual’s parents makes DNA-based human identification, and the subsequent reunification of remains with families, possible.

1.2  Types of DNA Analysis 1.2.1  Autosomal Short Tandem Repeats DNA in the nucleus of human cells is organized into 23 paired structures called chromosomes. One of those pairs is called the allosome pair (sex

Human Identification through DNA Analysis

P

5' End 3' End

C

G

P

A

P

T C

T

A

C P

A 3' End

C

P

P

G T

P

P

A

P

P

G

G

P

T

P

P

P

T

A

P

P

3

P

P

5' End

Figure 1.3  Molecular structure of DNA.

chromosomes), which determines the sex of an individual. The other 22 pairs are called autosomes. During fertilization, each parent randomly contributes one of each pair of their chromosomes to make up the unique chromosome set for their offspring. This blending of genomes means that each individual’s nuclear DNA is a composite, with half of the DNA (and, therefore, half of the DNA polymorphisms) donated by each parent, as illustrated in Figure 1.4.

Figure 1.4 (see color insert)  Autosomes are inherited half from the mother and half from the father.

4

DNA Analysis for Missing Persons in Mass Fatalities 8 Repeats = 8 allele AATT AATT AATT AATT AATT AATT AATT AATT TTAA TTAA TTAA TTAA TTAA TTAA TTAA TTAA AATT AATT AATT AATT AATT AATT AATT TTAA TTAA TTAA TTAA TTAA TTAA TTAA

7 Repeats = 7 allele

Figure 1.5  Example microsatellite alleles.

Worldwide, the most commonly used DNA test used for human identification is short tandem repeat (STR) analysis. STRs belong to a class of DNA length polymorphism called microsatellites. Microsatellites are short sequences of DNA, normally 2–6 base pairs in length, which repeat one after another (in tandem). Figure 1.5 gives an example of STR alleles, which are alternative forms of the same DNA section at a specific locus. Microsatellites are the product of slippage of complementary strands of DNA during replication and are found throughout the genome. It is believed that repetitive sequences account for as much as 3%, or approximately ninety million base pairs of the human genome. Autosomal STRs are microsatellite repeats found on any one of the 22 chromosome pairs that are not responsible for determining sex. The autosomal STR loci most commonly used for human identification have 4-nucleotide (tetranucleotide) or 5-nucleotide (pentanucleotide) sequence repeats. With alleles that vary from about 5–30 tandem sequence repeats, each of these STR loci have only a moderate power of discrimination. To overcome this limitation, STR kit manufacturers have developed kits that combine 7–15 loci into a single test, called a multiplex. A multiplexing approach allows more STR loci to be analyzed in a shorter time, while requiring less DNA and dramatically improving the overall power of discrimination. This makes STR loci perfect, in many cases, for identification and kinship association with the small quantities of degraded DNA commonly encountered with samples tested in mass fatality events. As with any testing system, STRs do have limitations. The loci used in most commercially available multiplex kits produce amplified fragments that range from 150 to 400 base pairs in length. In some cases, severely degraded DNA may not contain fragments of DNA large enough for the larger loci to successfully amplify. Such a failure to amplify, called “drop out,” can potentially hinder the identification and/or kinship association of a sample. To address this problem, STR amplification kit manufacturers have begun to produce multiplex kits that have reduced the size of the targeted amplified fragments (amplicons) to enable their use with more highly degraded samples.

Human Identification through DNA Analysis

5

Figure 1.6 (see color insert)  Y-STR haplotype is passed only from the father to the son.

1.2.2  Y-STR Haplotype The chromosomes responsible for determining the sex of an individual are called allosomes, or the sex chromosomes. Women inherit one X chromosome from their mother and one X chromosome from their father, while men inherit an X chromosome from their mother and a Y chromosome from their father, as seen in Figure 1.6. STR loci located on the Y chromosomes have also been adapted for use in human identification. They have now also been incorporated into multiplex kits. Unlike autosomal chromosomes, Y chromosomes do not undergo recombination, and thus the set of Y-STR loci are inherited as a single unit from father to son. This means that barring “new” intergenerational mutations, all paternally related males will have identical Y chromosome STR profiles, profiles inherited from one parent as a single unit are called haplotypes. This paternal pattern of inheritance makes Y-STR analysis particularly useful for kinship associations when only paternally related extended family members are available for reference comparisons. Figure 1.8 illustrates this. Although there are multiple features associated with Y-STR analysis that make it suitable, and in some instances recommended, for use in mass fatality events, it should be kept in mind that Y-STR analysis also has limitations. Since the Y chromosome is found only in males, Y-STR analysis cannot be used to identify female victims. Additionally, while Y-STRs are useful for kinship analysis, they are often not suitable for identification in cases involving paternally related males, and since families often travel together, it is not uncommon for multiple individuals killed in a mass fatality event to be paternally related. 1.2.3  Mitochondrial Polymorphism The types of DNA analysis discussed thus far have focused on DNA found in the chromosomes of the cell’s nucleus. However, contained within the mitochondria is another source of polymorphic DNA. Within the mitochondrial

6

DNA Analysis for Missing Persons in Mass Fatalities Paternal Grandfather

Paternal Grandmother

Y STR Father and mother deceased Y STR

Y STR

Paternal Uncle The Y STR haplotype of the mass fatality victim will match the Y STR haplotype of the paternal grandfather and uncle

Y STR Mass Fatality Victim

Figure 1.7  Y-STR haplotype inheritance.

chromosome’s control region are two regions called hypervariable region 1 (HV1) and hypervariable region 2 (HV2). HV1 and HV2 include approximately 550 base pairs of sequence that exhibit multiple single nucleotide polymorphisms (SNPs) or base pair variability between maternally unrelated individuals. The most common approach used for performing mitochondrial DNA analysis includes polymerase chain reaction (PCR) amplification of regions HV1 and HV2, followed by PCR-based cycle sequencing, which is outlined in Figure 1.8. Unlike nuclear DNA, which is inherited from both parents, mitochondrial DNA (mtDNA) is maternally inherited, as outlined in Figures 1.9 and 1.10. Just as Y chromosomes are passed only from father to son, each individual’s mitochondrial genome is passed intact only from a mother to her children. This maternal inheritance pattern makes mitochondrial DNA analysis useful for kinship associations when parents or siblings are not available, but other maternally related relatives are. Additionally, with as many as ten thousand mtDNA copies in each cell, mtDNA analysis is an excellent choice for testing highly degraded (and low quantity) samples such as hair shafts, where there are no nuclei, and the bones and teeth of skeletonized remains if profiles cannot be generated with STRs. Mitochondrial DNA analysis, much like Y-STR analysis, has its own set of limitations when used for human identification. In most instances, mitochondrial sequences cannot be used alone to conclusively identify an individual’s remains. Variation in the mitochondrial sequences is not unique to

Human Identification through DNA Analysis

Mitochondria

Nucleus

Mitochondrial Chromosome 16,569 bp

16024

7

Control Region 16365 1 73 340 HV2 HV1 PCR Amplification

Sequence Data A T A C T T G A C

Cycle Sequencing of PCR Product

Figure 1.8 (see color insert)  Overview of mtDNA analysis.

an individual, and many people often share the same sequence. Additionally, since it is only passed from mother to child without modification, it cannot be used to differentiate between maternally related individuals. For this reason, when multiple maternally related individuals are killed in a mass fatality event, mtDNA profiling cannot specifically identify them. Finally, the sequencing process used for mtDNA profiling yields data that is unsuitable for analyzing commingled soft tissue remains (blood and tissue) and potential mixtures encountered when profiling personal items such as hairbrushes or toothbrushes that have been used by multiple people.

Figure 1.9 (see color insert)  mtDNA is inherited only from the mother.

8

DNA Analysis for Missing Persons in Mass Fatalities Maternal Grandfather

Maternal Grandmother Mito

Father and mother deceased Mito

Mito Maternal Aunt

Mito

Mito

Mass Fatality Victim

Sibling

The mtDNA haplotype of the mass fatality victim will match the mtDNA haplotype of the sibling, the maternal aunt, and the maternal grandmother

Figure 1.10  The inheritance pattern of the mtDNA haplotype.

1.3  A History of DNA Profiling In 1984, Sir Alec John Jeffreys recognized that the molecular probes he had developed to examine muscle proteins in humans could be used to identify individuals by creating a unique “DNA fingerprint.” DNA fingerprinting which examined many locations at one time was first used to provide scientific evidence of a genetic relationship in a disputed immigration case. The complex patterns visualized by these multi-locus probes were inherited half from the mother and half from the father and could be used to assess both biological relationships and identity. When local police, unable to solve the murders of two young women who had been sexually assaulted, approached Dr. Jeffreys for assistance, the first forensic application occurred. Dr. Jeffreys used DNA fingerprinting techniques to provide scientific evidence that linked the semen found on the victims to a local man. When DNA was first used, human identification DNA was cut with a restriction enzyme, separated through an agarose gel, transferred to special paper, and detected with a radioactively labeled probe. The probe bound to multiple locations in the DNA (creating a DNA fingerprint) or to single locations in the DNA (creating a DNA profile). This technique, known as restriction fragment length polymorphism (RFLP) analysis, was very labor intensive. Typically a case with a small quantity of DNA could take over a month to process, although cases with ample DNA

Human Identification through DNA Analysis

9

could reveal information in under a week. While RFLP-based DNA profiling procedures were reliable and informative in determining biological relationships and identifications, they were also expensive, required skillful labor, did not lend themselves to automation, and the technique required at least 50 ng of fairly high molecular weight DNA to expect useful results. Over the years, the ability to obtain DNA profile results from samples with limited or degraded DNA were dramatically improved by looking at smaller areas of variation and making multiple copies through a technique called PCR. Today, all autosomal forensic DNA analysis and some of the sex-linked analyses are PCR based, amplifying STRs. Using this technology, human identifications can be reliably obtained from as little as 200 pg of nuclear DNA. STRs are smaller fragments of DNA with shorter repeat sequences than the DNA evaluated using the RFLP method and are therefore less influenced by degradation. Currently, DNA profiles for human identification typically contain 16–24 autosomal loci using a single, multiplexed amplification. Unfortunately, in many instances small, degraded samples are likely to be encountered when dealing with mass fatality human remains samples. With this in mind, DNA analysts need to carefully consider which methods to apply and in which order to apply them when analyzing mass fatality remains. See Chapter 8 for a more in-depth discussion on the technology used in current DNA profiling.

1.4  Using DNA for Identification of Human Remains Following a mass fatality, the entity or organization responsible for identifying the human remains will use DNA profiling to supplement traditional methods of human identification, which include anthropology, pathology, fingerprint, and dental records. While DNA is a powerful identification tool, DNA results are only part of an overall identification effort, which, depending on the condition of the human remains, also takes into account non-DNA evidence. In certain circumstances, where there is extensive fragmentation and/or decomposition of the bodies, DNA analysis may be the only method of identification. DNA analysis is often the only tool available that can be used to identify and reunite fragmented human remains. As with other identification technologies (anthropology, fingerprints, odontology), human identification through DNA analysis is, at its most basic, a biometric technique. Like these other technologies, DNA analysis uses a characteristic specific to the unknown sample (remains) to compare to a known sample from the deceased (reference). In order to identify human

10

DNA Analysis for Missing Persons in Mass Fatalities

Reported Missing (RM)

Toothbrush from RM

Reference Sample

Human Remains of Unknown Origin

Unknown Sample

Figure 1.11 (see color insert)  Using a reference sample to identify an unknown sample.

remains, a DNA profile from the human remains must be “matched” to one or more DNA profiles from biological samples of known origin (reference sample), as in Figure  1.11. However, unlike these other comparison techniques, DNA analysis offers the additional flexibility of allowing the comparison of a profile from the unknown sample to immediate and extended family members.

1.5  The DNA Analysis Process The DNA analysis process can be broken down into several primary phases. The initial phase, sample collection, involves collecting the biological material (sample) in a way that preserves the DNA, while also providing a level of documentation that will ensure the source can be properly identified. The DNA extraction/purification phase is the first hands-on laboratory processing step. Here the DNA is released and separated from the other material in the sample, making it available for further testing. Once extracted, the DNA is quantified and then profiled using PCR amplification of one or more of the types of loci discussed above (autosomal STRs, Y-STRs, or mitochondrial

Human Identification through DNA Analysis

11

sequence HV1 and HV2). The PCR-amplified sample must then be analyzed in a way that produces usable data. Finally, once the data is available, it must be interpreted in light of the desired end result, whether it is for identification or kinship association. Although most forensic and paternity testing laboratories already have validated procedures in place to address each of these stages in their current process, it is important to consider how the demands of the scale of a mass fatality event may affect pre-existing laboratory practices and procedures. 1.5.1  Organizing and Planning In the face of a mass fatality event, the first inclination of the scientist is to immediately begin acquiring and testing samples and then to attempt identifications. However, prior experiences by laboratories that have dealt with mass fatality events show that it is best to complete a preliminary assessment of the event in order to develop and select the appropriate procedures for sample collection and testing. In 2009, AABB (formerly the American Association of Blood Banks) published “Guidelines for Mass Fatality DNA Identification Operations” (PDF available for download on line). These guidelines present a comprehensive overview of the different factors involved in a mass fatality DNA identification operational response, including program management and oversight, technical considerations, data review, screening and statistics, reporting, and other considerations. Appendices containing detailed information on statistical analysis are also included. The guidelines are designed to be overarching and applicable to decision makers, as well as laboratory managers and scientists. Its sole focus is on DNA identification operations and should be carefully read prior to embarking on any mass fatality DNA identification operation. 1.5.2  Sample Selection and Collection Samples of known origin are called reference samples. There are several types of DNA reference samples used to identify human remains in mass fatality operations, such as direct references, personal items, and kinship samples. In order for the laboratory to produce meaningful results, each of the reference types must be properly collected and identified. The type of reference samples collected depends on accessibility and the ability of the laboratory or laboratories to test the samples. A direct reference is a sample that has some sort of paperwork or documentation linking its origin to a missing individual. Typically a professional (such as a doctor or nurse) will have collected these samples during a medical test. Direct reference samples can be attributed to the missing individual through a record of collection (e.g., medical records). Personal items are objects purported to have or to contain

12

DNA Analysis for Missing Persons in Mass Fatalities

Figure 1.12  Collection of buccal swab for a kinship reference sample.

DNA from the reported missing (RM) because they were used by the RM or came from the RM. However, personal items have no associated documentation linking the item to the RM. These samples are typically found at an RM’s home or place of employment and could be a hairbrush, toothbrush, or favorite coffee cup. Family references or kinship samples are often the references of choice for the identification of the RM. Because family references or kinship samples are standardized, the laboratory can process the samples in a timely and consistent manner. Additionally, a sample can be re-collected from a family member if the laboratory has any questions and wishes to perform additional testing. Collectors typically use a buccal swab (scraping from the inside of the mouth as illustrated in Figure 1.12) to collect these samples. Alternatively, some laboratories use bloodstains. The sample type depends on the laboratory’s automation and preference for testing. The DNA laboratory should be consulted prior to the collection of postmortem samples. Typically, the best postmortem sample is the biological specimen that is the least difficult to obtain, the least challenging for the laboratory to process, and the most likely to produce successful results. 1.5.3 Extraction/Purification Mass fatality human identification using DNA analysis can require the extraction of DNA from a variety of different sample types. Extraction protocols make the DNA contained within the cell available for subsequent genetic testing and can vary from the preparation of a crude solution of cellular extract to a preparation of high quality (large fragment), highly purified DNA. The preparation of a crude solution of cellular extract involves the release of DNA through the disruption of the cell’s membranes, producing free DNA in a mixture of cellular debris. DNA purification protocols yield a solution of DNA where all other cellular debris has been removed. Purification techniques also include a cell lysis step, where the cell’s membranes and other components are disrupted. However, unlike crude cell lysis techniques, purification protocols also remove the non-DNA components

Human Identification through DNA Analysis

13

within the mixture, both cellular (proteins, RNA, lipids) and non-cellular (salts and detergents). 1.5.4 Amplification Each method of DNA analysis discussed in this chapter relies on polymerase chain reaction (PCR) to amplify or copy the individual loci found in the initial source of DNA by several orders of magnitude. PCR performs this copying or amplification by reproducing in a test tube using the normal process of DNA replication that occurs in the cell. Samples that start with only a few complete copies of template DNA can, with the aid of PCR, produce tens of millions of copies of the sequence of interest suitable for genetic analysis. This occurs because as PCR progresses, each replicated copy of the DNA produced by a previous copying cycle can then be replicated in the subsequent cycle(s), resulting in an exponential increase in DNA copies, as outlined in Figure 1.13. The initial step in each cycle is denaturation, where the reaction mix is heated to near boiling (as shown in Figure  1.14), and the double-stranded DNA template separates (melts) into two single strands. The reaction mix is then cooled until the primer binds or anneals to the DNA template. Finally the sample is heated to the optimal temperature for the DNA polymerase to make a new copy of the DNA molecule. Depending upon the needs of the specific type of test being performed, this series of steps will be repeated for 28–32 cycles. During PCR amplification, the STR fragments or sequencing products are labeled with fluorescent dyes attached to the primers. Template DNA Prior to PCR – 1 copy Target Sequence 1st Cycle – 2 copies 2nd Cycle – 4 copies 3rd Cycle – 8 copies 4th Cycle – 16 copies

28th Cycle – 100 million copies

Figure 1.13 (see color insert)  Making copies of the target DNA sequence.

14

DNA Analysis for Missing Persons in Mass Fatalities Single Strand DNA

Double Strand DNA

DNA Extends by Single Bases (Green)

Primer (Blue) Binds DNA

28–32 More Cycles

Melting Temperature

One Cycle

Extension Binding Time

Figure 1.14 (see color insert)  The molecular biology of PCR amplification.

1.5.5  Data Generation/Analysis Regardless of the testing method used, once the isolated DNA samples are PCR amplified, the resulting product must be analyzed or characterized. While there are some minor differences between the processes for performing fragment analysis (autosomal STR and Y-STR) and DNA sequencing (mtDNA), the same basic principles and equipment are utilized in both cases. The amplified fluorescently labeled DNA fragments are separated based on their molecular weight (length). To perform this separation, most human identity testing laboratories use a process called capillary electrophoresis. During capillary electrophoresis, the amplified PCR products are injected into a capillary (hollow silica tube), which contains a denaturing polymer (usually POP4 or POP6). Electrical current is then applied across the polymer, forcing the negatively charged amplified products to migrate through the polymer-filled capillary toward the positively charged end of the capillary. Smaller DNA fragments migrate more rapidly than larger ones, and as a result the fragments separate based on the size. Near the end of their migration, the labeled fragments pass through a region of the capillary where they are struck by a laser beam, causing the associated dyes to fluoresce. A camera then detects the fluorescence and converts it to digital data. The digital data can then be analyzed by software that converts the digital data to STR profiles or mitochondrial sequences. This is outlined in Figure 1.15.

Human Identification through DNA Analysis

15

Capillary filled with polymer (POP4 or POP6)

Argon Laser –

+ Outlet (anode)

Inlet (cathode)

CCD Camera

DNA fragments separate as they migrate from cathode to anode Electrical Current

Figure 1.15 (see color insert)  Detection of DNA fragments using capillary electrophoresis.

1.5.6  Data Interpretation Given all of the potential sample collection issues, handling requirements, and complex processing procedures associated with DNA analysis, it would be easy to believe that the difficult portion of the analysis is done once the DNA from a sample has been converted to digital data. However, completion of the laboratory portion of the testing is just the first step in the analysis process. Once the laboratory portion is complete, the analyst must use that data to make inferences about the source of the sample. In the more common applications of human identity testing (parentage and forensic testing) this can be a relatively uncomplicated process. Mass fatality events, however, are seldom uncomplicated due to the challenges presented by the number of victims, the potential for fragmentation of the remains, the possibility of extensive sample degradation, and the difficulties associated with securing suitable victim and family reference samples. The true scope and complexity of data interpretation can be overwhelming when added to the potential use of multiple testing techniques that are not directly comparable and potentially performed by different laboratories, as well as the requirement that the associated remains be correctly reunited with a particular family.

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DNA Analysis for Missing Persons in Mass Fatalities

1.5.7  Profile Comparison Comparing two complete or mostly complete DNA profiles, from one unknown sample to another or from an unknown sample to a direct reference sample, is a relatively straightforward process. In most instances the analyst can make a locus-by-locus comparison to establish a match or non-match with little or no uncertainty. As pictured in Figure 1.16, an unknown sample can be compared to reference samples to determine potential matches. Even when dealing with moderately to severely degraded profiles, most forensic laboratories have developed match criteria that are sufficiently robust to allow the analyst to determine if the samples in question could have originated from a common source and to assign a probability statistic to any match that is identified. Direct unknown sample-to-sample or unknown sample to direct reference sample comparisons can usually be expressed as a random match probability. This is the probability that an individual in a random population would match an observed profile. However, when dealing with profile comparisons in a mass fatality event, standard match criteria and comparison statistics developed for use with straightforward criminal casework may no longer be sufficient or appropriate to perform the required task.

Reference Sample 1

6000

D3S1358 120

TH01

200

D21S11

4500 3000 1500

Unknown Sample

6000

D3S1358 120

TH01

200

0 D21S11

atch

M

15 555 17 604

4500

6 1629

30 609

7 1804

31 446

3000 1500 0

15 1008 17 1024

6 2682 7 2188

30 948 31 828

No

Reference Sample 2

Ma

tch 6000

D3S1358 120

TH01

15 599

9 1284

200

D21S11

4500 3000 1500 0

16 546

28 33 1131 973

9.3 1341

Figure 1.16 (see color insert)  Comparison of unknown profile to reference profiles. (Courtesy of Michelle Beckwith.)

Human Identification through DNA Analysis Alleles a, b

17

Alleles c, d

Alleles a, c

Figure 1.17 (see color insert)  Inheritance of alleles.

1.5.8  Kinship Analysis Kinship analysis is the comparison of genetic profiles to determine the potential biological relationship between two or more individuals. As noted at the outset of this chapter and in Figure 1.17, an individual’s genetic makeup is a unique blending of genetic material from each of their parents. This blending means that the degree of an individual’s “relatedness” to other individuals in his or her immediate and extended family can often be established through genetic testing. For this reason kinship analysis can be a very useful tool in mass fatality events. Additionally, kinship analysis can be useful when attempting to identify remains where no direct reference sample for the deceased is available, and when confirming the origin of a familial reference sample. This means that the profiling of immediate relatives (parents, offspring, and full siblings) usually provides the best opportunity for a kinship association with deceased persons. It is important to keep in mind, however, that even in the absence of immediate family members, a kinship association may be possible with comparisons to a sufficient number of extended family members or through mtDNA or Y-STR comparisons when appropriate. In mass fatality DNA operations, a genetic pedigree is drawn for the missing person, and samples may be collected from all available appropriate family members. There are limitations associated with kinship analysis. First, the process relies on the availability of immediate family members or a sufficient number of extended family members to be able to perform the comparison. With the possibility that all immediate family members could be victims in the event, and the extended family may not exist or be available, finding the necessary familial reference samples may be difficult. Additionally, even

18

DNA Analysis for Missing Persons in Mass Fatalities

with immediate and/or extended family members available, the statistical strength of the association can be negatively impacted by intergenerational genetic mutations that result in allele changes. Also there may be instances where individuals are not genetically related the way they believe due to adoption. Kinship analysis is limited in its ability to differentiate between victims who are siblings of the same gender where no direct reference sample or phenotypic characteristics are available. Finally, no current genetic profiling technique can differentiate identical twins. Even with these limitations, kinship analyses can play an important role in the identification of human remains and give the family the opportunity to participate in the identification of their family member(s). 1.5.9  Reporting the Profile Matches Analysts will look at the locations of the matches and determine how common or rare the match is based on the frequency of the matching alleles in the population. Best practices dictate that a match threshold is set for reporting matches. Typically this threshold may be set by the entity that is responsible for the identification of the human remains and is often set in conjunction with support from expert panels or groups. Once the match threshold is met, relationship and/or match reports are issued. These reports are used along with non-DNA identification evidence by the entity responsible for making the final identification determination of the human remains.

1.6 Explaining the DNA Process to Non-Technical Personnel In a mass fatality event, the need will arise to discuss DNA analysis with groups and/or organizations that will have an integral role in the process but do not have a technical background in DNA analysis. Whether these groups are made up of scientific or medical personnel, untrained laypersons, or, as is often the case, a mix of both, the analyst should be prepared to explain the process, how it is being carried out, and why it is being done. Without a clear understanding of the testing process, a mother may be hesitant to provide the laboratory with the missing child’s toothbrush, a governmental official may push for rapid identifications due to a lack of understanding of the effects of sample quality and sample numbers on testing times, and while collecting samples, a non-governmental organization employee may not understand what samples are useful for testing. It should be considered one of the laboratory’s prime responsibilities to provide the information necessary for each of

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these groups to understand how they can contribute to the ultimate goal in the most efficient and effective DNA identification effort possible. It is not surprising that when faced with the challenge of discussing a technical and detailed process, the scientist typically provides technical and detailed explanations that exceed the untrained listener’s ability to understand. This approach is understandable given that the scientist’s usual audience is both technically trained and understands the specific application of DNA analysis to questions of human identity. However, in most instances this is clearly the wrong approach to take. When dealing with discussions of the application and significance of DNA analysis during a mass disaster event, the analyst should remember that while all parties have the same ultimate objective, identifying the victims and reuniting them with their families, not all groups approach this goal with the same background and understanding of how DNA analysis can or will contribute to that goal. Know the audience and limit information to only what they need to know. When possible, it is best to try to determine the makeup of the audience and to focus the presentation on their role in the process. For example, it is appropriate to discuss the details of how the proper handling of fragmented remains may affect subsequent genetic testing with those individuals responsible for collecting the remains; however, family members of the victims need only know that the collected remains will be tested. This information is obviously related, but not identical. Avoid technical explanations and language unless absolutely neces­ sary. Remember that the purpose of presenting information about DNA analysis during a mass fatality event is entirely different from that when presenting the same information at other times. Even with a mixed audience of technical and non-technical people, it is better to simplify the technical aspects of the presentation as much as possible. While it is technically correct to say, “the cell is lysed using a detergent releasing the nuclear and mitochondrial DNA, which is then purified using an organic solvent,” it is unlikely that a non-technical listener will understand. However, the same person may have no problem understanding that “the DNA is released from the cell.” When a technical response is the only acceptable explanation, it is better to try to relate it to something the least technical person in the audience can understand. Relate the information to the audience. Even when tailoring the presentation to the audience and avoiding overly technical descriptions, never assume that the audience understands all of the points being made. It is always best for the presenter to take a moment to make sure that the audience understands how the information relates to

20

DNA Analysis for Missing Persons in Mass Fatalities

them than to assume that they understand this relationship. This can be accomplished by summing up the point in a “This means that…” statement. For example, after discussing the inheritance of nuclear DNA with kinship diagrams and STR profiles, the speaker can sum up the point by saying “this means that a couple’s offspring represents a unique blending of their DNA.” If you don’t know the answer, it is better to say so. Remember that no one wants incorrect or false information. If the speaker is unsure of or can’t answer a question, it is better for him or her to say so, offer to find the information if available, and provide it at a later time than to attempt a guess and risk being wrong. An obvious example of this is the question, “How long will it take to identify my loved one?” If, depending on the size and conditions of the event, it is possible that some victims may never be identified, it is better to say so than to make an optimistic guess or estimate.

1.6.1  Common Questions Note: The following answers are general and should only be used as examples. One should be prepared to answer questions that are specific to each mass fatality response operation. 1.6.1.1  Why Identify the Remains? Some families may desire the identification and reunification of their loved one’s remains for acceptance and understanding, while others may feel that the process interferes with their grief and eventual healing. However, identification is typically preferred to ensure that those families desiring reunification can be adequately addressed. 1.6.1.2 How Is the Testing Done? Identification and reunification are performed by comparing the genetic material collected from items used by the victim and their family to the genetic material from the remains. See https://www.ncjrs.gov/pdffiles1/ nij/209493.pdf for a wonderful guide for families. 1.6.1.3  How Long Will the Process Take? While the testing process for an individual sample can be relatively quick depending on the sample, mass fatality identifications are complex and often take a great deal of effort. Due to the complexity of the incident, and the availability of the samples, it may not be possible to identify all of the victims. (Depending on the size and scope of the incident, it is best to answer this question without specifics since complications in testing may increase analysis time.)

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21

1.6.1.4  How Can I Help Identify My Loved One? The best way to assist in identifying your loved ones is by working with the agencies involved in the identification process and providing all of the information possible about the victim. 1.6.1.5 What Are the Sources of DNA Samples that Can Be Used? DNA can often be obtained from the biological remains. This DNA will be compared to DNA known to be from the victim or to DNA from the victim’s relatives. 1.6.1.6 What Are Useful Sources of DNA from the Victim? DNA from the victim’s previously collected medical specimens or personal items can be used to make a direct match to remains. For example, if a loved one recently had surgery or blood work done, a specimen may have been stored at the hospital or clinic. You should provide any known medical specimens or ask for help in locating them. DNA from the victim may also be found on their personal items. A toothbrush or other items containing saliva are often good sources. However, it is very important that these items were used only by the victim or rarely used by anyone else. For example, a hairbrush used by the whole family would not be a good source of DNA from the victim. 1.6.1.7 How Can DNA from Relatives Be Used? If personal items or medical specimens are unavailable, or if the testing on these items does not work, DNA testing can be done on samples from blood relatives. The DNA from adoptive parents, adopted children, stepparents, or other non-blood relatives cannot provide information on the genetic identity of a victim. The ability to match victims to their relatives depends on how closely related they are to the victim. The most useful DNA samples are from close blood relatives such as the victim’s biological mother, father, children, brothers, or sisters. This is because DNA of close relatives is more similar than the DNA of more distant relatives. If DNA from the victim’s children is used, it is helpful to have DNA from the children’s other biological parent. Although DNA from more distant relatives can be used, this process is more difficult. In some cases, samples may be requested from specific relatives. For example, DNA samples could be requested from a maternal relative of the victim, such as the victim’s aunt, uncle, or half-brothers or half-sisters on the mother’s side of the family in order to help with specific genetic tests. 1.6.1.8  Why Might DNA Analysis Not Work? DNA testing might not be able to identify your loved one. The most likely reason would be that there is no usable DNA in the recovered remains. Some

22

DNA Analysis for Missing Persons in Mass Fatalities

victims’ remains may not be found. Also, DNA testing may not work if no usable DNA can be found on submitted personal items. 1.6.1.9  How Much Will Testing Cost? Costs will vary depending on the size and complexity of the incident. 1.6.1.10  Who Issues the DNA Identification Reports? The answer to this question will depend on the circumstances of the incident. However, it is recommended that the laboratories involved in the testing process establish a firm reporting procedure prior to the implementation of the project when the pressures of an actual incident are not present. 1.6.1.11 Are There Any Guidelines for Using DNA? AABB (formerly the American Association of Blood Banks) has developed “Guidelines for Mass Fatality DNA Identification Operations.” 1.6.1.12  Who Will Have Access to My DNA and Profile? Note: This answer will be incident specific and cannot be provided here. Before the operation begins, a policy should be developed on what information is released by the DNA operation. This policy should follow applicable laws and regulations.

Additional Resources American Association of Blood Banks. 2010. Sozer, A., Baird, M., Beckwith, S., et al., Guidelines for Mass Fatality DNA Identification Operations. www.aabb.org/ programs/disasterresponse/Documents/aabbdnamassfatalityguidelines.pdf (accessed September 30, 2012). Butler, J.M. 2012. Advanced Topics in Forensic DNA Typing: Methodology. New York: Elsevier Academic. Butler, J.M. 2010. Fundamentals of Forensic DNA Typing. New York: Elsevier Academic. Jefferys, A., et al. 1985. Hypervariable “minisatellite” regions in human DNA. Nature 314: 67–73. Lee, J. 2008. Recommendations for DNA laboratories supporting disaster victim identification (DVI) operations—Australian and New Zealand consensus on ISFG recommendations (letter). Forensic Science International: Genetics 3: 54–55. Prinz, M., Carracedo, A., Mayr, W.R., et al. 2007. DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI). Forensic Science International: Genetics 1: 3–12. The Scientific Working Group on Disaster Victim Identification (SWGDVI). Protocols, Guidelines, Forms, and Standard Operating Procedures. www.swgdvi.org/resources.html (accessed September 30, 2012).

2

Chapter Mass Fatalities

This chapter defines a mass fatality, provides select examples of past mass fatalities, and discusses the various factors affecting the response effort. A mass fatality response consists of the recovery, identification, and reunification of the deceased with their families. It is important that each individual involved in the response effort has an understanding of how he or she fits into the overall mass fatality response.

2.1  Definition of a Mass Fatality A mass disaster is an incident that results in great devastation to property and local infrastructure. When incidents involve numerous injuries to humans, the event is referred to as a mass casualty. A mass fatality is an event resulting in more deaths than the local available resources can process. See Figures 2.1, 2.2, and 2.3 for visual comparison of the differences between these types of incidents. All three types of events (mass disasters, mass casualties, and mass fatalities) impact local resources, thus reducing the response capacity or capability of local authorities. Therefore, local authorities must seek assistance from outside resources. Various agencies as well as governmental and non-governmental entities nationally and internationally may provide outside resources. The specific capabilities needed to facilitate a mass fatality response will be discussed in later chapters.

2.2  Jurisdictional Issues The cause of a mass fatality event may determine which agencies and authorities are involved in the identification and reunification process. For example, law enforcement agencies will conduct criminal investigations if the mass fatality is a result of criminal or terrorist-related events. Industrial incidents may involve regulatory agencies specific to the type of industry involved. A humanitarian response to the identification of human remains found in mass graves may be handled by a neutral and separate forensic operation with little or no ties to the government. 23

24

DNA Analysis for Missing Persons in Mass Fatalities

Figure 2.1  Mass disaster—large destruction of property.

Due to the complexity of a mass fatality incident, various agencies and jurisdictions must work together closely. Since most agencies have their own policies and procedures for investigating death-related incidents, planning and communication are paramount in helping to integrate the efforts of these various agencies and to ensure that both local and outside agencies are able to perform their duties effectively and efficiently. A

Figure 2.2  Mass casualty—large number of injuries.

Figure 2.3  Mass fatality—large number of deaths.

Mass Fatalities

25

balance between the command and control structures of the different agencies must be maintained.

2.3  Causes of Mass Fatality Incidents In general, there are two main causes of mass fatalities: natural and manmade. Natural disasters, shown in Figure 2.4, are caused by weather, dramatic changes in the earth’s surface (e.g., earthquakes, volcanoes), or disease. Due to their powerful and destructive nature, weather and earth-related disasters usually have a significant impact on local infrastructure. Mass disaster causes the destruction of homes, buildings, roads, bridges, water supplies, and electricity, all of which leads to a community’s loss of infrastructure. This loss of infrastructure further complicates the response effort. Often, local individuals who would typically respond to a disaster become victims and are unable to participate in the response, thus increasing the need for outside assistance. One such example is the December 26, 2004, Indian Ocean 9.1 magnitude earthquake. Spanning Southeast Asia to the coast of Africa, the earthquake created a massive tsunami resulting in the death of an estimated 230,000 people in more than fourteen countries. Disease-related disasters also affect local responders. If not directly affected, local responders may be concerned about the risk of disease and will refuse involvement in the response. The most serious type of diseaserelated disaster is a pandemic; a pandemic is an epidemic of infectious disease that spreads through human populations across a large region, such as a continent or even worldwide. Pandemic disasters have occurred around the world for thousands of years. Past pandemics include the Antonine Plague in AD 165, in which more than 5 million people died in the Near

Figure 2.4  Aftermath of Hurricane Katrina.

26

DNA Analysis for Missing Persons in Mass Fatalities

Figure 2.5  Aftermath of the 9/11 attack in New York City.

East, and the Bubonic Plague (a.k.a. the Black Death) in the 1300s, which began in Central Asia and spread to Europe, killing an estimated 75 million people. Man-made incidents are caused either intentionally or unintentionally by one or more individuals. Examples of intentional man-made disasters include terrorist attacks (foreign and domestic), acts of war, and other political conflicts including human rights violations. The September 11, 2001, events that caused destruction of the World Trade Center and the Pentagon, as well as the crash of the plane in Shanksville, Pennsylvania, are vivid examples of intentional man-made disasters. Figure 2.5 shows a building across the street from Ground Zero World Trade Center shortly after the terrorist attack. The terrorists who committed these disasters methodically planned and executed hijackings of airplanes, killing thousands of individuals and impacting many more worldwide. Another example of an intentional manmade disaster is the explosion of a truck in Amerli, Iraq, on July 7, 2007, which killed 115 people and injured almost twice that many. Again, terrorists planned and executed this event that resulted in the destruction of human life and property. In both of these examples, the incidents were criminal acts involving multiple jurisdictions in an identification effort that became a part of the overall criminal investigation process. Man-made unintentional mistakes also cause mass fatalities. These acts may include transportation accidents and industrial plant explosions. In 1856, two locomotives heading toward each other on the same track, unknown to the conductors, collided and killed more than 50 people in Pennsylvania. Although the conductors of these trains had no way of

Mass Fatalities

27

knowing about the impending danger, they were ultimately responsible for this horrible accident.

2.4  Mass Fatality Response Overview Historically, the identification of human remains following a mass fatality have been made through exhaustive investigative efforts (such as collecting and verifying eyewitness testimony) combined with careful anthropological examinations. More recently, with the advent of robust DNA profiling techniques, DNA identifications have become more prevalent in human identification efforts. Because of the recent popularity of forensics, it is becoming the identification method expected by families. The process of human identification following a mass fatality is complex and involves many forensic scientific disciplines. DNA is just one tool in the identification toolbox. At a high level, the identification process involves the recovery of human remains, examination of the human remains for identifying characteristics (postmortem information), collection of identifying characteristics of the individual who is presumed deceased (antemortem information), and comparison of the antemortem information to the postmortem information in order to identify the human remains, as outlined in Figure 2.6. In order to have a successful mass fatality response, all activities must be integrated into the planning and operational phases.

Body recovery

Collection of postmortem information from human remains (including DNA samples)

DNA profile testing and analysis

Comparison of postmortem and antimortem information and medico-legal identification of remains

Collect antemortem information about the Reported Missing (including DNA reference samples)

Repatriation of remains to families

Figure 2.6  Overview of a mass fatality response operation.

Family/friends report possible victim then investigations are conducted and Reported Missing (RM) named, if appropriate

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DNA Analysis for Missing Persons in Mass Fatalities

2.4.1  Field Operations/Body Recovery The proper location and recovery of human remains is critical to the correct identification of human remains. It is essential that the site of the mass fatality be properly identified and documented, and bodies exhumed. It is tempting for families and well-intentioned individuals to remove human remains. However, body recovery should only be performed by trained individuals who will maintain proper documentation of the handling of the evidence (human remains and associated forensic evidence) so that a sound chain of custody can be established and maintained. The scene and exhumation process should be fully reconstructed based on documentation. Individuals involved in recovery must gather and record data at the scene and then ensure the data accompanies the recovered remains to the morgue. Critical data collected during the recovery process includes, but is not limited to: • Precise location of the remains • Condition of the remains • Clothing and other personal effects related to the remains Improper exhumations such as the disarticulation of intact skeletal structures can turn a relatively simple identification effort into a very complex and time-consuming undertaking and negatively impact the entire identification effort. Many identification response efforts have been negatively impacted because well-meaning individuals without proper education and training were responsible for collecting human remains. Once the location and condition of the remains have been properly documented, each set of remains, or fragments of remains, should be considered and treated as separate recoveries, placed in its own body bag, and given a unique tracking or field recovery number. A well-designed numbering system should minimize confusion and support the unique designation of the human remains and associated items in the field. Once the recovery team recovers the body, it is transported along with the recovery documentation to a field collection point, morgue, or anthropological evaluation unit. 2.4.2  Morgue Operations Once the human remains have been recovered, they are taken to a mortuary facility for analysis (postmortem examination) which may include: • Examination of personal effects such as clothing and jewelry • Full description of the body • Collection of identifying information such as fingerprints, dental information

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• Pathological analysis to determine cause and manner of death • Anthropological analysis to gather identification information such as sex, age, race, and other characteristics that can be used as potential evidence in the identification • Articulation (or reassociation) of the remains based on bone morphology, anatomical completeness, position • Collection of DNA samples The postmortem information is documented, preferably into a computer program that will allow the comparison of ante- and postmortem information. A morgue operation typically includes the following units: intake, pathology, dental, fingerprint, radiology, DNA, anthropology, personal effects, management, and photography. Each unit examines the recovered bodies and gathers applicable postmortem information. Once the examination is complete, the body will be stored in an appropriate storage unit until it is identified and returned to the family, or if the body is identified but unclaimed, buried, cremated, or disposed of in a manner determined by the local authorities. 2.4.3  Reporting the Missing and Presumed Deceased In order to make identifications the postmortem information must be compared to antemortem information collected from the friends and family of the presumed deceased. Often family members do not know if their loved ones have died or if they have fled the area, for example, if their home was destroyed or if they were being persecuted and fled to another country. Often after a mass fatality, individuals will believe that their family member(s) has perished and will report their family member(s) to local authorities as missing and potentially deceased. Generating an accurate list of the deceased can be time consuming, is often overlooked in mass fatality responses, and is critical to the success of the identification effort. Often there are multiple agencies supporting the identification of missing individuals which results in multiple lists of the missing. Multiple lists cause confusion and can leave huge gaps in the identification effort as families feel that they provided information, when in reality the information has not gone to the correct entity in charge of the identification effort. If lists are combined there are often duplicate cases that need to be carefully compared and combined if appropriate into a single case. All individuals who are reported missing must be located alive or potentially identified among the dead. Therefore, there must be close coordination between a missing persons investigation and the identification effort. Close coordination ensures that when someone is found alive, his or her name is promptly removed from the list of the missing and presumed dead and the case closed. Ideally there should be a single list of the reported missing (RM). The RM names should be vetted to make sure:

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DNA Analysis for Missing Persons in Mass Fatalities

• The person is believed to be deceased (as opposed to temporally missing) • Each individual is represented only once on the list (often people will be reported missing several times by different family members) • Each individual is given a single unique RM number • No two individuals have the same RM number • There is an indication of which individuals are related to each other in RM cases Generating an accurate list of the deceased can be time consuming, but prompt removal of names from the missing persons list helps to develop an accurate list of the individuals who are most likely deceased. It is essential that there be an accurate list of individuals who may have perished. 2.4.4 Collection of Antemortem Information about the Deceased Family Assistance Operations (FAOs) interact directly with the families and friends of the missing and presumed deceased. FAO staff members collect antemortem data during interviews with family members. Antemortem (before death) data is information that describes a missing individual. This antemortem information is used to compare against the characteristics of the human remains. Forms should be used to collect the information to ensure that the information is consistent. Information obtained includes sex, height, weight, occupation, clothing, hair color, etc. Standard forms are comprehensive and can take several hours to complete. Forms can be modified for the specific circumstance so that the families are not subject to irrelevant questioning. However, it is important that the information is as comprehensive as possible. Seemingly unimportant antemortem information may be critical in differentiating two different sets of similar human remains during the identification process. Additionally, pictures may be collected to corroborate the information provided by the family. In addition to the physical description of the missing person, identification reference items are collected from professionals. These reference items include medical records, fingerprint records, and dental records. When DNA identifications are part of the identification process, the FAO staff may also collect DNA reference samples from the family members. A Family Assistance Center (FAC) is often formed at a location proximal to the mass fatality site. The FAC can meet the physical, mental, and spiritual needs of families as they wait to receive word on the positive identification of their loved ones. This is where the antemortem information is collected from the family members of the RM. There are situations where the public, including the surviving family members of the missing and presumed deceased,

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distrust government officials. Such distrust often makes the public reluctant to provide information about their loved ones and presents significant difficulties when FAOs collect antemortem information. As families are an integral part of the identification effort, it is important to address concerns with families from the outset. 2.4.5 Comparison of Antemortem and Postmortem Information Antemortem information gathered at the FAO is then compared to the postmortem data gathered at the morgue using computer programs. Using this comparison, local authorities must agree on the criteria for a positive identification. Establishing strict identification criteria and holding to these criteria will add continuity to the operation and prevent confusion.

2.5  Managing the Mass Fatality Response Operations A mass fatality response is typically complex and involves a large number of personnel and multiple concurrent operations. Well-defined roles and responsibilities, as well as clear lines of communication, supervision, and management are essential to an effective response. The success of the response is directly connected to advance planning, the organizational structure put in place to handle the response, and the use of properly trained and experienced professionals to conduct response operations. The full-time management of a response does not end until all operations have ceased and the event is considered closed. A single managerial entity, organization, agency, or office should initiate, manage, and have ultimate responsibility and authority for a mass fatality response. This managerial entity should provide clear leadership and supervision throughout the entire response. Additionally, this entity should provide written documentation of the acceptable, standard operating procedures for every aspect of a mass fatality response and distribute these procedures to all personnel. This management structure helps ensure that the necessary information is properly obtained, documented, coordinated, handled, and stored. The manner in which response personnel perform their work and how they obtain the data are important. When response personnel use incorrect methods for collecting and documenting their findings, the data may be legally unacceptable, thus negatively impacting identification efforts. The managerial entity should assign appropriate personnel to ensure there is daily supervision of all aspects of a mass fatality response and to make certain that standard operating procedures are followed. Additionally, this

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entity should choose the types of professionals needed to properly conduct response work. The managerial entity should also decide upon: • Overall management of a mass fatality response • Creation of standard operating procedures • Selection of the types of response personnel and their qualification requirements Providing correct information to the families and the public is extremely important. Only the managerial entity or its designee should be allowed to release information concerning the response to the public. Regardless of the type of mass fatality incident, all operations must be flexible enough to make adjustments for unique or special circumstances. Personnel from each operational unit (Family Assistance, Body Recovery, and Morgue) must readily share information and communicate openly among themselves as needed. Additionally, regular communication between the managerial entity and supervisors from field, morgue, and FAOs is important to ensure appropriate planning for the availability of adequate personnel and supplies, long-term planning for the ongoing incident, and planning for the operation’s eventual closure.

2.6  Factors Impacting a Mass Fatality Response When formulating a mass fatality response, there are several aspects of the incident that can impact the response effort. These factors, listed below, should be considered when planning the response effort. 2.6.1  Closed versus Open Events A closed mass fatality event is one where the identities of all the deceased are known, and the identification process involves matching the identity with the body. In a closed event, there is typically a list of the missing people known to be in the location of the fatality event. Airline crashes, where authorities receive a manifest with passenger names, are the best example of closed mass fatality events. In an open mass fatality event, the identity and the number of the missing and presumed dead is primarily unknown because there is no list or manifest. Open events, where a missing persons unit will be an integral part of the response effort and will help to create a list of the presumed missing, can be much more complicated than a closed event. Once a list of the presumed missing has been created, the list will need to be vetted so that a

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Closed • Steven Able • Mary Adell • Jeremy Archer • Kenneth Black • John Brown • Sally Brown • Don Cartwright • Doris Cartwright • Virginia Smith • Gloria Powers • Joseph Powers • Charles Pringle

Open • Steven Able • Mary Adell • Jeremy Archer • Kenneth Black • John Brown • Sally Brown • Don Cartwright • Doris Cartwright • Virginia Smith • Gloria Powers • Joseph Powers • Charles Pringle • Kristine Pringle • Mabel White • Marshall West • Cathy Whlie • Nigel Zed • Ian Zed • Margaret Zed • Catherine Zona

?

Figure 2.7  Closed versus open events—matching names to human remains.

true list of the missing can be assembled. For example, during the Hurricane Katrina/Rita response, there were over 13,000 reported missing persons cases filed in Louisiana. However, there were only 910 bodies from stormrelated deaths processed through the Louisiana morgue known as the Victim Identification Center. It took considerable resources to reduce the number of reported missing to the same number of unidentified bodies. In this type of situation, collecting antemortem information (and DNA samples) for individuals who are not deceased wastes precious resources. Figure 2.7 illustrates how a closed event requires less effort to match names with individuals when compared to an open mass fatality event. 2.6.2  Number of Deceased The number of deceased individuals impacts all components of the identification process and has a direct impact on resources needed to support the identification effort. As outlined in Figure 2.8, mass fatality events that involve fewer than 100 fatalities can be handled much more easily than those involving tens of thousands. As the size of an operation increases, local authorities often need to make substantial changes to their operations. As the number of bodies increases, information technology and automation become increasingly important. Five thousand bodies cannot be processed using the same policies and procedures used to process five bodies.

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DNA Analysis for Missing Persons in Mass Fatalities

Amount of Resources

International National Regional

ple

m

Co

y xit

of

e

Id

n

tio

ca

ifi nt

ss

ce

o Pr

Local 10

Number of Deceased

10000+

Figure 2.8  Impact of number of deceased on the required response resources.

2.6.3  Rate of Recovery The rate of recovery of the remains also impacts the identification effort. If the rate of recovery is somewhat fast and unimpeded, there will be a need for a large surge capacity early on in the response. If the rate of recovery is slow (because of weather conditions, topography of the affected area, rubble, or other impediments), resources may need to be scheduled over a longer period of time, as illustrated in Figure 2.9. The rate of recovery also affects morgue operations for the same reason. FAC operations are similarly affected by the rate of recovery. Family members and friends of the missing and presumed dead will interact directly with FAC staff and depend on them for information about the identification and reunification process. When the recovery rate is slow, families may become impatient and angry if they are not aware of the time needed to properly recover and identify bodies. Once a family accepts the reality that their loved one is most likely deceased, their greatest desire is to have the body returned to them for proper disposition. This period of waiting can be difficult emotionally and deepen the surviving family members’ grief. Managing expectations is essential to helping families understand Fast and Unimpeded

Large surge capacity early on in response

Slow due to weather conditions, topography, rubble or other impediments

Resources scheduled over a longer period of time

Figure 2.9  Rate of recovery.

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the issues involved in the recovery process and helps them cope with the waiting period. It is especially important to help families understand that everyone involved in the response wishes for an efficient identification process, one without mistakes. 2.6.4  Condition of Human Remains Fragmentation, decomposition, and commingling of the human remains add to the complexity of the identification effort. As the condition of the remains deteriorates, the identification becomes increasingly difficult, the recovery process more complicated, and the need for DNA identification resources greater. 2.6.5 Fragmentation Fragmentation poses challenges since the resources necessary for collecting body fragments are greater when the body is not intact. Each separate body fragment must be collected individually and treated as a separate recovery. Individuals involved in the recovery and identification process should never assume that two fragmented body parts found near each other are from the same body. Each fragmented remain must be collected separately and given its own unique case number. Collecting each fragment separately will increase the need for body bags, marking materials, body tags, and other necessary resources. The morgue will process and examine the fragmented remains through the various units separately. Likewise, as piece-to-piece identifications occur, DNA operations become increasingly complex. FAC interactions with families will also need to address the issues of fragmentation. As the FAO works with the families, decisions may have to be made about how to release fragments of bodies. Close coordination with local officials, families, and the FAO will help establish guidelines as to how and when fragmented remains will be released and will also help to honor family wishes. 2.6.6 Decomposition Decomposition will also have an impact on all aspects of the identification effort. Identifiers such as tattoos, scars, birthmarks, fingerprints, and other distinguishing features are not detectable in severely decomposed or skeletal remains. Because the physical handling of decomposed remains is more time consuming, recovery will be increasingly difficult. Families often want to know the specifics about the body’s condition, thus necessitating the dissemination of accurate information in a sensitive manner. Local authorities in charge of the response effort typically decide which information is

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DNA Analysis for Missing Persons in Mass Fatalities

appropriate to release to family members, how it will be released, and who will release the information. One central information outlet helps to mitigate misinformation and helps to ensure family members’ emotions will not be adversely affected by receiving conflicting reports of the dead. 2.6.7 Commingling Mass fatality events caused by localized high energy events such as explosions typically result in the fragmentation and subsequent commingling of remains. In addition, bodies (skeletons) found in makeshift mass graves are often commingled. Some perpetrators of mass killings move bodies from grave to grave, thus causing further commingling and the spreading of fragmented bones across different sites. In these situations, careful anthropological and pathological examinations are necessary. DNA testing is often the only method of reassociation and identification. See Figure 2.10 for a detailed drawing of commingled remains from Guatemala.

2.7  Finances and Politics Finances and/or political pressure often dictate the identification effort. The extent of the mass fatality identification effort is directly related to the availability of funds to support the recovery and identification effort. Decision makers often do not understand that fully funded operations are required for proper identification and thus have a great effect on the well-being of surviving family members. An identification effort can be expensive. Calculation of

Figure 2.10  Documentation of a mass grave through a detailed hand drawing

of commingled remains. (Courtesy of the Fundacion Antropologia Forense de Guatemala [FAFG].)

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an adequate budget and identification of appropriate financial resources are critical to the success of the identification project.

2.8 Availability of Antemortem Records and DNA Reference Samples The availability of antemortem records and DNA reference samples is critical to the identification process. If there is no antemortem information (e.g., fingerprints, dental records, or DNA profiles) to compare the human remains to, identifications cannot be made.

Additional Resources Adams, B.J., and Byrd, J. 2008. Recovery, Analysis, and Identification of Commingled Human Remains. New York: Humana Press. Dupras, T.L., Schultz, J.J., Wheeler, S.M., and Williams, L.J. Forensic Recovery of Human Remains: Archaeological Approaches. New York: Taylor & Francis. Mundorff, A.Z. 2012. Integrating forensic anthropology into disaster victim identification. Forensic Science Medicine and Pathology 8: 131–139. Teachen, P.R. 2012. Mass Fatalities: Managing the Community Response. New York: Taylor & Francis.

Chapter Postmortem Functions—Body Recovery and Morgue Operations

3

Postmortem functions focus on locating, documenting, examining, and storing human remains. The information in this chapter is presented as a broad overview of body recovery and morgue operations.

3.1  Field Operations (Body Recovery) After an incident is officially declared a mass fatality by the appropriate entity, the managerial entity will organize and activate a field operations team. Field operations (also known as body recovery) include locating and documenting human remains, as depicted in Figure 3.1. Only after proper documentation should the remains be recovered and transported to designated body processing sites or morgues. There may be tremendous pressure from surviving family members, public officials, the media, and others to rapidly recover and identify the deceased following a mass fatality event. However, it is important to properly recover the human remains in a safe and well-documented manner. Although the public will expect the process to begin immediately following a disaster, recovery operations do not begin until the disaster site has been assessed and declared safe and the recovery teams assembled and briefed or trained on the proper recovery procedures. 3.1.1  Locating Remains Since mass fatalities are often associated with localized high-energy events (e.g., plane crash, tornado), body recovery may involve working in every possible type of terrain and environment. Bodies may be found in a variety of places. Places bodies may be found include: • In trees • On top of roofs or buildings 39

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DNA Analysis for Missing Persons in Mass Fatalities

Figure 3.1 (see color insert) Exhumed graves showing documentation. (Courtesy of the Fundacion Antropologia Forense de Guatemala [FAFG].)

• Within bodies of water • Within or underneath collapsed structures and piles of debris (see Figure 3.2) • Within or underneath vehicles, trucks, trains, planes, boats, etc. • In storm drains or sewer systems • In wells • In mass graves

Figure 3.2  Collapsed rubble may need to be carefully removed in order to identify remains.

Postmortem Functions—Body Recovery and Morgue Operations

41

Additionally, the bodies may be whole or fragmented and found in a variety of conditions (e.g., burned, fresh, decomposed, and commingled). Based on the condition of the human remains, it may be difficult for an untrained individual to recognize human remains. Therefore, it is important to ensure that members of the recovery team are experienced in identifying remains in a wide range of circumstances. Only professionals who have been specially trained in body recovery should be involved in the process. Mistakes and omissions made during the recovery effort can be detrimental and potentially lead to false or misidentifications. The process for locating a potential mass fatality site depends primarily on the type of incident. Generally, information regarding a potential mass fatality site comes from people in the area who report the location of bodies to the authorities. If rescue of the living is a component of the incident, many reports will come from the rescuers as they work in the field. Eyewitnesses may report information about mass murders to relevant authorities, although information may also come anonymously or from those responsible for the deaths of the individuals. Trained investigators are important in collecting and distilling the eyewitness testimony so that it can be relied upon in the human identification process. Aerial photography or ground-penetrating radar may also be employed to find mass graves. 3.1.2 Personnel Members of the field operations team may include archaeologists, forensic anthropologists, forensic pathologists, forensic odontologists/dentists, investigators, photographers, and evidence specialists. Certain situations will require additional personnel, particularly armed security forces, ordnance/bomb specialists, and biological/chemical/nuclear/hazardous material specialists. Depending on the situation, it may be helpful to have an individual familiar with the area (a local resident) as a part of the recovery team. This local person should be able to identify streets and structures even when signage is missing. Single individuals may fulfill multiple roles on the team if they have the necessary skill sets. For example, a forensic anthropologist may also be a qualified investigator and photographer. Additional support personnel may be required depending on the needs of a given situation (e.g., heavy equipment operator to move large pieces of debris). The field operations team is responsible for locating, documenting, recovering, and transporting the dead bodies from a mass fatality incident to a designated processing site or morgue. Accurately recording the recovery location is often crucial in the identification of human remains. This team is responsible for documenting not only the bodies

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DNA Analysis for Missing Persons in Mass Fatalities

but also the entire scene. The team should fully document and investigate any inconsistencies encountered in the field (e.g., a mass fatality incident involves the crash of a large commercial airplane due to reported mechanical problems, but one of the recovered bodies has what appears to be a gunshot wound to the head). The team is also responsible for documenting and recovering associated personal property, ensuring that personal property remains with/on the bodies, and ensuring that both the personal property and the bodies are adequately secured. Detailed and complete documentation of all field activities is essential, as is ensuring the transfer of this data to the morgue operations team and identification unit, and cooperating with their potential requests to clarify or provide additional information. The field operations team should also assume responsibility by clearly noting any obvious unique features that may aid in identification of the deceased, as well as possible health risks, or other dangerous situations that the morgue operations team may face (e.g., bodies exposed to hazardous materials or bodies found with unexploded ordnances). A field operations supervisor should be assigned to the team. Table  3.1 provides a list of the responsibilities of the field operations supervisor. If there are multiple recovery sites working simultaneously, the field operations supervisor may need to assign team leaders to each site. Field operations require many of the same actions and types of data collection. However, exact procedures may vary slightly based on the type of mass fatality incident (e.g., natural disasters, acts of terrorism, large-scale transportation accidents, acts of war or genocide, and structural collapse of buildings or roadways). The field operations team should be aware of the emotional and psychological difficulties associated with the recovery of human remains. These difficulties can be especially troublesome when working with mass graves or when a large number of children are among the deceased.

Table 3.1  Responsibilities of the Field Operations Supervisor • Successful completion of body recovery • Safety of all team members • Inventory, allocation of, and accountability for all equipment and supplies • Team’s adherence to the standard operating procedures • Regular communication with the managerial entity • Performance of administrative duties and completion of necessary paperwork • Assurance that personnel follow the legal rules governing chain of custody and handling of evidence

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43

1. Determine the nature of the situation

2. Fully assess hazarads of all types, not just biological, chemical, and nuclear

3. Advise personnel of potential health and safety concerns and anything done to neutralize the hazard

4. Take necessary precautions, obtain protective gear, and determine appropriate specialists to employ

Figure 3.3  General health and safety considerations when preparing to locate human remains.

3.1.3  Health and Safety Concerns Before fieldwork begins, the individual in charge of the identification effort, working with local health officials and law enforcement, must determine the nature of the situation (e.g., biological contagion versus act of war), appropriate precautions, necessary protective gear, and appropriate specialists. Additionally, recovery personnel should clearly note any health risks, contagions, or other dangerous situations (e.g., bodies exposed to hazardous materials or bodies found with unexploded ordnance) and should communicate these risks to others involved in the recovery and identification efforts. All personnel, not just field operations personnel, responsible for handling the remains, must be advised of potential health and safety concerns. All personnel must also be made aware of any actions to try and neutralize the hazard (e.g., bodies were chemically treated to neutralize a contagion). This is outlined in Figure 3.3. Depending on the circumstances, the leader of the identification effort will need to fully assess hazards of all types, not just biological, chemical, and nuclear. For example, after the collapse of the World Trade Center in

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DNA Analysis for Missing Persons in Mass Fatalities

New York City, air quality was regularly monitored. If necessary, rescue and recovery workers must wear specific types of protective breathing gear. Caution should be exercised when handling situations involving terrorism, war, or genocide because there may be ongoing threats and/or danger for responders. Such threats may occur at the scene or from bodies via unexploded ordnance, hidden bombs, sniper fire, and/or presence of hazardous materials. Appropriately educated and trained personnel, as well as adequate safety measures, should be employed at all times. 3.1.3.1  Mass Graves Mass graves may present different challenges to the field operations team. Depending upon the circumstances, it may be necessary to address external dangers prior to and during excavation. Such dangers include subsurface mines or unexploded and/or purposely planted ordnance on the bodies. Workers could also face gunfire or other life-threatening dangers. 3.1.3.2  Environmental Hazards Normal environmental conditions must be taken into consideration and planned for (e.g., adequate hydration in extreme heat and monitoring individuals for signs of heat exhaustion). The presence of poisonous plants, high altitudes, dangerous insects and animals, and hazardous terrain must also be adequately addressed to ensure the safety of response personnel. 3.1.3.3  Families On-Site If a recovery site is not secured, families of the potentially deceased may be on-site. The field operations team should be aware of the health and safety of the families and cognizant of the potential for families to cause issues (even though they may be unintentional). Family members may be emotionally distraught or have mental health issues that may manifest themselves in physical responses. Sometimes these responses may be aimed at team members simply because they are present and therefore easy targets. Other family members may insist on helping with recovery, which could jeopardize the integrity of the site and/or cause safety issues. The field operations team should provide information about the exhumation and identification process to families. When families come to a site, it is a good opportunity to collect antemortem data and DNA samples. 3.1.4  Assessing a Disaster Site Once a disaster site has been deemed safe to enter, a scene evaluation team can assess the site, establish communication with the managerial entity, evaluate the scene to determine the size and complexity of body recovery operations, and develop a field action plan. The scene evaluation:

Postmortem Functions—Body Recovery and Morgue Operations

45

• Documents date, time, and location of incident • Determines size and scope of scene • Appraises accessibility of the incident scene and determines level of difficulty for body recovery • Obtains photo and/or video documentation (without sound) • Determines number of known fatalities • Estimates number of potential fatalities • Estimates number of remains for autopsy • Determines condition of human remains (bodies) • Identifies possible biological, chemical, physical, or radiological hazards (see above) • Determines types and numbers of personnel and equipment needed for body recovery Information obtained from the site evaluation can assist in developing and establishing not only the recovery operations, but also the morgue and Family Assistance Center operations. This information is also used to develop appropriate incident-specific recovery policies, procedures, and forms and will help determine the resources—equipment, supplies, and personnel—required to properly recover the human remains. 3.1.5 Documentation Accurate documentation of the recovery location, including a description of the scene and circumstances, is often crucial in correctly identifying bodies and determining the manner of death. The field operations team should fully document and investigate inconsistencies. It is important that standard procedures and nomenclature be used across the entire scene. If various recovery teams use different nomenclature to document the search for bodies, it will be difficult, if not impossible, to have a good understanding of the status of bodies (how many have been found or not found). During the recovery efforts after Hurricane Katrina, the use of different symbols in documenting the presence or absence of bodies located in buildings is an excellent example of how detrimental varied nomenclature can be. Following the initial recovery effort, it was not clear which symbols were used to indicate the result of the search. Therefore, subsequent body recoveries had to be performed several times. Recovery teams should only utilize terminology and forms approved by the managerial entity. Field documentation may include videography, photography, mapping, Global Positioning System (GPS) receiver readings, latitude/longitude, physical addresses or intersections, detailed measurements, grids, written field reports by team members, and/or other standard techniques. It should also

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DNA Analysis for Missing Persons in Mass Fatalities

Table 3.2  Information Documented at Time of Body Recovery • Location, including physical address, intersection, Global Positioning System (GPS) receiver reading, or latitude-longitude • Description of scene including measurements, photographs, video, grid number, situation (e.g., mass grave) • Description and condition of body including estimated age, sex, and race • Description of anything found on the body (clothing, jewelry, obvious tattoos or other identifiers, obvious evidence of trauma). Do not remove any items from the body— document what is present, and where it is located (e.g., yellow metal ring with one clear stone on left fourth finger). • Who found the body, when, and the person’s contact information • Names of recovery team members present at the recovery, as well as the date/time of actual recovery • How many photographs were taken on scene • Presumptive identification, if any, and what it is based on • What was done with the body—put into body bag, wrapped in a sheet or tarp • Where the body and personal items were stored or if transported, by whom and when • The unique field recovery identification number, bar code, or other unique code assigned • Health and/or safety concerns

involve assigning unique field numbers to each individual body and each unassociated item of interest. 3.1.5.1  Other Field Documentation Unique physical characteristics and personal items on the bodies are important clues used in the identification process. Due to the processes of decomposition and the possibility of personal items becoming disassociated from the bodies, it is important to document personal items in the field. Although field operations team members should document items in detail in the field, they should leave clothing and personal items on the bodies as they were found. Only obviously visible identifiers should be noted in the field. Nothing should be removed from the body before the body is taken to the morgue since detailed examinations of the remains should only be performed in the controlled environment of the morgue. As listed in Table 3.2, field operations team members collect and document information at the time of body recovery. All field documentation must be forwarded to the morgue operations team and identification personnel. Morgue or identification personnel may contact members of the field operations team to clarify or provide additional information. 3.1.5.2  Associated and Unassociated Property Associated items are those found on the remains (e.g., jewelry), worn by the deceased (e.g., backpack attached to the body or clothing on the body), or

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Figure 3.4  Unassociated item.

found within other associated items (e.g., wallet found in the pocket of the pants worn by the deceased). Unassociated items, such as that seen in Figure 3.4, are found near the bodies but cannot be directly connected to the bodies (e.g., a briefcase found two feet from a body). Unassociated items are typically documented and numbered, noting their location on the scene. Field operations team members must follow standard procedures to properly collect unassociated items, and the managerial entity should have procedures in place for how to handle unassociated items. In addition to properly documenting, numbering, and bagging each body separately, personnel should do the same for each unassociated item. These bags must be securely stored until they can be transported to the morgue or other designated processing site for evaluation. 3.1.6  Removal of Remains After bodies are located and thoroughly documented, they must be removed and either stored or transported to the morgue or other processing site. Cultural values and the managerial entity typically determine how bodies are handled. Some societies require that extreme care and deference be shown to the dead, while others require less. It is recommended that the recovery team use vinyl zipper-closure body bags for the human remains. Unless commingled masses are bagged together, there should be one body per bag. Alternatively, if bags are unavailable, clean sheets are an appropriate substitute; however, they must be securely sealed to prevent contents from escaping. A minimum of two labels with the individual’s field number, bar code, or other unique identifier should be used per bag— one placed inside the body bag and the other attached to or placed on the outside of the body bag. The type of tag must be predetermined, as many tags will not retain their information over time or in various environmental conditions.

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DNA Analysis for Missing Persons in Mass Fatalities

3.1.6.1  Mass Graves Since mass graves contain multiple individuals often in various stages of decomposition, the recovery team must carefully document the bodies prior to removal. The careful removal of each body ensures that all body parts are bagged together. The managerial entity should have standard procedures for addressing whether badly commingled bodies are to be separated in the field or bagged together for separation in the morgue. Some mass graves are secondary burial sites and may primarily contain body parts, not whole bodies. In certain cases, to purposely complicate the identification process, the perpetrators will move the bodies from grave to grave. 3.1.6.2  Commingled Remains Commingled remains require careful documentation prior to removal. As disarticulated remains may come apart during recovery, personnel should exercise extreme care when removing bodies. The managerial entity should have standard procedures for handling and documenting these situations. As commingled and incomplete remains can be more difficult to identify, personnel should employ appropriate and careful recovery techniques. The field operations team should also document whether a mass grave is a primary or secondary burial site. 3.1.6.3  Personal Items During removal and bagging, associated personal items should remain with the body. Field operations team members should thoroughly document personal items as part of the field report. Unassociated items should be placed in a separate bag and have their own unique field identification number. The managerial entity will determine whether these items are transported to the morgue or to a different processing site. 3.1.6.4  Chain of Custody In order to avoid later allegations of tampering or misconduct, field operations team members must handle possible evidence according to protocol. Written or electronic records must document all aspects of the collection, storage, and transfer of the evidence. 3.1.7  Transportation and Storage Field operations team members or other responsible personnel must follow designated protocols to ensure bodies, personal items, and possible evidence are appropriately secured, stored, and transported. See Attachment A for a sample Transportation Log.

Postmortem Functions—Body Recovery and Morgue Operations

49

Bodies and their associated personal items are typically stored in refrigerated units (trucks) to retard the rate of decomposition until the morgue operations team is able to process the bodies and associated remains. Depending upon the specific incident, bodies may be transported immediately from the recovery site to a collection site. At the collection site, bodies may be stored until a refrigerated unit is full and sent to the morgue. Alternatively, bodies may be transported directly to the morgue. Refrigerated trucks require constant fuel or other forms of power. Thus, maintenance personnel should be on contract and immediately available if problems are encountered. In remote areas, refrigeration may be impossible. The appropriate storage container will depend on the condition of the body. Maintaining accurate logs of the storage and transportation ensures that the whereabouts of every body is known at all times. Logs should contain the unique code assigned to each recovered body, a record of who accepted the body for storage or transportation, and all other details concerning transportation.

3.2  Morgue Operations Morgue operations involve the following key tasks: • Processing, analyzing, and documenting human remains and personal items from the mass fatality incident • Comparing postmortem information from the human remains to the antemortem information collected by the Family Assistance Operations to identify the human remains • Storing human remains and personal items • Releasing the human remains after they have been identified or releasing human remains for long-term storage or other disposition While mass fatalities can differ greatly in scope, morgue operations are fairly consistent. In order to address special circumstances (e.g., health hazards from explosive material), flexibility is necessary. 3.2.1 Personnel The morgue operations team typically includes photographers, forensic pathologists, forensic odontologists/dentists, forensic anthropologists, radiology technologists, fingerprint specialists, and DNA collection specialists. Additional personnel may include evidence specialists, investigators, ordnance/bomb specialists, biological, chemical, nuclear, and hazardous material specialists, radiologists, technicians, autopsy assistants, security

50

DNA Analysis for Missing Persons in Mass Fatalities

Table 3.3  Responsibilities of the Morgue Operations Team • Storing the remains, maintaining an accurate inventory of what has been received, and knowing exactly where the bodies are located at any given moment • Transporting the remains to and from the morgue facility • Collecting and documenting all information from processing the remains, personal property, and evidence in the morgue • Processing and adequately storing this data, whether in paper and/or computerized files • Providing security for the remains, recovered personal property, and evidence • Comparing antemortem data (as obtained by the Family Assistance Operations team) and postmortem data (including field recovery data) for recommendations on identification of remains • Obtaining additional information from any sources as needed for identification purposes, and appropriately documenting all identification-related work

personnel, fingerprint identification specialists, computer data entry personnel, file clerks, computer specialists, equipment maintenance personnel, supply clerks, and transportation/storage personnel. Individuals may fulfill multiple roles on a team if they possess the necessary skill sets (e.g., a forensic pathologist may also perform DNA collections). A supervisor should be assigned to assume responsibility for all morgue-related functions. This supervisor will assume responsibility for all morgue-related activities. It is important that the morgue operations supervisor be vigilant in ensuring that standard operating procedures are followed. Additionally, information necessary for the identification of remains and the documentation of injuries, evidence of torture, genocide, and terrorism must be properly documented. The morgue operations supervisor should also guarantee that the morgue follows legal rules governing appropriate forensic handling of evidence. This supervisor may also be assigned to oversee the storage of remains at the morgue facility, data management, and storage activities. The morgue operations supervisor needs to have regular communication with the managerial entity, field operations supervisor, and Family Assistance Operations supervisor. Communication among individual operations is crucial for the successful identification of the deceased. Table 3.3 outlines the responsibilities of the morgue operations team. The managerial entity may also task the morgue operations team with the responsibility of releasing remains and/or personal property after the official identification has been made. In order to ensure the appropriate individuals or agencies receive the bodies and personal property, morgue operations functions must be carried out according to established standard operating procedures.

Postmortem Functions—Body Recovery and Morgue Operations

51

3.2.2 Safety Morgue personnel should always work in a safe environment. If any individual in the morgue observes potential safety issues, they should be brought to the attention of the appropriate morgue supervisor. Human remains should be considered potentially infectious for HIV, HBV, and other blood-borne pathogens. All individuals coming into contact with human remains must use universal precautions, including: • Wearing gloves when touching blood, body fluids, body substances, or any item or article associated with deceased human remains • Using masks, goggles, or face shields when splattering or splashing of blood or body fluids is possible • Wearing gowns or aprons when splashing, splattering, smearing, or soiling from blood or body fluids is a risk • Carefully handling scalpels or other sharp objects to avoid injury • Washing hands and other body parts immediately if contaminated with blood or body fluids • Washing hands immediately after removing gloves It is a good idea to have a personal protective equipment (PPE) dressing/ gowning area established for use prior to entering the morgue processing stations, as well as an area designated for the removal and discarding of PPE upon exit. Biohazard bags should available to discard used PPE. 3.2.3  Numbering the Human Remains In order to account for both the unique field number/code and the unique morgue number/code, the record-keeping system for logs should be flexible. This is especially relevant when the morgue operations team separates remains from a single body bag into multiple individuals. Each individual identified by morgue personnel should have the same field number/code but a different morgue number/code. This way, specimens can be separated and associated with other specimens without causing confusion. The morgue operations team may also receive and store unassociated items or property and/or possible evidence. Appropriate logs and secure storage facilities must be maintained. As human remains arrive from the field, the morgue operations team should log human remains and personal items. Maintaining accurate records ensures immediate access to all information collected at the morgue.

52

DNA Analysis for Missing Persons in Mass Fatalities

Figure 3.5 (see color insert)  Evaluation of skeletal remains. (Courtesy of the Fundacion Antropologia Forense de Guatemala [FAFG].)

3.2.4  Morgue Examinations In the morgue, as seen in Figure  3.5, trained personnel perform the detailed examination and analysis of bodies. Data collected from these examinations is used in combination with the information collected in the field and by the Family Assistance Operations team. Such information helps identify deceased individuals, and if needed, their cause and manner of death. In order to ensure that postmortem data is accurate, reliable, and legally binding, data must be collected according to approved protocols and accepted industry standards. If data is improperly collected, identifications will be negatively affected. See Attachment B for postmortem data collection forms used by disaster mortuary operational response teams (DMORTs) (www.dmort.org). Table 3.4 lists the information that personnel will try to obtain during the postmortem examination. Unlike routine morgue casework where human remains are typically processed in one location, a mass fatality incident requires separate stations, and the remains are often moved from station to station. Typically in a mass fatality response, all human remains will be processed through the following stations: • • • • • • • • •

Decontamination (if needed) Admitting Triage Photography X-ray Fingerprint Odontology Personal effects Anthropology

Postmortem Functions—Body Recovery and Morgue Operations

53

Table 3.4  Information That May Be Collected during Morgue Operations • Detailed and complete description of remains with photographs: documenting the remains as received and after clothing is removed and/or any debris is removed from the remains (e.g., mud is washed off), as well as any personal identifiers (e.g., tattoos) • Autopsy findings (if autopsies are performed) • Documentation of possible evidence • Complete dental examination including radiographs and photographs • Full-body radiographs and medical/anthropological assessment of radiographs • Fingerprints, palm prints, and/or footprints may also be obtained • Fingerprint comparisons • Skeletal analysis of remains • Separation of commingled remains and determination of minimum number of individuals represented by the commingled remains • Detailed and complete description with photographs of recovered personal items and clothing • Proposed identification of deceased • Assignment of a unique morgue case number to each individual set of remains, especially if commingled remains were separated into individuals • DNA specimens extracted and sent to the DNA laboratory

• • • •

Pathology DNA Final processing Storage

The number of stations may vary depending upon the magnitude of the morgue operations. Figure 3.6 outlines the general flow of human remains through the morgue.

Triage Receipt

Nonhuman Remains Removed

Admitting Escort Assigned

Photography

Personal Effects

Odontology

Pathology

Anthropology

Fingerprint

Radiology

DNA

Final Processing

Remains Holding

Release

Figure 3.6  Common steps for processing human remains.

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DNA Analysis for Missing Persons in Mass Fatalities

3.2.4.1 Photography Upon arrival, morgue personnel should take detailed full-body photographs of the bodies. Additional photographs will help to document possible identifications on the bodies, such as tattoos. After pathologists remove clothing and associated personal items, bodies should be properly cleaned and documented in writing and photographs. Details from clothing and personal items may provide clues as to the identity of the dead (e.g., inscription on a ring). The management entity determines whether or not clothing and/or associated personal items are kept with bodies after removal and cleaning. If separated from the bodies, clothing and/or associated personal items should be packaged separately and clearly labeled with corresponding field and morgue numbers/codes. 3.2.4.2  Forensic Pathology Pathologists, particularly forensic specialists and medical examiners, will conduct external examinations of all bodies. Pathologists usually begin with a complete description of the body as received from the field, including clothing and associated personal items. Then pathologists typically remove clothing and associated personal items, updating their descriptions with findings from the now nude body. Depending upon the type of incident and mandates from the managerial entity, an autopsy may be performed. Pathologists are responsible for fully documenting all possible identifiers on the body, including tattoos, body piercings, surgical interventions, nonsurgical amputations, evidence of disease or injury, implants, and irregularities. As needed, pathologists will also remove implants and devices to provide serial numbers and additional descriptive information. Additionally, pathologists may work alone or in conjunction with forensic dentists and anthropologists to assess body radiographs obtained in the morgue. The forensic pathologist can make presumptive or positive identification. 3.2.4.3  Body Radiography It is recommended that full-body radiographs, including the extremities, be obtained for all bodies. X-rays, such as the one in Figure  3.7, can provide a wealth of information, such as remote and recent fractures, evidence of surgery, presence of implants and orthopedic devices, presence of foreign objects (e.g., bullets), and location of displaced teeth. Additionally, if antemortem radiographs are available, ante- and postmortem radiographs can be compared for identification purposes. The pathologist, dentist, and anthropologist should review all x-rays.

Postmortem Functions—Body Recovery and Morgue Operations

55

Figure 3.7  X-ray.

3.2.4.4 Fingerprints If there has not been a significant amount of deterioration, a large majority of positive identifications are made through fingerprint comparisons. In order to successfully obtain postmortem prints, special techniques should be employed, especially when working with decomposed or burned bodies. It is crucial that specially trained and experienced fingerprint personnel work in the morgue. Some jurisdictions will also obtain palm- and/or footprints. 3.2.4.5 Dental Dentists, particularly those with forensic training and experience, will perform dental examinations. Dentists will chart the presence and/or absence of teeth, restorations, and appliances. If necessary, dentists may obtain a full set of dental radiographs and photographs. Positive identifications can be made through ante- and postmortem dental comparisons. Postmortem dental examinations can differ from those performed on the living. Dentists may have to remove the upper and lower dentition and associated bone. Individual teeth may have been knocked out of the mouth and may be located in other parts of the body. If possible, missing teeth should be retrieved and assessed with the rest of the dentition. Additionally, if remains were burned, heat may have altered teeth and associated bone. Forensic dentists may also work with the pathologist and/or anthropologist to assess postmortem body radiographs. Forensic dentists are trained to make positive identifications based on ante- and postmortem dental comparisons. Some forensic anthropologists are trained in dental assessments and identification. 3.2.4.6  Forensic Anthropology Anthropologists, particularly those who specialize in forensics, analyze bones. Bone analysis is most helpful with highly decomposed, skeletonized,

56

DNA Analysis for Missing Persons in Mass Fatalities

burned, fragmented, or commingled remains. Anthropological assessments can provide estimates of age, sex, race, stature, and build, as well as documentation and analysis of trauma, pathology, time since death, and unique—possibly identifying—traits. Forensic anthropologists have experience assessing radiographs and may provide presumptive or positive identification through ante- and postmortem radiograph comparisons. 3.2.4.7 DNA In a mass fatality incident it is highly recommended that DNA specimens be collected from each body, even if DNA is not needed for a positive identification. DNA samples should only be collected after all other examinations have been conducted in the morgue. Additionally, only personnel properly trained in postmortem DNA sample collection should collect samples. Blood, bone (as seen in Figure 3.8), muscle, and teeth can be used for DNA testing. Specific procedures and protocols for the selection of appropriate specimens and their testing are set forth in subsequent chapters of this training manual. 3.2.4.8 Administrative During examinations, an administrative person should be assigned to the morgue. This person assumes responsibility for assigning unique morgue numbers/codes to individuals, assembling written documentation from the various examinations, and providing it to the professionals who will be comparing the postmortem information to the antemortem information. 3.2.5 Documentation When appropriate, work performed by morgue operations personnel should be fully documented in writing, with photographs, radiographs, body diagrams, and/or drawings. Some jurisdictions may allow for the dictation of reports that must then be transcribed, but this method should be

Figure 3.8 (see color insert)  A window of bone cut for DNA testing.

Postmortem Functions—Body Recovery and Morgue Operations

57

carefully considered because if there are a large number of bodies, this can be quite time consuming. 3.2.5.1  Data Management Proper data management should be a part of every operations unit from the beginning of the incident. Whether data is computerized or maintained in paper files, the managerial entity should have standardized procedures for processing, storing (short and long term), backing up, and accessing data. The effective management of very large amounts of information is only possible with the use of computers and specialized disaster management software. If there are more than 25–50 bodies to process, it becomes imperative that the managerial entity use information technology tools to collect data and manage the postmortem data. Some computer programs used in mass fatality responses for postmortem and antemortem data collection and comparison include: • Victim Identification Program (VIP) used by the U.S. federal Disaster Mortuary Operational Response Teams (DMORT). • FRED, the identification program used by the Florida statewide response team—Florida Emergency Mortuary Operations Response System (FEMORS). • Sapphire’s DataEase and Unified Victim Identification System (UVIS). These backups must be checked to ensure proper recording. Some jurisdictions have created their own applications to meet the specific needs of the mass fatality response. Regardless of the program’s origin, computer software should be fully tested to ensure it can handle the needs of all operations. Personnel required to use the chosen software should be trained and proficient in its use. All computers, networks, software, and databases will require on-site support and regular upkeep. The managerial entity should have appropriate staff in place to support the data management needs of the mass fatality response operation. Immediate computer entry of all data (documentation from morgue examinations, including photographs and radiograph) is highly recommended. Morgue personnel should maintain a copy of the original records and check data entries for accuracy. To save space, records can be scanned as non-alterable files. It is critical to control access to files and computers and to identify individuals with the privilege to enter and edit data. All data should be kept secure, and files should be backed up regularly.

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DNA Analysis for Missing Persons in Mass Fatalities

If computerization of the data is not possible, then copies should be made of paper records, and the copies should be stored at a secure off-site location.

Additional Resources Andrew, E. 2011. Mortuary provision in emergencies causing mass fatalities. Journal of Business Continuity & Emergency Planning 5(1): 430–439. Blau, S., and Briggs, C.A. 2011. The role of forensic anthropology in Disaster Victim Identification (DVI). Forensic Science International 205(10–3): 29–35. Okoye, M.I., and Wecht, C.H. 2007. Forensic Investigation and Management of Mass Disasters. Tucson, AZ: Lawyers and Judges. Schuliar, Y., and Knudsen, P.J.T. 2012. Role of forensic pathologists in mass disasters. Forensic Science, Medicine, and Pathology 8: 164–173.

Attachment A Transportation Log Departed from:               Arrived at:          Transported by:                             Date of Departure:              Date of Arrival:        Time of Departure:             Time of Arrival:        Field Case Numbers Transported:                                                                                                                                                                  

Attachment B (Forms 1–14)

                                                                                                                                                        

Postmortem Functions—Body Recovery and Morgue Operations

Form 1

59

60

DNA Analysis for Missing Persons in Mass Fatalities Tracking Form

Incident Incident Date

PM Victim Status:

Site Recovery #

Date Received by Admitting:

Morge Reference #

Date Processed In Morgue:

ME/C #

Tracker:

Presumptive ID:

Name

,

,

Last Name DOB

First Gender

Middle

Suffix

SSN

Section Leader MUST mark below when processing completed. “Yes” = Completed, “No” = nothing was performed at that station.

Morgue Station:

Start Time

Station Leader’s Name

Signature

Completed: Yes

No

Radiology

Yes

No

Pathology

Yes

No

Personal Effects

Yes

No

Fingerprints

Yes

No

Odontology

Yes

No

Anthropology

Yes

No

DNA

Yes

No

Embalming

Yes

No

Admitting/Exit

Yes

No

Admitting

Triage

From Site Recovery Description of Remains: Tracking Form Comments

Barcode Number:

This Bag Also Produced Morgue Reference No’s:

Place Barcode Sticker Here.

Form2

Postmortem Functions—Body Recovery and Morgue Operations Site Recovery # Put N/A in all unused fields.

Recovery Date

Incident Date Morgue Reference No.

Classification of Remains: MM/DD/YYYY

Choices: Complete HR (C/HR), Fragmented HR (F/HR) or Common Tissue (CT/HR) Recovery Grid #:

Time:

Incident

Victim Site Recovery Form

24 hour (00:00)

Condition: select all that apply

GPS of Recovery:

Place / Address of Recovery:

Autopsied Previously Burned-Partial Thickness

Decomposed Embalmed

Mummified Saponified

Skeletonized-Partial Skeletonized-Full

Burned-Full Thickness Cremains

Fragmented Fresh

Scavenged Skin Slippage

Wet-Environmental

Description of Remains: Position Remains Found In: Estimated Age:

Baby/Child

Estimated Sex:

Male

Clothing on Remains: (brief description)

Yes No

Personal Effects on Remains: (brief description)

Yes No

Adolescent

Female

Young Adult

Undetermined

Middle Aged

Elderly

No Estimate

Estimated Race:

Recovery Comments:

Presumptive FIELD ID: ID Based On:

Last

First

DOB (MM/DD/YYYY)

SSN

Middle ID# / Drivers license # / State

Recovered By: Name and Agency (if applies)

Delivered to Transport Staging: Site Recovery Report Completed by:

Phone #

Date Recovered

Time Recovered

Name and Agency (if applies)

Phone #

Date Recovered

Time Recovered

Name and Agency (if applies)

Phone #

Delivered to Morgue by: Team Leader:

Form 3

Agency

Phone # Date Delivered

Time Delivered

61

62

DNA Analysis for Missing Persons in Mass Fatalities

Radiology 1

Examining Radiologist Scribe

Incident Incident Date Morgue Reference No.

Exam Date: Classification of Remains:

This is Inital X-ray Exam:

This includes a Secondary X-ray Exam: Number of Additional Radiographs:

Number of Initial Radiographs:

Radiology Technologist(s): Name (list all who worked on THIS case):

Reason for Additional X-rays:

Pacemaker Present:

Yes

No Implants Present:

Yes

No

Notable Findings Per Technologist:

Technologist notified the following person of “notable findings”: Name of Specialist

Form 4

Morgue Section

Date Notified

Postmortem Functions—Body Recovery and Morgue Operations

Radiology 2 for DVP

Examining Radiologist Scribe Exam Date:

Incident Incident Date Morgue Reference No.

Assessment Done By: List Names

Radiologist

Type of Forensic Specialist: Estimated Gender: Estimated Age:

Male 0-2

Female 3-5

6-10

Pathologist

Anthropologist

Dentist

Undetermined 11-20

21-30

31-40

41-50

51-70

Radiology Specific Findings: 1

Location:

Side:

Type: Detailed Description:

2 Location:

Side:

Type: Detailed Description:

3 Location:

Side:

Type: Detailed Description:

4 Location:

Side:

Type: Detailed Description:

5 Location:

Side:

Type: Detailed Description:

Comments:

Form 5

71+

63

64

DNA Analysis for Missing Persons in Mass Fatalities Examining Pathologist Scribe Exam Date:

Gender:

Male Female

Estimated Race:

Estimated Age:

Undetermined

6-10 11-20

Asian

Hispanic

American Indian

Undetermined

cm: Auburn Black

Length:

Short

Medium Curly

Description:

Red

White

kg:

Other - specify

Shaved

Other - specify

Wig

Other - Specify

Facial Hair:

Yes

Facial Hair Color:

Auburn

Facial Hair Type:

Clean Shaven

Beard & Moustache

Goatee

Sideburns

Moustache

Beard

Stubble

Lower Lip

Eyes Info

No Blond Brown

Black

Color: Condition:

Gray

Salt & Pepper

Red

White

Blue

Green

Hazel

Brown

Grey

Undetermined

Missing-Right

Glass-Right

Glass-Left

Contaract-Left

None

Present:

Yes

Corneal Implant-Left

Glasses

Dental

No

Dentures:

Contaract-Right Other - specify Other - specify

Corneal Implant-Right Yes

Upper

Engraved/Labeled

No

Lower

Engraved/Labeled

Yes

Type and location:

No

Type and location:

Other - specify

Other - specify

Missing-Left

Contacts

Form 6

NA Other - Specify

Both Intact

Aids:

Appliance:

Male Pattern Baldness Undetermined

Bald

N/A

Hair Transplant

Jaw/Face Only Large Extremities Resolving

lbs:

If measured: cm inches Straight

Hair Piece

Unfixed

Absent Complete, all muscles Hands, Feet Fingers, Toes

Salt & Pepper

Wavy

Extension

Fixed

Large/Robust Undetermined

Rigor - check all that apply

Gray

Long

71+

Location of Lividity - required

Estimated Weight

Blonde Brown

41-50 51-70

Small/Gracile Medium/Intermediate

Lividity:

Saponified Scavenged Skin Slippage Skeletonized-Partial Skeletonized-Full Wet-Environmental

Height inches:

21-30 31-40 Other - specify

Build

Autopsied Previously Burned-Partial Thickness Burned-Full Thickness Cremains Decomposed Embalmed Fragmented Fresh Mummified

Hair Info

0-2 3-5

Black

Condition of Remains: check all that apply

Accessory:

Incident Date

Morgue Reference No.

Caucasian

Classification of Remains:

Color:

Incident

Pathology 1 Page 1 of 3

Postmortem Functions—Body Recovery and Morgue Operations

Pathology 2 for DVP

Incident

Natural

Artificial

Not known

Color

Long

Medium

Short

Examining Radiologist Scribe Exam Date:

Fingernails Type

N a i

Length

l

Extra Long

Toenails Color

s

External Genitalia (check all that apply)

Incident Date Morgue Reference No.

Description

Description

□ Female □ Circumcised □ Circumcision Undetermined □ Male □ Uncircumcised □ No Identifiable External Genitalia

Evidence of Possible Surgery: As Indicated By Scars, Sutures, etc. (check all that apply)

□ Amputation □ Appendectomy □ Brain □ Caesarean □ Cardiac

□ Gall Bladder □ Other - Specify □ Laparotomy □ Mastectomy □ Reconstructive □ Tracheotomy

Scars, Amputation, Birth Marks, Deformities: Category

Location

Side

Description

Location

Side

Description

Location

Side

Description

Location

Side

Description

Location

Side

Description

Scars: Amputation: Birth Mark: Deformity: Category Scars: Amputation: Birth Mark: Deformity: Category Scars: Amputation: Birth Mark: Deformity: Category Scars: Amputation: Birth Mark: Deformity: Category Scars: Amputation: Birth Mark: Deformity:

Form 7

Yes

No

Specify Other Surgeries here:

65

66

DNA Analysis for Missing Persons in Mass Fatalities

Examining Pathologist

Pathology 3 for DVP Page 3 of 3

Scribe Exam Date: Body Piercing and Tattoos

Body Piercing(s)

Total # Path Photos Taken

Yes

Incident Incident Date Morgue Reference No. No

Tattoo(s)

Yes

No

Image #’s:

Pathology Narrative:

Body Diagram Used Category

Yes

Location

No

Referred for Autopsy Position

Yes

No

Tox Collected

Yes

No

Description

Tattoo Piercing Category

Location

Position

Description

Location

Position

Description

Location

Position

Description

Location

Position

Description

Tattoo Piercing Category Tattoo Piercing Category Tattoo Piercing Category Tattoo Piercing Foreign Objects / Implants / Prosthetics / Orthopedics In Body Foreign Object Present: Type:

□ Pacemaker □ Prosthetic □ Other - Specify

Type Other:

Description: Type:

□ Pacemaker □ Prosthetic □ Other - Specify

Removed from Body: Type Other:

Description: Type:

□ Pacemaker □ Prosthetic □ Other - Specify

Description:

Form 8

Position:

Position:

Removed from Body: Type Other:

Position:

Removed from Body:

Yes

No Location:

Yes

No

Location:

Yes

No

Location:

Yes

No

Postmortem Functions—Body Recovery and Morgue Operations

PE Section Leader Photographer

Incident

Clothing for DVP Page ____of ___

Incident Date Morgue Reference No.

Exam Date:

CLOTHING INVENTORY: For additional items add pages. Clothing Item

Color

Description

Size

Unique Features

Clothing Item

Color

Description

Size

Unique Features

Clothing Item

Color

Description

Size

Unique Features

Clothing Item

Color

Description

Size

Unique Features

Clothing Item

Color

Description

Size

Unique Features

Clothing Item

Color

Description

Size

Unique Features

Anything Handwritten On Clothing Or Tags? (location and description)

Associated Personal Effects (found on the body): Backpack

Cellphone

Fanny Pack

Book Bag

Coin Purse

ID Bracelet

Jewelry Money Clip

Yes

Wallet

No

Other-Specify in box below.

Purse

Other PE: Description of Item(s):

Monetary Items: (cash, coin, travelers checks, foreign money)

Identification Sources: (credit cards, checkbook, Id’s, etc.)

Unassociated Personal Effects (with but not on the body):

Other Personal Effects:

Form 9

Yes

No

67

68

DNA Analysis for Missing Persons in Mass Fatalities PE Section Leader Photographer

Incident

Jewelry for DVP Page of

Incident Date

Morgue Reference No.

Exam Date:

Watch

Jewelry Inventory Type Make

Band Material Face Color

Jewelry

Jewelry/Type Style

Jewelry/Type Style

Jewelry/Type Style

Jewelry/Type Style

Jewelry/Type Style

Description

Photo taken:

Material Color Stone Color

Photo taken:

Size

Photo taken:

Size

Photo taken:

Size

Photo taken:

Size

Photo taken:

Material Color Stone Color

Material Color Stone Color

Material Color Stone Color

Inscription

Yes

Yes

Photo taken:

No

Photo taken:

No

Photo taken:

No

Photo taken:

No

Photo taken:

No

Yes

No

Yes

No

Inscription

Description Yes

Yes

Inscription

Description Yes

No

Inscription

Description Yes

Yes

Inscription

No

Description

Use this Space for Additional Info Regarding Jewelry:

Form 10

Photo taken:

No

Description

Size

Material Color Stone Color

Yes

Yes

No

Inscription Yes

No

Postmortem Functions—Body Recovery and Morgue Operations

Examiner

Fingerprinting

Incident Incident Date Morgue Reference No.

Date of Exam: Classification of Remains:

Condition of Hands: (burned, decomposed, skeletonized, scavenged, etc.) Condition of Right Hand:

Fingers Printed

Yes No

Condition of Left Hand:

If not printed why?

(Check all fingers printed below)

Right Hand Describe Condition if Needed:

Left Hand

□ Thumb □ Index □ Middle □ Fourth □ Little

□ Thumb □ Index □ Middle □ Fourth □ Little

1 2 3 4 5

Right Palm Printed: Footprints Taken:

Yes

Right Foot

Condition of Feet:

Fingerprint Exam Notes:

Form 11

No

Right Palm Printed: Yes

No

Left Foot

Describe Condition if Needed: 6 7 8 9 10

Yes

No Yes

No

69

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DNA Analysis for Missing Persons in Mass Fatalities

Examining Anthropologist

Anthropology 1. Page 1 of 2

Scribe Exam Date:

Estimated Age Lower Age Range

Upper Age Range

□ Male □ Female

Incident Incident Date Morgue Reference No.

Estimated Sex

□ Male possible □ Female possible

□ Unknown

Classification of Remains: Condition of Remains:

□ Autopsied Previously □ Burned-Partial Thickness □ Burned-Full Thickness

□ Cremains □ Decomposed □ Embalmed

Skeletal Race:

□ Fragmented □ Saponified □ Skeletonized-Partial □ Fresh □ Scavenged □ Skeletonized-Full □ Mummified □ Skin Slippage □ Wet-Environmental Skeletal Build:

Estimated Stature

Caucasian

Hispanic

Small/Gracile

(cm)

Black

Undetermined

Medium/Intermediate

(in)

Asian

Other - Specify

Large/Robust

American Indian

Undetermined

Missing Parts

□ None - Intact Body □ Cranium □ Partial Cranium □ Mandible □ Partial Mandible □ Torso □ Partial Torso

□ R Upper Arm □ Partial R Upper Arm □ R Forearm □ Partial R Forearm □ R Hand □ Partial R Hand □ L Upper Arm

□ Partial L Upper Arm □ L Forearm □ Partial L Forearm □ L Hand □ Partial L Hand □ R Upper Leg □ Partial R Upper Leg

□ R Lower Leg □ Partial L Lower Leg □ Partial R Lower Leg □ L Foot □ R Foot □ Partial L Foot □ Partial R Foot □ L Upper Leg □ Partial L Upper Leg □ L Lower Leg

□ Cranium □ Partial Cranium □ Mandible □ Partial Mandible □ Torso □ Partial Torso □ R Upper Arm

□ Partial R Upper Arm □ R Forearm □ Partial R Forearm □ R Hand □ Partial R Hand □ L Upper Arm □ Partial L Upper Arm

□ L Forearm □ Partial L Forearm □ L Hand □ Partial L Hand □ R Upper Leg □ Partial R Upper Leg □ R Lower Leg

□ Partial R Lower Leg □ L Foot □ R Foot □ Partial L Foot □ Partial R Foot □ L Upper Leg □ Partial L Upper Leg □ L Lower Leg □ Partial L Lower Leg

Unique Skeletal Features (Pathology, Healed Trauma, Unique Identifiers, etc.)

Unique Skeletal Features: (include location, type and description)

Form 12

Skeletal Diagram Used:

Yes

No

Postmortem Functions—Body Recovery and Morgue Operations Examining Anthropologist Scribe Exam Date:

Anthropology 2. Page 2 of 2

Evidence of Ante Mortem Fractures (Old Fractures)

Skeletal Trauma: (include location, type and description)

Race / Ancestry Based On:

Age Based On:

Stature Based On: (include measurements)

Anthropology Dental Comments:

Anthropology Miscellaneous Comments:

Form 13

Incident

Incident Date

Morgue Reference No.

Yes

No

71

72

DNA Analysis for Missing Persons in Mass Fatalities Examiner

DNA

Incident

Incident Date

Morgue Reference No.

Exam Date: Classification of Remains:

DNA Specimen Taken:

Yes

If no DNA Specimen taken, why?

Specimen Taken Type: □ Bone1 □ Bone2 □ Muscle1 □ Muscle2 □ Organ1 □ Organ2 □ Tooth1 □ Tooth2 □ Buccal Swab □ FTA Card LAB ID #

Side:

No

Entire Remains Taken

(If body bag contains less than complete body)

Description:

Size of Specimen:

LAB or AFIP/AFDIL label:

Place label here.

DNA Notes:

Form 14

Yes

No

Chapter Antemortem Functions—Family Assistance Operations

4

Collecting antemortem information about the missing person is a critical aspect of the identification process. Antemortem data is information about the deceased prior to death. Friends and family members of the missing person provide this information to Family Assistance Operation personnel. This chapter examines the responsibilities and capabilities of Family Assistance Operations (FAOs) in a mass fatality response. The FAO does not necessarily refer to an actual facility or center operating a FAO, but instead refers to the capabilities and services that should be made available to the affected community. The Family Assistance Center (FAC) is the physical location housing the FAO where family members can receive support and assistance. The FAO will vary greatly from disaster to disaster based on the specific needs of those affected by the incident and the available resources. As soon as possible after a mass fatality, it is important to immediately prepare for FAOs by identifying necessary services and capabilities and designing an appropriate service plan with available community organizations. Additionally, building an operational framework for the activation of FAOs will provide for better-organized plans. This ultimately saves time, reduces monetary costs, and relieves some of the grief suffered by the families of the deceased.

4.1  Function of Family Assistance Operations In a mass fatality response, the FAO provides an opportunity for information exchange between the authorities and the families and friends of the dead. The FAO also provides critical services to the public. Typically the FAO serves the following purposes: • Collection of antemortem data for identification • Provision of information to the public and specifically to the families of the deceased 73

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• Notification of death to the families and assistance in releasing the bodies to respective family members • Assisting with the physical, mental, and spiritual needs of the affected community in dealing with the mass fatality As indicated by the name, the FAO works closely with families, friends, coworkers, and others with knowledge of and information on the deceased. FAO team members interview the living to collect detailed descriptive information about the deceased, which will be compared against the postmortem information gathered in the morgue. The FAO team will also work with the families to obtain dental and medical records on the deceased, accurately document, process, and store all data whether in paper and/ or computerized files, as well as provide psychological, emotional, and/ or spiritual support to the families. FAO personnel may answer questions that arise during the postmortem evaluation and/or the identification process by reaching out to and obtaining additional information from family and friends of the missing person. Once an identification has been made, the FAO may notify families of the identification and coordinate the release of the human remains. In addition, the FAO team may collect DNA samples. Family Assistance Center operations may take place in one central location or multiple locations, or FAO personnel may travel to meet with the families.

4.2 Personnel The managerial entity in charge of the mass fatality response will activate personnel to work with families of those lost in the mass fatality incident. Since the functions of the FAO are extremely specialized and varied, typically for large mass fatality responses no single agency or organization can immediately provide all of the services required by families. Therefore, it is essential to identify appropriate agencies and organizations to support the FAO, and it is imperative to clarify the roles of each agency and organization in the response. Some agencies and organizations are very limited in their capabilities and can only provide one service needed by the response, while others provide numerous services. Members of the FAO team may include law enforcement personnel, investigators, social and/or psychological experts, grief/bereavement specialists, DNA collection specialists, dentists, doctors, anthropologists, religious clergy/leaders, personnel for computer data entry, file clerks, and computer specialists, as seen in Figure  4.1. Additional personnel may include genetic specialists, security personnel, and foreign language translators/interpreters. The FAO

Antemortem Functions—Family Assistance Operations

Religious Clergy Leaders

Social Psychological Experts

Grief Bereavement Specialists DNA Procurement Specialists

Law Enforcement

Investigators

75

Family Assistance Operations

Anthropologists

Health Professionals Doctors Dentists

Data Entry Clerks Computer support Personnel

File Clerks

Figure 4.1  The Family Assistance Operation (FAO) is complex with multiple specialists supporting the operations.

supervisor should maintain regular contact with the managerial entity and the morgue operations supervisor.

4.3  Creating a Reported Missing Case Trained personnel carefully interview surviving family members with detailed questions about the missing person. Typically, antemortem information is part of a reported missing (RM) case created for each individual believed to have perished. Each RM case should have a unique identifier. This unique identifier is typically a case number given to the RM by the agency responsible for the identifications. A name is not a unique identifier. Depending on the type of mass fatality (e.g., open versus closed, extremely large number of reported missing persons, mass fatalities that cross multiple jurisdictions, etc.), considerable resources may be required to ensure that a unique case number is assigned for each possible missing person. Related individuals should each have their own RM case number, and it is important to make sure the cases are linked.

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DNA Analysis for Missing Persons in Mass Fatalities

Missing Persons Unit

• Help find those who have been reported missing but are truly alive • Identify duplicate RM cases

Morgue or Other ID Site

• Compare it to

the postmortem (or after death) records to make positive indentifications

Death Record

• Aid in completing

death certificate or other official legal record of death

Figure 4.2  The use of antemortem data.

4.4  Antemortem Information The antemortem information is typically collected by the FAO through a series of detailed questions about the characteristics of the reported missing person. Antemortem information may be used in different ways, as shown in Figure 4.2. • The missing persons unit (if one has been established or involved in the response) uses antemortem information to find those who have been reported missing but are alive. Antemortem information is also used to identify duplicate RM cases. Inquiries from multiple family members often cause duplicate missing persons reports. • Antemortem information is used at the morgue or other identification site by comparing it to the postmortem (or after death) information collected by the morgue. • Antemortem information is also used to complete the information required for issuing a death certificate or other official legal record of death. When collecting antemortem information, such as the examples in Table 4.1, it is critical to maintain accurate records of who provided the data. This information should be recorded in the electronic identification program or in the RM case file and should become part of the case’s permanent record.

4.5  Information Technology Support The amount of data required and collected during a mass fatality victim identification effort can be enormous. The only way to successfully manage identifications is to capture the data electronically. For a mass fatality involving more than 20 people, computer programs are imperative to successfully capturing and managing data. There are commercial programs, as well as free

Antemortem Functions—Family Assistance Operations

77

Table 4.1  List of Antemortem Data That May Be Collected by the FAO • Name • Date of Birth • Place of Birth • Physical characteristics • Height • Weight • Eye and hair color • Race • Sex • Past surgeries • Scars • Birthmarks • Deformities • Medical implants • Prosthetics • Facial hair • Piercings • Tattoos • Social Security or other government issued number • Citizenship • Level of education • Parents’ names and places of birth • Next-of-kin information • Clothing and jewelry information • Medical records information • Dental records information • Fingerprints on file information • Biological donor information for DNA comparison

programs, to help manage the data and make identifications. One example of a freely available program is the Victim Identification Program (VIP) available at www.dmort.org. See Attachment A for the antemortem data collection forms used by DMORT (http://www.dmort.org/forms/AnteInterview.pdf). In addition to managing identification data, these programs provide valuable statistical data. Assembling statistical data such as age, race, educational level, and other information may be of use in future response efforts and/or preventative planning. While sophisticated computer programs are available to manage the identification data, the identifications will only be as accurate as the antemortem data provided. Maintaining data accuracy is a challenge for the FAO, and simple errors, such as transposed numbers, can cause missed or delayed identifications. For example, if the antemortem information indicates there

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is a missing woman born in 1993 when actually the woman’s birth year was 1939, the identification may be missed. The morgue will be looking for the remains of a younger woman when they should be looking for an older woman. Likewise, if the wrong race is entered, an identification could be missed as the morgue searches for a Caucasian woman when they should instead search for an African American woman. Information technology tools need to be supported. This support includes systems administration and operational support to ensure the data is secure, accessible to the appropriate individuals, accurate, and backed up routinely.

4.6  Providing Information to the Public The dissemination of accurate and timely information to the public is important to manage public expectations and to enlist support for the human identification effort. Basic terminology, such as “recovery,” “body identification,” and “Family Assistance Center” can be confusing to the general public. A well-informed pubic information spokesperson can help manage public expectation by providing accurate and timely information, as well as using terms the public can understand. In order to minimize confusion and grief, it is important to clearly explain the processes that will take place during the mass fatality identification response. Public announcements are not the principal responsibility of FAO individual staff members, but rather the responsibility of the authorities managing the overall mass fatality response. FAO staff members typically talk to individual families in order to provide information on a more personal basis. Though the information will vary from one incident to another, the FAO can address family concerns and may answer the questions listed below: Recovery process: • Who recovers the bodies? • Where are the bodies being transported? • What are the conditions of the bodies? Identification process: • What are the criteria for identification? • How long will it take? • What is involved with the DNA process? • Will cause and manner of death be determined? • Who is responsible for the positive identifications? Other: • How do I obtain death certificates or other legal records of death? • Is there mental/physical/spiritual help available?

Antemortem Functions—Family Assistance Operations

79

• Is there financial assistance? • When will bodies be released? It is imperative to keep communication open and honest. This will help diffuse misinformation, confusion, and added grief. Creating and distributing scripted answers to commonly asked questions can be very helpful in disseminating information about the identification process to families. As with the collection of antemortem data, every conversation with family members should be recorded. This record of communication will be critical if family members have subsequent questions regarding the processing of their case.

4.7  Financial Assistance There may or may not be financial aid available to family members. In some countries there are governmental agencies that provide financial assistance for the cost of death or for the final disposition of the bodies. In other areas, the government itself will take care of the burial or other disposition of the bodies. If assistance is available, determining which agency offers this assistance, and providing contact information, is another valuable resource for the families of the deceased. The FAO may also help families apply for financial assistance.

4.8  Notification and Release The confirmation of death and positive identification is extremely important to the families in understanding and accepting the death. Once a positive identification has been made and the authorities are prepared to release the body, the FAO may notify the appropriate family members that their relative has been identified and will coordinate the release of the body from the morgue. As this is a sensitive process, the FAO must handle it with care and decorum. It is of paramount importance that personnel assigned to this task possess the necessary skill sets to facilitate the release of a body in an appropriate manner. It is equally vital to have carefully designed and deliberate procedures in place to facilitate the release of human remains and the items associated with the body (e.g., jewelry, papers, etc.).

4.9  Grief Support When family members experience the death of a loved one, they naturally grieve the loss in their own manner. Often the ways in which families express

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DNA Analysis for Missing Persons in Mass Fatalities

grief are driven by cultural and religious factors and must be managed accordingly. It is important to remember there is no right or wrong way to express grief. This is an individual issue affected by family, culture, spiritual beliefs, and religion. Providing supportive services to grieving family members may help them accept their loss and allow them to move into the next stage of life without the presence of their deceased family member. Depending on individual need, there are several sources for support. Some will want a religious leader involved, while others are more comfortable speaking with a mental health professional or a compassionate friend. Identifying several sources of assistance for grief support is important in helping families deal with their loss. It may not be feasible to have grief experts actually in a physical FAC, but appropriate outside resources should be identified and appropriate referral procedures should be established.

4.10  Family Assistance Centers (FACs) If the FAO has a physical location for families to visit, as opposed to providing only remote support, the FAC may have additional components. Since the needs of surviving family members can and will be different in each mass fatality response, it is necessary to identify the capabilities and services needed to run the FAC. The following sections discuss the various components that would be part of an ideal FAC. 4.10.1 Reception As families enter the FAC, it is important that they be greeted and given clear instructions as to what they should do and the services they should expect. The first point of contact with the FAC is important, as it can help put the families at ease and help them feel safe in an unfamiliar setting. 4.10.2  Child Care/Play Area Often, families will arrive at the FAC with small children. The antemortem interview where data is collected can take several hours and as children become restless quickly, it is advisable to have a specially designated childcare area with toys, books, stuffed animals, and other age-appropriate activities. Trained child-care staff must be available to manage and ensure the safety of each child.

Antemortem Functions—Family Assistance Operations

81

4.10.3  Private Meeting Rooms Collecting antemortem information from families can be extremely emotional, and families will benefit from the availability of a private meeting room. 4.10.4  Office Space for FAC Staff Different office areas/spaces will be required for different units within the FAC: staff collecting antemortem data, staff using the phone to speak with family doctors and dentists, and for DNA operations (see below). 4.10.5  Nutritional Services Families who travel to the FAC often lack money and transportation to obtain food or other necessary personal items. Agencies typically responsible for providing food in disaster situations may provide food upon request. In order to meet dietary requirements, cultural expectations, and religious restrictions, cultural and religious specificities need to be taken into consideration when obtaining these services. In addition to feeding families at the center, meals must also be provided for the FAC staff. 4.10.6  Quiet Room A quiet, distraction-free place away from the crowds is recommended (e.g., a room without a television, radio, telephones, etc.). When a family member needs a place to be alone, the Quiet Room should serve as a place to which they can retreat. If appropriate, this room may also be used as a prayer room. If the room is to be used for regular religious practice in the FAC, religious leaders should be consulted regarding the physical requirements of the room. 4.10.7  Communications Center When individuals travel, particularly far from their homes, staying in communication with other family members is important. It is helpful if the FAC has an area with telephone and Internet capabilities for family use. Families may also need to contact other family members, a family physician, or a dentist in order to provide antemortem data requested by the FAO. 4.10.8 Security Family members and staff at the FAC must have a safe and secure place to interact with each other. Additionally, private data related to the missing person and stored at the FAC must be protected. Local law enforcement may

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DNA Analysis for Missing Persons in Mass Fatalities

provide security services, but often contracts are obtained with private security companies for this capability. 4.10.9  Medical Care Family members visiting the FAC often need medical attention. If a family member becomes injured, ill, or overwhelmed with stress while visiting the FAC, assistance from medical professionals will be required. 4.10.10  DNA Operations The DNA operations may take on a variety of different forms depending on the extent to which DNA is used in the identification effort and the location of the FAC relative to DNA laboratory operations. At a minimum there should be a private area for families to provide DNA samples. This may be a room where families can go after they have provided antemortem data. Alternatively, the DNA collector can meet with the families in the private meeting rooms where they were interviewed and provided antemortem data. DNA operations can also be quite expansive and may include the following functional areas: • • • • • •

Collection scheduling Sample accessioning Sample storage Family assistance specific to DNA testing Data analysis Report writing

Rather than working in isolation in the DNA laboratory, it is often easier for DNA analysts to perform the profile analysis where there is easy access to antemortem records and communication families.

4.11 Family Assistance Operations Relationship with the Morgue Questions arising throughout the mass fatality response identification process often require interaction between the FAO and the morgue/identification operation. Effective communication is important regardless of how the local authorities choose to gather, store, and compare antemortem and postmortem data. Additionally, the morgue and the FAO must coordinate the release process. Since these operations are typically separate (e.g., not in the

Antemortem Functions—Family Assistance Operations

83

same physical location), communication can occur via phone, e-mail, or real time via the computer system used to make the identifications.

Additional Resources DMORT. 2011. Ante Mortem Interview. http://www.dmort.org/forms/AnteInterview.pdf. INTERPOL. 2012. DVI Guide, Disaster Victim Recovery Form, and DVI Forms. http://www.interpol.int/INTERPOL-expertise/Forensics/DVI-Pages/Forms. National Transportation Safety Board and Office of Transportation Disaster Assistance. 2008. Federal Family Assistance Plan for Aviation Disasters. http://www.ntsb. gov/doclib/tda/Federal-Family-Plan-Aviation-Disasters-rev-12-2008.pdf. Reuniting the Families of Katrina and Rita: Louisiana Family Assistance Center. http://www.nfdma.com/kat.pdf.

Attachment A (Forms 1a–1i)

84

Form 1a

DNA Analysis for Missing Persons in Mass Fatalities

Antemortem Functions—Family Assistance Operations

Form 1b

85

86

Form 1c

DNA Analysis for Missing Persons in Mass Fatalities

Antemortem Functions—Family Assistance Operations

Form 1d

87

88

Form 1e

DNA Analysis for Missing Persons in Mass Fatalities

Antemortem Functions—Family Assistance Operations

Form 1f

89

90

Form 1g

DNA Analysis for Missing Persons in Mass Fatalities

Antemortem Functions—Family Assistance Operations

Form 1h

91

92

Form 1i

DNA Analysis for Missing Persons in Mass Fatalities

5

Chapter Identification of Remains

The primary objective in most mass fatality incidents is to locate and identify the dead. The information in this chapter is presented as a broad overview of the different types of identifications and the identification process. For each specific mass fatality response, the managerial entity should document and communicate the standards for what will be considered an acceptable identification. These standards may include the procedures used during Field, Morgue, and Family Assistance Operations to collect and document antemortem and postmortem information, the methods used to compare the information, and the thresholds used to make identifications.

5.1 Identification As field data is made available and morgue examinations are completed, members of the Identification Unit should review the postmortem data, as well as the antemortem data collected by the Family Assistance Operations team and/ or Missing Persons Unit on the missing/presumed dead. As comparisons are constantly occurring, the vast number of possible matches is narrowed down. Eventually the number of possible matches will be narrowed down to a single comparison, which will then be re-evaluated. If the match holds up under scrutiny, it will be passed on as a proposed identification. The Identification Unit should take detailed case notes while making comparisons. As expected, a mass fatality incident will produce a large number of deceased bodies. Depending upon the circumstances, there may be significantly more people reported as missing/possible fatalities by family and friends. For example, if a plane crashes in a deserted area, chances are that only the people on the plane need to be accounted for in the identification process. However, if a plane crashes into a highly populated commercial area, then people on the ground also need to be accounted for. The circumstances of the incident greatly affect the number of possible deceased whose records have to be culled through when making identifications. This is outlined in Figure 5.1.

93

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DNA Analysis for Missing Persons in Mass Fatalities Plane Crash Deserted Area

• In most situations,

only the people on the plane need to be accounted for in the identification process

Plane Crash Highly populated commercial Area

• People on the ground

also need to be accounted for in the identification process, including visitors and customers who are in the area on a daily basis

Figure 5.1  Incident circumstances.

5.2  Types of Identification There are three basic categories of “identification”: tentative, presumptive, and positive. Some scientists argue that there should only be one type of identification—the positive identification based on scientific criteria. For completeness, all three will be discussed. The reader may choose to consider the first two as leads rather than identifications and the terms should only be used internally in the operations and not discussed with the public. 5.2.1  Tentative Identification A tentative identification is the least reliable of the three and is based upon non-scientific, non-forensic information. Some experts do not even consider a tentative identification an “identification” at all, but rather a designation assigned by investigators as a starting point in the identification process. It helps to initially eliminate some of the identification options and is intended to provide an initial focus toward certain records. There may be multiple tentative identifications per deceased individual. Tentative identifications are typically based on general personal items, recovery location, and general physical descriptors. For example, the Field and Morgue Operations records both describe a particular deceased individual as an elderly black male with the right foot amputated above the ankle. Investigators search the antemortem records and find one reported missing individual who fits this initial description. So far, this is only a tentative identification. 5.2.2  Presumptive Identification A presumptive identification is typically based on a large amount of positive comparative data, none of which is scientifically or forensically based. Some jurisdictions accept presumptive identifications consisting of a certain quantity and specific types of positively comparable data. Positive comparable

Identification of Remains

95

Figure 5.2  Positive comparable data.

data may include unique physical features such as body art, the presence of non-surgical objects within the remains that are documented by family or friends of the deceased, and/or evidence of comparable surgical procedures, as shown in Figure 5.2. For example, the body of an Asian male approximately 20–30 years old was recovered from the inside of a completely secured home at 523 West 32nd Street, New Orleans, Louisiana. This male was wearing pink eyeglasses, there was a medical alert tag engraved with the name “Peter Miller” and “Diabetic” on the left wrist, and he had a vivid multicolored tattoo of a rose on his right inner forearm. The deceased was described as 171 cm in height with purpledyed streaks in otherwise black hair, with all his own teeth and the left upper central incisor overlapping the right upper central incisor. An antemortem record indicates a reported missing person by the name of Peter Miller, an Asian American who had refused to leave his home at 523 West 32nd Street, New Orleans, Louisiana, even though there was a mandatory evacuation order. This missing person is a 26-year-old college student who lives alone and is not known to have a partner. The missing person is also reported as 65 inches tall (165.1 cm), with black hair that is often dyed different colors and a multi-colored tattoo of a flower on one of his forearms, and is known to wear brightly colored eyeglasses. Additionally, the missing person has overlapping and slightly crooked teeth, especially the front teeth, and is a known diabetic. None of the other missing person reports indicate a diabetic Asian male under the age of 52 years. This is a tentative identification. 5.2.3  Positive Identification A positive identification is the most reliable and is based upon scientific and forensically based data, including DNA, fingerprint, and dental comparison results. Some jurisdictions will also accept positive identifications based on

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DNA Analysis for Missing Persons in Mass Fatalities

the comparison of radiographs or surgically placed (and medically documented with traceable serial numbers) devices (e.g., pacemakers) or implants (e.g., joint replacement hardware). In many countries the forensic communities have established standards that define what is acceptable as positive identification. In order to ensure identifications are more likely to be supported by the court, the best standards are established with the input of the legal system. Unfortunately there are no worldwide, universal standards, although the international forensic science organizations are a driving force behind moving in this direction.

5.3  Identification of Bodies One of the primary goals of a mass fatality incident response is to identify the dead. To accomplish this goal, appropriately trained individuals must, in accordance with protocols, review, compare, and process ante- and postmortem data collected by the Field, Morgue, and Family Assistance Operations personnel. Postmortem information is compared to antemortem information for potential matches, as outlined in Figure 5.3. 5.3.1  Appropriate Personnel Law enforcement officers, special investigators, and forensic specialists (namely forensic pathologists, medical examiners, forensic dentists, DNA analysts, fingerprint analysts, and forensic anthropologists) typically conduct comparisons and investigations necessary for identifying the dead.

Information collected from the human remains in the Morgue (postmortem)

Information collected from the victim’s families or other agencies (antemortem)

• Dental

• Dental

• Fingerprints • Medical devices

IT

• Fingerprints • Medical devices

• Surgeries

• Surgeries

• Body Art • Physical characteristics • DNA

• Body Art • Physical characteristics • DNA

Forensic Pathologists: Death Certificate and Release of Body

Figure 5.3  Postmortem and antemortem comparisons.

Identification of Remains

97

It is recommended that identification personnel prepare a report showing a comparison matrix and other supporting documentation or computer-aided comparison results when proposing an identification. As per standard protocol, the medical examiner, forensic pathologist, Medical Legal Institute, or Ministry of Justice personnel present this report for review. In some areas, the identification must be approved by a court of law in order to be legally binding. The final approval of an identification must be shared with the Data Management Unit of the Morgue Operations team and the Family Assistance Operations team/Missing Persons team. 5.3.2  Identification Standards and Guidelines Whenever possible, it is important to follow the applicable parts of the following guidelines: • FBI’s Scientific Working Group on Disaster Victim Identification (SWGDVI) (in development) • Recommendations Regarding the Role of Forensic Genetics for Disaster Victim Identification (DVI). DNA Commission of the International Society for Forensic Genetics (ISFG), 2007 • Disaster Victim Identification Guide. INTERPOL, 2009 • Missing People, DNA Analysis and Identification of Human Remains: A Guide to Best Practices in Armed Conflicts and Other Situations of Armed Violence. International Committee of the Red Cross (ICRC), 2009 • Guidelines for Mass Fatality DNA Identification Operations. AABB, 2010 These publications outline varying approaches with varying levels of detail. While each of these publications may not be applicable to every project, they can serve as good references.

5.4  Presentation and Review of Proposed Identification The managerial entity establishes the procedures for all aspects of identification work and the specific qualifications of the personnel who perform the investigations and comparisons. In many countries, the courts approve legal identifications, and their input regarding procedures, personnel, and required documentation is essential. Each team member should maintain adequate documentation of all identification-related work to clearly show cases that were ruled out. Requests for

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DNA Analysis for Missing Persons in Mass Fatalities

clarifications, additional information (what was requested, by whom, when, and any responses), and active comparisons should also be documented. Whenever a presumptive or positive identification is proposed, it is highly recommended that team members complete a comparison matrix for each case. The use of such a matrix clearly identifies all the consistencies and inconsistencies between the antemortem and postmortem records. Team members should attach copies of documents further supporting the proposed identification to the matrix (e.g., DNA identification report). (See Attachment A: Antemortem–Postmortem Comparisons.)

5.5  Acceptance/Authorization of Identification The managerial entity should assign appropriate personnel (preferably a medical examiner, forensic pathologist, or appropriate person(s) from agencies such as the Medical Examiner’s Office or Coroner’s Office in the United States or the Medical Legal Institute or Ministry of Justice in other countries to review the supporting documentation for the proposed identifications. (See Attachment B: Case Identification Approval.) If the identification is accepted these individuals may also support the processing of the information in the courts for legal authorization/certification of the identification(s).

5.6  Family Notification of Identification The managerial entity determines the appropriate personnel and operational branch for handling family notification of identifications once the identification has been finalized. Personnel should follow their standard procedures or special protocols established for the mass fatality incident when notifying the family of the identification and what information is released to the family. The handling of information is usually assigned to one of the following: the FAO team, a medical examiner, forensic pathologist, or appropriate person(s) from either the Medical Legal Institute or Ministry of Justice. When dealing with fragmented remains it is very important to discuss with the family if they want to (1) take the portion of the body that was identified and be notified when additional pieces are found, (2) take the portion of the body that was identified and not be notified when additional fragments are found, or (3) take possession of the body when all parts of the body have been found and identified (understanding that based on the circumstances, that may never happen). Sometimes the family will opt for the first option and then change to the second once they have been notified several times.

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5.7  Release of Remains and Personal Property Once identification has been finalized and the family has been notified, the body will be released to the legal next of kin or appropriate agency. The managerial entity determines the appropriate personnel and operational branch for handling the release of remains and personal property. Before releasing bodies, the managerial entity should assess existing laws that may govern or affect the activities that support the release. The Morgue Operations team usually stores bodies until proper arrangements are made for release. Great care should be taken to ensure that the correct body is released and that the Morgue Operations team appropriately documents the release. Sometimes a body will be identified but there will not be a relative who can claim the body. Additionally, there may be bodies that cannot be identified because no antemortem information was presented. The disposition of unclaimed and unidentified bodies and their associated personal items will be dictated by the managerial entity for the mass fatality response or by the courts.

Additional Resources American Association of Blood Banks. 2010. Sozer, A., Baird, M., Beckwith, S., et al., Guidelines for Mass Fatality DNA Identification Operations. http://www.aabb. org/programs/disasterresponse/Documents/aabbdnamassfatalityguidelines. pdf (accessed September 30, 2012). International Committee of the Red Cross (ICRC). 2009. Missing People, DNA Analysis and Identification of Human Remains: A Guide to Best Practices in Armed Conflicts and Other Situations of Armed Violence. http://www.icrc.org/ eng/assets/files/other/icrc_002_4010.pdf (accessed September 29, 2013). International Society for Forensic Genetics. 2007. DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations Regarding the Role of Forensic Genetics for Disaster Victim Identification (DVI). Forensic Science International: Genetics 1(1): 3–12. INTERPOL. 2009. Disaster Victim Identification Guide. http://www.interpol.int/ content/download/9158/68001/version/5/file/Guide.pdf (accessed September 29, 2013). The Scientific Working Group on Disaster Victim Identification (SWGDVI). Protocols, Guidelines, Forms, and Standard Operating Procedures. http://www. swgdvi.org/resources.html (accessed September 30, 2012)

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Attachment A Antemortem—Postmortem Comparisons by                  on                Name                Date:               Feature Sex Age Race Height Build Complexion Body Piercings Hair Length & Color Facial Hair Fingernails Opticals Prosthetics/Objects in Body Scars, Marks, Deformities, Amputations Surgeries Dentition Tattoos Fingerprints Obtained? Fingerprint Analysis DNA Collected? DNA Results Residence/Recovery Location Personal Effects Clothing Other:

Antemortem Case #

Postmortem Case #

Consistent? Y/N

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Attachment B Case Identification Approval                                Field Operations Case Number    Morgue Operations Case Number Identified as: Antemortem Record # (RM#)                                           First Name   Middle Name    Last Name   Jr./Sr./III                               Date of Birth        Federal ID #       Other # Method(s) of Identification ☐ Fingerprint ☐ Dental   Positive ID      Consistent      ☐ DNA    %

No Inconsistencies    

Pathology Findings (includes comparison of radiographs and surgical implants with serial numbers)   ☐ Description:                          ☐ Personal Effects   Description:                          ☐ Field Scene Information   Description:                        Autopsy Required: ☐ Yes  ☐ No  Autopsied By                               Name    Date Identification Approved By:                                Signature                Date                                Name (Print)

Chapter Identification and Collection of Biological Samples from Human Remains

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Sample collection from human remains for DNA profiling is a key component to a successful identification. Proper sample collection will provide the laboratory with the best possible sample. This chapter explains the necessary steps for collecting DNA samples from human remains and provides rationale for the various steps. The information in this chapter focuses on collection techniques for samples that will undergo nuclear DNA profiling protocols even though these same procedures are generally applicable to sample collection for mitochondrial DNA analysis.

6.1  Special Considerations for Sample Collections The options for postmortem sample collection can be severely limited by factors that alter the remains, such as environmental influences, postmortem trauma, and others (e.g., animal activity). With time, the viability of remains decreases along with sampling options. Decomposition of the human body begins shortly after death when bodily functions stop. Environmental conditions such as humidity and heat, and body exposure to water, and fire, may accelerate the decomposition process. The DNA in cells is destroyed as decomposition progresses. Since DNA sampling of human remains is a destructive process, the collector must ensure the sample does not destroy or alter the characteristics of remains critical for identification by another scientific means, such as dental or fingerprint identification. DNA samples should only be collected after the human remains have been thoroughly examined and documented by a qualified professional (e.g., anthropologist, pathologist, etc.) as to not impede the postmortem information collected by other disciplines. For example, if teeth are removed for DNA testing before the odontologist’s examination, then precious dental information may be lost.

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Typically a DNA profile is only as good as the biological sample that is collected. Errors in the collection or documentation of samples may adversely impact the identification process and could even result in a mistaken identification. Any mistakes made during sample collection may call into question the credibility of the entire identification process. Therefore, individuals responsible for sample collection must exercise extreme caution to ensure suitable samples are obtained and properly identified. If a complete set of human remains is available, the collector should make a decision on which sample to collect based on the ease of collection and the ability of the laboratory to successfully process the sample (see below). However, frequently unidentified human remains are fragmented, therefore limiting the selection possibilities. The challenge of DNA sample collection increases when human remains are highly fragmented and/or co-mingled, as it takes a trained individual to determine if biological material is of a single source. Protocols for these circumstances are discussed later in this chapter. Since DNA sampling of human remains is a destructive process, the collector must ensure the sample does not destroy or alter the characteristics of remains critical for identification by another scientific means, such as dental or fingerprint identification. In circumstances where the identification effort is limited to a closed population, DNA samples should also be taken from human remains identified by other conventional means, such as fingerprints or dental comparisons. Generating DNA profiles of identified casualties in closed population situations is for elimination purposes, and is a necessary step in the overall identification effort. A unique DNA profile from an identified set of remains will permit re-association of any potential fragmented remains and will also serve to exclude their DNA profile from others in the closed population, including siblings.

6.2  Determining the Best Sample to Collect DNA is found throughout the human body and therefore presents a variety of options for sample choice. While we know from experience which samples are most likely to produce good quality DNA testing results (blood, soft tissue, organs, bone, and teeth), these types of samples are not always available or in good condition. Less desirable sources of DNA include the liver, spleen, hair, perspiration, and blood serum. Therefore, the human remains must be carefully screened to determine the best available sample. In terms of mass fatality identification operations, the best available sample is the biological specimen that is (1) the least difficult to obtain, (2) the least challenging for the laboratory to process, and (3) the most likely to produce successful results.

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Often there is only one opportunity to obtain a DNA sample from a set of human remains, and selection criteria decisions must be based on an assessment of the circumstances. When deciding which type of sample to collect, it is preferable to discuss sampling options with the laboratory that will be performing the DNA testing. A laboratory may have particular expertise in a specific sample type, which will enable the laboratory to expedite the testing process. Providing samples that the laboratory cannot easily process may lead to delays and other difficulties. A common order of sample preference for a laboratory is blood, tissue, fingernail, bone, and teeth. This chapter focuses heavily on the collection of bone samples because bone is one of the most common sample types available when decomposition of human remains occurs. 6.2.1  Ease of Sample Collection It is important to consider the amount of time it will take to obtain a sample. For example, if an autopsy is being performed on an unidentified body that is not highly decomposed, a blood sample can be drawn with a syringe. With minimal time and effort, the blood can be put in a purple top (EDTA) tube and later transferred to a filter paper bloodstain card, as shown in Figure 6.1. If the body is moderately decomposed where the reliability of the blood is questionable, dense muscle tissue can be collected along with other toxicological samples obtained during routine autopsy procedures. Skeletal muscle, lung, and skin flap can also be collected, as seen in Figure 6.2. However, it is important to avoid severely decomposed tissue or pure adipose. Bone specimens are the preferred choice when decomposition has started and affects the tissues. Many laboratories prefer to test long weight-bearing dense bone. Experience demonstrates that when having a complete set of human remains, obtaining the DNA sample from the anterior surface of the tibia is preferable because it is so easy to collect. The tibia bone can be easily exposed using a scalpel. The tibia bone can also be quickly and safely cut with a Stryker saw. See Additional Resources at the end of this chapter for studies by Dr. Mundorff on DNA yields from bone samples. Whole Blood (~7 ml collected in EDTA tube then transferred to bloodstain card)

Figure 6.1 (see color insert)  Processing whole blood. (Courtesy of Dave Boyer.)

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DNA Analysis for Missing Persons in Mass Fatalities Soft Tissue Skeletal muscle, lung and skin flap (~10 grams). Avoid severely decomposed tissue or pure adipose.

Figure 6.2 (see color insert)  Processing of soft tissue. (Courtesy of Dave Boyer.)

6.2.2  Ease of DNA Profiling Another key consideration when collecting samples is the amount of effort it will take for the DNA laboratory to process the samples. As previously mentioned, blood and tissue are excellent sources of DNA. In terms of the effort required to process them, these are materials from which laboratories can quickly extract DNA and create the DNA profile. Fingernails are good sources of DNA and they are the sample of choice once body decomposition has occurred. They are more challenging to process than blood and tissue, but less challenging than bones and teeth. Bones and teeth are both excellent sources for DNA, but the extraction procedure requires more time and resources for processing. It is important to keep in mind that laboratory throughput may decrease when processing difficult samples. The laboratory processing of bone samples (as shown in Figure  6.3) requires more effort than tissue or blood, but these sample types are preferred Bone Diaphyses of long bones, cranial vault, ilium, and phalanges. Samples consist of “windows” of compact bone from large specimens of tubular segments. (~1–2 cm × ~4–6 cm × ~0.5–1 cm) (~15 g to ~25 g in weight)

Figure 6.3 (see color insert)  Processing of a bone sample. (Courtesy of Dave Boyer.)

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because bones retain DNA significantly longer than tissue or blood. The best bone samples historically have been obtained from long bones, cranial bones, and ribs. Due to their fragile bone structure, irregularly shaped bones such as vertebrae are less desirable as DNA sources. Teeth also are an excellent source for DNA, but the extraction process for teeth can be more tedious and time consuming than bone extraction. If teeth are collected, the preference is the back molars without dental work. The quantity of sample available from teeth is limited and therefore more susceptible to contamination. Also, some DNA laboratories are not equipped or qualified to process teeth as DNA samples. When more laboratory work is required to extract each DNA sample, there will be a fewer number of samples processed, which can result in a longer and more expensive DNA identification project. 6.2.3  DNA Profiling Success Biological sampling of human remains should be based upon those that have the greatest chance of producing successful laboratory results (informative DNA profiles). DNA testing is expensive and time consuming. It is more beneficial for the identification team and the victims’ loved ones if high-quality results can be generated faster. When submitting samples for DNA testing, general laboratory preferences must also be taken into consideration, such as what sources of DNA laboratories generally prefer to receive and process when dealing with mass fatality responses. Often, collecting a consistent sample type is preferable since it allows the laboratory to streamline its testing process. Not all laboratories have the technology or experience to process certain types of biological samples, especially in large volumes. The biological sample most likely to produce successful DNA identification results is that which provides the best nuclear DNA profile. Tissue and/or whole blood from a body that has been dead for more than a few days without refrigeration is not likely to yield good quality nuclear DNA. There are other influences to consider when choosing the body origin for DNA sampling. For example, if the remains are exposed to excessive heat (such as temperatures generated in an airplane crash fire or similar incident), it is likely the DNA in the blood and tissue has been destroyed. Additionally, in extreme cases with intense heat and prolonged exposure, the DNA in the skeletal material may also be destroyed. If questions arise as to the ability of a sample to provide a good nuclear DNA profile, it should not be collected and the collector should select another sample with a greater likelihood of success, or multiple sample types can be collected so that the laboratory can choose which type is the best.

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All three of the elements listed above (ease of collection, least challenging for the lab, and most likely to produce successful results) must be considered when choosing which biological specimen to collect for DNA testing. When deciding which type of sample to collect, it is preferable to discuss sampling options with the laboratory or laboratories who will be performing the testing on the human remains. Collecting a consistent sample type may be preferable to a laboratory since it will allow the laboratory to streamline their testing process.

6.3  Collecting Multiple Samples There are circumstances when collectors must obtain multiple DNA samples from a single set of human remains. In criminal cases, most laboratory procedures require that a sufficient amount of sample be obtained to conduct testing and stored for future blind testing, confirmatory testing, or other quality control measures. Laboratories accredited for civil relationship testing perform duplicate testing on all exclusionary results and often Combined DNA Index System are verified by a second test. Therefore it is prudent whenever possible to perform duplicate testing on mass fatality samples. International guidelines recommend that DNA testing in response to mass fatality identifications be performed in duplicate. Extra samples are referred to as duplicate samples. Secondary samples, also referred to as redundant samples, are DNA samples taken from different body locations. In situations where examination of the human remains suggests a tissue sample is sufficient but the tissue may not yield a sufficient amount of DNA, a bone sample may be collected as an alternate sample. The laboratory can first test the tissue, and if successful DNA results are obtained, the alternate bone sample need not be tested. However, if the tissue sample fails to result in successful DNA results, the laboratory can use the bone sample and achieve the desired result without having to return to the origin of the sample for more biological material.

6.4  Establishing DNA Sample Protocol Each mass fatality is unique and has special requirements that should be taken into consideration for DNA sample collection. Before DNA sample collection begins, decision makers need to determine: • What is the smallest piece of biological material to be tested? • What samples will be collected?

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• How will fragmented remains be handled? • Is the project goal limited to identification or does it include re-association? Circumstances influencing the aforementioned decisions include but are not limited to the following: • Time lapse between events causing the deaths and actual sample collection • Degradation of the human remains • Number of fatalities or projected number of fatalities • Laboratory standards of sample submission for analysis • Laboratory capacity for sample processing • Availability of medical/mortuary equipment and facilities for collecting samples • Funding constraints • Time constraints • Political considerations • Desired results • Cultural considerations • Safety issues Any combination of the above considerations can influence decision processes for DNA sample collection and have far-reaching ramifications on the DNA identification effort. Regardless of the considerations, decision makers should agree on a set of standards for DNA sample collection before sample collection begins, thus ensuring a consistent process. The chosen process should produce consistent results and should also remain flexible enough to handle issues such as partial, fragmented, and/or commingled remains. Entities who have a legitimate interest in the test results and who are responsible and liable for the outcome will determine relevant protocols. The resulting process decisions should be documented in a written protocol, which serves as an operating procedure for agencies responsible for the collection process. DNA sample collection is a team approach. To ensure that the process is mistake-free, a single individual should not work in an isolated environment to collect a DNA sample. Many mass fatality incidents involve situations where there are fragmented human remains. DNA samples can be collected from any fragmented piece of remains, and subsequent DNA profiles from those fragments can be matched for re-association. To properly account for each fragmented item of biological material, it must be given a separate and unique item number for tracking purposes. Consideration must also be given as to whether or not to collect a fragment, especially if the fragment is so small that it will be entirely

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Figure 6.4  Conical tube for storage of tissue, bone, and teeth. (Courtesy of Dave Boyer.)

consumed in analysis, leaving no sample for subsequent or duplicate testing. Small, degraded samples can take considerable time for the laboratory to process and often do not yield sufficient results for identification purposes. 6.4.1  Order of Sample Preference and Sample Quantity As stated earlier, the order of preference for DNA sample collection is determined by the least amount of effort and time necessary to achieve successful results. Most DNA laboratories prefer sample submission in the following order: blood, tissues, and bone. If there is refrigeration available, tissue, bone, and teeth samples should be containerized in a 50 ml, screw-top, conical tube as depicted in Figure 6.4. Table 6.1 lists some commonly used sample types, quantities, and containers for each type of sample. Collecting small/trace amounts of sample (e.g., hair root) or samples that have not been validated by the laboratory will lead to processing difficulties in the laboratory. 6.4.2  Safety Precautions and Contamination Issues The safety of all individuals involved in DNA sample collection must be a priority. It is advisable to treat all specimens as potentially infectious when Table 6.1  Common Preferences for Sample Types, Amounts, and Containers Sample Type

Typical Sample Quantity (Approx.)

Blood

7 ml

Tissue Bone Teeth

10 grams 15 to 25 grams 1–2 undamaged tooth with crown and roots

Typical Collection Container (if refrigeration is available) Purple top (EDTA) tube—transferred later to a DNA specimen card 50 ml, screw-top conical tube 50 ml, screw-top conical tube 50 ml, screw-top conical tube

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Table 6.2  Cross-Contamination Prevention Steps to Guard Against Cross-Contamination • Disposable bench-top sheets should be placed on the hard specimen cutting surface and changed between sample collections. • Scalpel blades should be changed between collections of individual remains. The Stryker saw blade should be cleaned between sample collections with a 10% bleach solution. • Gloves should be changed after handling each item of biological material.

conducting DNA collection from human remains. Individuals should always wear appropriate personal protection equipment (PPE), including Tyvek® suits or laboratory coats, gloves, and safety goggles. They also should complete the appropriate training on blood-borne pathogens and laboratory safe practices before being assigned to work with human remains. If collection will take place in a laboratory environment, the collectors should be familiar with existing Chemical Hygiene Plans in place. All disposable equipment and supplies used during the process should be treated as biohazard material and disposed of accordingly. Disposable scalpels should not be cleaned and re-used due to risk of injury; rather, they should be discarded into a sharps container. It is also important to use procedures that will minimize contamination. Contamination (also referred to as cross-contamination) is the inadvertent introduction of DNA from one sample into another sample. As shown in Table 6.2, there are several steps that can be taken to minimize the possibility of sample cross-contamination. 6.4.3  Packaging, Labeling, and Storage Maintaining sample integrity involves proper handling and storage of the human remains, as well as the samples taken from the remains. It is important to make sure that collection and packaging are well documented and that packages containing the biological samples are sealed with tamper-evident tape. Access to packages containing biological samples must be limited to only authorized individuals. The potential for DNA degradation should be minimized through proper environmental storage of the collected samples. The packaging of the sample will depend upon the type and condition of the sample and how long the sample will be stored. Plastic containers stored in a refrigerated environment are typically preferable for fresh samples. However, if reliable refrigeration is not an option, bone samples can be placed into other types of breathable containers, such as clean paper boxes. These boxes should then be stored in a

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Figure 6.5  Computer-generated label.

dry location. Prior to collection, always coordinate with the DNA laboratory to determine optimal storage containers and conditions. Biological samples collected from fresh human remains for DNA analysis should be placed in a primary container which is then placed into a secondary container. The primary container should be a sterile 50 ml conical tube with a screw cap or other similar container that will hold enough of the sample and withstand being placed in the freezer. Samples should not be stored in formaldehyde or other preservatives. The tube should be marked with information regarding the specimen type, case number, specimen number, date collected, and initials of collector using an indelible marker or computer-generated label. Figure 6.5 depicts the computer-generated labels. The secondary container should be an individual polyethylene bag, which is tape sealed or heat sealed. All collected biological samples should be stored in a manner that prevents further degradation of the samples. Note: Whole blood in an EDTA tube should be refrigerated but never frozen. Transportation of biological samples should conform to applicable local laws and regulations. Packaging materials should never be purposely mislabeled to disguise their contents. If the DNA identification project involves criminal procedures, DNA samples should be labeled and cared for as evidence. An established “chain of custody” documents the integrity of the samples. The chain of custody creates an audit trail of the location where the sample was obtained, information on who collected and sealed the sample, and the date and time the sample was collected. The custody documentation also provides a description of the item sampled and identifies its origin. A typical item description may be: “3 inch tibia window, from HR [human remains] bag number XXX, placed in a 50 ml screw cap conical tube, marked for identification, 1000 hrs, [name of person collecting the sample], 11/23/2012.” Multiple samples may be documented on a single custody document and should be numbered appropriately. If duplicate or redundant sampling is required, the sample numbering numbers should be identical with an added identifier (i.e., #113a and #113b). A specimen number should never be used more than once.

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When the responsible individual removes a biological sample from human remains for DNA analysis, it must be properly secured for safekeeping. Temporary storage can be accomplished through a number of mechanisms, but a storage method must include some form of security to maintain sample integrity. Typically, a refrigerated container that can be locked is used or the room containing the refrigerated container is locked. Access must be controlled, and methods used for control must be documented. The sample security trail must be traceable from the point of collection to entry into the laboratory. Upon receipt by the laboratory, the burden of sample security transfers to that facility. Most modern DNA laboratory operations use an automated sample tracking system, commonly referred to as a Laboratory Information Management System (LIMS). (See Chapter 10.) Long-term storage of DNA samples applies to biological samples remaining after the laboratory conducts the DNA analysis. The laboratory is responsible for the maintenance of DNA samples requiring long-term storage.

Additional Resources Armed Forces DNA Identification Laboratory. 2008. Guidelines for the Collection of Specimens Requiring DNA Analysis. Bailey-Wilson, J.E., Ballantyne, J., Baum, H., et al. 2006. Lessons Learned from 9/11: DNA Identification in Mass Fatality Incidents. National Institute of Justice. http://www.nij.gov/pubs-sum/214781.htm (accessed September 29, 2013). Biesecker, L.G., Bailey-Wilson, J.E., Ballantyne, J., et al. 2005. DNA identifications after 9/11 World Trade Center attack. Science 310: 1122–1123. Gonzales, A.R., Henke, T.A., and Hart, S.V., National Institutes of Health. 2005. Mass Fatality Incidents: A Guide for Human Forensic Identification. https://www. ncjrs.gov/pdffiles1/nij/199758.pdf (accessed September 29, 2013). International Committee of the Red Cross. 2005. Missing People, DNA Analysis and Identification of Human Remains—A Guide to Best Practice in Armed Conflicts and Other Situations of Armed Violence. ICRC Pub. 2005 ref. 0871. http:// www.icrc.org/eng/resources/documents/publication/p4010.htm (accessed September 29, 2013). Mundorff, A. and Davoren, J. 2014. Forensic Science International: Genetics 8:55–63. Mundorff, A. 2008. Anthropologist-directed triage: Three distinct mass fatality events involving fragmentation of human remains (Chapter 7), in Recovery, Analysis, and Identification of Commingled Human Remains, B. Adams and J. Byrd, Eds, Humana Press, Totowa, NY, pp. 123–144. National Transportation Safety Board. 2006. DMORT Standard Operating Procedures for NTSB Activations. Office of Justice Programs, U.S. Department of Justice Special Report: Mass Fatality. http://www.dmort8.org/DMORT%20NTSB%20 SOP%20Nov%202006.pdf (accessed September 29, 2013). Pan American Health Organization. 2004. Disaster Manuals and Guidelines Series, No. 5: Management of Dead Bodies in Disaster Situations. http://www.paho. org/English/dd/ped/DeadBodiesBook.pdf (accessed September 29, 2013).

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UNESCO. 2003. The International Declaration on Human Genetic Data. http:// unesdoc.unesco.org/images/0013/001331/133171e.pdf#page=45 (accessed September 29, 2013).

7

Chapter Identification and Collection of DNA Reference Samples

All biological samples obtained from human remains for identification of unknown persons are considered “questioned” samples or samples of unknown origin. When the identity of a questioned sample has been established, it may be used as a reference sample to identify another questioned sample. This may be a reference sample to identify fragmented remains or a kinship sample to identify a missing relative. In order to identify human remains, a DNA profile from the human remains must be “matched” to one or more DNA profiles from biological samples of known origin. This chapter discusses the different types of reference samples that can be used to identify a reported missing (RM) individual and the steps involved in collecting the samples.

7.1  The Reported Missing The first step in collecting reference samples is to identify the reported missing (RM). The RM is a person believed to be deceased. Each RM should have a unique identifier, which is typically a case number given to the RM by the agency responsible for identifications. A name is not a unique identifier. Related individuals should each have their own RM numbers, and proper documentation should connect or identify the two RMs as being related. Typically the assignment of an RM case number is performed by the Family Assistance Operations in coordination with the collection of antemortem information. However, it is not uncommon for family members to arrive at a collection site to provide a sample prior to completing the entire antemortem data collection process. In these situations, DNA samples should be collected with as much information about the missing person as possible. DNA collectors should never pass up the opportunity to collect reference samples, as the laboratory will decide which samples to test. Once the sample and associated information are collected, the RM case can be opened and the remaining antemortem information obtained at a later date. Each time family members provide information about missing individuals, it is important 115

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Figure 7.1 (see color insert)  Properly sealed sample.

to verify whether it is a new RM case or a variation of a case already maintained by the laboratory. The laboratory may miss potential identifications if there are multiple cases for the same RM, each with a different subset of the reference samples.

7.2  Chain of Custody To ensure the integrity of the sample, a chain of custody must be maintained. The chain of custody begins at the time of collection and is maintained throughout the entire testing process. From the moment the sample or evidence is collected, the sample must be sealed, as shown in Figure 7.1. Additionally, every time the sealed sample is transferred from one individual to another, it must be documented, as seen in Figure 7.2. The proper sealing of the evidence and the associated chronological documentation (paper trail) of the collection, transfer, analysis, and disposition of the sample are critical to the successful operation of a DNA identification laboratory. Because the report issued by the laboratory can be used in court, it must be handled in a meticulous manner to avoid later allegations of tampering or misconduct. A proper chain of custody establishes that the evidence relates to the crime scene and was not fraudulently planted. Maintaining a proper chain of custody is critical to the identification process and subsequent court proceedings.

Figure 7.2  Documenting the transfer of a sample.

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7.3  Reference Sample Types There are several types of DNA reference samples used to identify human remains in mass fatality operations: direct references, personal items, and kinship samples. In order for the laboratory to produce meaningful results, each of the reference types must be properly collected and identified. The types of reference samples collected depends on accessibility and the ability of the laboratory or laboratories to test the samples. 7.3.1  Direct References A direct reference is a sample that has some sort of paperwork or documentation linking its origin to a missing individual. Typically a professional (such as a doctor or nurse) collected these samples during a medical test. Direct reference samples can be attributed to the missing individual through some type of record of collection (e.g., a medical record). The DNA laboratory should discuss the potential availability of these samples with the family and then follow up with the organization that potentially has the sample. A medical or legal release may be necessary to obtain the sample. Direct reference samples are sometimes preferable because they have the potential to provide the full (complete) DNA profile from the RM, which can be easily and directly matched to the profiles from human remains. However, obtaining direct reference samples may be time consuming and in cases where tissues are embedded in paraffin or biopsy slides, it can be labor intensive and difficult to profile even for extremely experienced laboratories. When collecting direct reference samples, it is important to obtain the paperwork, which documents collection of the sample. If there is minimal, incomplete, or questionable paperwork documenting the origin of the sample, the laboratory should carefully consider whether to use precious resources to test the sample. The origin of the sample should be verified by comparing its profile to other reference samples. Therefore, even if the laboratory obtains a direct reference, alternative references may be needed. The handling of direct references should be limited, and if possible, the responsible individual should deliver the sample(s) to the DNA laboratory. In order to preserve the chain of custody, the direct reference samples should be delivered in their original containers. A DNA profile may be available to use as a direct reference in situations where DNA databases are commonly used for forensic or other identification purpose, such as in the military services. Such reference sample profiles may require special authorization and documentation. For example, the state of Louisiana maintains a DNA profile database for law enforcement purposes, but there are numerous rules and regulations protecting access to profiles in the database. During mass fatality responses, however, Louisiana law allows

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law enforcement agencies access to the database. After Hurricane Katrina, profiles from the law enforcement database were used to make identifications of the deceased. 7.3.2  Personal Items Personal items are objects purported to have or to contain DNA from the RM because they were used by the RM or came from the RM. Personal items, however, have no associated documentation linking the item to the RM. These samples are typically found at an RM’s home or place of employment (as seen in Figure 7.3) and could be a hairbrush, toothbrush, or favorite coffee cup. If the mass fatality is associated with a disaster that destroyed the individual’s home and place of work, or if the RM has been missing for many years, personal items will be unavailable. Table  7.1 lists common personal items (including biological material) and provides general guidelines for the degree of usefulness. The success in profiling may vary depending on the experience of the laboratory and the quantity and quality of DNA on the personal item. DNA from another person may be deposited on the RM’s personal item(s), possibly without the knowledge of the family member presenting the item for testing. Therefore, it is important to have a record of who may have used or handled the personal item. It is also imperative to collect elimination samples from individuals who may have left DNA on the personal item. Elimination samples are biological samples taken from individuals who could have potentially left their DNA on an item. In order to eliminate the possibility of another person other than the RM leaving their DNA on the item, elimination samples are profiled

Figure 7.3  Personal items found in a home.

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Table 7.1  Personal Items and Their Usefulness for Obtaining DNA Profiles Personal Item Hairbrush Letter sent by RM Toothbrush Clothing Razor Pipe or cigarette holder Nail file Teeth (biological material) Hair (biological material)

Usefulness High High (if RM licked the envelope seal or stamp) Moderate to high (especially if not cleaned after each use) High (when garment was worn next to the skin and not laundered) High High Moderate (if not cleaned) Moderate (teeth without fillings or dental work are preferable) Low to moderate (hair must contain the hair root)

by the laboratory and compared to the profile from the personal item. Figure 7.4, the Personal Items Submission Form, is helpful when collecting personal items. As with direct references, personal items have the potential to provide a complete profile of the RM. Typically personal items are stored in clean paper bags with the identification document attached to the outside. If possible, the collector should not handle or examine the personal item at the collection location, and the personal item should remain in its original container whenever possible.

Figure 7.4  Personal Items Submission Form.

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7.3.3  Family References/Kinship Samples Family references or kinship samples are often the references of choice for the identification of RMs. Because family references or kinship samples can be standardized, the laboratory can process the samples in a timely and consistent manner. Also, there is an abundance of samples, which means that additional testing can be easily performed if the laboratory has any question about the DNA profile. Collectors typically use a buccal swab (scraping from the inside of the mouth, as seen in Figure 7.5) to collect these samples. Alternatively, some laboratories use bloodstains. The sample type depends on the laboratory’s automation and preference for testing. There are several challenges to the laboratory when using family reference samples for identification purposes. First, as outlined earlier, it is extremely important that all kinship samples are placed in the same RM case. When family reference samples are scattered among several different cases, there is a strong likelihood for missed identifications because each family reference by itself may not show enough genetic similarities with the profile from the human remains. Often several family references are required to provide sufficient genetic information to make a DNA identification. Second, not all family members may be genetically related the way they think they are, or there may be genetic changes between relatives (mutations). In order to avoid raising these issues with the family after testing, the laboratory should make sure that every possible family reference is collected as outlined below, thus ensuring all samples are available to resolve the case.

Figure 7.5  A buccal swab collection.

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Male Female

Deceased

Unknown

? Monozygotic

Dizygotic

Unknown

Figure 7.6  Common pedigree symbols.

7.4 Pedigree Prior to sample collection it is important to generate a family pedigree for each RM case. A family pedigree helps to identify which family references should be collected for testing. Most often, a representative from the DNA Unit will talk with family members and create the family pedigree or family tree. Since there may be confusion as to how family members are biologically related, it may be necessary to draw a family pedigree several times. Family members may provide different or conflicting information on family biological relationships. Conflicting results may be resolved by speaking with other family members. Once genetic profiles from the families have been generated and compared, additional information about the family structure can be obtained. In order to maintain a clear understanding of the family structure throughout the DNA operations, standard genetic nomenclature should be used to document the family structure. See Figure 7.6 for common pedigree symbols. Figure 7.7 depicts an example family pedigree drawn using standard pedigree symbols indicating mating individuals and offspring.

M

Figure 7.7 (see color insert)  An example family pedigree.

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Follow all of the lines from the reported missing to each biological relative without passing through a person who is able to provided a sample.

STOP

STOP

RM

STOP

Figure 7.8 (see color insert)  Determining kinship samples to collect.

Once a family pedigree has been constructed, the pedigree should be examined carefully to determine which family reference samples to collect. The samples for each family will be unique based on their family structure and the availability of individuals providing the sample. To identify which samples to collect, start from the RM and follow the lines along the pedigree to each biological relative able to provide a sample. Samples from individuals that will not contribute additional genetic information should not be collected. Figure 7.8 depicts an example family pedigree. The individuals in blue represent individuals who should provide samples. If possible, samples should be collected from all living offspring. In the example in Figure 7.8, a sample from the only son would be collected. A sample from the son’s other parent would also be collected to eliminate that parent’s genetic contribution to the offspring. Samples from the child’s son and his mother would not be collected because they will not contribute any additional genetic information above what the son will contribute. Next, the collector should follow the lines to the RM’s biological parents, whose samples should both be collected if available. In the case shown in Figure 7.8, only a sample from the father would be collected since the mother is deceased. Since the father’s sample will be collected, samples from other family members passing through this paternal line, such as the father’s siblings or parents, do not add any additional genetic information about the RM and therefore should not be collected.

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If a parent is deceased or unavailable to provide a DNA sample, an attempt to gather his or her genetic information should be made by collecting samples from genetic relatives. In this case, samples would be collected from any of the mother’s living siblings, parents, and children as shown. Since family reference samples are collected from multiple individuals, it is important that all samples collected for an RM are placed in the same RM case.

7.5  Scheduling Collections If there is a Family Assistance Center, families may provide reference samples when they provide antemortem data. Alternatively, the DNA Unit may send collectors out into the community to perform sample collections at specified locations (for instance, near a recently exhumed mass grave). Family members may be dispersed all over the world and collections will need to be scheduled, instructions provided, shipping policies and procedures implemented, and samples tracked (samples can be held up in customs). This is no small task for the DNA laboratory. Usually, a collection kit created by the DNA laboratory ensures the standardization of samples and allows the processing of a large number of samples in an efficient and cost-effective manner. The kinship collection kit (often referred to as a family/door reference collection kit) should also be used when collecting samples from elimination donors. See Table 7.2 for additional details about the sample collection kit. An example buccal swab collection kit is shown in Figure 7.9.

7.6  Collecting Kinship Samples It is important to only collect a sample from one person at a time. Interacting with families can be overwhelming, and it is easy to mix samples up if a sample is collected from more than one person at a time or if multiple samples are labeled before the collection process. Best practices include the collection, labeling, and packaging of each sample before proceeding to the collection of the next sample. Whenever possible a sample collector should be trained and competency tested prior to collecting samples from family members. Figure 7.10 is an example kinship reference sample collection identification form. The form collects the information needed by the laboratory and documents the sample collection. As the DNA laboratory uses family donor reference information to verify the identity of the donors and their relationship to the RM, the collection form should be completed in full. Each individual providing a sample must sign the collection form authorizing the use of the sample to identify the

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DNA Analysis for Missing Persons in Mass Fatalities Table 7.2  Sample Collection Kit Contents A buccal swab collection kit may contain: • Information on the DNA testing process • Buccal swab collection instructions • Collection (identification) form • One pair of disposable gloves • Four sterile collection cotton swabs • One swab collection envelope with security seal • One specimen envelope • Mailing supplies (optional) A bloodstain collection kit may contain: • Information on the DNA testing process • Bloodstain collection instructions • Collection (identification) form • One pair of disposable gloves • One alcohol wipe • One lancet • One blood collection card • One sterile plastic bandage strip • One blood card envelope with security seal • One specimen envelope • Mailing supplies (optional)

Figure 7.9  An example buccal swab collection kit.

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Figure 7.10  Family and/or Donor Reference Collection Form.

missing individual. The laboratory should honor this agreement and not use the sample for any other reason. It is critical that family members have confidence in the laboratory’s integrity.

Additional Resources Donkervoorta, S., Dolana, S.M., Beckwith, M., et al. 2008. Enhancing accurate data collection in mass fatality kinship identifications: Lessons learned from Hurricane Katrina. Forensic Science International: Genetics 2(4): 354–363.

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Hartman, D., Drummer, O., Eckhoff, C., et al. 2011. The contribution of DNA to the disaster victim identification (DVI) effort. Forensic Science International 205(1–3): 52–58. Lewis, Ricki. 2005. Human Genetics: Concepts and Applications. New York: McGraw-Hill. Zinnikas, W.A., Chain-of-Custody Considerations. http://www.health.ny.gov/guidance/oph/wadsworth/chain_of_custody_consider.pdf (accessed September 29, 2013).

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Developing a DNA profile is an integral part of the DNA identification effort. The basic tenets of the DNA operations are the same whether the profile is generated by the DNA laboratory in charge of identification effort or by outsourcing samples to one or more independent laboratories. This chapter reviews the major steps in generating a DNA profile, expands on the general procedures for developing one, and discusses various production options for obtaining one. This chapter is not intended to be a technical reference for DNA profiling. The material presented here discusses general procedural aspects of DNA profile technology and how this capability can be useful in human DNA identifications. General considerations when outsourcing the testing of samples are also discussed. Since the science of DNA profiling is ever evolving, the technical aspects of the laboratory process are not discussed, but rather, an operational view of the profiling process is given.

8.1  DNA Profiling Process Overview Current laboratory procedures for obtaining DNA profiles for identification purposes include the following steps in chronological order: • Receipt of samples by the DNA laboratory; accessioning samples into the Laboratory Information Management System (LIMS) and assigning a case number and unique sample identifier • Evidence assessment and cutting or sampling the biological material for testing • DNA extraction from the biological sample • DNA quantification to assess quantity and often the quality of DNA obtained from the extraction process • Amplification of specific sequences in extracted DNA with the PCR, typically using one or more “multiplex” kits, which fluorescently label the amplified fragments 127

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• Separation and detection of polymorphic amplified DNA, commonly by electrophoresis and spectroscopic instrumental methods • Analysis of DNA results—profile results for comparison to either standards or other evidentiary results that were previously developed (most commonly done with the assistance of genetic analysis software; discussed in detail in Chapter 9) 8.1.1  Sample Receipt and Accessioning DNA laboratory personnel, contracted collectors, or other individuals from organizations participating in the identification effort may collect biological samples (see Chapters 6 and 7). After collection, samples are transported to the Family Assistance Operation or DNA laboratory for initial cataloging. When the DNA samples are received, they are inspected for integrity, including signs of tampering, intact seals, and completed documentation/ paperwork. The receiving DNA operation may use a checklist to document the condition of samples upon receipt. Often the DNA operations will have a sample acceptance policy addressing the condition and types of samples the laboratory will accept for testing. A comprehensive sample acceptance policy may include: • A minimum sample size accepted for DNA testing • Appropriate sample collection methods to document • Collector and date • Procedures followed • Preservation or storage since time of collection • Agencies submitting sample(s)/case for testing • Maximum number of samples to test per case Often a DNA operation may not initially test a sample but will reserve the right to keep it and possibly test it later depending on the circumstances. Appropriate personnel will assign the sample a reported missing (RM) case number. In addition, the metadata (collection information and any other data supplied when the sample was obtained) will be entered into an appropriate tracking system (see Chapter 10) which will generate a unique sample identifier. The following is an example numbering system. This system is proposed as a common syntax for victim remains, personal effects, and family references: [year].[agency].[sampletype1].[set#].[sequence#].[sampletype2].[cutting].[lab]

A supervisor may get involved if there are any questions about the metadata and/or the chain of custody associated with a sample. If necessary, the DNA operation may obtain additional documentation from the collector and/or

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Table 8.1  Example Numbering for Samples Sample Information Year sample collected Agency

Sample Type 1

Set# Sequence#

Sample Type 2

Cutting Lab-ID

Example/Description 2008 AG1 = Agency 1 For example, a tissue sample AG2 = Agency 2 from a human remains R = remains collected by Agency 1 in 2008 K = kin/family reference and tested by lab T might be: D = direct reference 2008.AG1.R.T.B1.A.B.T P = personal item Processing site for remains Unique pedigree/family for family Unique pedigree/family for personal effect Serially assigned sample number B = bone T = tissue O = other S = swab L = blood H = high copy A,B,C,D One or two characters will be assigned by the DNA operation for each laboratory processing a sample

the person providing the sample. In some cases, the supervisor may reject the sample and request another sample. Table 8.1 is an example of a numbering system that may be used by a laboratory when testing samples from a mass fatality. If the samples are received and accessioned at the Family Assistance Operation, they will be transferred to the laboratory(ies) for testing. All transfers should be under strict chain of custody. 8.1.2  Aliquotting and Sample Evaluation Since it is often unnecessary to consume the entire sample, when possible DNA analysts will aliquot a portion of the biological material for testing. For example, 100 ul of a whole blood sample or a small punch from a bloodstain card may be taken and processed by the laboratory. These aliquots are typically placed into microfuge tubes or onto 96 well plates. Other sample types such as paraffin embedded tissues or pathology slides should only be processed by laboratories with validated procedures for processing such samples. Personal items submitted for DNA testing may be rubbed with a cotton swab moistened with water to remove the biological material from the item; the head of the swab should then be placed into an appropriate container. A portion of one kinship buccal swab is typically placed into the proper container

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(either a tube or a plate). If the sample of human remains is large, the analyst will place a portion of the sample into the appropriate container for further processing. If the sample is small, the entire sample will be processed. If possible, it is preferable to keep part (ideally 50%) of the sample for further testing in the future, if needed. The remaining sample will be resealed and stored in a secure location, thus ensuring additional aliquots can be taken if there is a failure in obtaining an acceptable profile or if additional testing is required. 8.1.3  Preparation of the Sample Buccal swabs or swabs from personal items typically need no preparation prior to extraction. Direct references such as paraffin embedded tissue or biopsy slides will need special treatment to remove the sample from the surrounding material. Bone samples are often cleaned both physically and chemically, cryogenically ground (pulverized) to a fine powder, and then decalcified. Hydroxyapatite is a major constituent of bone that can interfere with DNA extraction. This is because calcium that is present in hydroxyapatite has a high affinity for the phosphate groups on the DNA backbone. The decalcification step should increase the amount of DNA that can be extracted from the bone. 8.1.4  DNA Extraction Methods There are a variety of extraction methods used to isolate DNA. Effective DNA extraction procedures and methods should minimize most inhibitors encountered in biological materials and maximize the yield of usable DNA. When dealing with degraded samples it may be necessary to employ a variety of different extraction protocols.

8.2  DNA Extract Assessment Once DNA has been extracted and stabilized, it is desirable especially with samples other than buccal swabs to get an assessment of the quantity and quality of the DNA obtained. The quantity and quality of the extracted DNA will determine the volume of DNA extract to use in the amplification reaction. Typical concerns in the forensic laboratory for determining the quality of the extracted DNA are: (1) whether inhibitors are present in the sample that could cause PCR amplification failure; (2) the extent of degradation present in the sample; and (3) how much usable DNA for profiling was extracted. If there are good indications that an inhibitor may be present in the sample, remediation processes may be tried before the PCR step, especially if the sample is somewhat limiting. For example, the isolated DNA may be

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subjected to an alternate procedure for DNA extraction, with an expectation that the inhibitor present may be removed by the new extraction.

8.3  Amplification Strategies and Considerations After the DNA has been assessed, it will be amplified using validated procedures. It is important only to use validated amplification procedures since primer ratios are optimized for a given set of thermal cycling conditions. The level of sensitivity of each DNA profiling procedure can differ (e.g., nuclear versus mtDNA testing). A careful evaluation of nuclear extraction procedures is necessary to determine if any contamination is present at lower detection levels that may affect mtDNA testing. If template DNA amounts for PCR amplification are limiting or of poor quality, it may be necessary to conduct replicate amplifications in order to get reliable and reproducable results. 8.3.1  General Considerations Typically the autosomal STR loci are used when the primary purpose of conducting the DNA analysis is to identify an unknown individual. Amplification kits have been enhanced so that they now are suitable for amplifying degraded DNA. If kinship samples are used for testing, then it is important to use test systems that provide the highest power of exclusion or power of discrimination with the lowest mutation rate. Table 8.2 lists the power of exclusion for some commonly used sets of commercially available amplification kits. In cases of limited DNA evidence or highly degraded DNA (caused by environmental factors), miniSTRs, or reduced-sized PCR products, can be used. MiniSTR kits use primers that anneal more closely to the repeats compared to normal STR primers. Therefore, the PCR product will be smaller, increasing the chances of success with degraded samples. Y-STR typing may be necessary to aid in mixture interpretation and may also be helpful as an additional system when male relationships are examined. Because all Y-STR markers in use are currently linked and inherited as a block, the resulting profile generates a haplotype that cannot be analyzed in the same manner as the independently inherited autosomal STR markers. The most common mtDNA sequencing regions are HV1: 16024–16365 and HV2: 73–340. However, the sequencing of the HV D-Loop is costly and time consuming when compared to STR autosomal analysis. mtDNA sequencing is performed when additional systems are needed to reach the statistical threshold for a DNA identification, to examine maternal relationships, or for degraded samples.

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Table 8.2  Commercially Available Forensic Amplification Kits Amplification Kit

POWERPLEX® 16 System Kit

Caucasian American 0.9999994 African American 0.9999996 Hispanic American 0.9999983 D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539

vWA TPOX D18S51 Amelogenin D5S818 FGA Penta E Penta D a

AmpFℓSTR® SGM Plus® Kit

Power of Exclusiona 0.99997 0.99998 Loci D8S1179 D21S11

D3S1358 TH01 D16S539 D2S1338 D19S433 vWA D18S51 Amelogenin FGA

AmpFℓSTR® Identifiler® Kit

AmpFℓSTR® MiniFilerTM Kit

0.9999992 0.9999996 0.9999990

0.99976 0.99985 0.99970

D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 Amelogenin D5S818 FGA

D21S11 D7S820 CSF1PO

D13S317 D16S539 D2S1338

D18S51 Amelogenin FGA

Power of exclusion is the fraction of individuals who would not have the DNA pattern presented in a typical paternity case.

Along with STRs, single nucleotide polymorphisms (SNPs) have been used as an add-on test to help identify samples that have very degraded DNA. SNPs are variations that occur between individuals at a specific nucleotide. Because they are smaller in size than STRs (about 100 bp), they can be useful for degraded samples seen in mass fatalities. However, they have a lower power of discrimination. The US National Institute of Standards and Technology has an excellent on-line resource with up-to-date information on forensic DNA testing. See: http://www.cstl.nist.gov/strbase/. Regardless of what testing systems are used, it is very important to be consistent with using those systems for all samples. Important information may not be able to be used in a comparative manner if different test systems are used for references and human remains as different tests may evaluate different locations.

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8.4  DNA Separation and DNA Profile Generation Separation of polymorphic amplified DNA is commonly done by electrophoresis or other spectroscopic instrumental methods. The demands for technology to produce DNA results fast and efficiently has increased, and the development of instrumentation has made it possible for laboratories to produce DNA results from multiple samples within a few hours. Though there are various methods to separate DNA results from PCR amplification, the common method used in forensic DNA laboratories is capillary electrophoresis. Once a DNA profile result is developed, the DNA analyst can compare unknown samples to either known reference standards or other evidentiary results that were previously developed. This is most commonly done with the assistance of genetic analysis software. 8.4.1  DNA Profile Generation Once the DNA results are obtained from the instrument used for separation and detection of the PCR products, a DNA profile is generated. Typically this is done using some type of genetic analysis software. The data from the electropherogram is translated into a DNA profile. The goal of a DNA laboratory or laboratories is to generate a DNA profile for each unknown sample of human remains and for each reference sample to compare for identification purposes.

8.5 Emerging DNA Technologies A mass fatality is not the time to introduce new or experimental procedures in the laboratory. Often mass fatality projects are high profile and identifications time sensitive. Companies developing new procedures will often offer their new products and services to the DNA laboratory at no cost or at a reduced rate for marketing purposes. Do not accept products just because they initially appear to be of good value. The cost of validating and implementing new procedures into a laboratory is enormous. Improperly implemented procedures resulting in misidentifications can put the entire identification effort in jeopardy. However, if the laboratory has exhausted all traditional testing methods, it must turn to emerging technologies to obtain results from compromised samples. In these circumstances, a careful and thorough validation must be performed prior to the implementation of the novel testing process.

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8.6  Duplicate Testing and Profile Verification Whenever possible, the DNA operation should verify the accuracy of a profile. The verification process may involve the use of multiplex amplification kits with overlapping loci comparison to other reference samples (see reference verification in Chapters 9 and 10) or complete duplicate testing of the samples. While duplicate testing may initially appear to be time consuming and costly, the integrity of the operations relies on the accuracy of the data provided by the DNA laboratory or laboratories.

8.7  Options for Testing Law enforcement and humanitarian organizations with a need for DNA human identification technologies can acquire the expertise and acquire the resources to provide the needed services (in-house), or they can send the needed work to one or more independent laboratories (outsourcing). These options are not mutually exclusive. For example, instead of investing in expensive capabilities for the short-term increase in testing, it may be more beneficial to outsource work for specialized technologies. Outsourcing is helpful in reducing spikes in workload not encountered often by the organization. Outside laboratories may be other public entities or fee-for-service commercial laboratories. When another laboratory performs the work, it is important to have a contract or memorandum of understanding to clearly specify the terms of testing and the roles and responsibilities of both the primary DNA laboratory and the outsourcing DNA laboratory. The use of contracting laboratories may greatly expedite the identification process if sound and binding agreements exist between the mass fatality DNA operation and outside laboratories. Regardless, the entity responsible for the identifications should have and maintain all of the testing data and results produced by the outsourcing laboratory. 8.7.1  Selecting a Laboratory At a minimum, mass fatality DNA operations should consider the following criteria when selecting an independent laboratory or multiple laboratories for outsourcing work: • Demonstrated expertise in desired testing capabilities. This should not be confused with the accreditation status of the laboratory, although this is of great assistance for monitoring and evaluating the current practices and procedures of an organization.

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• Turnaround time (length of time from sample receipt to reporting) for completion of work. • Sample capacity that may be worked by the facility. • A site inspection by qualified personnel of the laboratory’s facilities, including an assessment of employee qualifications and data quality. • Documentation of laboratory validated procedures, results, and a quality management system. This is of vital importance for data that will be introduced into the legal system. • Contract (written) specifications for desired performance measures, including: • Sample numbering • Extraction method • Test systems • Minimum amplification reaction volume • Analysis equipment • Data presentation parameters for reporting • Data format for reporting • Turnaround time • Requirements for public announcements While outsourcing can be an important tool in supporting an identification operation, it is critical that the agency in charge of the identification effort have control of what the laboratory is doing and have access to all of the data such that an independent assessment of the testing can be performed. See Attachment A for an example statement of work for outsourcing. This example would need to be altered to fit to the specific requirements of the organization responsible for the DNA identifications. The outsourcing laboratory should not be writing the contract. The technical specifications of the contract should be written by someone independent of the outsourcing laboratory and who has experience in large-scale outsourcing and understands the needs of the identification effort. This should happen regardless of whether there is money exchanged for the testing or not. Outsourcing should not be undertaken without a detailed contract even if there will be no charge for the testing. For example, there have been instances where laboratories have issued public press releases about the testing or DNA matches that have been made. This public announcement is inappropriate and is not in the best interest of the DNA identification effort as the DNA testing is not the only factor in determining identity. The repatriation of remains is the objective of the human identification effort and takes time. The premature announcement of DNA matches puts undue pressure on the identification effort to repatriate remains. This public announcement only serves the laboratory and gives false hope to the families.

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Outsourcing DNA testing is complex, and there are ethical and legal considerations surrounding the ownership of DNA samples and the data derived from it. Outsourcing should not be done in lieu of developing local forensic DNA profiling capacity within a country. Because family reference samples can be validated to verify accuracy, and often there is an abundance of material from human remains that can be tested in duplicate to verify accuracy, mass fatality DNA identification efforts allow the laboratory to build sustainable capacity and expertise that can then be used in testing samples for criminal cases, thereby supporting the local justice system.

Additional Resources Butler, J.M. 2012. Advanced Topics in Forensic DNA Typing: Methodology. San Diego: Elsevier Academic. Butler, J.M. 2006. Genetics and genomics of core STR loci used in human identity testing. Journal of Forensic Sciences 51(2): 253–265. Butler, J.M., Kline, M.C., Vallone, P.M., et al. 2010. Population Studies Conducted by the NIST Forensics/Human Identity Project Team. http://www.cstl.nist.gov/ div831/strbase/NISTpop.htm. Coble, M.D., Just, R.S., O’Callaghan, J.E., et al. 2004. Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians. International Journal of Legal Medicine 118. http://www. familytreedna.com/pdf/Coble_2004.pdf (accessed September 29, 2013). Giese, H., et al. 2009. Fast multiplexed polymerase chain reaction for conventional and microfluidic short tandem repeat analysis. Journal of Forensic Sciences 54: 1287–1296. Hill, C.R., Kline, M.C., Coble, M.D., et al. 2008. Characterization of 26 MiniSTR loci for improved analysis of degraded DNA samples. Journal of Forensic Sciences 53 (doi: 10.1111/j.1556-4029.2008.00595.x). Hoffman, L.W., Cramer, Jill, Euy Kyun Shin, B.S., et al. Validation of Promega’s Powerplex® 16 for Paternity Testing. http://www.promega.com/~/media/files/ resources/conference%20proceedings/ishi%2011/paternity%20minisymposium/hoffman.pdf?la=en (accessed September 29, 2013). Identifiler User’s Manual. 2006. Probability of Paternity Exclusion for the AmpFℓSTR Identifiler Kit STR Loci (Table 4.5). http://www3.appliedbiosystems.com/cms/ groups/applied_markets_support/documents/generaldocuments/cms_041201. pdf (accessed September 29, 2013). Mailloux, C.M., and LaBerge, G. Population Genetics and Statistics. INTERPOL Core Loci. http://www.nfstc.org/pdi/Subject07/pdi_s07.htm (accessed September 29, 2013). National Institute of Standards and Technology. 2006. Core STR Loci Used in Human Identity Testing. http://www.cstl.nist.gov/div831/strbase/coreSTRs. htm (accessed September 29, 2013). PowerPlex® 16 System Technical Manual. 2007. Power of Exclusion of the PowerPlex® 1.2 and 16 Systems in Various Populations (Table 9). http://www.promega.com/ tbs/tmd012/tmd012.pdf (accessed September 29, 2013).

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Attachment A Example Statement of Work Buccal Swab, Bone, Tissue, and Personal Item DNA Profiling Goal: To provide a humanitarian service to reunite families with the remains of their relatives who died as victims of a mass fatality. Purpose: To use DNA technology to identify the human remains of victims of mass fatalities. General Scope of Services: Contractor shall, in an efficient and timely manner, test and report DNA profiles generated with the AmpFℓSTR® Identifiler and/or PowerPlex®16 PCR amplification kit (or kits with similar loci) approved by SNA International (SNA). Contractor shall have the demonstrated experience in processing buccal swab, bone, and personal item samples necessary to receive and process samples provided by SNA. Such samples will be extracted with the expectation that mitochondrial testing will be performed on the extract. In addition to returning acceptable DNA profiles, if requested, the Contractor shall return DNA extracts suitable for mitochondrial testing. The Contractor shall use standard nomenclature and plate set-up such that the data can be uploaded to SNA’s Virtual Data Analysis Site (VDAS) and easily run through The National Center for Biotechnology Information’s OSIRIS quality assessment tool for quality assessment. The Contractor/Partner laboratory shall successfully process a sample through the laboratory with minimal complications such as repeat testing, re-amplifications, and re-injections. The Contract/ Partner Laboratories shall provide data that is easy to review and devoid of artifacts and questionable allele calls as well as if requested, extracted DNA ready for mitochondrial testing. The outcomes/tasks for this contract shall include:

Outcomes/Tasks/Performance Indicators: 1. Unless otherwise specified in writing by SNA, the contractor shall maintain accreditation by the American Society of Crime Laboratory Directors-Laboratory Accreditation Board (ASCLDLAB) or accreditation/certification by the National Forensic

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Science Technology Center for the testing for forensic DNA analysis and/or AABB for relationship testing. If there is a lapse in accreditation or certification, the laboratory shall immediately cease all testing of the SNA samples and notify SNA. 2. Contractor shall process samples in a secure and dedicated laboratory. 3. Contractor shall have and strictly adhere to procedures and practices for each step of the process that maintains sample integrity and results in accurate results. 4. Unless otherwise specified in writing by SNA, the contractor shall process samples in strict accordance with the Federal Bureau of Investigation’s National DNA Index System (NDIS) Standards for acceptance of DNA data and provide SNA with all CODIS/NDIS required documentation. 5. Unless otherwise specified in writing by SNA, the contractor shall adhere to the most current Federal Quality Assurance Standards for Forensic DNA Testing Laboratories for the processing of forensic samples. 6. Contractor shall have a full-time technical leader located on-site at the laboratory where the testing is being performed. 7. Unless otherwise specified in writing by SNA, the contractor shall participate in, maintain current, and successfully complete approved external proficiency testing programs in accordance with the Federal Quality Assurance Standards for Forensic DNA Testing Laboratories for all analysts performing any DNA analysis pursuant to this contract. The results of such testing must be made available upon request to SNA. 8. Contractor shall utilize the SNA numbering system throughout the testing, analysis, and reporting process. 9. Contractor shall allow for unannounced site visits/inspections, as SNA deems necessary. 10. Contractor shall return all written and electronic data to SNA at a time that is mutually agreed upon by the parties. Records shall be logically organized, understandable, and contain complete chain of custody documentation for the samples in each case. 11. At a minimum, the Contractor shall maintain the supporting documentation for the testing  of the DNA samples for a minimum of ten years after completion of the delivery order. This includes all records associated with testing of the samples including worksheets and notes, chain of custody of the samples, quality control records, and administrative records. Prior to the destruction of the documentation, the Contractor shall give SNA

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the opportunity to receive this documentation at no additional cost. The notification of document destruction and release of records to SNA shall be made in writing via overnight carrier 120 days prior to the destruction and shall include a cover letter describing the testing and why the notification has been sent. 12. At any time upon written notification, the Contractor shall, within 14 days, provide SNA a list of the location of all electronic and hard copy of all sample profile records (including but not limited to GeneMapper® ID, Excel, and .cmf files) containing the SNA profiles including those located in the Contractor laboratory, on tape back-up, or housed off-site. Upon written notification, the Contractor shall provide SNA the specified profile records and destroy the profiles as well as any copies of the records within 14 days. The Contractor shall provide a written certification of destruction to SNA. 13. All samples must be amplified using SNA acceptable kits, at a minimum amplifying all loci found in AmpFℓSTR Identifiler or PowerPlex 16 loci or other locations requested by SNA. 14. Unknown evidence samples (human remains) can be analyzed in groups of up to 24 samples; buccal swabs (kinship reference samples) can be processed in a 96 well plate. A group consists of at least one positive extraction control, one reagent blank control. The corresponding known reference samples should be organized similarly; however, groupings and controls must correspond to the grouping and order of the unknown evidence samples. Groups may be organized and analyzed in batches, as long as the two controls enclose each group. 15. The positive extraction and reagent blank controls shall be defined and analyzed as follows: a. The reagent blank control consists of the reagents used in the extraction process without any sample or substrate added. b. The positive and reagent blank controls shall always be the first and last samples, respectively, in a group of samples. c. The positive and reagent blank controls must be carried out through the entire extraction process, quantification, amplification, and genetic typing. d. If requested by SNA, the Contractor shall use a mitochondrial sequencing system approved by SNA to demonstrate that all extraction controls specified by SNA are devoid of contamination so that additional testing such as mitochondrial sequencing analysis can be performed on the samples. 16. All reported profiles shall be directly (for example, in the same Genotyper file) associated with a ladder (containing all

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appropriate peaks and no irregularities) and acceptable positive and negative controls devoid of artifacts. 17. The Contractor shall not consume more than 50% of the entire quantity of sample provided by SNA without prior approval by SNA. 18. All reported profiles shall have an acceptable reagent blank directly (for example in the same Genotyper file) associated with each amplification that can be easily traced to each sample. 19. All profiles generated by the laboratory must be returned to SNA along with a master list documenting the location of all data. 20. Samples containing unusual profiles (tri-allelic patterns, confirmed imbalances) must be re-amplified and re-analyzed by the Contractor, unless insufficient sample is available. The profile shall be clearly identified by the testing laboratory for review by SNA. 21. All samples must be analyzed using Genotyper® NT software unless otherwise specified by SNA. The Contractor shall perform a technical review of all profiles, and this review should be documented. 22. Samples with off-ladder allele peaks shall be re-amplified and analyzed by the Contractor, unless insufficient sample is available. An off-ladder allele is defined as an “OL allele” in Genotyper and/or GeneMapper ID-X and cannot be attributed to an amplification or electrophoresis artifact. SNA will provide a list of acceptable OL alleles that do not need to be re-run. 23. The Contractor shall provide documentation of the analysis of the biological samples in a printed format provided by SNA. The case documentation shall be organized as specified by SNA and may include reports of results listing: −− Accession information −− Analyst(s) responsible for results −− Evidence descriptions −− Dates analyzed −− Results in the chart format provided by SNA −− Technical leader or laboratory director signature −− DNA extraction protocols which show batch sample organization, procedure followed, QC information, and final volume recovered −− DNA quantification protocol showing sample organization, QC information, procedure, results, and data used to report quantity (standards, standard curve, etc.) −− Amplification protocols which show sample organization, quantity amplified, QC information, and thermal cycling parameters −− Genotyper and GeneScan and or GeneMapper ID-X or other files specified by SNA for all samples and controls. GeneScan

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printout will be provided for all blank controls with the maximum vertical scale set no greater than 200 RFU. 24. Additional tests or re-analysis should be placed after the first-run data in chronological order. 25. In reporting the genetic profiles obtained as a result of its analysis of the biological samples provided by the SNA, the Contractor shall adhere to the following reporting criteria and report controls and DNA profiles: a. Are devoid of spikes and artifacts above background in the allelic regions (gray shaded areas in Genotyper) unless otherwise specified. b. Are devoid of stutter above acceptable levels (called as peaks or bands), unless otherwise specified. c. Contain alleles for all data that are CODIS acceptable or are designated in a CODIS acceptable manner. d. Have internal standards (for example ROX) that are of good quality and devoid of irregularities. e. Are devoid of peaks (or bands) that are misshapen or show irregularities (i.e., profiles should be devoid of –A peaks that fall above background), unless otherwise specified. f. Are accurately reported. Any reported samples that do not meet the quality standards of SNA must be re-analyzed by the Contractor at the Contractor’s expense. Inaccurate reporting of samples may be grounds for termination of this contract. For buccal swabs, the profiles will meet criteria such as: −− Alleles in ladders, positive controls, and samples and the ILS shall have a signal at least 3× that of background. SNA will not be measuring the signal-to-noise ratio for every sample. However, if SNA feels that background is excessive, the Contractor laboratory shall be prepared to demonstrate signal-to-noise contract compliance if requested. −− Peak shape shall be bell shaped and devoid of split peaks. −− Minimum peak height shall be 150 RFU for heterozygote alleles and ladder and 300 RFU for homozygote alleles and 150 RFU for ILS. −− Maximum peak height shall be 5,000 RFU for 310, 7,000 for 377, and 3100 Genetic Analyzers unless otherwise specified by SNA. −− Heterozygote allele peak height ratio shall be within 50%. If sample is retested and peak height ratio at the same location is still less than 50%, the Contractor shall provide supporting documentation for the imbalance. The run data shall be provided in a manner such that all data

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−− −− −− −− −− −−

−−

−− −− −−

is provided in the data package of the reported profile. This means that SNA will be able to evaluate all data associated with the profile without going back to previously submitted data packages. Screen shots of the first analysis (containing the ladder that was used and the sample) will be acceptable. The screen shots shall be of both the entire sample and ladder and an enlargement of the locus of interest. The Contractor shall provide SNA with a proposed method of reporting and documentation, and SNA will notify the Contractor of the approved method of reporting documentation. Spikes shall not be acceptable in the allele calling region. Profiles shall not be reported with any artifacts that would originally be called alleles or “OL” by GeneMapper ID. Extraneous peaks shall not be acceptable in the allele calling region. Stutter called by GeneMapper ID set at 20% shall not be acceptable. –A called by GeneMapper ID set at 20% shall not be acceptable. Tri-alleles: Shall be re-extracted and the profile verified. Upon reporting, SNA shall be provided with data from both runs documenting the tri-allelic profile in the same manner as the alleles with confirmed imbalance. Microvariants and off-ladder alleles: The Contractor shall provide SNA with a list of proposed microvariants and off-ladder alleles (above, below, and within the ladder) that may be reported without retesting. SNA will notify the Contractor of the microvariants and off-ladder alleles that may be reported without retesting. All other microvariants and off-ladder alleles shall be retested and documentation provided in the same manner as the confirmed imbalances and tri-alleles. Contain alleles that are CODIS acceptable or are designated in a CODIS acceptable manner. Have internal standards that are of good quality and devoid of irregularities. Have peaks (or band intensities) that are balanced across all loci and peaks (or band intensities) within a locus that are balanced (within 50%). This applies to all alleles including those above and below the ladder. If an imbalance is seen the laboratory shall retest the sample. If the imbalance is still observed after the retesting the

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Contractor shall report the profile and report the imbalance in the sample comments on VDAS and shall provide a picture of the original profiling demonstrating the reproducibility of the imbalance. −− Are devoid of peaks (or bands) that are misshapen or show irregularities (i.e., profiles should be devoid of –A peaks that fall above background). −− Is accurately reported for every sample. Any reported samples that do not meet the quality standards of SNA shall be re-analyzed by the Contractor laboratory at the Contractor laboratory’s expense prior to reporting to SNA. SNA expects that all profiles will be reported according to the contract, and it is unacceptable for profiles not meeting the specifications of the contract to be reported to SNA. Inaccurate reporting of samples may be grounds for termination of this contract. g. Contain sufficient information and files so that SNA can reconstruct and re-analyze the GeneScan and Genotyper data reported by the contractor. h. Contain ROX peaks 75–400 for every sample. The Contractor shall utilize an automated tracking system for the processing of samples to ensure transcriptional errors do not occur and use the specified sample numbering dictated by SNA. The Contractor shall notify SNA immediately in writing if a problem is anticipated or detected in the testing of the samples. Contractor shall use only validated analytical procedures to generate DNA profiles from any biological samples. Contractor shall maintain a consistent turn-around time that allows for the reporting of data on a weekly or biweekly basis. Contractor shall meet with representatives of SNA to develop a credible implementation plan for this project. Contractor shall provide training to the SNA (data reviewers, staff at the SNA offices or a facility chosen by SNA) as requested. Contractor shall propose, provide, and support the use of tools to ease data review for SNA. Contractor shall provide analytical services which result in complete and accurate profiles. Provide quality electronic data that can be easily reviewed and uploaded into VDAS and OSIRIS in a format acceptable to SNA. Provide any additional data and records requested by SNA in a timely manner. The Contractor shall not record any identification information from the sample other than the specified identification number.

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37. Contractor shall follow procedures and policies to ensure that data from reported profiles shall not be intermixed with profiles not used for reporting and that each profile shall be identified with a unique number provided by SNA. 38. All profiles generated by the laboratory shall be returned to SNA along with a master list documenting the location of all data. 39. The Contractor shall adhere to the data packages specifications dictated by SNA, and said data packages shall contain: (a) Genescan and Genotyper or whatever program is specified by SNA data for all tested samples in NT format or format specified by SNA, and (b) raw data file for reported samples and associated references and controls in all data (.fsa) files including ladders and controls unless otherwise specified by SNA. 40. Contractor shall provide accurate and complete reporting of unusual profiles as specified above. 41. Contractor shall provide any data files that may be requested by SNA in order to expedite data review. 42. Contractor shall provide bi-weekly (or more frequent) reports in a format provided by SNA, indicating at a minimum: −− Number of samples received and the batch number. (This information shall be provided on a more frequent basis when requested.) This information may be requested more frequently. −− Number of samples reported −− Number of failed samples including reason and sample number −− Potential pitfalls −− Any other related information requested by SNA 43. Contractor shall upload data packages to SNA’S Virtual Data Analysis Site (VDAS) and according to a schedule set by SNA. If the data package provided by Contractor is not the data package required by SNA, the Contractor shall cease to receive additional samples for testing until the appropriate corrective action is taken. SNA will decide if the corrective action is appropriate. 44. Unless otherwise specified, the Contractor shall submit its Data Packages in an electronic format specified by SNA to VDAS. VDAS may be available via the Internet or through a Virtual Private Network (VPN). While the Contractor may elect to use a dial-up modem to access the Web site, a broadband connection is recommended. 45. The Contractor may be given one or more login accounts to the site and/or VPN. The Contractor shall be responsible for implementing appropriate measures to secure the distribution of login names and passwords and for maintaining their confidentiality.

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46. Contractor shall implement all SNA-specified security requirements for accessing the VPN (e.g., anti-virus software, etc.) at the request of SNA. 47. Contractor shall upload all Data Packages to the site. The Contractor shall also upload and/or enter any technical comments specific to the Data Package (e.g., tri-allelic profiles, offladder alleles, and other information that would be relevant to data review). This may involve additional file uploads or manually entering data. 48. The Contractor shall monitor the site and answer all Data Package and/or sample-specific technical questions using the features provided in the Web site. The Contractor shall respond to all questions within twenty-four (24) hours of the time the questions were posted on the site (Monday through Friday). 49. At the direction of SNA, the Contractor shall add any and all sample-level comments in a delimited text file. The name of the file and its format shall be specified by SNA at a later date. (SNA envisions either a comma or tab delimited text file.) 50. Contractor shall, at the direction of SNA, make reasonable modifications to the Data Package format. If the changes are not made in the required time frame the contractor may be suspended from receiving samples. 51. At the direction of SNA, the Contractor shall be required to submit Data Packages on CD-ROM or some other mutually agreed upon format. 52. At the direction of SNA, the Contractor shall be required to submit answers to Data Packages and/or sample-specific technical questions using email or some other mutually agreed upon medium. 53. If a data package is rejected, Contractor shall have seven (7) calendar days to make any necessary corrections and re-post the Data Package. If Contractor needs more than seven (7) calendar days to correct the problem(s), Contractor shall notify SNA to request more time to make corrections. Under no circumstances shall the corrective action take longer than fourteen (14) calendar days. All re-posted data packages shall contain the same data package identifier used by the Contractor in the initial posting with the designator “RP” at the end of the Data Package name. All comments and/or responses to said comments from the initial posting of a data package shall be included in any re-posting of the same data package. 54. SNA reserves the right to request any information produced by the Contractor during the processing of the samples from

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the laboratory that shall enhance SNA’s review of the data. The Contractor shall produce the data in a format that can be uploaded to VDAS within thirty (30) calendar days of the request. If the Contractor cannot provide the requested information, the contractor shall cease to receive additional samples for testing until the appropriate corrective action is taken. SNA shall decide if the corrective action is appropriate. 55. It is anticipated that there may be multiple awards. SNA shall monitor the profiling success rate for each Contractor. If the Contractor consistently falls below the other Contractors in obtaining successful profiles, SNA may discontinue the service of that Contractor until the Contractor has conducted appropriate remediation. SNA shall establish the definition of successful profiling. 56. Shipping and reporting batch sizes shall be determined by SNA. The Contractor shall pay for all shipping to and from the laboratory. All shipping will be within the continental United States. 57. Contractor shall notify SNA within five (5) business days of any incident that has the potential to affect the SNA samples including those that were previously reported. 58. The Contractor shall provide the DNA extracts to SNA or a third party designated by SNA along with: −− Documentation of DNA concentration for each extract as well as proof of the absence of contamination including: −− Acceptable reagent blank/reagent control −− Positive control −− Positive amplification control −− Negative amplification control 59. Contractor shall maintain processing throughput as agreed upon by SNA. 60. Contractor shall immediately direct any and all outside inquiries regarding this project to SNA and immediately notify SNA of the inquiry. The Contractors shall not issue press releases, submit publications, or talk to the public without first obtaining approval from SNA. 61. The Contractor shall not be able to use the samples or profiles for any purpose other than fulfilling this contract.

Chapter DNA Profile Analysis and Interpretation

9

This chapter provides an overview of autosomal STR DNA profile analysis and interpretation. The chapter focuses on procedures to ensure accuracy of the DNA profile and corresponding identification and the subsequent statistical analysis of the data.

9.1  Parameters for Acceptable DNA STR Profiles Whether the DNA laboratory generates DNA profiles “in house” or they are generated by a vendor (outside) laboratory, the resulting electropherograms must be evaluated for quality and accuracy prior to their use in the identification process. Acceptable profile parameters can be based on the laboratory’s validation studies (internal testing) demonstrating that the profiles can be properly interpreted. These parameters include characteristics such as minimum peak height, maximum peak height, maximum allowable stutter, allowable background, and peak balance between and across loci. When the DNA operation must review thousands of profiles, often produced by a number of different laboratories, the data presentation parameters should be set high enough to ensure very little data interpretation needs to be performed during the data review process. By setting the data acceptance parameters high enough, the DNA operations will not have to send questionable samples back for retesting because the allele calls generated from the electropherograms are questionable or subject to interpretation. This practice can be more costly for the laboratories generating the profiles but will result in clean and clear data that is unambiguous and efficient to review. After DNA amplification, an electropherogram is produced for each DNA sample analyzed using a capillary electrophoresis instrument (CE) and appropriate data collection and analysis software for the instrument. The electropherogram is a graph that displays the data. In the electropherogram, the baseline establishes a residual signal associated with an instrument’s blank response. A blank response is a time where no fluorescent dyes are detected. The fluorescently labeled primers for each DNA locus allow the CE to record the relative time and quantity of each DNA fragment as it passes 147

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Figure 9.1 (see color insert) Example electropherogram. (Courtesy of Del Price.)

through a window in the instrument during electrophoresis. Because normal STR testing is often completed using multiplexes, the primers are labeled with multiple colors of fluorescent dyes, which the instrument simultaneously collects and then programmatically separates into each individual dye, called dye channels, for analysis of each individual color. The CE records the intensity of the fluorescence and plots it against time of analysis. Figure 9.1 shows an example of an electropherogram. 9.1.1  Allele Sizing All of the STRs loci currently used for human identification detect either four or five base pair repeats. The accepted nomenclature used to describe the sizes obtained for STR loci is the number of repeats a certain allele possesses rather than its base pair size. However, it is necessary to identify the base pair size of each fragment of DNA in order to assign a number of repeats. An internal lane standard (ILS) is composed of a range of DNA fragments each with a known base pair size and is injected with each sample into the CE. The ILS can be thought of as a ruler and is used to size the unknown DNA fragments. The allelic ladder, which is run separately from each DNA sample, is composed of DNA fragments corresponding to the common alleles for each locus. Both the ILS and allelic ladders are typically used in assigning allele calls to the profiles generated from the tested samples. The ILS from the Identifiler® kit is shown in Figure 9.2.

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Figure 9.2  Example ILS (internal lane standard). (Courtesy of Del Price.)

The most common method used to assign base pair sizes to the DNA fragments is the Local Southern sizing method. This method uses the four points from the ILS closest to the unknown DNA fragment to determine the size. The computer software program will generate a best-fit curve from the ILS using two points above the unknown fragment and one below, then using one above and two points below. The position of the unknown DNA fragment on the curve determines the base pair size. The unknown fragment’s base pair size is then compared to the base pair sizes of the expected alleles on the allelic ladder to make an allele designation for the fragment. Figure 9.3 shows the AmpFℓSTR® Identifiler allelic ladder. In the case where a DNA fragment does not correspond with an allele on the allelic ladder, the fragment is given an off-ladder (OL) allele designation. Peak detection is

Figure 9.3 (see color insert)  AmpFℓSTR® allelic ladder.

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based on the minimum relative fluorescent unit (RFU) value at which a peak will be given an allele designation. The RFU value is a measure of the amount of fluorescence detected by the CE, which correlates to the amount of DNA present. Any DNA peak detected below this threshold will not be assigned an allele designation; however, it can still be visualized in the displayed profile. The parameters for collecting data and assigning allele calls typically only need to be established when the designated software is installed. Parameters will not need to be changed unless new methods are validated. Prior to use, the software needs to be validated for use. Validation refers to the process of demonstrating that a laboratory procedure (in this case sizing) produces successful results, that those results are accurate, and that the same or similar results are obtained each time the procedure is performed by the laboratory. A validation study is typically completed for changes to software or for every new instrument, procedure, and method introduced to a laboratory prior to its use. The results of the validation are used to establish the parameters and procedures that should be followed each time the new or updated software, new instrument, procedure, or method is used in the laboratory. The validation should also be documented in the laboratory protocols and procedure manual. 9.1.2  Peak Morphology The analyst evaluating a DNA profile should determine if all peaks have acceptable peak morphology. A quality peak is one that falls within the minimum and maximum RFU threshold, is taller than it is wide, and has a bellcurve shape with a rounded peak apex. DNA profiles may not exhibit ideal peak morphology if a problem occurred during amplification or injection of the DNA sample into the capillary electrophoresis instrument or if the quality of the original sample has been compromised. Below are some common causes of profile morphological abnormalities. 9.1.2.1 Spurious Peaks (Background, Stutter, Dye Blobs, Spikes, –A, Pull-Up) Electropherograms often exhibit spurious peaks that do not indicate the presence of a true amplified DNA location. These extra peaks are often called “artifacts” and are produced at various points by the DNA analysis process. The most common artifacts are background noise, stutter, spikes, –A, and pull-up. If the spurious peak falls within the allele calling range and is of sufficient RFUs, it may be assigned an allele call or it may be labeled with an OL designation, depending on the location of the artifact peak. Data interpretation is an essential component of DNA profiling. Data analysis allows the analyst to ensure that the results reported are truly attributed

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to the sample and not to artifacts or non-related sources. Additionally, analysts can check the results to determine potential instances of contamination by laboratory employees or between samples. In order to check for potential contamination, many laboratories have established employee reference databases that are compared to sample results. Typically, a laboratory has an established protocol that documents how data is reviewed and the acceptability of common artifacts. The terms of the protocol are laboratory specific and rely largely on the results from validation studies performed for implementation of the data analysis software, electrophoresis technology, and chemistry used during the amplification reaction. For example, a laboratory may establish a minimum peak height of 200 RFUs where peaks below this value will not be analyzed. Through a validation study, the laboratory determines that peaks below this value are not distinguishable from the baseline. Background noise is comprised of small peaks that occur along the baseline in all samples and is typically caused by the instrument, air bubbles, urea crystals, and sample contaminants. Peaks from the baseline may be large enough to reach threshold and may generate an allele call by the analysis software. Such peaks can sometimes be distinguished due to their location and shape during data analysis. Stutter peaks occur one repeat before or, less frequently, one repeat after a real peak. Stutter occurs as a by-product of the process used to amplify DNA and is identifiable by its peak size and position relative to the true allelic peak. The arrow in Figure 9.4 indicates a stutter peak. There are a number of factors that affect the occurrence of stutter. The percentage of stutter exhibits an inverse relationship with the number of base pairs in the repeat unit.

Figure 9.4  Stutter peak that is less than 15% of the true peak. (Courtesy of Del Price.)

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Penta- and tetra-nucleotide sequences exhibit less stutter than tri- or dinucleotide sequences. Similarly, as the number of repeat units increases, the occurrence of stutter also increases. For example, twenty repeats at D16 have a higher chance of stutter than six repeats at D16. The uniformity of the repeat sequence also plays a role in the occurrence of stutter. Uniformity refers to the sequence of repeat units at a locus. Repeat sequences that are strictly the same base pairs repeated the same way are more uniform than those that repeat the same base pairs but in a different way. For example, the repeat sequence (AGAT)n for CSF1PO is more uniform than the (TTTC)3TTTTTTCT(CTTT)nCTCC(TTCC)2 repeat sequence at FGA. Generally, non-uniform sequences, such as FGA, exhibit less stutter than uniform sequences. Since stutter is a predictable occurrence, laboratories have typically completed stutter studies as part of their validation studies to determine acceptable stutter percentages for a sample. For example, stutter peaks may be acceptable provided they are less than 15% of the true peak, as seen in Figure 9.4. Dye blobs are a result of unincorporated dyes from the primers in the amplification kit which migrate freely through the capillary. The peak morphology is typically smaller and broader than a true peak. Often dye blobs do not affect a DNA profile result because they are outside of the allele calling range. Typically dye blobs are noted by the primer manufacturer and are consistently the same size and appear at the same time in the electropherogram. The small bumps in the baseline in Figure 9.5 are examples of dye blobs. Spikes are narrow peaks usually attributed to fluctuation in voltage or the presence of minute air bubbles in the capillary; they can also be caused by urea crystals in the capillary or other impurities in the DNA sample. Spikes are characteristically seen in the same position in all dye channels as depicted in Figure 9.6. Incomplete nucleotide addition, also referred to as –A, occurs during PCR when the Taq polymerase fails to add an additional adenosine nucleotide during final extension to the 3' end of all the DNA fragments. This can result in a peak with a shoulder or a split at the apex of the peak. –A is one base pair less than the main peak and can be given an allele designation. Less frequently, a similar phenomenon occurs, resulting in an addition of an extra nucleotide and is referred to as +A. Typically, either can be resolved and not mistaken for a true DNA peak. It is important, for sizing purposes, that all DNA peaks be adenylated because ladder peaks incorporate the additional A. Figure 9.7 shows the minus A peak which occurs because of incomplete adenylation. Bleed-through (also referred to as pull-up or matrix failure) typically occurs when large amounts of DNA cause the failure of the analysis software to discriminate between the different dye colors. A very strong signal from a locus labeled with green dye might mistakenly be interpreted as a yellow or blue signal, thereby creating false DNA peaks in the yellow or blue channel. As

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Figure 9.5  Example of dye blobs. (Courtesy of Brian Harmon.)

with spikes, pull-up can usually be identified through careful analysis of the position of peaks across the different channels. Figure 9.8 depicts two pull-up peaks as the result of the green dye overlapping into the yellow dye. While some artifacts are clearly identifiable, there are no clear standards for determining whether a peak is the result of an allele or a technical artifact. This may result in disagreement among scientists, so profiles need to be reviewed carefully. Having strict data interpretation standards will minimize the time DNA analysts involved in the identification process must spend reviewing and interpreting data. Interpretation standards revolve around the acceptability of an artifact. The acceptability of an artifact is laboratory specific and is based on the impact of the artifact on the profile. If an artifact interferes with the interpretation of the profile, it is not acceptable. For this reason, many laboratories place limits on the maximum number of acceptable artifacts in a profile. For example, it may be acceptable to have two or less peaks exhibiting incomplete adenylation in the sample.

9.2  Data Review 9.2.1  Evaluation of Controls In order to save time, typically the controls associated with a capillary electrophoresis run are evaluated first. Controls are samples with known results.

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Figure 9.6 (see color insert)  Example of spikes. (Courtesy of Brian Harmon.)

Figure 9.7  A peak. (Courtesy of John Butler.)

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Figure 9.8 (see color insert)  Example of pull-up. (Courtesy of Del Price.)

Controls are used to help access the quality and accuracy of each step of the DNA profiling process. The use of controls is generally required as a part of maintaining quality assurance (QA) and quality control (QC). QA refers to the planned or systematic actions necessary to provide confidence that a product or service will satisfy given requirements. QC involves the daily operational techniques and activities used to fulfill the requirements to maintain excellence. Using controls allows the laboratory to ensure that its procedures, methods, and instrumentation are working as expected and that the generated results are reliable. Table 9.1 outlines some of the common testing controls associated with forensic DNA typing. Controls can also be evaluated to determine instances of contamination and identify specific steps in the profiling process where an error may have occurred. For example, if the amplification positive produced the correct profile but the extraction positive did not, then it can be determined that there may be an issue with the extraction. Any questioned sample showing no results may actually have a detectable DNA profile. Similarly, peaks in

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Table 9.1  Common Testing Controls Control Sample Reagent blank

Extraction positive

Amplification negative

Amplification positive

Description A tube (without a sample) that is introduced at the beginning of the extraction process and treated in the same manner as the other tubes containing samples. The contents undergo the same testing process and are extracted concurrently, amplified utilizing the same primers, instrument model, and concentration conditions as required by the sample(s) with the most sensitive volume in the extraction set and typed utilizing the same instrument model, injection conditions, and most sensitive volume conditions of the extraction set. This control is designed to detect contaminants in reagents used to process samples. A sample with a known profile that is introduced and processed in the same manner as an extraction blank. The control sample undergoes the same testing process as the other associated samples. If the correct profile is obtained, the control serves as evidence that the profiling process was performed correctly. PCR reaction with all components except DNA. The amplification negative is amplified concurrently in the same instrument with the samples at all loci and with the same primers as the tested samples. The amplification negative provides evidence that there was no DNA contamination in the amplification reaction and analysis reagents. DNA of a known profile introduced at amplification set-up and processed in the same manner as all other samples and controls—this includes profile analysis. If the laboratory obtains the correct profiles for the amplification positive, the laboratory has evidence that the correct techniques were used during the amplification, detection, and analysis procedures.

Anticipated Result No profile detected

Correct profile

No alleles detected

Correct profile

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any negative controls could point to a contaminated reagent or a pipetting error. Evaluation of the control samples provides invaluable information and also aids in maintaining sample integrity and quality. Once the controls are evaluated and found to be acceptable, the data is further evaluated. 9.2.2  Evaluation of Allelic Ladders The analyst will evaluate the allelic ladders to ensure that there are no extraneous peaks and that all expected alleles are present and assigned the correct allele designation. The analyst will examine the location, intensity (RFU), and shape of the peaks to make sure that the injections were acceptable. 9.2.3  Evaluation of Internal Lane Standard (ILS) An ILS is injected along with each sample in order to facilitate assigning base pair sizes to unknown DNA fragments. The analyst shall evaluate the ILS for each sample to ensure there are no extraneous peaks, all expected alleles are present, and that the known base pair sizes were assigned correctly to each peak. Similar to the use of controls to access the quality of the DNA profiling process, the ILS is also used to access the quality of the injection. Since an ILS is composed of a range of DNA fragments each with a known base pair size, the quality of the ILS peaks is an indication of the quality of the injection. If the results for the ILS do not meet expectations, and, for example, the ILS has extra, missing, mislabeled, or imbalanced peaks, a poor injection could potentially be to blame. Since the ILS shows injection issues, then it can also be inferred that the sample may not have injected properly. Poor injections can lead to improper sample detection, peak sizing, and allele calls. 9.2.4  Evaluation of the DNA Profiles from Tested Samples The analyst evaluating each DNA profile should determine if the peaks have acceptable peak morphology. In addition, careful evaluation of each peak’s height and shape relative to other peaks within the same profile is necessary. Typically heterozygous peaks are similar in height and shape. Homozygous peaks are typically twice the height of heterozygous peaks. Ideally, all peaks within a profile should be of the same approximate height (with a homozygous peak’s height being twice that of a heterozygous peak), although a slight decline in the larger alleles can be observed. An example of homozygous and heterozygous peak height balance in a good-quality DNA profile can be seen in Figure 9.9. Profiles may not exhibit the ideal morphology if there was a problem that occurred with injecting the DNA into the electrophoresis process, the testing process, or if the sample’s DNA had been compromised in some manner (DNA is degraded or subjected to inhibitors). If the ILS appears correct,

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Figure 9.9 (see color insert)  Example of peak height balance. (Courtesy of Del Price.)

potential injection issues can be excluded. Below are some common causes of profile morphological abnormalities. 9.2.5 Degradation DNA degradation is often encountered when testing samples from human remains that have been exposed to the environment or are particularly old. Since degradation affects the entire DNA strand, degradation is more likely to interfere with the detection of the larger pieces of DNA, therefore the profile from degraded DNA often has taller peaks at the smaller loci and smaller peaks at the larger loci, thus creating a ski slope effect. Larger loci appear at the end of a channel. Figure 9.10 depicts the ski slope effect.

Figure 9.10  Example of the ski slope effect. (Courtesy of Brian Harmon.)

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Repeating the testing using alternative test systems such as Minifiler (designed for use on degraded DNA) can help eliminate some of the problems caused by degradation. Methods to obtain results from degraded samples include increasing PCR cycles and implementing data interpretation protocol that allows for a lower minimum RFU value. 9.2.6  Allelic Dropout Amplifying small amounts of DNA can result in profiles with peak height imbalances and potential loss of allelic peaks. This occurs when the amount of DNA is at the minimum threshold of the reaction components and one allele amplifies preferentially over the other allele. Typically, the laboratory sets a peak balance threshold of 50%. If the two peaks differ by more than 50%, the sample is retested. Allelic dropout can also be caused due to sequence polymorphisms that occur around or within STR repeat regions. The sequence variation can occur within the repeat unit, in the region flanking the STR repeat region, or in the primer-binding region. If the polymorphism occurs in the primer-binding site for the locus, primer hybridization can be affected, preventing amplification of the allele. Alleles that have failed to amplify due to a mutation in the primer-binding region are termed null alleles. Using a different primer set in the amplification reaction can complete verification of a null allele. 9.2.7  Tri-Allelic Patterns In some profiles, three peaks are observed at a single locus. Sometimes these peaks are not the result of a mixture or contamination, but are reproducible artifacts of the sample. Tri-allelic patterns can be caused by an extra chromosomal occurrence and duplication of the locus. Two types of tri-allelic patterns are observed. In the first type, the sum of the height of the two smallest peaks equals the height of the tallest peak. In the second type, the peak heights of all three peaks are balanced. Research and contributions from laboratories in the United States and around the world are compiled by the Short Tandem Repeat DNA Internet Data Base created by John Butler. It documents tri-allelic patterns at each of the CODIS core loci (http://www.cstl.nist.gov/strbase). 9.2.8 Microvariants Microvariants are alleles that contain a sequence variation in the form of incomplete repeat units. For example, a common microvariant is allele 9.3 at locus TH01. This repeat contains nine repeats of the tetranucleotide sequence AATG and an incomplete tenth trinucleotide repeat ATG.

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9.3  Documentation of Data Review Developing procedures for documentation can minimize variability in how results are evaluated and data is reported. Ultimately, the goal is to have two different individuals review the same data and receive the same results. Having a validated procedure can ensure the quality of the DNA profile results. This can be used as a guide for data review. The reviewer can focus on specific aspects of analysis that are critical to the reliability of a reported result. Management can be alerted of any issues and determine if a problem is systematic or the result of the analyst methods. The process can assist an analyst with troubleshooting. Documentation provides a record of the work an analyst performed. Most importantly, criteria can be established to maintain a consistent quality and format for uploading a DNA profile to a DNA archive. An archive is a file containing a collection of DNA profiles from a specified sample type. This will be discussed later in the chapter. Documentation for analysis is typically done in an electronic format, and a checklist is used to ensure proper procedures were followed. Use of various software programs can determine the format of the DNA profile for exporting. Using a checklist can guide the analyst through proper sample evaluation. The documentation at a minimum can include the following criteria: • • • • • • •

Specific parameters used to develop a DNA profile Analyst and case information Evaluation of controls noting any problems Sample edits or failures Any necessary rework Case information for saved edited projects Disposition of the electronic data

Proper data review helps to ensure the quality of the DNA profile. Data should be reviewed a second time by a peer or by a supervisor. The review process needs to occur prior to releasing reported results or adding a DNA profile to an archive. A peer or supervisor typically conducts the review process. This process should be a complete review of the case file: • • • •

Sample disposition was properly documented. Analyst initials and signature are included where appropriate. Proper controls were used and specific guidelines were met. Positive controls were typed as expected, and negative controls showed no signs of contamination. • Size standards were assigned correctly.

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• • • • • •

Peak height and resolution of DNA peaks were acceptable. Artifacts were noted and properly addressed. Any low-level DNA profiles or partial profiles were properly assessed. The reviewer agrees with the analyst DNA profile results. DNA profile results were transcribed correctly onto worksheets. The proper report table format was used, including a list of all evaluated loci, as well as a list of the proper alleles for the given sample. • Electronic files are completed and in the proper location. The checklist below is an example of a review checklist to use when evaluating samples. DNA Run Review Checklist Vendor              Submission Packet          Run                Controls and Ladders     Run contains at least one acceptable ladder, positive control, and negative control      Plots checked for quality including heterozygosity, peak height, morphology, and electrophoretic events     In Lane Size Standards called correctly and acceptable     Ladder peaks all labeled and correctly called     No called peaks in reagent blanks and negative amplification controls      Positive controls labeled correctly; no extraneous peaks labeled     Plots printed and placed in Submission Packet file Samples     Number of samples in submission packet concordant with number of samples noted in the VDAS Data Package Summary. Comments section of Summary checked for vendor notes on any sample issues.      Plots checked for quality including heterozygosity, peak height, morphology, and electrophoretic events     In Lane Size Standards called correctly and acceptable     No evidence of contamination or other extraneous labeled peaks     Plots printed and placed in Submission Packet file: One set to remain in file and second set for vendor file (one sample per page)     Place the copy of the sample in the vendor file     A ll samples compliant with the vendor contract (refer to Contract Compliance Summary) Initials:     Date:           Second Analyst (Controls):     Date:          

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Once a full and acceptable DNA profile has been developed, sample comparisons can be made.

9.4 Case Evaluation, Kinship Screening, and Kinship Calculations Once DNA profiles have been found to be of sufficient quality and have been imported into the data analysis portion of the LIMS, individual cases can be evaluated. The profiles from the family or personal items of a RM without any “hits” or similarities to human remains are evaluated to determine if the stated relationships are correct and if additional family reference samples are needed to identify the missing person. Evaluation of cases with no hits is a critical task as this identifies problems, allowing them to be corrected quickly and concurrently with other analyses. While it is tempting to analyze only those cases with hits, evaluation of “hitless” cases is an essential component of quality control in the human identification effort. Cases with hits to human remains undergo a more thorough process whereby the alleged relationship between the human remains and the family reference samples is tested in a rigorous fashion. As an example, Figure 9.11 depicts the flow of data for the analysis that was conducted for the Hurricane Katrina identifications. The figure also gives an overview of the entire process for one approach to DNA identifications. While both the flowchart in Figure  9.11 and this chapter appear to be linear, it is important to recognize that many of these functions/topics occur simultaneously as the laboratory generates profiles for each RM case and for the human remains. See Chapter 10 for a discussion on how work lists can be helpful in managing data. Checklist documents should be incorporated into the DNA standard operation procedures to help ensure that any step requiring verification was completed. 9.4.1  Population Databases Imagine that we have a set of human remains with a DNA profile, and that early analysis suggests that this set of remains belongs to a specific family. Because parents pass their DNA on to their children, relatives of a missing person will share much of that missing person’s DNA. Purely by chance, unrelated persons may also have DNA in common with that family. Kinship analysis compares the relative likelihood that this set of human remains shares alleles with family members because they are biologically related, versus the likelihood this set of remains shares DNA with the purported family members purely by chance. This calculation requires information about how

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Figure 9.11  DNA data analysis chart for Hurricane Katrina identifications.

common individual alleles are in the general population. This then allows a determination of how likely it would be for a randomly selected, unrelated person to contain alleles common to a given family. The collection of data on how common alleles are in the general population is often organized into an Allele Frequency Database or Population Database. Population databases are necessary in order to obtain allele frequencies. Because different racial groups have varying frequencies of alleles, each racial group should have its own allele frequency database. Through the DNA profiling of a large number (>100) of unrelated people from the same racial group and counting the number of times each allele size is encountered within the tested group, a population database can be created. The actual frequency of each allele within an ethnic group is determined by dividing the total number of each allele size by the total number of all alleles identified within the group. For example, the frequency of an allele that is seen 20 times in a group of 500 alleles (250 people) is 20 ÷ 500, or 0.04. Population databases for many races are published in journals and are available for public use, but it is also acceptable to create, validate, and use a population database within your laboratory. This may be advisable when focusing on small or unique populations who may not be well represented by published databases. Most kinship analysis software will come with standard

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published databases already installed. Additional population databases can be added to the software as necessary. Choosing the proper population database is critical. Alleles that are rare in one population may be quite common in another. This can cause purely random allele sharing with a family to appear significant. As a result, some purely coincidental allele sharing between a set of human remains and a family can appear significant. When the mass fatality victims are from different ethnic populations, it is common to report the results using several different relevant population databases. 9.4.2  Sample Checks As a first step in case evaluation, samples need to be examined to ensure that the acceptable DNA profile is not a result of contamination from personnel or the result of a sample switch. 9.4.2.1  Elimination Sample Check Once a DNA profile has been approved and verified, the profile will be compared to an elimination database consisting of the DNA profiles of the laboratory staff and the relevant collection staff to ensure the source of the DNA profile is not a result of inadvertent contamination. Using data management software (see Chapter 10), this task can be automated for accuracy and efficiency. 9.4.2.2  Duplicate Sample Verification Sample switches can happen in any large-scale scientific testing effort. They occur with sufficient frequency that many drug-testing programs retain a duplicate sample for retesting, just to verify that a sample switch has not occurred. A similar approach is useful for large-scale human identification projects. Rather than profile each sample once, DNA samples are typed multiple times. In order to guard against sample switches and other potential problems with quality assurance, there must be verification that the DNA profile from the sample is the same each time it has been tested. Through the use of data management software (see Chapter 10), this task can be automated for accuracy and efficiency. Alternatively, a DNA profile can potentially be verified by comparing it to other known reference samples in order to establish the appropriate relationships within a family. 9.4.2.3 Amelogenin The gender marker Amelogenin should be used to verify that the gender profile generated from the sample is consistent with the gender of the individual providing the sample. If two samples are switched, often the amelogenin marker will not match the reported gender of the sample donor. This check

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should be performed both for family reference samples, and if possible, for human remains samples as well. 9.4.3  Reference Sample Validation After verifying the accuracy of the DNA profile, it is necessary to verify that provided reference samples will offer sufficient information to make a DNA identification. 9.4.3.1  Kinship Reference Samples Kinship reference samples are samples of DNA collected from multiple, close biological relatives and are used to construct or infer the victim’s profile. These samples are typically collected in the form of a buccal swab or a blood sample and can thus be standardized. Analysis of these samples is more complex because the evaluation of a match typically does not produce high statistics. Issues can arise with the use of kinship samples when assumed biological relationships are found to be inaccurate. When using kinship samples, it is important to generate a family history diagram or pedigree. This diagram depicts the reported missing and how they are related to other family members. This diagram also helps to determine the most useful kinship samples to collect. 9.4.3.2  Relationship Verification Using the kinship analysis software provided as part of the data management software, the analyst should verify that DNA profiles generated from reference samples are consistent with the alleged relationship to other family members. 9.4.3.3  Kinship Simulation Kinship simulation is necessary to ensure that DNA profiles generated by the reference samples will provide adequate genetic information to generate a match during the screening process. This can be accomplished with a computer simulating possible DNA profiles for the reported missing person and calculating the relationship indices (discussed in Section 9.4) for each simulated profile. Simulation was first introduced through DNA-View, a well-known kinship analysis tool. If a sufficient number of the simulated profiles produce a relationship index greater than the minimum required threshold for identification, it is reasonable to conclude that the reference samples provided by the family will identify the missing person if their remains are found. If the minimum average threshold by the laboratory is not met for a sufficient number of the simulated profiles, there are multiple avenues for further analysis. The first is to simulate additional family members who could help identify the missing person. It is important to only simulate family members who are alive, available, and useful in identifying the missing person.

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The antemortem case data should provide some information about other potential relatives. The second avenue is to request appropriate additional testing (Y-chromosome, mitochondrial, additional STR loci, etc.) that will add weight to the original STR analysis. 9.4.3.4  Direct Reference Samples Direct reference samples are reference samples collected with paperwork documenting their origin to a missing individual. These samples usually have been collected by a professional (e.g., medical doctor or nurse) in the course of diagnostic testing and have been retained by the collecting agency. While these samples have the potential to provide complete profiles, they may be time consuming to test. 9.4.3.5  Relationship Verification All direct reference samples should be compared to family member samples in order to ensure the expected relationship within the family unit. 9.4.4  Personal Item Samples 9.4.4.1  Profile Quality The DNA profile must be reviewed for completeness. For DNA profiles with allele dropout, preferential amplification, or when any other abnormality is suspected, one must either only include heterozygous loci or have the capability to search for a single allele with anything for the second allele (13,?). 9.4.4.2  Elimination Samples In addition to comparing the samples to the elimination database, personal items need to be compared to RM-specific elimination samples. As discussed in Chapter 7, testing elimination samples from persons who may have handled personal items is extremely important. The DNA profile from the personal item should be compared with the elimination sample profiles to ensure that the personal item’s DNA profile does not match the relevant elimination samples. This aids in identifying potential situations of contamination. 9.4.4.3 Mixtures Mixtures are routinely encountered on personal items. Samples that produce mixtures cannot be used unless an elimination sample allows subtraction of sufficient alleles and/or genotypes from the mixture to produce the DNA profile of the reported missing person.

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9.4.4.4  Relationship Verification As with direct references, the profile from a personal item should be compared, when possible, to the kinship profiles for the RM to ensure the expected relationship within the family unit. 9.4.5 Screening A screen of unknown profiles from human remains against known profiles from RMs should be completed each time new sample profiles are added to the data management software. Each comparison will involve the calculation of kinship indexes that evaluate the likelihood of a biological relationship between the remains sample and reference samples. Indices should be grouped by family in order to create a “match index.” This can cause families with large numbers of reference samples matching against the human remains to generate a higher match index than families with a few samples or with only one relative possibly related to the human remains. Personal items and direct references will also produce large match indices. Each RM with a match index higher than the predetermined limit set by the laboratory should be individually investigated. “False” or coincidental hits occurring by chance increase with the number of comparisons performed, therefore all samples should only be initially searched against the relevant incident or site of remains recovery. Despite the increased possibility of coincidental hits, the entire database should be periodically searched to identify all possible hits. Once a direct reference or personal item profile has been successfully verified against kinship profiles, it will be used as the primary means of identification. A direct reference will typically provide a higher statistical value than testing any family members. 9.4.6  Direct Matches Direct matches are the most informative and are generated from personal items and direct reference samples provided from the reported missing. A sample provided by the RM can be matched at both alleles, which provides powerful statistical information and reduces the complications that can be observed in kinship testing (e.g., mutations, non-relatedness). In some cases, identity is considered certain after two samples match at a predetermined number of loci. 9.4.7 Calculations When a match has been identified, an estimate of the probability of the match having occurred by coincidence can be determined by the analyst. The

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Hardy-Weinberg principle should be applied to determine the match probability for direct matches. For heterozygous loci, the frequency of the match in the population is determined using the formula 2 × p × q = 2pq where p and q are the frequency of each allele in the population. Frequency of PQ = 2 × p × q = 2pq



For homozygous loci, the frequency of the match in the population is determined using the formula p × p = p2, where p is the frequency of the allele in the population. A correction factor (CF) is often included in homozygous calculations to correct for the possibility of subpopulations. Theta, a value chosen to represent the true non-randomness in the population, is often assumed to be 0.01.

Correction Factor = (p × (1 – p) × 0.01)



Frequency of PP = (p × p) + correction factor = p2 + p × (1 – p) × 0.01

The combined frequency of matching at multiple loci within a racial group is determined by the mathematical product of the frequency of each locus relevant to the match. Dividing 1 by the combined frequency of all relevant loci will determine the likelihood of a match. Combined Frequency = Freq(FGA) × Freq(TPOX) × Freq(vWA) × …...

Likelihood = 1/Combined Frequency

See Table 9.2 for an example.

Combined Frequency = 0.065 × 0.299 × 0.137 × 0.051 = 0.00014



Likelihood = 1/0.00014 = 7143

Table 9.2  Locus Frequency Calculations Locus FGA TPOX D8S1179

Alleles

Allele 1 Frequency

Allele 2 Frequency

21, 22 8 13, 14

0.173 0.544 0.339

0.189 0.202

Formula 2 pq p2 + CF 2 pq

Locus Frequency 0.065 0.299 0.137

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Approximately 1 in 7,143 individuals of the same race would be expected to match this profile. 9.4.8  Incomplete Loci Samples from human remains or personal items may produce incomplete profiles. All loci must be evaluated for consistency between the profiles even if the locus is of insufficient quality to be included in the statistical calculation or reported. This means that a 13,? could match a 13,17, but a 13,? could not match a 14,15.

9.5  Kinship Analysis 9.5.1  Likelihood Ratio Likelihood ratios are typically used to statistically define the significance of matching DNA loci in relationship testing. A likelihood ratio is a number representing how many times more likely one scenario is versus a different scenario. For example, Jim is 15 times more likely to be Ann’s uncle than an unrelated individual. Kinship indexes are a form of statistical calculation known as a likelihood ratio. For the purposes of this publication, the terms “kinship index” and “likelihood ratio” are often used interchangeably. A likelihood ratio is a comparison of how likely a result would be given two different, mutually exclusive hypotheses (hypothesis 1 and hypothesis 2). Formal evaluation of the apparent relationship requires calculation of kinship indexes comparing the likelihood that the human remains is the reported missing person versus the likelihood that the human remains is instead from an unrelated person. For example, a likelihood ratio in a paternity case compares how likely it would be for a child to receive paternal alleles assuming that a known person is the biological father of the child (hypothesis 1) versus the child receiving the paternal alleles assuming that the true biological father is an unknown man (hypothesis 2). The likelihood for hypothesis 1 depends on the alleles of the mother, child, and alleged father only. The likelihood for hypothesis 2 depends on the alleles of the mother, child, and how common the paternal alleles are in the relevant population (see Section 9.4.1). Figure  9.12 illustrates a likelihood ratio calculation for an individual locus. This calculation is for a mother AB, a child BC, and an alleged father CD, where the lowercase letters represent allele frequency, and 0.5 is the transmission chance for the required allele. All formulas for likelihood ratios can be calculated manually, although this is often inconvenient, particularly for less direct relationships such as siblings, half-siblings, and distant relatives (i.e. grandchildren, cousins).

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Numerator–Expected Father 2ab

AB 0.5

CD BC

Denominator–Unrelated Man

2cd

2ab

0.5

AB 0.5

Probability = 2ab × 2cd × 0.5 × 0.5

CD BC

2cd c

Probability = 2ab × 2cd × 0.5 × c

Paternity Index Index =

Index =

2ab × 2cd × 0.5 × 0.5 2ab × 2cd × 0.5 × c 0.5 c

Figure 9.12 (see color insert)  Likelihood ratio calculation for an individual locus.

Ideally, kinship software (in the laboratory information management system, see Chapter 10 for additional information) will calculate the required formula for any scenario. A selection of simple kinship formulas can be found in Tables 9.3 and 9.4. AABB relationship guidance documents or other standard publications also contain standard formulas for most common kinship indexes. Since the final evaluation of kinship will be evaluated at many loci, often as many as 15 (or possibly more), the combined likelihood ratio is the mathematical product of the likelihood ratios of each individual locus reported:

Combined Index = Index(FGA) × Index(TPOX) × Index(vWA) × …...

9.5.2  Prior Odds DNA-based human identifications make use of a statistical concept called Bayes’ Theorem to combine information from the DNA analysis with nonDNA data. This non-DNA data is often based solely on the number of persons missing from a given mass fatality or other event. The non-DNA data is quantified into the prior odds, while the DNA data is quantified into the kinship index.

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Table 9.3  Missing Child Where Both Parents Provided a Sample RM #49875 Mother A A A AB AB BC BC BD A AB B BC AB AB AB

Possible Child A AB AB A A AB AB AB A A AB AB AB AB AB

RM#49875 Father AB AB BC AB AC AB AC AC A A A A AC A AB

Formula 1/2a2 1/4ab 1/4ab 1/4a2 1/4a2 1/8ab 1/8ab 1/8ab 1/a2 1/2a2 1/2ab 1/4ab 1/8ab 1/4ab 1/4ab

Table 9.4  Missing Father Where the Child and the Child’s Mother Provided Samples RM # 60987 Wife A A A AB AB BC BC BD A AB B BC AB AB AB

RM# 60987 Child A AB AB A A AB AB AB A A AB AB AB AB AB AB AB AB A A

Possible Father AB AB BC AB AC AB AC AC A A A A AC A AB AC AB A AC A

Formula 1/2a 1/2b 1/2b 1/2a 1/2a 1/2a 1/2a 1/2a 1/a 1/a 1/a 1/a 1/2(a + b) 1/(a + b) 1/(a + b) 1/4a (a + b)/4ab 1/2a 1/2a 1/a

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Kinship evaluation in mass victim identification projects utilizes nonDNA information. At a minimum, the number of persons missing from a disaster sets the context for the kinship evaluation. Because the number of persons missing is a numerical quantity, this allows a formal expression of this non-DNA information. For example, a flood with 1,000 persons reported missing means that before any scientific analysis is conducted, the identification effort has an approximately 1-in-1,000 chance of correctly identifying a given set of human remains. Prior odds may be based on the number of missing, gender, or other available information and should be a predetermined value for each incident. The DNA information in a DNA-based human identification is quantified in a calculation called the kinship index. The non-DNA information is quantified in a DNA-based human identification in a calculation called the prior odds. The mathematics for the combination of the kinship index and the prior odds is:

Posterior Odds = Likelihood Ratio × Prior Odds

Posterior odds is merely the kinship index multiplied by the prior odds. The posterior odds provide a numerical weight to the opinion of identification. A posterior odds of one means that considering all of the available information, it is equally likely that the identification is correct or wrong. Posterior odds of less than one means it is more likely that the identification is incorrect. Posterior odds of greater than one means it is more likely that the identification is correct. 9.5.3 Mutations A mutation occurs when there is a change in DNA from one generation to the next at a particular locus. A mutation can result in a false exclusion. It is important to recognize the possibility of mutation when a difference occurs at only one or two loci and is accompanied by a large residual index. The residual index is a combined index of all matching loci for a case. When the residual index is large, it is likely that you are viewing either a true mutation or a relative of the RM that you are investigating. Explore the possibility that the inconsistency may be a result of inaccuracies in the reference sample relationships or possible DNA profiling error. Table 9.5 shows an example of a single inconsistency identified at D13S317. A statistic must be included in the combined index calculation for loci that exhibit inconsistencies. There are several different methods that may be used in order to try to estimate the likelihood of mutation. Fimmers method identifies the likelihood of the individual mutational event by using mutation rates for the specific DNA mutation (example: FGA 19 ≥

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Table 9.5  Example of a Single Mutation

Locus D8S1179 D21S11 D7S820 CSF1P0 D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51 Amelogenin D5S818 FGA

Item 1 Unidentified Remains

Item 2 Reference Sample Mother

Item 3 Reference Sample Father

11,12 30,30 7,10 11,13 17,18 6,6 11,14 13,16 22,24 11,14 18,19 9,9 16,17 X,Y 10,13 21, 22

11,12 30,30 9,10 11,13 17,18 6,6 11,12 13,16 21,24 11,14 18,19 9,9 17,18 X,X 10,13 21,21

10,11 30,31 7,8 11,12 15,18 6,9.3 13,15 12,16 22,23 12,14 19,20 9,9.3 16,18 X,Y 11,13 22,23

20). Brenner’s method accounts for the change in the number of repeats that the apparent mutation exhibits where smaller changes are more probable (example: FGA 19 ≥ 20 is more probable than 18 ≥ 20). Some laboratories estimate the mutation likelihood by using the mutation rate divided by the average power of exclusion for the locus or simply stating the mutation rate. Regardless of the method chosen to estimate the likelihood of a mutational event, the mutation must be statistically taken into account in the combined likelihood index. See Table 9.6 for the AABB’s 2003 summary of mutation rates. 9.5.4  Unexpected Non-Relatedness During the family verification or the identification process, it could become apparent that one or more individuals in a family are not related in the manner that the provided pedigree states. When apparent non-relatedness is encountered, all of the provided relationships must be verified. In some cases, it is possible to eliminate samples that do not directly affect the identification or may hinder the identification if their reported relationship can be definitively determined to be inaccurate. Additional family reference samples may be required to try to identify the source of the inconsistency.

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Chromosomal Location

TPOX D2S1338 D3S1358 FGA D5S818 CSF1PO D7S820 D8S1179 TH01 vWA D13S317 D16S539 Penta E D18S51 D19S433 D21S11 Penta D

2p25.3 2q35 3p21.31 4q28 5q23.2 5q33.1 7q21.11 8q24.13 11p15.5 12p13.31 13q31.1 16q24.1 15q26.2 18q21.33 19q12 21q21.1 21q22.3

Mutation Rate 0.01% 0.12% 0.12% 0.28% 0.11% 0.16% 0.10% 0.14% 0.01% 0.17% 0.14% 0.11% 0.16% 0.22% 0.11% 0.19% 0.14%

9.6  Reporting Matches Once a match has been identified and all of the relevant data, statistical calculations, and information about the case have been reviewed, a DNA identification report provides the medical examiner with the DNA information regarding specific human remains. After careful review by multiple DNA analysts, a DNA identification report will be generated for any sample that meets a predetermined posterior odds threshold for kinship and any direct sample matches. 9.6.1  Special Situations A match could be identified through screening in which there may not be enough available family members to meet the predetermined index threshold set by the laboratory. It is also possible that the medical examiner could request specific human remains be compared with a certain family that could result in less than stellar statistics. In these situations, it may be necessary to provide the medical examiner with whatever DNA information is available, as well as help the medical examiner understand its value. The medical examiner must also be informed if unexpected non-relatedness was discovered during the testing process. All information assumed by the DNA analyst should be shared in the identification report.

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Additional Resources Bieber, F.R., Brenner, C.H., Lazer, D. 2006. Finding criminals through DNA of their relatives. Science 312:1315–1316. Brenner, C. Mutations in Paternity DNAVIEW website. http://dna-view.com/mudisc. htm (accessed October 6, 2013). Butler, J.M. 2006. Genetic and Genomics of Core STR Loci Used in Human Identity Testing. Journal of Forensic Sciences 51(2): 253–265. http://www.cstl.nist.gov/ div831/strbase/pub_pres/Butler_coreSTRloci_JFS_Mar2006.pdf. Committee on DNA Forensic Science, NRC. 1996. The Evaluation of Forensic DNA Evidence. Atlanta, GA: National Academies Press. Fimmers, R., Henke, L., Henke, J., Baur, M. How to deal with mutations in DNA testing. In: Rittner, C., Schneider, P.M., eds. Advances in Forensic Haemogenetics 4. Berlin: Springer Verlag, 1992:285–7. Life Technologies Corporation. 2012. AmpFℓSTR® Identifiler® PCR Amplification Kit. Applied Biosystems. http://www3.appliedbiosystems.com/cms/groups/ applied_markets_support/documents/generaldocuments/cms_041201.pdf. Life Technologies Corporation. 2010. GeneMapper® ID-X Software Version 1.2. Applied Biosystems. http://www3.appliedbiosystems.com/cms/groups/ applied_markets_support/documents/generaldocuments/cms_090043.pdf. National Center for Biotechnology Information. Open Source Independent Review and Interpretation System User Guide version 2.04. http://www.ncbi.nlm.nih. gov/projects/SNP/osiris/OsirisUserGuide-2.04.pdf. National Institute of Standards and Technology, STRBase. http://www.cstl.nist.gov/ div831/strbase/index.htm Promega Corporation. 2012. PowerPlex® 16 System. http://www.promega.com/~/ media/files/resources/protocols/technical%20manuals/101/powerplex%20 16%20system%20protocol.pdf?la=en. Sozer, A., Baird, M., Beckwith, M., et al. 2010. Guidelines for Mass Fatalities DNA Identification Operations. Bethesda, MD: AABB.

Chapter DNA Sample, Case, and Data Tracking Using Information Technology Tools

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A Laboratory Information Management System (LIMS) manages the data associated with the collection, testing, and reporting of samples. A LIMS is critical to any medium to large DNA identification operation. This chapter reviews the important features of a LIMS used in a mass fatality DNA identification effort. It also focuses on reported missing (RM) case management, sample collection scheduling, sample collections and tracking, data analysis, and reporting.

10.1  Laboratory Information Management System (LIMS) The LIMS is the core component of a quality DNA identification effort. The amount of data generated during the DNA operation is overwhelming and cannot be managed accurately on paper. The LIMS reduces opportunities for human error and supports the managerial entity of the response in organizing, planning, and executing laboratory operations. It enables individuals supporting the DNA identification effort to capture information electronically throughout the testing process. Specifically, the LIMS supports the DNA operations by: • • • • • •

Providing appropriate security to the case records and data Minimizing manual/paper record keeping and data entry Performing automatic error checking Incorporating quality control processes Generating work lists Acting as a single repository for all DNA information and data with immediate yet controlled access • Storing DNA profiles for easy access and retrieval • Performing DNA searching and statistical analysis computations

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A properly designed and implemented LIMS will support efficient and effective operations by tracking samples and profiles. This will allow users, with appropriate security, to easily access the data and visualize the data in a meaningful way. Data collection begins with the assignment of the RM and the collection of samples from reference samples and human remains, and continues throughout the testing process. The LIMS captures and validates resulting DNA profiles and also screens appropriate data from unknown samples against samples of known origin. The LIMS further supports the DNA analyst by performing complex comparisons and analyses, and it provides an electronic mechanism for reporting the data.

10.2  Assigning a Reported Missing Case One of the first steps in the DNA identification effort is the assignment of a reported missing (RM) case. This assignment involves inputting information about the RM and the associated references. Typically, the RM case is central to the entire identification effort, and it is assigned by the agency managing the overall identification effort. The DNA unit should get the RM case information directly from the identification software used by the Family Assistance Center Operations and morgue. The DNA unit will talk with the family to identify the reference samples available for testing and will document these reference samples in the LIMS. Data stored in LIMS supports construction of a family pedigree. Figure  10.1 portrays a family pedigree stored in the LIMS LISA (Laboratory Information System Application), produced by Future Technology Incorporated (FTI). The LIMS keeps track of which reference samples are needed and which have been collected, as well as any relevant associated testing information. Most importantly, the LIMS will be able to track related RMs, thus ensuring that an identified RM can be used to identify a related RM.

10.3 Collection of Samples from Unidentified Human Remains Typically the collection of samples from human remains takes place in the morgue. Ideally the DNA laboratory accessioning system should be used at the time the sample is collected. For example, LISA has a mobile unit that can be taken into the morgue and samples can be recorded electronically at the time of sample collection. Recorded information typically includes sample type, morgue numbers, name of sample collector, date, time of collection,

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Figure 10.1 (see color insert)  A family pedigree stored in the LIMS LISA. (Courtesy of Joel Galloway.)

and location where remains were found. Files, such as pictures taken during the specimen collection process, may also be associated with the sample file in the LIMS. Figure 10.2 shows a screenshot of LISA Mobile where sample information can be collected. When the data from the unidentified human remain is entered into the computer, unique bar-coded labels are printed and placed on the chain-of-custody paperwork in the morgue case file, as well as on the sample tube. When samples are taken to the laboratory, the electronic collection file is transferred to the LIMS, thereby eliminating the need to enter data by hand. The laboratory technician will simply verify that all of the samples are present and accounted for.

10.4  Collection Reference Samples A laptop extension to the LIMS may be used to collect samples from family members. For example LISA has a subset of the program called LISA FRC (Family Reference Collection) that can be used to collect, document, and catalog reference samples. The use of LIMS for reference sample collection is typically only cost-effective when a large number of samples are collected at one location. The use of the LIMS eliminates having to complete long antemortem forms by hand and eliminates handwriting interpretation

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Figure 10.2 (see color insert)  Screenshot of LISA Mobile. (Courtesy of Joel Galloway.)

“challenges.” The LIMS automatically error checks reference sample collection documents to identify errors in obtaining the collection information. For example the age of a father has to be at least 12 years older than the son or a message will be given to the user to double check the birthdates. Often the inadvertent switching of relatives is a common mistake in sample collection. In addition, the LIMS prints out a bar code so that the sample and associated chain-of-custody paperwork can be uniquely identified to minimize switching errors. A fully automated system uses a tablet computer to ensure that the family references and donors sign their consent electronically. During the reference sample collection process, the collector must correctly identify the RM. Figure 10.3 shows a screenshot of the form used for collecting information about the DNA kinship donor for the RM. In addition, information about the sample donor is collected. The LIMS typically uses several formats to ask the relationship of the donor to the missing person. This ensures that the donor’s reason for presenting a sample is clearly defined (e.g., kinship reference, elimination sample, etc.). As with postmortem samples, the electronic collection file is transferred to the LIMS when the reference samples are taken to the laboratory, thereby eliminating the need to enter the data associated with each of the samples collected by hand.

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Figure 10.3 (see color insert)  Screenshot of the DNA kinship donor form. (Courtesy of Joel Galloway.)

10.5 Tracking the Sample during Testing and Data Analysis When a sample arrives at the DNA operation, it is immediately accessioned into the LIMS. Accessioning involves critically examining the outer packaging of the collected sample as well as the associated paperwork and recording the pertinent information into the LIMS. The steps involved in receiving and documenting a sample into the laboratory are depicted in Figure 10.4. This Sample Receipt & Accessioning

Sample arrives

Outer package and associated paperwork examined Unique identifier– not RM number but links to RM case

Figure 10.4  Sample receipt and accessioning.

Accessioned into LIMS

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Sample removed and placed in appropriate container for testing

Process documented in LIMS and a sample number is assigned

Lables printed out and placed on sample aliquots

Figure 10.5  Aliquotting a sample.

is for the purpose of tracking and sample management, labeling the sample, and maintaining associated paperwork for chain of custody. The laboratory should have a sample acceptance policy and procedures to follow if samples are received without the proper paperwork or documentation. The LIMS will give each sample a unique identifier (which should not contain the RM number) that will link the sample to the RM case or RM cases electronically. If the RM number is included as part of the reference sample number, it will cause confusion if the sample is used for multiple cases. A RM will typically contain multiple reference samples. Unidentified human remains will also be logged into the LIMS. During the aliquotting, or partitioning, process, shown in Figure 10.5, a portion of the sample is removed and placed into the appropriate container for testing. This process is documented in the LIMS, and a daughter sample number is assigned to the sample. For example, if two aliquots are made from sample 2008LC2098 so that the sample can be sent to two different laboratories for testing, the sample aliquots may be recorded in the LIMS as 2008LC2098.1 and 2008LC2098.2. The LIMS will print out the sample labels that can then be placed on the sample aliquots. If an outside laboratory will test the samples, the DNA analyst will prepare the samples for shipping and the LIMS will track which samples were sent to which laboratory and will also record the overnight shipping company and the carrier’s air bill number. The analyst can set an expected completion date for the samples to be profiled by the testing laboratory, and the LIMS can notify the DNA operation when DNA profiles are overdue. If the DNA operations will test the samples in their own laboratory, the LIMS will be used to track each sample transfer and will interface with any large pieces of equipment that require the analyst to enter a sample number (e.g., genetic analyzers). Ideally the DNA analyst only needs to record the sample number one time into the LIMS. Throughout the testing process the LIMS will generate all paperwork and tube labels needed during the testing and analysis process.

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Nuclear DNA Testing and Analysis Data Flow Through LISA Specimen Availability from Casework

Organic Extraction with Optional Grinding

Import Duplex or Triplex Quantitation (Creates Lab Sheet)

No

Quantitation Availability in LISA? Yes AmpFISTER Profiler and/or Cofiler Amplification Import Quantitation Results into Amp. Sheet

Load File into Preconsensus

Analyst Performs, Data Reviews Edits and Signs Off on Preconsensus (Automatically Generating Consensus List) Technical Reviewer Performs Review, Edits and Signs Off on Consensus (Profiles Agreed Upon between P/C Are Pushed To Searchable State)

All Profiles Searchable?

Yes

Ready for Final Reporting/Kinship Analysis

No ABI 3100 Genetic Analyzer Setup Yes Reinjection/ Rerun?

Analyst/Reviewer Must Both Signoff Consensus for Disagreed Upon Profiles Between P/C (Remaining Consensus Profiles Pushed to Searchable State)

No Create Export File from ABI 3100 Sheet Run ABI Instrumentation Process to Generate Preconsensus Load File

Figure 10.6  General outline for processing samples.

The proper use of a LIMS greatly reduces the possibility of a sample switch or the mislabeling of a sample. Figure 10.6 is an example flowchart for nuclear DNA STR testing and profile analysis. The LIMS can electronically maintain laboratory standard operating procedures, maintain lot numbers of reagents, and electronically generate laboratory worksheets. Figure 10.7 depicts the layout of how the LIMS documents the sample information from the various testing processes in the DNA laboratory.

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Figure 10.7 (see color insert)  Documenting a sample in LIMS. (Courtesy of Joel Galloway.)

When utilizing data analysis software, the DNA analysts typically read the electropherograms generated by the genetic analyzers two times independently (see Chapter 9). The LIMS electronically captures the allele calls from the two independent data reads and incorporates the profile values into the LIMS databases. The allele calls from the two reads are electronically compared by the LIMS, and the DNA laboratory is alerted when the allele calls are discrepant. The laboratory then documents in the LIMS the appropriate corrective action taken to resolve the discrepancy. Figure 10.8 depicts a profile comparison performed by the LIMS. Likewise, the duplicate DNA profiles returned from the outside laboratory vendors are compared with the LIMS, and the DNA operation is alerted to discrepant results. The LIMS will also compare all profiles against the profiles in an elimination database (see Chapters 7 and 9) to look for known potential DNA contaminations.

10.6  DNA Profile Interpretation and Management The DNA operation will use the LIMS to perform the many complex functions of data interpretation (see Chapter 9). The LIMS manages the screening of profiles, contains the allele frequency databases, and applies the mathematical calculations for determining the relevance of matches and shared characteristics. It is virtually impossible to accurately and completely screen profiles by hand. The LIMS allows the DNA analyst to

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Figure 10.8 (see color insert) DNA profile comparison. (Courtesy of Joel Galloway.)

perform complex screening tasks to re-associate human remains and to investigate if any of the victims are related. A well-constructed LIMS will allow the DNA analyst to perform multiple operations without having to re-enter data or change the set parameters of a RM case. For example, the DNA analyst should be able to modify possible genetic relationships in the pedigree or screen potential victims of one mass grave against the human remains of another mass grave just to be complete in the analyses and interpretations performed. Figure  10.9 provides a screenshot of the data displayed in LIMS for kinship analysis.

10.7  Report Writing The report is the final work product of the laboratory. It is the documentation that defines the laboratory process. Reports containing even slight typographical errors can cause the testing to appear to be flawed, which may cause the community to lose confidence in laboratory operations. It is imperative that the final work product of the laboratory is without error. One way to ensure the accuracy of a final report is to generate the report in LIMS. The LIMS will contain report-writing templates and eliminate the need to hand type information. Once information in LIMS is verified, the laboratory can quickly and easily issue a report with a few keystrokes. In addition, the LIMS keeps a record of each time a report is generated and issued. See Attachment A for an example report created in LIMS, courtesy of Joel Galloway and FTI.

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Figure 10.9 (see color insert)  Kinship analysis. (Courtesy of Joel Galloway.)

10.8  Communication Logs Often there will be communications and decisions made about the testing of a sample and/or RM case. The LIMS is a repository for case- and samplespecific information to ensure that individuals participating in the testing process can access relevant information. This communication is essential to the swift processing of cases. For example, if there was some indication that the person submitting a kinship sample for a RM case might not be related to the RM, the laboratory would document this information in the LIMS case notes file. Upon processing the references for the RM case, the laboratory may find that not all of the relatives are related as originally claimed. If the notes regarding the questionable relationship are in LIMS, the sample profiles can be easily resolved, and the case can proceed through the data interpretation process without delay.

10.9 Security A well-designed LIMS will have different security access levels. This prevents someone from inadvertently changing data in the LIMS. Typically, each individual with the ability to access the LIMS has their own username

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and password and is allowed to perform the tasks that directly relate to their position in the identification effort. For example, individuals who examine the samples when they are received by the DNA operation and enter the associated metadata do not have access to the profile interpretation screens. Likewise, analysts who read DNA profiles may have access to view the samples’ metadata but do not have access to change the information. Security levels are established by management and typically maintained by the LIMS administrator.

10.10  Quality Control The LIMS can be used as a quality tool by the DNA operations. Lot numbers of reagents can be maintained by the LIMS. Lot numbers help to ensure that only critical reagents (e.g., reagents successfully passing quality control testing) are used in the testing process (see Chapter 11). The LIMS will only allow a DNA analyst to proceed with setting up laboratory reactions using reagents that have been properly recorded in the LIMS. For example, the LIMS stores the expiration dates for lots of reagents. If a reagent lot number is expired, the analyst will be alerted by the LIMS not to use the expired chemical.

10.11  Work Lists Besides managing data, one of the essential aspects of a LIMS is to alert the end user of what work needs to be performed in the DNA identification effort. Samples are continually being processed and data generated on a daily basis. Without the help of the work lists generated by the LIMS, it is virtually impossible for the DNA analysts to stay on top of what needs to be accomplished each day. By using the work lists generated in LIMS, the DNA operations will minimize duplication of efforts, identify appropriate resources to target choke points, and manage production by accessing progress. Table  10.1 outlines some of the DNA identification tasks that should be dictated by the LIMS.

10.12  Maintaining Fiscal Responsibility One of the obligations of the DNA identification effort is to maintain efficient and effective operations. This includes the proper use of funds, which can be complex if subcontractors and vendors are used in supporting the operations. A large DNA identification operation may require the evaluation and approval of hundreds of invoices. The LIMS can be used to track payment of goods and services and minimize inadvertent and duplicate payments to

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Table 10.1  DNA Identification Tasks Operational Area Sample collection

Sample testing

Profile interpretation

Reporting

Work List Verification that RM case is unique and truly missing Cases needing pedigrees References to be scheduled Kits to be sent to collection locations Samples to be collected Samples to be received by the DNA operations Samples to be accessioned Samples to be aliquotted Samples to be sent to vendors for testing Samples to be tested in house: • Extraction • Quantification • Amplification • Separation • 1st read • 2nd read Profiles to be received from the vendor Profile comparisons to be run RM cases to be validated Potential hits to be resolved Potential identifications to be verified Cases needing additional testing Cases to be reported Cases requiring technical review Cases requiring administrative review

a vendor. For example, sample collection agencies may be used to assist the DNA operations in scheduling and collecting the reference samples located outside the immediate vicinity of the Family Assistance Center. The DNA operations may negotiate a rate for each sample collection based on collection location. The vendor should be paid only if the sample was collected properly (chain of custody maintained) and the sample was received by the DNA operations. When the vendor submits an invoice for a sample collection, the laboratory administrative staff can verify in LIMS that the sample was collected and received properly by the laboratory. Administrative staff will often document that an invoice for the collection service was received and approved along with the date of approval. If a subsequent invoice is received by the DNA operation with a duplicate charge for the collection, the DNA operation can alert their accounts payable that a previous invoice contained the same charge.

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10.13  Acquiring the LIMS The DNA laboratory may purchase a LIMS commercially or use one constructed in-house. LIMS software is often expensive and complex. After a mass fatality, organizations may offer to donate their software to the DNA laboratory. While this may initially appear to be a good acquisition, the laboratory must be prepared to incur later fees associated with the maintenance and customization of the LIMS, which can sometimes be significant. A welldesigned LIMS will support the many aspects of the DNA operations and provide flexibility for customization to the individual identification operations. If the laboratory does not already have a LIMS, the LIMS should be one of the first purchases when setting up DNA operations, preferably even before sample collections begin.

Additional Resources Joyce, J.R. 2011. What Constitutes a Good Forensics LIMS? Scientific Computing. http://www.scientificcomputing.com/articles-IN-What-Constitutes-a-GoodForensics-LIMS-100411.aspx (accessed September 29, 2013). Olsen, A.N., Christiansen, L.C., Nielsen, S.J., et al. 2009. Customizing a commercial laboratory information management system for a forensic genetic laboratory. Forensic Science International: Genetics Supplement Series 2(1): 77–79. Parson, W., and Steinlechner, M. 2001. Efficient DNA database laboratory strategy for high through-put STR typing of reference samples. Forensic Science International 122(1): 1–6. Singer, D.C. 2001. A Laboratory Quality Handbook of Best Practices and Relevant Regulations. Milwaukee, WI: ASQ Quality Press.

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Chapter Implementing and Maintaining a Quality DNA Program

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Developing a quality program is an integral part of a DNA laboratory operation. A quality program illustrates a laboratory’s commitment to developing quality products, maintaining the integrity of the samples processed, and ensuring credibility of its scientific contributions and achievements in the global forensic community. Quality assurance and quality control standards guide the laboratory quality program. Quality control is the testing a laboratory performs to ensure procedures and methods are monitored, and to verify that products meet specified standards that provide confidence in results. Quality assurance is the process a laboratory employs to assess the quality of products or services by review of work, problem identification, corrective action to remedy deviations, and evaluation of remediation. A quality program gives a laboratory a framework for continuous improvement of its system, services, and testing. This chapter describes accreditation and standards as well as implementation of the major elements of a DNA quality program.

11.1 Accreditation and Its Role in International Recognition Accreditation is based on standards and guidelines established by collaborating agencies and is the blueprint for enabling laboratories to develop and implement quality programs. Accreditation is a process in which an unbiased third-party organization verifies that a laboratory conforms to specific standards, is competent, and can achieve a level of quality. It is a voluntary program for laboratories that want to achieve high standards for their services. Accreditation also helps a laboratory examine its quality program and assess the overall structure and function of its laboratory operations. It provides recognition, credibility, and confidence. Accrediting bodies are agencies that may conduct laboratory evaluations and perform accreditation. Accreditation Cooperation Bodies are groups of 195

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ILAC Regional Accreditation Co-operations

IAAC

EA

APLAC

European Accreditation Bodies

Asia Pacific Accreditation Bodies

Accrediting Bodies North/South American Accreditation Bodies

Figure 11.1  Accreditation cooperation bodies.

accrediting bodies that have signed mutual recognition arrangements (MRAs) signifying that their practice of accreditation is essentially uniform. The International Laboratory Accreditation Cooperation (ILAC) is the organization that represents the top level of MRAs, recognizing both regional accreditation cooperation bodies and individual accrediting bodies. Regional accreditation cooperation bodies such as the European Cooperation for Accreditation (EA), the Asia Pacific Laboratory Accreditation Cooperation (APLAC), and the Inter-American Accreditation Cooperation (IAAC) are the current ILACrecognized regional bodies with acceptable MRAs. These bodies, outlined in Figure 11.1, have MRAs with both ILAC and the accrediting bodies in their regions. ILAC and the Regional Accreditation Cooperations (EA, APLAC, IAAC) evaluate the quality of accreditation provided by ISO (International Standards Organization) 17025 and ISO 15189 accrediting bodies. 11.1.1 Standards There are a number of different organizations with established standards for DNA testing laboratories. Conformance with these standards demonstrates a laboratory’s competence and its ability to minimize variability. Each set of standards addresses management and technical aspects of a lab system that can impact the quality of the testing and the service provided. It is important

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to realize that standards are minimum requirements that laboratories can and frequently do exceed in their quality programs. The ISO partnered with the International Electrotechnical Commission (IEC) to establish the globally recognized ISO/IEC 17025:2005 Standards for accreditation of all types of testing and calibration laboratories. ISO/ IEC 17025:2005, together with the ILAC G19:2002 Guidelines for Forensic Science Laboratories addressing specifics particular to forensic laboratory testing, are the internationally recognized standard for forensic DNA testing laboratories wanting to achieve accreditation. In the United States, the Federal Bureau of Investigation (FBI) has established Quality Assurance Standards (QAS), developed by an independent group of scientists appointed by the FBI. These standards must be met by forensic DNA testing laboratories and convicted offender DNA laboratories participating in or providing data to the U.S. National DNA Index System (NDIS). The QAS’ goal is to ensure the quality of data entered into the national DNA database, enabling laboratories in the United States to share information. The AABB (formerly the American Association of Blood Banks) Relationship Standards Program Committee is composed of experts in the field of relationship testing. This committee developed the Standards for Relationship Testing Laboratories. These standards address issues and requirements specific to relationship testing laboratories conducting familial comparisons, as well as management, and technical issues common to other DNA laboratories. The ultimate goal of conformance to standards is to ensure that laboratories demonstrate competency, provide reliable results, handle samples with integrity, and ensure the validity of the laboratory equipment and testing methods. 11.1.2  Accreditation Bodies There are approximately 35 agencies that conduct quality assessments to ensure that a laboratory is in conformance with ISO standards. Often these can supply guidance documents and provide accreditation for international forensic DNA testing laboratories. Both agencies can provide additional support, including an external auditing service. The AABB provides an international accreditation program for relationship DNA testing laboratories. 11.1.3  Other Resources for Guidance INTERPOL has an international law enforcement DNA database and provides regional support, training, workshops, and information to member states. The European Network of Forensic Science Institutes (ENFSI) DNA Working Group, whose aims and objectives are to bring together

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organizations, actively pursues forensic DNA analysis methods for the purpose of exchanging and disseminating information on forensic applications. After 9/11, based on a request from the agencies involved in DNA identification efforts, the U.S. government convened a group of subject matter experts. This group published Lessons Learned from 9/11: DNA Identification in Mass Fatality Incidents, a document about large-scale DNA identification efforts (massfatality.dna.gov). After the Asian tsunami, the International Society for Forensic Genetics similarly published recommendations for DNA identification laboratories in Forensic Science International: Genetics.* See Attachment A. The International Committee of the Red Cross published Missing People, DNA Analysis and Identification of Human Remains, an overview of forensic human identification and the use of DNA analysis in both small and largescale identification programs (http://www.icrc.org/eng/resources/documents/publication/p4010.htm). At the request of the AABB, the organization that accredits DNA relationship testing laboratories worldwide, a group of mass fatality DNA identification subject-matter experts convened to create the Guidelines for Mass Fatality DNA Identification Operations. The American Association for the Advancement of Science (AAAS), an international non-profit organization whose goal is to promote cooperation among scientists and encourage scientific responsibility for the betterment of humanity, is developing guidelines for scientists working on human rights initiatives. These guidelines are designed to facilitate and promote cooperation between scientists and human rights organizations. 11.1.4  Quality System Elements 11.1.4.1  Organization and Management The basis for a quality program is the Quality Manual. The Quality Manual is composed of the documented policies, processes, and procedures that describe, define, and control the operation of the organization. The organization and complexity of Quality Manuals vary from laboratory to laboratory, but they should always be designed to help the organization function effectively. It is important to take into account standards and regulations when designing a Quality Manual to ensure that deviations or nonconformities do not occur through lack of appropriate description or instruction in the Quality Manual. *

Prinz, M., Carracedo, A., Mayr, W.R., et al. 2007. DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI). Forensic Science International: Genetics 1: 3–12.

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Internal Audits

Management Review

Organization & Management

Control of Records

Deviations, Corrective Action, & Improvement

199

Quality Systems

Management Quality System Elements

Customer Service & Complaints

Document Control

Review of Requests & Contracts Purchasing Services & Supplies

SubContracting

Figure 11.2  Management quality system elements.

The starting point for a Quality Manual is to define a laboratory’s mission (purpose), goals, and organizational structure. Figure 11.2 depicts the many elements of the management quality system. The mission and goals describe what the laboratory wants to accomplish in regard to the quality of its testing and level of service to its customers, and describes whether the mission and goal are being met. The laboratory must document the top management’s commitment to achieving the mission and goals and following standards to which it is accredited. It is critical for the laboratory to clearly delineate the roles, chain of command, and responsibility within the laboratory, from the top level of management to the lowest level of staff. This is often done effectively with one or more organizational charts. Management must appoint a member of the staff as a quality manager and give appropriate authority to staff to perform their duties. Management must empower the quality manager with the right level of responsibility and authority to ensure the quality system is implemented and followed. The quality manager may have other duties as well, but must

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Personnel Laboratory Accommodation/ Environmental Conditions

Reporting Results

Assuring Quality/ Monitoring Results

Technical Quality System Elements

Handling of Samples

Test Methods

Equipment

Sampling

Measurement Traceability/ Calibration

Figure 11.3  Technical quality system elements.

have direct access to the top level of management. These elements must be defined in the Quality Manual. Management policies, including a quality policy statement and the management’s commitment to comply with standards and continuously improve their system, must be documented in the Quality Manual. Figure 11.3 depicts the different elements of the laboratory technical quality system. This helps laboratory staff to operate effectively in their implementation of the quality program. 11.1.5  Document Control Documents include all policies, procedures, instructions, and other written and electronic information that the laboratory depends on for performing testing and implementing the quality program. An important element of the quality system is document control. Implementation of new documents and modification or archiving of old documents must be controlled to ensure that authorized, current documents are in use in the laboratory to prevent

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policies and procedures from unauthorized changes. Documents must have a version number or other identification that helps to determine whether the document is the most current version. Policies and procedures must describe the method of document creation, modification, review, authorization, personnel training, and document implementation. Policies must define the responsibility and authority for these tasks. 11.1.6  Review of Requests, Tenders, and Contracts The laboratory must have procedures in place that address requests for testing, offers for contracting (tenders), and contracts. These procedures must ensure the laboratory: • • • • •

Employs methods to meet customer expectations Has contracts agreed to by both the laboratory and the customer Has the capability and resources to perform the testing Notifies the customer if they deviate from the contract Documents requests, tenders, and contracts

11.1.7 Subcontracting When subcontracting testing to outside laboratories, whether for reasons of workload or the need for additional types of testing, subcontractors must comply with the same standards as the in-house laboratory. This is critical when using the DNA profile results for searching and comparison of missing persons and unidentified remains. If laboratories subcontract testing, they must notify the customers of the arrangements in writing and (as appropriate) get the customer’s approval. In a mass fatality, the customer for the DNA operations may be the management organization responsible for the overall DNA identification effort. The DNA operations must take responsibility for the work of the subcontractor. It is common practice for DNA laboratories to use the services of subcontractor laboratories when overwhelmed by workload surpassing the primary laboratory’s capacity. 11.1.8  Purchasing Services and Supplies The laboratory needs policies and procedures to ensure that appropriate quality supplies and services are purchased. Quality supplies and services are integral to maintaining a high quality of testing. The laboratory must evaluate its suppliers for quality and consistency. Services, such as installation and maintenance of equipment, and supplies must meet requirements specified by the laboratory. The laboratory must have procedures for receiving and inspecting reagents and supplies that could affect the quality of the

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test. Procedures for receiving and inspecting supplies help to ensure reagents and supplies meet the laboratory’s requirements before they are used. 11.1.9  Customer Service and Complaints The laboratory should have a goal of delivering high-quality service for the customers of the DNA identification operation. Customers may be internal customers, such as other departments of a parent agency, or external customers, such as other agencies and families of the victims. The laboratory should seek feedback from customers regarding its performance (e.g., turnaround time and communication) to improve the management system, testing, and customer service. The laboratory must document complaints and investigate to determine the cause of the complaint. It must also take appropriate corrective action to resolve complaints. When properly managed, complaints are an effective opportunity to improve the quality program and the service of the laboratory. 11.1.10  Deviations and Corrective Action A critical element of a quality program is a system for identifying deviations, errors, and non-conforming work, and then correcting problems so they will not happen again. This is a fundamental aspect of both quality assurance and continual improvement of the laboratory system. The quality plan should include policies and procedures for identifying, documenting, and correcting errors, and deviations from all policies and procedures. Nonconforming services or materials, such as samples, services, or reports that do not meet the laboratory’s specifications must be handled similarly. When a laboratory discovers an error or deviation, the underlying reason for the issue is analyzed (root cause analysis). Root cause analysis is sometimes misunderstood and thought to be the determination of who was responsible for a deviation. However, root cause analysis is supposed to determine why the deviation happened. If a staff member makes an error, why did it happen? Is it an issue of insufficient training? Is the protocol incorrect? Is the environment distracting? Good root cause analysis allows the underlying cause to be remedied, thereby reducing or eliminating the problem. A poor root cause analysis does not address the underlying cause, thus ensuring the problem will eventually recur. After the laboratory determines the cause, it must identify and implement the appropriate corrective action to correct the cause. The laboratory may recall incorrect reports or non-conforming samples and evaluate the corrective action after an appropriate time to ensure effectiveness of the action.

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11.1.11  Control of Records The laboratory must have procedures for maintaining quality program and technical records. Records must be stored securely and in a condition that will help to preserve and allow timely access to records as required. The laboratory must define the period of time that records are retained. This retention period should follow applicable laws and regulations. For a DNA identification effort, the records may be retained by the agency in charge of managing the entire mass fatality response. Technical records must be clear and include sufficient information to audit the testing. The laboratory must create a record at or about the time work is performed, and records must be detailed enough to determine how a DNA profile result was obtained, how the analyses were performed, and how the interpretations were made. The laboratory must have procedures to define the contents and organization of the case file. The case file contents can vary depending on the function of the laboratory but typically include items listed in Figure 11.4. The chain of custody documents the receipt and disposition of evidence samples at the laboratory. The communication log tracks communications in regard to the case, including the date, time, name of person, and a description of the conversation. Analyst notes should describe the work performed, including a description of how a sample was taken (if done at the laboratory), sample accessioning, processing, the results obtained, and the interpretation. The laboratory case report is typically the document containing the results obtained, and it is provided to all agencies involved with a case. Analyst notes should include a description of the evidence received. This is usually a written description, with a supporting diagram, photo, or photocopy, if applicable. The notes should include a description of how the evidence was sampled (if performed at the laboratory) and should detail how much was sampled for DNA analysis and how much remains for additional testing. The case file should include or reference the procedures used to develop a DNA profile from a biological sample. It is important to record the information listed in Figure 11.5. Example Case File Contents

• A communication log, if applicable • Chain-of-custody documentation • Analyst case notes • The official test report • If applicable, hardcopy or electronic data copied to a CD

Figure 11.4  Example case file contents.

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Example Analyst Case Notes

• Case number and item numbers for each evidence sample • Dates and names or initials of the analyst performing any part of the process

• Reagent lot numbers and when applicable expiration dates of reagents

• Any negative and positive controls used, and the results • Times and temperatures, if appropriate • Instrument operating parameters, or references to them • Serial number or laboratory tracking number of critical equipment (such as Thermocyclers)

• The order in which the samples were processed • Testing observations Figure 11.5  Example analyst case notes.

Case review is necessary to identify transcription errors and detect any problems with the quality of the DNA results or the interpretations. Data transcription, calculations, interpretations, and reports must be reviewed for accuracy. The documentation of the review must indicate that each critical element was reviewed. This can be done either by marking reviewed elements, utilizing a checklist, or through the use of a statement indicating review of all critical elements. Review of data and interpretations can detect issues overlooked by primary analysts, and it is an essential part of a quality assurance program. Required elements of reviews must be documented to ensure consistent reviewing. Data review can dramatically affect the overall quality of the laboratory and confidence in results. Figure 11.6 lists the things a data review should ensure. These records document the work performed and produce traceability of results and an auditable trail, thereby demonstrating the validity of the testing. Often it is best to maintain records in the Laboratory Information Management System. Data Review

• Appropriate procedures were correctly followed

• Evaluation of quality controls • Interpretation of results was correct • Replicate or repeat testing was consistent • Data was properly transcribed • Appropriate documentations • Reports Figure 11.6  Example data review elements.

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11.1.12  Internal Audits Auditing is an effective way to ensure policies and procedures are followed, to monitor the effectiveness of a laboratory operation, and to assess any problems in the testing process. Audits are an essential part of a quality assurance program and are required for a laboratory to achieve and maintain accreditation. The laboratory should conduct internal audits at least once a year to ensure compliance with standards and requirements. Targeted audits of a section or a process can be used to determine proper functioning or to identify systemic problems. The laboratory’s accrediting body determines the required schedule for external and accreditation assessments (audits). Accreditation assessments are conducted by the accrediting body. When the laboratory demonstrates compliance with the standards and has corrected any non-conformance with the standards, it will achieve accreditation. Laboratories are required to be reaccredited on a schedule set by the accrediting body. 11.1.13  Management Reviews The laboratory’s management must conduct a review of the management system and testing activities on a defined schedule to ensure their suitability and to make necessary changes and improvements. Management should address issues that arise after the review and should document any actions taken in response. Actions to resolve issues must be taken in a timely fashion as agreed by management. Management reviews must address the following:

Key Components of Management Quality Review • Overall objectives of the quality system • Results of interagency comparisons or proficiency tests • Suitability of policies and procedures • Changes in the volume and type of work • Reports from managerial and supervisory personnel • Client feedback • Outcome of recent internal audits • Complaints • Corrective and preventive actions • Recommendations for improvement • Assessments by external bodies • Other relevant factors, such as quality control activities, resources, and staff training

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11.1.14 Personnel The laboratory must maintain up-to-date records of staff education, training, and evaluation of competency. Personnel involved in the DNA identifications must possess appropriate education, training, and experience before performing testing. The laboratory must define the required qualifications for testing personnel, and the laboratory must maintain up-to-date job descriptions for all managerial, technical, and support personnel involved in testing. The laboratory should document policies and procedures that outline the training requirements for personnel who conduct testing and should also document policies and procedures for completing these requirements. These policies must address re-training and skills/expertise maintenance. Training must ensure that personnel are competent to perform testing (e.g., possess the necessary knowledge, skills, and abilities). When the training is technique or test specific, the laboratory must define criteria for determining successful completion. This can be observation of the test performance by an experienced analyst, correct results for known samples, or correlation of results with trained staff. Written tests must also have defined criteria for successful completion. After training is complete and a formal assessment of personnel training and competency is successful, the laboratory must document a statement of competency. This can take the form of a memorandum, letter, certificate, or other documentation. The date of declared competency must be recorded, and the laboratory must authorize all staff to do the jobs they are assigned. Many laboratories confer authorization and competency at the same time. Depending on the laboratory’s needs and training, competency may be modular, for specific tasks, or comprehensive, for the entire range of tasks performed by a staff member. If training and competency are modular, competency must be carefully tracked, such as with an up-to-date competency matrix. Tracking helps to ensure personnel are not assigned tasks for which they have not yet completed their competency. 11.1.15  Accommodation and Environmental Conditions Figure 11.7 depicts the division of different procedures in a laboratory. Layout is a critical part of the quality program and is essential to keeping equipment, reagents, and facilities used for manipulation of amplified DNA separate from those used for storage and manipulation of unamplified DNA. Pre- and postPCR activities should be carried out in separate rooms with closed doors. It is desirable to have airflow that prevents PCR product from traveling via air into pre-PCR areas of the laboratory. Extremely small amounts of amplified DNA can contaminate equipment, samples, or reagents if brought into areas

Implementing and Maintaining a Quality DNA Program

Humanitarian/Civil Sample Accessioning and Storage Bone (Unknown Aliquotting Extraction PCR Setup)

Buccal Swab/References Aliquotting Extraction PCR Setup

Other Unknowns Other Accessioning Screening Storage Other Aliquotting Extraction PCR Setup

Amplification and Data Collection

Pre-PCR

Identification and Collection of Knowns (References)

Post PCR

Collection From Skeleton (Unknowns)

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Interpretation and Reporting

Figure 11.7 (see color insert)  Division of laboratory procedures.

where unamplified DNA is processed or stored. The laboratory must have policies and procedures in place for the separation of equipment, reagents, consumable supplies, samples, and personal protective equipment (PPE) (lab coats, gloves, etc.) in these two areas. The laboratory must also have policies and procedures in place for routine cleaning and decontamination of the amplified DNA laboratory, as well as equipment that must be moved out of the amplified DNA laboratory. In addition to separating pre- and post-PCR operations, consideration should be given to separating limited biological samples with small quantities of DNA from samples containing higher levels of DNA. For example, while a known blood sample, taken as a reference from a family member, will be expected to contain a high level of DNA, a small portion of degraded bone might contain low levels of DNA. Testing areas for samples with low levels of DNA should be separated from the testing areas for samples with higher levels of DNA. This can be accomplished by having different rooms for processing these samples or different areas or containment hoods in the same room. If low copy number (LCN) or mitochondrial DNA testing is performed in the laboratory, then there must be complete separation of those activities from routine DNA testing laboratory rooms, with additional requirements and specifications. The goal of separating the processing of these samples is to prevent contamination and reduce the chance of laboratory errors. The laboratory must control, to the greatest extent possible, and monitor the room temperature, quality and availability of electricity and water, and other factors that can adversely affect the quality of testing. Room

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temperature should be controlled to the specifications of equipment and procedures that may be affected (e.g., capillary electrophoresis analyzers). Room temperatures should be monitored, if they will impact the analysis, and if the temperature passes out of acceptable range, the impact on testing must be evaluated. The laboratory can address the quality of electricity by using uninterruptible power supplies (UPS) and backup generators. Laboratory safety is an essential function of daily operations and protects both the staff and the evidence. Laboratory safety should include personal protective equipment (PPE) for staff, literature and training on handling and proper disposal of chemicals, building safety, and proper handling of biological samples. Safety policies must be documented. Equipment must be monitored for safety, and chemical storage must be appropriate to reduce the potential of a dangerous event. The laboratory must develop a documented emergency or disaster plan that takes into account possible natural disasters and man-made emergencies. The types of emergencies or disasters addressed in such a plan will depend on the laboratory’s individual situation and geographical location but can include storms, earthquakes, fire, power and water stoppages, and threats to the facility or personnel. The laboratory should have plans for emergencies that may arise, with particular attention given to emergencies that are likely to become threats to the laboratory. The plan must address the safety and protection of the staff, the samples and evidence, reagents, and equipment. It must also address long-term stoppages of power to protect samples and reagents, thus ensuring that samples can be moved to appropriate storage conditions either on site or off site. Copies of the emergency plan should be kept off site, in case the laboratory is inaccessible. An important component of the emergency plan is a communication plan that helps ensure that emergency contact information and phone numbers are not trapped in a burning building. 11.1.16  Test and Calibration Methods and Method Validation The laboratory must choose appropriate methods for all tests and must fully document such methods in clearly written procedures. Procedures must address sampling (if performed), handling, storage, processing, quality controls, analysis, interpretation, statistical calculations, and reporting. If the laboratory performs screening of items for biological materials, such as using chemical tests for biological fluids, microscopy, or alternative light sources, these procedures must also be documented. Documentation must include clear instructions for the use and maintenance of equipment. These instructions should be appropriate to the complexity of the equipment and should consider whether lack of instructions will jeopardize the testing. For example, microcentrifuges require

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minimal instructions, but capillary electrophoresis analyzers require extensive detailed instructions. Procedures, instructions, and manuals must be up to date and available to testing personnel in locations where the testing takes place. Testing will deviate from procedures only if the deviation is technically and scientifically justified, if the deviation has been authorized, and if the deviation has been documented in the case record. Particular attention should be paid to developing detailed procedures for the analysis and interpretation of contaminated samples, very low level and degraded DNA samples, mixed samples, and partial DNA profiles. These types of samples and profiles frequently occur in mass disasters and are much more difficult to interpret in familial comparisons than in straight comparisons to reference samples. Similarly, it is critical to develop detailed and appropriate procedures for familial comparisons and their associated statistical comparisons. Familial comparisons differ substantially from standard criminal casework comparisons and are subject to additional complexities, such as mutations, null alleles, and non-paternity (see Chapter 9). Appropriately detailed procedures increase the consistency of analyses and interpretations among laboratory staff. All technical methods must be fully validated before use on casework. Any method that has been validated by another laboratory or a commercial company must still be verified by an appropriate internal validation to demonstrate reliability. The quality and type of standard materials and reagents used in the testing must be adequate for the procedure being used. The laboratory must take care to order and use the appropriate grade, type, and quality of reagents, and the correct types of consumable supplies, where they could have an effect on the quality of the testing. The lot number or batch number of reagents or standards used in testing must be recorded, and the laboratory must test all critical reagents for their reliability prior to use. Standards and reagents must be labeled with the following information: name, concentration where appropriate, preparation and/or expiration date, identity of preparer, storage conditions where relevant, and a hazard warning if necessary. Lot and batch numbers allow traceability in case of quality problems. Preparation/expiration dates prevent the use of expired reagents. Generally, expiration dates are preferable to preparation dates for preventing use of expired materials. No reagents or reagent aliquots should be unlabeled. Similarly, water, detergent, and bleach “wash bottles” must be labeled and must have at least a preparation or a fill date. If water wash bottles are not periodically cleaned, they may develop microbial growth over long periods of time.

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11.1.17  Validation of Methods A laboratory needs to document validation to demonstrate the accuracy, reliability, and reproducibility of the testing procedures it uses. Validation is also critical for a laboratory to gauge the limitation of the procedures, equipment, and the analysts performing tests. The validation studies will provide a degree of confidence with testing procedures implemented in a laboratory. Documentation shows how the laboratory developed specific guidelines and procedures and demonstrates how the laboratory maintained the quality of the DNA tests. Developmental validations are necessary in cases where the laboratory develops its own method—a method that has not undergone developmental validation at another laboratory or at a company developing the method. Internal validation studies are essential to demonstrate the accuracy, reliability, and reproducibility of a method in its own system. These studies are conducted when new methods, technologies, or significant changes in methods are implemented in the laboratory. Validation studies must be completed, summarized, and reviewed before the method is authorized for use in casework. It is important to realize that additional validation studies may become necessary or desirable after the initial validation is completed. This does not invalidate the method and previous results. It is crucial for the laboratory to keep and maintain validation records for their quality program. Validation studies are an important training tool for analysts to understand the capabilities and limitations of the methods they use. 11.1.18  Uncertainty of Measurement DNA profiling is a qualitative measurement, so it is not subject to estimation of uncertainty of measurement. A qualitative measurement is one that involves a description. If the laboratory reports a numeric DNA quantity, then it must estimate its uncertainty for that measurement. 11.1.19  Control of Data The laboratory must appropriately check electronic calculations and data transfers in a systematic fashion to ensure proper function. Software used for testing and analysis is appropriately validated prior to use in casework. Analysis software is typically validated during method validation. All software must be validated prior to its use in testing. This applies LIMS, databases, and macros or other software that perform statistical calculations, profile matching, or database searches. This does not apply to software applications not used in analysis, such as word processing and spreadsheet applications used for calculations.

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The laboratory must have procedures in place for the maintenance of computers and automated equipment, as well as the protection of data integrity and confidentiality. 11.1.20 Equipment The laboratory should be furnished with appropriate equipment necessary to perform its testing. A laboratory must evaluate, test, calibrate, and maintain equipment to ensure validity of results. The laboratory should identify critical equipment (equipment that has a significant effect on the results of the test). Critical equipment must undergo a program of maintenance and calibration. Maintenance, performance checks, and calibration of critical equipment must be scheduled at regular intervals, performed, and recorded to show that instruments are working properly. The maintenance and calibration schedule can take many forms, including a list, a table, or electronic scheduling software. Generally, calibration or calibration verification must not be less frequent than recommended by the manufacturer. Reference materials should be provided, where appropriate, for calibration or calibration verification. General lab equipment (hot plates, stirrers, etc.) does not need calibration or performance checks, just routine cleaning and maintenance. Microscopes must also be routinely cleaned and maintained. Use and maintenance of instruments and equipment on a daily basis should be recorded and documented in a usage log. Equipment that has a significant effect on the results of testing must be calibrated before being put into use. Calibration, calibration checks, or performance checks are typically necessary after service or maintenance that could affect performance. The laboratory must possess documented procedures for calibration and performance checks. Equipment records provide measurement traceability and will support the detection of any problems, which may necessitate re-analysis of samples. 11.1.21 Sampling If the laboratory collects samples or takes samples from recovered remains or other items, the laboratory must have procedures in place for training and competency testing of any sampling methods utilized by the laboratory. Sampling must be documented as previously described. 11.1.22  Handling of Test and Calibration Items The laboratory must have procedures for the transportation, receipt, handling, protection, storage, retention, and/or disposal of samples. Samples that do not conform to requirements or acceptance policies or appear to not

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• Identity of the item of equipment and its software • Manufacturer’s name, type identification, and serial number or other unique identification

• Checks that equipment complies with the specification • Current location, where appropriate • Manufacturer’s instructions, if available, or reference to their location

• Dates, results and copies of reports and certificates of all

calibrations, adjustments, acceptance criteria, and the due date of next calibration • Maintenance plan, where appropriate, and maintenance carried out to date • Any damage, malfunction, modification or repair to the equipment

Figure 11.8  Records for equipment.

match their description must be documented upon receipt and discussed with the customer before processing. Samples must be uniquely identified, and the identification system should allow subdivision of samples. A documented chain of custody must be maintained for all samples. Laboratory security ensures the integrity of samples by controlling access to storage areas, maintaining confidentiality, and preventing evidence tampering. It is critical to properly secure and store both physical samples and case information. Figure 11.8 lists some of the laboratory policies pertaining to security of the samples. 11.1.23 Assuring the Quality of Test and Calibration Results (Quality Assurance, Quality Control, Proficiency Testing) The laboratory must have a program of quality control to ensure that its testing is “under control.” This requires the analysis of quality control samples with every batch of tested samples. Tested samples include positive and negative amplification controls, negative extraction controls, and may include positive extraction controls. These control samples ensure that reagents and procedures worked correctly and help to monitor contamination. Additionally, the laboratory can analyze certified reference materials, if available, and internally generated reference materials. The laboratory must record the performance and results of quality control testing and remediation done if the expected results are not obtained. Quality control results should be regularly analyzed to detect trends.

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If available, the laboratory must participate in a proficiency testing program that is acceptable to its accrediting agency. If a proficiency testing program is unavailable, then the laboratory must participate in regular interlaboratory sample exchange to correlate results with peer laboratories. The results of proficiency testing or inter-laboratory exchange must be reviewed, and appropriate records should be maintained (e.g., records and documentation of the proficiency “case,” records of the review of performance, and details of any corrective action necessary). Proficiency testing must be undertaken at least once a year. Good practice dictates that each analyst be proficiency tested. Internal proficiency testing generated by the staff of the laboratory— where the staff member performing the testing does not know the outcome before completion of the testing—can also monitor performance. If analysts testify in court, the testimony of each analyst should be evaluated on a regular basis to include appearance, performance, and effectiveness of presentation. The monitoring procedure should describe the remedial action if the evaluation is unsatisfactory. 11.1.24  Reporting the Results The laboratory must establish procedures and guidelines for reporting to ensure accurate, clear, and objective test reports (Figure 11.9). The reports should describe the samples tested, methods used, any results, interpretations, and statistical calculations as appropriate, and the signature or other identification of the individual taking responsibility for the report. Procedures must address which elements are required in the report, and reporting guidelines or templates should specify the conditions under which various interpretation statements can be made, thus ensuring uniformity of reporting among analysts.

Laboratory Security Policy

• Receipt and accessioning (logging) of samples • Storage of samples to prevent deterioration, loss or damage

• Documentation of storage • Tracking sampled portions of evidence while being processed

• Sample return to the submitting agency Figure 11.9  Laboratory security policy.

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Additional Resources American Society of Crime Laboratory Directors. 2012. International Terminal. http://www.ascld.org/content/international-terminal (accessed September 29, 2013). Butler, J.M. 1997. Short tandem repeat DNA Internet DataBase. http://www.cstl.nist. gov/div831/strbase/index.htm (accessed September 29, 2013). FBI. 2012. FBI Laboratory Services. http://www.fbi.gov/hq/lab/labhome.htm (accessed September 29, 2013). International Laboratory Accreditation Cooperation. 2002. Guidelines for Forensic Science Laboratories. http://www.ilac.org/documents/g19_2002.pdf (accessed September 29, 2013). International Organization of Standards. 2012. http://www.iso.org/iso/home.htm (accessed September 29, 2013). INTERPOL. 2012. Forensics—DNA. http://www.interpol.int/Public/Forensic/DNA/ Default.asp (accessed September 29, 2013). INTERPOL. 2012. Forensics—DVI (Disaster Victim Identification). http://www. interpol.int/INTERPOL-expertise/Forensics/DVI (accessed September 29, 2013). National Institute of Justice. 2008. DNA Initiative. http://www.dna.gov (accessed September 29, 2013).

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Attachment A ISFG Recommendations Regarding the Role of Forensic Genetics in Disaster Victim Identification* Recommendation #1. Every forensic DNA laboratory should make an effort to contact the relevant authority dealing with emergency response and establish involvement in a possible mass fatality preparedness plan. Policy decisions about sample collection, scope, and final goals of the effort will affect the victims’ families and the work stream and should be decided as early as possible. Recommendation #2. The internal response plan needs to address throughput capacity, sample tracking, and must have names of supervisors responsible for different tasks that are updated as personnel changes. Recommendation #3. Several sample types…for DNA testing should be taken at the earliest possible stage of the investigation provided traceability is guaranteed. Samples must be collected from each body or recognizable body part, even if identity is already established. Proper storage must be assured. Recommendation #4. Multiple direct references and samples from first-degree relatives should be collected for each missing person. Scientists with a background in genetics should be available for training or for consultations in the family liaison group. Recommendation #5. DVI DNA testing should only be performed by laboratories with demonstrated successful capabilities and continuous experience with these specified sample types. Recommendation #6. The set of loci to be analyzed has to be identified as soon as possible in concordance with the scientific community in the countries mostly involved. A minimum of 12 independent loci should be selected as standard set, but an even greater number of loci is preferred. Recommendation #7. All allele calls and all candidate matches have to be reviewed thoroughly. Composite DNA profiles can be generated if derived from the same specimen and consistent for overlapping loci. The duplication policy should consider the logistics and circumstances of the mass fatality incident. Recommendation #8. If the standard autosomal STR typing fails to give sufficient information, additional typing system such as *

From Prinz, M., Carracedo, A., Mayr, W.R., et al. 2007. DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI). Forensic Science International: Genetics 1: 3–12.

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mtDNA, Y-chromosomal STRs, or SNP markers may be used in selected cases. Recommendation #9. A centralized database is required for all data comparison. Electronic upload is recommended to avoid transcription errors. Recommendation #10. Especially if multiple family members are involved, DNA-based identification should whenever possible be anchored by anthropological and/or circumstantial data, a second identification modality, or multiple DNA references. Recommendation #11. In DVI work, DNA statistics are best represented as likelihood ratios that permit DNA results to be combined among multiple genetic systems or with other non-DNA evidence. Likelihood ratio thresholds should be determined for when DNA data alone can suffice for an identification; this will be based on the size and circumstances (e.g., closed versus open) of the event. All evidence and/or circumstances should be checked in making an identification, even if DNA provides the primary or sole evidentiary factor. Recommendation #12. The preparedness plan of the laboratory needs to include policies for family notification, long-term sample disposition, and data archiving.

Chapter Laboratory Development

12

This chapter defines the important steps in setting up a laboratory operation, including planning, staffing, identifying types of equipment and supplies, and the activities necessary to ensure that laboratory equipment performs appropriately. Although several types of laboratories (service, research, education, and/or training laboratories) support forensic DNA operations, they may operate independently or coexist. Table 12.1 provides a brief description of the different types of laboratories supporting forensic DNA profiling operations. As outlined in Figure 12.1, a solid laboratory is built on a foundation of a clear mission, vision, goals, and objectives. The policies and procedures outlining the functions of the laboratory support the people working in the laboratory. Finally, accreditation (see Chapter 11) provides the high-level management and administration required for a laboratory to provide consistent and reliable results. It is important to note that while the information presented in this chapter provides a high-level overview of the steps necessary to set up a laboratory, it should be supplemented with the advice and expertise of organizations and consultants who possess practical experience in establishing laboratory operations.

12.1  Laboratory Operations Strategy The definition of requirements is a key aspect in the successful development of a laboratory. Taking the time to define requirements helps to ensure that a laboratory will be appropriately staffed, equipped, and competent to operate. The following sections outline the key steps involved in planning for a new laboratory. 12.1.1  Mission, Vision, Goals, and Objectives of Laboratory The first step in defining requirements is to determine the mission, vision, goals, and objectives of the laboratory operation. By defining the mission, vision, goals, and objectives, the planning team will be able to determine the types of capabilities or functions that must be in place to become fully operational. 217

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Table 12.1  Laboratory Types Laboratory Type Service

Research

Education Training

Description Supports the collection, accessioning, examination, aliquotting, and extraction of samples, the quantitation and amplification of DNA, and the data analysis, interpretation, and reporting of DNA profiles to answer a question(s) of sample identification. This may include the testing of known and questionable samples for the identification of human remains from mass fatalities or testing to determine the contributor of a piece of evidence at a crime scene. Supports the development of new methodologies, including instrumentation and technology. Examples of investigation can include: • Defining limits of current technologies • Developing population databases • Investigating novel applications of current technology Provides laboratory courses on forensic laboratory techniques to undergraduate and graduate students Provides training of laboratory policies and procedures to individuals who will be working in operational laboratories (e.g., newly hired technicians or forensic DNA analysts)

The mission of a laboratory operation is its primary purpose: why it exists and what it does. A laboratory’s vision statement typically defines what a laboratory hopes to achieve in a given time period (e.g., 3 to 5 years). The goals and objectives describe specific, measurable activities that will help a laboratory achieve its mission and vision. Table  12.2 provides examples of the mission, vision, goals, and objectives for each of the four types of laboratory facilities.

Figure 12.1  Laboratory design components.

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Table 12.2  Examples of Mission, Vision, Goal, and Objectives Type of Laboratory

Service

Research

Education

Mission, Vision, Goal, and Objectives Examples Mission Statement: To conduct timely, accurate, and cost-effective DNA identifications to support human identification investigations. Vision Statement: To become the leading international forensic DNA laboratory supporting human rights. Goal: To establish a comprehensive DNA identification operation that will begin functioning by mid-2011 and offer differentiated services to the citizens in Iraq by 2013. Objectives: By midyear 2012, the laboratory will establish a functional DNA identification operation that has the capability to: • Profile 400 skeleton and 3,000 reference samples/year • Report identifications using industry best practices Mission Statement: To conduct timely, accurate, and cost-effective research to evaluate current and future instrumentation and technologies for forensic DNA testing. Vision Statement: To become the leading national forensic DNA research laboratory in the Middle East. Goal: To establish a comprehensive DNA research capability that will begin functioning by mid-2012 and conduct research, which enhances the DNA forensic operations. Objectives: By midyear 2011, the laboratory will establish a functional DNA research capability which supports the: • Development of a new methodology for sample extraction from bone • Evaluates emerging capabilities for laboratory information systems Mission Statement: To provide undergraduate and graduate students with a laboratory to support courses offered by the university forensic sciences department. Vision Statement: To become the leading university laboratory in the Middle East for forensic science education. Goal: To establish a comprehensive, fully staffed and equipped laboratory to support undergraduate and graduate student forensic DNA coursework by 2012. Objectives: By midyear 2010, the university will establish a functional DNA laboratory that supports student learning about best practices in forensic DNA profiling. Accordingly, students will participate in: • One pipetting laboratory exercise • Two laboratory exercises on DNA extraction and profiling • One 5-week laboratory exercise on DNA profile interpretation (Continued)

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Table 12.2  Examples of Mission, Vision, Goal, and Objectives (Continued) Type of Laboratory

Training

Mission, Vision, Goal, and Objectives Examples Mission Statement: To conduct timely, accurate, and cost-effective training on policies and procedures which will guide the handling of evidence for DNA identification testing. Vision Statement: To become the best forensics training facility for DNA operational laboratories in the Middle East. Goal: To establish a comprehensive forensics training laboratory by mid-2012. Objective: By midyear 2011, the laboratory will establish a capability to provide training courses. Training courses will prepare law enforcement officers to: • Collect and transfer evidence in accordance with the best industry standards.

12.1.2 Needs of the Individuals and Organizations with Interest in the Laboratory Operations Once the planning team has defined its mission, vision, goals, and objectives, it is important to identify individuals and organizations with an interest in the laboratory. These individuals and organizations have a vested interest in the success of a laboratory and are impacted by decisions made by the laboratory, including staff, victim’s families, funding organizations, and universities. Table  12.3 describes some examples of the needs of individuals and organizations with a vested interest in a laboratory.

12.2  Laboratory Functions Once the mission, vision, goals, objectives, and stakeholder needs are identified, it is important to define the functional areas of the laboratory. Identifying the functional areas will drive requirements for laboratory processes, staffing and training, and equipment and supplies. Each laboratory will function differently. For example, the primary functional areas of an operational forensic DNA laboratory may include the following: • • • • • • • •

Sample collection Accessioning and sample tracking and storage Sample evaluation and aliquotting Extraction Quantitation Amplification Data collection Analysis and reporting

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Table 12.3  Individuals and Organizations with a Vested Interest in the Success of a Laboratory Individual/Organization

Needs Service Laboratory

Law enforcement Funding organizations Victims

Police department DNA analysts

Defendants and suspects Citizens

Politicians

Education system Vendors

• Accurate and timely results • Appropriate use of funds in accordance with guidelines • Resolution and closure of cases • Accurate and timely results to identify and prosecute perpetrators • Accurate and timely results to support investigations • Accomplish quality work products that are scientifically sound and conform to acceptable practices of the forensic science community • Access to work areas, equipment, and supplies when needed to perform testing • Accurate and timely results to support appropriate prosecutions and exonerations • Provide resolution to victims, their families, prosecutors, and the entire criminal justice system • “Accountability” for use of public funds • Confidence in laboratory results • Eliminate backlog of forensic cases • Confidence in laboratory • Good use of funds • Comprehension of laboratory needs for the development of education and research programs that would support employment of matriculated students • Feedback on the use of equipment, reagents, and supplies • Laboratory purchase of reagents/supplies Research Laboratory

Service laboratories Laboratory staff Funding organizations Vendors

• Research projects that support enhanced operations • Interesting and challenging projects that result in publications • Projects that are completed on time and within budget, resulting in novel methods and publications • Feedback on the use of equipment, reagents, and supplies • Laboratory purchase of reagents/supplies (Continued)

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Table 12.3  Individuals and Organizations with a Vested Interest in the Success of a Laboratory (Continued) Individual/Organization

Needs Education Laboratory

Students Service laboratories Vendors

• Education that properly prepares students for work in the laboratory • Rigorous program that will provide a pool of highly educated potential employees • Feedback on the use of equipment, reagents, and supplies • Laboratory purchase of reagents/supplies Training Laboratory

Service laboratories

Students Vendors

• Appropriately trained staff who understand the process and can pass a competency test • Effective use of training funds • Training that allows students to pass a competency test • Feedback on the use of equipment, reagents, and supplies • Laboratory purchase of reagents/supplies

Table 12.4 provides a description of each of the primary functional areas. In addition to the primary areas of a forensic DNA laboratory, there are key support functions in the lab. Support functions may include administration, information technology, facility management, security, training, and quality management. Research, education, and training laboratories will have similar functions, but their policies, procedures, and sample flow will differ. Regardless of policies and procedures, understanding the various aspects of a laboratory will facilitate planning for laboratory design and layout, equipment, and staffing.

12.3  Sample Types and Number Estimates Before developing the facility design, it is important to estimate the volume of tests and the type of tests the laboratory will be conducting. By estimating the number and type of samples the laboratory operation will process, as well as the test systems the laboratory will employ, the planning team will be able to forecast the workload and identify requirements for facility design, staffing, equipment, supplies, and computing/communications infrastructure. This will assist the planning team in developing a budget for the laboratory’s initial and recurring costs. Without a proper budget, a laboratory will not be successful.

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Table 12.4  Primary Functional Areas of a Laboratory Functional Area Sample collection Accessioning and sample tracking Sample and record storage

Sample evaluation and aliquotting Extraction

Quantitation Amplification Data collection Analysis and reporting

Description of Function Evidence and samples from references are collected. For tracking purposes, samples and identifying data are logged into the laboratory with a Laboratory Information System. Samples are stored in a secure area to maintain sample integrity and to minimize deleterious change or contamination. Records and case files are stored in a secure location. Samples are screened, examined, and documented, and a portion of the sample is placed in a new container (tube) for testing. DNA is extracted from the sample following a validated protocol. The procedure may involve one of the following: • High salt manual extraction • Phenol chloroform • Extraction kits manual • Extraction kits automation The quality and quantity of the isolated DNA are measured. Various locations are amplified for analysis. Capturing and analyzing data electronically. Interpretation of data and preparation of reports.

Specifically, the laboratory will need to provide estimations for the types of samples it plans to process. For example, a service laboratory performing testing to identify victims of mass fatalities may need to process the following types of samples: • • • •

Human remains (bone, teeth, fingernails, tissue, blood) Family reference (buccal swabs) Direct references (biopsy slides, bloodstain cards) Personal items (hairbrushes, toothbrushes, envelopes)

All laboratory facilities will need to estimate the number of samples it will process for validation, training, controls, and standards. Additionally, it is important to incorporate the repeat rate (sample failure) since this may significantly impact laboratory operations. As it will take time for the laboratory to reach its full operational capability, it is helpful to estimate sample volumes over a given period of time (e.g., 5 years). This will allow the laboratory to gradually increase the number of samples tested, as equipment is installed, tested, and validated, and staff are trained and certified.

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12.4  Laboratory Design and Layout After identifying the sample number, sample types, and test systems the laboratory plans to process, the next step is to design and lay out the laboratory operation. First, it must be decided whether the laboratory will operate as a stand-alone facility or be located in an existing organization. If the laboratory shares space with other facilities, it is important to decide how the functional areas of the laboratory will operate—which areas will share physical space and which will occupy their own space. A safe, secure, and comfortable work environment should be the primary consideration when designing and laying out the laboratory. The layout must ensure that staff will be able to complete their work efficiently and effectively. Furthermore, areas for breaks and meetings are a necessity. Table 12.5 provides an overview of some important considerations for designing and laying out a laboratory.

12.5  Staffing and Training An important aspect of laboratory development is the planning, selection, and training of staff. The planning team should develop a staffing plan to document the approach for hiring, orienting, training, and certifying laboratory personnel. The plan should account for employment requirements, anticipated turnover, and training methodology. For example, a service laboratory will go through the accreditation process. Therefore, a staffing plan for this type of laboratory must include enough staff to acquire and maintain accreditation, as well as report accurate results in a timely manner. Prior to the start of laboratory operations, a detailed training program should be developed. As it will take considerable time for staff to become trained and proficient, planning for training early in the development of the laboratory is imperative. The training plan should address: • • • • •

Textbook and classroom training Observation of the testing being performed by trained scientists Technique practice on training samples Task performance under close supervision Written and practical competency testing

12.6  Quality Assurance and Quality Control Quality assurance and quality control (QA/QC) are paramount in maintaining sample throughput and accurate results. An important component

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Table 12.5  Important Considerations for Laboratory Design and Layout Considerations • Workflow must enable samples to flow throughout the laboratory, while minimizing the potential of contamination and supporting the absolute certainty of results. • Layout supports tours and visitors while minimizing operational disruption and traffic through the laboratory. • Pre- and post-PCR testing areas must be physically separated, and the relative pressurization of the spaces must prevent the migration of amplified DNA from the post-PCR spaces into pre-PCR spaces. • Separate areas for processing samples of “Known” origin from forensic “Unknowns” are preferable whenever possible. • Shared spaces should allow technicians to effectively complete paperwork or perform other job functions. • Supervisors should have office space away from the laboratory with sufficient privacy for meetings with staff. • Forensic analysts and staff performing data interpretation will need space outside of the laboratory to analyze the data. • The heating, ventilation, and air conditioning system should maintain the temperature and humidity within the laboratory at levels consistent with successful instrumentation operation. • Electrical power to the laboratory should be reliable. Surge protectors should be in place for all computers and uninterruptible power supplies for laboratory instrumentation. • An appropriate number of computer workstations are needed to sufficiently support the laboratory staff and equipment. • Adequate supplies and consumables should be readily available. • Sample storage at the laboratory should minimize deleterious change and/or cross-contamination. • All testing procedures must have a back-up in case of equipment failure. This back-up should be a manual procedure or a secondary, but equally comparable piece of equipment.

of laboratory development is the development of policies and procedures that provide written guidance and instructions for carrying out tasks. These policies and procedures should be based on appropriate tried and tested methods. An important aspect of QA/QC is to ensure all staff members receive correct training on properly operating equipment in the laboratory. In order to evaluate overall laboratory performance and individual staff performance, proficiency tests should be routinely conducted. The proficiency tests should span the normal operation of the laboratory and be conducted on a recurrent basis. The equipment must be calibrated, maintained regularly, and if errors are detected, taken out of service and upgraded or replaced.

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An important aspect of laboratory development and QA/QC is the initiation and maintenance of a robust validation process. Validation is the documented process of demonstrating that a laboratory’s methods and procedures consistently produce expected results with the staff performing testing on the equipment currently in use. Validation will be discussed in more detail below.

12.7  Equipment and Supplies Another key requirement for the development of a laboratory operation is the identification and ordering of supplies and equipment. The required equipment and supplies depend on the function of the laboratory operation and the type of testing occurring at the laboratory. A key consideration for the planning team is to determine how long it will take to purchase supplies and equipment, implement new technology, and ensure the equipment is operational. Attachment A illustrates some of the types of materials used to support a forensic DNA laboratory and provides rough (within an order of magnitude) cost estimates. It is important to work with your local vendor to obtain pricing for the materials and equipment needed (including service contracts). When preparing a budget, one needs to recognize that prices fluctuate, and there may be taxes and other fees associated with the purchases. Budgets should be carefully developed to cover all costs.

12.8 Validation Validation is the documented process of demonstrating that a laboratory’s methods and procedures consistently produce expected results. Validation is an important part of the process for implementing and integrating new laboratory technologies and ensures confidence in the quality of data and the reliability of results. The validation process is designed to evaluate new or modified procedures and methods using a specific set of criteria. The results of the validation studies help define a laboratory’s testing limitations with regard to testing systems, equipment, and staff performance. Validation provides a high degree of certainty that a procedure will meet predetermined specifications and quality attributes. To avoid implementing a flawed or incomplete plan, validation has to be performed before any new process or procedure is put into use. The results of the validation studies provide the laboratory with an understanding of how the procedures work and the limits of a process. For example, validation studies help to define the expected quality of the data from the equipment and testing method and the limits of

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detection. Validation also helps to develop guidelines for data interpretation and provide the laboratory with information on when data should not be used. Validation demonstrates whether a laboratory procedure is robust, reliable, and reproducible in the hands of the staff performing the test. A robust method frequently produces successful results. Reliable results are accurate results. Reproducible results give the same or similar results each time the sample is tested. 12.8.1  Stages of Validation There are two stages of validation: developmental validation and internal validation. Table 12.6 describes the two stages. 12.8.2  Guidelines and Organizational Standards It is recommended that laboratories work with their local or regional accrediting body to determine which sets of validation criteria are appropriate for their laboratory. Meeting such criteria is the best way to ensure the quality of the data and the competence of the staff performing the testing. Validation standards and guidelines are provided by both national and international organizations. Table 12.7 provides examples of several organizations. 12.8.3  The Validation Process Validation of methods increases an understanding of instrument/procedure thresholds and limitations. Before implementation for casework, the laboratory should validate all procedures, evaluate equipment performance, and evaluate staff performing the testing. Validation takes a considerable amount of effort and requires planning. Table 12.8 provides considerations for a validation strategy. 12.8.4  Testing Methods and Procedures Methods may be validated by the comparison of method outcomes and results with other established methods using either known samples or standard reference materials. There are several different types of measurements taken during validation. The measurements will depend on the method being validated. Table 12.9 provides a description of each validation criterion.

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Table 12.6  Types of Validation Stage

Developmental validation

Internal validation

Description Initial testing and evaluation performed on new technologies. Typically performed by the manufacturer or the entity initially implementing the new method. For example, the validation of new technologies for detecting short tandem repeat (STR) alleles and the development of commercial STR kits may include: • Testing of species specificity • Sensitivity and stability studies • Reproducibility • Effectiveness on various sample types • Population studies • Mixture studies • Precision and accuracy measurements • Testing of various testing parameters Process laboratories use to verify and demonstrate that established procedures, which have undergone developmental validation in other laboratories, work effectively in one’s own laboratory. Internal validation is conducted by each forensic DNA testing laboratory and is the in-house demonstration of the reliability and limitations of the procedure. For example, if the laboratory implements a new computer program for the interpretation of DNA profiles, the laboratory may perform the following analyses before program implementation: • Program settings will be inspected and verified • Visual inspection of the allele frequencies in the population databases to verify that they are correct • Screening will be performed on predefined groups of profiles to verify that correct matches are identified • Kinship analysis on various predetermined cases with different scenarios will be performed and compared to hand calculations and calculations performed with other software packages to confirm that the calculations are accurate. These cases can include: • Classic trio (mother, child, father) • Motherless • Grandparent • Full sibling • Half sibling • Case with a mutation All results will be accounted for, explained, and documented. Typically, the laboratory’s technical leader will review and sign the validation study. The technical leader’s signature demonstrates his or her review of the validation and approval for the laboratory to use the new method. Often accreditation agencies will review the validation studies during a site visit.

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Table 12.7  Examples of National and International Standard Organizations Providing Guidelines on Validation Organization

Description

International Organization for Standardization (ISO) and its partner, the International Electrotechnical Commission (IEC) 17025:2005

• International guidelines for calibration and testing laboratories • Includes forensic laboratories

International Laboratory Accreditation Cooperation (ILAC) G19, 2002

Scientific Working Group on DNA Analysis Methods (SWGDAM) Revised Validation Guidelines, 2004

AABB Standards for Relationship Testing Laboratories, 9th Edition, 2010

• Specific to forensic science laboratories • Generally references the ISO 17025:2005 standards • Provides guidelines and standards addressing validation of methods and procedures, equipment, and the competence of staff • Established in United States by Federal Bureau of Investigation • Quality Assurance Standards (FBI QAS) developed by an independent group of scientists • Includes minimum validation requirements for laboratories participating in the U.S. National DNA Index System • Provides relationship testing standards and guidance for standards for biological relationship testing laboratories • Provides guidelines on the use of DNA in mass fatality response operations which refer to the SWGDAM Guidelines

Table 12.8  Validation Strategy Validation Strategy Develop a validation plan addressing the following: • Type of testing • Number of samples to be tested • What is being evaluated with the specific testing method • How data interpretation will be affected • How equipment calibration and maintenance will be affected • How data results should be documented and reported • How conclusions will be documented and used • Criteria for a final evaluation of staff performing the test

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Table 12.9  Validation Measurements Validation Criteria Linearity range Dynamic range Sensitivity Limits of detection Selectivity of the method Matrix effects and interferences Limit of repeatability and/or reproducibility Sizing precision Stability of measured compounds Population distribution Measurement uncertainty

Description The extent to which an analytical procedure produces a signal directly proportional to the concentration or mass of the sample. The range of sample quantities that can be detected, from the highest and lowest. Minimum concentration of the particular substance that can be quantified with an acceptable level of precision and accuracy. The lowest concentration or smallest amount of sample that can be statistically differentiated from baseline noise. The extent to which an analytical procedure is free from interferences arising from non-analytes, including matrix components. Ability to distinguish between true results and artifacts or mixture components. Evaluates the reproducibility of results from repetition of the testing as appropriate to the type of method being evaluated. Determined by repeating a measurement over a specified time frame appropriate for the intended analytical procedure use. Mixture interpretation, mimicking, and evaluating detectable mixture ratios. Statistical evaluation (e.g., population studies and allele frequencies). Value given to quantitative measurements to identify that the value is not precise. Since DNA profiling is a qualitative measurement (rather than a quantitative measurement), it is not subject to an estimation of uncertainty value.

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Additional Resources ASCLD/LAB. 2010. Program Overview. An ISO/IEC 17025 Program of Accreditation. http://www.ascld-lab.org/documents/AL-PD-3041.pdf (accessed September 29, 2013). FBI. 2011. The FBI Quality Assurance Standards Audit for Forensic DNA Testing Laboratories. http://www.ascld-lab.org/documents/AL-X-012-08262011.doc (accessed September 29, 2013). International Organization for Standardization. ISO/IEC 17025:2005. General Requirements for the Competence of Testing and Calibration Laboratories. http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail. htm?cs​number​=39883 (accessed September 29, 2013). SWGDAM (Scientific Working Group of DNA Analysis Methods). 2011. Quality Assurance Standards for DNA Databasing Laboratories. http://www.swgdam.org/FBI%20Director%20Databasing%20Standards%20Revisions%20 APPROVED%20and%20final%20effective%209-1-11.pdf (accessed September 29, 2013).

Attachment A

X

X

Gloves

Laboratory coats, reusable

Data analysis software Profile quality assessment software Data interpretation software

Computer workstation

Item

X

X

X

X

X

X

Informatics X

X

X

Laboratory Consumables X X

X

X

Powder-free gloves are cleaner. Because people are often allergic to latex, labs use nitrile gloves for individuals with latex sensitivities Lab coats minimize exposure and contamination. Must be laundered, frequency depends on contamination hazards. Must send to a specialized laundry service

LSAM/DNAVIEW

X

Printers, ink, paper, barcode readers, barcode printers, Laboratory Information Management System GeneMapper ID-X OSIRIS

X

X

Associated Materials/Comments

X

X

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies

232 DNA Analysis for Missing Persons in Mass Fatalities

X

X

Office supplies

Bench paper

Kimwipes® EX L Delicate Task Wipers Small

X

Paper towels

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Protective eyewear

Laboratory coats, disposable Face masks

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

(Continued)

Disposable coats must consider increase in solid waste Ear-loop face mask helps to prevent contamination Safety lens protects eyes from UV light exposure, hazardous chemicals, and materials that could get into the eyes Used for cleaning benches and hands, and for bench protection Pens, highlighters, rulers, staplers, staples, tape, tape dispensers, paper clips, paper punch (3 holes), organizer, folders, Post-it® Notes, mouse pad, pen holder, calculator, calendars, batteries, paper, binder clips Absorbent bench paper roll, 19 in. W × 250 ft. L (2 rolls/case) Delicate, low lint, antistatic, non-abrasive wipes

Laboratory Development 233

Ethanol squeeze wash bottle Sterile ultrapure water deionized/ distilled Distilled/ deionized water squeeze wash bottle

4.5" × 8.4" in. pop-up box. 280 wipes per box, 30 boxes/ case Kimwipes Delicate Task Wipers, 14 7/10 × 16 6/10 Cleaning supplies Ethyl alcohol

Item

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X X

X

X

X

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X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

500 ml total volume

500 ml total volume (6 bt)

Laboratory should follow a set cleaning schedule 95% Denatured 500 ml (preparation 125 ml ethanol to 375 ml of distilled/deionized water) 500 ml total volume

Delicate, low lint, anti-static, non-abrasive wipes 140 sheets/box, 15 boxes/case

Associated Materials/Comments

234 DNA Analysis for Missing Persons in Mass Fatalities

Sterile cotton-tip swabs with applicator; individually wrapped

“Decon Bacdown” antimicrobial hand soap; pump bottle Sharpie® Ultra Fine Point markers Bleach squeeze wash bottle DNA AWAYTM squeeze bottle Bleach

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(Continued)

Cleaning supply for decontamination of tools and work area (1 part bleach to 9 parts water), 60 oz. Because bleach degrades in light, bleach mixture should be made fresh only when needed. Used for collecting buccal swabs, swabbing evidence items and select stains Sterile 2 pack

Drip free and prevents spilling; easy to control with label Decontamination of surfaces

Writing on tubes, plates, and evidence packaging

For hand cleansing

Laboratory Development 235

X

X

Evidence collection kits

Sexual assault collection kits

Item

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

Sterile plastic stick swabs, swab boxes, swab box integrity seals, sterile water ampoules, large evidence label, large evidence seal, biohazard label, pair of nitrile gloves, antimicrobial towelettes, evidence box Authorization for collection and release of evidence and information form; medical history and assault form; a 20" × 30" white paper sheet; 2 outer clothing bags and 1 panties bag; debris col­lec­tion for nail scraping; towel and comb for pubic hair combing; envelope for pulled pubic hairs; slides, swabs, and boxes for vaginal swabs and smear; slides, swabs, and boxes for rectal swabs and smear; slides, swabs, and boxes for oral swabs and smear; envelope for pulled head hairs, paper disk and envelope for saliva sample, 2 sterile swabs, swab boxes, and anatomical drawings chart

Associated Materials/Comments

236 DNA Analysis for Missing Persons in Mass Fatalities

Biohazard waste bags for benchtop Benchtop biohazard bag holder Sharps container

Biohazard waste containers Biohazard waste bags

X

X

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VWR® Premium Aluminum Foil Saran Wrap or VWR lab wrap plastic Sticky mats

X

X

X

Parafilm

X

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X

Must be present for disposal of any sharps instrument (Continued)

Benchtop holder for benchtop bags

Cleanline 24" × 36" Blue/Blue Sticky Mat removes trace contamination from shoes 6-gal red biohazard poly waste container w/foot lever Biohazard waste bag (10 gal, 50/bx, red) for biohazard and infectious waste 8.5 × 11 biohazard disposal bag, HDPE for benchtop holder

Minimizes evaporation of a liquid, reinforces bottle seals, and has a vacuum hose Roll W × L: 4 in. W × 250 ft. L Used for sealing and storage 12" × 25" roll 12" × 100"

Laboratory Development 237

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X

Labels for original bone storage bag white inkjet shipping labels, 2" × 4" Plastic quart size bag Sterile specimen containers Biohazard specimen bags Scalpel with replaceable blade Sterile stainless steel scalpel blades

X

X

Weigh boats

Item X

X

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

No. 20, fits handle no. 4, (www. carolina.com)

Scalpel, replaceable blade, no. 4 plastic handle (www.carolina.com)

For bone samples

For bone samples

For bone samples

Weigh boats weighing dish, three sizes 1", 3", and 5" used to weigh evidence and/or for reagent preparation 250/pack

Associated Materials/Comments

238 DNA Analysis for Missing Persons in Mass Fatalities

X

X

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X

Post-it® Notes

Coin envelopes, 2-1/4" × 3-1/2" brown Kraft coin envelopes

Kraft envelopes, 9" × 12", brown 96-well optical plate 96-well optical plate, full plate cover (PCR) MicroAmp® clear adhesive film

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Tamper-evident tape Weigh paper

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Full plate cover, prevents evaporation during thermal cycling Seals plate to prevent evaporation, cross contamination (products. invitrogen.com) (Continued)

Sample analysis on genetic analyzer

6" × 6" paper is smooth, lightweight, and dust free used for sample evaluation and/or reagent preparation Post-it® Note Pads, 3" × 3", yellow, 100 sheets per pad. Can assist with evaluating hair samples For packaging evidence that should be separated, repackaging evidence, or packaging evidence that was created in the lab; 500 per box Packaging evidence samples

Maintains integrity of evidence seal

Laboratory Development 239

Septa Strip for buffer and water trays 3130 and 3100-Avant capillary array 310, 3100, and 3130 3130 and 3100-Avant 310 electrode 3130 and 3100-Avant 3130 and 3100-Avant & 310 310 running buffer 3130 and 3100-Avant

Item

1 buffer and 2 water trays 3100 and 3130-Avant 16 capillary array Anode buffer reservoir jar Array ferrule sleeves 310 Platinum cathode electrode Array ferrule knob Anode buffer jar 25 ml 10 × running buffer 3100/3100-Avant polymer block assembly

X X X X X X X X

Associated Materials/Comments

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

240 DNA Analysis for Missing Persons in Mass Fatalities

POP-4TM polymer for the 3130 and 3100-Avant Genetic Analyzer POP-4TM polymer for the 310 Genetic Analyzer 310 48-tube sample tray 310 Genetic Analyzer sample tubes (0.5 ml) 310 Genetic Analyzer septa for 0.5 ml sample tubes 310 1.0 ml glass syringe Genetic analyzer buffer vials (4.0 ml) AB 310 pump block

3130 POP-4

310 POP-4

48-well sample tray 500 per bag

500 per bag

AB 310 Reservoirs for AB 310 AB 310 polymer block

X

X

X X

X

X X X

(Continued)

Laboratory Development 241

96 well plate retainer Hi-Di formamide GeneScan-500 LIZ AmpFℓSTR® Identifiler® (200 rxn kit) AmpFℓSTR® Yfiler® GS500 ROX size standard MicroAmp® 8-cap strip PCR strip tubes and caps for strip tubes, for optical plates

Reservoir, buffer/ water waste MicroAmp® 96-well base

Item

X X

X X

X X

X X X

X

X

Yfiler kit, Profiler Plus, COfiler kits 800 rxns Storage of post-PCR plates to prevent evaporation (300 strips N8010535 Invitrogen)

100 rxn/kit

Standard size for Identifiler kit 800 rxns 200 rxn/kit

Base is needed to stabilize the optical plates for sample preparation (4 bases/box) 96 well plate for 3100 and 3130 (4 bases/box) 25-ml bottle

X X

Reservoirs for AB 3130

Associated Materials/Comments

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

242 DNA Analysis for Missing Persons in Mass Fatalities

Micropipette tips—Aerosol resistant (filtered) Micropipette tips—Aerosol resistant (filtered) Micropipette tips—Aerosol resistant (filtered) Micropipette tips—Aerosol resistant (filtered) Micropipette tips—Aerosol resistant (filtered)

96-well plate septa 3100 and 3130-Avant VWR® gelloading pipet tips  10 ul barrier tips X

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(Continued)

Sterile tips 1,000 μl (960/pk) 96/ rack; 10 racks/box

Sterile tips 200 μl (960/pk) 96/rack; 10 racks/box

Sterile 100 μl (960/pk) 96/rack; 10 racks/box

Sterile tips 20 μl (960/pk) 96/rack; 10 racks/box

Sterile tips 0.5–10 μl 96/rack; 10 racks/box Sterile tips 0.5–10 μl (960/pk) 96/ rack; 10 racks/box

Septa covers plate for analysis on genetic analyzer 3100 and 3130 (10 mats) Pack of 1,000 (200 μl)

Laboratory Development 243

Sterile 15 ml conical tubes Sterile 50 ml conical tubes Sterile microfuge tubes 0.2 ml PCR Sterile microfuge tubes 2.0 ml Sterile microfuge DNA storage tubes 1.7 ml Axygen Sterile microfuge tubes 2.0 ml with screw cap 5 ml sterile plastic pipettes 10 ml sterile plastic pipettes 200/case 25 ml sterile plastic pipettes

Item

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X X X

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Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

Serological pipettes individually wrapped 200/case

Individually wrapped serological pipettes 200/case Serological pipettes individually wrapped

2.0 ml screw cap tubes 500/bag

1.5 ml 500/pk Eppendorf® microcentrifuge tubes Tube for storage; minimizes DNA adherence to tube walls 500/pk

0.2 ml thin-wall PCR tube with flat cap 1,000/pk DNase, RNase free

20 bags of 25 polypropylene plastic

20 bags of 25 polypropylene plastic

Associated Materials/Comments

244 DNA Analysis for Missing Persons in Mass Fatalities

QIAamp DNA Investigator Kit Organic extraction: To include phenol, chloroform, and isoamyl alcohol reagents Quantifiler kit TE Buffer (100 ml)

X

X

X X

X

Protective face shield Liquid nitrogen storage dewars

X

X

X

X

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Cryo gloves

Barcode printer labels Barcode label printer Barcode scanner Liquid nitrogen

X

X

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X

X X

X

X

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X

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X

400 rxns/kit [10 mM tris-HCl containing 1 mM EDTA•Na2] pH 8.0 (Continued)

Static evaporation rate of 0.15 L/ day; for transport and transfer of liquid nitrogen per use DNA extraction kit (50 DNA preps/ kit) Lysis buffer, chloroform, phenol with tris-HCL, phenol chloroform isoamyl alcohol PCI 25:24:1, 100% ethanol, 70% ethanol

Freezing bone and teeth to prepare sample for pulverizing Heaviest-duty waterproof cryogenic gloves for handling liquid nitrogen For handling liquid nitrogen

1,375 labels 1.25" × 1.0" and resin ribbon

Laboratory Development 245

Clinical Centrifuge Hermle Z 300

AB 3500 Capillary Array Service contract for 3130 Biohazard hood

Power protection system PCR System 9700 (base and block) AB 310 AB 3100-Avant capillary array AB 3130xl

DS-33 (dye set G5) Matrix Standard Kit

Item

X

X

X

X

X

X

X

X

X

X

X

X

X X

X

Equipment X

X

X

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

Laminar Flow, Biohazard-HoodClass II Universal Laboratory Centrifuges, 13,500 rpm, rotors for tubes and plates

310 Capillary 5 Pack 47 cm Applied Biosystems (4 capillary array genetic analyzer) Applied Biosystems (4 capillary array genetic analyzer) Applied Biosystems (8 capillary array genetic analyzer)

1200 V, 500 uA, 300 W

8 runs

Associated Materials/Comments

246 DNA Analysis for Missing Persons in Mass Fatalities

X

X

X

X X

X

X

X

X

X

X

X

Spectrafuge 24D Digital Microcentrifuge SpectrafugeTM Mini Centrifuge  2 × 0.2 ml strip tubes, individual adapters for 0.5 ml tubes, pack of 6 top loading balance Water bath/heat block Micropipettes, adjustable: 2, 10, 20, 200, and 1000 µl Pipette calibration system Multi-channel pipettes 8

X

X

Chemical hood

X

(Continued)

Pipette tracker software, reagents, photometer, barcode readers, barcode printers 8-Channel manual pipette, 5–50 µl

X X

Magnetic assist LTS Ranin

X

X

Heat block

Sartorius ELT Series

X X

Labconco fiberglass 30 laboratory hoods, Labconco 30300-00 hood with blower, 1/10 hp 24 × 1.5/2.0 ml rotor, 500 rpm to 13,300 rpm, with adapters 0.5/0.6 ml tubes, pack of 6 6 × 1.5/2.0 ml tubes

Laboratory Development 247

Refrigerators Microliter centrifuge

X

X X X

X

X

X

X

Timers “Diagger” Freezers

X

X

Graduated cylinder Glass beakers X

X

X

6-Place pipette holder PCR “dead” hoods

Erlenmeyer flask

X

Mixer (nutator)

Item

X X

X

X

X

X

X

X

X X

X

X

X

X

X

X

X

X X

X

X

X

X

X

X

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

Various sizes for cleaning tools, preparing reagents, making gels Various sizes for preparing and measuring reagents Magnetic clip and stand use on benchtop or clip to lab coat Summit chest freezer –30°C non-frost-free Laboratory refrigerator 10.1 ct 24 × 1.5/2.0 ml tubes 14,000 rpm,15Kx g

Labconco Purifier Filtered PCR Enclosures, Labconco 3970202 enclosures 0.6 m (2') nominal width Measuring reagents

For gentle mixing of liquids, sample tubes

Associated Materials/Comments

248 DNA Analysis for Missing Persons in Mass Fatalities

Paperwork bin Seal-film roller

Tweezers, fine point Test tube/conical tube rack Microfuge rack PCR tube rack

Multi-channel disposable solution basin Scissors Tweezers, set

MicroCentrifuge, Vortex Mixer, “two in one” WellAware GPS Pipetting System BioTX Autoclavable polypropylene carboys Thermometers

X

X

X

X

X

X

X X

X

X X

X

X

X

X

X

X X

X

X

X

X X

X

X

X X

X

X

X X

X

X

X X

X X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

Sealing PCR plate films (Continued)

1.5 ml and 2.0 ml tubes, 5 × 16 rows Capacity 8 × 12 rows for 0.2 ml tubes

8 × 50 ml conical centrifuge tubes

Stainless-steel; sharp points General Tool C421 Tweezer Set, 6-piece Fine point, stainless steel

VWR ASTM Partial Immersion, –20 to 150°C White reservoir pk of 10

20 L with spigot, used to hold purified water for lab bench

2400 rpm at 700 × G, vortex speed also 2400 rpm, 2 rotor-discs for 12 × 0.5 ml and 0.2 ml and 12 × 1.5 ml tubes Can also be used for laboratory training in pipetting

Laboratory Development 249

X

X

X

X

X

Dremel tool and blades Maxwell 16 System Maxwell® 16 Blood DNA Purification Kit BioRobotEZ1 Advanced

X

X

X

X

X

Bone grinder

Autopsy saw with 10' electrical cord Saw replacement blade Cutting board (plastic) Skull breaker

Item

X

X

X

X

Sample Extraction Post-PCR Analysis Reagent Sample Evaluation and and PCR (Amplification and Preparation Collection Accessioning Aliquotting Setup and Detection) Reporting and QC

Example List of Equipment and Supplies (Continued)

Automated extraction robot (1–6 samples/run)

3.5" long handle, 5/8" wide blade stainless steel Freezer mill, bone grinding Fisher 6770 7700, multiPro cordless set, for bone cutting and sanding Automated extraction robot, Maxwell® 16 SEV instrument 48 preps

Cutting bone

Associated Materials/Comments

250 DNA Analysis for Missing Persons in Mass Fatalities

Case—12 packs of 72 Slide mounting fluid; 4 ml, used for mounting slide covers and long-term storage Two dropper bottles of Nuclear Fast Red Stain and Picroindigocarmine (green); 30 ml each; a differential stain for sexual assault evidence slides Automated extraction robot (1–12 samples/run) 96 well PCR block Service contract for 7500

X X

X X X

X

XMAS Tree Stain

QIAcube

Note: X, typically used.

Real-time PCR system 7500 Real-time PCR service contract

40×–400×

20×–40×, sample evaluation

X

X

Stereo microscope 20×/40× Bino Head Halogen Lamp Compound light microscope Slides and slip covers Permount

Laboratory Development 251

Chapter Delivering Effective Training

13

Because a mass fatality human identification response requires a surge in personnel, proper training will be critical. A “training the trainer” approach is commonly used when there is a need to train large numbers of personnel. This approach to developing capabilities is twofold. First, trainers must master the knowledge and skills taught in the preceding chapters. Second, trainers must deliver effective training courses to meet the learning needs of the individuals who will play key roles in forensic DNA identification efforts. The trainer should focus on the knowledge and skills needed for delivery of effective training—planning the “right” training and delivering it in the “right” way. The information provided in this chapter will help the trainers prepare and conduct trainings. Key steps in the preparation, delivery, and evaluation of the training sessions are addressed.

13.1  Defining Stakeholder Learning Needs The primary outcome of this training information is to build on the existing knowledge and skills of individuals and groups, thus enabling a clearer understanding of the use of DNA in the identification of missing persons. Trainers must plan and deliver training material in a way that maximizes learning. Training must also meet the needs of trainees, motivating them to develop the competencies needed to successfully fulfill their roles and responsibilities in the various aspects of forensic DNA identification. Several groups have differing roles and responsibilities in forensic DNA analysis. Table 13.1 provides a high-level overview of organizations/groups and identifies their primary roles in DNA analysis. Once stakeholder roles and responsibilities have been identified, the next step is to determine what stakeholders need to learn to be fully capable of supporting forensic DNA identification. For example, Table  13.2 lists the learning needs for stakeholder groups and provides a crosswalk to the topics covered in Chapters 1 through 12 in this manual. The chapters in this book can be the foundation for training the many individuals who will take part in a human identification DNA effort following a mass fatality. 253

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Table 13.1  Roles and Responsibilities of Stakeholders Organization/Group

Role

University scientists

Education and research

Medical examiner/ coroner/medical legal institute

Human remains identification

Non-governmental organizations (NGOs) Politicians and government leaders Medicolegal professionals and anthropologists

Psycho-social support

Law enforcement and legal community

Legal implications for use of DNA identification in crimes and court cases

Medical community

Support missing person efforts

Decision and policy makers; funding Body recovery, storage, and identification

Responsibility Education of students who could be potentially employed in laboratories that will support the identification of missing persons Development of plans, policies, and procedures to support identification of human remains Responsible for the identification effort Provision of support for families of missing persons Dissemination of policies and directives Identification and recovery of human remains Re-association of human remains to minimize the number of DNA identifications Identify the individuals who are truly missing and whose bodies may need to be identified Identification of the legal boundaries for identification efforts Prosecution of perpetrators of crime and fraud Resolution of family disputes Maintain accurate and complete medical records so that they can be used in the identification process

13.2  Key Factors for Successful Training 13.2.1  Identify Participants and Schedule Training Understanding the learning needs of the individuals in the different stakeholder groups will determine how to organize the training and determine who needs to attend which training sessions. As noted, stakeholders have varying learning needs and therefore will need to attend different sessions. Once participants have been identified, it will be necessary to put a training schedule together. Multiple sessions of a chapter may be necessary if there are more than 10 individuals training in any given chapter. By scheduling multiple sessions, class size can be limited to no more than 10 trainees per trainer. This will provide a training environment conducive to interactive discussions and hands-on assistance. In addition to the types and numbers of trainees, the trainer must consider two other key factors for development

Delivering Effective Training

255

of the training schedule. One is the time frame for the training, and the second is the location of the training, which may require participants to travel. Finally, the length of time needed for both classroom and hands-on training must be factored in. For example, Table 13.3 lists the approximate times needed to conduct the training for each of the chapters. Attachment A provides a template for scheduling of the chapters.

13.3  Getting Ready for Training Preparation for the delivery of training can begin once trainers have identified trainees and developed a training schedule. Taking the time to prepare for delivery of the training material can help ensure the success of training sessions. Trainers should focus on three key areas: • Training materials and delivery methods • Training environment • Training evaluation methods The following sections discuss each of these areas. Attachment B provides a “Readiness Checklist” for trainers. 13.3.1  Training Materials and Delivery Methods Once the training schedule is complete, the next step is to review the training chapters and determine which materials are needed to support the training and which delivery method is most appropriate for the chapter. It will also be necessary to ensure that appropriate supplies and equipment are available. Attachment D lists the supplies and equipment necessary for chapters requiring hands-on experience. While the training schedule serves as a guide for the sequence of training, it might also be helpful to prepare a lesson plan listing the objectives, teaching methods, materials, supplies, and equipment needed for each chapter. Attachment C provides a template lesson plan. Trainers should also understand the key principles of adult learning. These principles should be kept in mind during the planning and delivery of training. Table 13.4 provides a general summary of key adult learning principles. Maintaining trainees’ interest can be a challenge when facilitating training sessions. Trainers should encourage participation and foster interactive discussion. Trainees will bring their own experiences and expectations to the sessions, all of which could impact the schedule. Therefore, it is imperative that trainers use skillful facilitation techniques to ensure the main points

Politicians and government leaders

Nongovernmental organizations (NGOs)

Select medical legal institute staff

University scientists

Organization/ Group

Develop courses and training materials to support DNA identification efforts of missing persons Understand how DNA testing supports accurate and timely identifications Understand the most efficacious way to provide assistance and psychosocial support to meet family needs Understand the importance and complexity of DNA identification to ensure funding and resources are allocated in a way that removes roadblocks to testing

Learning Need

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

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

Table 13.2  Crosswalk Stakeholder Learning Needs to Training Manual Chapters

256 DNA Analysis for Missing Persons in Mass Fatalities

Medicolegal and Understand the anthropological impact body experts fragmentation has on the DNA identification effort Law Understand DNA enforcement testing and its role and legal in courts of law community Medical Understanding their community important role in finding individuals alive and using medical records for presumptive identification

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

Delivering Effective Training 257

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DNA Analysis for Missing Persons in Mass Fatalities

Table 13.3  Time Estimates for Training by Chapter Chapter Chapter 1: Human Identification through DNA Analysis Chapter 2: Mass Fatalities Chapter 3: Postmortem Functions—Body Recovery and Morgue Operations Chapter 4: Antemortem Functions—Family Assistance Operations Chapter 5: Identification of Remains Chapter 6: Identification and Collection of Biological Samples from Human Remains Chapter 7: Identification and Collection of DNA Reference Samples Chapter 8: Application of DNA Technology for Human Identification Chapter 9: DNA Profile Analysis and Interpretation Chapter 10: DNA Sample, Case, and Data Tracking Using Information Technology Tools Chapter 11: Implementing and Maintaining a Quality DNA Program Chapter 12: Laboratory Development Chapter 13: Delivering Effective Training Mock Field Exercise Preparation and Execution a

Lengtha (hours) 2.5 2.5 3.5 5 5 5 5 5 5 5 5 4 4 16

Includes time for a 10 minute break per hour taught, 30 minutes for “questions and answers,” and 1.5 hours for review and competency testing for Chapters 4 through 13.

Table 13.4  Key Adult Learning Principles Typically the adult learner: • Needs to understand the relevancy of the learning material. Why is it necessary for them to learn the material and skills, and how will this help them in their jobs? • Likes to be actively involved in the learning. • Likes to build on their existing experiences and knowledge. • Learns in different ways (e.g., visual, auditory, hands-on/haptic). • Needs to clearly understand how they will be evaluated.

of the lesson are presented and discussed. Examples of effective facilitation techniques include: • Get everyone involved from the start (e.g., introductions, ice-breaker exercise, game, puzzle) • Create interest in the topic • Relate lesson to real-life experiences • Explain objectives • Let trainees know how the lesson will benefit them in their profession and organization

Delivering Effective Training

• • • • •

259

Ask questions—who, what, why, when, and how Summarize and reinforce main points Restate trainee comments or questions to ensure understanding Give positive feedback, especially for skill training Be aware of nonverbal messages—yours and theirs (e.g., body language, tone of voice, facial expression, eye contact)

13.3.2  Training Environment The training location should be conducive to learning and must be distraction-free. While it is not always possible, the location should be “off-site” and away from the trainees’ work location. This will ensure trainees are not distracted by work duties and responsibilities. The room where learning will take place must be large enough to accommodate all of the participating students, and seating arrangements should ensure trainees can easily see teaching aides, hear the trainer, and easily participate in discussions. A U-shaped arrangement of tables and chairs is highly recommended for the classroom portion of the training. Arrangements must also be made for conducting skill training and participating in mock exercises. For this portion of the training, there should be sufficient supplies and equipment. 13.3.3  Training Evaluation Methods Trainees may be evaluated for knowledge and skills gained during training. The time allotted for the training of each model has time built-in for preparation and review of course material, and, if necessary, remedial training. Each trainee should achieve a level of competency at the end of the course. In order to ensure that the personnel can properly complete their duties, a competency exam should be administered and the trainee should be able to pass. This demonstrates that the trainee is competent to perform the duties he or she was trained on.

Additional Resources Blanchard, P.N., and Thacker, J. 2012. Effective Training. Upper Saddle River, NJ: Prentice Hall. Davis, J.R., and Davis, A.B. 1998. Effective Training Strategies: A Comprehensive Guide to Maximizing Learning in Organizations. San Francisco: Berrett-Koehler. Philips, J., and Stone, R. 2002. How to Measure Training Results: A Practical Guide to Tracking the Six Key Indicators. New York: McGraw-Hill. Silberman, M., and Auerbach, C. 2006. Active Training: A Handbook of Techniques, Designs, Case Examples, and Tips. San Francisco: Pfeiffer.

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Attachment A

DNA Analysis for Missing Persons in Mass Fatalities

Delivering Effective Training

Attachment B

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262

Attachment C

DNA Analysis for Missing Persons in Mass Fatalities

Delivering Effective Training

Attachment D

D (continued)

263

264

D (continued)

DNA Analysis for Missing Persons in Mass Fatalities

Delivering Effective Training

265

Terminology

A AABB Standards for Relationship Testing Laboratories:  Standards that dictate laboratory practices for paternity, biological, and other family relationship testing ABO Blood Typing:  A test that uses antibodies to detect variations on the surface of human red blood cells; individuals are typed as having A-, B-, O-, or AB-type blood due to the presence or absence of agglutinogens, type “A” and type “B” Accreditation:  A process in which an unbiased third-party organization verifies that a laboratory conforms to specified standards, is competent and can achieve and maintain a level of quality; demonstrates compliance with standards, and assures a quality control program Accrediting Bodies:  Agencies that may conduct laboratory evaluations and determine if a laboratory meets specified standards related to maintaining a level of quality Allele:  Alternative form of the same DNA section at a specific locus Allele Frequency: A measure of the commonness of DNA fragments of variable length and/or sequence at a particular locus in a population; the proportion of alleles of a particular type in a sampled population Allele Frequency Databases or Population Databases:  Data that has been collected by sampling a racial population to determine how often (common or rare) alleles are in the sampled population; used when performing a statistical analysis on the match results generated from DNA profiling to determine the significance of the match Allelic Dropout:  Failure to detect an allele within a DNA profile because of one or more of the following reasons: mutations in the DNA nucleotide sequence that prevents optimal binding of the primer, failure of the sequence to amplify during PCR due to low template DNA or degradation Allelic Ladder:  An artificial mixture of the common alleles in the human population for a particular locus used in the determination of alleles in a DNA sample; analogous to a ruler

267

268

Terminology

Amelogenin:  A gene that codes for tooth enamel present on the X and Y sex chromosomes that is used in DNA identification testing to determine the gender of the contributor of a biological sample American Society of Crime Laboratory Directors/Laboratory Accredita­ tion Board (ASCLD/LAB):  A not-for-profit cooperation that offers voluntary accreditation to public and private laboratories in the United States and internationally for general forensic disciplines; see www.ascld-lab.org Amplification:  Biochemical reaction that is used to multiply the quantity of specific sequences in a DNA sample; see Polymerase Chain Reaction Anneal:  Second step of the polymerase chain reaction where primers bind to the primer binding site on the 3' end of the target sequence creating a double strand whereby DNA polymerase can begin extension Antemortem:  Before death Antemortem Information:  Information or data collected about a reported missing during the opening and investigation of a missing person case; is typically collected from family members in order to identify human remains; often this information involves the characteristics of the person, the circumstances of when he or she was last seen, and may include information on medical records and potential DNA reference samples Artifact:  A non-allelic product observed in a DNA profile, which is caused by the amplification reaction or the detection process Autosomal Chromosomes (autosome): Chromosomes that are not sex chromosomes; human genome contains 22 autosomes

B Background Noise:  Small peaks that occur along the baseline in the electropherogram of a DNA profile, typically caused by the instrument, air bubbles, urea crystals, and sample contaminants that do not necessarily indicate the presence of DNA Bases:  The four building blocks of DNA: adenine, cytosine, guanine, thymine; commonly referred to as A, C, G, T, respectively Biohazard Container:  A container for materials potentially contaminated with blood-borne pathogens or other biohazardous material Biological Evidence:  Evidence commonly recovered from crime scenes in the form of blood, semen, saliva, tissue, hair, bones or teeth Biological Fluids: Fluids that have human or animal origin, commonly encountered at crime scenes (e.g., blood, semen, saliva, urine)

Terminology

269

Body Recovery: Involves the location, documentation, collection, and transportation of human remains to designated body processing sites/morgues

C Cambridge Reference Sequence (CRS): Also known as the Anderson sequence; a mitochondrial type that is used as a standard sequence to compare results of a mitochondrial DNA analysis where results are reported as differences from the CRS in position and base; example, the CRS contains a T at position 16126 (16126T) and an analyzed sample contains a C at the same location; the result of the analysis for the sample would be reported as 16126C; if no other base changes are reported, then it is assumed that the analyzed sample and the CRS contain the same sequences Capillary Electrophoresis (CE):  A method used in genetic analysis of DNA that uses an electrokinetic injection to introduce negatively charged DNA molecules to narrow silica capillaries containing a polymer solution; under the influence of a high voltage electrical field these DNA molecules labeled with florescence migrate based on size and are detected with a camera Case File:  A record of information kept together that documents the characteristics and disposition of evidence related to a single case; may include data on source, collection, testing results, and reporting for all tests and procedures performed on the evidence and track chain of custody Case Identifier:  The unique numeric or alphanumeric characters assigned to a single case tested by the laboratory Cell:  The smallest component of life capable of independent reproduction; contains the organelles from which DNA is extracted Chain of Custody:  Key aspect of maintaining sample integrity; written or electronic record documenting the location where or from whom the sample was collected, the individual who collected the sample, date and time of collection, and the description of the item; also documents movement of the evidence between persons or storage to establish a record of possession for the item Chromosome:  Dense structures composed of nuclear DNA and histones; human genome is composed of 22 matched pairs of autosomal chromosomes and two sex-determining chromosomes Clean/Sanitize:  The process of removing biological and/or chemical contaminants from equipment, tools, supplies and surfaces

270

Terminology

Closed Mass Fatality Event:  An event resulting in more deaths than the local available resources can process where the identities of the individuals in the deceased population are known; example: a crash of a large airplane would be closed because the airline maintains a manifest of the passengers and crew CODIS Core Loci:  Thirteen STR (short tandem repeat) sequence locations identified for use in the Combined DNA Index System (CODIS); the locations are TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, THO1, VWA, D13S317, D16S539, D18S51, and D21S11 Collection:  The process of identifying, documenting, gathering, and packaging physical evidence Combined DNA Index System (CODIS): U.S. Federal Bureau of Investigation sponsored database that links DNA evidence obtained from crime scenes to the potential perpetrators of crime by comparing crime scene evidence to DNA profiles obtained from offenders, also contains profiles from missing persons, unidentified human remains, and relatives of missing persons; three levels of CODIS: the Local DNA Index System (LDIS), used by individual laboratories; the State DNA Index System (SDIS), used at the state level to serve as a state’s DNA database containing DNA profiles from LDIS labs; and the National DNA Index System (NDIS), managed by the FBI as the nation’s DNA database containing all DNA profiles uploaded by participating states; there are strict rues and regulations pertaining to the use and dissemination of the information in CODIS Combined Relationship Index:  The mathematical product of the likelihood ratios (or relationship indexes) of each individual locus reported; if genetically linked loci are used then haplotype frequencies must be employed Commingling: A result of multiple fragmented remains from different individuals in a single space; if not carefully examined by a specifically trained professional, the remains may be mistaken as coming from a single individual Contamination: An undesirable transfer of DNA from one source to another source; for example, contamination can occur between samples, from samples to reagents, and from samples to equipment and supplies Control Sample:  Sample with known results introduced during DNA profiling process to determine if the reagents and methodology used were performed accurately to ensure that the results obtained from the sample are robust, reliable, and reproducible; negative DNA control: samples without DNA to show that the reagents do not have contaminating DNA; positive DNA control: contains a sample of DNA with a known profile; if controls produce expected results,

Terminology

271

it is established that the analysis was performed properly and the reagents performed as expected Cytoplasm: The jelly-like material and cell organelles (excluding the nucleus) that are contained withing the plasma membrane of a cell

D Degradation:  The fragmenting or breakdown of DNA by chemical or physical means including exposure to environmental conditions that may prevent a sample from yielding a complete or usable DNA profile Developmental Validation: The testing and determination of conditions and limitations of a novel DNA methodology for use with DNA samples; typically completed by the manufacturing company Dideoxy Sequencing: Dideoxynucleotide sequencing, also known as the “Sanger method”—technique to synthesize complementary strands of DNA to determine the order of nucleotides in a DNA sample that uses polymerase incorporation of fluorescently labeled dideoxyribonucleotide triphosphates (ddNTPs) as chain terminators instead of deoxyribonucleotide triphosphates to halt the synthesis of a complementary DNA strand at various points, creating strands that differ in length by a single base Differential Extraction:  Extraction technique that separates lysed epithelial cells from sperm cells in a sample; typically performed in sexual assault cases to separate the sperm fraction from the victim’s DNA Direct Reference:  A sample that has supporting documentation linking its origin to a missing individual; examples include samples taken for medical purposes Disposable Instruments:  Items that will be used only once to collect or examine biological samples then discarded (e.g., scalpel, razor blade, tweezer) Deoxyribonucleic Acid (DNA): Molecule often referred to as the “blueprint of life;” genetic material present in all cells of the body with the exception of mature red blood cells; DNA codes for replicating the cell and generating proteins; also determines physical attributes; nuclear DNA is inherited equally from each biological parent and mitochondrial DNA is inherited from the mother DNA Analysis:  Also known as DNA profiling, a process of testing to identify DNA patterns or types to determine or confirm the identity of a source of DNA; in the forensic setting, this testing is used to exclude or not exclude individuals as possible sources of biological materials and can also be used to indicate relationships such as parentage

272

Terminology

DNA Mixtures:  A DNA sample that contains the genetic material from more than one individual DNA Profile:  A set of numbers that are determined in the laboratory based on the characteristics at particular locations in an individual’s DNA; the numbers are a measure of repeat sequences at several locations on the DNA molecule Dye Blobs:  The result of unincorporated dyes (fluorophores) from the primers in the amplification kit that are detected during capillary electrophoresis; typically exhibit poor peak morphology (smaller and broader peaks) and generally do not affect interpretation of a DNA profile (appear outside of the allele calling range)

E Electropherogram:  A plot of results from an analysis done by electrophoresis automatic sequencing, may be used for deriving results from genealogical DNA testing, paternity testing, DNA sequencing, genetic fingerprinting, etc., and shows a sequence of data that is produced by an automated DNA sequencing machine Electrophoresis:  The application of an electrical current to an appropriate medium for the purposes of separating charged molecules; separation can also be based on size Elimination Sample:  A sample of known source that is provided with consent and used to exclude the donor as a possible source of DNA that may be present in a sample Epithelial Cells:  The outside layer of cells that covers the internal and external body of organs including skin and mucus membranes; buccal swabs are a collection of epithelial cells from the inside of the cheek in the mouth Evidence:  Something that can be scientifically analyzed to prove or disprove a fact or theory in question in legal matters Excluded:  A determination made after a scientific analysis that determines that an individual does not contain the genetic characteristics necessary to be the source of a biological sample because the two samples are dissimilar Exclusion:  A test result indicating that an individual is shown to be eliminated as the source of the DNA evidence or as a parent of a child Exogenous DNA:  Contaminating of DNA unrelated to the sample or reagent

Terminology

273

F Family Assistance Center:  A physical location that houses capabilities and services to provide a place for family members to seek support and assistance regarding a missing person including the opening of a missing person case and the collection of reference samples Family References or Kinship Samples:  DNA samples from close biological relatives of a missing person used to construct/infer the victim’s profile Forensic Science:  The application of science to law; the use of proven scientific fact for the evaluation of evidentiary samples to prove or disprove a theory in legal matters Fragmentation:  Human remains in multiple pieces and separated, typically requires that each fragment is treated as a separate case

G Gel:  Semisolid matrix (usually agarose or acrylamide) used in electrophoresis to separate molecules Gene:  The basic unit of heredity; a functional sequence of DNA that typically codes for a protein Gene Frequency:  The relative occurrence of a particular allele in a population Genetic Loci:  Physical location on a chromosome for a particular gene or DNA sequence Genetics:  The study of inherited characteristics Genome:  All of the genetic material of a particular organism; humans contain nuclear (chromosomal) and mitochondrial genetic material Genotype:  The genetic constitution of an organism Grief:  A multi-faceted response to loss, particularly to the loss of someone or something of importance although often focused on the emotional response to loss, it also has physical, cognitive, behavioral, and social implications

H Haploid:  Having a single set of chromosomes Haplotype: A particular combination of alleles in a defined region of a chromosome Hardy-Weinberg Equilibrium:  Principle that states in large random interbreeding population, barring disturbing forces such as non-random

274

Terminology

mating and mutations, the gene and genotype frequencies will remain constant over time Heredity:  The transmission of traits from one generation to the next Heteroplasmy:  The presence of more than one mitochondrial DNA type within a single individual Heterozygote: An individual exhibiting two alleles at a particular locus which is detected and exhibited as two distinctive peaks in an electropherogram Homozygote: An individual exhibiting indistinguishable alleles at a particular locus; exhibited as a single peak for a particular locus in an electropherogram Hypervariable Region:  Location within the D-loop of mitochondrial DNA that exhibits a high degree of variation in nucleotide sequence known as HV1 and HV2

I Included (Inclusion): A determination made after the scientific analysis of a biological sample that concludes that an individual cannot be excluded as a potential contributor Inconclusive:  A determination after a scientific analysis in which no conclusion can be reached; inconclusive results can be caused by results that do not meet the laboratory’s testing standards Inter American Accreditation Cooperation (IAAC): An association of accreditation bodies whose mission is to promote cooperation among accreditation bodies and other interested parties in North and South America for the development of conformity assessment structures to achieve the improvement of products, processes, and services Internal Lane Standard (ILS):  Is composed of a range of DNA fragments each with a known base pair size and is injected with each sample into the CE to use in the sizing of sample allele peaks, analogous to a ruler Internal Validation:  The testing and determination of conditions and limitations of a novel DNA methodology for use with DNA samples to show that established methods and procedures perform as expected within the laboratory International Laboratory Accreditation Cooperation (ILAC): Inter­ national organization with the goal of facilitating trade by promoting the acceptance of accredited test and calibration results through a network of mutual recognition agreements among accreditation bodies

Terminology

275

K Known Samples:  A DNA sample with known origin; also referred to as reference samples that are used to determine the identity of a missing person; see Reference Samples

L Laboratory:  A facility with the capability to perform the DNA analysis of samples while maintaining sample integrity Laboratory Information Management System (LIMS): Computer program, typically tailored for a specific laboratory, with the capability to aid in common laboratory actions including case file management and sample tracking Likelihood Ratio:  Also referred to as kinship index or relationship index; is typically used to statistically define the significance of matching DNA loci in relationship testing; expresses the chance of obtaining the DNA profiles under two mutually exclusive hypotheses—the first hypothesis is that the sample from the remains is related to the family as reported, and the second hypothesis is that the sample from the remains is unrelated to the family but that the family pedigree is otherwise accurate Linkage:  The affinity of certain loci or alleles to be inherited together Linkage Disequilibrium:  Genes or genetic markers that are not in random association Locus (plural loci):  The physical location of a gene or DNA region of interest on a chromosome Low Copy Number DNA: A DNA sample with a presumed low quantity of DNA; typically requires amplification using a very sensitive reaction and very careful interpretation of results

M Major Contributor Profile: The most apparent DNA profile within a DNA profile where multiple individuals have contributed biologic material Marker:  A DNA sequence of a known location on a chromosome that is used to identify a particular gene or trait Mass Casualty:  Incident involving numerous human injuries Mass Disaster: Destructive incident that results in great devastation to property and local infrastructure

276

Terminology

Mass Fatality:  An event resulting in more deaths than the local available resources can process and requires assistance from outside agencies and organizations Match:  A determination made after the scientific analysis that concludes that two samples are similar and may share the same source; the statistical significance of the match is determined by evaluating the frequency of the matching alleles by matching alleles in the relevant population Measurement Scale:  An object showing standard units of length (e.g., ruler) used in photographic documentation to quantify a piece of evidence Mitochondrial DNA (mtDNA):  Circular DNA found in the mitochondria of eukaryotic cells that is inherited solely from the biological mother; mtDNA sequences are less discriminating than STR profiles Mixed Profile: DNA sample with multiple sources resulting in a profile commonly exhibiting three or more alleles at a single loci Morgue Operations:  Component of a mass fatality response involving the key tasks of processing and analyzing human remains and personal items, processing and determining postmortem information, and storing the bodies until release or other disposition; it also may (but not always) include making identifications by analyzing post- and antemortem information Multiplexing:  The use of multiple primer sets in a single reaction to amplify multiple target sequences during PCR Mutation:  A change in DNA nucleotide sequence from one generation to the next at a particular locus Mutation Frequency: A measure of the occurrence of changes in DNA nucleotide sequence from one generation to the next

N No Results:  A determination made after the scientific analysis indicating that it is not possible to determine a profile or if two samples are similar or dissimilar due to a lack of interpretable results Non-match:  The elimination of an individual as the source of a biological sample when one or more types from a specific location in the DNA of a known individual are not present in the type(s) for that specific location in the DNA obtained from an evidence sample Nuclear DNA:  DNA found in the nucleus of a cell Nucleated:  Having a nucleus or nuclei Nucleotide: Basic component of a nucleic acid, including DNA, whose sequence encodes the informational content; four types, adenine, thymine, cytosine, and guanine commonly represented by A, T, C, and G, respectively

Terminology

277

Nucleus: The eukaryotic cellular organelle that houses genetic material organized into chromosomes

O Off Ladder (OL):  A DNA fragment length that does not correspond with an allele on the allelic ladder Open Mass Fatality Event:  An event resulting in more deaths than the local available resources can process where identities of the individuals in the deceased population are not all known

P Partial Profile:  The result of a DNA analysis that does not yield complete results for all loci analyzed Paternal Inheritance:  Genetic material that is inherited from one’s biological father only Paternity Index (PI):  Statistical value used in questioned paternity cases that is the likelihood that the genetic alleles obtained by the child support the assumption that the tested man is the true biological father instead of an untested randomly selected man; combined paternity index (CPI): determined by calculating the product of the individual paternity indexes for each tested loci PCR Inhibitors: A substance that interferes with the polymerase chain reaction (PCR) including dyes, soil, and heme (from hemoglobin); see Polymerase Chain Reaction Personal Items:  Objects purported to have or contain the DNA from the reported missing because they are thought to have been used by or came from them; unlike direct references, these items have no associated documentation linking them to the reported missing Personal Protective Equipment (PPE):  Articles such as disposable gloves, face masks, eye protection (goggles), and shoe covers that are used to provide a barrier to keep biological or chemical hazards from contacting the skin, eyes, and mucous membranes and to avoid contamination of a biological sample Polymerase Chain Reaction (PCR):  An in vitro biochemical process that results in millions of copies of desired DNA sequences through repeated cycling of a reaction in three steps: first: denaturation of the template DNA; second: annealing of primers to complementary sequences at a predetermined temperature; and third: extension of the bound primers by a DNA polymerase

278

Terminology

Polymorphic:  Variable, more than one kind; different forms of the same locus Polymorphism: Variations in DNA sequences in a population that are detected in human DNA identification testing Positive Identification:  Most reliable form of identification; based upon scientific and forensically based data that includes DNA, fingerprints, and dental records Posterior Odds (PO):  Posterior Odds = Likelihood Ratio × Prior Odds Postmortem Information:  Information collected from human remains at the morgue including x-rays, anthropological and dental information Precision:  Refers to the agreement or similarity in value of a series of individual measurements or results; obtaining the same result for a sample from the same source after multiple analysis when using the same or equivalent equipment and supplies Presumptive Identification:  Identification based on comparative data, but none of which is scientifically or forensically based Primer:  Short pre-existing polynucleotide chain to which new nucleotides can be added by DNA polymerase Prior Odds:  Prior Odds = Prior Probability/(1 – Prior Probability) Prior Probability (Pr):  The strength of the evidence that the individual is related as specified in the pedigree based only on the non-DNA evidence including the number of missing persons and gender Probability:  The chance of an event occurring Probability of Exclusion:  A statistical assessment used when interpreting samples with multiple contributors which provides an estimate of the portion of the population that has a genotype composed of at least one allele not observed in the mixed profile Probe:  Single-stranded DNA or RNA of a specific base sequence, labeled either radioactively or immunologically, that is used to detect the complementary base sequence by hybridization Product Rule:  Calculates the expected chance of finding a given STR profile comprised of unlinked loci within a population by multiplying the frequency of occurrence of the combination of alleles (genotype) found at a single locus, by the frequency of occurrence of the genotype found at the second locus, by the frequency of occurrence, in turn, of each of the remaining genotypes in the profile Proficiency Tests:  An assessment of an analyst’s performance in conducting DNA analysis; can be classified as internal, where the test is produced by the agency taking the test, or external, where the test is obtained from an approved proficiency test provider; external tests can be open where the agency is aware of the test, or blind where the agency is unaware of the test

Terminology

279

Pull-up (Bleed-Through):  A peak seen in one or more color channels of an electropherogram that is not due to the presence of DNA, but to the inability of the collection software to discriminate between a signal from a different dye color

Q Quality:  Measure of excellence Quality Assurance:  Refers to those planned or systematic actions necessary to provide adequate confidence that a product or service will satisfy given requirements for excellence Quality Control:  Day-to-day operational techniques and the activities used to fulfill requirements of maintaining excellence including monitoring procedures and methods to verify that products meet specified standards that provide confidence in results Quality System: Organizational structure, responsibilities, procedures, processes, and resources for implementing quality management Quantitative Real-Time PCR (qPCR):  A method to determine the concentration of DNA in a sample using the polymerase chain reaction; determines the cycle number in which the concentration of specific amplified sequences meets a minimum threshold by measuring fluorescence after each cycle to calculate concentration

R Reagent Blank: An analytical control sample that contains no template DNA and is used to monitor contamination from extraction to sequence analysis; this sample is treated the same as any other forensic or reference sample being analyzed Recombinant DNA Technology:  A collection of techniques that combines DNA from multiple sources to make new DNA; used in the cloning of genes and the genetic modification of organisms Recombination:  The process in which chromosomes or DNA molecules are broken and the fragments are rejoined in new combinations typically during meiosis Reference Material:  A material for which values are certified by a technically valid procedure and accompanied by, or traceable to, a certificate or other documentation that is issued by a certifying body Reference Samples:  Samples with known origin used to determine the identity of unknown human remains; typically one of three types: direct: a biological sample that has some sort of paperwork documenting

280

Terminology

its origin to a missing individual; personal items: objects thought to contain DNA from the deceased but without associated documentation; family or kinship: biological samples originating from relatives of the reported missing used to infer the DNA profile of the deceased Reported Missing (RM):  A person believed to be deceased as a result of a mass fatality Reproducibility: The ability to obtain the same result when the test or experiment is repeated Restriction Enzyme: A protein extracted from bacteria that recognizes specific, short nucleotide sequences and cuts DNA at or near the sequence Restriction Fragment Length Polymorphism (RFLP): Technique used to examine variable number tandem repeats (VNTR) sequences in which a restriction enzyme is used to cut the regions of DNA surrounding the VNTR; see also VNTR Review:  An evaluation of documentation to check for consistency, accuracy, and completeness RFU value:  Relative Fluorescent Unit; A measure of the amount of fluorescence detected by the CE, which correlates to the amount of DNA present in capillary electrophoresis analysis using a genetic analyzer

S Sequencing:  Determination of the order of nucleotides in a DNA or RNA molecule or the order of amino acids in a protein Sex Chromosomes (X and Y chromosomes):  Chromosomes that may be present or absent, or present in variable copies according to the gender of an individual; in humans, sex chromosomes represented by X and Y where “XX” corresponds to a genetic female and “XY” corresponds to a genetic male Short Tandem Repeat (STR) Typing:  DNA analysis method that targets regions on the chromosome which contain multiple copies of a short DNA sequence repeated in tandem Short Tandem Repeats (STRs):  Multiple copies of a small identical DNA sequence arranged in tandem; variations that occur between individuals are typically due to the number of repeats Single Nucleotide Polymorphisms (SNPs):  DNA sequence variations that occur at a single nucleotide (A, T, C, or G) Single Source Profile:  A DNA profile that has originated from only one individual Spikes:  Narrow peaks characterized by sharp narrow peaks appearing at the same time in multiple dyes usually attributed to fluctuation in

Terminology

281

voltage or the presence of minute air bubbles or urea crystals in the capillary Standard Operation Procedures (SOPs):  A prescribed set of steps that are to be followed routinely by all laboratory personnel for various functions in the laboratory including sample handling/testing, reagent preparation, decontamination, data review, and reporting; SOPs are often unique to each laboratory Stutter Band or Peak:  A minor peak appearing in an electropherogram one repeat unit smaller than a primary STR allele and/or occasionally, one repeat unit larger than the primary allele; each location typically has its own expected stutter values based on the unique characteristic of each locus Substrate:  The material upon which a biological sample has been deposited (e.g., upholstery, clothing, wood, carpeting, glass)

T Technical Review:  An evaluation of reports, notes, data, and other documents to ensure there is appropriate and sufficient basis for the scientific conclusions Tentative Identification:  Least reliable form of identification, based upon non-scientific, non-forensic information Threshold Value:  A minimum value that must be exceeded; in the review of data from an analysis performed on a capillary electrophoresis instrument, threshold value is the minimum relative fluorescent unit (RFU) that must be met or exceeded which detected peaks can be reliably distinguished from background noise, typically determined through in-house laboratory validation studies that will vary among laboratories Traceability:  The property of a result of a measurement where it can be related to an appropriate standard through an unbroken chain of comparisons typically reserved for items used for calibrations or analytical procedures Trace Evidence:  Physical evidence that results from the transfer of small quantities of materials (e.g., textile, fibers, glass fragments, paint chips, hair) Trauma:  An event that is perceived by an individual as being threatening to the life or integrity of oneself or others and must be accompanied by feelings of intense terror, horror, helplessness, or powerlessness causing the nervous system to be overwhelmed, be unable to adequately process the event, and fail to operate normalizing responses

282

Terminology

U Unknown Sample:  Biological sample for which its origin is not known; in mass fatality events, this sample is taken from unidentified human remains U.S. Federal Bureau of Investigation Quality Assurance Standards for DNA Databasing Laboratories:  Standards that describe the quality assurance requirements that laboratories performing DNA testing on database, known, or casework reference samples for inclusion in the Combined DNA Index System (CODIS) shall follow to ensure the quality and integrity of the data generated by the laboratory UV Light Source:  Light source that emits light in the ultraviolet region, 10–400 nm, used to visualize or enhance visualization of potential items of evidence including biological fluids, fingerprints, and fibers by causing these items to fluoresce or absorb light

V Validation:  Refers to the process of demonstrating that a laboratory procedure produces successful results, that those results are accurate, and that the same or similar results are obtained each time the test is performed by the laboratory performing the test Variable Number of Tandem Repeats (VNTRs):  Certain regions of DNA that are repeated multiple times that may vary between individuals who are tested using the Restriction Fragment Length Polymorphism (RFLP) technique; see Restriction Fragment Length Polymorphism Variant:  Different form—an alternate sequence from the most commonly occurring sequence Virtual Data Analysis System (VDAS):  Web-based computer program permitting direct posting, download, and tracking of DNA data

W Work Product:  Materials that are generated as a function of analysis including extracts, amplified product or microamplification plates, electropherograms, reports, and worksheets

Y Y-STR:  A short tandem repeat (STR) located on the Y chromosome

FORENSICS & CRIMINAL JUSTICE

DNA Analysis for Missing Person Identification in Mass Fatalities Advances in DNA technology have expanded such that forensic DNA profiling is now considered a routine method for identifying victims of mass fatalities. Originating from an initiative funded by a grant from the U.S. Department of State, DNA Analysis for Missing Person Identification in Mass Fatalities presents a collection of training modules that supply comprehensive instruction in these complex techniques. The book begins with a concise overview of DNA analysis methods and their use in identifying victims of mass fatalities. It then goes on to explore: • Mass fatality response operations, including body recovery, mortuary operations, family assistance, the identification of human remains, and psychosocial support for families • Best practices in DNA sample collection and the different types of reference samples that can be used to identify a reported missing (RM) individual • Autosomal short tandem repeat (STR) DNA profile analysis and interpretation, and procedures to ensure data accuracy • Major steps involved in generating a DNA profile and the complex aspects of data analysis and interpretation • The importance of data management using information technology tools, and tips for maintaining quality operations • Accreditation and standards and the major elements of a DNA quality program • Setting up a laboratory operation, including planning, staffing, identifying types of equipment and supplies, and the procedures for ensuring that laboratory equipment performs appropriately The book includes a discussion of the key steps in the preparation, delivery, and evaluation of training sessions for personnel responding to a mass fatality human identification event. It also provides a comprehensive vocabulary list with terms related to mass fatality DNA identification. This text is a must-read for organizations contemplating the use of DNA in human identification initiatives following mass fatalities. It is also a tremendous value to emergency manager/planners, medical legal authorities, and forensic DNA laboratories. K14972

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