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The AGT Cytogenetics Laboratory Manual, 4th Edition

Marilyn S. Arsham (Editor), Margaret J. Barch (Editor), Helen J. Lawce (Editor)
ISBN: 978-1-119-06122-9
1218 pages
April 2017, Wiley-Blackwell
The AGT Cytogenetics Laboratory Manual, 4th Edition (1119061229) cover image

Description

Cytogenetics is the study of chromosome morphology, structure, pathology, function, and behavior. The field has evolved to embrace molecular cytogenetic changes, now termed cytogenomics.
Cytogeneticists utilize an assortment of procedures to investigate the full complement of chromosomes and/or a targeted region within a specific chromosome in metaphase or interphase.  Tools include routine analysis of G-banded chromosomes, specialized stains that address specific chromosomal structures, and molecular probes, such as fluorescence in situ hybridization (FISH) and chromosome microarray analysis, which employ a variety of methods to highlight a region as small as a single, specific genetic sequence under investigation.

The AGT Cytogenetics Laboratory Manual, Fourth Edition offers a comprehensive description of the diagnostic tests offered by the clinical laboratory and explains the science behind them. One of the most valuable assets is its rich compilation of laboratory-tested protocols currently being used in leading laboratories, along with practical advice for nearly every area of interest to cytogeneticists. In addition to covering essential topics that have been the backbone of cytogenetics for over 60 years, such as the basic components of a cell, use of a microscope, human tissue processing for cytogenetic analysis (prenatal, constitutional, and neoplastic), laboratory safety, and the mechanisms behind chromosome rearrangement and aneuploidy, this edition introduces new and expanded chapters by experts in the field.  Some of these new topics include a unique collection of chromosome heteromorphisms; clinical examples of genomic imprinting; an example-driven overview of chromosomal microarray; mathematics specifically geared for the cytogeneticist; usage of ISCN’s cytogenetic language to describe chromosome changes; tips for laboratory management; examples of laboratory information systems; a collection of internet and library resources; and a special chapter on animal chromosomes for the research and zoo cytogeneticist. The range of topics is thus broad yet comprehensive, offering the student a resource that teaches the procedures performed in the cytogenetics laboratory environment, and the laboratory professional with a peer-reviewed reference that explores the basis of each of these procedures. This makes it a useful resource for researchers, clinicians, and lab professionals, as well as students in a university or medical school setting.

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Table of Contents

Contributing authors xxvii

Preface xxix

Acknowledgments xxxi

1 The cell and cell division 1
Margaret J. Barch and Helen J. Lawce

1.1 The cell 1

1.2 The cell cycle 14

1.3 Recombinant DNA techniques 19

1.4 The human genome 21

References 22

2 Cytogenetics: an overview 25
Helen J. Lawce and Michael G. Brown

2.1 Introduction 25

2.2 History of human cytogenetics 25

2.3 Cytogenetics methods 29

2.4 Slide‐making 49

2.5 Chromosome staining 58

2.6 Chromosome microscopy/analysis 59

2.7 Laboratory procedure manual 69

References 70

Contributed protocols 75

Protocol 2.1 Slide‐making 75

Protocol 2.2 Slide‐making 76

Protocol 2.3 Making wet slides for chromosome analysis 78

Protocol 2.4 Slide‐making 82

Protocol 2.5 Slide preparation 82

Protocol 2.6 Slide preparation procedure 84

3 Peripheral blood cytogenetic methods 87
Helen J. Lawce and Michael G. Brown

3.1 Using peripheral blood for cytogenetic analysis 87

3.2 Special uses of peripheral blood cultures 88

3.3 Peripheral blood constituents 89

3.4 Specimen handling 91

3.5 Cell culture equipment and supplies 93

3.6 Harvesting peripheral blood cultures 95

3.7 Chromosome analysis of peripheral blood 95

3.8 Storage of fixed specimens 95

Acknowledgments 95

References 95

Contributed protocols 98

Protocol 3.1 Blood culture and harvest procedure 98

Protocol 3.2 High‐resolution peripheral blood method 100

Protocol 3.3 Constitutional cytogenetic studies on peripheral blood 108

Protocol 3.4 Blood culture and harvest procedure for microarray confirmation studies 115

4 General cell culture principles and fibroblast culture 119
Debra F. Saxe, Kristin M. May and Jean H. Priest

4.1 Definitions of a culture 119

4.2 Basic considerations in cell culture 121

4.3 Fibroblast culture 128

4.4 Lymphoblastoid cell lines 132

Glossary 132

Reference 133

Additional readings 133

Contributed protocols section 134

Protocol 4.1 Solid tissue collection for establishing cultures 134

Protocol 4.2 Solid tissue transport and sendout media 135

Protocol 4.3 Tissue culture reagents 138

Protocol 4.4 Phosphate buffer solution deficient in Ca2+ and Mg2+ 141

Protocol 4.5 Solid tissue and fibroblast culture setup 141

Protocol 4.6 Solid tissue setup and processing 142

Protocol 4.7 Flask and coverslip setup for POC/fibroblast cultures 145

Protocol 4.8 Coverslip setup for solid tissue biopsy specimens 147

Protocol 4.9 Solid tissue (fibroblast) culturing and harvesting 150

Protocol 4.10 Fibroblast culture maintenance: media feeding and changing 154

Protocol 4.11 Routine subculture of fibroblast cultures 155

Protocol 4.12 Manual harvest for flasks 157

Protocol 4.13 Treated media for contamination 158

Protocol 4.14 Fungizone–mycostatin solution for treatment of fungus/yeast contaminated cultures 158

Protocol 4.15 Mycoplasma testing 159

Protocol 4.16 Plating efficiency of serum 160

Protocol 4.17 Routine replication plating for human diploid cells 160

Protocol 4.18 Cell counting chamber method 161

Protocol 4.19 Cell viability by dye exclusion 161

Protocol 4.20 Mitotic index 161

Protocol 4.21 Growth rate‐estimation of mean population doubling time during logarithmic growth 162

Protocol 4.22 Maintenance of fibroblast cultures as non‐mitotic population 163

Protocol 4.23 Synchronization at S‐phase with BrdU 163

Protocol 4.24 Making direct FISH preparations from abortus tissue 164

Protocol 4.25 Cryopreservation 165

Protocol 4.26 Cryopreservation with Nalgene cryogenic container 166

Protocol 4.27 Lymphoblastoid lines 167

Protocol 4.28 Freezing tissue cultures (cryopreservation) 171

5 Prenatal chromosome diagnosis 173
Kristin M. May, Debra F. Saxe and Jean H. Priest

5.1 Introduction 173

5.2 Amniotic fluid 173

5.3 Culture of amniotic fluid 175

5.4 Analysis of amniotic fluid 178

5.5 Chorionic villus sampling 180

5.6 Analysis of chorionic villi 184

References 186

Contributed protocols section 188

Protocol 5.1 Amniotic fluid culture setup and routine maintenance 188

Protocol 5.2 Coverslip (in situ) harvest procedure for chromosome preparations from amniotic fluid, CVS, or tissues (manual method) 191

Protocol 5.3 Harvest of flask amniocyte cultures 193

Protocol 5.4 Amniotic fluid culturing, subculturing, and harvesting (flask method) 195

Protocol 5.5 Criteria for interpreting mosaic amniotic fluid cultures 198

Protocol 5.6 Chorionic villi sampling – setup, direct harvest, and culture 199

Protocol 5.7 Chorionic villus sampling 204

Protocol 5.8 G‐Banding with Leishman’s stain (GTL) 208

Protocol 5.9 Cystic hygroma fluid protocol 209

6 Chromosome stains 213
Helen J. Lawce

6.1 Introduction 213

6.2 Chromosome banding methods 220

6.3 5-bromo-2′-deoxyuridine methodologies 246

6.4 T‐banding/CT‐banding 252

6.5 Antibody banding and restriction endonuclease banding 252

6.6 Destaining slides 252

6.7 FISH DAPI bands 252

6.8 Sequential staining 253

Acknowledgments 253

References 253

Contributed protocols section 266

Protocol 6.1 Conventional Giemsa staining (unbanded) 266

Protocol 6.2 Leishman’s stain 266

Protocol 6.3 Quinacrine mustard chromosome staining (Q‐bands) 266

Protocol 6.4 C‐banding 268

Protocol 6.5 C‐banding 270

Protocol 6.6 C‐banding 271

Protocol 6.7 C‐banding of blood slides 272

Protocol 6.8 Giemsa‐11 staining technique 274

Protocol 6.9 Distamycin A/DAPI staining 275

Protocol 6.10 Chromomycin/methyl green and chromomycin/distamycin fluorescent R‐banding method 277

Protocol 6.11 Bone marrow and cancer blood G‐banding 278

Protocol 6.12 Trypsin G‐banding 280

Protocol 6.13 Giemsa‐trypsin banding with Wright stain (GTW) for suspension culture slides and in situ culture coverslips 281

Protocol 6.14 G‐banding blood lymphocyte slides 284

Protocol 6.15 Cd staining 285

Protocol 6.16 CREST/CENP antibody staining 286

Protocol 6.17 AgNOR (silver staining) 287

Protocol 6.18 Sister chromatid exchange blood culture and staining 289

Protocol 6.19 Sister chromatid exchange fibroblast culture and staining 291

Protocol 6.20 T‐banding by thermal denaturation 294

Protocol 6.21 CT‐banding 295

Protocol 6.22 Lymphocyte culture and staining procedures for late replication analysis 295

Protocol 6.23 Destaining and sequential staining of slides 298

Protocol 6.24 Restaining permanently mounted slides 299

7 Human chromosomes: identification and variations 301
Helen J. Lawce and Luke Boyd

7.1 Understanding the basics 301

7.2 Description of human chromosome shapes 302

7.3 Determination of G‐banded chromosome resolution 355

Acknowledgments 356

Glossary 356

References 357

8 ISCN: the universal language of cytogenetics 359
Marilyn S. Arsham and Lisa G. Shaffer

8.1 Introduction 359

8.2 Language 359

8.3 Karyotype 364

8.4 Numerical events 378

8.5 Structural events 380

8.6 Derivative chromosomes (der) 394

8.7 Symbols of uncertainty 397

8.8 Random versus reportable 403

8.9 Multiple cell lines and clones 404

8.10 Fluorescence in situ hybridization 408

8.11 Microarray (arr) and region‐specific assay (rsa) 420

8.12 Conclusion 422

Acknowledgments 422

Addendum for ISCN 2016 updates 426

References 426

9 Constitutional chromosome abnormalities 429
Kathleen KaiserRogers

9.1 Numerical abnormalities 429

9.2 Structural rearrangements 444

References 472

10 Genomic imprinting 481
R. Ellen Magenis

10.1 Introduction 481

10.2 Human genomic disease and imprinting 488

10.3 Germ cell tumors – UPD and imprinting 493

Glossary 494

References 496

11 Cytogenetic analysis of hematologic malignant diseases 499
Nyla A. Heerema

11.1 Introduction 499

11.2 Myeloid leukemias 508

11.3 Myelodysplastic syndromes 514

11.4 Myeloproliferative neoplasms 515

11.5 B‐ and T‐cell lymphoid neoplasms 517

11.6 Lymphomas 522

11.7 Laboratory practices 525

Acknowledgments 533

Glossary of hematopoietic malignancies 533

References 535

Contributed protocols section 553

Protocol 11.1 Cancer cytogenetics procedure 553

Protocol 11.2 Bone marrow/leukemic peripheral blood setup and harvest procedure 558

Protocol 11.3 Bone marrow and leukemic blood culture and harvest procedure using DSP30 CPG oligonucleotide/interleukin‐2 for B‐cell mitogenic stimulation 560

Protocol 11.4 Culture of CpG‐stimulated peripheral blood and bone marrow in chronic lymphocytic leukemia 562

Protocol 11.5 Plasma cell separation and harvest procedure for FISH analysis 567

Protocol 11.6 Plasma cell separation and harvest procedure for FISH 569

Protocol 11.7 Bone marrow GTG‐banding 571

Protocol 11.8 GTW banding procedure (G‐bands by trypsin using Wright stain) 573

12 Cytogenetic methods and findings in human solid tumors 577
Marilu Nelson

12.1 Introduction 577

12.2 Processing tumor specimens 579

12.3 Recurrent cytogenetic abnormalities 592

12.4 Molecular genetic and cytogenetic techniques 608

12.5 Conclusion 612

Glossary 612

References 613

Contributed protocol section 631

Protocol 12.1 Solid tumor cell culture and harvest 631

Protocol 12.2 Solid tumor cell culture and harvest 637

Protocol 12.3 Solid tumor culture 643

Protocol 12.4 Solid tumor harvest: monolayer and flask methods 644

Protocol 12.5 Solid tumor culturing and harvesting 646

13 Chromosome instability syndromes 653
Yassmine Akkari

13.1 Introduction 653

13.2 Fanconi anemia 656

13.3 Bloom syndrome 658

13.4 Ataxia–telangiectasia 658

13.5 Nijmegen breakage syndrome 659

13.6 Immunodeficiency, centromeric instability, and facial anomalies syndrome 660

13.7 Roberts syndrome 661

13.8 Werner syndrome 661

13.9 Rothmund–Thomson syndrome 662

13.10 Proficiency testing 662

Glossary 662

References 667

Contributed protocol section 671

Protocol 13.1 Fanconi anemia chromosome breakage procedure for whole blood 671

Protocol 13.2 Supplemental procedure; Ficoll separation of whole blood 675

Protocol 13.3 Fanconi anemia fibroblast set up, culture, subculture, and harvest procedure 676

Protocol 13.4 Fanconi anemia chromosome breakage analysis policy 681

Protocol 13.5 Table for breakage studies result interpretation 682

Protocol 13.6 Fanconi anemia 684

14 Microscopy and imaging 687
Margaret J. Barch and Helen J. Lawce

14.1 The standard microscope 687

14.2 Brightfield microscopy 695

14.3 Fluorescence microscopy 697

14.4 Specialized microscopy 699

14.5 Capturing the microscopic image 701

References 703

15 Computer imaging 705
Christine E. Haessig

15.1 Introduction 705

15.2 Techniques to improve karyogram image quality 705

15.3 Metaphase preparation 706

15.4 Microscopy 706

15.5 Image capture 707

15.6 Enhancement 710

15.7 Advanced contrast 710

15.8 Macro programming 712

15.9 FISH imaging 713

15.10 Printing 715

15.11 Quality control 715

15.12 Archiving 715

Acknowledgments 715

References 715

16 Fluorescence in situ hybridization (FISH) 717
Helen J. Lawce and Jeffrey S. Sanford

16.1 Introduction 717

16.2 Clinical applications of FISH probes 722

16.3 Deletion/duplication probes for constitutional abnormalities 730

16.4 Hematology/oncology and solid tumor probes 734

16.5 Sources and characteristics of probes available to the clinical cytogenetics laboratory 736

16.6 Special uses of probes 738

16.7 Important FISH probe adjuvants 739

16.8 Principles of FISH 741

16.9 FISH methods – an overview 744

16.10 FISH analysis and reporting 757

16.11 FISH probe testing and validation 765

16.12 FISH for special investigation 768

16.13 Preimplantation genetic FISH 771

16.14 Other applications 776

16.15 Variants in FISH signal patterns 777

16.16 Conclusion 777

Acknowledgments 778

Glossary 778

References 780

Contributed protocols 790

Protocol 16.1 FISH (fluorescence in situ hybridization) methods 790

Protocol 16.2 LSI, CEP, and paint probe protocol 796

Protocol 16.3 FISH protocol for multiprobe® FISH panels 799

Protocol 16.4 Slide pretreatment with pepsin for FISH 800

Protocol 16.5 Interphase FISH for amniotic fluid specimen aneuploidy 801

Protocol 16.6 FISH on direct preparations from abortus tissue 803

Protocol 16.7 FISH on cultured non‐mitotic abortus tissue 804

Protocol 16.8 FISH on smears 806

Protocol 16.9 FISH on very small samples 808

Protocol 16.10 Paraffin‐embedded tissue FISH method 810

Protocol 16.11 VP2000 automated slide processor method for FFPE FISH 811

Protocol 16.12 Plasma cell targeted FISH 814

Protocol 16.13 Plasma cell separation for interphase FISH using easy SEP magnet method 815

Protocol 16.14 Preimplantation genetic testing (PGD) for aneuploidy 818

Protocol 16.15 Preimplantation genetic testing (PGD) FISH for translocations 821

Protocol 16.16 Post‐FISH BrdU antibody detection 823

Protocol 16.17 Same‐day HER2 IQ‐FISH pharmDx™ for breast tissue 824

17 Multicolor FISH (SKY and MFISH) and CGH 833
Turid Knutsen

17.1 Introduction 833

17.2 Multicolor FISH (SKY/M‐FISH) 834

17.3 Comparative genomic hybridization 849

17.4 Conclusion 859

Acknowledgments 859

References 859

Contributed protocols section 864

Protocol 17.1 Spectral karyotyping (SKY) 864

Protocol 17.2 Spectral karyotyping (SKY) 877

Protocol 17.3 DNA spectral karyotyping 878

Protocol 17.4 Multicolor‐FISH method (M‐FISH) I 881

Protocol 17.5 Multicolor FISH (M‐FISH) or 24‐color FISH II 884

Protocol 17.6 Multicolor FISH (M‐FISH) III 888

Protocol 17.7 Comparative genomic hybridization I 891

Protocol 17.8 Comparative genomic hybridization II 898

18 Genomic microarray technologies for the cytogenetics laboratory 903
Bhavana J. Davé and Warren G. Sanger

18.1 Introduction 903

18.2 Applications 907

18.3 Genomic microarray in a cytogenetics laboratory 913

18.4 Conclusion 922

Acknowledgment 922

Authors’ note 923

References 923

19 Mathematics for the cytogenetic technologist 937
Patricia K. Dowling

19.1 General concepts 937

19.2 Solutions 942

19.3 Statistical tools 956

19.4 Using a hemacytometer 968

19.5 Quantification and purity determination of DNA using spectroscopy 973

Reference 974

Additional readings 974

20 Selected topics on safety, equipment maintenance, and compliance for the cytogenetics laboratory 975
Helen Jenks and Janet Krueger

20.1 Introduction 975

20.2 Biological hazard safety 975

20.3 Chemical safety 980

20.4 Fire safety 986

20.5 Electrical safety 987

20.6 Disaster plan 988

20.7 Equipment operation, maintenance, and safety 988

20.8 Ergonomics 996

20.9 Regulatory considerations 998

Acknowledgments 1001

References 1001

Contributed protocols section 1003

Protocol 20.1 Autoclave sterilization, liquid nitrogen, pro‐par 1003

Protocol 20.2 Dishwashing procedure 1003

Protocol 20.3 Eppendorf pipette calibration 1004

Protocol 20.4 NIST thermometer calibration 1006

Protocol 20.5 Thermometer calibration 1008

Protocol 20.6 Timer calibration 1008

21 A system approach to quality 1011
Peggy J. Stupca and Sheryl A. Tran

21.1 Quality system 1011

21.2 Process management 1013

21.3 Documents and records 1015

21.5 Continual improvement 1022

21.6 Summary 1023

References 1023

Contributed protocols section 1025

Protocol 21.1 Quality control overview document 1025

Protocol 21.2 Monitoring specimen quality from off‐hill sites 1030

22 Laboratory management 1031
Mervat S. Ayad and Adam Sbeiti

22.1 Introduction 1031

22.2 Management concepts and functions 1032

22.3 Personnel management 1033

22.4 Quality management and control 1036

22.5 Budget development and monitoring 1039

22.6 Conclusion 1043

References 1043

Suggested reading 1043

23 Laboratory information system 1045
Peining Li and Richard Van Rheeden

23.1 Historical perspective 1045

23.2 General description of LIS 1045

23.3 LIS in cytogenetics laboratories 1048

23.4 Trends for the future LIS 1051

Acknowledgments 1052

References 1052

24 Animal cytogenetics 1055
Marlys L. Houck, Teri L. Lear and Suellen J. Charter

24.1 Introduction 1055

24.2 Domestic animal fertility 1056

24.3 Captive management 1057

24.4 Wildlife conservation 1059

24.5 General sample collection considerations 1060

24.6 Fibroblast cell culture 1062

24.7 Peripheral blood culture 1063

24.8 Chromosome analysis 1064

24.9 Molecular and comparative cytogenetics 1070

Acknowledgments 1071

Glossary 1072

References 1072

Contributed protocol section 1078

Protocol 24.1 Blood feather collection 1078

Protocol 24.2 Avian lymphocyte culture (for large birds) 1078

Protocol 24.3 Lymphocyte culture using whole blood 1084

Protocol 24.4 Lymphocyte culture using autologous plasma/buffy coat (AP/BC) 1085

Protocol 24.5 Horse lymphocyte culture method 1087

Protocol 24.6 Rhino blood culture 1089

Protocol 24.7 Organ tissue collection protocol from carcass 1090

Protocol 24.8 Skin biopsy procedure 1090

Protocol 24.9 Placenta biopsy procedure 1091

Protocol 24.10 Freezing of fibroblast cell cultures 1092

Protocol 24.11 Freezing tissue biopsy samples for later initiation of cell culture (tissue piecing) 1094

Protocol 24.12 Preparation of primary cultures from feather pulp 1095

Protocol 24.13 Preparation of primary cultures from solid tissue (explants) 1096

Protocol 24.14 Preparation of primary cultures using enzyme digestion 1097

Protocol 24.15 Harvesting of fibroblast cell cultures 1098

Protocol 24.16 Preparation of competitor DNA for FISH hybridization 1099

Protocol 24.17 In situ hybridization of BAC clones labeled with spectrum fluorochromes: probe and slide preparation 1100

Protocol 24.18 Labeling DNA with spectrum fluorochromes 1102

25 Online genetic resources and references 1103
Wahab A. Khan

25.1 Introduction 1103

25.2 Resource information 1103

Index 1113

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Author Information

About the Editors
Marilyn S. Arsham, (retired) Cytogenetic Technologist II, Western Connecticut Health Network, Danbury Hospital campus, Danbury, Connecticut, USA

Margaret J. Barch, (formerly) Frank F Yen Cytogenetics Laboratory, Weisskopf Child Evaluation Center, University of Louisville, USA

Helen J. Lawce, Clinical Cytogenetics, Oregon Health & Science University Knight Diagnostics Laboratory, USA

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