Details

The AGT Cytogenetics Laboratory Manual

4. Aufl.

von: Marilyn S. Arsham, Margaret J. Barch, Helen J. Lawce
209,99 €
Verlag:	Wiley-Blackwell
Format:	EPUB
Veröffentl.:	03.03.2017
ISBN/EAN:	9781119061281
Sprache:	englisch
Anzahl Seiten:	1168

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Titelbeschreibung

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.

Inhaltsverzeichnis

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 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 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 Kaiser‐Rogers 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 chroniclymphocytic 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 M‐FISH) 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 21.4 Assessments 1018 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

Autorenportrait

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

Back cover copy

Cytogenetics is a segment of the evolving cytogenomic branch of genetics that explores the genetic makeup of a cell, in particular chromosomes both in metaphase and in interphase cycles, as well as at the molecular level. It utilizes an assortment of procedures to illuminate both the full complement of chromosomes and a targeted region within a specific chromosome. These investigative tools include routine analysis of G-banded chromosomes, specialized stains that address specific chromosomal structures, and molecular probes, such as fluorescence in situ hybridization 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 treatment of the diagnostic tests offered by this clinical laboratory environment and explains the scientific theories behind those procedures. 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 that reflect the intuitive experience of its authors. 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; 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 with a resource that teaches the procedures performed in the cytogenetics laboratory environment, and the laboratory professional with a peer-reviewed reference that explores the multiple facets behind each of these procedures. Topic coverage is rich in detail, yet presented with the student in mind, making it a useful resource for researchers, clinicians, and laboratory professionals, as well as students in a university or medical school that offers introductory or laboratory-related courses exploring cytogenetics' role within medical genetics. Reviews for the third edition: "A collection of protocols and explanations for cytogenetic techniques from the common to the peculiar." "An excellent book. Looking forward to an updated version."