The AGT Cytogenetics Laboratory Manual

deficient in Ca2+ and Mg2+ 141Protocol 4.5 Solid tissue and fibroblast culture setup 141Protocol 4.6 Solid tissue setup and processing 142Protocol 4.7 Flask and coverslip setup for POC/fibroblast cultures 145Protocol 4.8 Coverslip setup for solid tissue biopsy specimens 147Protocol 4.9 Solid tissue (fibroblast) culturing and harvesting 150Protocol 4.10 Fibroblast culture maintenance: media feeding and changing 154Protocol 4.11 Routine subculture of fibroblast cultures 155Protocol 4.12 Manual harvest for flasks 157Protocol 4.13 Treated media for contamination 158Protocol 4.14 Fungizone-mycostatin solution for treatment of fungus/yeast contaminated cultures 158Protocol 4.15 Mycoplasma testing 159Protocol 4.16 Plating efficiency of serum 160Protocol 4.17 Routine replication plating for human diploid cells 160Protocol 4.18 Cell counting chamber method 161Protocol 4.19 Cell viability by dye exclusion 161Protocol 4.20 Mitotic index 161Protocol 4.21 Growth rate-estimation of mean population doubling time during logarithmic growth 162Protocol 4.22 Maintenance of fibroblast cultures as non-mitotic population 163Protocol 4.23 Synchronization at S-phase with BrdU 163Protocol 4.24 Making direct FISH preparations from abortus tissue 164Protocol 4.25 Cryopreservation 165Protocol 4.26 Cryopreservation with Nalgene cryogenic container 166Protocol 4.27 Lymphoblastoid lines 167Protocol 4.28 Freezing tissue cultures (cryopreservation) 1715 Prenatal chromosome diagnosis 173Kristin M. May, Debra F. Saxe and Jean H. Priest5.1 Introduction 1735.2 Amniotic fluid 1735.3 Culture of amniotic fluid 1755.4 Analysis of amniotic fluid 1785.5 Chorionic villus sampling 1805.6 Analysis of chorionic villi 184References 186Contributed protocols section 188Protocol 5.1 Amniotic fluid culture setup and routine maintenance 188Protocol 5.2 Coverslip (in situ) harvest procedure for chromosome preparations from amniotic fluid, CVS, or tissues (manual method) 191Protocol 5.3 Harvest of flask amniocyte cultures 193Protocol 5.4 Amniotic fluid culturing, subculturing, and harvesting (flask method) 195Protocol 5.5 Criteria for interpreting mosaic amniotic fluid cultures 198Protocol 5.6 Chorionic villi sampling - setup, direct harvest, and culture 199Protocol 5.7 Chorionic villus sampling 204Protocol 5.8 G-Banding with Leishman's stain (GTL) 208Protocol 5.9 Cystic hygroma fluid protocol 2096 Chromosome stains 213Helen J. Lawce6.1 Introduction 2136.2 Chromosome banding methods 2206.3 5-bromo-2'-deoxyuridine methodologies 2466.4 T-banding/CT-banding 2526.5 Antibody banding and restriction endonuclease banding 2526.6 Destaining slides 2526.7 FISH DAPI bands 2526.8 Sequential staining 253Acknowledgments 253References 253Contributed protocols section 266Protocol 6.1 Conventional Giemsa staining (unbanded) 266Protocol 6.2 Leishman's stain 266Protocol 6.3 Quinacrine mustard chromosome staining (Q-bands) 266Protocol 6.4 C-banding 268Protocol 6.5 C-banding 270Protocol 6.6 C-banding 271Protocol 6.7 C-banding of blood slides 272Protocol 6.8 Giemsa-11 staining technique 274Protocol 6.9 Distamycin A/DAPI staining 275Protocol 6.10 Chromomycin/methyl green and chromomycin/distamycin fluorescent R-banding method 277Protocol 6.11 Bone marrow and cancer blood G-banding 278Protocol 6.12 Trypsin G-banding 280Protocol 6.13 Giemsa-trypsin banding with Wright stain (GTW) for suspension culture slides and in situ culture coverslips 281Protocol 6.14 G-banding blood lymphocyte slides 284Protocol 6.15 Cd staining 285Protocol 6.16 CREST/CENP antibody staining 286Protocol 6.17 AgNOR (silver staining) 287Protocol 6.18 Sister chromatid exchange blood culture and staining 289Protocol 6.19 Sister chromatid exchange fibroblast culture and staining 291Protocol 6.20 T-banding by thermal denaturation 294Protocol 6.21 CT-banding 295Protocol 6.22 Lymphocyte culture and staining procedures for late replication analysis 295Protocol 6.23 Destaining and sequential staining of slides 298Protocol 6.24 Restaining permanently mounted slides 2997 Human chromosomes: identification and variations 301Helen J. Lawce and Luke Boyd7.1 Understanding the basics 3017.2 Description of human chromosome shapes 3027.3 Determination of G-banded chromosome resolution 355Acknowledgments 356Glossary 356References 3578 ISCN: the universal language of cytogenetics 359Marilyn S. Arsham and Lisa G. Shaffer8.1 Introduction 3598.2 Language 3598.3 Karyotype 3648.4 Numerical events 3788.5 Structural events 3808.6 Derivative chromosomes (der) 3948.7 Symbols of uncertainty 3978.8 Random versus reportable 4038.9 Multiple cell lines and clones 4048.10 Fluorescence in situ hybridization 4088.11 Microarray (arr) and region-specific assay (rsa) 4208.12 Conclusion 422Acknowledgments 422Addendum for ISCN 2016 updates 426References 4269 Constitutional chromosome abnormalities 429Kathleen Kaiser-Rogers9.1 Numerical abnormalities 4299.2 Structural rearrangements 444References 47210 Genomic imprinting 481R. Ellen Magenis10.1 Introduction 48110.2 Human genomic disease and imprinting 48810.3 Germ cell tumors - UPD and imprinting 493Glossary 494References 49611 Cytogenetic analysis of hematologic malignant diseases 499Nyla A. Heerema11.1 Introduction 49911.2 Myeloid leukemias 50811.3 Myelodysplastic syndromes 51411.4 Myeloproliferative neoplasms 51511.5 B- and T-cell lymphoid neoplasms 51711.6 Lymphomas 52211.7 Laboratory practices 525Acknowledgments 533Glossary of hematopoietic malignancies 533References 535Contributed protocols section 553Protocol 11.1 Cancer cytogenetics procedure 553Protocol 11.2 Bone marrow/leukemic peripheral blood setup and harvest procedure 558Protocol 11.3 Bone marrow and leukemic blood culture and harvest procedure using DSP30 CPG oligonucleotide/interleukin-2 for B-cell mitogenic stimulation 560Protocol 11.4 Culture of CpG-stimulated peripheral blood and bone marrow in chronic lymphocytic leukemia 562Protocol 11.5 Plasma cell separation and harvest procedure for FISH analysis 567Protocol 11.6 Plasma cell separation and harvest procedure for FISH 569Protocol 11.7 Bone marrow GTG-banding 571Protocol 11.8 GTW banding procedure (G-bands by trypsin using Wright stain) 57312 Cytogenetic methods and findings in human solid tumors 577Marilu Nelson12.1 Introduction 57712.2 Processing tumor specimens 57912.3 Recurrent cytogenetic abnormalities 59212.4 Molecular genetic and cytogenetic techniques 60812.5 Conclusion 612Glossary 612References 613Contributed protocol section 631Protocol 12.1 Solid tumor cell culture and harvest 631Protocol 12.2 Solid tumor cell culture and harvest 637Protocol 12.3 Solid tumor culture 643Protocol 12.4 Solid tumor harvest: monolayer and flask methods 644Protocol 12.5 Solid tumor culturing and harvesting 64613 Chromosome instability syndromes 653Yassmine Akkari13.1 Introduction 65313.2 Fanconi anemia 65613.3 Bloom syndrome 65813.4 Ataxia-telangiectasia 65813.5 Nijmegen breakage syndrome 65913.6 Immunodeficiency, centromeric instability, and facial anomalies syndrome 66013.7 Roberts syndrome 66113.8 Werner syndrome 66113.9 Rothmund-Thomson syndrome 66213.10 Proficiency testing 662Glossary 662References 667Contributed protocol section 671Protocol 13.1 Fanconi anemia chromosome breakage procedure for whole blood 671Protocol 13.2 Supplemental procedure; Ficoll separation of whole blood 675Protocol 13.3 Fanconi anemia fibroblast set up, culture, subculture, and harvest procedure 676Protocol 13.4 Fanconi anemia chromosome breakage analysis policy 681Protocol 13.5 Table for breakage studies result interpretation 682Protocol 13.6 Fanconi anemia 68414 Microscopy and imaging 687Margaret J. Barch and Helen J. Lawce14.1 The standard microscope 68714.2 Brightfield microscopy 69514.3 Fluorescence microscopy 69714.4 Specialized microscopy 69914.5 Capturing the microscopic image 701References 70315 Computer imaging 705Christine E. Haessig15.1 Introduction 70515.2 Techniques to improve karyogram image quality 70515.3 Metaphase preparation 70615.4 Microscopy 70615.5 Image capture 70715.6 Enhancement 71015.7 Advanced contrast 71015.8 Macro programming 71215.9 FISH imaging 71315.10 Printing 71515.11 Quality control 71515.12 Archiving 715Acknowledgments 715References 71516 Fluorescence in situ hybridization (FISH) 717Helen J. Lawce and Jeffrey S. Sanford16.1 Introduction 71716.2 Clinical applications of FISH probes 72216.3 Deletion/duplication probes for constitutional abnormalities 73016.4 Hematology/oncology and solid tumor probes 73416.5 Sources and characteristics of probes available to the clinical cytogenetics laboratory 73616.6 Special uses of probes 73816.7 Important FISH probe adjuvants 73916.8 Principles of FISH 74116.9 FISH methods - an overview 74416.10 FISH analysis and reporting 75716.11 FISH probe testing and validation 76516.12 FISH for special investigation 76816.13 Preimplantation genetic FISH 77116.14 Other applications 77616.15 Variants in FISH signal patterns 77716.16 Conclusion 777Acknowledgments 778Glossary 778References 780Contributed protocols 790Protocol 16.1 FISH (fluorescence in situ hybridization) methods 790Protocol 16.2 LSI, CEP, and paint probe protocol 796Protocol 16.3 FISH protocol for multiprobe(r) FISH panels 799Protocol 16.4 Slide pretreatment with pepsin for FISH 800Protocol 16.5 Interphase FISH for amniotic fluid specimen aneuploidy 801Protocol 16.6 FISH on direct preparations from abortus tissue 803Protocol 16.7 FISH on cultured non-mitotic abortus tissue 804Protocol 16.8 FISH on smears 806Protocol 16.9 FISH on very small samples 808Protocol 16.10 Paraffin-embedded tissue FISH method 810Protocol 16.11 VP2000 automated slide processor method for FFPE FISH 811Protocol 16.12 Plasma cell targeted FISH 814Protocol 16.13 Plasma cell separation for interphase FISH using easy SEP magnet method 815Protocol 16.14 Preimplantation genetic testing (PGD) for aneuploidy 818Protocol 16.15 Preimplantation genetic testing (PGD) FISH for translocations 821Protocol 16.16 Post-FISH BrdU antibody detection 823Protocol 16.17 Same-day HER2 IQ-FISH pharmDx(TM) for breast tissue 82417 Multicolor FISH (SKY and M-FISH) and CGH 833Turid Knutsen17.1 Introduction 83317.2 Multicolor FISH (SKY/M-FISH) 83417.3 Comparative genomic hybridization 84917.4 Conclusion 859Acknowledgments 859References 859Contributed protocols section 864Protocol 17.1 Spectral karyotyping (SKY) 864Protocol 17.2 Spectral karyotyping (SKY) 877Protocol 17.3 DNA spectral karyotyping 878Protocol 17.4 Multicolor-FISH method (M-FISH) I 881Protocol 17.5 Multicolor FISH (M-FISH) or 24-color FISH II 884Protocol 17.6 Multicolor FISH (M-FISH) III 888Protocol 17.7 Comparative genomic hybridization I 891Protocol 17.8 Comparative genomic hybridization II 89818 Genomic microarray technologies for the cytogenetics laboratory 903Bhavana J. Davé and Warren G. Sanger18.1 Introduction 90318.2 Applications 90718.3 Genomic microarray in a cytogenetics laboratory 91318.4 Conclusion 922Acknowledgment 922Authors' note 923References 92319 Mathematics for the cytogenetic technologist 937Patricia K. Dowling19.1 General concepts 93719.2 Solutions 94219.3 Statistical tools 95619.4 Using a hemacytometer 96819.5 Quantification and purity determination of DNA using spectroscopy 973Reference 974Additional readings 97420 Selected topics on safety, equipment maintenance, and compliance for the cytogenetics laboratory 975Helen Jenks and Janet Krueger20.1 Introduction 97520.2 Biological hazard safety 97520.3 Chemical safety 98020.4 Fire safety 98620.5 Electrical safety 98720.6 Disaster plan 98820.7 Equipment operation, maintenance, and safety 98820.8 Ergonomics 99620.9 Regulatory considerations 998Acknowledgments 1001References 1001Contributed protocols section 1003Protocol 20.1 Autoclave sterilization, liquid nitrogen, pro-par 1003Protocol 20.2 Dishwashing procedure 1003Protocol 20.3 Eppendorf pipette calibration 1004Protocol 20.4 NIST thermometer calibration 1006Protocol 20.5 Thermometer calibration 1008Protocol 20.6 Timer calibration 100821 A system approach to quality 1011Peggy J. Stupca and Sheryl A. Tran21.1 Quality system 101121.2 Process management 101321.3 Documents and records 101521.5 Continual improvement 102221.6 Summary 1023References 1023Contributed protocols section 1025Protocol 21.1 Quality control overview document 1025Protocol 21.2 Monitoring specimen quality from off-hill sites 103022 Laboratory management 1031Mervat S. Ayad and Adam Sbeiti22.1 Introduction 103122.2 Management concepts and functions 103222.3 Personnel management 103322.4 Quality management and control 103622.5 Budget development and monitoring 103922.6 Conclusion 1043References 1043Suggested reading 104323 Laboratory information system 1045Peining Li and Richard Van Rheeden23.1 Historical perspective 104523.2 General description of LIS 104523.3 LIS in cytogenetics laboratories 104823.4 Trends for the future LIS 1051Acknowledgments 1052References 105224 Animal cytogenetics 1055Marlys L. Houck, Teri L. Lear and Suellen J. Charter24.1 Introduction 105524.2 Domestic animal fertility 105624.3 Captive management 105724.4 Wildlife conservation 105924.5 General sample collection considerations 106024.6 Fibroblast cell culture 106224.7 Peripheral blood culture 106324.8 Chromosome analysis 106424.9 Molecular and comparative cytogenetics 1070Acknowledgments 1071Glossary 1072References 1072Contributed protocol section 1078Protocol 24.1 Blood feather collection 1078Protocol 24.2 Avian lymphocyte culture (for large birds) 1078Protocol 24.3 Lymphocyte culture using whole blood 1084Protocol 24.4 Lymphocyte culture using autologous plasma/buffy coat (AP/BC) 1085Protocol 24.5 Horse lymphocyte culture method 1087Protocol 24.6 Rhino blood culture 1089Protocol 24.7 Organ tissue collection protocol from carcass 1090Protocol 24.8 Skin biopsy procedure 1090Protocol 24.9 Placenta biopsy procedure 1091Protocol 24.10 Freezing of fibroblast cell cultures 1092Protocol 24.11 Freezing tissue biopsy samples for later initiation of cell culture (tissue piecing) 1094Protocol 24.12 Preparation of primary cultures from feather pulp 1095Protocol 24.13 Preparation of primary cultures from solid tissue (explants) 1096Protocol 24.14 Preparation of primary cultures using enzyme digestion 1097Protocol 24.15 Harvesting of fibroblast cell cultures 1098Protocol 24.16 Preparation of competitor DNA for FISH hybridization 1099Protocol 24.17 In situ hybridization of BAC clones labeled with spectrum fluorochromes: probe and slide preparation 1100Protocol 24.18 Labeling DNA with spectrum fluorochromes 110225 Online genetic resources and references 1103Wahab A. Khan25.1 Introduction 110325.2 Resource information 1103Index 1113