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.

Contributing authors xxvii Preface xxix Acknowledgments xxxi 1 The cell and cell division 1Margaret 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 25Helen J. Lawce and Michael G. Brown 2.1 Introduction 25 2.2 History of human cytogenetics 25 2.3 Cytogenetics methods 29 2.4 Slidemaking 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 Slidemaking 75 Protocol 2.2 Slidemaking 76 Protocol 2.3 Making wet slides for chromosome analysis 78 Protocol 2.4 Slidemaking 82 Protocol 2.5 Slide preparation 82 Protocol 2.6 Slide preparation procedure 84 3 Peripheral blood cytogenetic methods 87Helen 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 Highresolution 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 119Debra 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 rateestimation of mean population doubling time during logarithmic growth 162 Protocol 4.22 Maintenance of fibroblast cultures as nonmitotic population 163 Protocol 4.23 Synchronization at Sphase 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 173Kristin 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 GBanding with Leishman's stain (GTL) 208 Protocol 5.9 Cystic hygroma fluid protocol 209 6 Chromosome stains 213Helen J. Lawce 6.1 Introduction 213 6.2 Chromosome banding methods 220 6.3 5-bromo-2-deoxyuridine methodologies 246 6.4 Tbanding/CTbanding 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 (Qbands) 266 Protocol 6.4 Cbanding 268 Protocol 6.5 Cbanding 270 Protocol 6.6 Cbanding 271 Protocol 6.7 Cbanding of blood slides 272 Protocol 6.8 Giemsä11 staining technique 274 Protocol 6.9 Distamycin A/DAPI staining 275 Protocol 6.10 Chromomycin/methyl green and chromomycin/distamycin fluorescent Rbanding method 277 Protocol 6.11 Bone marrow and cancer blood Gbanding 278 Protocol 6.12 Trypsin Gbanding 280 Protocol 6.13 Giemsätrypsin banding with Wright stain (GTW) for suspension culture slides and in situ culture coverslips 281 Protocol 6.14 Gbanding 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 Tbanding by thermal denaturation 294 Protocol 6.21 CTbanding 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 301Helen J. Lawce and Luke Boyd 7.1 Understanding the basics 301 7.2 Description of human chromosome shapes 302 7.3 Determination of Gbanded chromosome resolution 355 8 ISCN: the universal language of cytogenetics 359Marilyn 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 regionspecific assay (rsa) 420 8.12 Conclusion 422 Acknowledgments 422 Addendum for ISCN 2016 updates 426 References 426 9 Constitutional chromosome abnormalities 429Kathleen KaiserRogers 9.1 Numerical abnormalities 429 9.2 Structural rearrangements 444 References 472 10 Genomic imprinting 481R. 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 499Nyla 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 Tcell 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/interleukin2 for Bcell mitogenic stimulation 560 Protocol 11.4 Culture of CpGstimulated 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 GTGbanding 571 Protocol 11.8 GTW banding procedure (Gbands by trypsin using Wright stain) 573 12 Cytogenetic methods and findings in human solid tumors 577Marilu 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 653Yassmine 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 687Margaret 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...