Methods in Human Cytogenetics

This volume was originally intended to be an English translation of the book MetllOden in der medizinischen Cytogenetik, published in 1970. Just about then, however, a number of new techniques were introduced in human cytogenetics and soon acquired the utmost importance, parti cularly in clinical diagnosis, so that the English edition had to be con siderably enlarged. As a result, there are now twelve chapters instead of eight, and two additional authors have been called upon, Dr. KRONE and Dr. SCHNEDL. In addition to the up-to-date presentation of con ventional methods of cell culture and techniques for the preparation and identification of human chromosomes, this text covers the various tech niques of producing banding patterns and applying them in chromo some identification. Further, it deals with the culture of amniotic fluid cells and gives instructions for handling tissue-culture cells for bio chemical analysis; it thus meets the ever-increasing requirements of a modern cell-culture laboratory. To paraphrase the aims of this book, we quote part of the preface to the German edition: "It was intended to collect the various methods so as to make them accessible for laboratory use. Furthermore, it is hoped that the reader faced with current research problems will be stimulated to modify and supplement the techniques described, instead of merely applying them automatically. In a rapidly developing field, some methods are still preliminary, and no final presentation seems possible.

Contenu
I Cell Cultures from Blood and Bone Marrow.- 1. Introduction.- 2. Bone Marrow.- 2.1 Collection of Material.- 2.2 Direct Preparation of Chromosomes from Cells in Metaphase or Prometaphase.- 2.2.1. Direct Preparation of Cells Immediately Following Biopsy.- 2.2.2. Cell Preparation Following Short Term Incubation with Colchicine.- 2.3. Storage of Bone Marrow Biopsies.- 2.4. Comments on the Preparation of Chromosomes.- 2.5. Long Term Cultures of Bone Marrow Cells.- 3. Culture of Peripheral Blood Cells.- 3.1. Whole Blood Cultures.- 3.2. Leukocyte Culture after Separation of Erythrocytes.- 3.2.1. Separation of Erythrocytes.- 3.2.2. Separation of Mononuclear and Polymorphonuclear Leukocytes.- 3.2.3. Choice of Medium.- 3.2.4. Supplements to Culture Medium.- 3.2.5. Storage of Culture Media.- 3.2.6. Duration of Culture.- 3.2.7. Increasing the Number of Mitoses.- 3.3 Storage and Transport of Blood Samples.- 3.4. Procedure with Leukemias.- 3.5. Long Term Cultures from Peripheral Blood Cells.- 3.6. Lymphocytes from Lymph Nodes, Thymus, and Spleen.- 4. Phytohemagglutinin: Properties and Mode of Action.- Appendix: Laboratory Procedures.- References.- II Cell Cultures from Tissue Expiants.- 1. Introduction.- 2. Areas of Application.- 3. Material.- 4. Introduction to Methods.- 5. Techniques for Collecting Tissue.- 6. Setting up Tissue Expiants and Culturing Cells.- 6.1. Enzymatic Dissociation of Tissue Expiants.- 6.2. Setting up Solid Expiants.- 6.2.2. Direct Deposition on the Glass Surface.- 6.2.2. Plasma Method.- 6.2.3. Sandwich Method.- 6.2.4. Cellophane Method.- 6.3 Subculturing.- 6.4. Culturing in a CO2 Incubator.- 6.5. Abortus Material.- 7. Harvest of Cultures for Chromosome Preparation.- 8. Long Term Preservation of Tissue Culture Cells.- Appendix I: Summary of Some of the Methods.- Appendix II: Catalog of Some Reagents, Media and Culture Vessels.- References.- III Culture and Preparation of Cells from Amniotic Fluid.- 1. Introduction.- 2. Collecting and Transportation of Amniotic Fluid.- 3. X- and Y-Chromatin.- 4. Cell Culture.- Appendix: Protocol for Amniotic Fluid Cultures.- References.- IV Preparation of Metaphase Chromosomes.- 1. Introduction.- 2. Preparation of Cell Suspensions.- 2.1. Material.- 2.2. General Observations on Preparation Procedure.- 2.3. Preparation by Air Drying.- 2.4. Preparation by Squashing.- 3. Direct Preparation from Monolayer Cultures.- 4. Direct Preparation of Embryonic Tissue.- 4.1. Field of Application.- 4.2. Material.- 4.3. Technique.- 5. Staining.- Appendix: Laboratory Guide.- References.- V Fluorescence Microscopy of Chromosomes and Interphase Nuclei.- 1. Introduction.- 2. Fluorescent Dyes for Chromosomes and Cell Nucle.- 2.1. Acridine Orange.- 2.1.1. Procedure of Staining with Acridine Orange.- 2.2. Quinacrine Derivatives.- 2.2.1. Application.- 2.2.2. Quinacrine Dyes.- 2.2.3. Preparation of Specimens.- 2.2.3.1. Chromosome Preparations.- 2.2.3.2. Interphase Cells.- 2.2.4. Staining Procedures.- 2.2.5. Restaining with Other Dyes.- 3. Other Fluorochromes.- 4. Technique of Fluorescence Microscopy.- 4.1. Principle.- 4.2. Microscope Equipment.- 4.3. Microphotography.- 5. Analysis of Preparations.- 5.1. General Comments.- 5.2. Chromosome Preparations.- 5.3. Interphase Cells and Y-Chromatin.- Appendix 1: Staining with Quinacrine-Dihydrochloride and Quinacrine-Mustard.- Appendix 2: Suppliers of Quinacrine Stains.- Appendix 3: Phosphate Buffer Solution (Sörensen).- References.- VI Banding Patterns in Human Chromosomes Visualized by Giemsa Staining after Various Pretreatments.- 1. Introduction.- 2. General Comments on the Different Methods.- 3. Methods for Demonstrating the Centromeric Heterochromatin.- 3.1. The Method of Arrighi and Hsu (1970).- 3.2. The Method of Yunis et al. (1971).- 3.3. Meiotic Chromosomes.- 3.4. Methods for Demonstrating the Secondary Constriction of Chromosome No. 9.- 4. Methods for Demonstrating Banding Patterns over the Entire Chromosome.- 4.1. Methods Using NaOH Treatment Followed by an Incubation in Buffer.- 4.1.1. The Method of Schnedl (1971).- 4.1.2. The Method of Gagné et al. (1971).- 4.1.3. The Method of Drets and Shaw (1971).- 4.2. Methods Using Various Incubation Procedures without NaOH.- 4.2.1. The Method of Sumner et al. (1971).- 4.2.2. The Method of Patil et al. (1971) Giemsa-9-Teehnique.- 4.3. Methods Using Enzymatic Digestion (Pronase, Trypsin).- 4.3.1. The Method of Dutrillaux et al. (1971).- 4.3.2. The Method of Seabright (1971).- 4.3.3. The Method of Wang and Fedoroff (1972).- 4.3.4. The Method by Sun, Chu and Chang (1973).- 4.4. The Reverse Bands.- 4.4.1. Reverse Bands Using G'emsa (Dutrillaux and Lejeune, 1971).- 4.4.2. The Acridine Orange Reverse Bands (for instance Bobrow et al., 1972b).- Appendix: Laboratory Procedures.- References.- VII Autoradiography of Human Chromosomes with 3H-Thymidine.- 1. Introduction.- 2. Theoretical Basis.- 2.2. The DNA Synthesis Cycle.- 2.2. Incorporation of Thymidine into DNA.- 2.3. Tritiated Thymidine.- 2.4. Autoradiographic Film Material.- 2.4.1. Emulsions.- 2.4.2. Stripping Films.- 3. Techniques.- 3.1. Labeling with Tritiated Thymidine.- 3.1.1. Continuous Labeling.- 3.1.2. Pulse Labeling.- 3.2. Harvest of Cultures and Chromosome Preparations.- 3.3. The Autoradiographic Technique.- 3.3.1. Covering the Preparation with Autoradiographic Film Material.- 3.3.2 Exposure.- 3.3.3. Development and Fixation of the Autoradiographic Film.- 3.3.4. Staining.- 3.3.5. Evaluation of Chromosomes in Autoradiographs.- 3.3.6. Preparation of Control Pictures by Removal of Silver Grains and Dissolving Autoradiographic Film.- Appendix: Summary of the Procedure.- References.- VIII The Human Karyotype Analysis of Chromosomes in Mitosis and Evaluation of Cytogenetic Data.- 1. Introduction.- 2. Chromosomes at Normal Metaphase.- 2.1. The Material for Analysis.- 2.2. Location of Individual Chromosomes.- 2.3 Effect of Ageing on the Karyotype.- 2.4. Influence of Culture Conditions on the Number and Structure of Chromosomes.- 3. The Standard Karyotype.- 3.1. The Individual Chromosomes on Routine Stain.- 4. Identification of Individual Chromosomes by Special Methods.- 4.1. The Individual Banding Patterns (Q and G Bands).- 4.2. C Bands.- 4.3. T Bands.- 4.4. Autoradiography.- 4.5. Chromosome Measurement.- 5. Variability of the Karyotype.- 5.1. General Properties of Chromosomal Variants and Their Basis for Recognition.- 5.2. Sites and Frequencies of Chromosomal Variants.- 6. Evaluation of Cyto…