Methods in Human Cytogenetics

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 Cytogenetic Data.- 6.1. Microscopic and Photographic Analysis.- 6.2. Diagnosis of Chromosomal Mosaics.- 6.3. Analysis of Chromosomal Breaks.- 6.4. Chromosomal Analysis by Computer.- 6.5. Storage and Retrieval of Cytogenetic Data.- 7. Nomenclature.- 7.1. Designation of the Normal Karyotype and of Numerical Alterations.- 7.2. Designation of Structural Alterations (Chicago Conference, 1966).- 7.3. Alterations of the Chicago Nomenclature and New Symbols of the Paris Conference (1971).- 7.4. Chromosome Band Nomenclature.- 7.4.1. Identification and Diagrammatic Representation of Landmarks and Bands.- 7.4.2. Designation of Band and Region Numbering.- 7.5. Designation of Structural Changes According to Break Point.- 7.5.1. Simple Breaks.- 7.5.2. Two-break Rearrangements.- 7.5.3. Three-break Rearrangements.- 7.5.4. Four-break Rearrangements.- 7.5.5. Marker Chromosomes.- 7.5.6. Derivative and Recombinant Chromosome.- Appendix: Tables.- References.- IX Analysis of Interphase Nuclei.- 1. Introduction.- 2. Y-Chromatin.- 3. X-Chromatin.- 4. Techniques for Preparing Interphase Nuclei.- 4.1. Introductory Remarks.- 4.2. Obtaining the Material.- 4.3. Fixation.- 4.4. Preparing Different Tissues.- 4.4.1. Buccal Smears.- 4.4.2. Smears from Other Mucosae.- 4.4.3. Teasing Preparations.- 4.4.4. Hair Roots.- 4.4.5. Amniocentesis Material and Other Cell Suspensions.- 4.4.6. Preparations from Membranes.- 4.4.7. Tissue Cultures.- 4.4.8. Peripheral Blood Cells.- 4.4.9. Spermatozoa.- 4.4.10. Section Preparations.- 5. Staining.- 5.1. Fluorescence Staining with Quinacrines.- 5.2. Specific Staining Methods for X-Chromatin.- 6. Evaluation of the Preparations.- 6.1. Y-Chromatin.- 6.1.1. Oral Mucosa.- 6.1.2. Hair Root Cells.- 6.1.3. Blood Cells.- 6.1.4. Spermatozoa.- 6.1.5. Other Tissues.- 6.2. X-Chromatin.- 6.2.1. General Considerations.- 6.2.2. Mucosa Smears.- 6.2.3. Tissue Cultures.- 6.2.5. Membrane Preparations from Amnion.- 6.3. Section Preparations.- 6.4. Prenatal Sex Diagnosis.- 6.5. Polyploid Cells.- 6.5. Malignant Tissues.- Appendix: Laboratory Procedures.- References.- X The Diagnosis of X-Chromatin by the Leukocyte Test.- 1. Introduction.- 2. Methods.- 3. Evaluation.- References.- XI Chromosomes in Meiosis.- 1. Introduction.- 2. Review of the Meiotic Process in Man.- 3. Preparations for Meiotic Prophase Figures.- 3.1. Male.- 3.2. Female.- 4. Preparations for Meiotic Metaphase Figures.- 4.1. Male.- 4.1.1. Squash Method.- 4.1.2. Air-drying Method.- 4.1.3. Staining for Banding Patterns of Meiotic Chromosomes.- 4.1.4. Recovery of Meiotic Figures from the Ejaculated Seminal Fluid.- 4.2. Female.- 5. Interpreting the Findings.- 5.1. Polyploid Germ Cells?.- 5.2. Chiasma Frequency.- 5.3. Precocious Disjunction.- 5.4. Trivalents.- 5.5. Quadrivalents.- 6. Summary.- References.- XII Marginal Notes on the Handling of Tissue Culture Cells for Biochemical Analysis.- 1. Introduction.- 2. Selected General Notes.- 2.1. Some Peculiarities of Homonuclear Fibroblasts.- 2.1.1. Limitation of the Amount of Material.- 2.1.2 Fibroblasts Do Not Grow in Suspension.- 2.1.3. Growth of Human Homonuclear Fibroblasts is Impossible or Unsatisfactory in Chemically Defined Media.- 2.2. Composition of the Medium.- 2.3. Stage within the “Lag-log-stationary” Cycle.- 2.4. Contamination of Cultures with Mycoplasma.- 3. Discussion of Some Procedures.- 3.1. Establishment of Cultures and Method of Subculturing.- 3.2. Harvest of Fibroblast Cultures.- 3.3. Homogenization.- 4. Presentation of Results.- References.