Table Of ContentMethods in Human
Cytogenetics
Edited by
H. G. Schwarzacher and U. Wolf
Coeditor of the English Version
E. Passarge
With Contributions by
W. Gey, W. Krone, S. Ohno, E. Passarge,
R. A. Pfeiffer, W. Schnedl,
H. G. Schwarzacher, M. Tolksdorf, U. Wolf
With 59 Figures
Springer-Verlag
Berlin· Heidelberg· New York 1974
Translated from a revision of the German edition" Methoden
in der medizinischen Cytogenetik", edited by H. G. Schwar
zacher and U. Wolf. Springer-Verlag Berlin Heidelberg New
York 1970.
ISBN-13: 978-3-642-65789-4 e-ISBN-13: 978-3-642-65787-0
DOl: 10.1007/978-3-642-65787-0
This work is subject to copyright. All rights are reserved, whether the whole or
part of the material is concerned, specifically those of translation, reprinting,
re-use of itIustrations, broadcasting, reproduction by photocopying machine or
similar means, and storage in data banks.
Under § 54 of the German Copyright Law, where copies are made for other than
private use, a fee is payable to the publisher, the amount of the fee to be
determined by agreement with the publisher.
© by Springer-Verlag Berlin' Heidelberg 1974. Library of Congress Catalog
Card Number 74-6556.
Softcover reprint ofthe hardcover 1st dition 1974
The use of registered names, trademarks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the
relevant protective laws and regulations and therefore free for general use.
Typesetting, Printing and Binding: Universitatsdruckerei H. StOrtz AG, WOrzburg
Preface
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."
In preparing the English edition, the German edition was first
translated by Mr. YArER COHEN, whose work is gratefully acknowledged.
However, it would have been difficult to edit the book without the help
of EBERHARD P ASSARGE, who revised all chapters except the last two (XI
and XII) and offered many valuable comments. We are much indebted
to him.
Wi en and Freiburg i. Br., March 1974
H. G. SCHWARZACHER
U. WOLF
Contents
CHAPTER I Cell Cultures from Blood and Bone Marrow
RUDOLF ARTHUR PFEIFFER
1. Introduction . . . . .
2. Bone Marrow . . . . . 1
2.1. Collection of Material. . 2
2.2. Direct Preparation of Chromosomes from Cells in
Metaphase or Prometaphase . . . . . . 2
2.2.1. Direct Preparation of Cells Immediately Following
Biopsy. . . . . . . . . . . .... 3
2.2.2. Cell Preparation Following Short Term Incubation
with Colchicine. . . . . . . . . . . . . . . 3
2.3. Storage of Bone Marrow Biopsies. . . . . . . 5
2.4. Comments on the Preparation of Chromosomes 5
2.5. Long Term Cultures of Bone Marrow Cells 6
3. Culture of Peripheral Blood Cells . . . 7
3.1. Whole Blood Cultures. . . . . . . . 8
3.2. Leukocyte Culture after Separation of Erythrocytes 10
3.2.1. Separation of Erythrocytes. . . 11
3.2.2. Separation of Mononuclear and Polymorphonuclear
Leukocytes. . . . . . . . . . 12
3.2.3. Choice of Medium . ..... 14
3.2.4. Supplements to Culture Medium 15
3.2.5. Storage of Culture Media . . . 16
3.2.6. Duration of Culture. . . . . . 17
3.2.7. Increasing the Number of Mitoses 17
3.3. Storage and Transport of Blood Samples. 17
3.4. Procedure with Leukemias. . . . . . . 18
3.5. Long Term Cultures from Peripheral Blood Cells 18
3.6. Lymphocytes from Lymph Nodes, Thymus, and Spleen 20
4. Phytohemagglutinin: Properties and Mode of Action 21
Appendix: Laboratory Procedures 26
References . . . . . . . . . . . . . . . . . . . 28
CHAPTER II Cell Cultures from Tissue Explants
ULRICH WOLF
l. Introduction . . . . 39
2. Areas of Application 39
3. Material ..... . 41
4. Introduction to Methods. 41
5. Techniques for Collecting Tissue 42
VIII Contents
6. Setting up Tissue Explants and Culturing Cells 44
6.1. Enzymatic Dissociation of Tissue Explants 44
6.2. Setting up Solid Explants . . . . . . 45
6.2.1. Direct Deposition on the Glass Surface 45
6.2.2. Plasma Method. . . 45
6.2.3. "Sandwich" Method 46
6.2.4. Cellophane Method. 47
6.3. Subculturing . . . . 48
6.4. Culturing in a CO Incubator. 49
2
6.5. Abortus Material . . . . . . 49
7. Harvest of Cultures for Chromosome Preparation. 49
8. Long Term Preservation of Tissue Culture Cells 50
Appendix I: Summary of Some of the Methods.. 51
Appendix II: Catalog of Some Reagents, Media and
Culture Vessels 55
References . . . . . . . . . . . . . . . . . . . 57
CHAPTER III Culture and Preparation of Cells from Amniotic Fluid
ULRICH WOLF
1. Introduction . . . . . . . . . . . . . . . . 59
2. Collecting and Transportation of Amniotic Fluid 64
x-
3. and Y-Chromatin ........... . 64
4. Cell Culture . . . . . . . . . . . . . . . . 65
Appendix: Protocol for Amniotic Fluid Cultures 67
References. . . . . . . . . . . . . 69
CHAPTER IV Preparation of Metaphase Chromosomes
HANS GEORG SCHWA RZACHER
1. Introduction . . . . . . . . . 71
2. Preparation of Cell Suspensions. 72
2.1. Material .......... . 72
2.2. General Observations on Preparation Procedure 72
2.3. Preparation by Air Drying. . . . . . . . . 73
2.4. Preparation by Squashing . . . . . . . . . 74
3. Direct Preparation from Monolayer Cultures . 75
4. Direct Preparation of Embryonic Tissue 76
4.1. Field of Application. 76
4.2. Material. 76
4.3. Technique .... . 77
5. Staining ..... . 77
Appendix: Laboratory Guide. 78
References . . . . . . . . . 81
CHAPTER V Fluorescence Microscopy of Chromosomes and Interphase
Nuclei
HANS GEORG SCHWA RZACHER
1. Introduction . . . . . . . . . . . . . . . . . . 83
2. Fluorescent Dyes for Chromosomes and Cell Nuclei 83
Contents IX
2.1. Acridine Orange . . . . . . . . . . . . 83
2.1.1. Procedure of Staining with Acridine Orange 84
2.2. Quinacrine Derivatives 84
2.2.1. Application . . . . . . 84
2.2.2. Quinacrine Dyes . . . . 86
2.2.3. Preparation of Specimens 86
2.2.3.1. Chromosome Preparations 86
2.2.3.2. Interphase Cells 87
2.2.4. Staining Procedures . . . 87
2.2.5. Restaining with Other Dyes 89
3. Other Fluorochromes . . . 89
4. Technique of Fluorescence Microscopy 89
4.1. Principle ...... . 89
4.2. Microscope Equipment 90
4.3. Microphotography . . 90
5. Analysis of Preparations . 91
5.1. General Comments . . . 91
5.2. Chromosome Preparations 91
5.3. Interphase Cells and Y-Chromatin 92
Appendix 1: Staining with Quinacrine-Dihydrochloride
and Quinacrine-Mustard. . . . . . . . . . . . 92
Appendix 2: Suppliers of Quinacrine Stains 92
Appendix 3: Phosphate Buffer Solution (Sorensen) 93
References . . . . . . . . . . . . . . . . . . 93
CHAPTER VI Banding Patterns in Human Chromosomes Visualized by
Giemsa Staining after Various Pretreatments
WOLFGANG SCHNEDL
1. Introduction . . . . . . . . . . . . . . 95
2. General Comments on the Different Methods 97
3. Methods for Demonstrating the Centromeric Hetero-
chromatin . . . . . . . . . . . . . 98
3.1. The Method of Arrighi and Hsu (1970) 99
3.2. The Method of Yunis et al. (1971) 99
3.3. Meiotic Chromosomes. . . . . . . . 99
3.4. Methods for Demonstrating the Secondary Constric-
tion of Chromosome No.9. . . . . . . . . . . . 100
4. Methods for Demonstrating Banding Patterns over the
Entire Chromosome. . . . . . . . . . . . . . . 101
4.1. Methods Using NaOH Treatment Followed by an In-
cubation in Buffer . . . . . . . 101
4.1.1. The Method of Schnedl (1971) . . . . 101
4.1.2. The Method of Gagne et al. (1971) . . 104
4.1.3. The Method of Drets and Shaw (1971) 104
4.2. Methods Using Various Incubation Procedures with-
out NaOH. . . . . . . . . . . . 104
4.2.1. The Method of Sumner et al. (1971). . . . . . . . 104
x
Contents
4.2.2. The MethodofPatiletal. (1971) "Giemsa-9-Technique" 105
4.3. Methods Using Enzymatic Digestion (Pronase, Tryp-
sin) . . . . . . . . . . . . . . . . 105
4.3.1. The Method of Dutrillaux ef at. (1971) 105
4.3.2. The Method of Seabright (1971) 106
4.3.3. The Method of Wang and Fedoroff (1972) 107
4.3.4. The Method by Sun, Chu and Chang (1973) 107
4.4. The Reverse Bands. . . . . . . . . . . 107
4.4.1. Reverse Bands Using G;emsa (Dutrillaux and Lejeune,
1971) ................... 107
4.4.2. The Acridine Orange Reverse Bands (for instance
Bobrow et aI., 1972b) . . . . . . 107
Appendix: Laboratory Procedures 108
References. . . . . . . . . . . 116
CHAPTER VII Autoradiography of Human Chromosomes with
3H_ Thymidine
WOLFGANG GEY
I. Introduction . . 119
2. Theoretical Basis 120
2.1. The DNA Synthesis Cycle 120
2.2. Incorporation of Thymidine into DNA 122
2.3. Tritiated Thymidine. . . . . . 122
2.4. Autoradiographic Film Material 124
2.4.1. Emulsions... 124
2.4.2. Stripping Films. . . . . . . . 125
3. Techniques. . . . . . . . . . 125
3.1. Labeling with Tritiated Thymidine 125
3.1.1. Continuous Labeling . . . . . . 125
3.1.2. Pulse Labeling . . . . . . . . . 126
3.2. Harvest of Cultures and Chromosome Preparations 126
3.3. The Autoradiographic Technique . . . . . . . . . 127
3.3.1. Covering the Preparation with Autoradiographic Film
Material. . . . . . . . . . . . . . . . . . .. 127
3.3.2. Exposure ................... 129
3.3.3. Development and Fixation of the Autoradiographic
Film . . . . . . . . . . . . . . . . . .. 129
3.3.4. Staining.................... 130
3.3.5. Evaluation of Chromosomes in Autoradiographs . . 130
3.3.6. Preparation of Control Pictures by Removal of Silver
Grains and Dissolving Autoradiographic Film 130
Appendix: Summary of the Procedure . 132
References . . . . . . . . . . . . . . . . 132
CHAPTER VIII The Human Karyotype
Analysis of Chromosomes in Mitosis and Evaluation of Cyto
genetic Data
EBERHARD P ASSARGE
1. Introduction . . . . . . . . . . . . . . . . . . 135
Contents XI
2. Chromosomes at Normal Metaphase 136
2.1. The Material for Analysis 136
2.2. Location of Individual Chromosomes 137
2.3. Effect of Ageing on the Karyotype 140
2.4. Influence of Culture Conditions on the Number and
Structure of Chromosomes. 140
3. The Standard Karyotype. 143
3.1. The Individual Chromosomes on Routine Stain. 145
4. Identification of Individual Chromosomes by Special
Methods. 147
4.1. The Individual Banding Patterns (Q and G Bands) 151
4.2. C Bands. 158
4.3. T Bands. 160
4.4. Autoradiography 160
4.5. Chromosome Measurement 166
5. Variability of the Karyotype 167
5.1. General Properti.es of Chromosomal Variants and
Their Basis for Recognition 168
5.2. Sites and Frequencies of Chromosomal Variants 169
6. Evaluation of Cytogenetic Data. 177
6.1. Microscopic and Photographic Analysis 177
6.2. Diagnosis of Chromosomal Mosaics. 180
6.3. Analysis of Chromosomal Breaks . 180
6.4. Chromosomal Analysis by Computer 181
6.5. Storage and Retrieval of Cytogenetic Data 183
7. Nomenclature 183
7.1. Designation of the Normal Karyotype and of Numeri-
cal Alterations 184
7.2. Designation of Structural Alterations (Chicago Con-
ference, 1966) 185
7.3. Alterations of the Chicago Nomenclature and New
Symbols of the Paris Conference (1971) 187
7.4. Chromosome Band Nomenclature. 187
7.4.1. Identification and Diagrammatic Representation of
Landmarks and Bands. 188
7.4.2. Designation of Band and Region Numbering. 188
7.5. Designation of Structural Changes According to Break
Point 189
7.5.1. Simple Breaks 189
7.5.2. Two-break Rearrangements 190
7.5.3. Three-break Rearrangements 191
7.5.4. Four-break Rearrangements 192
7.5.5. Marker Chromosomes . 193
7.5.6. Derivative and Recombinant Chromosome. 193
Appendix: Tables. 194
References . 199
XII Contents
CHAPTER IX Analysis of Interphase Nuclei
HANS GEORG SCHW ARZACHER
1. Introduction . 207
2. Y-Chromatin. . . . . . . . 208
3. X-Chromatin. . . . . . . . 208
4. Techniques for Preparing Interphase Nuclei 209
4.1. Introductory Remarks . 209
4.2. Obtaining the Material 210
4.3. Fixation . . . . . . . 210
4.4. Preparing Different Tissues. 211
4.4.1. Buccal Smears . . . . . . 211
4.4.2. Smears from Other Mucosae 211
4.4.3. Teasing Preparations 212
4.4.4. Hair Roots. . . . . . . . 212
4.4.5. Amniocentesis Material and Other Cell Suspensions 212
4.4.6. Preparations from Membranes 212
4.4.7. Tissue Cultures. . . . 213
4.4.8. Peripheral Blood Cells. 214
4.4.9. Spermatozoa 214
4.4.10. Section Preparations 214
5. Staining . . . . . . 214
5.1. Fluorescence Staining with Quinacrines 214
5.2. Specific Staining Methods for X-Chromatin 215
6. Evaluation of the Preparations 217
6.1. Y-Chromatin. . 217
6.1.1. Oral Mucosa. . 218
6.1.2. Hair Root Cells 219
6.1.3. Blood Cells 219
6.1.4. Spermatozoa. 220
6.1.5. Other Tissues. 221
6.2. X-Chromatin. 221
6.2.1. General Considerations 221
6.2.2. Mucosa Smears. . . . 222
6.2.3. Tissue Cultures. . . . 225
6.2.4. Membrane Preparations from Amnion. 225
6.2.5. Section Preparations 225
6.3. Prenatal Sex Diagnosis 226
6.4. Polyploid Cells . . . . 226
6.5. Malignant Tissues. . . 228
Appendix: Laboratory Procedures 228
References . . . . . . . . . . . 232
CHAPTER X The Diagnosis of X-Chromatin by the Leukocyte Test
MARLIs TOLKSDORF
1. In trod ucti on 235
2. Methods. 235
3. Evaluation 236
References 242
