Flow Cytogenetics

312 Pages·1990·12.693 MB·English

by  Joe W. Gray and Johan S. Ploem (Eds.)

ANALYTICAL CYTOLOGY SERIES
Editor-in-Chief
J. S. Ploem
University of Leiden
Leiden, The Netherlands
Editorial Board
D. Arndt-Jovin M. R. Melamed
Max Planck Institute for Biophysical Chemistry Memorial Sloan—Kettering Cancer Center
Gottingen, Federal Republic of Germany New York, New York, USA
K. R. Castleman M. L. Mendelsohn
California Institute of Technology Lawrence Livermore National Laboratory
Pasadena, California, USA Livermore, California, USA
O. D. Laerum M. A. Van Dilla
University of Bergen Lawrence Livermore National Laboratory
Bergen, Norway Livermore, California, USA
S.A. Latt M. van der Ploeg
The Children's Hospital Medical Center University of Leiden
Boston, Massachusetts, USA Leiden, The Netherlands
B.H. Mayall I. T. Young
Lawrence Livermore National Laboratory Technische Hogeschool Delft
Livermore, California, USA Delft, The Netherlands
Marvin A. Van Dilla, Phillip N. Dean, Ole D. Laerum, and Myron R. Melamed (eds)
Flow Cytometry: Instrumentation and Data Analysis, 1985
JoeW. Gray (ed.)
Flow Cytogenetics, 1989
IN PREPARATION:
Kohen: Cell Structure and Function by Microspectrofluorometry
Laerum/Bierknes: Flow Cytometry in Haematology
Flow Cytogenetics
Edited by
Joe W. Gray
Biomedical Sciences Division
Lawrence Livermore National Laboratory
Livermore, California, USA
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Contributors
Κ. L. ALBRIGHT, Los Alamos National Laboratory, Life Sciences Division, Los Alamos,
New Mexico 87545, USA
N. A. ALLEN, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
J. A. ATEN, Laboratory for Radiobiology, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
MARTY F. BARTHOLDI, Los Alamos National Laboratory, US Department of Energy,
Los Alamos, New Mexico 87545, USA
J. G. J. BAUMAN, TNO Radiobiological Institute, Lange Kleiweg 151, 2288 GJ Rijswijk,
The Netherlands
N. C. BROWN, Los Alamos National Laboratory, Life Sciences Division, Los Alamos,
New Mexico 87545, USA
G. BRUNS, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
E. W. CAMPBELL, Los Alamos National Laboratory, Life Sciences Division, Los Alamos,
New Mexico 87545, USA
Α. V. CARRANO, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
MARY CASSIDY, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
M. CHRISTENSEN, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
L. M. CLARK, Los Alamos National Laboratory, Life Sciences Division, Los Alamos, New
Mexico 87545, USA
J. G. COLLARD, The Netherlands Cancer Institute (Antoni van Leeuwenhoekhuis), Department
of Experimental Cytology, H6 121 Plesmanlaan, 1066 CX Amsterdam, The Netherlands
L. SCOTT CRAM, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
P. N. DEAN, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
L. L. DEAVEN, Los Alamos National Laboratory, Life Sciences Division, University of
California, Los Alamos, New Mexico 87545, USA
T. DONLON, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
G. J. VAN DEN ENGH, Biomedical Sciences Division, Lawrence Livermore National
Laboratory, Livermore, California 94550, USA
JOHN H. EVANS, MRC Human Genetics Unit, Western General Hospital, Crewe Road,
Edinburgh , EH4 2XU, Scotland, UK
JUDITH A. FANTES, MRC Human Genetics Unit, Western General Hospital, Crewe Road,
Edinburgh EH42XU, Scotland, UK
xii CONTRIBUTORS
J. J. FAWCETT, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
A. FLINT, The Children's Hospital, Department of Pediatrics, Harvard Medical School, Harvard
University, Cambridge, Massachusetts 02138, USA
J. C. FUSCOE, Center for Environmental Health, U-40, The University of Connecticut, Storrs,
Connecticut 06268, USA
JOE. W. GRAY, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
DARYLL K. GREEN, MRC Human Genetics Unit, Western General Hospital, Crewe Road,
Edinburgh EH42XU, Scotland, UK
P. HARRIS, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
C. E. HILDEBRAND, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
P. J. JACKSON, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
J. W. G. JANSSEN, Deutsches Rotes Kreuz, Blutspendezentrale, Ulm, Oberer Easelberg 10,
Postfach 1564, 7900 Ulm/Donau, FRG
J. H. JETT, Los Alamos National Laboratory, US Department of Energy, Los Alamos, New
Mexico 87545, USA
P. DE JONG, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
S. KOLLA, Los Alamos National Laboratory, Life Sciences Division Los Alamos, New Mexico
87545, USA and Stanford University Graduate School, Stanford, California, USA
PAUL M. KRAEMER, Los Alamos National Laboratory, US Department of Energy, Los
Alamos, New Mexico 87545, USA
L. KUNKEL, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
D. KURNIT, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
M. LALANDE, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
RICHARD G. LANGLOIS, Biomedical Sciences Division, Lawrence Livermore National
Laboratory, Livermore, California 94550, USA
S. A. LATT,t The Children's Hospital, Department of Pediatrics and Genetics, Harvard Medical
School, Harvard University, Cambridge, Massachusetts 02138, USA
ROGER V. LEBO, Department of Obstetrics, Gyneacology and Pediatrics, University of
California, San Francisco, California 94143, USA
J. L. LONGMIRE, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
CHARLOTTE LOZES, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
JOE LUCAS, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
J. S. McNINCH, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
M. L. MENDELSOHN, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
t Deceased.
CONTRIBUTORS xiii
L. J. MEINKE, Los Alamos National Laboratory, US Department of Energy, Los Alamos, New
Mexico 87545, USA
J. MEYNE, Los Alamos National Laboratory, US Department of Energy, Los Alamos, New
Mexico 87545, USA
DAN H. MOORE II, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
R. K. MOYZIS, Los Alamos National Laboratory, US Department of Energy, Los Alamos, New
Mexico 87545, USA
U. MULLER, The Children's Hospital, Department of Pediatrics, Harvard Medical School,
Harvard University, Cambridge, Massachusetts 02138, USA
J. MULLIKIN, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
A. C. MUNK, Los Alamos National Laboratory, US Department of Energy, Los Alamos, New
Mexico 87545, USA
R. NEVE, The Children's Hospital, Department of Pediatrics, Harvard Medical School, Harvard
University, Cambridge, Massachusetts 02138, USA
L. PEDERSON, 4421 Vine Street, Vancouver, British Columbia V4P5W6, Canada
J. PERLMAN, Center for Environmental Health, U-40, The University of Connecticut, Storrs,
Connecticut 06268, USA
DONALD C. PETERS, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
D. PINKEL, Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore,
California 94550, USA
M. VAN DER PLOEG, Sylvius Laboratory, 72 Wassenaarseweg, Leiden, The Netherlands
F. ANDREW RAY, Los Alamos National Laboratory, US Department of Energy, Los Alamos,
New Mexico 87545, USA
A. J. SILVA, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
U. TANTRAVAHI, The Children's Hospital, Department of Pediatrics, Harvard Medical
School, Harvard University, Cambridge, Massachusetts 02138, USA
BARB J. TRASK, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
Livermore, California 94550, USA
A. TULP, The Netherlands Cancer Institute (Antoni van Leeuwenhoekhuis), Department of
Experimental Cytology, H6 121 Plesmanlaan, 1006 CX Amsterdam, The Netherlands
M. A. VAN DILLA, Biomedical Sciences Division, Lawrence Livermore National Laboratory,
University of California, Livermore, California 94550, USA
BRYAN D. YOUNG, Medical Oncology Laboratory, Imperial Cancer Research Fund,
St Bartholomew's Hospital, 45 Little Britain, London EC1, UK
Preface
Flow Cytogenetics describes the application of flow cytometry and sorting to the special tasks of
mammalian chromosome classification and purification. The first publications on flow cytogenetics,
which appeared in 1975; promised accurate, statistically precise chromosome classification using
flow cytometry (called flow karyotyping) as well as high purity chromosome fractionation. The 17
chapters in this book illustrate that the early promise of flow cytogenetics has been realized and
exceeded. Application areas for flow karyotyping now include; quantification of radiation induced
chromosome damage for biological dosimetry, determination of chromosome normality in cell
cultures to suport somatic cell genetic studies and detection of numerical and structural aberrations
associated with genetic diseases such as Down's syndrome. Applications for sorted chromosomes
include gene mapping and the construction of chromosome-specific recombinant DNA libraries.
This book covers both the technical aspects and application areas of flow cytogenetics.
The first chapter serves as an introduction and overview and should be consulted by readers not
familiar with the field.
Chapters 2 to 6 are devoted to the technical aspects of flow cytogenetics; specifically, instru
mentation, cell culture, chromosome isolation, chromosome staining and flow karyotype analysis.
These chapters cover the technical developments that have allowed flow cytogenetics to become a
practical tool in numerous laboratories.
Chapters 7 and 8 cover the use of univariate and bivariate flow karyotyping for chromosome
classification and for detection of homogeneously occurring aberrations (i.e. aberrations that occur
in most or all of the cells from which chromosomes were isolated). These chapters should be
consulted for examples of the use of flow karyotyping for clinical detection of disease-linked
chromosome aberrations or for characterization of cultured cell lines (including human-rodent
hybrids).
Chapters 9 and 10 cover the progress that has been made on the use of flow cytometry for detection
of the random structural aberrations produced by clastogenic agents such as ionizing radiation.
The next three chapters cover specialized developments in flow cytogenetics. Chapter 11
describes the use of silt-scan flow cytometry for chromosome classification and aberration detection
based on chromosome shape. Chapter 12 summarizes the use of velocity sedimentation for
chromosome enrichment prior to sorting. Chapter 13 describes the use of high speed sorting for
chromosome purification.
Chapters 14 through 16 summarize the applications of flow sorted chromosomes. Chapter 14 is
devoted to gene mapping using sorted chromosomes. Chapter 15 focuses on techniques for
construction of recombinant DNA libraries using sorted chromosomes and on the use of these
libraries in genetics research. Chapter 16 continues the theme of the previous chapter; summarizing
the current status of the human-chromosome-specific libraries produced by the National Laboratory
Gene Library Project.
The last chapter focuses on the use of fluorescence in situ hybridization to stain specific
chromosomes, genes or gene transcripts in interphase cells or nuclei so that their presence and
normality can be assessed flow cytometrically. The techniques covered here are rapidly developing
and represent one of the important future directions in flow cytogenetics.
xvi PREFACE
The chapters in this book describe the substantial progress that has been made in flow cyto
genetics during the past 15 years. In addition, they suggest areas where development is continuing.
At least three forces are acting to drive future work: 1. The availability of commercial instru
mentation that is capable of high resolution chromosome analysis and sorting. 2. The need for
chromosome characterization and for chromosome specific DNA generated by the International
Genome effort. 3. The increasing availability of chromosome-specific probes to support
chromosome specific staining using in situ hybridization. This book is aimed at investigators just
entering the field as well as those interested in the continued development or application of Flow
Cytogenetics.
JOEW. GRAY
Livermore, California
July 1989
1
Human Chromosome Analysis by Flow Cytometry
BRYAN D. YOUNG
Medical Oncology Laboratory
Imperial Cancer Research Fund
St Bartholomew's Hospital
45 Little Britain, London EC1, UK
I. Introduction 1
II. Factors Affecting Resolution 3
A. Source of Cells 3
B. Preparative Techniques 3
C. Choice of DNA-specific Stains 5
D. Flow Instrumentation 6
III. Application of Flow Systems to Karyotype Analysis 6
A. Chromosome Classification 6
B. Aberration Detection 7
C. Flow Analysis of a Human "Microchromosome" 8
D. Flow Karyotype Analysis of Tumor Cells 10
IV. Flow Sorting 11
V. Applications of Chromosome Sorting 11
VI. Future Prospects 12
Acknowledgements 13
References 13
I. INTRODUCTION
is not only highly labor intensive but is
The application of banding techniques to the dependent on the skill and objectivity of the
study of metaphase chromosomes has revealed cytogeneticist.
many chromosomal aberrations associated with The automation of karyotype analysis would
genetic disease and cancer. As techniques bring great benefits to the study of human
have improved, the cytogenetic analysis of disease, both in terms of efficiency and
the human karyotype has assumed greater objectivity. Automatic slide-based computer
diagnostic value. In a routine service, it is analysis of the karyotype has been in
generally reckoned that a cytogeneticist can development for a number of years. Although
analyze about 250 samples per year. However, these efforts have not been entirely successful,
in cancer cytogenetics, where more complex they have shown that measurement of the DNA
karyotypes are often encountered, this figure content of chromosomes provides a reliable
may be considerably less. This area of diagnosis basis for karyotype analysis because, unlike
FLOW CYTOGENETICS Copyright © 1989 Academic Press Limited.
ISBN 0-12-296110-2 AH rights of reproduction in any form reserved.
2 BRYAN D. YOUNG
9,10,11,12
RELATIVE FLUORESCENCE
Fig. 1. Flow karyotype measured for chromosomes isolated from a peripheral blood sample of a normal
individual. Chromosomes were prepared from 10 ml of blood using a previously described protocol (49).
Chromosomes were stained with ethidium bromide at 4°C and analyzed on a FACS 440 cell sorter with the
laser tuned to 488 nm. Background noise was reduced by electronic gating (38). The peaks due to each
chromosome are indicated.
length or banding measurements, DNA content population. The coefficient of variation (CV) of
measurements are independent of the degree the peak is an indication of the measurement
of contraction (32,33,34). Flow chromosome precision. Flow cytometry offers the advantages
analysis is an alternative to conventional of high resolution (measurement CVs of less
analytical techniques that are applied to than 2% are common) and statistical precision
chromosomes in metaphase spreads mounted (measurement of 105 chromosomes can be
on microscope slides. In the flow approach, accomplished in a few minutes). Measurement
chromosomes are isolated from mitotic cells, of chromosomal DNA content by flow
stained with one or more fluorescent dyes cytometry has a number of advantages over
(usually DNA specific) and analyzed as they slide-based systems and could, in the future,
flow one by one through the beam(s) of a flow form the basis of an alternative system for
cytometer. Instrumentation for chromosome abnormality detection.
analysis is discussed in detail in Chapter 2. The Flow sorting offers the possibility of purifying
results of analyzing several hundred thousand chromosomes according to their fluorescence
chromosomes are accumulated to form a flow intensity. With proper care, this technology
karyotype like that shown in Fig. 1 for human allows collection of microgram quantities of
chromosomes stained with ethidium bromide. DNA from one or a few chromosome types.
Each peak is produced by chromosomes that Purities above 90% are often achieved. The
yield the same fluorescent signals. The mean DNA collected in this manner has proved useful
of the peak is proportional to the relative for gene mapping (28,29) and for production of
chromosome DNA content. The area of the recombinant DNA libraries (21,45).
peak is proportional to the frequency of It is intended in this chapter to provide an
occurrence of that chromosome type in the overview of the extent to which flow systems