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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY


    ENVIRONMENTAL HEALTH CRITERIA 46






    GUIDELINES FOR THE STUDY OF GENETIC EFFECTS
    IN HUMAN POPULATIONS







    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Orgnization
    Geneva, 1985


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CONTENTS

GUIDELINES FOR THE STUDY OF GENETIC EFFECTS IN HUMAN POPULATIONS

CONTRIBUTORS

PREFACE

1. INTRODUCTION

2. METHODOLOGICAL AND EPIDEMIOLOGICAL ISSUES

    2.1. General considerations
    2.2. Methodological issues
    2.3. Epidemiological considerations - common components
         2.3.1. Study samples
         2.3.2. Comparison samples
         2.3.3. Confounding variables
         2.3.4. Interaction
         2.3.5. Methods for estimating exposure
         2.3.6. End-points
         2.3.7. Dose-response relationships
         2.3.8. Time-response relationships
         2.3.9. Problems in sample size and interpretation of 
                findings
    2.4. Long-term medical follow-up
         2.4.1. Relevance and limitations
         2.4.2. Personal identification
         2.4.3. Starting-point records
         2.4.4. End-point records
         2.4.5. Methods required for integrating source files

3. MUTATIONS IN SOMATIC CELLS

    3.1. General considerations
         3.1.1. End-points
         3.1.2. Study requirements
    3.2. Chromosomal end-points
         3.2.1. Structural aberrations
                3.2.1.1  Methods of culture
                3.2.1.2  Fixation and slide preparation
                3.2.1.3  Analysis of cells
                3.2.1.4  Data analysis
                3.2.1.5  Radiation-induced aberrations and 
                         estimates of exposure
                3.2.1.6  Chemically-induced aberrations and 
                         estimates of exposure
                3.2.1.7  Conclusions
         3.2.2. Sister-chromatid exchange (SCE)
                3.2.2.1  Formation of sister-chromatid exchange
                3.2.2.2  Relevance of sister-chromatid exchange

                3.2.2.3  Factors potentially influencing SCE 
                         frequency
                3.2.2.4  Methods for sister-chromatid exchange 
                         analysis
                3.2.2.5  Data processing and presentation
                3.2.2.6  Conclusions
    3.3. Gene mutations
         3.3.1. Principles and basis for the methods
                3.3.1.1  Autoradiographic method
                3.3.1.2  Cloning method
         3.3.2. Relevance and limitations
                3.3.2.1  Relevance
                3.3.2.2  Limitations
         3.3.3. Procedures for assay of TGr T-lys arising  in vivo 
                in human beings
                3.3.3.1  Autoradiographic method
                3.3.3.2  Cloning method
         3.3.4. Data presentation and analysis
                3.3.4.1  Autoradiographic method
                3.3.4.2  Cloning method
         3.3.5. Conclusions

4. GERMINAL MUTATIONS

    4.1. Introduction
         4.1.1. Approaches for detecting germinal mutations
                4.1.1.1  Detection of chromosomal mutations
                4.1.1.2  The biochemical approach to detecting 
                         point mutations
                4.1.1.3  Indicator phenotypes
         4.1.2. Methodological considerations and strategies
                4.1.2.1  Sample acquisition and storage
                4.1.2.2  Timing of studies
         4.1.3. Summary
    4.2. Germinal chromosomal abnormalities
         4.2.1. Principles and basis of the method
         4.2.2. Relevance and limitations
                4.2.2.1  Studies of induced abortions
                4.2.2.2  Studies of fetal deaths
                4.2.2.3  Studies of prenatal diagnosis specimens
                4.2.2.4  Studies of live births
                4.2.2.5  Studies of indicator phenotypes of 
                         chromosomal abnormalities
         4.2.3. Procedures
                4.2.3.1  Fetal specimens
                4.2.3.2  Live births and other offspring
                4.2.3.3  Detection of "indicator" phenotypes for 
                         germinal chromosomal mutations - trisomies
                4.2.3.4  Data presentation
    4.3. Biochemical approaches to detecting gene mutations in 
         human populations
   
         4.3.1. Biochemical methods for monitoring for gene 
                mutations
                4.3.1.1  One-dimensional electrophoresi
                4.3.1.2  Two-dimensional electrophoresi
                4.3.1.3  Enzyme activity
                4.3.1.4  Other biochemical approaches

         4.3.2. Analytical strategy and methodological 
                considerations
                4.3.2.1  One-dimensional electrophoresis
                4.3.2.2  Two-dimensional electrophoresis
                4.3.2.3  Enzyme activity
                4.3.2.4  Sample acquisition and storage
         4.3.3. Data management
         4.3.4. Considerations in screening for germinal mutations
                4.3.4.1  Sample size
                4.3.4.2  Distinction between "true" and "apparent" 
                         mutations
                4.3.4.3  Implementation of gene mutation screening 
                         programmes
         4.3.5. Summary
    4.4. Sentinel phenotypes
         4.4.1. Introduction
         4.4.2. Basis of method
         4.4.3. Relevance and limitations
         4.4.4. Procedures
                4.4.4.1  Surveillance of sentinel anomalies
         4.4.5. Data intrepretation
                4.4.5.1  Surveillance of sentinel anomalies
         4.4.6. Conclusions
    4.5. Fetal death
         4.5.1. Introduction
         4.5.2. Procedures
         4.5.3. Data processing and presentation
         4.5.4. Conclusions

REFERENCES

CONTRIBUTORS

The following experts participated in the preparation of this 
monograph: 

Dr R.J. Albertinib, University of Vermont, College of Medicine, 
   Burlington, Vermont, USA

Dr K. Altlandb, Institute for Human Genetics, University of 
   Giessen, Giessen, Federal Republic of Germany

Dr A.V. Carranoa,b, Biomedical Sciences Division, L-452, Lawrence 
   Livermore National Laboratory, Livermore, California, USA

Dr A. Czeizelb, Department of Human Genetics, National Institute of 
   Hygiene, Budapest, Hungary

Dr G.R. Douglasa,b, WHO Collaborating Centre, Mutagenesis Section, 
   Department of National Health and Welfare, Ottawa, Ontario, 
   Canada

Dr H.J. Evansb, Medical Research Council, Clinical and Population 
   Cytogenetics Unit, Western General Hospital, Edinburgh, 
   Scotland, United Kingdom

Dr E.B. Hooka,b, Bureau of Maternal and Child Health, New York 
   State Department of Health, Albany, New York, and Department of 
   Pediatrics, Albany Medical College, Albany, New York, USA

Dr J.R. Millera,b,c, Central Research Division, Takeda Chemical 
   Industries, Ltd., Osaka, Japan

Dr H. Mohrenweisera,b, Department of Human Genetics, University of 
   Michigan, Ann Arbor, Michigan, USA

Dr H.B. Newcombeb, Deep River, Ontario, Canada

Dr R.J. Prestona,b, Biology Division, Oak Ridge National 
   Laboratory, Oak Ridge, Tennessee, USA

Dr. E.M.B. Smithb, International Programme on Chemical Safety, 
   World Health Organization, Geneva, Switzerland

Ms M. Smitha,b, Vital Statistics and Disease Registries Section, 
   Statistics Canada, Ottawa, Ontario, Canada

Dr D. Warburtonb, Department of Human Genetics and Development, 
   Columbia University, New York, New York

-------------------------------------------------------------------
a   WHO/DNHW Consultation, July 27-28, 1981, Ann Arbor, Michigan, 
    USA
b   WHO/DNHW Task Group Meeting, September 18-21, 1984, Ottawa, 
    Ontario, Canada
c   Chairman, Ann Arbor and Ottawa meetings

NOTE TO READERS OF THE CRITERIA DOCUMENTS

    Every effort has been made to present information in the 
criteria documents as fully and as accurately as possible.  In 
the interest of all users of the environmental health criteria 
documents, readers are kindly requested to communicate any errors 
found to the Manager of the International Programme on Chemical 
Safety, World Health Organization, Geneva, Switzerland, in order 
that they may be included in corrigenda, which will appear in 
subsequent volumes. 

    In addition, experts in any particular field dealt with in the 
criteria documents are kindly requested to make available to the 
WHO Secretariat any important published information that may have 
inadvertently been omitted, so that it may be considered in the 
event of updating and re-evaluation of the conclusions contained in 
the criteria documents. 

PREFACE

    Monitoring and assessment of effects on human health from 
exposure to environmental agents of all types, with monitoring to 
confirm that control measures are working effectively, are key 
aspects of the World Health Organization's Environmental Health 
Programme.  Without objective data on effects, attempts to assign 
causes can only be speculative.  Speculation does not provide a 
reliable basis for remedial action and the control of health 
hazards.  Knowledge of the presence and identity of mutagenic 
agents in the environment and the extent of exposure to them is 
therefore extremely important in interpreting and using the results 
obtained from monitoring populations for genetic effects. 

    Within the World Health Organization, there are a number of 
programmes that are concerned with the monitoring and assessment of 
human exposures and effects on health. 

    Control of environmental health hazards is one such programme.  
This has the objective that, by 1989, the countries actively 
involved will have formulated and developed national policies and 
programmes for the protection of the health of their populations 
against environmental hazards.  A number of projects on the 
monitoring of air and water quality, food contamination, and of 
selected pollutants in human tissues are carried out by the World 
Health Organization.  These activities are complemented by a more 
recent component, the Human Exposure Assessment Location (HEAL) 
project, which is devoted directly to the monitoring of human 
exposure to certain pollutants in all environmental media.  All of 
these monitoring projects are implemented in conjunction with the 
United Nations Environment Programme's Global Environmental 
Monitoring System (GEMS). 

    The International Programme on Chemical Safety is a cooperative 
activity of the World Health Organization, the International Labour 
Office, and the United Nations Environment Programme.  It resulted 
from concern expressed on the hazards of exposure to chemicals and 
the pressing need to identify and assess them, so that control 
measures could be applied and safe use achieved.  One of the means 
of achieving safe use is the preparation and dissemination of 
documents of practical use to those involved in implementing 
chemical safety.  Publication in the International Programme on 
Chemical Safety environmental health criteria series emphasizes the 
importance of a systematic approach to monitoring human populations 
for genetic effects as an integral part of environmental health 
management. 

    These guidelines are intended as a source of practical 
information on the design and conduct of genetic studies on human 
populations exposed, or suspected of being exposed, to mutagenic 
agents.  As they are guidelines, they do no include comprehensive 
protocols for studies.  However, attention is directed to important 
details that must be included, as well as pitfalls to be avoided.  
It is envisaged that this document will be of use both to those 
already involved in the assessment of human genetic hazards and to 

those who wish to become better informed on this subject.  Although 
future requirements are not ignored, these guidelines principally 
encompass methods that are considered practicable at present. 

    The original concept for these guidelines arose from the 
recommendations of a WHO Consultation on Genetic Monitoring held in 
Ottawa, Canada, on October 17, 1980, organized by the WHO 
Collaborating Centre on Environmental Mutagenesis, Department of 
National Health and Welfare (DNHW) Canada, and held in conjunction 
with an International Conference on Chemical Mutagenesis, 
Population Monitoring, and Genetic Risk Assessment.  The drafting 
of the monograph, implemented initially by the late DR K.C. BORA 
(DNHW), and subsequently directed by DR G.R. DOUGLAS (DNHW), who 
was also responsible for editing the final draft, began with a 
WHO/DNHW Consultation held in Ann Arbor, Michigan, USA, on 
July 27 - 28, 1981.  This meeting produced a draft document 
containing material that formed the basis for this monograph. 
Subsequently, a joint WHO/DNHW Task Group Meeting, held in Ottawa, 
Canada, on September 18 - 21, 1984, completed the project. 


                            * * *

    Financial and other support for the meeting was provided by the 
Department of National Health and Welfare, Canada.  The United 
Kingdom Department of Health and Social Security covered the costs 
of printing. 


1.  INTRODUCTION

    The extent to which human somatic and germinal mutation 
frequencies may be increased by exposure to ionizing radiation 
and to the variety of chemicals that characterize modern societies 
has been a matter of concern in recent years.  Somatic mutations, 
either genic or chromosomal, are not transmitted to the offspring 
of an exposed individual.  However, increases in the frequency of 
these mutations may contribute to an increase in the frequency 
of acquired disorders, for example, cancer.  Increases in the 
frequency of germinal mutations, genic or chromosomal, are likely 
to contribute to inherited defects in the offspring of individuals 
exposed to mutagenic agents.  There is, therefore, a clear need to 
develop and apply methods to study exposed populations at risk of 
increased levels of somatic or germinal mutations. 

    Any effort to determine whether an increase in mutation rate 
has occurred in a given population must contend with formidable 
problems, one of the most significant of which is defining the 
genetic end-points suitable for study.  Germinal mutations, either 
genic or chromosomal, can give rise to a plethora of phenotypes, 
only a few of which are useful and meet the criteria required 
for studies in mutation epidemiology.  Likewise, using current 
techniques, only a subset of somatic-cell mutations are amenable 
to study.  However, since it is much easier to obtain large samples 
of somatic rather than germinal events, it would be ideal if some 
measure of germinal mutations and the health effects of their 
consequent phenotypes could be conveniently extrapolated from 
studies of somatic events.  Although such an extrapolation has 
been attempted for the effects of ionizing radiation (Brewen et 
al., 1975), it is not possible, at present, to attempt the same for 
chemical effects.  Despite these difficulties, it is essential that 
efforts be made to initiate studies aimed at measuring somatic and 
germinal genetic changes and assessing the relationships between 
the two. 

    Although similar mutational changes will occur in somatic 
and germ cells,the methods for detecting them are quite different.  
An increase in the frequency of somatic mutations, genic or 
chromosomal, can be established from relatively few individuals, 
provided that a large number of cells is analysed from each sample 
(Bloom, 1981).  In contrast, determining an increase in the 
frequency of mutation in germ cells by examining affected offspring 
involves large study populations; the smaller the increase to be 
detected (or excluded), the larger the sample needed (Neel, 1980; 
Vogel & Altland, 1982).  International cooperation may be necessary 
to obtain adequate samples.  However, because of the costs involved 
in mounting large-scale epidemiological surveys, it is essential 
that they should be designed to make the most efficient use of 
resources and that the tests used should be designed to give 
meaningful results.  The general principles are the same for 
all countries, and comparability can be achieved through 
standardization of many of the procedures.  Since the practical 
details of the strategy and tactics in any one country will be 

influenced by local circumstances, the emphasis in these guidelines 
will be placed on general principles and examples of possible 
procedures. 

2.  METHODOLOGICAL AND EPIDEMIOLOGICAL ISSUES

2.1.  General Considerations

    Certain general principles influence the design of 
epidemiological studies for genetic effects and the nature of 
the procedures for data gathering and analysis.  The information 
requirements are basically similar, whether changes in the genetic 
make-up of somatic cells or germ cells in the exposed individuals, 
or inherited changes affecting the offspring of such individuals 
are being investigated.  The key elements for any such study should 
include: 

    (a)  means of identifying the exposed (or affected) population 
         to be studied; 

    (b)  availability of a reasonably similar comparison group, to 
         serve as the unexposed (or unaffected) controls;

    (c)  an idea of either the nature of the presumed mutagen, or, 
         at least, of the general source of the anticipated harm;

    (d)  prior knowledge of the likely end-points that could serve 
         as indicators of genetic damage; 

    (e)  where possible, some separation into different levels of 
         exposure with which to investigate a likely dose-response 
         relationship; and 

    (f)  a means of observing the time course of the response.

    Not all of these will be equally available for any study. 
However, none of the elements that are available should be 
overlooked in the initial documentation or the subsequent 
analysis.  In general, the need to guard against oversights in 
necessary information will tend to increase with the interval of 
time between the causal events and the resulting expressions of 
harm. 

    Wherever long-term follow-up is involved, particular attention 
should be paid to the personal identification of the individuals so 
that starting-point data and end-point data will be appropriately 
and unambiguously matched. 

2.2.  Methodological Issues

    Not all studies will have a broad information base at the 
outset.  However, useful work can still be done, even where there 
is no prior indication of harmful exposures, no suggestion of which 
persons might be exposed, and no clear idea of the end-point 
effects to expect.  In principle, watch can be kept over a wide 
range of potential indicators of genetic damage, among large 
populations, such as the new-born, and this should be continued 
over a long period of time.  In this broad kind of continuing 
observation, it is the temporal changes in the frequency of 

end-points, above familiar background rates, that alert the 
investigator to a possible mutagenic effect.  The term 
"surveillance" is used to describe this type of activity. 

    Methods for determining the frequency of a number of end-points 
are discussed in this monograph.  Various techniques are used for 
determining germinal mutations.  Indicators include potential 
germinal effects, adverse reproductive outcomes, or other health 
problems that may have a genetic basis. 

    Because of size considerations, it will often be necessary to 
combine data from many studies.  This will be greatly facilitated 
if all investigators adhere to standardized laboratory procedures 
and record-keeping practices including identification of the 
individuals, with standard nomenclature for causes of death, 
congenital anomalies, and protein and chromosomal variants. 

2.3.  Epidemiological Considerations - Common Components

    The following comments apply to all methods and end-points. 

2.3.1.  Study samples

    The appropriate epidemiological study design for examining the 
possible hazards of agents must be determined.  A cohort design 
will be used in most studies where there is known exposure to a 
suspected mutagenic agent.  Alternatively, a case-control study 
design may be used when exposure is common (e.g., smoking, 
caffeine), or rare phenotypes are the end-points of interest 
(e.g., new mutant phenotypes).  In a case-control study, groups 
of individuals are selected on the basis of whether they have the 
genetic end-point under study.  Also, in a case-control study, a 
number of exposures can be evaluated in relation to a selected end-
point, in contrast to a cohort study in which a number of diseases 
are evaluated in relation to one or more exposures. 

    In determining the population to be studied, several different 
approaches can be taken.  For example, questionnaires can be used 
to determine if individuals in a particular setting have been 
exposed to a putative hazard, and to gather data on possible 
controls, or existing data sources (e.g., vital records, census 
data, employment and work history records, special registries, 
populations of specific geographical locations) may be used.  
Regardless of the approach used, sufficient personal identifying 
information must be recorded regarding the individuals to allow for 
follow-up and for searching any large files. 

2.3.2.  Comparison samples

    The identification and use of appropriate controls is 
essential.  In both cohort and case-control studies, a basis for 
comparison is required, in order to evaluate whether the outcome 
observed differs from that expected, had there not been any 
increased risk from the agent under study.  In certain cases a 
population may serve as its own control by sampling before, during, 

and after exposure (internal controls in a longitudinal study). 
Since the frequency of a given end-point may vary according to age, 
sex, socioeconomic status, and other variables, it is important 
that this is taken into consideration when making the comparisons. 

2.3.3.  Confounding variables

    Confounding can be described as the mixing of effects caused by 
variables that are associated with both the exposures and end-
points to be studied, or a distortion of the apparent effect of an 
exposure on risk brought about by the association with other 
factors that can influence the outcome.  In this regard, the 
following should be considered: 

    (a)  variables known to cause mutagenic effects should be
         identified as far as possible (e.g., cigarette smoking, 
         individuals known to have undergone radiotherapy);

    (b)  cases and controls should be appropriately matched (e.g., 
         for age and sex) in such a way that they have the same 
         distribution with respect to known confounding variables; 
         and 

    (c)  appropriate statistical analysis to partition confounding 
         variables should be used. 

2.3.4.  Interaction

    If an exposure is associated with an increased risk of a 
particular end-point, it is important to determine whether the 
risk is additive to that of other known causes of disease, or 
synergistic in its effect. 

2.3.5.  Methods for estimating exposure

    The following methods can be used to estimate either the levels 
of exposure, or values that are proportional to the levels of 
exposure: 

    (a)  estimates of length of exposure as well as ambient levels 
         of the agent may be the best or only estimate of dose in a 
         working environment; it is recognized that such estimates 
         are crude, but, in some situations, they may be all that 
         is available; 

    (b)  direct measurement of an agent in the environment (e.g., 
         air or water sampling) may be used; 

    (c)  direct measurement of an agent or its metabolites in body 
         fluid and tissues to estimate body burden (e.g., blood, 
         urine, hair, teeth); 

    (d)  observation of other pathological evidence of organ or 
         tissue damage (e.g., liver damage, chloracne) may be 
         helpful in estimating doses; and 

    (e)  the use of questionnaires related to work histories and 
         lifestyles may assist in determining the exposure of 
         concern or other exposures. 

    In some cases, there may be ample time to obtain exposure 
data, but, with emergency situations, the information may be only 
transiently available.  Thus, it must be collected early and the 
people involved, recruited, or identified before they disperse. 

2.3.6.  End-points

    Effects of somatic or germinal origin are considered in
sections 3 and 4 of this document, respectively.

2.3.7.  Dose-response relationships

    The existence of a dose-response relationship - that is, an 
increase in disease incidence with increase in amount of exposure - 
supports the view that an association is a causal one.  Thus, it is 
desirable to attempt to quantify exposure as far as possible.  
However, under certain circumstances, in order to make the best use 
of limited resources, initial studies could be restricted to those 
with high exposure doses.  Individuals so exposed would be compared 
to an unexposed population.  If no differences were found, the 
study could be terminated, but it should be noted that, at high 
doses, excess cell lethality might mask genetic effects.  If 
differences were observed, effects at intermediate dose levels 
should be studied, paying special attention to possible confounding 
synergistic effects. 

    Estimates of doses for populations that have been exposed 
accidently will most likely be less accurate than for those exposed 
occupationally and medically.  In such cases, it may be more useful 
to estimate the upper boundary of the dose (i.e., maximum possible 
dose) rather than the mean, or to stratify the population into 2 or 
3 groups whose bounds, though wide, probably do not overlap enough 
to wipe out the suspected differences between them. 

2.3.8.  Time-response relationships

    Consideration of the optimal sampling time is critical for 
quantifying the somatic end-points in the exposed individual. 
Appropriate sampling times should be selected to maximize an 
effect, whenever this is possible.  This is discussed further in 
section 3. 

2.3.9.  Problems in sample size and interpretation of findings

    The ability to detect an effect of a given incidence will 
depend on the sample size that can be studied and the base-line 
frequency of a given end-point in the unexposed population.  The 
more frequent the end-point, the smaller the sample size needed to 
detect a given effect.  The required sample size will also depend 
on the chosen values of alpha, the probability of falsely rejecting 
the null hypothesis, and beta, the probability of falsely accepting 

the null hypothesis.  These are sometimes known as Type 1 and Type 
2 errors.  It is customary, in most experimental situations, to 
choose alpha = 0.05 and beta = 0.20 (2-tailed test).  However, in 
order to be more certain that a real effect is not missed, then an 
alpha = 0.10 (or 0.05 for a one-tailed test) and beta = 0.05 could 
be chosen.  However, this choice will increase the sample size 
required to rule out a given effect incidence. 

    It is possible, for given values of alpha and beta, to 
calculate the sample size required to rule out a given effect 
incidence, for an end-point of known frequency in the exposed 
population.  Conversely, the effect size detectable with a given 
sample size can be calculated.  Table 1 shows the relationship 
between sample size and effect incidence for a given degree of 
assurance that the effect is real. 

    In any study, a negative result should be expressed in terms of 
the size of the effect that can be ruled out at a given power.  For 
example, "this study rules out a 2-fold increase in the frequency 
of end-point X, with 80% confidence" or "this study has a 50% power 
to detect a doubling of the frequency of end-point X". 

    Further considerations of sample size will be discussed under 
the sections devoted to each specific test end-point. 

Table 1.  Sample sizes needed to detect a given increase in 
frequency, with alpha = 0.05a
-----------------------------------------------------------
Frequency   Sample size to detect    Sample size to detect
of                a doubling               a tripling           
end-points  beta = 80    beta = 95   beta = 80  beta = 95
-----------------------------------------------------------
.000001     23 5551 082  39 001 131  7 850 356  13 000 369
.00001      2 355 076    3 900 058   785 021    1 300 011
.0001       235 475      389 951     78 487     129 976
.001        23 515       38 940      7834       12 972
.01         2319         3839        769        1272
.05         435          719         141        232
.10         200          329         62         102
.15         121          199         36         58
-----------------------------------------------------------
a Two-tailed probability without correction for continuity.  
  For discussion of continuity correction, and computation 
  formula, see Fleiss (1981).

2.4.  Long-Term Medical Follow-up

    People need to be identified uniformly on various files in 
order to monitor human populations for delayed effects caused by 
exposure to environmental agents.  Epidemiological studies are 
greatly facilitated by the existence in many countries of 
centralized, computerized, accessible national registries relating 
to health outcomes. 

    The kinds of data required are concerned with establishing 
statistical associations, which may serve as pointers to possible 
"troublespots", or with investigating suspected exposed groups.  An 
investigation involves defining the population cohort for study, 
the end-points of interest, and the appropriate study methods for 
carrying out individual long-term follow-up efficiently (WHO, 
1983). 

    A number of existing population-based files, concerned with 
health events, are available as end-point records that can be used 
to measure various health effects among a cohort under study 
(Bloom, 1981).  Normally, follow-up will consist simply of using a 
starting-point record, in its machine-readable form, to search by 
computer for an end-point record relating to the same individual or 
family.  Procedures have been developed for doing this and for 
analysing the results, without loss of personal privacy (Smith & 
Newcombe, 1980). 

2.4.1.  Relevance and limitations

    Lack of adequate recorded information and of organization and 
use of historical data in a consistent fashion are two major 
stumbling blocks in long-term epidemiological or genetic research.  
The term "record linkage" has been used to describe the process 
whereby two or more records relating to the same individual, 
family, or event are brought together.  The success of this 
procedure depends on the quality, quantity, and discriminating 
power of the items of personal identification on the machine-
readable records that are to be brought together. 

    Existing historical data should be more readily accessible 
for statistical analysis, but with built-in safeguards so that 
confidentiality of health records and personal privacy are not 
compromised.  Examples of relevant data are:  a) mortality data; 
b) birth defect monitoring programmes; c) health surveillance 
programmes; d) vital records, such as marriages and births; e) 
morbidity records; and f) cancer registries.  In addition, where 
new data are being collected (e.g., on suspected high-risk groups, 
or on individuals who exhibit "early indicator" conditions), this 
should be done in such a fashion that it is possible to make 
international comparisons and to pool data. 

2.4.2.  Personal identification

    Each record needs to contain enough information to indicate 
unambiguously the particular individual and/or family to whom it 
refers (Table 2).  If a universal numbering system were available 
for each individual in the population, and were in general use on 
all medical and vital health records, the problem of matching would 
almost disappear.  In the absence of such a universal number, the 
record should ideally contain full birth names, birth date, 
birthplace, sex, mother's maiden surname, and place of residence.  
Other useful items are full current surname and current address.  
Since records being linked will seldom have been generated at 
exactly the same time, and, since some of the items are subject to 

change with time, it is desirable that the date of the event and 
the type of event be known.  For example, the date of event (e.g., 
hospital visit) may infer a "last-known alive date", which could 
substantially reduce the amount of scanning where a mortality 
search is being carried out. 

    To identify the family involved, the marital status of the 
individual, and, if applicable, the spouse's birth surname, 
forenames, and birthplace should be recorded.  In the case of 
birth, marriage, death, cancer registries, and especially genetic 
registries, it is desirable to have parental birth names, 
birthplaces, and birth dates (or ages). 

Table 2.  A list of items to be included on starting- and end-point
records to facilitate follow-up studiesa
-------------------------------------------------------------------
*  1.    Surname
*  2.    Previous surname (if any)
*  3.    First given name
*  4.    Second and other given names
*  5.    Usual name (or nickname)
*  6.    Sex
*  7.    Birth date (year, month, day)
*  8.    Birth province or country
*  9.    Birth city or place
*  10.   Father's surname
*  11.   Father's first given name
*  12.   Father's second given name
+  13.   Father's birth province or country
   14.   Father's birth city or place
+  15.   Father's birth date (or age)
*  16.   Mother's maiden name
+  17.   Mother's first given name
+  18.   Mother's second given name
+  19.   Mother's birth province or country
   20.   Mother's birth city or place
+  21.   Mother's birth date (or age)
*  22.   Marital status
*  23.   Spouse's birth surname
*  24.   Spouse's first given name
   25.   Spouse's second given name
   26.   Spouse's birth province or country
   27.   Spouse's birth city or place
   28.   Social Security Number or equivalent
   29.   Health Insurance Number
*  30.   Place of residence - province or country
   31.   Place of residence - complete address including postal 
         code
*  32.   Date of event
*  33a.  Last known alive date (e.g., date of last contact)
   33b.  Date hired by company
   33c.  Date left company
   34.   Principal lifetime occupation - type of work, type of
         business, length of time worked
-------------------------------------------------------------------

Table 2.  (contd.)
-------------------------------------------------------------------
   35.   Other items where applicable (e.g., birth order of child,
         status of birth, religion, race, etc.)
*  36.   Control code to indicate the kind of event
*  37.   A control code digit to indicate whether alternate entries
         for the same event are being recorded (e.g., where an
         individual may have alternate spellings for surname)
*  38.   A unique number (where no other suitable number is 
         available)
   39.   Where applicable, an indicator to denote whether the
         individual is known to be dead,the date of death, and the
         province or country of death
-------------------------------------------------------------------
a From:  Smith (1977).
*,+ Top priority would be given to collecting the items identified 
    with asterisks.  For genetic studies, additional parental 
    variables will be required, particularly those indicated with 
    +.  Information relating to diagnostic procedures, work 
    histories, exposure histories, etc., plus updates, are added to 
    the basic record.  For marriages, the record should identify 
    the groom, bride, groom's parents and bride's parents.

    Records in files can  contain erroneous information and 
omissions.  Thus,in order to reduce the frequency of matching 
errors, it is necessary to introduce a measure of "redundancy" 
into the identifying information.  Alternative entries can also be 
created, where the surname of an individual may have an alternative 
spelling (e.g., adoptions).  It may also be useful to record 
nicknames, race, principal lifetime occupation, and birth order. 

2.4.3.  Starting-point records

    Where information is sought concerning the likely health 
effects of possible harmful agents in the environment, and of 
various social and economic circumstances, the chief limiting 
factor is a shortage of starting-point records in a form suitable 
for searching death records, cancer registries, etc., for evidence 
of harm to the exposed individual or his/her offspring. 

    Collaboration and cooperation is required among a variety 
of organizations to help resolve this problem (e.g., Regulatory 
Agencies, Departments of Labour and Health, and research workers).  
Various laboratory tests and studies are being carried out to 
predict whether particular chemicals would be likely to cause 
mutations or cancers in human beings exposed over long periods to 
low or moderate doses.  Human data relating to subpopulations can 
serve as a potential starting point for a variety of studies, but 
the records need to be uniformly stored and readily accessible.  
Studies involving large cohorts can be carried out at a national 
level.  International cooperation is necessary in cases where the 
numbers are small, and pooling of data is required.  An example 
would be to follow up people who show "early indicator" conditions.  
For instance, individuals who show chromosomal breakage could be 
identified, and followed up to see whether they subsequently 

developed cancer or had reproductive problems (e.g., stillbirths) 
or defective children.  Similarly, children found by survey to have 
protein variants could be followed up to see whether they developed 
special health problems or died early. 

    Starting-point records can comprise a wide assortment of 
administrative and other kinds of microdata files.  These may 
include  ad hoc local files (e.g., nominal rolls created using 
personnel, pay, and pension records), specialized centralized 
registers (e.g., health surveillance registers), records of 
exposure to certain agents, records of employment, special 
survey records, vital registrations, or other sources. 

2.4.4.  End-point records

    A number of health events are routinely documented in forms 
that are centralized to a certain extent and are in a machine-
readable form that lends itself to studies in which a measure of 
the health impact on a particular cohort is required.  Included 
among such sources are death registrations (Kinlen, 1980; 
Patterson, 1980; Smith & Newcombe 1980), and special registers of 
cancer, congenital anomalies, and other handicapping conditions.  
Secondary sources that might be helpful include marriage records, 
to give changes of name for follow-up and family composition, and 
birth records to indicate offspring at potential risk.  Both birth 
and stillbirth records are of particular value, where fertility is 
of interest, for indicating a potential health effect.  Hospital 
and medical insurance records, may be useful to help convert 
locally-based registers into population-based ones, and to give 
a measure of the social burden of disease. 

2.4.5.  Methods required for integrating source files

    Computer methods have been developed to derive family 
statistics relating to hereditary and congenital disease and terms 
of other ill health (Newcombe, 1967, 1969, 1977; Smith, 1977, 
1980).  For example, combining records from the vital registration 
system, certain specialized disease registers, and records from a 
universal hospital insurance scheme made multi-generation and 
cousin studies possible (Table 3 and 4).  However, individual 
follow-up is in much greater demand, primarily for detecting 
delayed cancers. 

    For linking an individual with health and other relevant 
records, a unique identity number assigned at birth would be 
extremely useful.  These are not available in many countries.  
Where no unique number is available, the searching procedures 
to be used in bringing records together may involve probabilistic 
matching techniques.  A generalized record linkage system and 
probabilistic matching procedures have been developed and used for 
such studies (Smith et al., 1980; Howe & Lindsay, 1981; Smith & 
Newcombe, 1982). 

    Linkage projects have been developed or are being planned in a 
number of international centres (Mi, 1967; Acheson, 1968; Skolnick, 
1980; Beebee, 1981; Fox & Goldblatt, 1982; Smith, 1982), which 
facilitate the establishment of:  (a) mortality data bases; (b) 
cancer incidence reporting systems; (c) death clearance of cancer 
registers; (d) procedures for supplementing cancer registers with 
hospital and medicare data; and (e) procedures for constructing 
better handicap registers. 
Table 3.  Identifying information available in the magnetic tape files of vital and health records example from British Columbiaa
--------------------------------------------------------------------------------------------------------------------------------------
Event and its date and place           Father                     Mother                                    Child                     
Event        Date   City &   Sur- Initials Age Province Maiden  Initials Age Province Sur- Given Sex Birth Full  City  Province Otherb
             (or    Province name              of birth surname              of birth name names     order birth of    of birth
             year*)                                                                                        date  birth                 
--------------------------------------------------------------------------------------------------------------------------------------
Marriage     X      X        X    X        X   X        X       X        X   X                                                  (1)
Livebirth    X      X        X    X        X   X        X       X        X   X        X    X     X   X     X     X     X        (2)
Stillbirth   X      X        X    X        X   X        X       X        X   X        X    X     X   X     X     X     X        (2)
Death        X      X        X    X            X        X       X        X            X    X     X   X     X     X     X        (3)
Handicap     X*                                                                       X    X     X         X     X              (4)
Congenital   X*                                                                       X    X     X         X     X              (4)
malformation                                                            
surveillance
Hospital
Separation   X      X                                                                 X    X     X         X                    (5)
--------------------------------------------------------------------------------------------------------------------------------------
a From: Newcombe (1977).
b (1)  Marital status of groom and bride.
  (2)  Legitimate or illegitimate status; singleton, twin, triplet, etc.
  (3)  If married women, own maiden name surname instead of mother's; marital status.
  (4)  Coded address representing city, school district and provincial health unit area; whether born in home province.
  (5)  Hospital code and admission number: maiden surname if different; marital status; address as city, street, and number; religion; 
       three initials of head of family, or spouse, or guardian; plus codes for relationship and whether address is the same.

Table 4.  Example of the file organization for a sibship history of disease (fictitious)a,b
---------------------------------------------------------------------------------------------------------------------------------
Event (birth     Year              Father                           Mother                               Child                      
order in               Surname Initials Province  Age  Maiden  Initials Province  Age  Given     Sex     Birth      Disease
parentheses)                            of birthc      surname          of birthc      names         year month day
---------------------------------------------------------------------------------------------------------------------------------
Marriage         1948  Cox     JW       07        25   Bell    MA       09        21
Live birth (1)   1950  Cox     JW       07        27   Bell    MA       09        23   Annie May  F  50   01    09
Live birth (2)   1952  Cox     JW       07        29   Bell    MA       09        25   Brian John M  52   04    14
Reg. handicap    1952  Cox     -        -         -    -       -        -         -    Brian John M  52   04    14  Spina bifida
Hospitalization  1955  Cox     -        -         -    -       -        -         -    Brian John M  52   04    14  Spina bifida
Death            1956  Cox     JW       07        -    Bell    MA       09        -    Brian John M  52   04    14  Spina bifida
Stillbirth (3)   1954  Cox     JW       07        31   Bell    MA       09        27   Mary Jane  F  54   07    26  Anencephaly
---------------------------------------------------------------------------------------------------------------------------------
a From: Newcombe (1977).
b Obtained by merging the individual histories of birth, ill health and death, with the sibship histories of parental marriages 
  and births.
c Coded from a 2-digit geographical code for all Canadian provinces and all major countries; 07 = Saskatchewan 09 = British 
  Columbia.

3.  MUTATIONS IN SOMATIC CELLS

3.1.  General Considerations

    General reasons for studying somatic mutation in human 
populations include: 

    (a)  to determine if an unsuspected introduction of new (or 
         increase in already present) mutagens has occurred;

    (b)  to evaluate mutation frequencies in populations known (or 
         suspected) to be exposed to mutagens; 

    (c)  to monitor for changes in frequency in populations as a 
         consequence of the removal of mutagens; 

    (d)  to identify groups in the general population with high 
         frequencies of somatic mutations, so that studies can be 
         undertaken to attempt to detect the responsible factors; 
         and 

    (e)  to define the heterogeneity of susceptibility to genotoxic 
         agents within the population. 

3.1.1.  End-points

    Genetic damage can be assayed at the molecular, functional 
gene, and chromosomal levels.  At the molecular level, DNA damage 
can be assessed in terms of adduct formation, strand breakage and 
repair, or base sequence alterations.  At present, the measurement 
of adduct formation requires either prior knowledge of the nature 
of the adduct or limited population sizes, and is, therefore, not 
currently applicable as an assay for monitoring large populations.  
DNA strand breakage and repair are usually transient phenomena 
initiated at the time of insult and completed shortly afterwards, 
making them less than ideal for population monitoring purposes. 

    Recent research developments have made it feasible to use 
restriction enzyme mapping techniques to analyse alterations in 
DNA base sequence (Botstein et al., 1980; Skolnick & Francke, 
1982; Southern, 1982).  This approach is being used to define gene 
structure and identify polymorphisms for linkage analysis.  The 
same approach could equally well be used to screen for mutational 
changes, but its application must await the availability of 
appropriate batteries of DNA probes. 

    With current techniques, changes in gene function can be 
detected by the acquisition of resistance to selective agents or 
by the alteration or loss of cellular constituents detected by 
immunological methods.  Although these immunological approaches 
hold considerable promise, the available data are insufficient 
for application to routine monitoring.  On the other hand, 
methods for the detection of thioguanine-resistant peripheral 
blood T-lymphocytes have been established and are being refined.  
A detailed description of the principles and methods of this 
approach is presented in section 3.3. 

    At the chromosomal level, genetic damage is observed as an 
alteration either in chromosome number or in chromosome structure.  
Although numerical changes represent a significant proportion of 
human heritable genetic diseases, their consequences in somatic 
cells are less well characterized.  Moreover, estimates of 
numerical changes in somatic cells are heavily influenced by 
technical artifacts.  Alterations in chromosome structure are more 
accurately assessed in somatic cells and are observed cytologically 
as chromosomal aberrations or sister chromatid exchanges (SCEs).  
These are currently the most readily-available and widely-used end-
points for evaluating somatic mutations in human beings.  There is 
extensive literature concerning these methods (Office of Technology 
Assessment, US Congress, 1983), which are discussed in section 3.2.  
Micronuclei, which can result from structural or numerical 
chromosome aberrations, have been used as an indicator of such 
damage in human studies.  Micronuclei have been enumerated in 
cultured lymphocytes to detect the effects of ionizing radiation 
(Krepinski & Heddle, 1983) and in the exfoliated epithelial cells 
of persons exposed to chemical carcinogens (Stich et al., 1982).  
However, this method, though showing potential for monitoring 
genetic damage in human beings, has not yet been accepted or used 
widely and so is not considered in detail. 

3.1.2.  Study requirements

    Any study attempting to evaluate human populations for somatic 
cell mutations should satisfy the following requirements: 

    (a)  the study should include exposed groups and appropriate 
         concurrent controls; 

    (b)  specimens from exposed and control individuals should be 
         gathered, handled, transported, and evaluated at the same 
         time and in the same way, using for example, the same 
         culture medium, serum, and reagents; 

    (c)  samples should be coded and evaluated in such a way that 
         the the origin of the sample is not known, at the time of 
         evaluation; 

    (d)  attempts should be made to quantify levels of exposure and 
         to determine dose-response relationships. 

    However, in some instances, concurrent controls may not be 
necessary, for example, in longitudinal studies to determine 
temporal changes in mutation frequencies.  In these cases, any 
possible confounding variables, such as changes in batch or source 
of media, sera, or reagents used in the study should be minimized.  
Reagents should be stored and, when there is a batch change, 
comparative studies of selected individuals (or stored samples) 
with new and old reagents should be undertaken to determine if 
there are differences.  It is recognized that it may be extremely 
difficult to find appropriate controls in all instances, because of 
various confounding factors that can influence somatic mutation 
frequencies (e.g., age, sex, exposure to ionizing radiation, 

medication, recent viral disease, smoking, etc).  An appropriate 
control group is one that is matched as closely as possible with 
the exposed group for these confounding factors. 

    If it is impossible to find appropriate matched controls for 
everyone in an exposed group, but it is necessary to study all 
those exposed, the following alternatives are suggested: 

    (a)  to obtain the best controls available and avoid the most 
         significant confounding factors; 

    (b)  to stratify the cases, prior to analysis, into those with 
         well-matched controls and those with less well-matched 
         controls; 

    (c)  to give the greatest weight in the analysis and 
         interpretation of results to the group with the well-
         matched controls. 

    In emergency situations, when appropriate matched controls 
are not available, it is important to obtain some control specimens 
that will be sampled, transported, and cultured at the same time 
as those from exposed individuals.  Sources of such specimens 
are members of study teams, assuming that they have not been 
contaminated.  Each batch of specimens, collected in emergency 
circumstances and transported to the laboratory, should include a 
specimen from at least one unexposed individual. 

    The questions arise as to when, and for how long after exposure 
to a mutagen, individuals can usefully be studied.  It may be 
important to follow populations with elevated mutant frequencies 
sequentially, to determine if such frequencies are changing.  
Because of the long life span of the circulating peripheral 
lymphocyte, some types of mutation can be detected many years after 
the exposure.  However, the persistence of these events cannot be 
predicted with certainty and will vary with exposure duration, 
agent, and other factors.  Thus, for chromosomal aberration and 
sister chromatid exchange measurements, samples should be taken as 
soon as possible after exposure.  For example, persons receiving 
chemotherapy can have high frequencies of chromosomal aberration, 
sister chromatid exchange (SCE), and gene mutation in the 
lymphocytes.  However, in the months following cessation of 
treatment, the chromosomal aberration frequency decreases, often to 
a level that may not be significantly different from that in the 
controls.  The frequency of SCE also diminishes in time.  For gene 
mutations, there may be a time interval between the mutational 
event and the expression of resulting mutants.  This interval will 
vary from days to weeks and may vary with the markers.  Thus, 
following exposure, somatic mutant frequencies may remain the same, 
decrease, or increase as a result of cell division, selection, or 
migration  in vivo.  Similarly, persons therapeutically exposed to 
ionizing radiation show elevated frequencies of chromosomal 
aberration and gene mutation, but not SCE.  In these patients, the 
frequency of aberrations may remain elevated for many years after 
exposure.  At present, there are few data relevant to the 

persistence of specific locus somatic mutants.  It is recommended 
that more than one end-point for somatic mutational change be 
considered in any population study, because each end-point has 
limitations with regard to the conclusions that may be drawn.  
For example, negative findings for chromosomal aberrations do not 
preclude the possibility of increases in somatic mutations of other 
types.  In a population presumably exposed to ionizing radiation, 
chromosomal aberration studies are most appropriate, but SCE 
could also be analysed as an indicator of exposure to confounding 
chemical agents.  Conversely, in a population exposed to chemicals, 
SCE studies are more appropriate in terms of assay sensitivity, but 
chromosomal aberration studies provide additional important 
information. 

    Considerable effort can be expended in subject identification 
and sample collection; therefore, sufficient blood should be 
collected at one time to assay several somatic cell mutational end-
points.  Even though it may be necessary to evaluate a number of 
end-points in a particular study, it is worthwhile setting 
priorities for their analysis.  For example, slides could be 
prepared for the determination of both chromosomal aberrations and 
SCE, but only one end-point scored initially.  This choice should 
be based on the type of end-point most likely to be elevated.  The 
remaining slides can be scored later, according to the needs of the 
study. 

    In addition, blood collected from mutagen-exposed and control 
individuals should be sufficient to allow cryopreservation of 
cells and other components for later study.  This will make 
more extensive evaluations possible as well as contribute to the 
creation of a repository of material from mutagen-exposed human 
beings for subsequent research. 

3.2.  Chromosomal End-Points

3.2.1.  Structural aberrations

    Chromosomal aberrations can be studied in any cycling cell 
population, or in any non-cycling cell population that can be 
stimulated by a mitogenic agent to enter the cell cycle.  In 
animals, there are several cell types that fit these criteria, 
but for human studies, to all intents and purposes, there are only 
two cell types that are practically suitable.  These are the bone 
marrow cells, which are a cycling population, and the peripheral 
blood lymphocytes, which are normally non-cycling, but can be 
stimulated to enter the cell cycle by  in vitro culturing with a 
mitogen such as phytohaemagglutinin (PHA).  However, because of 
the ease of obtaining blood samples, in contrast to bone marrow 
samples, lymphocyte assays have been used in the majority of 
studies on the induction of chromosomal aberrations in human 
beings. 

    Since the observation by Moorehead et al. (1960) that 
peripheral lymphocytes could be stimulated by PHA to enter the cell 
cycle and be observed at metaphase, an enormous amount of data has 
been obtained on the induction of chromosomal alterations by 
radiation and chemical agents using this lymphocyte assay system 
(Preston et al., 1981).  It is often assumed that this is a simple 
and informative assay for human population monitoring, providing 
information on potential clastogenic (or mutagenic) exposures.  
However, it should be emphasized that such a view should be 
regarded with caution in the case of supposed chemical exposures, 
and the obvious distinction from its use for radiation exposures 
should be appreciated. 

3.2.1.1.  Methods of culture

    There are numerous methods for culturing human lymphocytes.  
Many components of the methods are optional, and depend on 
individual preference.  Those that are less variable are described 
here.  More complete methods can be found in Evans & O'Riordan 
(1975), Bloom (1981), and Preston et al. (1981). 

    It appears to be advantageous to set up cultures from fresh 
blood samples.  However, this is not always possible, usually 
because samples are taken at some distance from the laboratory and 
have to be transported.  It is still possible to achieve good 
growth from samples taken several days before culturing.  Although 
samples can be maintained at 4 C during shipping and storage, it 
appears to be better to maintain them at room temperature whenever 
possible (Dzik & Neckers, 1983). 

    Cultures can be established from whole blood (approximately 
0.5 ml per culture), buffy coats, or purified lymphocytes.  
However, if a large number of samples are to be handled at one 
time, it is clearly advantageous to use whole blood.  For buffy-
coat cultures, it is preferable to use the white-cell layer from 
about 3 ml of centrifuged whole blood, thus larger total blood 
samples are needed.  One of the advantages of buffy-coat cultures 
is that the cell fixation procedure is not hampered by the large 
volume of red cells, present in whole-blood cultures, which are 
often difficult to disperse.  A possible disadvantage of whole-
blood cultures is that a small quantity of the agent under study, 
or another contaminating agent, could be carried over with the 
donor's serum.  However, there seems little probability that this 
would influence the results, and thus, it would not negate the use 
of whole-blood cultures.  It is also important to establish 
cultures in duplicate, as a precautionary measure against culture 
failure or sparseness of analysable cells. 

    A large number of different tissue culture media have been 
successfully used, and the choice is a matter of personal 
preference.  This is also true for the serum type used, with the 
added proviso that it should be virus-free.  Several different 
T-cell mitogens are also suitable, PHA being the most frequently 
used.  One additional point concerns the use of tissue culture 
media containing a low concentration of folic acid.  It has been 

noted that the growth of human lymphocytes in such media (notably 
TC 199) can result in the appearance of specific chromosome breaks 
at so-called "fragile sites" (Jacky et al., 1983), or in an 
increase in aberrations at possible fragile sites (Reidy et al., 
1983).  It is recommended that, for population-monitoring studies, 
low folate media should be avoided, though it is suggested that 
parallel cultures with low folate media could still be used to 
provide additional or different types of information. 

    While it is appropriate for each laboratory to use a medium and 
serum that provides good lymphocyte growth conditions, it is most 
important that each laboratory should determine the rate of 
progression of mitogenically-stimulated cells through their first 
and subsequent cell cycles with their specific culture conditions, 
using blood samples from several individuals.  The rate of cell 
progression can be variable, depending on the mitogen, culture 
medium, serum, and temperature (usually 37 C in a 5% C02/95% air 
environment). 

    The progression of cells can be measured by culturing in the 
presence of bromodeoxyuridine (BrdU), sampling cells over a range 
of times (for example, every 2 h, from 42 h to 54 h after mitogenic 
stimulation), and staining the fixed preparations using a technique 
for obtaining differentially-stained chromosomes (Goto et al., 
1978).  In this way, the cells that have replicated their DNA once, 
in the presence of BrdU, will contain evenly- and darkly-stained 
chromatids.  Those that have replicated twice, in the presence of 
BrdU, will contain differentially-stained chromatids, one light 
blue and one dark blue, when Giemsa stain is used.  Cells that 
progress through 3 or more cell cycles, in the presence of BrdU, 
will contain some differentially-stained and some evenly but 
lightly-stained chromosomes.  In studies on chromosome aberrations, 
it is important to analyse cells in their first metaphase after 
mitogenic stimulation; thus, a fixation time should be chosen when 
a high proportion of analysable cells are at the first division 
stage.  Generally, it is not feasible to use a fixation time when 
all the cells are at this stage, because this requires very early 
fixation (approximately 42 h after stimulation), when the number of 
mitotic cells is too low.  A compromise time (for example, 48 h) is 
selected, when, for the average individual, about 90% of the cells 
are at the first division stage.  There is considerable variation 
from individual to individual in the percentage of cells at the 
first or subsequent division, even 48 h after culture initiation, 
and it is good practice to check the proportion of first mitoses 
in a series of cultures containing BrdU, separate from the series 
established for aberration analysis.  In this way, the possible 
effects on aberration frequencies of analysing different 
proportions of first division cells from different samples can be 
ascertained.  It is also possible to analyse chromosome aberrations 
from cultures grown with BrdU, where preparations have been 
differentially stained:  cells showing no differentiation, and 
cells that are clearly first metaphase (M1) can then be analysed. 

    The decision on selecting or rejecting samples with a high 
proportion of 2nd divisions is optional, but should, at least, be 
consistent.  If resampling is possible, then this should be done 
using an earlier fixation time than that for the first sample.  
There is also a possibility that, at the standard fixation time for 
any particular laboratory, there may be insufficient analysable 
mitotic cells, as a result of an unusually long cell cycle, either 
inherent or induced.  The only solution to this problem is to 
resample using a later fixation time. 

3.2.1.2.  Fixation and slide preparation

    Many different methods are available for obtaining 
conventional, banded or harlequin-stained metaphase preparations, 
and essentially any one that produces well-spread complete 
metaphases is acceptable (MacGregor & Varley, 1983).  There is 
little likelihood of this step of the assay influencing the 
results. 

3.2.1.3.  Analysis of cells

    There are many schemes available for the classification of 
chromosome aberration types, and comprehensive descriptions can be 
found in Savage (1975) and Bloom (1981).  The following section 
includes descriptions of the abberations most commonly observed in 
control samples or samples from individuals exposed to radiation or 
chemical agents. 

    (A)   Chromosome-type aberrations

         (1)  Terminal and interstitial deletions

FIGURE 1A

    It is not possible to distinguish between chromosome-type 
terminal deletions and non-sister union isochromatid deletions 
(Fig. 2D), and so, in cases of radiation exposure, when induced 
aberrations are of the chromosome-type, it is appropriate to 
classify all paired acentric fragments as terminal deletions 
(Fig. 1A). 

    The small interstitial deletions appearing as paired dots are 
classified as "minutes" (Fig. 1A).  The larger interstitial 
deletions in which there is a clear space in the centre of the ring 
are usually classified as acentric rings.  The distinction is not 
particularly clear-cut, and, in general, is merely an indication of 
the different sizes of interstitial deletions.  Acentric fragments 
associated with inter- or intrachanges are not classified as 
terminal deletions. 

         (2)  Asymmetrical interchanges (usually dicentrics)

FIGURE 1B

FIGURE 1C

    It is assumed that a dicentric, analysed at the first  in 
 vitro metaphase will be accompanied by an acentric fragment 
(Fig. 1B) and a tricentric by 2 acentric fragments (Fig. 1C).  A 
cell with a dicentric and two acentric fragments is, by convention, 
classified as a dicentric with its accompanying fragment and a 
terminal deletion.  The two acentric fragments could be the 
result of incomplete rejoining during the formation of the 
dicentric.  However, although this cannot be ascertained, it has 
been shown experimentally that its probability of occurrence is 
rather low (< 10%) (Schmid & Bauchinger, 1980). 

    Asymmetrical interchanges, i.e., dicentrics, can be analysed 
with greater efficiency than any other aberration type (> 95%), 
and it is their frequency that is generally used for estimations of 
radiation dose.  For such determinations, a tricentric, for 
example, is assumed to be equivalent to 2 dicentrics. 

         (3)  Asymmetrical intrachange (centric ring)

FIGURE 1D

    In asymmetrical intrachanges as in interchanges, a centric ring 
is accompanied by an acentric fragment, and the same classification 
scheme applies to these as for dicentrics ((2), above) (Fig. 1D). 

         (4)  Symmetrical interchanges (reciprocal translocations)

FIGURE 1E

    Symmetrical interchanges (Fig. 1E) are particularly difficult 
to observe in conventionally-stained preparations, unless the 
exchanged pieces produce 2 chromosomes, very different from the 
normal karyotype.  However, it is suggested that obvious 
symmetrical interchanges should be recorded, but giving less weight 
to their frequency than to that of most other aberration types. 

    It is not usually possible to observe these symmetrical 
intrachanges (Figs. 1F, 1G) in conventionally-stained preparations, 
unless a pericentric inversion produces a chromosome that is 
distinctly different from the normal karyotype.  Any obvious 
symmetrical intrachanges should be recorded, but less weight given 
to their frequency than to that of most other aberration types. 

         (5)  Symmetrical intrachanges (peri- and paracentric 
              inversions) 

FIGURE 1F

FIGURE 1G

    (B)   Chromatid-type aberrations

    Chromatid-type aberrations are generally classified in the 
same way as chromosome-type aberrations; the apparent unit of 
involvement in a chromatid-type aberration is, in most cases, the 
single chromatid, and not the whole chromosome, as seen for 
chromosome-type aberrations. 

        (1)  Terminal deletions

    A terminal deletion is a distinct displacement of the chromatid 
fragment distal to the lesion, or, if there is no displacement, the 
width of the non-staining region between the centric and acentric 
regions is greater than the width of a chromatid (Fig. 2A).  The 
latter definition is used to distinguish between terminal deletions 
and achromatic lesions or "gaps" (section (2)). 

FIGURE 2A

         (2)  Interstitial deletions

    Chromatid-type interstitial deletions (Fig. 2B) are not as 
readily observable as their chromosome-type counterpart, partly 
because the small deleted fragment is often separated from the 
deleted chromosome, and is not observed. 

FIGURE 2B

         (3)  Achromatic lesions ("gaps")

    Achromatic lesions or gaps are non-staining or very lightly 
stained regions of chromosomes, present in one chromatid (single) 
or, in both sister chromatids, at apparently identical loci 
(double).  If the non-staining region is of a width less than that 
of a chromatid, the event is recorded as an achromatic lesion (Fig. 
2C).  This is clearly only a working definition.  It is generally 
suggested that achromatic lesions should be recorded, but always 
separately from chromatid deletions.  Their frequency should not 
be included in the totals for aberrations per cell, since their 
significance and relationship to other "true" aberration types is 
not clear at present. 

FIGURE 2C

         (4)  Isochromatid deletions

    Isochromatid deletions appear as exceptions to the class of 
chromatid-type aberrations, since they involve both chromatids, 
apparently with "breaks" at the same position on both.  However, in 
suitable material they can be shown to be induced by radiation in 
the S and G2 phases of the cell cycle, as is the case for other 
chromatid-type aberrations. 

    There are several possible types (Fig. 2D), depending on 
the nature of the sister unions.  If sister union occurs, it is 
possible to distinguish isochromatid aberrations from chromosome-
type terminal deletions. 

FIGURE 2D

In mammalian cells, however, sister union is a rare event, and most 
of the isochromatid deletions are of the non-union proximal and 
distal type.  The acentric fragment is most often not associated 
with the deleted centric part of the chromosome.  The convention 
for analysis (discussed for chromosome-type terminal deletions, 
above) is that, for radiation exposures, paired acentric fragments 
are recorded as chromosome-type terminal deletions, but, for 
chemical exposures or control samples, they are most appropriately 
recorded as isochromatid deletions. 

         (5)  Asymmetrical interchanges (interarm interchanges, 
              asymmetrical chromatid exchanges) 

    Asymmetrical interchanges are the chromatid-type equivalent to 
chromosome-type dicentrics (Fig. 2E). 

FIGURE 2E

         (6)  Symmetrical interchanges (symmetrical chromatid
              exchanges)

    Symmetrical interchanges are the chromatid-type equivalent of 
chromosome-type reciprocal translocations.  In the case of 
chromatid-type symmetrical exchanges, somatic pairing maintains an 
association between the chromosomes involved in the exchange, and 
thus they can be readily observed in the absence of any chromosome-
banding procedures (Fig. 2F). 

FIGURE 2F

         (7)  Asymmetrical and symmetrical intrachanges (interarm 
              intrachanges) 

    There are 2 forms of symmetrical and asymmetrical interarm 
intfachanges but, when analysing metaphase cells, only one of each 
is distinguishable (Fig. 2G).  The symmetrical intrachange can be 
observed in somatic pairing. 

FIGURE 2G

         (8)  Triradials

    A triradial (3-armed configuration) can be described as the 
interaction between one chromosome having an isochromatid deletion 
and a second having a chromatid deletion.  Many types of triradials 

can be formed but, in mammalian cells, where the frequency of 
sister union is low, the 2 illustrated are the most common (Fig. 
2H). 

FIGURE 2H

It is important to note that the different types of chromosome 
aberration should be recorded separately.  The results for any 
sample should be presented as the aberration frequency per cell 
for each aberration type, or frequency of aberrant cells for each 
aberration type.  It is not appropriate to express the results as 
total aberrant cells or total breaks per cell, as this method of 
presentation can hide important facets of the data.  For the 
statistical analysis of data, it is legitimate to combine 
aberration classes into the general categories of chromatid-type 
deletions, chromosome-type deletions, chromatid exchanges, and 
chromosome exchanges.  Occasionally, cells are seen with multiple 
aberrations, and an attempt should be made to analyse such cells in 
detail, rather than to record them simply in a category "multiple 
aberration", since this classification will mean a loss of 
information.  If it proves impossible to analyse a cell, it should 
be recorded in a category of "too many aberrations to analyse", and 
not included in the total of cells scored. 

    The selection of cells to be analysed is particularly 
important, to avoid observer bias.  Such bias can result in either 
a higher or lower measure of the unbiased aberration frequency, 
depending on the nature of the selection.  The generally accepted 
method of cell selection is to scan slides at low magnification; 
cells that appear to be suitable are then analysed at high 
magnification.  Once a cell is observed at high magnification, 
it should only be rejected if:  (a) the cell cannot be analysed 
because of the number or complexity of aberrations; in this case, 
it should be appropriately recorded, but not included in total 
cells analysed; (b) the cell is not sufficiently well spread 
when seen at high magnification, and the overlap of chromosomes 
prohibits an accurate analysis; (c) non-chromosomal material, such 
as dirt or stain crystals, not discernible at low magnification, 
prevent complete analysis; (d) the cell contains fewer centromeres 
than the observer's acceptable cut-off point; this may be the 
analysis of cells with 46  2 centromeres or only cells with 46 
centromeres, either cut-off point is acceptable, provided that the 
criterion is consistently applied. 

    It is particularly important in population-monitoring studies 
for the samples to be analysed under code, and for the slides from 
exposed and control individuals to be randomized in such a way that 

the observer is unaware of the identity of the sample.  There are 
strong arguments in favour of more than one laboratory, with at 
least 2 individuals in each laboratory, being responsible for the 
analysis, in order to remove possible laboratory or individual 
scoring bias, which could influence the results.  Microscope 
co-ordinates of every suspected aberrant cell should be recorded.  
In order to provide further consistency, the classification of any 
cell as aberrant by one individual should be confirmed by a second, 
and it is advantageous to keep a photographic record of all 
confirmed aberrant or otherwise suspect cells. 

    The number of cells to be analysed from any sample cannot be 
fixed with any certainty, because it can be argued that this number 
depends on the aberration frequency, and thus is retrospective.  A 
general suggestion would be to analyse 200 cells per sample. 

    The pros and cons of analysing banded preparations have been 
discussed frequently, and do not warrant repetition.  The amount of 
time expended on the preparation and analysis of banded samples 
does not seem to be justified by the increase in sensitivity of 
analysis for aberration types detectable by banding.  However, the 
choice of scoring conventially-stained or banded preparations 
should be left to the discretion of each laboratory. 

3.2.1.4.  Data analysis

    Many statistical methods are available for the analysis of 
data obtained from human population-monitoring studies.  The 
appropriateness of any particular method will depend, to some 
extent, on the design of the study and the nature of the data 
collected.  Two examples of general approaches are given in Brewen 
& Gengozian (1971) and Evans & O'Riordan (1975). 

3.2.1.5.  Radiation-induced aberrations and estimates of exposure 

    For a variety of reasons, it is sensible to discuss the use of 
the lymphocyte assay for estimating exposure to ionizing radiation 
separately from cases of chemical exposure.  In some ways, it was 
fortunate, but, in retrospect, perhaps also unfortunate, that all 
the original studies on the analysis of chromosomal aberrations in 
the lymphocytes of exposed or possibly-exposed persons applied to 
radiation exposure.  The fact that, in these cases, estimates of 
exposure could be made with some accuracy has often led to the 
assumption that similar exposure-estimating procedures could be 
applied to persons, groups, or populations exposed environmentally 
or occupationally to chemical agents.  A clear note of caution in 
accepting this assumption is made here, and will be discussed 
further. 

    Ionizing radiation and a small number of chemical agents (e.g., 
streptonigrin, bleomycin, neocarzinosatin, cytosine arabinoside, 
and 8-methoxycaffeine) are able to produce aberrations in all 
stages of the cell cycle including chromosome-type aberrations in 
G1 and chromatid-type aberrations in S and G2. 

    The peripheral lymphocytes, a subpopulation of which is 
mitogenically stimulated when blood samples are placed in culture, 
are essentially non-cycling, and are in the G0 stage of the cell 
cycle, the term usually applied to non-cycling G1 cells.  Thus, 
following radiation exposure, chromosome-type aberrations will be 
induced in these non-cycling G1 lymphocytes, and can be observed at 
the first metaphase following mitogenic stimulation in culture.  
The fact that aberrations can be produced in G1 cells means that 
their frequency can be directly related to the dose received (i.e., 
aberration frequency is proportional to dose).  In addition, it has 
been shown for many species (Brewen & Gengozian, 1971; Preston et 
al., 1972; Clemenger & Scott, 1973), including man (Brewen et al., 
1972), that the frequency of aberrations induced  in vitro is the 
same as the frequency induced by the same dose delivered  in vivo.  
This means that a standard dose-response curve, for any particular 
radiation type, can be obtained for  in vitro exposures, and can 
then be used for estimating doses received by individuals as a 
result of radiation accidents during medical or other environmental 
exposures.  The frequency of aberrations (usually dicentrics) is 
measured in cultured blood samples from exposed individuals, and 
then converted into a dose estimate from the standard dose-response 
curve.  There is extensive literature on the use of the lymphocyte 
assay as a biological measurement of dose for many different types 
of radiation (Bender & Gooch, 1966; Lloyd et al., 1983).  However, 
there are still some limiting factors in the use of the assay.  
These include the length of time of blood sampling after exposure, 
partial-body exposure, and non-homogeneous exposures. 

    It is also apparent that the lymphocyte assay can be used for 
estimating doses following long-term radiation exposure (Evans et 
al., 1979).  Again, this is possible because the aberrations are 
induced in G0 cells, and their frequency will be directly related 
to exposure.  Cells containing radiation-induced aberrations as 
well as normal cells, will gradually be lost from the peripheral 
lymphocyte pool, as a result of lymphocyte turnover.  However, the 
cells repopulating the peripheral pool will be derived mainly from 
normal precursor cells, and not from those containing aberrations.  
Many chromosome-type aberrations are lethal to the cell as the 
result of loss of acentric fragments at division, or because of the 
mechanical interference of the aberration with division.  Thus, 
during long-term exposures (months or years), the aberration 
frequency will not be additive with time (or dose), but will reach 
an equilibrium where new aberrations are formed and existing ones 
are lost from the sampled population. 

    A similar argument holds for determining the length of time 
after short-term exposure when samples can provide a measure of 
the maximum aberration frequency or true induced frequency.  
The lymphocyte turnover will result in loss of cells from the 
peripheral pool, both normal and abnormal, and repopulation largely 
by normal precursor cells.  The aberration frequency appears to 
stay constant for about 6 weeks after exposure (Brewen et al., 
1972). 

    Samples have been taken many years after exposure, for 
example, with radiation-treated ankylosing spondylitis patients 
(Buckton et al., 1978) and human beings exposed to the atomic bombs 

in Hiroshima and Nagasaki (Awa et al., 1978).  The fact that some 
lymphocytes are very long-lived, in excess of 20 years (Awa et 
al., 1978; Buckton et al., 1978), means that radiation-induced 
aberrations can still be observed in cells that were present as 
peripheral lymphocytes at the time of exposure.  If it is assumed 
that the aberration frequency declines exponentially with time, an 
approximate estimate of the dose received can be made; the word 
approximate is emphasized. 

3.2.1.6.  Chemically-induced aberrations and estimates of exposure

    On the basis of the success of the lymphocyte assay for 
estimating radiation exposures, it seemed appropriate to analyse 
chromosome aberrations in blood samples from individuals, 
occupationally exposed to single chemicals or complex mixtures 
(Office of Technology Assessment, US Congress, 1983), to determine 
whether there had been exposure to a clastogen, and to relate 
aberration frequencies to dose level.  However, the ability to 
estimate dose levels from aberrations in populations exposed to 
chemical mutagens is not reliable at present.  More research is 
necessary to make the assay more reliable and applicable, and to 
make the results more readily interpretable. 

    The present low sensitivity of the assay for measuring the 
frequency of chromosome aberrations following chemical exposure is 
due to the mechanisms of induction of aberrations by chemical 
agents.  These mechanisms may also result in induced aberration 
frequencies being only indirectly related to exposure dose, in 
contrast to radiation-induced aberrations. 

    Chromosome aberrations induced by chemical treatments are 
almost exclusively produced during the S-phase, irrespective of the 
cell-cycle stage treated.  Thus, the majority of aberrations will 
be of the chromatid-type, although there are exceptions to this 
general hypothesis (Evans & Vijayalaxmi, 1980; Preston & Gooch, 
1981).  Therefore, it is more appropriate to consider that the 
probability of producing aberrations in G1 or G2 cells following 
chemical treatments is low, but is considerably increased when 
treated cells are in, or pass through, the S-phase. 

    Chemically-induced DNA damage in non-cycling lymphocytes will 
not generally be converted into aberrations until the cells are 
stimulated to re-enter the cell cycle  in vitro, and undergo DNA 
replication.  Since repair of DNA damage can take place in G0 cells 
as well as during the long first  in vitro G1 stage, the aberration 
frequency will not necessarily be proportional to the amount of 
induced DNA damage but rather to the amount of DNA damage remaining 
at the time of DNA replication.  It is clear that the amount of DNA 
damage, present at the time of replication, which has the potential 
to be converted into aberrations, will depend on several factors 
including:  (a) the dose received; (b) the induced amount of the 
particular types of DNA damage that can result in aberrations (a 
value that can vary with the agent); (c) the amount of DNA repair 
in G0 cells before sampling (i.e., time between exposure and 
sampling); and (d) the amount of repair in G1 cells from mitogenic 
stimulation to the first  in vitro S-phase.  Many of these will also 

be subject to individual variation.  The outcome is that, for most 
chemical agents, only a proportion of induced DNA damage can be 
converted into aberrations, at the time of replication.  These DNA 
repair factors all work to reduce the sensitivity of the lymphocyte 
assay for measuring chemical exposure, and will result in the 
aberration frequency being, at best, only indirectly proportional 
to exposure. 

    If an increase in chromosome aberrations is observed in a 
potentially exposed group when compared with a matched control 
group, it is reasonable to conclude that there has been exposure to 
a clastogenic agent, but no estimate of exposure level or of 
subsequent adverse health effects (genetic or somatic) can be made.  
On the other hand, if there is no difference in aberration 
frequency between the possibly-exposed group and a matched control 
group, it is not possible to rule out an exposure.  However, in 
this case, it may be possible, on the basis of previous experience, 
to deduce a maximum exposure level that is not associated with a 
detectable difference in aberration frequency. 

3.2.1.7.  Conclusions

    Future research should enhance the sensitivity of the assay 
system.  In the meantime, it is suggested that care be taken in 
the choice of groups to be considered for monitoring studies, with 
particular regard to the usefulness of the information that might 
be obtained, and to the likelihood of being able to draw definitive 
conclusions.  Cytogenetic monitoring can be an appropriate method 
for the detection of chemical exposure, but, clearly, it cannot, at 
present, be considered universally decisive.  It should be used in 
conjunction with other end-points. 

3.2.2.  Sister-chromatid exchange

3.2.2.1.  Formation of sister-chromatid exchange

    SCE results from the breakage and rejoining of DNA at 
apparently homologous sites on the 2 chromatids of a single 
chromosome.  They were first visualized by Taylor et al. (1957) 
who used tritiated thymidine to differentially label the DNA of 
replicating cells and autoradiography to distinguish the silver 
grain pattern on the 2 sister chromatids.  Advances in 
cytochemistry over the subsequent 15 years led to greatly 
simplified procedures for visualizing these exchanges.  Latt 
(1973) demonstrated that bromodeoxy-uridine (BrdU), an analogue 
of thymidine, when incorporated into DNA, could quench the 
fluorescence of the fluorochrome Hoechst 33258.  Perry & Wolff 
(1974) found that incorporated BrdU also diminished the uptake 
of Giemsa stain into the chromatin.  In principle, SCEs can be 
observed in any cell that has completed two, or the first of two, 
replication cycles in the presence of BrdU.  The most common 
procedure for human lymphocytes is to grow the cells in BrdU for 
two replication cycles. 

    SCEs are most efficiently induced by substances that form 
covalent adducts with the DNA, or interfere with DNA precursor 
metabolism or repair (Perry & Evans, 1975;  Wolff, 1977; Perry, 
1980; Carrano & Thompson, 1981; Latt, 1981).  It is well documented 
that SCEs are produced during DNA replication (Wolff et al., 1974) 
and that the polarity of DNA is maintained in the process of 
exchange (Taylor, 1958; Wolff & Perry, 1975).  The molecular 
mechanism by which the exchange is formed is not known but, since 
there is an absolute requirement for DNA synthesis, hypotheses have 
implicated the mechanics of this process in SCE formation (Bender 
et al., 1974; Painter, 1980; Cleaver, 1981, Shafer, 1982).  
Baseline SCE frequencies are increased in some human diseases 
such as Bloom's syndrome (Chaganti et al., 1974) and multiple 
sclerosis (Sutherland et al., 1980; Vijayalaxmi et al., 1983a).  
Cells from patients with the inherited disease Xeroderma 
pigmentosum are hypersensitive to the induction of SCEs by 
ultraviolet radiation and alkylating agents (Wolff et al., 1975, 
Wolff, 1977).  There is no clear relation, however, between the 
repair capacity of these cells and their sensitivity to SCE 
induction (de Weerd-Kastelein et al., 1977). 

3.2.2.2.  Relevance of sister-chromatid exchange

    An SCE represents the breakage of 4 strands of DNA (2 double 
helices), a switch of the strands from one to the other arm of the 
same chromosome, and the rejoining of the strands in their new 
locations.  The question is whether the breakage and rejoining 
events occur without producing any modification in the genetic 
code.  There is evidence suggesting that this exchange process 
might not always be error-free.  In particular, a considerable 
amount of information has been gathered concerning the relationship 
between induced SCEs and other genetic effects  in vitro and  in 
 vivo. 

    Because the technique for enumerating SCEs is relatively 
simple, this assay has been used quite extensively in genetic 
toxicology studies.  It has been amply demonstrated that the 
frequency of SCE is dramatically increased when cells, or 
animals, including human beings, are exposed to known mutagens 
and carcinogens (Perry & Evans, 1975; Latt et al., 1981; Lambert 
et al., 1982).  Moreover, in early experiments using Chinese 
hamster cells in culture, it was found that the induction of SCEs 
was linearly related to the increase in single-gene mutations (HPRT 
locus), when the cells were exposed to several substances each of 
which differed in the type of lesion produced in DNA (Carrano et 
al., 1978; Carrano & Thompson, 1982).  The ratio of induced SCE to 
induced mutation differed for each of the agents, suggesting that 
each type of lesion formed is processed differently by the cell 
favouring either the formation of SCEs or mutations.  It is 
possible that the same lesion is capable of producing both an SCE 
and a mutation and/or that the lesions that form mutations are a 
subset of those that form SCEs.  The induction of mutations has 
been shown recently to be correlated with the induction of SCEs in 
mice for one chemical, ethyl nitrosourea (Jones et al., in press).  
In this study, the induction of SCEs in mouse splenocytes, 
following intraperitoneal administration of the chemical, was 

linearly proportional to the increase in mutations at the HPRT 
locus in the same splenocyte population, suggesting that the 
mutation-SCE relation is not unique to  in vitro  studies. 

    Other investigations compared the induction of SCEs to either 
the induction of transformation  in vitro or induction of tumours 
 in vivo.  In a series of experiments, the induction of SCEs was 
shown to be linearly and positively correlated with the induction 
of transformation of Syrian hamster embryo cells  in vitro (Popescu 
et al., 1981).  This relationship held for exposure to 5 chemicals, 
but not for X-irradiation.  The ratio of induced SCEs to induced 
transformation frequency varied for each chemical.  This is 
analogous with the results found for the SCE-to-mutation ratio 
described above.  Cheng et al. (1981) examined the induction of 
SCEs in mouse bone marrow, regenerating liver, and alveolar 
macrophages following intraperitoneal injection of ethyl carbamate 
and related chemicals.  The relative potencies in inducing SCE 
parallelled the reported activities for the induction of lung 
adenomas in mice.  For ethyl carbamate, the doses for the 
significant induction of SCEs in alveolar macrophages and lung 
adenomas were similar.  Further, the slopes of the dose responses 
showed that the two end-points were of comparable sensitivity. 

    The results of early studies on rabbits demonstrated that it 
was possible to measure an increase in SCEs in peripheral blood 
lymphocytes for many months following repeated low-level exposure 
to mitomycin C  in vivo (Stetka et al., 1978).  Increased SCEs in 
human beings have been observed in cigarette smokers (Lambert 
 et al., 1978; Carrano, 1982) and in individuals undergoing 
chemotherapy (Lambert et al., 1978; Musilova et al., 1979; Gillian 
Clare et al., 1982).  The results of these studies suggest that a 
subpopulation of lymphocytes with high SCE frequencies may be 
present for long periods after termination of exposure.  These 
cells may represent persistent damage in long-lived lymphocytes 
or a sensitive subpopulation (Carrano & Moore, 1982).  If this 
is true, they may offer a more appropriate approach to the 
quantification of persistent damage  in vivo. 

3.2.2.3.  Factors potentially influencing SCE frequency

    The baseline SCE frequency in human peripheral lymphocytes 
averages about 7 - 10 per cell, but has been reported to range from 
about 2 to 45 per cell in unexposed individuals (Alhadeff & Cohen, 
1976; Lambert et al., 1976; Crossen et al., 1977; Galloway, 1977; 
Carrano et al., 1980).  Even within the same laboratory, the 
baseline frequency may vary by a factor of two or more.  Several 
potential sources of variation have been identified and they 
generally fall into 2 categories:  a) culture factors associated 
with the  in vitro growth of the lymphocytes; and b) biological 
factors associated with the genotype, lifestyle, or general health 
of the individual.  The biological factors usually cannot be 
mitigated and must be accounted for in the selection of appropriate 
control cohorts or ultimately in the statistical analysis. 

    (a)   Culture-associated factors

    A major source of variation can be attributed to the 
concentration of BrdU relative to the number of lymphocytes in the 
culture.  The frequency of SCE has been shown to increase by as 
much as 50%, when the BrdU concentration is raised 10-fold (Carrano 
et al., 1980).  The ability of BrdU to induce SCE has been well 
documented in human and other cell systems (Wolff & Perry, 1974; 
Lambert et al., 1976; Latt & Juergens, 1977; Mazrimas & Stetka, 
1978) and it has been further shown that the SCE frequency can 
increase if the BrdU concentration is held constant, but the 
cell number decreased (Stetka & Carrano, 1977).  The number of 
lymphocytes that respond to mitogen and eventually incorporate 
the analogue is generally not controllable, and, therefore, the 
effect of BrdU can best be minimized by standardizing both the 
concentration of BrdU and white blood cells at culture initiation.  
Since the SCE increase above baseline following most long-term 
exposures may be small, careful consideration of the influence of 
BrdU is warranted. 

    Other culture-associated factors have been examined to 
determine whether they influence the SCE frequency.  One concern 
has been the optimal time to harvest cells.  In at least three 
studies, it has been concluded that the baseline SCE frequency 
does not depend on the time of harvest.  Carrano et al. (1980) 
found that, for any individual, the SCE frequency could vary by 
as much as 30% as a function of culture time, but that the pattern 
was not consistent among donors.  Similarly, Beek & Obe (1979), 
Becher et al. (1979), and Morimoto & Wolff (1980) did not observe 
any significant alteration in the baseline SCE frequency as a 
function of culture time.  In a study by Snope & Rary (1979), for 
4 individuals, the baseline SCE frequency observed in 58-h cultures 
was about 30% less than in 70-h cultures.  The authors interpreted 
this to indicate that there were at least 2 subpopulations of 
lymphocytes that differed in SCE frequency.  Studies by Lindblad & 
Lambert (1981) and Das & Sharma (1984) also revealed that the more 
slowly cycling lymphocytes had a higher SCE frequency.  The 
possibility that this effect might be due to T- and B-lymphocyte 
subpopulations has not been completely resolved.  Santesson et al. 
(1979) found that B-lymphocytes had a significantly lower SCE 
frequency than T-lymphocytes, but Lindblad & Lambert (1981) could 
not confirm this.  In order to optimize an experimental design to 
account for such an effect, all samples should, where possible, be 
harvested at the same time. 

    Two other factors have been shown to influence the baseline SCE 
frequency, i.e., the serum used and the culture temperature.  Using 
sera from 4 different sources, McFee & Sherrill (1981) showed that 
the SCE frequency could increase by 50% as the serum concentration 
increased.  Kato & Sandberg (1977) also demonstrated an effect of 
sera on the production of SCEs.  The frequency of SCE in amphibian 
cells increased almost 5-fold with increasing culture temperature 
(Speit, 1980).  In Chinese hamster cells, both lower (33 C) and 
higher (40 C) temperatures increased the frequency of SCE by 
approximately 60%.  Because the alteration in temperature was 
only effective in altering SCE frequencies while BrdU was being 

incorporated, the author concluded that SCEs might arise via a 
temperature-dependent disturbance of DNA replication.  In a study 
by Das & Sharma (1984), the frequency of SCEs in human lymphocytes 
increased as a function of culture temperature and reached a 
maximum at 40 C.  The authors concluded that temperature-dependent 
DNA replication enzymes might be responsible for this effect. 

    In many human population studies, the cohort to be sampled 
may be far away from the laboratory performing the cytogenetic 
analysis, and it will often be necessary to ship the samples.  
If blood is shipped commercially, recording thermometers in the 
shipping container will provide some quality control.  The effect 
of blood storage on SCE frequency has been examined (Carrano et 
al., 1980).  No consistent alteration in SCE frequency was observed 
for blood held at either 4 or 22 C for up to 48 h.  This was 
confirmed by Sharma & Das (1984) for blood stored at 5 C for up to 
7 days, at 22 C for 3 days, and at 37 C for 2 days.  However, it 
should be noted that such storage can lead to an overall decrease 
in lymphocyte numbers and modification of lymphocyte subclasses 
(Dzik & Neckers, 1983).  Thus, the samples should be placed in 
culture as quickly as possible. 

    Finally, Deknudt & Kamra (1983) evaluated 4 different 
mitogens for their effects on SCE frequency.  They found that 
PHA, concanavalin A (Con A), Wistaria floribunda (WFA), and lentil 
lectin (LcH-A) extracts did not result in different baseline SCE 
frequencies in the same donors.  However, significant differences 
among mitogens were noted for lymphocytes that had been exposed 
to cyclophosphamide or mitomycin C.  The use of a single T-cell 
mitogen, such as PHA, for all studies is therefore recommended.  
The experience accumulated from the use of lymphocyte cultures 
suggests that there are many culture-associated factors that could 
influence the SCE frequency.  In order to minimize variation and 
maximize sensitivity, the use of standardized procedures is 
essential. 

    (b)   Biological factors

    Factors, unique to the individual being sampled, include such 
confounding variables as sex, age, diet, genotype, medication, and 
smoking.  Each of these potentially plays a role in the induction 
or expression of SCEs.  Although some information is available on 
the influence of these factors, the consequences are not always 
clear-cut.  Moreover, it is likely that they represent only a small 
number of possible confounding factors. 

    Several investigators have compared the baseline frequency of 
SCEs in male versus female donors and have not observed significant 
differences (Galloway & Evans, 1975; Alhadeff & Cohen, 1976; 
Crossen et al., 1977; Latt & Juergens, 1977; Morgan & Crossen, 
1977; Cheng et al., 1979; de Arce, 1981; Waksvik et al., 1981; 
Carrano & Moore, 1982; Livingston et al., 1983).  The absence 
of clear documentation of a sex difference for baseline SCE 
frequencies suggests that this factor may not have to be matched in 
a population study.  However, that there is evidence for increased 
SCE frequencies in women taking oral contraceptives (see below).  

No studies relating SCEs to cyclic hormonal variation in the female 
have been reported. 

    Another variable of concern in population studies is the age of 
the donor, since it is not always possible to obtain accurate age 
matching.  The independence of age and baseline SCE frequency in 
lymphocytes in adults has been observed (Galloway & Evans, 1975; 
Morgan & Crossen, 1977; Hollander et al., 1978; Cheng et al., 1979; 
Lambert & Lindblad, 1980; Carrano & Moore, 1982; Livingston et al., 
1983).  Lower SCE frequencies have been found in infants (Seshadri 
et al., 1982), in cord blood (Ardito et al., 1980), and in children 
with a mean age of 1.5 years (Husgafvel-Pursiainen et al., 1980).  
De Arce (1981) reported that people in the age range of 30 - 40 
years had a higher number of SCEs per cell than those between the 
ages of 0 - 10 or 60 - 70 years.  Schmidt & Sanger (1981) found a 
significant increase in SCE with age when comparing age groups of 
1 - 18 months, 10 - 13 years, 26 - 32 years, and 63 - 85 years.  
However, this might be due to differential BrdU incorporation 
between cultures.  With the exception of lower SCE frequencies in 
new-born infants, the weight of evidence suggests that baseline SCE 
frequencies do not change with age (at least up to 60 years). 

    It has been suggested that genetic factors play a role in the 
baseline SCE frequency.  When Cohen et al. (1982) compared SCE 
frequencies, both within and among 12 pedigrees (2 generations), 
they found significant differences between, but not within, the 
families, suggesting a genetic contribution to baseline SCEs.  
Pedersen et al. (1979) examined the cultured lymphocytes from 11 
monozygotic and 9 dizygotic twins of the same sex.  They did not 
find any significant differences in the SCE frequency in 
monozygotic versus dizygotic twins and concluded that genetic 
factors do not play a role in baseline SCE frequency.  Waksvik et 
al. (1981) also did not find any genetic contribution to SCE 
frequencies in their studies on twins.  In order to determine 
whether baseline SCE frequencies were related to ethnic origin, 
SCEs were measured in adults of both sexes with no interracial 
family backgrounds from Caucasian, American black, oriental, and 
native American races (Butler, 1981).  There was no significant 
difference in the average frequency of SCEs in the 4 races.  
Further research is needed to resolve the issue of genetic factors. 

    The potential influence of other factors on the frequencies of 
human SCEs are worth considering.  Because a large number of women 
use oral contraceptives, care must be taken that this does not 
confound the consequences of exposure to other agents.  A study of 
oral contraceptive users demonstrated a significant increase in 
lymphocyte SCE associated with the daily intake of d-norgestrel or 
ethenyl estradiol over a period of 6 - 24 months (Balakrishna 
Murthy & Prema, 1979).  Additional studies are needed to confirm 
and extend these findings. 

    Despite knowledge of natural mutagens in food and the role of 
food processing in creating mutagens (Nagao & Sugimura, 1981; Ames, 
1983), little attention has been given to the role of diet in 
causing baseline SCE variation.  It has been reported that severe 
protein calorie malnutrition in children is associated with 

increased lymphocyte SCE frequencies (Balakrishna Murthy et al., 
1980).  Following nutritional rehabilitation, SCE frequencies were 
seen to decrease.  Increased SCEs have also been reported in 
chronic alcoholics (Butler & Sanger, 1981).  For most human 
studies, however, the nutrition factors are more likely to involve 
long-term, low-level intake of caffeine, saccharin, other 
additives, or well-cooked foods.  Each of these factors has been 
shown to be genotoxic in microbial, mammalian  in vitro, or animal 
assays, but there are no data on how these might influence SCE 
frequency in human beings.  Dietary information should be collected 
in the initial questionnaire.  Such information may be useful in 
interpreting a population difference or can be used in large 
composite data bases to search for diet-related factors.  The use 
of prescription and non-prescription medication, or vitamins, 
should also be identified and appropriate information should be 
collected before blood sampling, so that matched controls can be 
employed. 

    Cigarette smoking is, perhaps, the most important confounding 
factor in the interpretation of human SCE frequencies.  The 
preponderance of evidence indicates that individuals who smoke 
have elevated SCE frequencies (Lambert et al., 1978; Ardito et al., 
1980; Hopkin & Evans, 1980; Lambert & Lindblad, 1980; Carrano, 
1982; Husum et al., 1982; Livingston et al., 1983).  However, this 
effect has not been observed in all studies (Hollander et al., 
1978; Hedner et al., 1983).  Since the elevation can be so marked, 
it is important to control for cigarette smoking in any population 
study. 

    Taken together, the culture and biological factors that 
potentially confound the baseline SCE frequency in human beings 
are numerous and contribute to individual variation. 

3.2.2.4.  Methods for sister-chromatid exchange analysis

    The procedures for SCE analysis in human populations will 
depend, to some extent, on the specific circumstances of the study.  
The following generalized outline of a procedure may have to be 
modified to fit special situations. 

    In order to identify any of the factors that may confound the 
analysis of SCEs, it is important to obtain relevant information on 
the background of each individual in the study population.  This 
information is best obtained through a questionnaire.  The 
questionnaire should attempt to determine the obvious exposure and 
potential confounding factors.  In addition to the questions 
related to personal and family cancer incidence, birth defects, 
known genetic disorders, and current health, there are specific 
questions related to the interpretation of the SCE results.  These 
include:  a) smoking history; b) medication and drug use; c) known 
or suspected exposures to physical or chemical agents occurring 
at work, at home, or during the pursuit of various hobbies or 
recreation; and d) nutrition, particularly the adherence to special 
diets or the use of artificial sweeteners or caffeine.  Because a 
complete list of agents that may directly or indirectly affect SCE 
frequencies has not been established, it is better to collect too 
much information than too little. 

    When the blood is collected, it should be mixed with heparin, 
acid-citrate-dextrose, or another appropriate anticoagulant.  As 
already stated, the cultures should be established as soon as 
possible after the blood is withdrawn, preferably within 24 h.  
Just prior to establishing the culture, a total white blood count 
and a slide for a differential should be made from the blood 
sample.  These parameters can serve as indicators of abnormally 
high or low lymphocyte counts, factors that can have a bearing on 
the observed frequency of SCEs. 

    It is important that a standard procedure be employed and, 
although the exact procedure may differ among laboratories, there 
are some common requirements.  Because BrdU can induce SCEs in 
human lymphocytes, both the BrdU concentration and the number 
of lymphocytes potentially incorporating the drug, should be 
stabilized.  In order to ensure this, it is important that each 
laboratory uses a fixed BrdU concentration and a consistent number 
of lymphocytes per culture.  Selection of culture medium, serum 
type and concentration, whole blood versus purified lymphocyte 
cultures, and duration of cultures is somewhat arbitrary and should 
be based on experience.  The serum lot should be pre-tested for its 
potential to support lymphocyte growth and the reproducibility of 
SCE frequencies in duplicate samples from the same donor.  It is 
recommended that the same serum lot be used for each population 
study.  The duration of culture should be such that it yields a 
high proportion of second division cells so that the SCE frequency 
is representative of a major fraction of the lymphocytes.  Exposure 
of the cultures to other than yellow or red light should be 
avoided.  Replicate cultures should be established for each 
individual, and, when a large number of individuals is processed, 
the inclusion of blood cultures from historical controls is useful. 

    At the appropriate culture time, the cells can be arrested in 
mitosis with Colcemid(R), colchicine, or other suitable agents.  
Several slides should be prepared from each culture and the 
remaining cell pellet can be stored in absolute methanol:glacial 
acetic acid (3:1) at a temperature of 4 C or lower, for future 
recall.  The slides can be stained for analysis by fluorescence 
alone (Latt, 1973), fluorescence plus Giemsa (Perry & Wolff, 1974), 
or a suitable alternative.  Once a staining procedure has been 
standardized and adopted, it should be used throughout the whole 
course of the study. 

    Slides should be coded and scored blind.  The coded slides can 
be scanned under low magnification (100 - 200X) and selected for 
scoring on the basis of good staining and chromosome number.  Only 
well-differentiated metaphases should be accepted for scoring.  It 
is not essential for all the chromosomes in a cell to be counted.  
Overlapping chromosomes can be disregarded and the remaining 
chromosomes scored.  At least 43 chromosomes should be analysed per 
cell.  The SCEs are counted for each chromosome in the cell and 
expressed as SCE per chromosome or converted to the number of SCEs 
per diploid cell.  Although the precise number of cells to be 
scored per individual will ultimately depend on the desired 
statistical sensitivity, a minimum of 50 cells should be scored, 
with 25 cells from each replicate culture. 

3.2.2.5.  Data processing and presentation

    The standard statistical descriptors such as mean SCE per cell 
or per chromosome, standard deviation or standard error, and number 
of cells scored should be determined for each individual.  For 
heteroploid cells, the SCE frequency should be normalized to a 
diploid cell.  Population means and their errors should be 
presented similarly and information on the number of cells scored 
per sample as well as the number of people per group should be 
given. 

    Student's t-test is commonly used for the statistical analysis 
of population data.  When comparing population groups, however, the 
sample size is the number of people in each group, not the number 
of cells scored for each group.  Caution is advised with the use of 
t-tests.  Individual and/or population means may not be normally 
distributed and hence the t-test may be inappropriate.  Analysis of 
variance or appropriate non-parametric statistics may be more 
rigorous for the particular population under study  (Carrano & 
Moore, 1982).  Data transformation can be useful in the statistical 
analysis, if the mean and the variance are independent (DuFrain & 
Garrand, 1981).  No single statistical procedure can be recommended 
for universal application.  The investigators must carefully 
examine the data and apply the most appropriate test for the 
questions being addressed. 

3.2.2.6.  Conclusions

    There are many examples of populations exposed to mutagens in 
which large increases in SCE have been demonstrated.  These studies 
show that the end-point can be a very sensitive indicator of 
chemical exposure.  In contrast, SCEs are not a useful indicator of 
human radiation exposure.  From the many reported studies, it is 
evident that further quantitative information on variations in 
SCE level between people, and the factors that influence these 
frequencies, are required.  For this reason, marginal increases 
above a baseline SCE frequency must be interpreted with caution.  
Significantly increased levels are an indication that the 
population has been exposed to a genotoxic agent; on the other 
hand, failure to observe increased levels does not necessarily 
indicate the absence of exposure.  Any increase in SCE frequency 
for an individual or group cannot be interpreted as an indication 
that the individual or group is likely to suffer adverse health 
consequences. 

3.3.  Gene Mutations

    New procedures are being developed to detect mutations that 
lead to the production of abnormal haemoglobins (Bigbee et al., 
1981), the loss of cell surface proteins (Bigbee et al., 1983), or 
the loss or modification of enzymes used in the synthesis of DNA in 
human somatic cells.  The last of these procedures has been used 
for human population studies and is considered in detail. 

    Resistance to 6-thioguanine is a marker that has been used 
widely to detect somatic mutations occurring  in vitro in cultured 

mammalian cells (Hsie et al., 1979).  Since similiar somatic 
mutations occur  in vivo, quantification of 6-thioguanine resistant 
(TGr) cells in peripheral blood provides a measure of specific-
locus somatic mutations occurring  in vivo. 

    In human beings, a naturally-occurring germinal mutation 
of the X-chromosomal gene for hypoxanthine-guanine 
phosphoribosyltransferase (HPRT) (EC 2.4.2.8) provides a prototype 
for 6-thioguanine resistance at the somatic level (DeMars, 1971).  
HPRT normally converts hypoxanthine and guanine to inosine 
monophosphate, and it phosphorylates some purine analogues (e.g., 
6-thioguanine) to produce cytotoxic products (Caskey & Kruh, 1979).  
Mutant cells, deficient in this enzyme, are unable to use exogenous 
hypoxanthine as a purine source and are resistant to killing by 
these purine analogues. 

FIGURE 3

    TGr somatic cells arising  in vivo can be shown to be mutants 
by the stability of their phenotype and deficiency of HPRT activity 
(Albertini, 1982; Albertini et al., 1982a; Morley et al., 1983b).  

Also, molecular studies have demonstrated changes in the HPRT locus 
at the DNA level (Albertini et al., in press). 

3.3.1.  Principles and basis for the methods

    TGr T-lymphocytes (T-lys), present in human blood, can be 
measured either by autoradiography to determine TGr T-ly variant 
frequencies (Vfs), or by cloning to determine TGr T-ly mutant 
frequencies (Mfs).  Autoradiography is the earlier method (Strauss 
& Albertini, 1977, 1979) and has been used for some human 
population studies (Strauss & Albertini, 1979; Strauss et al., 
1979; Cole et al., 1982; Lange & Pranter, 1982; Morley et al., 
1982; Vijayalaxmi et al., 1983b; Albertini, 1983; Albertini et al., 
1983a); it is simple, inexpensive, and can be automated (Anmeus et 
al., 1982; Zetterberg et al., 1982).  Cloning is a more recent 
development by which it is possible to recover the TGr cells for 
characterization of the nature of the mutations (Albertini, 1982; 
Albertini et al., 1982; Strauss, 1982; Morley et al., 1983a; 
Vijayalaxmi & Evans, 1984).  This characterization may be  
necessary in order to quantify the different types of somatic 
 mutations responsible for a collection of  mutants. 

3.3.1.1.  Autoradiographic method

    The autoradiographic detection of rare TGr T-lys is based on 
the ability of these cells to incorporate tritiated thymidine 
(HTdR)  in vitro following short-term lectin stimulation in the 
presence of cytotoxic concentrations of 6-thioguanine.  The vast 
majority of human peripheral blood T-lys are in the G0 stage of the 
cell cycle  in vivo.  When stimulated  in vitro with lectins, such 
as phytohaemagglutinin (PHA), T-lys are activated to early G1, 
which is characterized by the acquisition of T-cell growth factor 
(TCGF) receptors on their surfaces (Maizel et al., 1981).  These 
receptors are also called interleukin-2 (IL-2) receptors or Tac 
antigens (Deeper et al., 1983).  A series of cellular events 
results in the production of interleukin-1, (IL-1) and IL-2, and 
other factors in short-term peripheral blood mononuclear cell 
cultures, with IL-2 driving the activated T-lys through G1 to DNA 
synthesis and subsequent events in the cell cycle. 

    The initial round of lectin-stimulated T-ly DNA synthesis 
 in vitro is inhibited by 6-thioguanine.  Inhibition probably is 
at the activation step in 6-thioguanine sensitive cells and 
occurs without incorporation of the analogue into DNA.  The 
interval of lectin stimulation before the addition of HTdR in 
the autoradiographic assay is short, so that cell division  in 
 vitro does not occur (Strauss & Albertini, 1979).  Under proper 
conditions, the T-lys that incorporate this label in the presence 
of 6-thioguanine are TGr variants.  These cells can be enumerated 
by autoradiography and the TGr variant frequency (Vf) calculated. 

3.3.1.2.  Cloning method

    Human T-lys are easily propagated for extended periods  in vitro  
by supplying TCGF to cells that have been activated with lectin or 
antigen (Paul et al., 1981).  Thus, mass populations derived from 

peripheral blood can be developed and maintained.  Moreover, 
freshly-obtained human T-lys may be cloned directly  in vitro, in 
the presence or absence of 6-thioguanine.  In the first case, large 
cell innocula must be used, to ensure the presence of rare TGr 
cells (Albertini, 1982; Albertini et al., 1982a; Strauss, 1982; 
Morley et al., 1983a,b; Vijayalaxmi & Evans, 1984).  Activating 
intervals are short so that cell division does not occur  in vitro 
prior to exposure to cytotoxic concentrations of 6-thioguanine, 
thus ensuring that the TGr cells recovered  in vitro actually arose 
 in vivo.  The number of TGr T-lys determined by cloning is used to 
derive  in vivo TGr T-ly mutant frequencies (Mfs). 

3.3.2.  Relevance and limitations

3.3.2.1.  Relevance

    The ability to measure TGr T-lys arising  in vivo is important, 
because it provides a genetic end-point for assessing human 
genotoxicity that may be qualitatively different from somatic cell 
chromosomal damage.  Thus, the ability to measure gene mutation 
provides a broader basis for human monitoring.  Furthermore, gene 
mutations, as assessed by TGr T-lys, are end-points that are 
qualitatively different from other measures of  in vivo DNA damage.  
Measuring human somatic cell gene mutations occurring  in vivo has 
the following advantages: 

    (a)  The effects of metabolic and kinetic factors are included 
         in measurements of somatic cell mutation occurring  in vivo.

    (b)  Mutagenicity assays for  in vivo events assess the genetic 
         effects of given environments, rather than the genotoxic 
         potential of targeted chemicals or other agents.  Thus, 
         these assays can be used to measure the effects of 
         environmental mixtures in which the components and/or 
         their synergistic actions may not be known.

    (c)  Mutagenicity assays,  in vivo, may define human population 
         heterogeneity in susceptibility to various mutagens/
         carcinogens (Vijayalaxmi et al., 1983b).  Such individual 
         susceptibilities may be important in assessing human 
         genotoxic risks. 

    (d)  Mutagenicity assays,  in vivo, have the potential for being 
         developed into a means of making quantitative human 
         genetic health risk assessments, provided that appropriate 
         correlations can be made between individual assay results 
         and individual health outcomes (Fowle et al., 1984).

3.3.2.2.  Limitations

    Although progress has been made in demonstrating that TGr T-lys 
are mutant somatic cells, at least 3 major problems remain for 
human  in vivo somatic cell mutation assays.  Two are concerned 
with quantification and apply primarily to gene mutation assays.  
The third applies to all human somatic cell mutation assays and the 
relevance of the results for making estimates of human health 
risks. 

    (a)  The  in vivo sensitivities of the human TGr T-ly assays in 
         terms of dose-response characteristics are not known at 
         present.  However, because mutant cells are also 
         detectable in animals (Garcia & Couch, 1982; Gocke et al., 
         1983; Recio et al., 1983; Jones et al., in press), these 
         studies may provide useful information.

    (b)  Although somatic cell mutations are the events of 
         relevance for human population monitoring, TGr T-ly assays 
         measure the frequency of somatic cell mutants, not the 
         number of mutations.  In order to derive mutation 
         frequencies from mutant frequencies, it is necessary to 
         have information including cell pool sizes and 
         distributions  in vivo, representativeness of test samples, 
          in vivo cell kinetics, and  in vivo positive or negative 
         selection of mutants.  One method for circumventing this 
         problem is to determine whether the mutants scored in a 
         mutagenicity assay are qualitatively heterogeneous, which 
         will yield a minimum number of independent mutational 
         events giving rise to the observed collection of mutants. 
         The ability to recover TGr T-lys from clonal assays makes 
         these determinations possible.

    (c)  Assays of genetic damage occurring  in vivo in human 
         somatic cells have not been validated in that it has not 
         been possible to use the results as predictors of human 
         genotoxic health risks.  Before such results can be used 
         as surrogate markers for human health outcomes, high-risk 
         populations will  have to be monitored and correlations 
         made between the results from mutagenicity assays and 
         epidemiological outcomes.  The importance of other factors 
         (e.g., immunotoxicity) in influencing human genotoxic
         disease risks remains to be assessed.

3.3.3.  Procedures for the assay of TGr T-lys arising  in vivo in 
human beings

3.3.3.1.  Autoradiographic method

    TGr T-ly Vfs for normal, control adults, as determined 
autoradiographically by the method described here, rarely exceed 
10 x 10-6. By contrast, chemotherapy-treated cancer patients 
frequently have values several times higher (Strauss & Albertini, 
1977, 1979; Lange & Pranter, 1982; Albertini, 1983).  Other 
laboratories report somewhat higher normal values, as well as a 
positive correlation with age (Morley et al., 1982), indicating 
that local ranges of normal values must be developed.  In one 
laboratory, approximately 75% of placental cord blood values fell 
within the normal range for adults, while the remainder were 
elevated (Albertini et al., 1983).  Whether this reflects maternal 
exposure to genotoxic agents, or an altered ratio between mutants 
and mutations in the fetus remains to be determined. 

    The basic autoradiographic method for assessing TGr T-lys has 
been described in detail by Albertini et al. (1982b) and Albertini 
& Sylwester (1984).  Peripheral blood mononuclear cells (MNCs), 

which include the T-lys, are separated from whole blood by the 
Ficoll-Hypaque method (Boyum, 1968).  After washing, the MNCs are 
suspended in a dimethyl sulfoxide-containing medium and aliquoted 
into ampules for controlled freezing and storage.  Freezing is 
important for holding and shipping samples and serves also as one 
method for eliminating the phenocopy effect of cycling cells as 
described below.  For assaying, MNCs are thawed, washed, and 
suspended in appropriately supplemented medium for short-term 
tissue culture.  A minimum of 5 x 106 MNCs are cultured without 
6-thioguanine (control culture), while several times this number of 
cells are cultured with 2 x 10-4 M 6-thioguanine (test cultures).  
All cultures contain phytohaemagglutinin (PHA), for activation of 
T-lys, and are incubated under standard conditions.  After 
approximately 24 h (Strauss & Albertini, 1979), 3HTdR is added 
to all cultures, and incubation continued for an additional 18 h.  
Cultures are terminated by adding cold 0.1 M citric acid, to 
prepare suspensions of free nuclei.  The nuclei are then washed 
and suspended in methanol-acetic acid fixative, counted, diluted 
if necessary, and added to coverslips affixed to microscope slides.  
Slides are dried, stained with aceto-orcein and subsequently 
autoradiographed by standard methods.  Slides are scored and Vfs 
are calculated as described in section 3.3.4.1. 

    A subclass of 6-thioguanine-sensitive T-lys in human peripheral 
blood may become labelled and scored as TGr (i.e., as variants), 
when using the autoradiographic method, unless certain precautions 
are taken (Albertini et al., 1982a,b; Albertini, 1982).  These 
phenocopies occur because, at any given time, a small minority of 
human T-lys are in an activated state,  in vivo.  These cells have
the Tac antigen, when initially put into culture, and do not 
require the activation step before proceeding to  in vitro DNA 
synthesis.  Although ultimately 6-thioguanine sensitive, these 
cells probably are not effectively blocked from accomplishing at 
least one round of DNA synthesis  in vitro, even in the presence 
of 6-thioguanine.  Thus, activated T-lys in the peripheral blood 
constitute a potential source of phenocopies, when TGr T-ly Vfs are 
determined by autoradiography.  Cryopreservation appears to remove 
this phenocopy effect by forcing the  in vivo activated T-lys to 
proceed to DNA synthesis  in vitro at a time when a label is not
present (Albertini et al., 1982a,b).  These cells are not scored as 
variants.  Cryopreservation is a critical step, although other 
methods, such as prolonged incubation in 6-thioguanine, or 
immunological elimination of Tac positive T-lys at the initiation 
of culture, may accomplish the same purpose. 

3.3.3.2.  Cloning method

    From the limited data available, normal control adults have 
shown peripheral blood TGr T-ly Mfs of < 20 x 10-6 when based on 
clonal assays in which single-cell cloning efficiencies achieved 
values > 0.10.  Similar findings from a number of laboratories 
have been reported (Strauss & Albertini, 1979; Morley et al., 
1983a; Vijayalaxmi & Evans, 1984).  Results for placental blood and 
for mutagen-exposed adults remain to be determined.  TGr T-ly Mfs
have been slightly higher than TGr T-ly Vfs, concurrently 
determined (Albertini et al., 1984). 

    The cloning method for assessing TGr T-lys has been described 
by Albertini et al. (1982a).  Peripheral blood MNCs are obtained 
from whole blood as for the autoradiographic method, but freezing 
is not required.  The MNCs are incubated with PHA in order to 
activate the T-lys.  The duration of activation is sufficiently 
short to ensure that cell division does not occur.  Activated cells 
are then innoculated in limiting dilutions into the wells of 
microtitre plates, in the presence or absence of 6-thioguanine 
(approximately 10-5 M).  In addition to the appropriate medium, 
wells contain an optimum concentration of crude TCGF and feeder 
cells.  X-irradiated B-lymphoblastoid cells are well suited for use 
as feeders.  A TGr lymphoblast line is used to avoid interference 
with subsequent HPRT enzyme assays of the recovered TGr T-lys.  An 
average of 1 activated cell per well is innoculated into the non-
selection wells (containing no 6-thioguanine); 105 activated cells 
per well are innoculated into selection wells (containing 10-5 M 
6-thioguanine).  Wells are scored after approximately 2 weeks of 
culture with one change of medium, by microscopy, by scintillation 
spectrometry of HTdR incorporation (added during the last day of 
culture), or by cell transfer and clonal expansion.   In vitro  
cloning efficiencies (CEs) of T-lys are determined from wells 
receiving, on average, 1 cell per well.  By assuming a Poisson 
distribution of cells in wells, the average number of clonable 
cells per well is derived from the P0 class of that distribution, 
i.e., the observed fraction of wells without growing cells.  
Similarly, the incidence of TGr cells in wells receiving 105 
activated T-lys in 6-thioguanine is determined from the P0 class of 
wells in the plates containing 6-thioguanine.  The TGr T-ly Mf is 
the incidence, divided by 105, corrected for the CE. 

    TGr T-lys obtained by cell transfer and clonal expansion may be 
characterized for T-ly subset markers, stability of the TGr 
phenotype, HGPRT enzyme activity, and, by using suitable cDNA 
probes, for the nature of the molecular lesion at the DNA level 
(Albertini et al., 1982a, in press). 

    For reasons not known at present, human T-lys, activated  in 
 vitro as described, may fail to clone as efficiently when present 
as single cells than when present in large numbers (i.e., 105 
cells/well), even when the latter are selected in 6-thioguanine 
(Albertini et al., 1984).  Clonal assays with a single cell CE 
of < 0.10 have the potential for yielding falsely elevated Mf 
values, because of an underestimate of clonable cells in selection 
wells.  Current research is directed at increasing and/or 
standardizing single-cell CEs.  At present, however, only Mfs 
determined in clonal assays achieving single cell CEs of 0.10 or 
more should be considered valid. 

3.3.4.  Data presentation and analysis

3.3.4.1.  Autoradiographic method

    Slide scoring is done by one of 2 methods that give almost 
identical results.  One method involves determining labelling 
indices (LIs) for both control (no 6-thioguanine) and test 
(6-thioguanine containing) cultures for each individual.  The LI of 

test cultures (LIt) is determined by counting all labelled nuclei 
on all slides made from test cultures, and dividing this number by 
the total number of nuclei (determined in suspension) added to all 
slides. 

           Number of labelled nuclei on all test slides
    LIt =  --------------------------------------------
           Number of nuclei on all test slides

    The LI of control cultures (LIc) is determined from a 
differential count of 2500 nuclei on slides from control cultures. 

           Number of labelled nuclei per 2500 nuclei
    LIc =  -----------------------------------------
                             2500

    The TGr T-ly Vf for each individual is calculated from the LI 
of test cultures (LIt) divided by the LI of the control culture 
(LIc). 

           LIt
     Vf =  ---          
           LIc

    An alternative method of scoring autoradiographic assays has 
recently been described for mouse TGr T-lys (Gocke et al., 1983), 
and is equally applicable to human assays.  Statistical methods for 
deriving confidence intervals for variant frequencies are described 
by Sylwester & Albertini (1984). 

3.3.4.2.  Cloning method

    Cloning efficiencies (CEs) are calculated from the P0 class of 
the Poisson distribution: 

    P0 = e-x, or

    x = -ln P0 (control)

where x is the average number of clonable cells per well.  When CE 
is determined by single cell innocula, x = CE. 

    The incidence of TGr T-lys in selection wells, receiving on 
average 105 cells per well, is also determined from the P0 class of 
the Poisson distribution: 

    P0 = e-y, or

    y = -ln P0 (test)

where y is the average number of clonable TGr T-lys per well.

    The TGr T-ly Mf is determined from the incidence of TGr
T-lys per well divided by the average cell innocula, corrected
by the CE.  When 10 cells are innoculated into selection wells:

          -ln P0 (test)
    Mf =  -------------                     
          CE x 105

    Appropriate statistical procedures for handling Mf data, 
differences between Mfs, etc. are being developed by methods 
analogous to those referred to in section 3.3.4.1. 

3.3.5.  Conclusions

    TGr T-lys arise  in vivo, are present in human peripheral
blood, and their frequencies are measurable.  Known mutagen 
exposure increases the frequencies of these cells and their 
characterization  in vitro has shown them to be somatic mutants.  
Thus, methods are available for measuring specific locus somatic 
mutants occurring  in vivo in human beings, for purposes of human 
genetic monitoring.  The 2 assays described here continue to be 
developed.  There are potential technical sources of error.  Among 
the limitations in the interpretation of the results of these 
assays is the fact that the numbers of somatic mutants, and not the 
numbers of somatic mutations, are being measured.  The latter may be 
of most interest for human population monitoring.  However, 
characterization of the recovered mutant cells, which is possible 
in the cloning assay, may make it possible to estimate the minimum 
number of mutations responsible for a given number of mutants. 

    Methods described here can be applied to the detection of 
mutations, occurring  in vivo at other genetic loci in human T-lys, 
in order to broaden the base for human monitoring. 

4.  GERMINAL MUTATIONS

    Methods and technical aspects of approaches available for 
detecting germinal mutations in human populations are reviewed in 
this section.  It should serve as a guide, when such studies are 
initiated, and as a reference for defining specific methods for 
estimating human mutation rates. 

4.1.  Introduction

    Germinal mutations include a spectrum of alterations in 
either the structure or quantity of DNA in germinal cells.  The 
study of germinal mutations is the quantification of transmitted 
genetic damage.  Within the context of monitoring for induced 
genetic damage, this is an important consideration, because it is 
the offspring of exposed individuals, rather than the exposed 
individuals themselves, that are the focus of concern.  It is 
generally assumed that a significant proportion of all mutations 
have deleterious effects on both the health and the genetic 
constitution of future generations.  Thus, the increased genetic 
risk and associated health effects are of ultimate concern, 
following exposure to a putative mutagen, when possible increases 
in germinal mutation rates are being ascertained. 

    Germinal mutations are usually classified in 2 categories.  
Chromosomal mutations are operationally defined as changes in 
either chromosome number or structure observable with standard 
karyotypic techniques.   More minute changes in DNA structure are 
classified as gene mutations, often referred to as "point" 
mutations. 

4.1.1.  Approaches for detecting germinal mutations

    The 3 approaches to monitoring for germ cell mutations are 
generally divided as follows:  (a) chromosomal, (b) biochemical, 
and (c) the indicator phenotype; each detects a different set of 
end-points. 

4.1.1.1.  Detection of chromosomal mutations

    Germinal chromosomal mutations occur in about 5% of recognized 
conceptions (Hook, 1981a,b), making them more amenable than the 
rarer specific locus mutations for study in small exposed 
populations.  Unbalanced chromosomal complements are almost always 
associated with deleterious phenotypic effects, leading to fetal 
death, livebirths with anomalies and mental retardation, and/or 
sterility.  Balanced chromosome complements may occur as mutations 
without phenotypic effect.  However, their deleterious effects will 
be seen primarily in the next generation in individuals with 
unbalanced chromosomal complements. 

    Chromosome abnormalities can originate in four different ways: 
a) they may be inherited from a parent; b) they may result from 
errors during gametogenesis (e.g., meiotic non-disjunction); c) 
they may result from events during conception (e.g., dispermy 

resulting in triploidy); d) they may result from events after 
conception (e.g., mitotic non-disjunction).  Only the second 
category listed above is regarded as a germ-cell mutation in the 
strict sense, but to avoid unnecessary complexity in the discussion 
that follows, triploidy and tetraploidy will be considered in the 
review of germinal events. 

    Chromosomal mutations can be subdivided into numerical and 
structural anomalies. 

    (a)   Numerical aberrations

    Numerical chromosomal abnormalities include trisomy, monosomy, 
triploidy, and tetraploidy.  Most instances result from events that 
occur during gametogenesis in a parent, or at the time of 
fertilization, although strict proof of the time of origin is often 
lacking. 

    Apart from triploidy and tetraploidy, numerical chromosome 
abnormalities involving sex chromosomes without a mosaic 45, X cell 
line, or involving autosomes are usually presumed to have resulted 
from a germinal mutation.  The only exceptions are situations in 
which numerical abnormalities occur in only a single tissue, as 
for example in some malignancies.  It is often difficult, however, 
to exclude formally the possibilities that they:  a) have been 
inherited from a parent who is a cryptic mosaic for the abnomal 
line; or b) have resulted from a somatic event, mitotic non-
disjunction, early in the development of the organism. 

    The strongest risk factor known for numerical abnormalities is 
older maternal age, which is highly associated with the frequency 
of trisomy (Hassold et al., 1980, 1984).  Paternal age seems to 
have little, if any, effect (Hook, in press).  In studies using 
chromosomal markers, it has been demonstrated that at least 60% of 
trisomy 21 appears to be the result of maternal 1st division non-
disjunction (Juberg & Mowrey, 1983). 

    Numerical abnormalities almost always occur in offspring 
of parents who have normal chromosomal complements, and thus, 
such abnormalities are presumably the result of germinal 
mutations.  Although there is not a great deal of experimental or 
epidemiological evidence to link numerical chromosome abnormalities 
with environmental agents, increases in such anomalies must be 
considered as possible outcomes of exposure to possible mutagens.  
An unknown proportion of trisomy or monosomy may result from post-
zygotic non-disjunction, or, in the case of trisomy, may be 
inherited from parents who are cryptic carriers, and thus may not 
be the result of germinal mutations in a strict sense.  For 
trisomy, the results of studies of the parental origin of the extra 
chromosome suggest that a maximum of 25% of trisomies are due to 
post-zygotic non-disjunction, and it may well be considerably less 
(Juberg & Mowrey, 1983). 

    Evidence suggests that mosaic trisomies (47/46) do not 
originate from post-zygotic events any more often than non-mosaic 
trisomies (Hassold, 1982). 

    Ninety percent of numerical anomalies in recognized conceptuses 
terminate as fetal deaths; thus, a study restricted to live births 
will miss a major proportion of detectable abnormalities. 

    Only a few kinds of numerical anomalies commonly survive to 
birth.  The most common is Down's syndrome, trisomy 21, occurring 
in about 1 in 1000 live births.  Other numerical anomalies such as 
trisomy 18 and trisomy 13 sometimes survive to occur as live 
births, but these are much less common. 

    (b)   Structural aberrations

    The proportion of individuals with detected structural 
cytogenetic  abnormalities is likely to vary with technical 
factors.  Recent advances in the resolution of chromosome 
substructure (high resolution banding) have reduced significantly 
the size of detectable lesions and such advances appear likely to 
continue.  Thus, it is difficult to specify precisely the 
proportion of recognized conceptuses with a structural abnormality.  
With currently available techniques, these are much less frequent 
than those with numerical abnormalities.  Among live births, the 
ratio of detected structural to numerical abnormalities is about 
1:4 to 1:5 (Hook & Hamerton, 1977).  Among fetal deaths, the ratio 
is much lower, about 1:30 (Warburton et al., 1980).  Unlike 
numerical abnormalities, a significant fraction of detected 
structural abnormalities are known to be inherited, so that 
inferences concerning mutation are not possible unless both parents 
are studied and found not be be carriers of the aberration present 
in the offspring.  The possibility of false assignment of paternity 
must also be considered.  Unlike trisomies, structural chromosome 
abnormalities, in general, show little if any association with 
parental age, although markers and X-isochromosomes may represent 
exceptions.  In instances in which the parental origin of a 
structural mutation has been investigated, many have been found to 
be predominantly paternal in origin, unlike trisomies (Magenis & 
Chamberlin, 1981). 

    In general, mosaicism, involving a normal cell line and a cell 
line with a structurally abnormal chromosome, is not attributable 
to germinal cell mutation, but may be safely inferred to be of 
post-zygotic origin (supernumerary markers are, however, 
exceptions). 

    In terms of sensitivity to environmental factors, mutant 
germinal structural chromosome aberrations are likely to be more 
similar to germinal specific locus mutations than numerical 
abnormalities, as both involve alterations in the structure of the 
genetic material.  Germinal chromosomal rearrangements, unlike 
specific locus mutations, show little association with advanced 
paternal age in human beings (Hook, in press) indicating that there 
are etiological differences between these effects. 

4.1.1.2.  The biochemical approach to detecting point mutations

    Many biochemical approaches for the study of germinal mutations 
have been proposed (Bloom, 1981), but most are not feasible at 
present.  Two general approaches are currently used to study the 
proteins in offspring of selected individuals.  Electrophoresis is 
used to detect a significant proportion of amino acid substitutions 
while enzyme activity measurements are used to detect major losses 
in protein function or protein quantity. 

    Studies involving genetic typing of large numbers of 
individuals, either to analyse population structure or to develop 
mutation monitoring programmes, have demonstrated the feasibility 
of large-scale screening using one-dimenional electrophoretic 
techniques (Harris et al., 1974; Neel et al., 1980a; Altland et 
al., 1982).  Through the introduction of high-resolution two-
dimensional electrophoresis (Klose, 1975; O'Farrell, 1975), the 
number of proteins that can be studied in a single sample has been 
increased to several hundred.  The search for mutational events 
resulting in loss of a functional gene product, using quantitative 
techniques, is complementary to the electrophoretic assay, in that 
this assay detects genetic events not normally detectable by 
standard electrophoretic assays.  Both the electrophoretic and 
enzyme activity approaches have been used in studies to determine 
the induced mutation rate in exposed mice (Johnson & Lewis 1981; 
Johnson et al., 1981; Bishop & Feuers, 1982) and  Drosophila (Racine 
et al., 1980).  Thus, it should be possible to acquire data on the 
mutation rate in human populations and also to examine directly 
some of the problems associated with extrapolation from 
experimental animals. 

    One other biochemical approach has been proposed for detecting 
mutational events in human populations.  It is possible to study 
mutations at the DNA level by employing the restriction enzyme 
mapping approach.  One potential advantage of the restriction 
enzyme mapping approach (or other techniques that directly monitor 
specific alterations in DNA structure) is the ability to examine 
larger portions of the genome and thereby acquire increasing 
amounts of data from each individual.  Since this approach is 
currently in the developmental stage, appropriate samples should be 
retained, as far as possible, to take advantage of this or other 
techniques that may be developed in the near future. 

    The major constraints in relation to the biochemical approach 
include:  (a) the large number of determinations and, to date, the 
large populations necessary to detect statistically-significant 
increases in the mutation rate; (b) the distinction between 
"apparent" mutations and nonparentage; and (c) the problem of 
confirming that a suspected variant is the result of an alteration 
in DNA structure, i.e., is a transmissible trait. 

4.1.1.3.  Indicator phenotypes

    Three types of indicator phenotypes are considered in this 
section:  Down's syndrome, fetal death, and sentinel phenotypes.  
Down's syndrome will be discussed in section 4.3, which deals with 
chromosomal mutations.  The potential use of fetal death as an 
expression of genetic damage arising from genic mutations will be 
discussed in section 4.5.  Although the term "sentinel phenotype" 
is relatively new (Sutton, 1971), the notion of studying mutations 
in human beings by counting the frequency of dominant traits is 
credited to Danforth (1921).  Mulvihill & Czeizel (1983) have 
reviewed the current status of the concept. 

    As indicators of germinal genic mutations, sentinel phenotypes 
are a significant health problem and are recorded in a variety of 
health facilities.  The sentinel phenotype is a clinical disorder 
that:  (a) occurs sporadically as a consequence of a single, highly 
penetrant mutant gene, (b) is a dominant or X-linked trait of 
considerable frequency and low fitness, and (c) is uniformly 
expressed and accurately diagnosable with minimal effort, at or 
near birth.  Individuals with such traits are important for the 
surveillance and monitoring of germinal genic mutations because 
affected persons of unaffected parents arise from a new mutation.  
Their use has severe disadvantages connected with the difficulty 
of accurate nosological diagnosis arising from their genetic 
heterogeneity and a general lack of clinical expertise.  The 
sentinel phenotype approach, like other strategies in mutation 
epidemiology, is burdened with problems created by the inability 
to link separate data files, the necessity to maintain 
confidentiality, and the difficulty in collecting sufficiently 
large study populations or study samples.  The best course of 
action for the present is to obtain field experiences and to 
sustain critical discussion of the approach. 

4.1.2.  Methodological considerations and strategies

4.1.2.1.  Sample acquisition and storage

    Future access to study subjects may be limited; therefore, the 
maximum quantity of sample, usually blood, should be obtained on 
the initial contact.  When more than one test is to be performed, 
attempts should be made to coordinate the appropriate collection 
procedures and minimize the number of different acquisitions.  In 
situations where fetal tissue is being collected for chromosomal 
studies, it is suggested that, where possible, a sample of kidney 
tissue should be collected and stored for possible biochemical 
analysis. 

    Adequate samples should be stored for confirmation of any 
observation and also for future analysis using new and/or refined 
techniques.  Lymphocytes should be stored in a manner that allows 
for the retrieval of viable cells and the subsequent expansion of 
the number of cells, thus providing material for future studies. 

4.1.2.2.  Timing of studies

    With specific regard to mutation studies, following suspected 
exposure to mutagens, women of child-bearing age who have been 
exposed to a mutagen and wives of exposed men should be identified 
and surveyed as soon as possible.  Questionnaires or interviews 
should be undertaken and used to obtain data on basic biological 
and demographic variables (and information on other possible 
mutagenic factors).  The factors include, but are not necessarily 
limited to, age, race, previous pregnancy history, smoking, and 
drug use.  Women should be encouraged to participate in a 
continuing evaluation of future pregnancy outcomes.  If a woman 
thinks she is pregnant but is uncertain, pregnancy testing should 
be carried out as soon as possible.  A control population of 
similar ethnic and socioeconomic background should be identified.  
This could be done at the time of identification of women at risk 
of pregnancy, or could be done after pregnancy is confirmed, using 
as a control group, women pregnant at the same gestational age. 

    Each of these approaches has difficulties.  To wait until the 
exposed woman is pregnant before selecting a matched (pregnant) 
control may result in the pregnancy of the exposed woman being 
terminated by the time the control is chosen.  It may be extremely 
difficult to avoid biasing the results towards a more favourable 
outcome in the controls.  If controls are selected prior to the 
pregnancy of the exposed woman, there is no certainty that the 
exposed and control women will become pregnant at the same time, 
if in fact they conceive at all.  One possible approach is to 
select several possible controls for each exposed woman, before 
pregnancy, and follow the reproductive history of all of them.  
Ideally, these controls should be matched as far as possible in 
age, race, socioeconomic status, and previous reproductive history. 

    Infants, children, and young unmarried males, who have been 
exposed should also be identified early and followed for possible 
inclusion should a subsequent long-term study be undertaken (see 
section on record linkage). 

    Women recruited into the study should be interviewed by 
investigators,  at least once a month, to ascertain if they are 
pregnant or think they may be pregnant.  If there is any question, 
pregnancy testing should be carried out. 

4.1.3.  Summary

    Given the current state of knowledge in the area of human 
mutations, all 3 approaches, namely indicator phenotype, 
chromosomal, and biochemical, to estimating germinal mutational 
damage in human populations should be considered complementary.  
They detect different types of genetic "damage" or "end-points", 
have different degrees of relevance for estimating potential health 
effects and probably different sensitivities.  Therefore, a 
collaborative monitoring exercise should employ all 3 in order to 
obtain the maximum amount of data possible. 

4.2.  Germinal Chromosomal Abnormalities

4.2.1.  Principles and basis of the method

    Chromosomal aberrations associated with germinal cell mutation 
have already been discussed in section 4.1.1.1. 

    Depending on their origin and consequences, chromosomal 
mutations may be detected in gametes, the embryo or fetus, in 
live births, or at later stages in life. 

    If a population is exposed to a mutagen, the most direct method 
of detection of germinal chromosomal mutants would be examination 
of the gametes themselves.  At present, there are no methods for 
evaluating human ova.  Preparations of human sperm chromosomes can 
be made, but these methods are difficult and time-consuming, and 
few laboratories have yet been successful with this technique 
(Martin et al., 1983).  It is possible that improved techniques for 
studying sperm chromosome constitution will be available in the 
future, but, at present, this method cannot be regarded as 
practical for most situations involving mutagenic exposures.  
Also, at the present time, alterations in sperm morphology or 
function cannot be regarded as a reliable index of germinal cell 
mutation, nor can changes in the proportion of sperm showing double 
"Y" bodies.  Thus, the following discussion focuses on detection of 
chromosomal mutations in the offspring of those exposed to known or 
suspected mutagens. 

    A clinically-recognized pregnancy is generally diagnosed after 
the first missed menses, at about 4 weeks of gestation.  The 
frequency of chromosome abnormalities at this point in gestation 
has been estimated to be about 5% (Hook, 1981a).  About 15% of 
recognized pregnancies terminate in fetal death, approximately one-
third having a chromosome aberration (Harlap et al., 1980; Hook, 
1981a).  The rate of loss between conception and recognized 
pregnancy is not known, but it is generally agreed to be very high 
(Kline et al., 1980).  The proportion of these early losses that is 
associated with chromosomal aberration is unknown.  The bulk of the 
evidence from the use of sensitive human chorionic gonadotropin 
serum assays, capable of diagnosing pregnancy immediately after 
implantation (usually 7 days after conception), suggests that loss 
between implantation and the first missed menses may be at least as 
common as loss afterwards (Miller et al., 1980; Edmonds et al., 
1982), but not all results are in agreement (Whittaker et al., 
1983).  Preimplantation losses are still unmeasurable.  Only 
clinically-recognized pregnancy in the usual sense will be dealt 
with here, since the other types of study are unlikely to be 
feasible on the necessary scale, and chromosome studies cannot 
be performed on these early losses. 

    The proportion of chromosome abnormalities in fetal deaths 
varies with gestational age, being highest (about 50%) in the 
8 - 12 weeks range, and then decreasing with gestational age (to 
about 7%) at more than 22 weeks (Warburton et al., 1980).  The 
kinds of chromosome abnormalities found in fetal deaths include 

many rarely, or never, seen in live births, e.g., triploidy, 
tetraploidy, and trisomy for most whole chromosomes.  Monosomy X, 
though occurring in only about 1 in 20 000 live births, occurs in 
about 7% of early fetal deaths; triploidy occurs with about equal 
frequency, as does trisomy 16, an anomaly never seen at term and 
compatible only with very rudimentary embryonic development 
(Warburton et al., 1980). 

    Chromosome abnormalities are found in about 7% of late fetal 
deaths (including still births) (Sutherland et al., 1978).  At 
birth, the proportion has been found to be 0.6% (Hook & Hamerton, 
1977).  About 90% of all recognized conceptuses with chromosome 
abnormalities terminate as fetal deaths. 

    Most recognized abnormalities involve numerical aberrations 
that are presumptive mutants.  Structural chromosome abnormalities 
make up only about 5% of the chromosome abnormalities seen in fetal 
deaths (Warburton et al., 1980).  About 40% of structural 
abnormalities found in fetal deaths or at amniocentesis are  de novo 
mutational events:  the rest are inherited from a carrier parent 
(Jacobs, 1981; Hook et al., 1984). 

4.2.2.  Relevance and limitations

4.2.2.1.  Studies of induced abortions

    Chromosome studies of induced abortions are a useful source of 
data.  Such investigations are, of course, only possible where 
induced abortion is a legal option.  Advantages are that the 
frequency of chromosomal abnormalities is still high at the point 
in gestation when most induced abortions are performed, that the 
specimens are likely to be viable in culture, and that the 
procedure can be scheduled for collection purposes.  A disadvantage 
is that it is likely to be more difficult to obtain specimens from 
the controls than from the exposed population.  If, for example, 
exposed women were more likely than controls to provide specimens 
of early abortions, the unadjusted rate of chromosome abnormalities 
would appear, incorrectly, to be higher. 

4.2.2.2.  Studies of fetal deaths

    Studies in which the products of conception from fetal deaths 
are examined cytogenetically are likely to be the most productive 
in terms of the proportion of chromosome abnormalities that are 
detected.  However, such studies are difficult because of problems 
in specimen retrieval, culture failure, and the high cost of the 
tissue culture procedures required.  Usable specimens will be 
obtainable from only a portion of cases, even with the best 
retrieval systems, since viable fetal tissues are sometimes not 
present at the time of expulsion.  Organization of retrieval 
systems can be a challenge, even in such ideal situations as a 
teaching hospital, and are extremely difficult outside a medical 
setting.  Through special education, attempts can be made to 
collect specimens from women having early abortions, where the 
tissue is passed at home, and medical attention may not be sought.  

In New York City, an interview study suggested that 40% of women 
having a first trimester fetal death did not seek medical attention 
(Kline et al., 1981).  Factors affecting the probability of 
specimen retrieval must always be examined to rule out biases that 
would affect the outcome of the study. 

    When cytogenetic analysis is performed, further case loss 
occurs because not all will be successfully karyotyped.  This might 
produce differences in studies done in different laboratories or at 
different times.  Thus, cases and controls should be studied at the 
same time, in the same laboratory.  Ideally, the laboratory 
carrying out the study should not know if a specimen is from a 
control or a case. 

    One further problem in interpreting studies of fetal death is 
the possibility that an environmental agent might influence not 
only the rate of occurrence of a genetic abnormality, but also the 
probability that a conceptus with an abnormality can survive to a 
particular point in gestation.  If an exposure reduces the 
viability of a conceptus with a chromosome abnormality, such as 
monosomy X, so that it is lost even earlier in gestation, before 
recognizable pregnancy, a reduction in that anomaly will be seen in 
all recognized conceptuses.  An exposure that postpones the fetal 
death of a conceptus with abnormality from before the usual 
recognition of pregnancy to later in gestation would result in 
an increased proportion of detected abnormalities. 

4.2.2.3.  Studies of prenatal diagnosis specimens

    In some locations, amniocentesis for prenatal diagnosis is 
widely available.  This procedure is usually done at 16 - 20 weeks 
of gestation.  In such areas, many women exposed to putative 
mutagens might seek such procedures, though a much smaller 
proportion of controls might do so.  While data available from 
such an outcome should be used in any analysis, this cannot be 
regarded as a plausible sole source of pertinent data on chromosome 
abnormalities.  It should be noted that, if all women in a 
population undergo amniocentesis, the expected proportion of 
chromosomally abnormal fetuses is 1%. 

4.2.2.4.  Studies of live births

    Studies of live-born infants and older children are easiest to 
undertake, and cheapest to perform.  However, because only a small 
proportion would be expected to be affected (0.6%), a relatively 
large-scale study would have to be undertaken to detect a 
statistically-significant increase in the proportion affected.  A 
sample of 25 000 births is needed to detect a doubling of the 
trisomy rate at birth ( P < 0.05)a. 
                                                              
-------------------------------------------------------------------
a For the purposes of discussion, cases with an extra sex
 chromosome have been included.

    Some inferences are possible in relation to large exposed 
populations, based, in part, on phenotypic evaluation.  Cytogenetic 
study for mutation investigation might, for example, be carried out 
only on children with major malformations at birth, i.e., about 2%.  
In any event, such studies would be indicated on this group of 
children for clinical reasons.  This approach would reveal 
primarily only unbalanced autosomal abnormalities, about 1/3 - 1/2 
of all chromosomal abnormalities in live births (both structural 
and numerical abnormalities would be detected).  Such a restriction 
in a cytogenetic study would result in perhaps 10 - 20% of those 
evaluated being found to have an abnormality.  This reduces 50-fold 
the total number of cytogenetic studies required in the population, 
but does not avoid the need for a large original exposed population 
from which the selected group with malformations would be drawn. 

4.2.2.5.  Studies of indicator phenotypes of chromosomal abnormalities

    Inferences in large populations might be possible on the basis 
of simple enumeration of Down's syndrome individuals, 98% of whom 
are likely to be trisomic or carry a  de novo chromosome 
rearrangement.  The expected frequency is about 1 in 1000 births.  
Similarly, cases of Patau syndrome and Edwards syndrome (resulting 
from 47, +13 and 47, +18, respectively) might also be enumerated.  
The expected frequencies, however, are much smaller (each about 1 
in 10 000 live births (Hook & Hamerton, 1977), and the phenotypes 
are not as useful an indication of karyotypic abnormality. 

    Some conclusions on the probability of karyotypic abnormality 
can be reached through morphological examination and classification 
of specimens from fetal death.  A useful classification scheme, 
suggested by Byrne (1983), provides categories reflecting the 
degree of organization of development, as well as developmental 
age.  These categories range from an "empty sac" with no visible 
embryo, to an apparently normal fetus greater than 30 mm.  Studies 
associating specimen morphology with karyotype have shown that, 
while about 1/2 of the most poorly-developed specimens have a 
chromosome abnormality, less than 2% of fetuses greater than 30 mm 
in length and with no visible malformations externally, will have 
a chromosome abnormality (Byrne, 1983).  Thus, if resources are 
limited, such specimens might be left unstudied without much loss 
of information. 

    True hydatidiform moles represent a category of conception 
where the chromosome constitution is usually 46,XX, reflecting 2 
identical male haploid complements in an egg with no maternal 
chromosomes.  Although other rare karyotypes occur, single 
morphological examination is sufficient to indicate the chromosome 
abnormality with over 98% accuracy. 

4.2.3.  Procedures

4.2.3.1.  Fetal specimens

    In a prospective study, women can be provided with sterile 
containers containing a balanced salt solution for collection of 
specimens from early fetal deaths.  Later fetal deaths are likely 

to reach medical attention, and collection systems must be 
organized at likely treatment areas.  Specimen containers should 
also be available at such possible collection points as clinics, 
emergency rooms, and wards. 

    (a)   Morphological examination

    Specimens should be examined externally and described in a 
classification scheme that reflects the degree of development 
as well as developmental age.  Fetal autopsies can be performed 
on specimens of 30 mm or more.  A careful description of the 
classification scheme must be provided, with photographs, whenever 
possible, of abnormal specimens. 

    (b)   Karyotyping

    Successful cultures can be obtained, up to 5 days after 
expulsion, though such a delay is not recommended.  Thus, transport 
of samples over some distance is possible.  As the products of 
conception are almost never obtained in a sterile state, the use of 
culture medium for storage is not recommended, since it will 
encourage the growth of contaminants.  Specimens should be kept 
refrigerated (but not frozen), during storage or transport over 
long distances. 

    Specimens from induced abortions should be obtained in a 
sterile container from the operating room or clinic.  Care must be 
taken to prevent the usual routine fixation of such specimens. 

    A most important aspect of the procedure is the careful 
examination of the specimen to ensure that only fetal tissues are 
taken for culture.  Some specimens will contain only decidua, and 
cannot be used.  Any fetal tissue can be used for karyotyping; if 
available, fresh tissues from the embryo or fetus are most 
desirable; however, the actual embryo may be very small for the 
length of gestation, very macerated, or absent altogether.  In this 
case, fetal membranes (amnion and/or chorion), or placental villi, 
carefully dissected away from maternal tissues, may be used.  
Maternal cell contamination is an inevitable possibility in such 
studies, but evidence suggests that it is infrequent in experienced 
hands (Warburton et al., 1980; Hassold, 1982). 

    Fetal tissues may take from a few days to several weeks to 
reach the stage where they can be karyotyped.  Cover slip 
preparations with  in situ chromosome preparations allow faster 
karyotyping, but may not yield as large a number of analysable 
cells as cultures in flasks, which must be trypsinized before 
harvesting (Byrne, 1983). 

    Banded chromosome analysis should be carried out on all 
specimens, and on at least 10 cells from each culture analysed.  
Mosaicism is not uncommon among cultures from spontaneous 
abortions.  More cells must be counted if a non-modal cell, not 
accounted for by random loss, is found among the first 10 cells, 
e.g., if a normal cell is found in an otherwise trisomic culture, 

or a trisomic cell or a 45,X cell is found in an otherwise normal 
culture.  In general, the proportion of mosaics among all 
abnormalities detected, is expected to be small (Warburton, 1980).  
For the purposes of initial analyses, it is suggested that they be 
classified with non-mosaic aberrations with the same abnormal line. 

    Almost all instances of numerical chromosome abnormalities 
are presumably the result of a mutation in the most recent 
generation.  Thus, if monosomy, trisomy, triploidy, or tetraploidy 
is discovered, then study of the parents to confirm a mutation is 
not necessary, as the likelihood of mutation is perhaps 0.99.  
However, this is not the case for structural cytogenetic 
abnormalities.  At about 18 weeks of gestation, for instance, 
only about 40% of structural abnormalities are the result of a 
recent mutation, 60% being inherited from carrier parents.  Thus, 
if a structural rearrangement is observed, study of the parents 
should be undertaken to determine if this is the result of a 
mutation first manifest in the offspring. 

4.2.3.2.  Live births and other offspring

    Chromosome studies will normally be performed on PHA-stimulated 
lymphocyte cultures from specimens of peripheral blood.  Specimens 
should be collected as soon as possible after birth in order to 
minimize losses of neonatal deaths.  Cord blood can be used, and is 
easy to collect routinely. 

    Preservation of whole blood samples by deep-freezing (-70 C.) 
in DMSO will make possible a good retrieval of cells for chromosome 
analysis (or other kinds of studies) for up to a year (Nakagone et 
al., 1982).  Furthermore, specimens can be collected and stored 
before laboratory facilities have been organized for the study, 
and, if necessary, can be transported to a laboratory some distance 
away. 

    If a structural rearrangement is found, parental studies are 
necessary. 

4.2.3.3.  Detection of "indicator" phenotypes for germinal chromosomal 
mutations - trisomies

    Cases of Down's syndrome can be identified through medical 
records, vital statistics, registries, or education settings. 
Identification is likely to be incomplete through any of these 
routes, and care must be taken that both exposed and unexposed 
women are equally likely to be discovered. 

4.2.3.4.  Data presentation

    Results in those exposed should initially be presented 
according to broad categories of events (Table 5) and also 
within subcategories, when sample size allows, because different 
chromosomal abnormalities may reflect different etiological 
factors.  Trisomy 21 and trisomy 16 may well result from different 
mechanisms, for example, while increased triploidy might indicate 
increased dispermy. 

    Data should be presented on "conceptions" followed from 
detection of pregnancy, and results compared by category between 
exposed and control groups.  Results should be expressed as the 
proportion of either the exposed or control group with a specific 
abnormality.  Data should be stratified by maternal age, or other 
adjustment made for this variable, because of the very strong 
association of maternal age with trisomies (Hassold et al., 1980, 
1984).  In addition, exploratory analysis should be undertaken 
of other possible confounding variables such as ethnicity, 
socioeconomic status, cigarette smoking, and alcohol consumption.  
Any differences between exposed and control groups in these 
variables should be adjusted for in the analysis by sample 
stratification, or multivariate analyses.  Results should also 
be analysed according to whether the father, the mother, or both 
parents have been exposed.  If sufficient data are available by 
exposure category, a dose-effect relationship should be sought.  
With regard to structural rearrangement, cases in which one or 
both parents cannot be studied should be scored separately, and 
regarded as possible mutants.  The mutation rate for structural 
rearrangements should be reported as a range.  The minimum boundary 
excludes such cases as being of unknown status.  The maximum 
boundary includes such cases. 

    It should also be recognized that lack of an observed 
difference during the first few years after exposure does not 
preclude the possibility of late manifestations of effects.  In one 
study, it has been claimed that a radiation effect on trisomy 21 
frequency first manifested itself 10 years after exposure (Alberman 
et al., 1972). 

    A problem that may arise in the evaluation of results is that 
of missing data.  Pregnant exposed and control individuals may die, 
or withdraw before the outcome of the pregnancy is established.  
Moreover, if a fetal death occurs, tissue may not be collected and, 
if collected, the specimen may not include fetal tissue.  Even if 
fetal tissue is set up in culture, the karyotyping procedures may 
not be successful.  In experienced laboratories, the proportion of 
successful cultures has varied from 65% to 90% (Warburton, 1980). 
Moreover, culture failures are likely to be preferentially higher 
for fetuses with chromosome abnormalities because the greatest 
proportion of culture failures occurs in specimens with poorly 
"organized" morphogenesis, which have been dead for some time  in 
 utero.  Among specimens cultured successfully, the proportion of 
cytogenetic abnormalities in specimens with poorly organized 
morphologies is at least 50%, at least twice as high as that of 
specimens whose morphogenesis is more normal (Byrne et al., in 
press).  Assuming that this occurs also among unsuccessful 
cultures, then those of unknown outcome will contain a higher 
proportion of chromosomally abnormal specimens than successful 
cultures. 

    Another problem arises if the effect of exposure to a suspect 
substance is not to increase the proportion of conceptuses with 
abnormalities but only to alter the proportion of conceptuses whose 
karyotypes can be determined.  If, for example, such a detection 

effect occurs selectively more often on fetuses of abnormal 
karyotype, then there will be a bias towards an apparent mutagenic 
effect of the substance. 

Table 5.  Types of germinal chromosomal abnormalities
-------------------------------------------------------------------
 Numerical Aberrations:  all presumed mutants

    Monosomy (almost entirely X monosomy)

    Trisomy  16
             21
             others

    Triploidya

    Tetraploidya

 Structural aberrationsb

    Unbalanced: Robertsonian

        deletions and rings
        markers and fragments
        other

    Balanced:

        Robertsonian
        inversions
        reciprocal
-------------------------------------------------------------------
a Most triploidy results from dispermy, and tetraploidy from 
  mitotic non-disjunction.  Thus, they might not be regarded 
  strictly as germinal cell mutations.
b Both parents must be shown to have normal chromosomes, before a 
  case can be  regarded as new mutant.

    For these reasons, inferences about differences between exposed 
and control populations (on the basis of comparison of the observed 
proportion of the abnormal among those studies) require explicit 
assumptions about the conceptuses of unknown karyotype.  The 
characteristics of this group, for example, the proportion of them 
among the total number of conceptuses in the sub-populations, their 
fetal pathology, and their maternal age association, should be 
investigated closely.  If there are striking differences in the 
nature of the conceptuses of unknown karyotype between exposed 
and unexposed populations, great caution is necessary in drawing 
conclusions from comparisons involving conceptuses of known 
karyotype. 

4.3.  Biochemical Approaches to Detecting Gene Mutations in Human 
Populations

4.3.1.  Biochemical methods for monitoring for gene mutations

    Two approaches have been used to study the proteins in 
offspring of selected individuals.  One is based on the detection 
of differences in charge, shape, or size of proteins by one- or 
two-dimensional electrophoretic techniques.  In the other, 
quantitative enzyme activity measurements are used to detect enzyme 
variants associated with either the loss of enzyme function or the 
absence of the enzyme protein. 

4.3.1.1.  One-dimensional electrophoresis

    The introduction of new electrophoretic technology has 
increased the proportion of variants detectable at the protein 
level and the number of gene products that can be studied.  Using 
these techniques, it has been shown that, in contrast to previous 
assumptions that only one-third of the amino acid substitutions 
could be detected, many neutral change substitutions can also be 
detected.  This reflects the alterations in higher-order protein 
structure associated with these later substitutions, which change 
the charge distribution on the molecule.  Experimental data suggest 
that 80 - 90% of all amino acid substitutions may be detectable 
with refined techniques (Johnson, 1976, 1977; Ramshaw et al., 1979; 
Fuerst & Ferrell, 1980).  The use of narrow-range pH gradients for 
isoelectric focusing has also increased the detection and 
resolution of the variants at many loci (Altland et al., 1979; 
Chramback et al., 1980). 

    The number of gene products that can be studied by 
electrophoretic analysis depends on the availability of techniques 
to demonstrate selectively the position of the allele products 
following electrophoretic separation.  In addition to previously 
developed protein specific stains (Harris & Hopkinson, 1976), 
immunological techniques (e.g., immunoblotting and immunofixation) 
(Chapuis-Cellier et al., 1980; Tsang et al., 1983) have become more 
widely used, especially with the recent modifications.  One 
additional approach has involved several sequential steps of 
electrophoresis to separate the protein of interest from the 
remaining proteins, so that standard protein-staining techniques 
can be used for visualization (Altland & Hackler, 1984). 

    The results of many studies involving genetic typings of large 
numbers of individuals, either for population structure studies 
or in the context of the development of mutation monitoring 
programmes, have demonstrated the feasibility of sizeable screening 
efforts using blood samples as the source of material (Harris et 
al., 1974; Neel et al., 1980a,b).  The products of at least 40 - 50 
loci can be routinely screened by electrophoresis with either 
starch and/or polyacrylamide as the support medium.  For many 
proteins, either support can be used, with polyacrylamide having 
the advantage of requiring smaller sample aliquots and potentially 
increased resolution.  Isoelectric-focusing is feasible for many of 

these proteins, especially when the potential for increased 
resolution of some proteins is considered.  Electrophoretic 
techniques have been used in several human mutation screening 
programmes (Neel et al., 1980a,b; Altland et al., 1982a,b).  
They have also been used for estimating the mutation rate in mice 
(Pretsch & Narayanan, 1979; Johnson & Lewis, 1983; Neel, 1983) and 
 Drosophila (Mukai & Cockerham, 1977; Voelker et al., 1980). 
Electrophoretic mobility variants have been identified and the 
genetic transmission of the trait to subsequent generations has 
been confirmed. 

4.3.1.2.  Two-dimensional electrophoresis

    The high resolution two-dimensional (2-D) electrophoretic 
techniques described by O'Farrell (1975) with many subsequent 
modifications (Dunn & Burghes, 1983a,b) has been used to separate 
the large array of proteins found in cells and tissues.  The 
positions of the several hundred proteins in many types of samples 
can be visualized by protein staining, especially when the highly 
sensitive silver stains (Merril et al., 1981; Sammons et al., 1981) 
or isotopic labelling of proteins in nucleated cells followed by 
autoradiography or fluorography (McConkey et al., 1979; Thomas et 
al., 1984) are used. 

    The ability of the 2-D system to resolve electrophoretic 
mobility variants has been shown by Warner et al. (1982).  In 
other studies, the level of heterozygosity for polypeptides has 
been examined in several types of human cells and tissues (McConkey 
et al., 1979; Walton et al., 1979; Comings, 1982; Hamaguchi et al., 
1982).  More recently, extensive studies have been reported on the 
level of genetic variation in plasma proteins and other blood 
proteins (Rosenblum et al., 1983, 1984).  One advantage of the 2-D 
approach over the single-dimension electrophoresis techniques is 
the ability to study the gene products of at least several  hundred 
loci in each sample (individual).  The 2-D electrophoresis 
technique has been used to estimate the induced mutation rate 
in mice (Klose, 1979; Marshall et al., 1984). 

4.3.1.3.  Enzyme activity

    Much of the background to the search for enzyme deficiency 
variants is derived from the study of metabolic diseases.  The 
extensive lists of inborn errors of metabolism indicate not only 
the prevalence of genetic events associated with loss of enzyme 
function but also the usefulness of quantitative enzyme assays as a 
tool for studying this class of genetic events (Beutler, 1979; Kahn 
et al., 1979; Miwa, 1979). 

    The search for mutational events resulting in loss of function, 
owing to either loss or nonfunctionality of the gene product, is 
complementary to the electrophoretic assays, in that it detects 
genetic events, not normally detectable by standard electrophoretic 
assays.  Current data suggest that inherited rare enzyme deficiency 
variants occur more frequently in human populations than rare 
electrophoretic variants (Mohrenweiser, 1981; Mohrenweiser & Neel, 

1981; Satoh et al., 1983).  In addition, mutations resulting in 
loss of enzyme function occur at least as frequently as 
electrophoretically identifiable mutations in mice and  Drosophila,  
following exposure to mutagenic agents, either radiation or 
chemical (Racine et al., 1980; Charles & Pretsch, 1981; Johnson & 
Lewis, 1981). 

4.3.1.4.  Other biochemical approaches

    Alterations in base sequence can be analysed using the 
restriction enzyme mapping techniques (Botstein et al., 1980; 
Skolnick & Francke, 1982; Southern, 1982).  Most of the current 
effort, in addition to defining gene structure, has been directed 
towards identification of polymorphisms at restriction enzyme sites 
and subsequent linkage analysis, but similar techniques could be 
employed for screening for mutational events (Beaudet, 1983; Cooper 
& Schmidtke, 1984 for recent listings of cloned human DNAs).  
Although it is clear that it is now technically possible to 
detect base sequence changes, incorporating this approach into 
a monitoring protocol must await further developments that will 
facilitate the generation of the quantity of data necessary for a 
mutation screening programme. 

    In addition to the sensitivity of the method, another potential 
advantage of an approach that studies mutations by examining the 
DNA directly, is the ability to obtain very large quantities of 
data from each individual.  Thus, this method can be employed for 
the study of small populations. 

4.3.2.  Analytical strategy and methodological considerations

    Biochemical analyses of the samples collected from the F 
population (offspring of exposed individuals) will be carried out 
usually in a number of laboratories while population and health 
data will be derived from various sources.  It is unlikely that all 
laboratories will be able to complete the entire battery of assays 
or that the techniques will be identical in each laboratory or for 
each study, thus each laboratory should receive samples from both 
exposed and control groups.  The key for the successful completion 
of the analytical aspects of any study will be for each laboratory 
to establish and maintain high standards of technical competence 
and performance.  Provision should be made for the exchange of 
samples, as this will increase the amount of data obtained from 
each individual and also serve to confirm the existence of 
interesting observations.  The basic technical procedures for 
electrophoresis and enzyme activity assays, are available.  These 
techniques form the basis for establishing a new laboratory effort.  
The methods described in each section are current routine 
laboratory procedures; thus, the biochemical techniques for 
monitoring germinal mutations in a human population are available. 

4.3.2.1.  One-dimensional electrophoresis

    Approximately 40 - 50 blood proteins have been examined for 
electrophoretic variation in many laboratories including the 
laboratories at the University of Michigan, USA (Neel et al., 

1980a) and in Japan at the Radiation Effects Research Foundation 
(RERF) (Neel et al., 1980b).  Approximately half of the proteins 
studied at the University of Michigan use polyacrylamide as the 
support medium while the laboratory at RERF relies more on starch 
as the support medium.  Many of these proteins are also being 
studied by isoelectric focusing techniques as a component of 
mutation screening programmes in other laboratories (Altland et 
al., 1982a,b). 

    Techniques are available for the electrophoretic analysis 
of approximately 20 proteins, when the sample is obtained from 
dried blood (Altland et al., 1979, 1982b; Metropolitan Police 
Forensic Science Laboratory, 1980).  The general principals of 
electrophoretic separation and isozyme (protein) identification 
using samples collected as dried stains are as described for other 
blood samples. 

    High-speed one-dimensional electrophoretic screening 
procedures have been developed for vertical polyacrylamide gel 
electrophoresis, flat bed isoelectric focusing, and the sequential 
combinations of these procedures.  With these techniques, using 
multiple sample handling procedures, 96 samples are analysed 
simultaneously (Altland et al., 1982a).  This is particularly 
useful when large numbers of samples are being analysed. 

    An electrophoretic mobility variant is identified by an 
alteration in the standard profile that is consistent with the 
appearance of a new allele product.  This decision process must 
include previous knowledge of the protein subunit structure, 
chromosomal location (e.g., hemizygous males) and factors such as 
age, sex, etc., which are important in interpreting the data.  The 
new protein should have characteristics that indicate that it 
differs from the original protein by an amino acid change.  It 
is important to confirm, using as many additional techniques as 
possible, that any new variant is not the result of an artifactual 
or secondary alteration in protein structure.  Ultimately, such 
confirmation would require amino acid or DNA sequencing studies.  
This is important because of the low probability of obtaining data 
to confirm genetic transmission of a new mutation in human 
populations.  The proteins that are being studied in the above-
mentioned studies have been selected because of the low frequency 
of artifactual findings.  But, as with any technique, it is 
important for each laboratory to undertake appropriate control 
experiments. 

4.3.2.2.  Two-dimensional electrophoresis

    The 2-D electrophoresis techniques, used for human mutation 
monitoring studies, have been described by Neel et al. (1983, 1984) 
and modifications of the technique of O'Farrell (1975), by Anderson 
& Anderson (1977) and Anderson et al. (1980).  The blood sample is 
fractionated into various components, e.g., plasma, erythrocyte 
membranes, haemolysate, platelet, etc., the proteins of which are 
then analysed.  The proteins in the cell fraction to be studied are 
solubilized in 4 - 8 M urea, which dissociates the multimeric 

proteins into component subunits.  The component polypeptides are 
separated on the basis of charge by isoelectric-focusing in the 
first dimension.  Electrophoresis in the second dimension is in 
the presence of sodium dodecylsulfate, so that the proteins are 
separated in this dimension on the basis of apparent molecular 
size.  The sensitive silver-based staining techniques are most 
often used for identifying the positions of the separated 
polypeptides (Merril et al., 1981; Sammons et al., 1981).  
Analysis of the results of 2-D electrophoresis can be scored by 
visual inspection (Hanash et al., 1982; Rosenblum et al., 1983, 
1984), though recently, significant progress has been achieved 
in automating and computerizing the analysis of these gels 
(Skolnick, 1982; Skolnick et al., 1982; Skolnick & Neel, in press).  
Computerized analysis can increase the amount of data obtained from 
each gel (the number of locus tests completed for each sample) and 
also reduce the workload at this step. 

4.3.2.3.  Enzyme activity

    The methods for detecting enzyme deficiency variants, defined 
as a level of enzyme activity that is less than 65% of the mean 
for the population and more than 3 standard deviation units below 
the mean, have been described by Fielek & Mohrenweiser (1979), 
Mohrenweiser & Fielek (1982), and Mohrenweiser (1983a).  Additional 
or alternative methods have been described by Bulfield & Moore 
(1974), Krietsch et al. (1977), Eber et al. (1979), and Satoh et 
al. (1983).  The general analytical strategy for analysing large 
groups of samples has been outlined by Mohrenweiser (1983b).  
Enzymes that are:  (a) present with reasonable levels of activity; 
(b) primarily the gene product of a single locus; (c) not 
influenced by environmental factors (e.g., nutritional status); 
(d) easy to assay; and (e) exhibit little total variation among 
individuals, are good candidates for inclusion in this approach 
(Mohrenweiser, 1982; 1983a,b).  The gene products of at least 
12 - 14 loci can be routinely monitored for the presence of null 
variants in erythrocytes. 

    Each of these biochemical approaches either has been, or is 
being, used to obtain data for the estimation of radiation-induced 
mutation rates in human populations.  Detailed techniques are 
available for each component in this section.  The most significant 
problem is obtaining the commitments (funding, study and control 
populations, etc.) necessary to initiate a major mutagenic 
study.  Obtaining agreements on general approaches, necessary 
standardization of technical aspects, and scoring of significant 
events is a lesser problem. 

4.3.2.4.  Sample acquisition and storage

    There are two general strategies for sample acquisition.  The 
first involves obtaining a large volume (up to 20 ml, although 5 ml 
is an adequate sample) of whole blood.  All members of a nuclear 
family are sampled to the maximum extent possible, so that the 
material for family studies to determine heritability of the 
characteristic is routinely available.  Experience has shown that 

when the gene products of 40 - 50 loci are studied, family studies 
are necessary in at least 10% of the families, even when only a 
single child per family is studied.  With the number of loci 
studied with the 2-D electrophoresis technique, obviously the 
percentage of family studies increases to include almost every 
family.  In either case, when the number of locus tests per sample 
or family unit increases to this level, the work involved in 
recontacting families for additional samples becomes a considerable 
task. 

    The blood sample is fractionated into at least plasma, buffy 
coat, and erythrocyte fractions.  Each part is subdivided into a 
fraction for routine analysis as well as a fraction for long-term 
storage and potential additional analysis.  An aliquot of red 
cells is stored in glycerol-sorbitol solution and retained in 
liquid nitrogen for blood group analysis in the event of a 
potential mutation being observed.  The "white cell" component 
could be fractionated into several components, e.g., platelets, 
polymorphonuclear leukocytes, etc., which could be used as samples 
for 2-D electrophoretic studies.  Alternatively, either all or 
fractions of the lymphocyte samples could be retained as a source 
of DNA for future restriction enzyme mapping studies.  Another 
alternative to the above protocol would be to store the white cell 
fraction under such conditions that intact, viable cells could be 
recovered and, with appropriate cell culturing techniques, the 
number of cells expanded for use in future studies.  In certain 
situations, where a specific population is of special interest and 
the potential for follow-up studies is limited, it may be advisable 
to establish and store transformed lymphoblast cell lines in order 
to maintain the genetic information contained in this population 
for future studies.  The second acquisition strategy involves 
obtaining a smaller volume of sample, often in the form of a dried 
blood stain, which is usually obtained in conjunction with other 
studies, usually with new-born metabolic screening programmes. 
Procedures have been established for screening for gene mutations 
at 20 loci using a blood sample obtained on a Guthrie Test card.  
With these techniques, 300 samples or 6000 locus tests per day can 
be completed (Altland et al., 1982a,b; Vogel & Altland, 1982).  
As the new-born metabolic screening programme in many countries 
encompasses the total new-born population, this strategy appears 
useful for establishing a surveillance progamme for determining the 
human germinal mutation rate.  The number of locus tests from each 
individual is somewhat less than with the larger samples, thus the 
frequency of family studies is less, though obviously as the number 
of tests per sample increases, it may be important, whenever 
possible, to obtain concurrent parental samples. 

    It has been suggested that children with congenital birth 
defects could be of special interest for monitoring studies because 
of a high gene mutation rate in such children (Dubinin & Altukhov, 
1979).  However, study has not been able to confirm a similar 
increased or high mutation rate among a group of children with 
apparently similar congenital birth defects (Neel & Mohrenweiser, 
1984).  The usefulness of the approach of targeting specific F 
individuals within a population for detailed study, rather than 
all F individuals, needs further study. 

4.3.3.  Data management

    Neel et al. (1979) have developed a computer-based data 
management system for handling the types and volume of data 
generated in a population monitoring programme.  This includes 
a sample identification system with a letter designation for the 
geographical site of sample acquisition, a number code for family 
identification, and a single digit indicating family position.  
This identification, which is the first entry into a computerized 
data management system, is used for identification of all data 
generated, thus it is used for linkage to the electrophoretic 
and enzyme activity files, which are additional components of 
the computer-based data management system.  It is linked to family 
identification (e.g., name) only when follow-up is necessary, in 
order to maintain confidentiality of records.  A similar data 
management system is currently in operation at RERF.  It is 
important that the data-management system should be complete 
and compatible with all aspects of the study (section 2). 

4.3.4.  Considerations in screening for germinal mutations

4.3.4.1.  Sample size

    The logistic problems associated with a germinal mutation 
screening programme are significant because of the scale of the 
effort.  The magnitude of the effort reflects both the rarity of 
mutational events (background mutation rates of 105 - 106) and 
the need for statistical methods to ascertain generally small 
(10 - 100% increase) differences between background and induced 
mutation rates.  The sample sizes necessary for any of these 
approaches to monitoring depend on the baseline rate of the effect 
under consideration and the magnitude of the increase in mutation 
rates that can be left undetected. 

    Vogel (1970), Neel (1980), and Vogel & Altland (1982) have 
calculated the sample sizes necessary for a research design 
involving the contrasting of 2 samples.  The samples can be either 
collected consecutively, as when studying the possibility of a 
changing mutation rate in a defined population, or simultaneously, 
when contrasting a control group of children with a group at high 
risk of mutation because of the occupational or other exposure 
of their parents.  The minimum sizes of the 2 samples are 
determined by the magnitude of the errors, type I (alpha) and type 
II (phi) that are considered acceptable.  The minimum increase in 
mutation rate that is to be detected (or the maximum to be deemed 
insignificant) also influences the sample size necessary to detect 
an increased mutation rate.  Most geneticists would agree that 
any monitoring programme should detect an increase of 50% above 
background.  Using type I and type II error limits of 0.05 and 0.20 
and a mutation rate of 1 x 10-5, it would require some 7.5 million 
observations (Neel, 1980).  There is considerable disagreement on 
how much effort should be devoted to detecting smaller increases in 
mutation rate.  Given the exponential nature of the relationship, 
detecting an increase of 20% involves 2 samples 5 times as large 
as those required to detect a 50% increase.  It is obvious that, 

although the set of assumptions and limits may change, generating 
a data base necessary for estimating changes in human mutation 
rates (or finding that at some limit of power, the rate has not 
changed) requires many observations and considerable analytical 
effort. 

    With the biochemical approach, in which many proteins are 
examined in any given child, and with 2 loci at risk of mutation 
for each polypeptide included in the study, the number of children 
will be only a small fraction of the number of determinations.  
For example, in a pilot study (Neel et al., 1980a), 50 proteins 
were examined for mutations influencing electrophoretic behaviour.  
Thus, the demonstration of a difference of 50% between the 2 groups 
could be accomplished with 2 samples of approximately 90 000 
children (it is assumed that each protein of a child is an 
independent test of mutation and that each child constitutes 2 
tests of each protein, i.e., both parents exposed).  With the 
two-dimensional gel electrophoresis method, which could permit 
simultaneous monitoring of 500 (or more) different proteins of 
the blood serum, erythrocytes, and/or leukocytes, with the same 
assumptions as above, the  size of the 2 samples is reduced to 
approximately 7500 individuals. 

4.3.4.2.  Distinction between "true" and "apparent" mutations

    Whenever a genetic trait is encountered in a child, neither 
of whose parents is affected, the possibility that it is due to a 
discrepancy between legal and biological parentage rather than the 
result of mutation must be considered.  This is less likely when 
the mutation involves a major chromosomal abnormality rather than 
a biochemical marker, because individuals carrying the former are 
often sterile or have a severly reduced life expectancy.  With the 
sentinel phenotypes or biochemical variants, when an apparent 
mutant is encountered it is always necessary to carry out extensive 
genetic typings on both parents, and the child.  At present, with 
the battery of test traits usually available, including enzymes, 
blood groups, and HLA, a child whose legal father is not the 
biological parent can be detected with an assurance approaching 
98%.  Thus, in a situation characteristic of many countries, such 
as the USA, where 2 - 3% of the children born in wedlock have 
fathers other than the legal one and where variants of the type 
under consideration have a frequency of 2 - 4/1000 examinations, 
"false" mutations (i.e., undetected parentage exclusions) will have 
a frequency not very different from the present expectation for 
mutation. 

    It should be pointed out that recent developments, which could 
be used in a study such as this mutation protocol, are also useful 
for paternity studies.  Many gene products studied using two-
dimensional electrophoretic techniques are characterized by 
genetic polymorphisms useful in parentage studies, resulting in 
an improvement in the ability to detect discrepancies between 
legal and biological parentage.  The study of DNA restriction 
site polymorphisms will also decrease the frequency of undetected 

non-parentage, thus the probability of an apparent mutation being 
due to non-parentage should become very small with the increased 
number of loci that can now be studied. 

    In the final analysis, there may always be some uncertainty as 
to whether a particular apparent mutant is due to an undetected 
discrepancy between legal and biological parentage.  If the 
control population has been properly chosen, however, the amount 
of non-parentage could be demonstrated to be the same in the two 
populations, so that this factor would be a constant diluent in the 
2 samples.  Furthermore, with a statistical approach, recently 
developed by Rothman et al. (1981), under suitable conditions, 
probabilities can be developed for parentage, and apparent 
mutations ranked according to these probabilities. 

4.3.4.3.  Implementation of gene mutation screening programmes

    Final decisions regarding analytical strategy and technical 
approaches, beyond the points described above, must await further 
information regarding the population of interest.  The magnitude 
of the study helps determine the number of laboratory centres 
necessary to complete the study within a reasonable time.  
Similarly, the size of the study population (and estimated mutation 
rate) will dictate the amount of data that must be obtained from 
each individual studied for the generation of a statistically 
significant data base.  The size of the study population (as well 
as the age of the individuals from whom blood is obtained) will 
also need to be considered when blood fractionation and allocation 
procedures are finalized.  Furthermore, techniques are continually 
being refined and new approaches being developed, and it is unwise 
to dictate the specific technical details until such time as the 
commitment to proceed with a study has been made.  At this point, 
laboratories should be able, with relative ease, to define the 
technical details for the laboratory and analytical protocols. 

4.3.5.  Summary

    In many respects, with the limitations of the current state of 
knowledge regarding the detection and subsequent consequences of 
gene mutations, all practical approaches to estimating the germinal 
mutation rate in human populations, following exposure to a 
putative mutagen, should be considered to be complementary.  
This is important as each technique differs in sensitivity, detects 
different types of genetic damage or end-points, and has different 
degrees of relevance for estimating potential health effects.  
Therefore, it is logical in a collaborative monitoring exercise to 
use all of the approaches in order to obtain the maximum possible 
amount of data. 

    The technical aspects of a collaborative effort to use 
biochemical methods to monitor selected human populations for 
germinal mutation frequencies are available.  Such programmes have 
been conducted in the Federal Republic of Germany (Altland et al., 
1982b), Japan (Neel et al., 1980b; Satoh et al., 1983), and the USA 
(Neel et al., 1980a; Mohrenweiser, 1981).  Thus, feasibility has 

been tested at a significant level of effort.  There is no 
technical reason for not expanding such an effort to other 
populations of interest. 

4.4.  Sentinel Phenotypes

4.4.1.  Introduction

    A sentinel phenotype is a clinical disorder that occurs 
sporadically as a consequence of a single, highly penetrant 
mutant gene, which is a dominant or X-linked trait of considerable 
frequency and low fitness, and is uniformly expressed and 
accurately diagnosable with a minimal clinical effort, but a 
relatively high probability of ascertainment (Mulvihill & Czeizel, 
1983). 

    A new dominant mutation can manifest itself at any time in the 
human life-span.  In practice, it is worth distinguishing three 
separate groups of sentinel phenotypes:  sentinel anomalies, 
easily observed at birth and generally recorded in birth defects 
registries (Table 6); sentinel childhood tumours, which occur in a 
well defined age-group and are recorded in childhood tumour 
registries; and genetic disorders with delayed onset, which are 
readily diagnosed and recorded in various types of genetic disease 
registries (Table 7).  For practical reasons, sentinel anomalies 
are the easiest to handle. 

4.4.2.  Basis of method

    Certain essential requirements for the surveillance or 
monitoring of sentinel anomalies include: 

    (a)   Sensitivity and specificity

    Theoretically, all dominant phenotypes might be suitable for 
these purposes.  McKusick (1983) catalogues 1828 autosomal dominant 
traits in man.  These probably occur at a total birth prevalence 
of 1% (Matsunaga, 1982).  Nearly half of autosomal dominant 
disorders may be caused by new mutations (Holmes et al., 1981).  
Nevertheless, at present, no single dominant phenotype suits all, 
or nearly all, the criteria of the above definition.  However, the 
22 anomalies listed in Table 6 comply satisfactorily.  The main 
problems are the validity of a diagnosis and the relatively 
uncommon occurrence of all these candidate anomalies.  If the 
baseline prevalences are too low, both the specificity and 
sensitivity of population screening are decreased (Hook, 1981b). 

    (b)   Reliable and accurate diagnosis

    A sentinel phenotype must be clearly distinguishable from 
related disorders.  However, each of the anomalies in Table 6 has 
features that overlap with so many other anomalies that highly-
trained clinical experts must be involved in the diagnosis.  In 
general, these well-known genetic anomalies have a considerable 
genetic heterogeneity.  In addition, genocopies and sometimes 

phenocopies exist for all sentinel anomaly candidates.  The task 
is to make a precise nosological diagnosis, i.e., to identify the 
effect of the specific locus or sometimes of the given allele of 
the specific locus. 

Table 6.  Candidate sentinel anomalies and the figures of the
Hungarian Programme per 10 000 births
-------------------------------------------------------------------
Congenital anomaly                            McKusick  Expected
(s = syndrome)                                number    rate
                                                        (1983)
-------------------------------------------------------------------
Achondroplasia                                10 080    1.08            
Acrocephalosyndactyly type I (Apert s.)       10 120    0.07           
Acrocephalosyndactyly type V (Pfeiffer s.)    10 160    0.02           
Aniridia, isolated                            10 620    0.11           
Van der Woude s.                              11 930    0.02           
Cleidocranial dysplasia                       11 960    0.04           
Contractural arachnodactyly                   12 105    0.04           
Crouzon s.                                    12 350    0.11           
EEC s.                                        12 990    0.04           
Holt-Oram s.                                  14 290    0.07           
Treacher-Collins s.                           15 450    0.04           
Moebius s.                                    15 790    0.04           
Nail-patella s.                               16 120    0.04           
Oculo-dento-digital (ODD s.)                  16 420    0.02           
Osteogenesis imperfecta, type I               16 620    0.22           
Polysyndactyly preaxial IV.                   17 420    0.22           
Split hand and/or foot, typical               18 360    0.15           
Spondyloepiphyseal dysplasia cong.            18 390    0.07           
Thanatophoric dwarfism                        18 760    0.04           
Whistling face (Freeman-Sheldon s.)           19 370    0.02           
Incontinentia pigmenti (Bloch-Sulzberger s.)  30 830    0.11           
Oral-facial-digital s. (Gorlin-Psaume s. or   31 120    0.02           
 OFD I.)                                   
-------------------------------------------------------------------
Total                                                   2.59
-------------------------------------------------------------------
Cataracta                                     11 620    0.71
Ptosisa                                       17 830    0.66
-------------------------------------------------------------------
Grand total                                             3.96
-------------------------------------------------------------------
a Excluded from the final list because ptosis was rarely 
  ascertained and the nosological diagnosis of cataract was 
  impossible.


   
Table 7.  Candidate sentinel phenotypes without reliable nenonatal
manifestations
--------------------------------------------------------------------
Disorder                                      McKuskick  Inheritance
                                              number
                                              (1983)
--------------------------------------------------------------------
Amelogenesis                                  10 450     ADa           
Exostoses, multiple                           13 370     AD            
Fibrodysplasia ossificans progressiva         13 510     AD            
Marfan's syndrome                             15 470     AD            
Myotonic dystrophy (Steinert disease)         16 090     AD            
Neurofibromatosis (Recklinghausen disease)    16 220     AD            
Polycystic kidneys                            17 390     AD            
Polyposis coli, I (familial)                  17 510     AD            
Polyposis intestinal, II (Peutz-Jeghers       17 520     AD            
 syndrome)                                                      
Polyposis intestinal, III (Gardner syndrome)  17 530     AD            
Retinoblastoma (hereditary)                   18 020     AD            
Tuberous sclerosis                            19 110     AD            
von Hippel-Lindau syndrome                    19 330     AD            
Waardenburg syndrome                          19 350     AD            
Wilms' tumour (hereditary)                    19 407     AD            
Haemophilia A (Classical)                     30 670     XRb           
Haemophilia B (Christmas disease)             30 690     XR            
Muscular dystrophy                            331 020    XR            
 (Duchenne type)                                                 
Martin-Bell                                   30 955     XR            
--------------------------------------------------------------------
a AD = autosomal dominant.
b XR = X-linked.

    (c)   Complete ascertainment

    In countries where most births occur in hospitals and there are 
neonatal check-ups, the easily-recognized congenital anomalies of 
autosomal dominant origin can be used as sentinel anomalies, 
particularly if a population-based registry or surveillance of 
congenital anomalies is functioning. 

    (d)   The knowledge of pedigree data

    Since only a new occurrence of sentinel phenotype in a family 
is a mutation, obtaining data from and/or clinical examination 
of both parents is necessary to verify that neither is affected.  
In practice, it is necessary to obtain clinical data on the 
grandparents in order to exclude the possibility of non-penetrance 
in the parents. 

    (e)   Possibility of further action

    After a significant increase in the rate of occurrence of one 
or more mutations, it is essential to initiate an analytical 
epidemiological study to identify the responsible mutagens and to 
achieve the ultimate goal, i.e., prevention of further genetic 
damage. 

4.4.3.  Relevance and limitations

    The theoretical advantage of using sentinel phenotypes is 
considerable.  When a sentinel anomaly or sentinel childhood tumour 
is recognized in an infant or child, in the absence of an affected 
parent, there is little question that a germinal mutation has 
occurred. 

    From the practical point of view, there are three advantages: 

    (a)  Since sentinel phenotypes are diseases rather than 
         innocuous physical or biochemical traits or variants, the 
         affected person in a developed country will enter the 
         health-care system.  Thus, affected persons will almost 
         certainly be registered in a recording system for reasons 
         other than their potential use in surveillance.  Thus, the 
         so-called opportunistic approach is feasible (this term is 
         used to describe studies that are built on data, already 
         collected for other reasons). 

    (b)  In the case of sentinel anomalies and sentinel childhood 
         tumours, parents with an affected infant or young child 
         are likely to be willing to cooperate in additional 
         investigations, with regard, for example, to family or 
         environmental histories. 

    (c)  In sentinel phenotypes in which there is a low fitness, 
         there is a low probability of false paternity. 

    The main theoretical disadvantage is that the phenotype is not 
an immediate gene product, but a distant manifestation of altered 
DNA.  Hence, intervening developmental events could obscure the 
relationship between a mutagenic event and its expression as a 
sentinel phenotype.  In addition, dominant traits tend to have 
greater variation in clinical manifestations than most recessive 
and chromosomal syndromes.  The main practical difficulty is that 
most clinicians lack experience in the accurate diagnosis of these 
rare disorders. 

    Since the expected incidence of mutant candidate sentinel 
anomalies is 2 - 3 per 10 000 live births, a very large study 
population is required in order to be able to detect any effects 
in exposed groups of individuals. 

4.4.4.  Procedures

4.4.4.1.  Surveillance of sentinel anomalies

    A programme may be based on existing congenital anomaly 
registries and surveillance systems.  The target population 
comprises all the new-born infants and, when possible, those up 
to the age of one year.  Several sources of ascertainment are  

desirable.  Three criteria of congenital anomaly surveillance 
should be continuously checked: 

    (a)   Completeness of ascertainment

    In Hungary, the total birth prevalence of congenital 
anomalies included in Chapter XIV "Congenital Anomaly" of the 
International Classification of Diseases was estimated to be about 
60 out of every 1000 new-born infants at, or after, birth (Czeizel 
& Sankaranarayanan, 1984).  Individual types of congenital 
anomalies show a wide range of completeness of ascertainment. 

    (b)   Validity of diagnosis

    The validity of diagnosis may be known as a result of  ad hoc  
epidemiological studies.  However, there may be a wide range of 
misdiagnosis for different types of congenital anomalies. 

    (c)   Base-line frequency

    The baselines of all pregnancy outcomes (e.g., spontaneous and 
induced abortions, still births, low-birthweight infants, etc.) and 
other potentially confounding demographic variables (e.g., paternal 
and maternal age) are also taken into consideration in evaluating 
sentinel anomalies. 

    The surveillance of sentinel anomalies should consist of three 
steps: 

    (1)  Indexed patients with sentinel anomalies, notified to the 
         congenital anomalies registries and surveillance systems, 
         depending on their type of sentinel anomalies, should be 
         referred, with the help of their parents, to selected 
         participating paediatric, orthopaedic, or ophthalmological 
         institutions. 

    (2)  Specialists in these institutions should examine these 
         indexed patients to confirm (or exclude) the nosological 
         diagnosis, to clarify family history (whether a sporadic 
         or familial case), to obtain data on environmental history 
         (with the help of a specifically-designed questionnaire), 
         and to give genetic counselling (if parents plan to have 
         babies in the future). 

    (3)  The data obtained by experts at medical institutions 
         should be sent back to the programme director to be 
         evaluated. 

4.4.5.  Data interpretation

4.4.5.1.  Surveillance of sentinel anomalies

    Preliminary Hungarian experiences, based on data obtained 
during 1980 - 82, are inconclusive.  The registered prevalence at 
birth of 24 sentinel anomalies was 2.6/10 000 births.  The rate of 

participation was only 41% (the main causes of case loss were the 
death or severe condition of certain indexed patients) and the 
diagnosis was confirmed in only 60% of the cases examined.  
Nevertheless, as an average of 7 possible new mutations per year 
was found over the 3-year period, during which nearly 430 000 
births occurred, it is hoped that improving the completeness of 
notification, participation rate, and diagnostic skills will 
increase the number of new mutations identified in the future. 

    The possible causes of new mutations can be studied by 
obtaining the environmental histories of the parents.  The 
environmental histories of the parents of familial cases can 
provide adequate control data. 

    The main problem is that the basic data concerning even common 
candidate sentinel anomalies are not known completely.  Knowledge 
of their frequency in different populations, the rate of prenatal 
loss, definite figures on their penetrance,and a timetable of the 
expression of their manifestations would improve the efficient use 
of these sentinel anomalies. 

    The total prevalence at birth of the 24 sentinel anomalies 
studied is not low.  Assuming a Poisson distribution of events, 
a background rate of 4.0 sentinel anomaly cases per 10 000 births, 
and a 50% proportion of sporadicity, a sample size of 38 000 would 
be needed to detect a doubling of mutation rate with probabilities 
of type I and II errors at the 0.05 level.  The null hypothesis 
would be rejected, if 22 or more new mutations were seen. 

    The surveillance of sentinel anomalies has some other practical 
benefits.  Experts at referral centres have the opportunity to 
improve their diagnostic skills, and to suggest prognosis and 
treatment.  From this aspect, verifying a genocopy or phenocopy is 
important.  Furthermore, surveillance may promote the detection of 
teratogens.  One of the main practical advantages of surveillance 
is economy.  In the Hungarian programme, an opportunistic and cost-
effective approach is used (Mulvihill & Czeizel 1983). 

4.4.6.  Conclusions

    At present, most sentinel anomalies are not suitable for 
surveillance, because of the inability to validate the diagnosis 
and the incomplete ascertainment of some types.  However, the 
reliability of the diagnosis of sentinel anomalies will improve as 
the number and skill of clinical geneticists increases.  Thus, the 
material in national or regional surveillance systems with a large 
and unselected data base will become usable for studying sentinel 
anomalies.  The main argument for surveillance is that data are 
already available in the registries and surveillance systems used 
for other important medical purposes.  In addition, the existence 
of such programmes will improve existing medical care. 

    The monitoring of sentinel phenotypes has a specific purpose.  
Defined populations, such as the offspring of self-poisoned 
persons, epileptics, cancer patients (e.g., patients with acute 

lymphoid leukaemia), or workers exposed to chemical compounds, 
would be worth studying.  However, the sentinel phenotype approach 
is not useful for the monitoring of a small circumscribed new-born 
population (< 50 000), exposed to known or suspected mutagens, 
because such a small sample cannot yield statistically significant 
results.  Nevertheless, the importance of a opportunistic approach 
should be stressed, because some types of industrial and disease 
registries may provide data for this purpose. 

    There are a number of possibilities for improving the 
feasibility of the surveillance and monitoring of sentinel 
phenotypes.  Because the birth prevalence of individual sentinel 
anomalies is relatively low, international collaboration should be 
encouraged.  For example, the International Clearinghouse for Birth 
Defects Monitoring Systems (Flynt & Hay, 1979) represents a huge 
surveillance programme involving more than 24 countries and the 
records of more than 2 million births per year.  The majority of 
the national and regional systems are able to evaluate the 
occurrence of sentinel anomalies.  However, the fact that most 
sentinel anomalies do not have separate codes in the International 
Classification of Disease is a serious technical limitation.  
Unique codes need to be given to them so that international 
comparisons of rates of sentinel anomalies can be made. 

    An important approach involves the use of sentinel childhood 
tumours, mainly hereditary retinoblastomas and Wilms' tumours.  
Patients with these disorders are diagnosed and, in general, 
hospitalized in central institutions; thus, their records can 
also be used for monitoring purposes.  Although retinoblastoma 
and Wilms' tumour are not usually detectable at birth, they have a 
specific feature beyond those mentioned above.  The vast majority 
of cases of either type occur sporadically, and all sporadic 
bilateral cases have an autosomal dominant origin, while most 
sporadic unilateral cases are non-hereditary, persumably arising 
from somatic mutation.  Hence, these two types of tumour can be 
used for surveillance of both germinal and somatic mutations. 

    Registries of individual genetic disorders (e.g., Huntington, 
neurofibromatosis, polyposis coli) (Table 7), in several countries, 
also offer a possibility for the surveillance of sentinel 
phenotypes. 

    Improvement in diagnostic laboratory procedures has resulted 
in progress in the surveillance of sentinel phenotypes (Vogel & 
Altland, 1982).  Recently, screening for the lipoproteinaemias has 
become widespread and affords an opportunity for detecting common 
familial hypercholesterolaemias of autosomal dominant origin.  
Finally, some X-linked recessive traits and disorders, such as 
Duchenne muscular dystrophy, Martin-Bell syndrome, or haemophilia 
A, would be useful sentinel phenotypes, particularly if:  (a) a 
considerable fraction of cases were new mutations, and (b) 
heterozygosity in the mothers could be detected reliably and 
inexpensively by laboratory techniques. 

    Although, the monitoring of sentinel phenotypes cannot compete 
with the efficacy of studying protein variants and the methods of 
the new molecular genetics, simultaneous approaches can provide 
possibilities for comparison and, in addition, the sentinel 
phenotype approach may be more feasible in certain countries. 

4.5.  Fetal Death

4.5.1.  Introduction

    The proportion of human fetal deaths normally attributable to 
germinal genic mutations is not known.  If rare recessive lethals 
are a common cause, studies on inbreeding have failed to show the 
increase in fetal deaths expected (MacCluer, 1980).   Dominant 
lethal mutations acting in fetal life can occur, but their 
contribution to fetal death is unknown and difficult to study with 
present techniques.  If this contribution were large, an increase 
in fetal death with increasing paternal age might be expected.  
Such an effect was seen in one study (Selvin & Garfinkel, 1976), 
but was not seen in another study of late fetal deaths (Hatch, 
1984). 

    The occurrence of a fetal death, particularly an early fetal 
death, without any investigation of the products of conception, can 
be considered as an indicator of germinal genic mutation.  However, 
any increase in FD could be due also to exposure to a teratogen 
during pregnancy, or to interference with normal maternal hormonal 
or immunological functions during pregnancy. 

    Teratogenic effects could be separated from genetic effects, if 
the exposure could be allocated according to the sex of the exposed 
parent, and whether it was pre-conceptional or post-conceptional.  
Although the possibility exists that maternal exposure can occur 
indirectly through paternal contact, most paternal exposures are 
likely to affect fetal health via genetic changes transmitted in 
the sperm.  Pre-conceptional exposures only, in females, are also 
most likely to induce genetic damage.  However, it is not possible 
to distinguish between germinal chromosome aberrations and genic 
mutation using exposure data. 

    If morphological examination of the products of conception can 
be carried out (section 4.2), further information can be gained.  
Genetic effects, either chromosomal or genic mutations, are likely 
to result in developmentally abnormal specimens, whereas factors 
acting via interference with the maternal physiological response in 
pregnancy are likely to result in developmentally normal specimens.  
A change in the usual distribution of morphological types among 
specimens of FD can be used to document an effect of an exposure:  
a doubling of the number of "intact empty sacs" compared with 
controls would confirm the validity of an observed rise in overall 
rate of FD. 

    If the products of conception can be examined cytogenetically 
(section 4.2), it may be possible to distinguish a class of FD 
most probably due to genic mutation.  A rise in developmentally 
abnormal, but chromosomally normal specimens, after maternal 
preconception, or paternal, exposures, might possible be attributed 
to an increase in germinal genic mutations, lethal for fetal life. 

4.5.2.  Procedures

    If exposed and unexposed populations are to be compared for 
overall frequency of fetal death, the population(s) to be studied 
must be clearly defined, and data collected on the total number of 
births over 28 weeks of gestation, i.e., late fetal death (still 
birth) and the total number of FDs.  For purposes of comparison 
between an exposed and unexposed population, bias should not be 
introduced, if the ascertainment of either of these variables is 
not complete, as long as the exposure status does not influence the 
ascertainment.  However, the higher the observed rate of early and 
intermediate fetal death (spontaneous abortion) the greater the 
power of the study, and the less likely it is to miss effects 
limited to only particular subsets of the total (as is likely for 
genetic effects).  As most genetic effects will probably be 
observed among the early spontaneous abortions, it is particularly 
important to ascertain these as completely as possible.  This is 
not usually possible with most existing vital statistics sources 
of fetal deaths.  In a retrospective study, it will probably be 
necessary to use either medical records or interviews to ascertain 
reproductive history. 

    In a case-control study, efforts would be made to ascertain 
spontaneous abortions through a medical treatment centre, and to 
compare exposure histories between women who have spontaneous 
abortions and women having live births.  If the sample were 
relatively small, cases and controls would probably have to be 
matched for age, in order to control for this source of bias.  
Finding controls at a point in gestation that matches the cases of 
spontaneous abortion will also help to control for differences in 
time of ascertainment, and differences in recall of events 
occurring at the beginning of the pregnancy. 

    Data must be collected as carefully as possible on factors 
known to influence the frequency of fetal death (e.g., maternal 
age, socio-economic status, previous reproductive history, and 
gestational age at abortion).  Gestational age is usually estimated 
from menstrual history.  Even if data on the developmental stage of 
the fetus is available, it should not be used to estimate 
gestational age, since development often ceases some time before 
abortion.  Smoking and alcohol consumption histories would also be 
useful, since these common exposures are associated with increased 
abortion risk and might differ in exposed and unexposed 
populations. 

4.5.3.  Data processing and presentation

    Frequency is usually expressed as the number of fetal deaths 
divided by the number of births over 28 weeks plus the number of 
fetal deaths.  Maternal age is the most important variable to be 
controlled in any analysis, since it will influence the overall 
frequency of abortion, and also the distribution of the kinds of 
chromosome anomalies expected.  Trisomies in spontaneous abortions 
increase with maternal age in the same way as trisomies in live 
births.  On the other hand, monosomy X shows a decrease in 

frequency with maternal age in spontaneous abortions.  The risk 
of abortion increases with the number of previous abortions in a 
woman's history, and this variable should also be examined for 
possible confounding effects. 

4.5.4.  Conclusions

    (a)  Fetal death can be used as a possible indicator of 
         germinal genetic mutation.  Because of the high background 
         frequency, and concentration of anomalies, an increase can 
         be detected in a small sample size, e.g., a sample from 
         only 200 women will detect a doubling with 95% power.

    (b)  Without cytogenetic studies, it is not possible to define 
         the kind of genetic damage that has occurred (i.e., 
         chromosomal or genic mutation) or to separate mutagenic 
         from teratogenic effects.  However, defining the exposure 
         of the parent (pre-conceptional versus conceptional) may 
         make more inferences possible about the cause of a rise in 
         the frequency of fetal deaths.

    (c)  Estimates of the rate of fetal death may be imprecise 
         because of difficulties in identifying all causes, and in 
         determining the relevant population denominator of term 
         births.  Data on factors known to influence rates of fetal 
         death, such as maternal age, socio-economic status, and 
         previous reproductive history, need to be collected, so 
         that possible biases can be explored in the analysis.

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    See Also:
       Toxicological Abbreviations