Chromosome aberration analysis is a promising complement to conventional radiation dosimetry, particularly in the complex radiation fields encountered in the space environment. The capabilities of a recently developed automated karyotyping system were evaluated both to determine current capabilities and limitations and to suggest areas where future development should be emphasized. Cells exposed to radiometric chemicals and to photon and particulate radiation were evaluated by manual inspection and by automated karyotyping. It was demonstrated that the evaluated programs were appropriate for image digitization, storage, and transmission. However, automated and semi-automated scoring techniques must be advanced significantly if in-flight chromosome aberration analysis is to be practical. A degree of artificial intelligence may be necessary to realize this goal

Prichard, Howard M.

English

NASA Technical Reports Server

N87-25896
1936
NASA/ASEE SUMMER FACULTY FELLOWSHIP PROGRAM
3ohnson Space Center
University of Houston
Evaluation of an Automated Karyotyping System for
Chromosome Aberration Analysis
Prepared by:
Academic Rank:
University and Department:
Howard M. Prichard, Ph.D.
Associate Professor
The University of Texas School of'
Public Health
Occupational Health
NASA/3SC
Directorate:
Division:
Branch:
3SC Colleague:
Date:
Contract #:
Space and
Directorate
Medical Sciences
Life Sciences
Gerald Taylor, Ph.D.
December, 1986
NGT=##=005-803
University of Houston
26-1
https://ntrs.nasa.gov/search.jsp?R=19870016463 2020-03-20T10:50:51+00:00Z
ABSTRACT
Chromosome aberration
conventional radiation
radiation
aberration
subjective
studies
ground facilities
require a major
the quality of
required is of great importance.
fields encountered in the space environment.
analysis is time and labor intensive, and
analysis is a promising complement to
dosimetry, particularly in the complex
Manual
requires
evaluations by trained personnel. In-flight chromosome
would therefore require either the transmission of images to
or automated scoring routines that would not
committment of flight personnel. In either case,
the digitized image and the amount of data storage
In this project, the capabilities of a
automated karyotyping system were evaluated
current capabilities and limitations and to
future development should be emphasized.
recently developed
both to determine
suggest areas where
Cells exposed to
radiomimetic chemicals and to photon and particulate radiation were
evaluated by manual inspection and by automated karyotyping. It was
demonstrated that the evaluated programs were appropriate for image
digitization, storage, and transmission. However, automated and
and semi-automated scoring techniques must be advanced significantly
if in-flight chromosome aberration analysis is to be practical. A
degree of artificial intelligence may be necessary to realize this
goal.
NASA Colleague: Gerald Taylor, Ph.D., S.D. 3
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INTRODUCTION
While short LEO (Low Earth Orbit) missions have not produced
crew radiation exposures at levels of concern, other projected
missions involve the possibility of high, even life-threatening
exposures. Lunar and deep space missions beyond the earth's
magnetosphere involve the risk of solar flare radiation, and some
earth orbits involve appreciable time in the Van Allen radiation
belts. Even extended low earth orbit missions such as those
envisioned for Space Station personnel involve some accumulated
exposure to Inner Van Allen Belt radiations, due primarily to
repeated passage through the South Atlantic Anomaly. Space
radiation environments are generally complex and qualitatively
different from terrestrial radiation fields that have produced the
bulk of our knowledge on the biologic effects of radiation. While
there is at present a great deal of interest in the biological
effects of the high energy heavy nuclei found in the galactic
cosmic ray flux, it appears at present that protons in the range of
(approximately) i0 to I000 MeV are the limiting form of radiation
for most comptemplated deep space and Low Earth Orbit (LEO)
missions. In terms of flux, energy deposition, and presumed
biological effect, protons are the single most important component
of galactic cosmic rays, solar flare radiation, and Inner Van Allen
belt radiation (Haffner, 1967). While such exposures are of only
moderate concern for the welfare of Space Station crew, it is
important for future programs to determine as much as possible about
the biological effects of space radiations administered in the
environment of a spacecraft.
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The
conduct of
confounding
condition,
Belt can
experimental
through the
between LEO
Space Station represents an invaluable resource for the
these types of radiobiological experiments. Potentially
effects such as weightlessness are the default
and access to the energetic protons of he inner Van Allen
be achieved with little energy expenditure. Small
packages
belt if
and GEO.
could be subjected to repeated passages
"piggybacked" on transfer vehicles operating
A relatively small delta yea could subject a
dedicated probe to a pre-programmed exposure to belt protons prior
to retrieval. The effects of various types of shielding and
differential responses between physical dosimeters and biological
systems under flight conditions could be studied much more readily
from the station than from a facility on the ground. Of particular
interest are the responses of human cells, such as lymphocytes,
exposed deliberately in vitro or incidently in vivo Chromosome
aberration analysis is one of the principle techniques used to
quantitate the response of such systems.
Chromosome aberration studies have long been used to determine
the extent of radiation exposure in cases in which conventional
dos£metry systems were either not in place or not appropriate for
the mode of irradiation (e.g. Collins 1980, Brewen 1982, Evans 1979,
Lloyd 1979). At
subjective aspect
chromosome spreads.
each spread
to whether
present, the most labor-intensive and potentially
of the procedure is the manual evaluation of
A trained cytological technician must examine
and make what are in many cases subjective decisions as
or not an observed feature represents a true anomaly.
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Many cells must be examined to obtain reliable statistics on a
single sub-lethal radiation exposure, the exact number being
dependent on the dose and the degree of precision required, among
other things. (A one Gray (i00 tad) dose produces an expected
yield of dicentric and acentric aberrations on the order of 1 per 20
cells, while the "spontaneous" background rate is about one per
hundred cells (Lloyd 1980).) While the Space Station is a very good
place to perform relevant experiments, it would be a misuse of
resources to dedicate one or more crew members to extensive manual
cytological examinations if a reasonable alternative were available.
One alternative is to digitize microscopic images of cell
preparations for transmission and reconstruction on the ground,
where conventional cytological analysis could be pursued. A related
option is to use appropriate software to process digitized images in
flight. The latter option is particularly attractive for situations
in which data links to the ground are at a premium, as might be the
case in a deep space mission. Both alternatives require that the
digitized be of adequate quality for analysis and that data storage
and transmission requirements not be excessive.
Slide Preparation
MATERIALS AND METHODS
Slides
radiation
(Prichard,
radiation
of human lymphocytes arrested at metaphase after various
and chemical treatments had been prepared previously
1986). Blood samples had been subjected to one of three
treatments: i). Control - no radiation, 2). Gamma - 0.662
26-5
MeV gamma rays from a Cs-137 irradiator, and 3). Proton - 40 MeV
protons from the University of Texas Health Science Center at
Houston Cyclotron Facility. Venous blood had been drawn into a
heparinized 15 ml vacutainer tube, which was then shaken and placed
into a 35 to 37 degree transfer case until treatment and/or culture.
Aliquots were given the appropriate radiation treatment and returned
to the sample case. Another aliquot was cultured for a predetermined
interval, irradiated, then returned to the incubator. One half ml
treated whole blood was placed in a culture tube along with 4.5
of modified RPMI culture medium and stimulated with the mitogen
of
ml
PHA. The culture tubes
desired incubation period
circumstances). One hour
0.I ug/ml) was added
medium was spun down
supernatent plasma and
were placed in a 37 C incubator for the
(72 hours or 48 hours, depending on
before harvest, Colcemide (final conc. =
to arrest dividing cells at metaphase. The
to separate the cellular mass from the
all but the lower half centimeter of plasma
was pipetted off and replaced by hypotonic KCI. The solution was,
mixed and allowed to stand for 30 minutes, after which three ml of
fixative solution was added, the solution mixed, and spun down.
After three successive washes, slides were prepared by allowing
several drops of the fixative containing suspended cells to fall ca.
one meter onto a clean slide still wet from storage in 4 C distilled
water. The slides were then air dried, stained with Giemsa, and
coverslipped after a zero point disk had been placed in the center
to facilitate mapping.
26-6
Metaphase Spread Selection
The
Zonax
chromosome
examination
power oil
previously prepared slides were mapped at low power with a
micro-computer driven microscope and the locations of
spreads were stored on a floppy disk to permit rapid
under high power objectives. Spreads which under high
lenses seemed suitable for metaphase aberration analysis
A set of spreads were selected forwere logged by map number.
automated and manual analysis.
Image Digitization
An analog image of the microscope field was generated by a
videocamera mounted on the microscope. The program Chromexec, by
Perceptive Systems, Inc, and associated hardware permitted a variety
of contrast adjustment routines prior to digitization of the image.
The digitized image consisted of a 256 x 256 array of 8 bit bytes.
When cameras and monitors of appropriate quality were used, the
visual quality of the image was judged by the author to be quite
adequate for gross abnormality or karyotype analysis. This
judgement was confirmed by a panel of experienced cytogeneticists
assembled to review the quality of the image and other features of
the system (Pathak, 1956). However, a four-fold reduction in pixel
number produced obvious degradation of the image, thus confirming
the appropriateness
to recall a mapped
digitize, document,
of the 256 x 256 array size. The time required
spread, adjust focus and gray levels, and to
and store an image was found to be on the close
26-7
order of two minutes. Most of this time could be eliminated by the
application of macros and program features not available on the
tested version of the program.
Automated vs Manual Image Analysis
In manual scoring of metaphase spreads, the trained
cytotechnician visually scans the microscopic images and identifies
gross abnormal'ities such as rings, dicentrics, gaps, chromatid
breaks, and chromosome breaks. In the sort of scoring traditionally
done for radiation exposure assessment, finer distinctions requiring
banding and full karyotyping are generally not performed. A trained
cytologist can perform this sort of evaluation on a good spread in a
minute or two. The automated karyotyping feature of the Chromexec
program produces a full karyotype of a digitized spread in about
four minutes, provided that few of the chromosomes touch or overlap.
While produces a display that is much easier to score
than spread, and makes possible a much more refined
class the current version of the program consumes
time than a manual scoring. Furthermore, the
manual scorer becomes greater as the spread gets
"messier", i.e., more touches and overlaps.
the program
an unsorted
of analyses,
considerably more
advantage of the
CONCLUSIONS AND RECOMMENDATIONS
The advantage in speed currently held by manual scoring can be
reduced on one or both of two fronts. In the first case, it is
recognized that the contest is unfair, in that the manual scorer is
26-8
not required to separate and sort chromosomes, but rather to detect
anomalies. If this were the goal of the scoring program, the
competition would be more direct. Pattern recognition routines such
as those under development in other fields would be one approach to
this effort. The other strategy would be to take full advantage of
the karyotyping abilities of the existing program and develop
dosimetric techniques based on the more sophisticated chromosome
analysis procedures. This approach would be especially attractive
if it could be shown that reliable dosimetry could be performed by
intensive study of a small number of cells, rather than by the
superficial investigation of a large number of cells, as is the
current practice.
In any event, it has been shown that even with the current state
of the art, it is possible to digitize, store, and transmit
cytogenetically useful images. Even if no further advances are made
in the area of automated scoring, it is seen that the unique
radiobiological environment represented by the Space Station could
be exploited by sending digitized images to the ground for analysis.
26-9
REFERENCES
Bettega 1981 - "Chromosome Aberrations Induced by Protons up to 31
MeV in Cultured Human Cells", D. Bettega, S. Dubini, A. M. F.
Conti, T. Pelucchi, and L. T. Lombardi, Radiat. Environ. Biophys.
19, 91-100, 1981
Brewen 1982 - "Chromosome Aberrations in Mammals as Genetic
Parameters in Determining Mutagenic Potential and Assessing Genetic
Risk." Progress in Mutation Research, Vol. 3, ed. by K. C. Bora, G.
R. Douglas, and E. R. Nestman, Elsevier Biomedical Press, Amsterdam,
Oxford, New York.
Collins 1980 - "A Case of Child Abuse by Radiation Exposure", V. P.
Collins and M. E. Gaulden, in The Medical Basis for Radiation
Accident Preparedness, ed by K. F. Hubner and S. A. Fry., Elsevier
North Holland Inc. 1980.
Evans 1979 - "Radiation Induced Chromosome Aberrations in Nuclear
Dockyard Workers", H. J. Evans, K. E. Buckton, G. E. Hamilton, and
A. Carothers. Nature 277, 531-534, 1979
Haffner 1967 - "Radiation and Shielding in Space", James W. Haffner,
Academic Press, New York, 1967
Lloyd 1979 - "Radiation Exposure and Chromosome Damage", D. C.
Lloyd, Occupational Health, Nov. 1979, pp 504-509.
Lloyd 1980 - "The Incidence of Unstable Chromosome Aberrations in
Peripheral Blood Lymphocytes from Unirradiated and Occupationally
Exposed People", D. C. Lloyd, R. J. Purrott, and E. J. Reeder,
Mutation Research 72, 523-532, 1980.
Pathak 1986 - Sen Pathak, Ph.D., M.D. Anderson Hospital and Tumor
Institute, personal communication, 1986.
Prichard 1986 - "Cytogenetic Analyses of Peripheral Lymphocytes
Subjected to Simulated Solar Flare Radiation", H. M. Prichard, NASA
Contractor Report 17931, July, 1986, pp 23-1 23-9.
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Evaluation of an automated karyotyping system for chromosome aberration analysis

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