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Original Communication
Image Analysis System
and its use in Cytogenetic Analysis
Md. Equebal Ahmad, Kiran Kucheria
Division of Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi-110029, India.

Cytogenetic analysis is an important tool in the

diagnosis of genetic disorders. It involves cell culture,
metaphase arrest, hypotonic treatment, cell fixation, slide
preparation and use of banding methods. Following the
banding, analysis is done by taking photomicrographs of
the metaphase spreads and developing and printing the
film in the dark room. Individual chromosomes are then
manually cut, paired, pasted and a karyotype is made. This
whole procedure is time consuming, labour intensive and
expensive.
With the increasing workload of the cytogenetic laborato
ries and availability of improved computer capabilities for
image processing, the Image Analysing System, with ap
propriate software, are being used for cytogenetic analy
sis. The system includes a high resolution research micro
scope with or without an automatic metaphase scanning
stage, charged couple device (CCD) camera and computer
with special software for image capturing, chromosome
counting, automatic karyotyping etc. The system is con
nected to an ordinary inkjet/laser printer. The present pa
per deals with our experience of one year on use of image
analysis system in routine cytogenetic analysis which has
cut down the cost and time of karyotyping and analysis by
non use of photographic film, developing, printing and dark
room facilities. Hence provides reports to the patients with
karyotypes using ordinary paper in one hour only. The most
important is that this system has made storing and retrieval
of data very easy.
Key Words: Image analysis system, cytogenetics, karyo
typing

Revolution in computer technology and its increasing

application in image analysis has made the interpreta
tion of chromosomal patterns an automated process.
Computer image processing in cytogenetics offers sev
eral advantages over conventional method. Image analy
sis systems are rapidly replacing conventional method
of photomicrography and dark room requirements. Im
provements in microprocessors, video cameras, moni

tors, printers and computer technology have contributed

to better image quality, resolutions and several other
functions.
In conventional method prepared slides are scanned
under microscope and well-spread metaphases are sub
jected to photography. The film is developed manually
and printed using the dark room facilities by the scien
tist. Dried prints are then assembled for cutting individual
chromosomes and homologous pairs are pasted for
making karyotype. Therefore a need was felt to have a
computer-assisted system which can make the process
of karyotyping simple, easier, rapid and cost effective.
Although work in this direction started in early 1960s,
the earlier systems developed were not so effective. In
recent years few systems are being developed using
which a metaphase can be captured, enhanced,
karyotyped and printed in less than an hour. Thus re
ducing the turn around time significantly. The cost of
laser black and white prints is considerably less than
the cost of photographic prints. Besides these all the
data and karyotypes can be stored using optical disks
for long-term storage and retrieval. Use of image analy
sis system for karyotyping replaces the storage of tradi
tional film negatives, require less storage space, and
are more expedient in reproducing a print if needed.
Materials and Methods
Image analysis system has four components.
1.

Binocular high resolution research microscope

2.

CCD camera

3.

Computer with appropriate software

Address for correspondence: Dr. Kiran Kucheria, Incharge, Division of Genetics and Head, Department of Anatomy, All India Institute of
Medical Sciences, New Delhi-110029, INDIA Fax: 91-11-6862663/6521041, E-mail: kkucheria@hotmail.com
Indian Journal of Human Genetics January-June 2002 Volume 8 Isuue 1

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16

Image Analysis System

4. High resolution quality printer

most of the softwares developed for karyotyping have follow

ing components (Josifek et al., 1971) .

Steps involved in karyotyping by image analysis system:

Slides were prepared as per the standard procedure for
conventional cytogenetic analysis and were subjected to chro
mosome banding G, R, Q or C as per the requirement. Rest
of the process was completed in the following way.
(a) Slides were scanned, and well-banded and well-spread
metaphases were selected and sharply focused. The
metaphase spreads visualized under 100X and their images
seen on the monitor were captured. The digitizing system
within the computer takes the signal from the microscopes
video camera and converts it to a numerical matrix. This ma
trix is composed of row and column indices that create a grid
like system (Popescu et al., 1999) . The intersecting points
on this grid are called as pixels. Each pixel or picture is
attributed a gray value. These gray values represent the entire
field of view, including chromosomes, debris, cytoplasm and
interphase nuclei. The gray levels or gray scale values
represent whatever is being looked at through the objective.
(b) To help the user in analysis of captured metaphases

Image segmentation to find the individual object

Procedures for eliminating non-chromosome-like ob

jects

Procedures to separate touching and overlapped

chromosomes

An accurate procedure for centromere determination

An intuitive but qualitative way to describe the

banding patterns

A context-sensitive classification procedure based

upon relative length, centromeric index and banding
description of the chromosomes

An accurate method for chromosome rotation

Option for straightening of the bent chromosomes

(c) The bands of the initial karyotype obtained were then

improved using contrast and enhancement features of the
system to get better resolution of the bands (Figure 1 & 2) .
(d) When optimal banding resolution was obtained and
analysis was done, print was taken in just one click of the
mouse within few seconds. Reports to the patients were given

Figure 1: Enhancing resolution of image

Figure 2: Sclaing of karyotype

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Equebal A et al

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2.Chromosome Counters determine the chromosome num

ber by automatically counting the number of chromosomes in
a given metaphase spread.

Figure 3: Karyotype of a human male 46XY

along with the karyotypes on different formats (Figure 3) .
(e) Description of Specific Features:
The following features/capabilities are representative of
image analysis system for cytogenetic analysis.
1. Metaphase Finders/Scanners aid the cytogeneticist to
locate rapidly suitable metaphases for analysis. These instru
ments automatically scan the microscope slide to locate likely
metaphase spreads. Metaphase finding/scanning instruments
are not always accurate. In some cases their use is limited to
specific types of staining and spreading.

3.Photomicroscopy eliminates the need of photomicrogra

phy, photographic film, photographic dark room processes,
printing paper and cutting and pasting of chromosomes when
performing karyotyping. The process uses digital image pro
cessing to digitize the metaphase images by dividing the pic
ture into a grid of pixels (Graham, 1987) . The resolution and
detail is determined by the number of pixels in the image and
the range in the level of contrast (grayness) . The level of
grayness may theoretically range from 0 to 256 (Stanley et
al., 1995) . Optical information about each pixel as well as its
location may be processed and stored in the computer data
base.
4. Image Analysis Systems exist with varying amounts of
decision-making ability. The metaphase chromosomes are
manipulated (cut) and arranged (pasted) in pairs on the karyo
type card (projected onto the computer monitor) . Chromo
somes are classified on the basis of chromosome dimension
(e.g., relative length of chromosomes, centromeric index i.e.
ratio of short arm to long arm) and banding pattern profile.
Assuming absorption imagery, bands are considered as dark
regions of the chromosomes by the system. An optical den
sity based threshold selects the dark parts as regions poten-

Figure 4: Image enhancing features

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18

Image Analysis System

Division of Genetics
Department of Anatomy
All India Institute of Medical Sciences
New Delhi 110029

Case Number:
Date:
Patient Name:
Date of birth:
Referral reason: Hypergonadotrophic hypogonadism
Specimen type: Blood

Result: Karyotypic analysis shows 47, XXY pattern

Figure 5: Proforma for giving results to the patients

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Equebal A et al
tially bearing a band. In this way the vague connections be
tween clearly separated bands are avoided. From this and
some more information a subset of the bands is extracted
and used for classification. Some of the systems use the cen
tral position of the following bands to get an initial karyotype
(Martin et al., 1990) :

Darkest band on the chromosome

Band having the largest area

First band on the p-terminal

Last band on the q-terminal

First band after the centromere position on the q-ter

minal.
After computing the a posteriori of probabilities of each
chromosome a context sensitive classifier assigns the chro
mosomes to the possible classes. It is assumed that the
metaphase contains two copies of each autosome and two
sex chromosomes, XX for females and XY for males. An ex
ception is made when there is any numerical anomaly. For
classification all chromosomes are ordered in decreasing
length, building a list with longest chromosomes first. Chro
mosomes unidentified by the system are left unclassified which
can be manually placed to its suitable position after analysis.
5. Enhancement, Alteration and Manipulative Features
There are features to enhance or contrast chromosome
images in metaphases and/or individual chromosomes to im
prove the banding characteristics of the chromosome (s) .
Other features alter or allow the operator to alter chromosome
appearance to make analysis better e.g. straightening, en
larging, trimming, selectively altering staining pattern within a
metaphase spread, and dodging/lifting the background (Fig
ure 4) .
6.Graphic presentations can also be done to observe any
minor chromosomal rearrangement in the metaphase. Any
deviation in the graph indicates deletion or addition in a par
ticular chromosome.
7. Specialized Analysis. A few instruments have the ability
to perform analysis of specialized studies such as Fluores
cent in situ hybridization (FISH) and Comparative Genome
Hybridization (CGH) analysis. Some chromosome analyzers
automatically count the number of satellites or SCEs per
metaphase cell or the number of hybridized probe sites per
interphase or metaphase cell.
8.Hard Copy Prints. Most systems are capable of produc
ing photographic quality; printer generated hard copy of the
metaphase images and karyotypes.
9.Generating Reports. Many instruments have capabilities
of generating (preparation & printing) a final report along with

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the karyotype in different formats (Figure 5) .

10.Computerized Patient Data Storage, Retrieval and Ar
chival Systems. A database includes metaphase images and
karyotypes, patient identifying information etc.
11.Training Feature. This feature permits the operator to
teach the system to recognize the chromosome preparations
particular to a given laboratory. It allows the operator to train
the instrument to recognize different staining preparations.

Discussion

Although the image analysis system makes the pro

cess of cytogenetic analysis rapid, cost effective and
eliminates labour involved in the conventional method,
the system cannot itself detect any abnormality (struc
tural or numerical) present in a metaphase. All its deci
sion-making features are just to help in improving the
resolution of the band and making the cytogenetic analy
sis correct and faster. Therefore a qualified, skilled and
well-trained cytogeneticist is must for the purpose of
cytogenetic analysis. Presently Image Analysis Systems
are easily available in India and can be obtained from
leading microscopes companies.
Acknowledgements
Authors are grateful to the Department of Biotechnology
(DBT) , New Delhi and All India Institute of Medical Sciences
(AIIMS) for providing financial assistance and necessary fa
cilities.

References
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Josifek K, Haessig C and Pantzar T (1991) . Evaluation of
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Martin AO, Isenslein BS, Thomas CW, et al (1990) . Stylized
chromosome images. Cytometry 11 (1) : 40-50.
Popescu M, Gader P, Keller J, et al (1999) . Automatic karyo
typing of metaphase cells with overlapping chromosomes.
Comput. Biol. Med. 29 (1) : 61-82.
Stanley R, Keller J, Gader P et al (1995) . Automated chromo
some classification limitations due to image processing.
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