This text is a report made on behalf of the training session organised by Dr M. Jeevan Kumar, PhD., Research Assistant Professor, Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur. The report covers the laboratory practices involved in karyotyping (GTG Banding) human chromosome from whole blood, the explanation to each step of karyotyping, the details about the functions of each reagent, reagent preparation protocols, etc. Karyotyping was done using GenASIs BandView software. The text involves invaluable information about the landmarks of each chromosome in a much simplified and organised way and several symbols approved by the ISCN used in karyotyping routine. Wishing all the very best to the readers and young scientists, for whom this text will find worthful.

1. 1
INTRODUCTION TO HUMAN CHROMOSOME KARYOTYPING
(This is a report made after a training session on Karyotyping and Human Chromosome Analysis, organized
by Dr M. Jeevan Kumar, PhD., Research Asst. Professor, Department of Genetic Engineering, SRM Institute of
Science and Technology, Kattankulathur)
Karyotyping: The laboratory technique involved in the generation of a pictorial representation of one’s
metaphase chromosome (from any tissue), based on the size of the chromosome, position of centromere and
its unique banding pattern.
Chromosomal aberrations are abnormalities in the number or microscopically observable structure of
chromosomes. The number of chromosomes in human cells is 46 with 22 autosomal pairs (one of each type
contributed by the mother and one of each type from the father) and 2 sex chromosomes - 2 X chromosomes
for females (one from father and one from mother) or an X and a Y chromosome for males (the X from the
mother and the Y from the father). The chromosomes visible only at the metaphase stage of mitosis, 22
homologous pairs of autosomes and two sex chromosomes. Each chromosome has a characteristic size and
shape in the “normal” cell. During most of the cell cycle, interphase, the chromosomes are somewhat less
condensed and are not visible as individual objects under the light microscope. Mitosis, or nucleus division,
is the first part of M-phase and in consists of four stages (prophase, metaphase, anaphase and telophase).
However during cell division, mitosis, the chromosomes become highly condensed and are then visible as
dark distinct bodies within the nuclei of cells. The chromosomes are most easily seen and identified at the
metaphase stage of cell division. Karyotyping involves separating and classifying the chromosomes based
on the length of the chromosome, location of the centromere, and the banding pattern.
Automated systems for karyotyping provide the user: ease of use, speed, image quality, and accuracy. The
banding of chromosomes by using dyes was discovered in the late 1960's and before that cytogeneticists
depended on chromosome length and position of a constriction to identify the individual chromosomes. The
band width and the order of bands is characteristic of a particular chromosome - a trained cytogeneticist can
identify each chromosome (1, 2, 3... 22, X and Y) by observing its banding pattern under the microscope.
Chromosomes are arranged and numbered by size, from largest to smallest. Identifying chromosomes has
become easier in recent years by using certain staining techniques. One of the most common staining
techniques involves Giemsa stain, which gives the chromosomes a banded appearance (hence Giemsa
banding or G-banding).
G-banding is the treatment of chromosomes in the metaphase stage with trypsin (to partially digest the
protein) and stain them with Giemsa. Each homologous chromosome pair has a unique pattern of G-bands,
enabling recognition of particular chromosomes. Karyotyping is the process by which doctors and
geneticists take pictures of the chromosomes while the cells are undergoing mitosis. The picture is then
enlarged. The pictures of the chromosomes are then cut up so that each chromosome is removed. The
chromosomes are matched up and attached to a paper according to size. The chromosome pairs are
numbered from largest to smallest. There are 22 pairs of chromosomes which match up exactly. Then
the sex chromosomes are paired. In the female (XX) the chromosomes match but in the male (XY) the
chromosomes do not match. This technique can be used to assess the “normalcy” of an individual’s
chromosomes and to assay for various genetic diseases such as Down syndrome and Klinefelter syndrome.
It is estimated that one in 100 live births have some kind of chromosomal abnormality. A chromosome
is divided by its centromere into short arm (p - petit) and long arm (q - queue).
Chromosomes can be classified by the position of their centromere. In humans, they are:
(i) Metacentric: If its two arms are equal in length.
(ii) Sub-metacentric: If arms' lengths are unequal.
(iii) Acrocentric: If the p arm is so short that is hard to observe, but still present.

2. 2
The diagrammatic representation of metacentric, sub-metacentric and acrocentric chromosomes are shown
below along with some characteristic examples.
The photograph is enlarged and cut out into individual chromosomes. The homologous chromosomes can be
distinguished by length and by the position of the centromere so that the chromosomes can be arranged in 7
groups (A, B, C, D, E, F and G). Karyotypes are arranged with the short arm of the chromosome on
top, and the long arm on the bottom. In addition, the differently stained regions and sub-regions are
given numerical designations from proximal to distal on the chromosome arms.
For example, Cri du chat syndrome involves a deletion on the short arm of chromosome 5. It is written
as 46,XX,5p-. The critical region for this syndrome is deletion of 15.2, which is written as
46,XX,del(5)(p15.2).
Alterations in chromosome number: Non-disjunction occurs when either homologue fail to separate
during anaphase I of meiosis, or sister chromatids fail to separate during anaphase II. The result is that
one gamete has two copies of one chromosome and the other has no copy of that chromosome (the other
chromosomes are distributed normally). If either of these gametes unites with another during fertilization,
the result is aneuploidy (abnormal chromosome number). A trisomic cell has one extra chromosome (2n
+1). For example: trisomy 21 (Down syndrome). A monosomic cell has one missing chromosome (2n - 1),
usually lethal except for one known in humans: Turner's syndrome (Monosomy XO). The frequency of
nondisjunction is quite high in humans, but the results are usually so devastating to the growing zygote that
miscarriage occurs very early in the pregnancy. If the individual survives, he or she usually has a set of
symptoms - a syndrome - caused by the abnormal dose of each gene product from that chromosome.
Less automated systems require the user to “cut out” the chromosomes using the mouse and then to place
them into a karyotype. In semi-automated systems, the system will “cut out” the chromosomes, and the user
classifies them into a karyotype. On the other hand, a fully automated imaging system will capture the
metaphase chromosomes (either bright-field for G-banding or fluorescent for Q-banding), separate or “cut
out” the chromosomes, classify them, and arrange them in a Karyotype. However, some metaphases contain
very complex clusters of overlapping chromosomes, and the user might still need to intervene and manually
separate the chromosomes using the mouse. A fully automated karyotyping system can also be used in
conjunction with a so-called metaphase finding capability. This means that the system will automatically
scan the slide in search of good metaphase spread that can be used for karyotyping. Based on several
parameters, the system images metaphase spreads (fluorescent or bright-field) and presents them to the user
for review and analysis. Key factors for a metaphase finding system are the ability to recognize appropriate

3. 3
metaphases or cells, accuracy of relocation to a metaphase of interest, speed of scanning and sensitivity (the
percentage of metaphase cells found by the system).
Software features important for metaphase finding include the following:
 Definition of rare event classification parameters to ensure optimum scan results. The user can define
the parameters that are utilized by the system to identify the rare event.
 Ability to quickly relocate to a metaphase or rare cell for review.
 Sort function to organize metaphases or cells after scanning based on specific parameters.
Because of the general nature of the scanning system, it can also be used in other applications that require
scanning for particular cells (rare events), such as FISH spot counting for detection of tumour cells in body
fluids. Laboratories are using scanning systems more and more for streamlining their workflows. The
systems are set up to continuously scan slides for metaphases or rare events while technologists are
analysing the detected metaphases or cells on remote review stations. This increases throughput while using
the technologists’ time where it is most valuable: analysing cases.
The metaphase chromosome spread is obtained by casting a fixed sample of lymphocytes which were
arrested in its metaphase. The convenience of peripheral blood as a source of human cells, the abundance of
mitotic cells and the simplicity of the cell culture technique make this the most convenient approach to study
human chromosomes for both clinical and research purposes. This method of chromosome preparation
provides metaphase cells that can be stained by a variety of methods or used for Fluorescence in situ
Hybridization [FISH]. The most common chromosome staining techniques involve exposing fixed
preparations to a protease [e.g., trypsin], followed by an appropriate semi-permanent stain. The
characteristic banding patterns obtained reflect both structural and functional differences in different parts of
the chromosomes. The staining procedure described here provides a Giemsa banding pattern using trypsin
with Giemsa stain [i.e., GTG banding]. This procedure is reliable and with only minor modifications,
suitable for preparing chromosomes from a variety of human tissues.

4. 4
DAY 1
SAMPLE COLLECTION AND CULTURING
The BSL-II cabinet was cleaned using 70% ethanol and the required equipment (except the blood sample)
was placed inside the cabinet under UV radiation for 15 minutes. The materials required were:
(i) EuroClone®
media
(ii) Falcon tube (15 mL) - 4 nos.
(iii) Micropipette and tips
(iv) Test tube stand, gloves, marker pen, etc.
The sample culturing started by aliquoting 5 mL of the media to four Falcon tubes. 1 mL of whole blood
sample was transferred to the media and mixed thoroughly (one sample culture). The ratio of media to blood
shown above is for babies (<5 years old). For donors >5 years of age, one sample culture means 500 L
whole blood to 3 mL of media. One to two threads of the tubes were loosened and kept in a slanting position
inside the CO2 Incubator at 37 °C, 5% CO2 concentration for about 68 hours.
Result: The blood sample was collected ethically from the donor using informed consent letter and sampled
appropriately for culturing the cells.
Conclusion and Points to Remember:
(i) It is always important to have an informed consent from the patient or the patient’s legal
guardian for collecting the sample for diagnostic purposes involving one’s identity like a
karyotype. A copy of the informed consent form used is attached in the report.
(ii) Sodium Heparin is a natural blood anti-coagulant. It is used in blood collection vials (labelled
as NH) for live cultures and the vial lined with sodium heparin has a light green seal cap.
Potassium EDTA or K-EDTA – lined blood collection tubes (labelled as K2E or K3E) are
also available, but the blood collected in those tubes are not viable for a live culture as EDTA
is a cytotoxin. K3E vials have a pink seal cap.
(iii) EuroClone®
media: The culture media used for the growth of lymphocytes has the brand
name EuroClone®
. The trademark of EuroClone®
is shown below:
The media is specific for blood cell culture and it mainly contains:
NH Blood
Collection tube
Vacutainer
KE Blood
Collection tube
Vacutainer

5. 5
 PHA-L: Phytohaemagglutinin – L (leukoagglutinin) is a lectin found in plants, especially in
certain legumes like the red kidney beans (Phaseolus vulgaris). It is a mitogen, which induces
mitosis and affects the cell membrane in regard to transport and permeability to proteins.
PHA-L triggers the T – lymphocytes especially to get activated from the quiescent phase (G0)
of cell cycle into G1 and M phase of cell cycle. PHA-L usually takes two days to actively
induce mitosis in T cells which is the reason for incubation for three days and the human cell
cycle take 24 hours to complete. PHA-E is used for erythrocyte culturing.
 FBS: Fetal Bovine Serum (FBS) comes from bovine fetal blood. It has very low level of
antibiotics but has very high proportions of growth factors for T-cell culturing. Bovine Serum
Albumin (BSA) is the major component of FBS.
 RPMI 1640: The growth medium used in cell culture developed by George E. Moore, Robert
E. Gerner and H. Addison Franklin in 1966 at the Roswell Park Memorial Institute (RPMI).
One litre of RPMI 1640 contains:
(a) Glucose – 2 g
(b) Phenol red pH indicator – 5 mg
(c) Micronutrients and electrolytes – NaCl (6 g), NaHCO3 (2 g), Na2HPO4 (1.512 g), KCl
(400 mg), MgSO4 (100 mg), Ca(NO3)2 (100 mg)
(d) Amino acids – Glutamine, arginine, asparagine, etc.
(e) Vitamins – i-inositol, Choline chloride, p-aminobenzoic acid, folic acid, nicotinamide,
etc.
The medium contains great deal of phosphates and is formulated for use in a 5% CO2
atmosphere.
(iv) Cells are cultured in CO2 incubator at 37 °
C at 5% CO2 concentration so as to maintain the
normal body temperature and the natural carbonic acid – by carbonate buffering system of the
blood.
(v) The threads of the Falcon tubes containing cell culture should be loosened up to one or two
threads so as to allow CO2 diffusion to the media. Placing the tubes in the slanting position
increases the surface area of CO2 diffusion. Also, it is important to place the tubes in different
positions to prevent cross-contamination, if in case happens.
(vi) Cell culture should be mixed by inversion daily morning until harvesting to avoid
sedimentation and to facilitate maximum absorption of nutrients by the cells.
Amniomed®
Plus is a medium specifically
developed for primary culture of amniotic
fluid cells and CVS for fetal karyotyping.
This medium promotes optimal in vitro
adhesion and growth of embryonic cells,
reducing the time required for prenatal
cytogenetic diagnosis; moreover it is
optimised for both in situ and flask culture.

6. 6
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DAY 2
PREPARATION OF COLCHICINE SOLUTION AND HARVESTING BUFFERS
Purpose: To prepare Colchicine solution, Ethidium Bromide (EtBr) solution, Phosphate Buffered Saline
(PBS) and Giemsa Buffer (GB) for the harvesting of the cultured cells.
Procedure:
1. Preparation of Colchicine solution
Materials required:
(i) Colchicine powder
(ii) Distilled water
(iii) Laboratory Weighing balance
(iv) 15 mL Falcon tube, gloves, spatula, etc.
The Colchicine solution was prepared in a 15 mL Falcon tube by dissolving 50 mg of Colchicine powder in
10 ml of distilled water. The solution was covered in foil and stored in refrigerator till harvesting.
2. Preparation of Ethidium Bromide (EtBr) solution
Materials required:
(i) Ethidium Bromide (s)
(ii) Distilled water
(iii) Laboratory Weighing balance
(iv) 15 mL Falcon tube, gloves, spatula, etc.
RPMI 1640 Medium FBS

7. 7
The EtBr solution was prepared in a 15 mL Falcon tube by dissolving 10 mg of EtBr (s) in 10 ml of distilled
water. The solution was covered in foil and stored in refrigerator till harvesting.
3. Preparation of PBS
Materials required:
(i) Potassium chloride (KCl)
(ii) Potassium dihydrogen phosphate (KH2PO4)
(iii) Sodium chloride (NaCl)
(iv) Disodium hydrogen phosphite (Na2HPO3)
(v) Distilled water
(vi) Laboratory weighing balance, Duran bottles, gloves, spatula, marker pen, etc.
Chemicals Amount for 5 L Amount for 250 mL
KCl 1 g 0.05 g
KH2PO4 1 g 0.05 g
NaCl 4 g 0.2 g
Na2HPO3 4.6 g 0.23 g
PBS was prepared by weighing the above chemicals and dissolving them in 250 mL of distilled water in a
Duran bottle. The Duran bottle was capped and kept for aging.
4. Preparation of GB - pH 7.0
Materials Required:
(i) Potassium dihydrogen phosphate (KH2PO4)
(ii) Distilled water
(iii) pH meter, Glacial acetic acid, sodium hydroxide
(iv) Laboratory weighing balance, Duran bottles, gloves, spatula, marker pen, etc.
Chemical Amount for 5 L Amount for 250 mL
KH2PO4 17 g 0.85 g
GB was prepared by weighing KH2PO4 and dissolving it in 250 mL of distilled water in Duran bottle. The
pH of the buffer was checked using a calibrated pH meter and was corrected to 7.0 by random addition of
glacial acetic acid and sodium hydroxide pellets. The Duran bottle was capped and kept for aging.
Result: For harvesting, Colchicine solution, Phosphate Buffered Saline (PBS) and Giemsa Buffer (GB)
were prepared and kept for aging.
Conclusion and Points to Remember:
(i) Colchicine is light sensitive so it should be covered with aluminium foil to prevent photo-
degradation. It is a carcinogen so should be handled carefully using gloves.
(ii) PBS is prepared to buffer the protease, trypsin at pH 7.5 – 8.5.
(iii) GB is prepared to buffer Giemsa stain so as to maximize the absorption of the stain by the
chromosome.
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8. 8
DAY 4
PREPARATION OF REAGENTS AND PERFORMANCE OF THE STEPS INVOLVED IN
HARVESTING AND POST – HARVESTING OF THE CULTURED CELLS
Purpose:
 To prepare 0.076 M potassium chloride (KCl) solution.
 To perform harvesting of the cultured cells.
 To prepare Carnoy’s fixation fluid and perform fixation of the cells.
Procedure:
1. Preparation of hypotonic potassium chloride (KCl) solution
Materials required:
(i) Potassium chloride (KCl)
(ii) Distilled water
(iii) 50 mL Falcon tube
(iv) Laboratory weighing balance, gloves, spatula, marker pen, etc.
Chemical Amount for 500 mL Amount for 50 mL
KCl 2.8468 g 284.68 mg
In a 50 mL Falcon tube, the above specified amount of KCl was dissolved in 50 mL of distilled water. The
solution was kept in the same incubator at 37 °C for a couple of hours before the commencement of the
harvesting procedures.
2. Harvesting of cultured cells
Harvesting is the procedure of isolating cultured lymphocytes from the whole blood culture using KCl.
Materials and Apparatus required:
(i) Prepared EtBr solution
(ii) Prepared Colchicine solution
(iii) Prepared Hypotonic potassium chloride solution
(iv) Cooling centrifuge
(v) Vortex mixer
(vi) Pasteur pipettes, Marker, Tissue roll, gloves, etc.
The steps involved in harvesting were:
(i) At around 67 hours 30 minutes of incubation, to two of the four culture tubes 50 µL of prepared
Ethidium Bromide (EtBr) solution was added forcibly.
(ii) The culture tubes were labelled appropriately and incubated at the same incubation conditions
with one to two threads of the caps loosened for 30 minutes.
(iii) After 30 minutes of incubation that is at the 68th hour, 75 µL of prepared colchicine solution was
added for forcibly to all the culture tubes.
(iv) The culture tubes were labelled appropriately and incubated at the same incubation conditions
with one to two threads of the caps loosened for 27 minutes.
(v) The incubated culture was centrifuged in a cooling centrifuge at 1800 rpm for 10 minutes.
Meanwhile, four Pasteur pipettes were marked for each culture tube, and extra pipettes were kept
ready to add KCl solution and to prepare Carnoy’s fixation fluid.

9. 9
(vi) Three-fourth of the supernatant was discarded carefully using different Pasteur pipette for each
culture tube.
(vii) While re-suspending the pellet using Vortex, approximately 10 mL of Hypotonic KCl solution
was added to each culture tube. The red colour of the culture changed to pinkish red due to
haemolysis by KCl solution.
(viii) After thorough mixing, the tubes were incubated at the same incubation conditions by keeping
the caps open for 15 minutes.
3. Preparation of Carnoy’s fixation fluid and fixation (post-harvesting) of cells at metaphase
Materials required:
(i) Methanol (CH3OH)
(ii) Glacial acetic acid (CH3COOH)
(iii) Falcon tubes – 2 nos. (15 mL and 50 mL)
(iv) Deep freezer and refrigerator
(v) Pasteur pipettes, tissue roll, gloves, test tube stand, etc.
The steps involved in post-harvesting (fixation) were:
(i) Preparation of Carnoy’s Fixation fluid (CFF) – prepared during KCl incubation step
Chemical Volume required
CH3OH 30 mL
CH3COOH 10 mL
To the 50 mL falcon tube, 30 mL of methanol was thoroughly mixed with 10 mL of glacial acetic acid. 2
mL from this mixture was aliquoted to the 15 mL falcon tube and kept in deep freezer for making it ice cold.
The rest of the mixture was saved for later.
(ii) After 15 minutes of incubation of culture tubes with hypotonic KCl solution, 0.5 mL of the ice
cold CFF was added to each tube and thoroughly mixed using the respective Pasteur pipette for
the culture tube. The deep red colour of the culture took an even deeper shade of red due to
coagulation and acidification of the proteins in the blood.
(iii) The culture tubes were centrifuged at 1800 rpm for 10 minutes in a cooling centrifuge after
properly labelled.
(iv) Most of the supernatant was carefully discarded and to the lymphocyte pellet was retained.
(v) While re-suspending the pellet using Vortex, approximately 10 mL of CFF at room temperature
was added to each culture tube.
(vi) The fixed sample in their respective culture tubes were arranged in test tube stand and stored in
refrigerator for aging.
Result: The culture samples were treated with hypotonic Potassium Chloride solution and was fixed using
Carnoy’s fixation fluid. The culture tubes were stored in refrigerator for aging.
Conclusion and Points to Remember:
(i) The chromosomes are highly condensed at metaphase stage with a diameter of 1400 nm.
(ii) EtBr was added to two of the culture tubes. EtBr intercalates with the DNA causing
elongation of condensed chromosomes for easy observation of the banding pattern.
(iii) Colchicine solution was added to the culture at the 68th
hour which corresponds to the late
metaphase stage of the cell cycle, thus hindering the with the spindle fibre formation causing

10. 10
the cells to get arrested at metaphase. Colchicine is a toxic alkaloid and secondary metabolite
originally extracted from plants of the genus Colchicum (C. autumnale) which affects the
polymerisation of tubulin proteins causing metaphase delay. The cells are fixed at this stage,
casted and banded for the observation of banding patterns in chromosomes.
(iv) Hypotonic KCl solution caused the cells to swell up by taking in more water from the
solution. This helps in the mechanical rupture of the cells during casting.
(v) CFF was prepared by mixing methanol and glacial acetic acid in the ratio 3:1. It is a
precipitating (or denaturing) fixative, also an acid fixative. It acts of the specimen by
reducing the solubility of protein molecules and often by disrupting their hydrophobic
interactions thus altering the tertiary structure. The methanol in CFF reduces protein
solubility, structural denaturation and thus overall coagulation of cytoplasmic proteins. It also
causes considerable shrinkage and hardening of tissue. The acetic acid in CFF acts as a
denaturant which weakens the hydrogen and ionic bonds between histone proteins and DNA.
It is also associated with tissue swelling, counteracting shrinkage due to methanol.
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Skeletal structure of Colchicine

11. 11
CASTING
PREPARATION OF SLIDES FOR CASTING AND CASTING PROCEDURE
Purpose: To prepare the slides for casting the fixed culture sample and baking.
Procedure:
1. Preparation of slides for casting
Materials and Apparatus required:
(i) Clean glass slides – 2 nos.
(ii) Distilled water
(iii) Gauze cloth
(iv) Beaker
(v) Refrigerator, etc.
The slides were prepared by washing the slides thoroughly in distilled water and wiping it with gauze cloth.
This step was repeated two to three times. After thorough cleaning, the slides were completely submerged in
a beaker full of distilled water in a slanting position and was kept in the freezer for 5 minutes. This step was
repeated two to three times and the slides are kept ice-cold before casting.
2. Casting Process
Casting is the procedure involving the spreading of the metaphase chromosomes on the slides by mechanical
shearing of the cells and baking the slides to dry and heat fix the chromosomes to the slide for staining.
Materials and Apparatus required:
(i) Freshly prepared Carnoy’s fixation fluid – 10 mL
(ii) Prepared glass slides
(iii) Cooling centrifuge, Vortex mixer and Hot air oven
(iv) Pasteur pipettes, gloves, forceps, test tube stand, tissue roll, etc.
The steps involved were:
(i) The culture tubes were centrifuged at 1800 rpm for 10 minutes in a cooling centrifuge.
(ii) Most of the supernatant was discarded and the pellet of fixed metaphase lymphocytes was
retained.
(iii) To the retained pellet, 2 mL freshly prepared CFF was added and the pellet re-suspended using
vortex mixer.
(iv) Prepared ice-cold slides were taken and the water drained but not wiped.
(v) The slides were labelled appropriately and handled using forceps.
(vi) Three to four drops of the fixed culture were dropped to the centre of the prepared slides from a
height.
(vii) A strong blow of air was given through the surface of the slides so as to maximize methanol
vaporisation.
(viii) The slides were then heat fixed in a hot air oven at 80 °C for one hour and the slides were
allowed to cool.
Expected Result(s): The fixed culture cells were casted on the slides by the mechanical shearing of the cells
and was heat fixed by baking techniques.

12. 12
Conclusion and Points to Remember:
(i) The KCl – treated cells are dropped from a height to the glass slide so as to facilitate rupture of
the cells on hitting the slide, which helps in spreading out the metaphase chromosomes and other
nuclear contents, thus obtaining a proper metaphase chromosome spread.
(ii) Evaporation of methanol from the spread culture by air blowing is necessary because this
technique spreads out the chromosome in the slide which enables easy identification of the
chromosomes during karyotyping.
(iii) The slides are heat fixed to avoid washing away of the chromosomes in the GTG banding step,
where a serial submerging strategy is used. Also it maximizes the stain absorption to the
chromosomes.
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BANDING
PERFORMANCE OF GTG BANDING OF THE CHROMOSOMES
Purpose: To perform GTG (G-banding using Trypsin with Giemsa) Banding technique using the casted
slides.
Procedure:
1. Preparation of Trypsin solution
Materials required:
(i) Protease trypsin powder
(ii) Prepared PBS
(iii) Couplin Jar(s) – 50 mL
(iv) Laboratory weighing balance, gloves, Pasteur pipettes, spatula, tissue roll, marker pen, etc.
Chemical Amount for 50 mL PBS
Protease Trypsin powder
20 mg
40 mg (for aged slides)
The Trypsin solution was prepared just before banding. In a 50 mL Couplin Jar, 20 mg of weighed protease
trypsin was dissolved in 50 mL PBS with thorough pipetting till the characteristic white froth built up in the
solution.
2. Preparation of Giemsa solution
Materials required:
(i) Stock Giemsa stain
(ii) Prepared GB
(iii) Couplin Jar(s) – 50 mL
(iv) Laboratory weighing balance, gloves, Pasteur pipettes, spatula, tissue roll, marker pen, etc.
Chemical Volume for 50 mL GB
Stock Giemsa Stain 3 mL

13. 13
The Giemsa solution was also prepared just before banding. In a 50 mL Couplin Jar, 3 Ml of the stock
Giemsa stain was added. The Couplin Jar was then filled with GB up to the 50 mL mark. The solution was
mixed thoroughly using Pasteur pipettes till the characteristic deep purple froth built up in the solution.
3. Setting up the Couplin Jars and Banding
Banding is the technique of staining the chromosome by which different intensities of a stain will be
absorbed by the different parts of the chromosome. Heterochromatin absorbs more stain and appear dark,
while euchromatin absorbs comparatively less stain and appear lightly stained. Banding is an important step
in Karyotyping as it reveals the final result which is to be recorded by the software. It also makes the
identification of the chromosome groups possible.
Materials required:
(i) Couplin Jar with prepared trypsin solution
(ii) Couplin Jar with prepared Giemsa solution
(iii) Couplin Jar(s)
(iv) Distilled water
(v) Pasteur pipettes, gloves, stop-watch, forceps, tissue roll, marker, etc.
The steps involved were:
(i) The Couplin Jars were arranged, from left to right, starting with trypsin solution, distilled water
and Giemsa solution.
(ii) The baked slides, which were cooled were submerged carefully to the inner racks of the Couplin
jar containing trypsin for 30 seconds.
(iii) The slides were taken one by one and submerged carefully to the inner racks of the Couplin jar
containing Giemsa solution for 5 minutes after a quick dip in the Couplin jar containing distilled
water. It is necessary that the slides should be fully submerged and it should be agitated slightly
to facilitate maximum activity of the reagents.
(iv) After proper staining, the slides were washed carefully in running tap water, upside down and
kept parallel to the flow.
(v) The underside of the slides are wiped with tissue and is observed under the microscope to analyse
the intensity of the staining. If the stain intensity is good to observe then the banding procedure is
complete, else the slides should be re-submerged in Giemsa solution for a couple of minutes
more.
This procedure of partially digesting the histone proteins and other cytoplasmic protein residues by trypsin
and staining the chromosome using Giemsa stain is known as GTG banding (G – banding using Trypsin
with Giemsa), where the heterochromatin is stained dark while the euchromatin is stained light.
Expected Result(s): The baked slides were treated with trypsin and Giemsa stain to perform GTG banding
of the metaphase chromosome spread for visualisation of the banding pattern of each chromosome while
karyotyping.

14. 14
Conclusion and Points to Remember:
(i) Trypsin is a serine protease enzyme, discovered by Wilhelm Kühne, which contain the catalytic
triad of His-57, Asp-102 and Ser-195. It is found in the digestive system of most vertebrates
where it hydrolyses proteins. Peptide cleaves peptide chains mainly at the carboxyl side of the
amino acids Lys or Arg, except when either is followed by Pro. The protein degradation
involving trypsin is called trypsinisation. Histone proteins are rich in Lys and Arg residues which
act as target sites for trypsin.
(ii) Giemsa stain, named after the German chemist and bacteriologist Gustav Giemsa, is a mixture
of methylene blue, eosin and Azure B. It is specific for the phosphate groups of DNA and
attaches itself to the regions of the DNA where high amounts of A – T bonding is present.
Structure of
Trypsin protease
Metaphase Chromosomes under
100X (oil immersion) lens
Courtesy: Dr M. Jeevan Kumar,
PhD.

15. 15
Skeletal structure of
Methylene Blue
Skeletal structure of
Eosin B
Skeletal structure of
Azure B
Couplin Jar Arrangement of Couplin Jars for Banding
Trypsin dH2O Giemsa

16. 16
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Stock Giemsa Stain Stock Trypsin Powder
Duran Bottles

17. 17
SOFTWARE ANALYSIS AND KARYOTYPING
KARYOTYPING USING SOFTWARE
Purpose: To perform karyotyping of the metaphase spread using GenASIs BandView®
Karyotyping
Software.
Procedure:
1. Capturing metaphase chromosome spread manually using ASI-CCD camera
(i) The banded slides were scanned under microscope at 10X and a perfect metaphase
chromosome spread was focused in 40X and 100X oil immersion.
(ii) After proper focusing, the image was captured using ASI-CCD (Applied spectral imaging-
Charged couple device) camera by following the steps of GenASIs Capture and Analysis
and GenASIs Spectral user guides.
(iii) The image was saved in a new file by entering the details of the patient like name, age,
gender, etc.
2. Processing of the captured image and generation of Karyotype Report
(i) The image was saved and the system automatically created boundary between chromosomes.
(ii) The chromosomes were identified, selected and placed according to the Chromosome
Groups.
(iii) The Karyotype was produced after the thorough analysis of chromosomes in their respective
groups.
(iv) It was studied to find chromosomal aberrations like deletions, duplications, translocations,
etc.
(v) The report was generated with appropriate ISCN notations GenASIs Spectral user guide.
Expected Result(s): The Karyotype of the patient was analysed and the report was generated.
Metaphase Chromosome spread
captured through ASI-CCD
camera
Courtesy: Dr M. Jeevan Kumar,
PhD.

18. 18
Conclusion and Points to Remember:
1. Complication arises in karyotyping when the chromosomes in the metaphase spread overlap each
other or if the system recognises cell debris as a chromosome.
2. International System for Human Cytogenetic Nomenclature
The International System for Human Cytogenetic Nomenclature (ISCN) is an international
standard for human chromosome nomenclature which includes band names, symbols and
abbreviated terms used in the description of human chromosome and chromosome abnormalities.
(a) Band Naming7
The centromere divides the chromosomes into a short arm (p; petit) and a long arm (q;
queue). It is defined by the first band on the short arm (p10) and the first band on the long
arm (q10). Telomeres are present at the termini (pter and qter).
Each chromosome arm is divided into regions based on landmarks (consistent and distinct
morphologic area of a chromosome). The regions immediately adjacent to the centromere are
designated as “1” (p1 and q1).The region numbers increase distally to the centromere.
Regions are divided into bands and bands into sub-bands.
For example, Xp22.3 = X chromosome, short arm, region 2, band 2, sub-band 3. This locus is
read as X q two-two (point) three, not X q twenty-two point three.
In case of deletion, duplication, translocation or any other chromosomal abnormalities, the
karyotype is compared with an ideogram of the human chromosome set and the bands are
identified. Ideogram is the pictorial representation of the chromosome, indicating the
prominent band names. For example, the ideogram of Chromosome 12 is shown below in low
resolution and high resolution.
Karyotype generation in
GenASIs BandView Panel
Courtesy: GenASIsTM
Cytogenetics Suite
Ideogram of
Chromosome 12;
low resolution
Ideogram of
Chromosome 12;
high resolution

19. 19
(b) Symbols used in karyotyping
After karyotyping the sample and prior to the generation of the final report or result, the
cytogeneticist should represent the condition based on some notations followed by the ISCN.
The following are some commonly used symbols for karyotype interpretation:
Symbol Remark(s)
t Translocation
rcp Reciprocal
rob Robertsonian translocation
del Deletion
r Ring Chromosome
dup Duplication
ins Insertion
inv Inversion
i Isochromosome
mar
Marker Chromosome – extra chromosome
of unknown origin
mos Mosaic
der Derivative Chromosome
dic Dicentric Chromosome
rec Recombinant Chromosome
add Additional material of unknown origin
cen Centromere
mat Maternal origin
pat Paternal origin
Sign (+) Gain of Chromosome
p Short arm of Chromosome
q Long arm of Chromosome
ish in-situ hybridisation
nuc ish Nuclear in-situ hybridisation
Sign (,)
Separates chromosome numbers, sex
chromosomes and chromosome
abnormalities
Sign (.) Denotes sub-bands
Sign (?)
Questionable identification of a
chromosome or chromosome structure
Sign (;)
Separates altered chromosomes and
breakpoints in structural rearrangements
involving more than one chromosome
Sign (/)
Separates cell lines – used in mosaic
karyotypes
3. Chromosome Identification
A chromosome is identified based on its unique banding pattern, size and position of centromere.
Based on this, human chromosomes are classified into seven groups: A (#1, #2 and #3), B (#4
and #5), C (#6, #7, #8, #9, #10, #11, #12 and X), D (#13, #14 and #15), E (#16, #17 and #18),
F (#19 and #20) and G (#21, #22 and Y).

20. 20
Chromosome
Group
Chromosome
Number
Chromosome Landmarks
A
1
#Largest metacentric
chromosome
#Lower 3 dark bands
(in q arm)
#Dark centromeric
band
#No dark bands in
the p arm
2
#Largest sub-
metacentric
chromosome
#Bands all over the p
and q arm
3
#Large metacentric
chromosome
#X-shaped band near
the centromere
B
4
#Large sub-
metacentric
chromosome
#Neck band
5
#Large sub-
metacentric
chromosome
#Belly band
C
6
#Medium-sized sub-
metacentric
chromosome
#Dark band at the tip
of p arm, rest
negatively stained
7
#Medium-sized sub-
metacentric
chromosome
#Eye band
8
#Medium-sized sub-
metacentric
chromosome
#Dome-shaped
centromeric band
#Lower dark band
9
#Medium-sized sub-
metacentric
chromosome
#Triangle-shaped
centromeric band

21. 21
10
#Medium-sized sub-
metacentric
chromosome
#3 prominent dark
bands in the q arm
11
#Medium-sized sub-
metacentric
chromosome
#Prominent dark
band away from the
centromere
12
#Medium-sized sub-
metacentric
chromosome
#Prominent dark
band towards the
centromere
X
#Medium-sized sub-
metacentric
chromosome #Dark
bands equally placed
from centromere
towards p and q arms
D
13
#Medium-sized
acrocentric
chromosome
#Dark bands in q
arm
14
#Medium-sized
acrocentric
chromosome
#Imaginary square
band in q arm
15
#Medium-sized
acrocentric
chromosome
#Prominent dark
band in the middle of
q arm, rest
negatively stained.
E
16
#Medium-sized
metacentric
chromosome
#Bands in both p and
q arms
#Dark centromeric
band
17
#Medium-sized sub-
metacentric
chromosome
#Prominent lower
band

22. 22
18
#Medium-sized sub-
metacentric
chromosome
#Imaginary square
band in q arm
#Teddy bear-shaped
structure
F
19
#Small-sized,
metacentric
chromosome
#Dark centromeric
band
#Negatively stained
p and q arms
20
#Small-sized,
metacentric
chromosome
#Bands in both p and
q arms
G
21
#Small-sized
acrocentric
chromosome
#Prominent dark
band from the
centromere.
#Fly-shaped
structure
22
#Small-sized
acrocentric
chromosome
#Dark band near the
centromere
Y
#Small-sized
acrocentric
chromosome
#Rich in
heterochromatin,
completely dark

23. 23
Some examples of karyotype and its interpretation are given below:
46,XY
Normal
male
46,XX
Normal
female
47,XY,+21
Trisomy
21: Down’s
syndrome

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45,X
Turner’s
syndrome
47,XXX
Triple X
syndrome
49,XXXXY
Klinefelter
Variant

25. 25
4. GenASIs Karyotyping Software
The karyotyping software used in Genetics Laboratory is GenASIs BandView®
Karyotyping
Software, developed by Applied Spectral Imaging (ASI), which identifies the chromosome
boundaries and provides tools and working panel to capture the metaphase chromosome spread
from the slide manually (using the ASI-CCD camera), process and align the chromosomes in a
systematic karyogram.
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CONCLUSION
A majority of the genetic abnormalities are directly related to the chromosomal aberrations. Cytogenetics is
the diagnostic study of the structure and properties of chromosomes and cell division, which employs
various methods, one of them being "karyotyping." It refers to a procedure of photographic representation of
a stained preparation in which the chromosomes are arranged in a standard manner. The development of
newer techniques in karyotyping has made it possible to visualize undetected chromosomal anomalies such
as microdeletions and microscopically undetected translocations. This report highlights the importance of
"karyotyping" and its importance in the diagnosis of human chromosomal disorders.
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49,XY,+3 mar
Marker
Chromosome