CYTOGENETIC SAMPLING

Cytogenetics is the study of chromosomal structure, location and function in cells. It

includes the study of chromosome number and appearance (karyotyping), the physical
location of genes on chromosomes, and chromosomal behaviour in processes such as
cell division.

The study of chromosomes, which are long strands of DNA and protein that contain most of
the genetic information in a cell. Cytogenetics involves testing samples of tissue, blood, or
bone marrow in a laboratory to look for changes in chromosomes, including broken, missing,
rearranged, or extra chromosomes. Changes in certain chromosomes may be a sign of a
genetic disease or condition or some types of cancer. Cytogenetics may be used to help
diagnose a disease or condition, plan treatment, or find out how well treatment is working.

There are 3 major methods of cytogenetic testing.

1. Routine karyotyping

2. Fluorescent in situ hybridization (FISH)

3. Comparative genomic hybridization (CGH) and microarray comparative

genomic hybridization (aCGH)

KARYOTYPING : Definition and Principle

Karyotyping is a laboratory procedure that allows examination of a patient’s set of

chromosomes. “Karyotype” also refers to the actual collection of chromosomes being
examined. Examining these chromosomes through karyotyping allows your physician
to determine whether there are any abnormalities or structural problems.

Chromosomes, which are in almost every cell of your body, contain the genetic
material inherited from your parents. They are composed of deoxyribonucleic acid
(DNA) and determine the way that every human being develops.

When a cell divides, it needs to pass on a complete set of genetic instructions to each
new cell it forms. Normally, when a cell is not in the process of division, the
chromosomes are arranged in a diffuse, unorganized way. However, during division,
the chromosomes in these new cells line up in pairs. In a karyotype test, which
examines dividing cells, these pairs are arranged by their size and appearance,
allowing a doctor to easily determine if any chromosomes are missing or damaged.

Test Applications

 If there are an unusual number of chromosomes, if they are arranged

incorrectly, or if they are malformed, this can be a sign of a genetic condition,
such as Down or Turner syndrome.

 Karyotyping can be used to detect myriad genetic disorders. For instance, if a

woman has a premature ovarian failure, she may have a chromosomal defect
that karyotyping can distinguish. The test is also useful for identifying the
Philadelphia chromosome, the presence of which can signal chronic
myelogenous leukemia.

 Babies can be karyotype tested before they are born to diagnose genetic
abnormalities that indicate serious birth defects, such as Klinefelter syndrome,
in which a boy is born with an extra X chromosome.

Materials Needed

• Chromosome medium -- KaryoMAX Peripheral Blood Karyotyping

Medium. These bottles usually come in groups of 10 (each bottle containing 5
mL of media).
• Colcemid solution (10 ml bottle) --- KaryoMAX Colcemid liquid
• Giemsa stain (100 mL bottle) --- KaryoMAX Giemsa Stain Stock
• Potassium chloride solution, 0.075M (about 10 mL per student) ---
KaryoMAX Potassium
Chloride Solution, 0.075M
• Sterile 1 or 5 mL syringe21-gauge needle for the syringeGreen-top
vacutube21-Guage multi draw needle . 15 mL centrifuge tube
• Pasteur pipette bulb for the Pasteur pipette 70 percent isopropyl alcohol pad

Equipment Needed

• Centrifuge
• Glacial acetic acid
• Absolute methanol
• Refrigerator
• Incubator
• Frosted glass slides
Procedure

A. Preparation of cell culture

1. Using a 21-gauge multi-draw needle and a green top vac tube, a qualified
technician withdraws blood from the patient (donor).
2. Prepare a sterile 5 mL syringe with a 21-gauge needle.
3. Wipe the top of the green top tube [Bottle with a needle] (containing blood)
with an isopropanol alcohol pad.
4. nsert the needle on the syringe into the green top and withdraw a few
milliliters of blood.
5. Open the bottle of chromosome medium and place five to ten drops of
blood into the medium. Sterile technique must be used because it is possible to
cause major contamination during this procedure.

B. Incubation

1. Mix the medium and blood by gentle inversion and place the bottle in a
preheated incubator at 37o C.
2. Incubation for 70 hours
3. Mix gently by inversion twice a day during incubation.

C. Stopping the cell division at metaphase :

1. Pre-warm the Colcemid in the incubator at 37 degrees Celsius. CAUTION:

Colcemid can be dangerous, so handle with care. Colcemid is a mitotic spindle
inhibitor. If splashed on the skin, rinse immediately and seek medical help.
2. Add 0.05 mL (50 microliters) of prewarmed 37oC Colcemid to the culture.
Mix gently and put the culture back into the incubator.
3. Incubate for 30 to 60 minutes.

D. Hypotonic treatment of the red and white blood cells

1. Remove the blood and Colcemid solution from the incubator and mix
gently.
2. Put the entire contents of the bottle into a conical centrifuge tube. If conical
tubes are not available, regular tubes can be used.
3. Centrifuge for six minutes at 500 - 900 rpm (see notes at the end of the lab
regarding centrifuge speed).
4. After six minutes, turn off the centrifuge and wait for a complete stop.
Carefully remove the tube.
5. Remove the supernate (clearish fluid on top) with a Pasteur pipette. Be very
careful not to disturb the button of cells on the bottom. Make sure that the bulb
of the pipette is depressed before it is inserted into the test tube. Leave some
fluid (anywhere from * to * mL) on the top of the button of cells. When
withdrawing fluid, keep the pipette tip against the side of the test tube to avoid
any shaky movements.
6. Add 1 mL of warmed 37oC hypotonic solution to the tube. Mix by flicking
the tube with your finger. Now add another nine mL of hypotonic solution.
The hypotonic solution should not be in contact with the cells for more than a
total of 24 minutes. Excess exposure may cause the rupture of the white blood
cells.
7. Thoroughly mix all the hypotonic fluid with the cells. This is done by
drawing all the mixture at the bottom of the tube into a Pasteur pipette and
forcing it out again. Do this two or three times to thoroughly mix.
8. Place the mixed solution into the 37oC incubator for nine minutes
9. The fixative solution must be made fresh. While the hypotonic solution is
working, make up the fixative solution as follows: add three parts chilled
absolute methanol (or as close as you can get) to one part glacial acetic acid.
Both chemicals should be as pure as possible.
10. After nine minutes, centrifuge for six minutes at 500 to 900 rpm.
11. Remove the supernate. leaving * or * mL of fluid on top of the button of
cells. At this time you probably have a small whitish or reddish film at the
bottom and slightly up the side of the tube. The film contains large quantities
of red blood cell debris and the enlarged white blood cells. Your entire
experiment, up to this point, has been to isolate that film at the bottom of the
tube.

E. Fixing the cells

1. Add 5 ml of the fixative solution to the centrifuge tube.

2. With a Pasteur pipette, mix the fixative and button of cells by drawing the
mixture into the Pasteur pipette and forcing it out again. Do this three or four
times. Place this solution of cells and fixative into a refrigerator for 30
minutes. Make sure the test tube is covered with aluminum foil because of the
smell. The 30 minutes in a refrigerator is a minimum; actually, it is possible to
keep cells in the refrigerator overnight. During this time, practice dropping
water on slides (instructions to follow).
3. After refrigeration, centrifuge the tube for six minutes at 500 to 900 rpm.
4. Remove the supernate and add another 6 mL of cold fixative and mix as you
just did in the instructions above.
5. Centrifuge the tube for six minutes at 500 - 900 rpm.
6. Repeat the above two steps.
7. Remove the supernate leaving about * mL of fluid at the bottom of the tube.
It is this remaining material that you will drop on your slides in the next
section. If you cannot see any material at the bottom of the test tube, do not
despair; proceed as though there is visible material present. It is often very
difficult to see.

F. Making the chromosome slides

1. The slide must be exceptionally clean. Use new, factory pre-cleaned, frosted
slide. The chromosome separation seems to work best if the slides are chilled
in the freezer first.
2. Lay five or six slides next to each other on paper toweling with no
separation between them.
3. Withdraw the entire contents of the centrifuge tube into a Pasteur pipette.
Be careful not to draw the fluid any farther than necessary into the pipette. The
cells have a tendency to attach to the sides of the pipette.
4. From a height of about 18 inches, drop two or three drops of fluid onto each
side.
5. Allow the slides to dry thoroughly. In fact, the best way to 'cure' the slides
are to place them in the incubator (37oC) overnight.
6. Stain the slide by immersion in fresh Giemsa stain for 7 - 10 minutes.
7. Remove the slides from the stain and rinse in distilled water until ALL the
excess stain is removed.