Karyotyping- Definition, Principle, Preparation and Technical Procedure

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 you have 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 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 (one bottle per student)--- 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-guage needle for the syringeGreen-top vacutube21-guage multidraw needle (for vactube)15 mL centrifuge tube
• Pasteur pipetteBulb for the pasteur pipette70 percent isopropyl alcohol pad

Equipments Needed

• Centrifuge
• Glacial acetic acid (ACS grade)
• Absolute methanol (ACS grade)
• Refrigerator
• Incubator (capable of 37oC)
• Frosted glass slides

Procedure

1. Preparation of cell culture

• Using a 21-guage multi-draw needle and a green top vacutube (both supplied by the local hospital, if you are lucky), a qualified technician withdraws blood from the students.
• Prepare a sterile 5 mL syringe with a 21-guage needle.
• Wipe the top of the green top tube [Bottle with needle] (containing blood) with an isoproponal alcohol pad.
• Insert the needle on the syringe into the green top and withdraw a few milliliters of blood.
• 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. 

2. Incubation

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

3. Stopping the cell division at metaphase

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

4. Hypotonic treatment of the red and white blood cells

• Remove the blood and Colcemid solution from the [Centrifuge] incubator and mix gently.
• Put the entire contents of the bottle into a conical centrifuge tube. If conical tubes are not available, regular tubes can be used.
• Centrifuge for six minutes at 500 - 900 rpm (see notes at the end of lab regarding centrifuge speed).
• After six minutes, turn off the centrifuge and wait for a complete stop. Carefully remove the tube.
• 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.
• Add one 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 rupture of the white blood cells.
• Throughly 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.
• Place the mixed solution into the 37oC incubator for nine minutes
• 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 galacial acetic acid. Both chemicals should be as pure as possible.
• After nine minutes, centrifuge for six minutes at 500 to 900 rpm.
• 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. 

5. Fixing the cells

• Add 5 mL of fixative solution to the centrifuge tube.
• 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 of 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).
• After refrigeration, centrifuge the tube for six minutes at 500 to 900 rpm.
• Remove the supernate and add another 6 mL of cold fixative and mix as you just did in the instructions above.
• Centrifuge the tube for six minutes at 500 - 900 rpm.
• Repeat the above two steps.
• 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.

6. Making the chromosome slides

• 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.
• Lay five or six slides next to each other on paper toweling with no separation between them.
• 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.
• From a height of about 18 inches, drop two or three drops of fluid onto each side.
• Allow the slides to dry thoroughly. In fact, the best way to 'cure' the slides are to place them in the incubator (37oC) overnight.
• Stain the slide by immersion in fresh giemsa stain for 7 - 10 minutes.
• Remove the slides from the stain and rinse in distilled water until ALL the excess stain is removed.

Sources

Kaplan, B.J. (1978) Preparation of the normal karyotype (workbook). Chicago: American Society of Clinical Pathologists.
Macgregor, H.C. & Narley, J.M. (1983). Working with animal chromosome. New York: John Wiley & Sons