Karyotype Analysis

Materials

• Fresh venous blood 8
• Heparinized syringes
• Eagle's spinner modified media with PHA
• Culture flasks
• Tissue culture grade incubator at 37° C
• 10 µ g/ml Colcemid
• Clinical centrifuge and tubes
• 0.075 M KCl
• Absolute Methanol and glacial acetic acid (3:1 Mixture, prepared fresh)
• Dry ice
• Slides, coverslips and permount
• Alkaline solution for G-banding
• Saline-Citrate for G-banding
• Ethanol (70% and 95% (v/v))
• Giemsa stain

Procedure 9

Draw 5 ml of venous blood into a sterile syringe containing 0.5 ml of sodium heparin (1000 units/ml). The blood may be collected in a heparinized Vacutainer, 10 and transferred to a syringe.

Bend a clean, covered 18 gauge needle to a 45° angle and place on the syringe. Invert the syringe (needle pointing up, plunger down), and stand it on end for 1-1/2 to 2 hours at room temperature. During this time the erythrocytes settle by gravity, leaving approximately 4 ml of leukocyte-rich plasma on the top, and a white buffy coat of leukocytes in the middle.

Carefully tip the syringe (do not invert) and slowly expel the leukocyte-rich plasma and the buffy coat into a sterile tissue culture flask containing 8 ml of Eagle's spinner modified media supplemented with 0.1 ml of phytohemagllutin (PHA).

BE EXTREMELY CAREFUL NOT TO DISRUPT THE RED BLOOD CELLS IN THE BOTTOM OF THE SYRINGE. RBC'S WILL INHIBIT GROWTH OF THE LEUKOCYTES.

Incubate the culture for 66-72 hours at 37° C. Gently agitate the culture once or twice daily during the incubation period.

Add 0.1 ml of colcemid (10 micrograms/ml) to the culture flasks and incubate for an additional 2 hours.11

Transfer the colcemid-treated cells to a 15 ml centrifuge tube and centrifuge at 225 xg for 10 minutes.

Aspirate and discard all but 0.5 ml of the supernatant. Gently tap the bottom of the centrifuge tube to resuspend the cells in the remaining 0.5 ml of culture media.

Add 10 ml of 0.075 M KCl, dropwise at first, and then with gentle agitation to the centrifuge tube. Gently mix with each ｄｒｏｐ.

START TIMING THE NEXT STEP IMMEDIATELY WITH THE FIRST ｄｒｏｐ OF KCL.

Let the cells stand EXACTLY 6 minutes in the hypotonic KCl.

THE HYPOTONIC SOLUTION SHOULD NOT BE IN CONTACT WITH THE CELLS IN EXCESS OF 15 MINUTES FROM THE TIME IT IS ADDED.

Centrifuge the cells at 225 xg for 6 minutes. Aspirate the KCl and discard all but 0.5 ml of the supernatant. Gently resuspend the cells in this small volume of fluid.

Add 10 ml freshly prepared fixative dropwise at first and then with gentle agitation. Gentle and continuous agitation is important at this step to prevent clumping of the cells. If the cells were not properly resuspended in step 10, the cells will clump beyond any further use.

Allow the cells to stand in fixative at room temperature for 30 minutes.

Centrifuge at 200 xg for 5 minutes and remove all but 0.5 ml of supernatant. Resuspend the cells in fresh fixative.

Wash the cells twice more in 10 ml volumes of fixative. Add the fixative slowly, recentrifuge, and aspirate the fixative as previously directed.

THE FIXED, PELLETED CELLS MAY BE STORED FOR SEVERAL WEEKS AT 4° C.

Resuspend the pellet of cells in just enough fixative to give a slightly turbid appearance.

Prop a piece of dry ice against the side of a styrofoam container and lace a clean slide onto the dry ice to chill the slide.

DRY ICE WILL CAUSE FROST BITE. HANDLE WITH TONGS ONLY.

Use a siliconized pasteur pipette to draw up a few drops of the suspended cells and ｄｒｏｐ the cells onto the surface of the chilled slide. Spreading of the chromosomes may be enhanced by dropping the cell suspension from a height of at least 12 inches. As soon as the cells strike the slide, blow hard on the slide to rapidly spread the cells. 12

FOR BEST RESULTS ALLOW ONLY ONE ｄｒｏｐ PER SLIDE.

Remove the slides from the dry ice and allow them to air dry. Perform the desired banding and/or staining procedures.

Procedure = Preparation of chromosomes for karyotype analysis can be performed in a number of ways and each will yield differing pieces of information. The chromosomes may be stained with aceto- orcein, feulgen or a basophilic dye such as toluidine blue or methylene blue if only the general morphology is desired.

Procedure = If more detail is desired, the chromosomes can be treated with various enzymes in combination with stains to yield banding patterns on each chromosome. These techniques have become common place and will yield far more diagnostic information than giemsa stain alone (the most commonly used process). A band is an area of a chromosome which is clearly distinct from its neighboring area, but may be lighter or darker than its neighboring region. The standard methods of banding are the Q, G, R, and C banding techniques. These are defined as follows:

• Q-banding
• Quinacrine stain
• Fluorescence microscopy

G-banding Giemsa stain Additional Conditions

• a. Heat hydrolysis
• b. Trypsin treatment
• c. Giemsa at pH 9.0

R-banding Giemsa or acridine orange Negative bands of Q and G reversed Heat hydrolysis in buffered salt

C-banding Giemsa stain Pretreatment with BaOH or NaOH followed by heat and salt.

The following directions are for a G-banding: 13

• Treat fixed and flamed slides in alkaline solution, room temperature for 30 seconds.
• Rinse in saline-citrate solution, 3 changes for 5-10 minutes each.
• Incubate in saline-citrate solution, 65° C for 60-72 hours.
• Treat with 3 changes of 70% ethanol and 3 changes of 95% ethanol (3 minutes) each.
• Air dry.
• Stain in buffered Giemsa for 5 minutes.
• Rinse briefly in distilled water.
• Air dry and mount.

Photograph appropriate spreads and produce 8 X 10 high contrast photographs of your chromosome spreads.

Cut each chromosome from the photograph and arrange the chromosomes according to size and position of the centromere.

Use Table 10.2 to identify the specific chromosomes.

Tape or glue each chromosome to the form supplied for this purpose.