Preparation Of Peripheral Blood Cells For Chromosome Analysis (Phytohemagglutinin Assay)

The blood cell karyotyping method was developed to provide information about chromosomal abnormalities. Although lymphocytes do not normally undergo mitosis, they can be stimulated to divide in the presence of a mitogen. In order to prepare lymphocytes for chromosome analysis, after 2 to 3 days of mitogen stimulation, a mitotic inhibitor is introduced to the culture to pause cells in the metaphase stage. Following exposure to a hypotonic solution accompanied by fixing and staining, chromosomes can be observed under a microscope and evaluated for potential abnormalities

Test procedure

1. Inoculate approximately 0.5 mL of heparinized whole blood into a glass or plastic tube with 10 mL of medium.
2. Incubate the culture at 37°C in 5% CO₂ atmosphere for 72 hr.
3. Add 0.5 µg/mL of KaryoMAX Colcemid Solution (Cat. No. 15212012 or 15210040) to each culture tube.
4. Incubate the culture for an additional 15–30 min.
5. Transfer the culture to a centrifuge tube and spin at 500 x g for 5 min.
6. Remove the supernatant and re-suspend the cells in 5–10 mL of hypotonic 0.075M KCl (Cat. No. 10575090).
7. Incubate at 37°C for 10–12 min.
8. Spin at 500 x g for 5 min.
9. Remove the supernatant, agitate the cellular sediment and add drop-by-drop 5–10 mL of fresh, ice-cold fixative made up of 1 part acetic acid to 3 parts methanol. Leave in 4°C for 10 min.
10. Repeat steps 8 and 9.
11. Spin at 500 x g for 5 min.
12. Resuspend the cell pellet in a small volume (0.5–1 mL) of fresh fixative, drop onto a clean slide, and allow to air dry.

At this stage, the preparation can be stained with Orecin or Giemsa. Giemsa banding has become the most widely used technique, and the most common method to obtain this staining is to treat slides with Trypsin-EDTA (0.5%) 10X (Cat. No. 15400054).

Giemsa staining

Banding of chromosome with enzymes and stains is essential to identifying normal and abnormal chromosome structures.

Reagents

1. Gurrs 6.8 buffer tablets: (Cat. No. 10582013) one tablet (100 mL, 500 mL, 1 liter size) dissolved in distilled water
2. 0.9% sodium chloride: 4.5 grams NaCl made up to volume of 500 mL distilled water
3. Gurrs stain (R66) Giemsa:
   • 3.0 mL Giemsa stain (Cat. No. 10092013) added to 48.5 Gurrs 6.8 buffer (unless otherwise stated on C of A)
   • 1 mL acetone
   • Mix in Coplin jar
4. Trypsin 2.5%:
   • 2.5 mL Trypsin (10X) (Cat. No. 15090046)
   • 49.5 mL 0.9% NaCl
   • Mix in Coplin jar

Equipment

• Coplin jars or larger containers
• Forceps
• Coverslips (24 mm X 50 mm)
• 5 mL serological pipets (Cat. No. 170355)
• 1 mL serological pipets (Cat. No. 170353)
• 50 mL graduated cylinder
• 50°C warming oven 

Procedure

1. Six Coplin jars are used for the banding sequence
   1. First jar—0.125% trypsin/0.9% NaCl mixture
   2. Second jar—0.9% NaCl for rinsing
   3. Third jar—0.9% NaCl for rinsing
   4. Fourth jar— Gurrs Giemsa stain (R66) mixed with Gurrs 6.8 buffer and acetone
   5. Fifth jar— Gurrs 6.8 buffer for rinsing
   6. Sixth jar— Gurrs 6.8 buffer for rinsing
2. A slide is placed in the first Coplin jar containing the trypsin/NaCl mixture for a prescribed amount of time. This time may be as short as 10 sec or as long as 2 min, depending on the activity level of the trypsin being used.
3. After the trypsin time has elapsed, the slide is removed and rinsed by sequential dipping into the 0.9% NaCl rinsing jars.
4. The slide is then placed in the staining jar containing the Gurrs stain and buffer for 5 minutes. This time may vary somewhat, depending on the strength of the stain used.
5. After the staining time has elapsed, the slide is removed from the jar and rinsed by sequential dipping into the two Gurrs buffer rinsing jars.
6. The slide is removed from the last rinse and air dried and coverslipped with Cytoseal 60. It is allowed to dry in the oven (50°C) after which it is ready for metaphase scanning under the microscope.

Learn more about Gibco media, reagents, and supplements for cytogenetic analysis