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

Lymphocytes are differentiated cells which normally do not undergo subsequent cell divisions. By culturing lymphocytes in the presence of a mitogen (KARYOMAX® Phytohemagglutinin (M-Form) (PHA), Cat. No. 10576), they are stimulated to replicate their DNA and enter into mitosis. After an optimum time of the cells being cultured (46 h for a newborn and 68 h for an adult), a mitotic inhibitor, KARYOMAX COLCEMID® Solution (Cat. No. 15210 or 15212), is added to the lymphocyte culture for 20 min.

The addition of COLCEMID to dividing cells acts to prevent the synthesis of spindle fibers and, therefore, to stop mitosis in metaphase. Metaphase is the optimum phase of mitosis for microscopically visualizing the chromosomes. By submitting cells to a hypotonic solution and a series of fixation steps, metaphase chromosomes can be microscopicallyobserved and analyzed.

As a quality control measure, each lot of Phytohemagglutinin is tested as a chromosome reagent for the examination of metaphase spreads used for cytogenetic studies. This reagent is evaluated by supplementation to an approved, non-phytohemagglutinin containing chromosome medium. These samples are then supplemented with freshly collected human peripheral blood and have been found to be acceptable in their ability to produce blastogenesis with human lymphocytes when compared to a previously tested control.

Test Procedure

  1. The required volume of peripheral blood is collected aseptically in a sodium heparinized vacutainer tube or syringe.
  2. Add 10 ml of either PB-MAX Karyotyping Medium or KARYOMAX Peripheral Blood Karyotyping Medium to each sterile T-25 flask to be set up for the assay.
  3. Add 0.75ml blood to each tube.
  4. Incubate flask in CO2 incubator for 48 to 68 h with caps loose.
  5. Add 0.05-0.1 ug/ml COLCEMID to each flask for a 15 minute incubation.
  6. After 15 min, transfer flask contents to a 15 ml centrifuge tube and spin down at 1,200 rpm for 5 min.
  7. Remove supernatant and resuspend pellet.
  8. Add 10 ml of 0.068 M KCl to pellet and gently mix. Allow to sit at room temperature for 15 min. Add 0.5 ml of fixative (three parts absolute methanol to none part glacial acetic acid). Gently mix with pipette.
  9. Centrifuge for 5 min at 1,200 rpm. Aspirate off supernatant. Add 10 ml of fixative, mix, and let sit at room temperature for 10 min.
  10. Repeat centrifugation step. Add 5 ml of fixative, mix, and let sit for 10 min atroom temperature.
  11. Centrifuge and aspirate off supernatant. Add 5 ml of fixative and incubate for 10min at 4°C.