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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. 

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