Evaluation of techniques for increasing mitotic yield and chromosome length in leukemic cells

Previously reported techniques for the culture of bone marrow and unstimulated peripheral blood for cytogenetic analysis were evaluated in patients with acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML). Samples were treated in the following manner: (1) cold synchronization (exposure to 4 C for eight hours, then incubation at 37 C for 14.5 hours [CML] or 16.5 hours [AML]); (2) cold synchronization plus Hoechst 33258 (as first treatment, with Hoechst 33258 added to a final concentration of 50 micrograms/ml for the last five hours of incubation; (3) methotrexate synchronization (methotrexate added to a final concentration of 10(-7) M, incubation at 37 C for 17 hours, then washing of cells and addition of thymidine to a final concentration of 10(-5) M for an additional 5 hours 15 minutes of incubation); and (4) controls (untreated, incubation at 37 C). All cultures were harvested simultaneously in the usual manner. Our results suggest that cold synchronization provides improvement in mitotic yield and chromosome length as well as in chromosomal morphology, as compared to the results obtained with cold synchronization plus addition of the fluorochrome Hoechst 33258, with methotrexate synchronization or with standard direct culture techniques. Cold synchronization can readily be applied to the routine laboratory culture of hematologic cells for cytogenetic analysis.