Flow cytometric analysis of megakaryocyte ploidy. Comparison with Feulgen microdensitometry and discovery that 8N is the predominant ploidy class in guinea pig and monkey marrow

Flow cytometry (FCM) was adapted to measure ploidy values in megakaryocytes isolated from normal guinea pigs and African green monkeys. The FCM approach was validated by microscopic identification of electronically sorted 8N and higher ploidy cells and by comparisons with Feulgen microdensitometry on the same samples. Sorted 8N populations contained only megakaryocytes and were completely free of cell clumps; clumping was also absent in cell suspensions examined prior to FCM. Discrete megakaryocyte peaks were found at 8N, 16N, and 32N but not at intermediate values. The ploidy patterns of isolated megakaryocytes were similar by both methods (approximately 15% 8N, 58% 16N, and 27% 32N in guinea pigs). Unexpectedly, FCM analysis of unseparated marrow cell suspensions found that the most frequent ploidy class of megakaryocytes was 8N (50%). In contrast, by microdensitometry, the 8N class accounted for only 21% of the megakaryocytes in unseparated marrow cell suspensions. Since the traditional method depends on measurement only of easily recognizable megakaryocyte nuclei, a microdensitometric analysis of unselected marrow cells was carried out. Many more smaller 8N megakaryocytes (48%) were found by this approach, confirming the FCM data and the bias of the traditional sampling method. The decrease in 8N megakaryocytes (50%–15%) with enrichment was accounted for by the consistent finding that cells lost during isolation were predominantly 8N. with relative sparing of the 32N class. FCM provides more rapid and objective analysis of ploidy patterns in much larger samples of megakaryocytes than previous methods.