Quantitation of megakaryocyte size, number and distribution during altered megakaryocytopoiesis in mice

The objective of this study was to validate quantitative methods for determining megakaryocyte diameter and number in the bone marrow and spleen following stimulated- rabbit antimouse platelet serum (RAMPS)- and inhibited-isobaric hypoxia at 5% ambient oxygen for 14 days-megakaryocytopoiesis in mice. In addition, cell size and number were compared between bones, cell diameter was compared between smear and histological section preparations and two general quantitative methods for calculating megakaryocyte diameter and number were compared for precision and ease of use.

Following treatment, blood was collected for the measurement of common hematologic parameters. The mice were then sacrificed and the sternum, femur, humerus, tibia and spleen were collected and processed for light microscopic quantitation. Megakaryocyte diameter was calculated from frequency distributions of cell diameters after correction for optically lost profiles. The number of megakaryocytes per unit volume of bone marrow or spleen was then calculated from mean cell diameter and an estimate of the number of cell profiles per unit of section area after correcting for errors due to tissue section thickness. Both RAMPS and 14 days of continuous hypoxia produced increased mean megakaryocyte diameter suggesting stimulation of megakaryocyte maturation. However, megakaryocyte number was significantly increased after RAMPS and significantly decreased after hypoxia, suggesting that RAMPS stimulates and hypoxia inhibits stem cell differentiation into megakaryocytes. The relative diameters of the megakaryocytes in the bone marrow were similar to the spleen, but the spleen had significantly lower numbers than in the bone marrow. Based on statistical estimates of precision, megakaryocytes in section preparations can be modeled as spherical for all treatments (hypoxia, RAMPS and control) with minimum error. Megakaryocytes flatten out on smear preparations, thus increasing their diameters. Also, fewer megakaryocytes were found using the smear compared to the section preparations. Under normal conditions, a 63% increase in diameter of the megakaryocytes was found in smear preparations compared to the section preparations. However, smear preparations showed constant and reproducible results suggesting that megakaryocyte diameters may be interconverted from smear preparations to section preparations by multipling the smear diameter by a factor of 0.63.