Comparison of stereologic techniques for the quantification of megakaryocyte size and number

This work describes the relationship between megakaryocyte size and number in bone marrow, based on stereologic theory, and compares methods of megakaryocyte quantitation from bone marrow sections. Mice were assigned to four treatment groups and received a single injection of either saline (S), thrombocytopoiesis-stimulating factor (TSF), normal rabbit serum (NRS), or rabbit anti-mouse platelet serum (RAMPS). Nine mice from each group were killed daily for three days, and one femur was removed from each mouse and sections prepared for light microscopy. Megakaryocytes were quantified using two general methods. In method 1, diameters of megakaryocyte section profiles were estimated from measurements of cell perimeter, major and minor axes, and profile area. Profile-size distributions, corrected for errors due to section thickness and optically lost profiles, were used to calculate mean megakaryocyte diameter (D). Megakaryocyte diameter and an estimate of the number of profiles per unit section area (NA) were used to calculate the number of megakaryocytes per unit volume (NV) of bone marrow. In method 2, estimates of the cell volume fraction and NA were used to calculate megakaryocyte NV and D. All calculations were made in accordance with the principles of stereology, a branch of morphometry based on geometric probability. Both methods provided satisfactory precision in estimating megakaryocyte D and NV. In general, cell D and NV were unchanged in S- and NRS-treated mice and were significantly greater after TSF and RAMPS treatment. This study confirms the biological effects of RAMPS and TSF on megakaryocytes and presents practical, precise methods by which megakaryocytes can be quantified from bone marrow sections.