QUANTITIZATION OF MEGAKARYOCYTOPOIESIS IN CULTURE BY COMPUTERIZED AUTOMATIC IMAGE-ANALYSIS

A computer-assisted automatic image procedure was established for the analysis of murine megakaryocytopoiesis in culture. This analysis syst em was based on acetylcholinesterase staining, a specific staining for murine bone marrow megakaryocytes, and an image capturing instrument with a computer program. Two kinds of routine megakaryocyte culture me thods were used, the plasma clot and the serum-free agar systems. A co mparison between manual counting and the instrument was made. The imag e analysis software was able to distinguish between megakaryocytes (MK ) at different stages of maturation. The results show that this analys is system can simultaneously detect not only the number of megakaryocy tes and their colonies in each dish, but also the surface area of indi vidual megakaryocytes, In addition, this analysis system functions aut omatically 24 hours a day and the results obtained are reproducible. U sing this system, we have confirmed previous observations that thrombo poietin (TPO) and heparin stimulate both proliferation and maturation of megakaryocytes. In addition, we found that platelet factor 4 (PF-4) significantly reduced the number of megakaryocytes but not their cell surface area, whereas TGF beta 1 decreased both number and surface ar ea of megakaryocytes, suggesting that PF4 and TGF beta 1 negatively re gulate megakaryocytopoiesis by different mechanisms. We noticed that m egakaryocytes grown under agar culture conditions regularly had an inc reased size in comparison with those grown in a plasma clot system, wh ich may be an indication that the plasma clot culture media contains a n inhibitor(s) of megakaryocyte maturation. Our data indicate that thi s image analysis system, in addition to its automatic and reproducible features, is more efficient and allows detection of more parameters t han routine manual microscopic detection.