Characterization of megakaryocytic cell lineage on man : normal and pathological situations

1.PL1 PLI which is produced as an ascitis in rat from spleen cells immunized with mononuclear blood cells and platelets was the target of this study to understand clearly its characteristics and its field of application. It was found to be one of the few antiplatelet monoclonal antibodies which does not recognize other nucleated blood cells. This was proved by the indirect immunofluorescence assay and by the electron microscopic study. The indirect immunofluorescence assay proved that PL1 does recognize thrombastenic platelets. From the effect of PL1 on the aggregation test, it appeared more clearly that neither GPIb nor the GPIIb/IIa complex are the antigen recognized by PL1 as it abrogates platelet aggregation with all the inducers. This indicates that the receptor site for PL1 might be another glycoprotein or another protein component of the glycocalyx which has a very important role in allowing in vitro aggregation to take place and which was not identified until recently, or PL1 might recognize an identical part of both GPIb and GPIIb/IIIa complex which appear to be responsible in allowing aggregation to take place by different inducers. The electron microscopy study using PL1 coupled with immunogold particles indicated that the antigen recognized by PL1 located on the outer surface of platelet (glycocalyx) emphasizing the fact that PL1 recognizes only the platelets in the blood. PLA was found to recognize the megakaryocyte at en earlier stage than other antiplatelet monoclonal antibodies. This stage might be the CFU-M as indicated by studies using the complement with PL1 in in vitro megakaryocyte colony growth. All the above findings prove that PL1, the new antiplatelet monoclonal antibody, can be useful in identifying the human platelets and megakaryocytes and might help in studying in vitro regulation of megakaryocytopoiesis by immunological techniques. For example, in our laboratory, PL1 has been to study human megakaryocytopoiesis in normal conditions and in some pathological conditions by the ELISA (enzyme-linked immunosorbent assay) techniques. The preliminary results are encouraging. In this technique, monuclear cells are incubated for 14 days in liquid medium in Titertek microtitre culture plates. Afterwards, cells are fixed to the plastic plates and PL1 is allowed to react for 30 minutes at 37°C. Later, an antibody specific to PL1 (rat immunoglobulin linked to peroxidise enzyme) is added to the plates for 30 minutes. Then, an enzyme substrate (Orthophenylene diamine dihydrochloride) is converted by peroxidase into a yellowish compound in the presence of H2O2. The intensity of reaction is measured by a spectrophotometer (Multiskan plate reader). The optical density is proportional to the concentration of the antigen recognized by PL1 and, therefore, to the amount of megakaryocytes arising in culture from their progenitors. This technique offers a new way to estimate human megakaryocytopoiesis in normal and pathological situations. 2. CMML According to the results obtained by our study, it appears that CMML is a disease of the pluripotent hematopoietic stem cell leading to abnormalities in all myeloid lineages and not only in the monocytic lineage which is the most significant feature of the disease. This feature is emphasized more through the differential counting of colonies for samples of blood and bone marrow from patients with CMML cultured in vitro where it has been shown that the most prominent type of cells is these colonies were the monocyte-macrophage cells. As stated above, ALISA in association with PL1 can be used to determine the efficiency of megakaryocyte production in different pathological conditions and to compare it with normal cases by applying it to in vitro cultures of megakaryocytes. Actually, ELISA can be used to understand more about megakaryocytopoiesis in different diseases such as CMML and add to the information we already obtained in the second part of our study.Thèse de doctorat en sciences médicales (physiologie) -- UCL, 198