Thrombopoietin Level and Bone Marrow Megakaryocyte Colony Formation in Various Diseases with Thrombocytopenia / 대한소아혈액종양학회지

PURPOSE: Thrombocytopenia is a serious life threatening consequence in patients with bone marrow failure syndrome. Thrombopoietin (TPO), recently cloned by several groups has been shown to be a key regulation of megakaryopoiesis and thrombopoiesis. Recent studies have demonstrated a positive or negative relationship between TPO levels and platelet counts due to underlying disease states. To clarify the role of TPO in thrombocytopenic condition we determined plasma TPO levels and megakaryocyte colony assay. METHPDS: TPO levels were measured in thrombocytopenic patient with aplastic anemia, chemotherapy induced bone marrow failure, idiopathic thrombocytopenic purpura (ITP) and in newborn by ELISA (QuantikineTM, R&D System, USA). Controls were short statured normal children with normal platelet counts. Plasma was preserved in 20oC until test. CFU-mega was determined by MegaCultTM (Stem Cell Tech. Inc., Canada). Ficoll separated mononuclear cells were cultured for 10~12 days with TPO or stem cell factor (SCF) in 37degrees C 5% CO2 atmosphere, colonies were fixed, stained and examined with inverted microscope. Results were analysed by Student-t test. RESULTS: TPO levels were markedly increased in aplastic anemia and chemotherapy induced thrombocytopenia compared to those of normal controls. Patients with ITP had decreased level of plasma TPO. There was inverse relationship between platelet count and TPO levels for patients with aplastic anemia and chemotherapy induced thrombocytopenia. There was no definite relationship between platelet counts and TPO levels but inverse relationship between platelet counts and PDW levels in neonates was noted. The levels of TPO were increased after improvement of platelet in thrombocytopenic neonate. Megakaryocyte colonies were increased in the mononuclear cells of the patients with ITP and chemotherapy induced thrombocytopenia. There was little colony formation in aplastic anemia. TPO had no definite effect in megakaryocyte colony formation but SCF increased colony formation. CONCLUSION: TPO levels were increased in aplastic anemia and chemotherapy induced thrombocytopenia but decreased in ITP. There was inverse relationship between platelet count and TPO levels in aplastic anemia and chemotherapy induced thrombocytopenia. Thus TPO could be useful for differentiate the etiology of thrombocytopenia. Megakaryocyte colony was increased in ITP and chemotherapy induced thrombocytopenia, but decreased in aplastic anemia. SCF was effective in megakaryocyte colony formation. TPO and SCF will be helpful to increase platelet in thrombocytopenic patients. However, further study will be needed.

Megakaryocyte Colony Formation According to the Origin of Hematopoietic Stem Cell / 대한소아혈액종양학회지

BACKGROUND: Development of hematopoietic growth factor made it possible to treat anemia and granulocytopenia following intensive chemotherapy and for thrombocytopenia, recently found thrombopoietin(TPO) is being applied experimentally in several countries. The megakaryocyte colony assay can assess the effect of TPO on the thrombocytopenia resulted from cancer chemotherapy or hematopoietic stem cell transplantation. In vitro colony assay procedures for detecting human erythroid and granulocyte macrophage progenitors have been in widespread use for many years. However, reproducible assay methods for human megakaryocyte progenitors have lagged considerably behind especially in Korea. Duration platelet recovery following transplantation depends on the origin of the hematopoietic cells. Usually thrombocyte recovery is delayed following cord blood stem cell transplantation because of the small amount of cells administered. This study was carried out to investigate and establish the megakaryocyte colony assay of hematopoietic stem cells obtained from the various origin of the hematopoietic stem cells with or without TPO. METHOD: Mononuclear cells of bone marrow, peripheral blood and cord blood were collected following Ficoll density gradient centrifugation and megakaryocyte colony assay was done using MegaCultTM(Stem Cell Tech. Inc., Canada). After liquifying the agarose, mononuclear cells were added and then agarose and cell mixture were dispersed into the two wells of the chamber slide. These slides were incubated for 18~21 days at 37oC, 5% CO2. The megakaryocyte colonies were detected by staining of the cells with a primary antibody to the GPIIb/IIIa antigen, secondary antibody, alkaline phosphatase and Evans Blue in order. Changes of CD34 and GPIIb/IIIa positive cells were also analysed in flask culture using flow cytometry. RESULTS: CD34 positive cells were most abundant in the mononuclear cells of the bone marrow, meanwhile the number of CFU-GM and megakaryocyte colony were greater in the mononuclear cells of the cord blood. After administration of TPO, the cell number of megakaryocyte colony was increased dose dependently, but CFU-GM colony did not show any response to TPO. With flask culture, the cell number was decreased with or without TPO. However adding GM-CSF, IL3 and TPO to cord blood mononuclear cell, the number of the cord blood mononuclear cells was increased on the 5 th day. The amount of CD34 positive cells was increased dose dependently to TPO in one of two cord blood and one peripheral blood. The amount of GPIIb/IIIa positive cells was increased dose dependently to TPO following incubation of all the mononuclear cells. CONCLUSION: This study revealed successful result of megakaryocyte colony assay using MegaCultTM in various kinds of mononuclear cells and suggested that TPO was useful for CFU-mega colony formation. The amount of GPIIb/IIIa positive cells was increased with TPO in the flask culture. Therefore TPO could be useful for assessment of CFU- mega, and could be applied for the in vivo and in vitro expansion of megakaryocytes and platelets.