Study On The Mechanism Of Thrombocytopenia In Acute Graft-versus-host Disease Status

[Objective]Some patients may suffer from one or two lines cytopenia after allogeneic stem cell transplantation,and the reduction of red blood cell(RBC)and megakaryocytes(Mk)are in the majority.The condition may result from acute graft versus host disease(aGVHD),the infection of cytomegalovirus(CMV),the use of ganciclovir antiviral drugs,etc.Prolonged cytopenia suggests poor prognosis,and it is one of the major reason of transplantation related complications and death.The mechanism of hematopoiesis dysplasia is not very clear,and it has the limited method of treatment.Patients with hematological malignancies who underwent allogeneic stem cell transplantation(allo-HSCT)in our center were included.We were dynamically observed the change of their white blood cell(WBC),absolute neutrophil count(ANC),hemoglobin,platelet count,and the cytokine level in bone marrow and blood.Moreover we analyses the clinical characteristics and risk factors of prolonged thrombocytopenia,and use colony forming unit(CFU)assay and multiparameter flow cytometry to analyse hematopoietic stem and progenitor cells.We aim to research the mechanism of cytopenia after allo-HSCT,especially thrombocytopenia,which may provide a new strategy for clinical.[Methods]Clinical characteristics of patients with thrombocytopenia afterallo-HSCT From August 2015 to February 2017,a total of 56 patients with hematological malignancies who underwent allo-HSCT and had complete medical records were included in the study(patients with disease recurrence were excluded).The complete blood count were observed before transplantation and at each time point after allo-HSCT,including 15 days,30 days,60 days,90 days,180 days,270 days and 360days.According to the onset of aGVHD and thrombocytopenia(TP),we divided patients into three groups:First)aGVHD/TP group:successful hematopoiesis reconstitution in 30days after allo-HSCT,the occurrence of more than II°aGVHD after 30 days,followed by persistent thrombocytopenia(<20x10~9/L)far more than 7 days,low hyperplasia of megakaryocyte in bone marrow,need for blood transfusion;Second)good hematopoiesis group:successful hematopoiesis reconstitution in 30 days,blood count increased to normal levels;Third)hematopoiesis dysplasia group:no aGVHD after allo-HSCT,slowly platelet recovery,platelet count remain less than 90x10~9/L in 90 days;Forth)normal control group:the healthy donor.Colony forming unit(CFU)assay of bone marrow Gain mononuclear cell from the each time point after allo-HSCT,and perform CFU assay,including colony forming unit-Granulocyte(CFU-G),colony forming unit-Macrophage(CFU-M),colony forming unit-Granulocyte/Macrophage(CFU-GM),colony forming unit-Mix,colony forming unit-Erythrocyte(CFU-E),Burst-forming unit-Erythroid(BFU-E),colony forming unit-Megakaryocyte(CFU-Mk).Every kind had 6 holes,and colony units was calculated under an inverted microscope at12 to 14 days of culture.Changes of hematopoietic stem/progenitor cell and hematopoietic microenvironment in bone marrow Gain mononuclear cellfrom each time point after allo-HSCT,and perform flow cytometry analysis.The immunophenotype of hematopoietic cells were defined as following:hematopoietic stem/progenitor cell(HSC/HPC,CD34~+),hematopoietic stem cell(HSC,CD34~+CD38~-),hematopoietic progenitor cell(HPC,CD34~+CD38~+),common myeloid progenitor(CMP,CD34~+CD38~+CD135~+CD10~-)and common megakaryocyte/erythrocyte progenitor(MEP,CD34~+CD38~+CD135~-CD10~-).The immunophenotype of endothelia cells,pericytes and megakaryocytes were defined as CD45~-CD34~+CD309~+and CD45~-CD34~-CD146~+,respectively.Statistical analysis Analyses the relationship between hematopoiesis recovery and aGVHD.IBM SPSS Statistics 20 and Graphpad Prism 5 was used to do satististical analysis.The survival rate was compared by using Kaplan-Meier survival curve.[Results]Firstly,a total of 13 patients developed II°or deeper aGVHD,10 of which were included into aGVHD/TP group.According to the onset of aGVHD and thrombocytopenia in 100 days after allo-HSCT,we divided patients into three groups:there were 17.86%(10/56)in the aGVHD/TP group,66.07%(37/56)in the good hematopoiesis group and 16.07%(9/56)in the hematopoiesis dysplasia group.The median onset time of a GVHD was 52(24 to 92)days.The overall survival(OS)in aGVHD/TP group were significantly lower than good hematopoiesis group(P<0.0001).The platelet count of the aGVHD/TP group was significantly lower than that of the good hematopoiesis group,and together with the decreased hemoglobin(HB)count,and no obvious reduction in ANC.The morphology of bone marrow showed that the number of megakaryocytes in aGVHD/TP group was decreased after aGVHD,and there was no significant statistical difference in the number of megakaryocytes in aGVHD/TP group and hematopoietic group.IL-2R significantly increased at the time of aGVHD,and this may be in inverse trend to platelet counts.Secondly,compared with the normal bone marrow from donor,the colony-forming ability of patients underwent allo-HSCT was decreased,and the MEP were the main one.After 60 days,the cells were restored to normal level gradually and slowly.The frequency of CFU in the aGVHD/TP group was significantly lower than that in the hematopoiesis dysplasia group,but there was no significant statistical difference between the two groups.Compared with good hematopoiesis group,the frequency of CFU was reduced in both lines of hematopoiesis cell in aGVHD/TP group,and the difference was statistically significant on 90 days after allo-HSCT,and megakaryocytes showed differentiation and developmental abnormalities.Thirdly,the proportion of HSC/HPC in the good hematopoiesis group was fluctuating,which was not completely returned to normal within 1 year after transplantation.The relative proportions of HSC/HPC,CMP and MEP in good hematopoiesis group were higher than those in other groups,but the proportions of HSC,HPC and MEP were lower than those in normal group,especially on the proportion of MEP.The difference between the aGVHD/TP group and the good hematopoiesis group was mainly on the proportions of HPC,CMP and MEP,and its overall level was significantly higher than that of the aGVHD/TP group 30 days later after allo-HSCT.Forthly,the poroportions of endothelia cells showed no significant difference between the aGVHD group and the good hematopoiesis group,the aGVHD/TP group and the normal control group respectively.The frequency of Mk and pericytes in aGVHD/TP group was significantly lower than the other two groups.Fifth,the porporation of Th1 in aGVHD/TP group was higher than good hematopoiesis group which was similar to that of the hematopoiesis dysplasia group.[Conclusion]Firstly,patients showed slowly recovery of hemoglobin and platelet counts after allo-HSCT.Comparied with good hematopoiesis group,the platelet count and megakaryocytes in bone marrow were significantly lower in aGVHD/TP group.From the recovery of various cells,hematopoiesis dysplasia group was different from aGVHD/TP group,which can be slowly hematopoiesis recovery.Secondly,the colony-forming ability of patients after allo-HSCT decreased,and CFU-E/BFU-E and CFU-Mk were the main ones.Megakaryocytes showed a small round of giant nucleus abnormalities and decreased platelet production,in addition to the obviously reduction number of erythrocyte and megakaryocytic colonies in aGVHD/TP group.This may be associated with the second strike of aGVHD on hematopoiesis cell.Thirdly,the porallo-HSCT after flow detection of bone marrow hematopoietic cells decreased to HSC/HPC and MEP-based,hematopoietic good group HSC/HPC ratio 1year after transplantation has not yet fully recovered to normal,suggesting that hematopoietic system was damaged by allo-HSCT.And the proportion of hematopoietic cells in aGVHD/TP group was more obvious,suggesting that the degree of hematopoietic inhibition of dry/progenitor cells in aGVHD environment was deepened.Fourthly,the number of megakaryocytes in aGVHD/TP group was significantly lower than that in the other two groups,and the abnormality of megakaryocytic cells might be related to the hematopoietic microenvironment abnormality have contributed.Fifthly,the porporation of Th1 may be associated with the development of thrombocytopenia after aGVHD.