Correlation Between CD34~+CD61~+ Megakaryocyte Precursors And Platelet Engrafment In Allogeneic Hematopoietic Stem Cell Transplantation

ObjectiveThis study was to quantify subpopulations of CD34~+ cells such as CD34~+CD61~+ that might represent megakaryocyte(MK)precursors in peripheral blood stem cell (PBSC)and bone marrow(BM)collections from normal,recombinant human granulocyte-colony stimulating factor(rhG-CSF)primed donors and to determine whether there is a statistical association between the dose infused megakaryocytic precursors and the time course of the platelet recovery following an allogeneic PBSC and/or BM transplantation.Materials and MethodsMaterials1.reagentsRat IgG2a-PE,IgG2a-FITC isotype control antibodies,CD34(FITC-labeled HPCA-2),and CD61(PE-labeled)rat anti-human antibodies(100 tests,Exalpha Biologicals Inc.USA);Ficoll-hypaque(1.077g/1,Shanghai,China),Hemolytic buffer(100ml,Baiyi Biotech,China),Bovine serum albumin(50g,Baiyi biotech, China).2.EquipmentsFlow blood cell separator(CS3000 plus Baxter CO.).Flow Cytometry(Epics XL, Coulter,USA) Methods1.Patient Characteristics24 patients with various hematologic malignancies took PBSCT/BMT from their HLA matched related or unrelated donors and haploidentical siblings in April 2007 and October 2007.20 evaluated patients were assigned into 2 groups according to different tansplant schemes.HLA matched group received PBSCT regime and haploidentical group were performed PBSCT combined with BMT.2.Mobilization Procedure,PBSC and BM HarvestNormal healthy donors were given rhG-CSF 5ug/kg/day subcutaneously twice daily and PBSCs were collected on day 4 and 5 in HLA identical group.In haploidentical group PBSCs were collected on day 4 and BMs were done on day 5.3.Flow CytometryThe total quantity and differential of collected white blood cells(WBC)were calculated for each PBSC and BM sample,CD34~+CD61~+ subpopulations in sample from PBSC/BM were measured by flow cytometry immediately or after storage over night.5×10~6 cells were stained(30min,wet ice,darkness)with different mono clonal antibodies.4.Conditioning Regimen,GVHD Prophylaxis and EngraftmentBased on Bu/CY/Ara-c or CY/TBUAra-c as preparative regimen,Antithymocyte globulin/cyclosporine/methotrexate/mycophenolate mofetil used for GVHD prophylaxis,rhG-CSF were intially administrated two group at day 7-10 following trasplantation for accelerating hematopoiesis.Engraftment was defined as following the nadir and first of 7 consecutive days on which the patient's unsupported platelet count was greater than 20×10~9/L.5.StatisticsSPSS13.0 software were used for data analysis,The absolute values of CD34~+ cells and its subsets were expressed as mean±s.d./kg(recipient body weight),minimum, maximum and median were also recorded.Non parametric t test(Mann-Whitney test) was used for comparing means of two groups,spearman method was used to evaluate the correlations between CD34~+,CD34~+CD61~+ cells and platelet engraftment,the Kaplan-Meier test was used to evaluate the difference in engraftment kinetics patients who received below or above the threshold dose;statistical significance was determined by log rank.For all the tests performed,a P-value of p＜0.05 was considered significant.Results1.The median number of infused CD34~+,CD34~+CD61~+ and CD34~-CD61~+ cells in haploidentical group were 6.24×10~6/kg(1.53～20.48),66.19×10~4/kg(8.16～493.83),and 34.38×10~6/kg(14.71～109.16)respectively,in HLA matched group those were 4.88×10~6/kg(1.00～8.24),14.16×10~4/kg(11.63～96.87),and 13.50×10~6/kg(1.74～35.61),resp ectively.CD34~+CD61~+ cells in two group presented a significant difference(p=0.031).2.Median days ofplatelet up to 20×10~9/L was 16.5(9.0～35.0)in haploidentical group respectively;in HLA matched group which was 10.5(6.0～37.0)respectively.No significant difference to platelet engraftment was encountered between two groups.3.For CD34~+ cell dose＞2×10~6/kg patients in HLA matched and haploidentical groups,The median number of CD34~+ cell dose infused was 14.93×10~4/kg in HLA matched group,and that was 76.76×10~4/kg in haploidentical group,A significant difference of median number for cells dose infused was presented between two groups (p=0.006),singnificant difference of time for platelet engrafment was also aquired (p=0.006).4.In this study,fair correlation to platelet engraftment was encountered with two subsets namely total CD34~+,CD34~+CD61~+cell dose,the number of CD34~+CD61~+ cells infused in 12 haploidentical patients or in 8 HLA matched patients was much better correlated to the time of platelet recovery up to 20×10~9/L than the number of CD34~+ cells infused in total 20 patients who have received an allogeneic PBSCT and/or BMT was.(r=-0.768 and p=0.004 for Haploidentical CD34~+CD61~+ cells,r=-0.747 and p = 0.033 for HLA matched CD34~+ CD61~+ cells,r=-0.449 and p=0.047 for CD34~+ cells). There was an inverse correlation between the number of infused CD34~+CD61~+ cells and time duration ofplatelet engraftment.Therefore,as the number of CD34~+ CD61~+ cells increased,duration of platelet engraftment(time to reach platelet count of 20×10~9/L)shortened significantly.ConclusionAfter allogeneic hematopoietic stem cell transplantation,a significant correlation of the number of infused CD34~+CD61~+ cells/kg with platelet recovery indicates that determining the number of megakaryocytic precursor cells by flow cytometry may predict the platelet reconstitutive capacity,and so it was in haploidentical PBSCT and BMT.For CD34~+ cell dose＞2×10~6/kg,patients in haploidentical group aquired much more CD34~+CD61~+cell dose than those in HLA matched group,time to reach platelet count of 20×10~9/L in haploidentical group was not prior to that in HLA matched group.Time for platelet engrafment shortened significantly in HLA matched group.