Comparative Evaluation of CD34+/CD33+/CD3+/CD19+ Microparticles in Peripheral Blood of Patients during Allogeneic Stem Cell Transplantation

Introduction Allogeneic hematopoietic cell transplantation (allo-HCT) has been widely used as a potential curative strategy of hematological malignancies. In principle, the conditioning and the immunological response of the graft are basic mediators of the procedure. Cell-derived microparticles (MPs) are small membrane vesicles considered as markers of cell activation and apoptosis. Recently it was shown that they participate in important biological functions at pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. The potential role of MPs from hematopoietic cells during HCT has not been addressed yet. In our previous work we have shown that CD34+ cells from cord blood transplants release MPs that carry microRNAs. The aim of this study was to quantify the MPs of CD34+/CD33+/CD19+/CD3+ cells in the peripheral blood of patients undergoing allo-HCT, compare them with the MPs of the graft that they have received and investigate their relationship with various parameters.

Methods Samples were collected from the graft and from the peripheral blood of 7 patients who underwent allo-HCT in our department. Four patients were diagnosed with acute myeloid leukemia (AML) and 3 with acute lymphoblastic leukemia (ALL). Informed consent was obtained from all patients. Patients received mobilized blood stem cell transplant from sibling donors. All patients were at complete remission before allo-HCT. Regarding the conditioning 5 patients received myeloablative regimen and 2 reduced intensity conditioning (RIC). All patients achieved successful and prompt engraftment. The MPs were isolated after centrifugation of the plasma and immunophenotyped by four-color fluorescence using antibodies against CD34, CD33, CD19 and CD3 as well as Annexin V (AnnV+). The number of MPs has been estimated in the graft as well as in the peripheral blood of the patients at days 0, 4 and 14 (day0, +4d, +14d) from the transplantation. Statistical analysis was performed using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables. Alterations in the number of MPs after day0 might be the result of the conditioning's effect and regarding the CD34+ MPs might be a correlation with the engraftment.

Results Myeloid-derived MPs(CD33+MPs) were decreased significantly from day0 to +14d (p<0,019,Spearman's rho =0,836) as well at +4d to +14d (p<0,049,Spearman's rho =0,757). Interestingly, stem cell derived-MPs (CD34+MPs) were significantly associated with B-lymphocyte-derived MPs (CD19+MPs) at +4d and +14d respectively (p<0,007,Spearman's rho =0,890; p<0,001,Spearman's rho =0,944). CD3+cells correlated significantly with CD3+MPs at +14d( p<0,034, Spearman's rho=0,791). The number of all measured AnnV+/CD34+/CD33+/CD19+/CD3+ MPs at +d14 was decreased compared to the number of AnnV+/CD34+/CD33+/CD19+/CD3+ MPs at day0 and +4d.

Conclusions In this study we have identified and monitored hematopoietic stem cell and MPs in the peripheral blood of patients undergoing allo-HCT. We have to mention that the number of MPs at day 0 represents the profile of the patient immediately after conditioning and before receiving the graft. The possible role of MPs in the engraftment could serve as a valuable parameter.