Identification Of CD34+ Microparticles In Cord Blood Units
Authors: Xagorari Angeliki E , Samalidou Maria , Bougiouklis Dimitris , Kotta Konstantina , Sioga Antonia , Ekonomou Luiza , Anagnostopoulos Achilles , Sotiropoulos Damianos 
Published: October 2019

Introduction
Cell-derived microparticles (MPs), small membrane vesicles, are considered as markers of cell activation, as well as apoptosis and recently was shown to participate in important normal biological functions and pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. Recently platelet MPs have been detected in patients undergoing allogeneic stem cell transplantation. Umbilical cord blood is an alternative stem cell source for transplantation where the presence of MPs has not been extensively studied. The aim of this study is to quantify the MPs of CD34+ cells in cord blood units and to investigate their relationship with different parameters.

Methods
Cord blood units (CBUs) (n=41) were processed using the Sepax automated method (Biosafe). After processing, cryopreservation was performed using 10% DMSO in a controled rate freezer (IceCube, Sy-lab). Units were thawed after two weeks. Adjusted variables of the CBUs included their net volume, the absolute number of CD34+ per graft and their viability as determined by flow cytometry, the number of leukocytes as recorded with an hematology analyzer and the absolute number of erythroblasts as estimated by optical microscope after Giemsa staining. Mononuclear cells were seeded in semisolid cultures in the presence of a cocktail of growth factors for colony forming unit granulocyte/macrophage (CFU-GM), burst forming unit erythroid (BFU-E) and Colony-Forming Unit-Granulocyte, Erythrocyte, Monocyte/macrophage, Megakaryocyte (CFU-GEMM) colony growth. The MPs were isolated after centrifugation of the plasma and their number was determined after incubation with Annexin V and CD34 by flow cytometry. Electron microscope photographs were obtained, after the proceedings for TEM. Statistical analysis was done using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables.

Results
The mean number of AnnV+/CD34+MPs increased during post-processing (76,7±15,9) compared to the number of pre-processing AnnV+/CD34+MPs (73±22 ) and decreased during post-thawing (52,4 ±16,7). On univariate analysis found that the number of AnnV+/CD34+MPs (pre-processing vs. post-processing) has statistically significant mean positive correlation (p<0,012, Pearson's rho =0,823) whereas the number of MPs post-thawing was not statistically significant. Pre-processing AnnV+/CD34+MPs has statistically significant correlation with the absolute number of pre-processing CD34+ cell per unit (p<0,0001, Spearman's rho =0,835), post-processing (p<0,0001, Spearman's rho =0,814) and post-thawing CD34+ cells per unit (p<0,0001, Spearman's rho =0,993).
Correlation of post-processing CFUs and AnnV+/CD34+MPs showed that the number of AnnV+/CD34+MPs has statistically significant mean negative correlation to the number of CFU-GEMM (p<0,028, Spearman's rho = -0,348).

Conclusions
These data confirm the identification of stem cell-derived MPs from cord blood samples. Moreover, the number of pre- and post- processing AnnV+/CD34+MPs was statistically correlated. In addition, the absolute number of CD34 (pre-, post- processing and post-thawing) was associated with the AnnV+/CD34+MPs pre- processing. Thus it remains to be shown if the stem cell derived MPs could be a useful parameter to predict the quality of the cord blood unit.

Disclosures:
No relevant conflicts of interest to declare.