Erythrocyte-derived microvesicles may transfer phosphatidylserine to the surface of nucleated cells and falsely 'mark' them as apoptotic

Lysis of erythrocytes using hypotonic solutions is one approach to remove red blood cells (RBCs) from umbilical cord blood (UCB), bone marrow (BM), and peripheral blood (PB) before flow cytometric analysis or sorting of nucleated cells (NCs). Our team employed this separation step to prepare UCB-, BM-, or PB-derived cells to sort very small embryonic-like stem cells (VSELs). We noticed that depletion of RBCs from UCB by hypotonic lysis resulted in a significant increase in the number of NCs including VSELs that bind Annexin-V (Ann-V). Surprisingly, these cells were not apoptotic and displayed normal proliferative potential. To explain this discrepancy, we show that RBC-derived microvesicles (RMV) released during erythrocyte lysis may transfer phosphatidylserine (PS) to the surface of NCs and 'mark' them falsely positive as apoptotic cells. This observation should be considered whenever Ann-V binding viability assays are employed to evaluate the quality of NCs depleted from erythrocytes via hypotonic lysis.