Generation and flow cytometric quality control of clinical-scale TCRαβ/CD19-depleted grafts

Abstract

Background: The depletion of TCRαβ+ T cells and CD19+ B cells is a graft purification method for haploidentical stem cell transplantation (HSCT) retaining stem cells, NK cells and TCRγδ+ T cells. To avoid treatment-related occurrence of severe GvHD a precise quantification of residual TCRαβ+ T cells in the graft is of essential importance. Methods: Nine stem cell grafts were purified immunomagnetically on a CliniMACS device and flow cytometric quality control (QC) was performed before and after TCRαβ/CD19-depletion. Results: As a challenge a new 10-color QC-panel was established, which enables accurate quantification of the graft composition. The binding sites of residual TCRαβ+ T and CD19+ B cells were at least partly occupied by depletion antibodies impeding flow cytometric analysis. Based on respective controls and an assumed variation coefficient of 18%, the detection limit of residual TCRαβ T cells was 1 cell/µl. and 0.002% of CD45+ cells. Log-depletion of TCRαβ and CD19 cells was −3.9 and −3.3, respectively. The recovery was 82.1%, 67.1% and 72.7% for stem cells, NK cells and for TCRγδ+ T cells. Conclusions: In clinical use this method may help to improve transplantation outcome, due to the correct application of the desired stem cell and the limited T cell dose. The panel is designed for the QC following TCRαβ/CD19-depletion but is adaptable to other depletion strategies as well. © 2015 International Clinical Cytometry Society.