Depletion of TCRαβ+ T cells and CD19+ B cells using the miltenyi clinimacs cell separation system

Abstract

Introduction: T cell depletion of haemopoietic progenitor cell (HPC) collections has been demonstrated to reduce the incidence of acute and chronic graft versus host disease (GVHD) following allogeneic transplantation using alternative donors such as haploidentical or voluntary unrelated donors. Clinical systems for T cell depletion include the depletion of TCRαβ+ T cells and CD19+ B cells using the Miltenyi CliniMACS cell separation system. The increasing complexity of processing associated with the depletion of TCRαβ+ T cells and CD19+ B cells presents several challenges for the processing facility. Materials (or patients) and methods: TCRαβ+ T cells and CD19+ B cells were depleted from HPC, Apheresis (HPC(A)) collected from 3 haploidentical donors and a matched unrelated donor using the Miltenyi CliniMACs cell separation system. The depletion was scheduled for day 0 of the transplant protocol with the procedure taking from 14 to 17 h to perform. Results: The median number of CD34+ cells pre depletion was 45.8 x 106 /kg, TCRαβ+ T cells was 15.9 x 108/kg and TCRgd+ T cells was 3.6 x 107/kg. The median log depletion of TCRαβ+ T cells was -4.1 (-3.9 to -4.3) and CD20+ B cells was - 2.6 (-2.4 to -2.7). When 410 x 106 CD34+ cells per kg recipient weight were recovered, the infusion of TCRαβ+ / CD19+ depleted HPC(A) was limited to a maximum of 5 x 104 TCRαβ+ T cells per kg recipient weight (n=3). All patients engrafted rapidly (ANC> 0.5 x 109/L between days 9-14 and Plt>50 x 109/L between days 9-14) with minimal acute GVHD observed post transplant. One recipient of a haploidentical transplant using TCRαβ+ & CD19+ depleted HPC(A) developed EBV associated post transplant lymphoproliferative disorder. Conclusion: Challenges for the processing laboratory include flow cytometric analysis for very low numbers of viable TCRαβ+ T cells and CD20+ B cells post selection that necessitates the analysis of large numbers of CD45+ cells (43 x 106 CD45+ cells) for a statistically significant result, the time taken to complete the procedure including the cryopreservation of excess HPC(A) collected but not depleted, and excess TCRαβ+ T cell & CD19+ B cell depleted HPC(A) not infused, and the maintenance of staff competency in an infrequent but complex processing procedure and the associated flow cytometric analyses.L718302822015-04-10 <br />