Transfusion, September 2016, Vol.56(9), pp.2336-2345
Byline: Sabine Huenecke, Melanie Bremm, Claudia Cappel, Ruth Esser, Andrea Quaiser, Halvard Bonig, Andrea Jarisch, Jan Soerensen, Thomas Klingebiel, Peter Bader, Ulrike Koehl BACKGROUND Excessive T-cell depletion (TCD) is a prerequisite for graft manufacturing in haploidentical stem cell (SC) transplantation by using either CD34 selection or direct TCD such as CD3/CD19 depletion. STUDY DESIGN AND METHODS To optimize graft composition we compared 1) direct or indirect TCD only, 2) a combination of CD3/CD19-depleted with CD34-selected grafts, or 3) TCD twice for depletion improvement based on our 10-year experience with 320 separations in graft manufacturing and quality control. RESULTS SC recovery was significantly higher (85%, n=187 vs. 73%, n=115; p〈0.0001), but TCD was inferior (median log depletion, -3.6 vs. -5.2) for CD3/CD19 depletion compared to CD34 selection, respectively. For end products with less than -2.5 log TCD, a second depletion step led to a successful improvement in TCD. Thawing of grafts showed a high viability and recovery of SCs, but low NK-cell yield. To optimize individualized graft engineering, a calculator was developed to estimate the results of the final graft based on the content of CD34+ and CD3+ cells in the leukapheresis product. CONCLUSION Finally, calculated splitting of the starting product followed by CD3/19 depletion together with CD34+ graft manipulation may enable the composition of optimized grafts with high CD34+-cell and minimal T-cell content. Article Note: This project was supported by the Frankfurter Stiftung fur krebskranke Kinder and Hilfe fur krebskranke Kinder Frankfurt e.V., Alfred und Angelika Gutermuth-Stiftung, and the LOEWE Center for Cell and Gene Therapy Frankfurt funded by Hessisches Ministerium fur Wissenschaft und Kunst (HMWK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supporting information: Additional Supporting Information may be found in the online version of this article Additional Supporting Information may be found in the online version of this article at the publisher's website: CAPTION(S): Material S1. Calculator for separation planning. Prior to manipulation PBSC data were entered: volume, CD3+ T-cells/A[micro]l as well as CD34+ SCs/A[micro]l (grey field). Based on aggregate quality data, defined limits for SC recovery of 85%, lower (10th percentile) and median CD3 log depletion with -3.1 and -3.6 were fixed in the calculator to calculate how much of the product to process for CD34 selection and how much for CD3/CD19-depletion. For instance, when the calculation indicated a T-cell dose〉300x103/kg bodyweight with log depletion -3.6, the decision was to split the product and to additionally do a CD34 selection. Fig. S1. Chronology of depletion quality for CD3/CD19-depletion and CD34-selection. In all flowcytometric assays the adapted ISHAGE protocol (Koehl et al.) in single platform with 7-AAD was used. In the years 2005 and 2006, quality control of grafts was measured on an EPICS XL 4-color, followed by 5-color FC500 flowcytometer. Optimizations included the implementation of the CD3 antibody for detection (SK7 clone), fluorescence minus one (FMO) control for CD3 region, criteria of measurement duration (minimum 100 events of CD3+ cells, maximum time stop 300s) and use of CD14 staining as dump channel. We could not find any statistical difference when analyzing depletion quality in regard to the year in which the purification was performed.
Stem Cells – Analysis ; T Cells – Analysis ; Stem Cell Transplantation – Analysis;