One-step check purity of CD3+sorted cells during monitoring of hematopoietic chimaerism after HSCT
Résumé
Hematopoietic chimerism assays on blood is the optimal biological test for monitored engraftment and to quantify % of donor vs recipient cells in post-hematopoietic stem cell transplantation (HSCT) recipients. Focusing on selected CD3+ cells chimerism involves determining purity of the studied cell population according to European Federation of Immunogenetics standards. Flow cytometry is the reference method for determining CD3+ subpopulations purity, however, most molecular biology laboratories do not have access to this technology. In this study, we propose an alternative method to determine CD3+ cell purity simultaneously with a chimerism assay performed. Twenty nine samples sorted using EasySep HLA Chim WB CD3 Pos Kit (StemCell, France) were both analyzed by labelling CD2-PC7, CD3-APC A750, CD45-KO in flow cytometry (Navios, Beckmann Coulter, France) and by quantitative PCR (Light Cycler, Roche, France) using a non-T Genomic Detection kit (Accumol®, Canada) concomitantly during the chimerism run (Qtrace® qPCR assay, Jeta Molecular, Netherlands). The mean of CD3+ purity % using flow cytometry test was 99.12 (+/-1.12), non significantly different from that observed with the qPCR test: 98.47 (+/- 2.1) (P = 0.15). One sample showed an unexplained discrepancy between flow cytometry and qPCR assays with 99.97% vs 89.4% CD3 purity respectively. One other sample showed a difference of more than two standards deviations between these two assays although purity was higher than 95% (validation threshold in our laboratory) (95.48% vs 99.7% respectively).
In conclusion, checking purity of CD3+ sorted cells using a qPCR kit can be a feasible and robust alternative to flow cytometry assay. The advantage is time saving by performing both hematopoietic chimerism analysis and control purity of CD3+ sorted cells in one-step amplification, which allowed us to improve our results availability