One-step check purity of CD3+sorted cells during monitoring of hematopoietic chimaerism after HSCT

Archive ouverte

Desoutter, Judith | Joris, Magalie | Usureau, Cedric | Costa, Cecilia, Da | Marolleau, Jean-Pierre | Guillaume, Nicolas

Edité par CCSD ; Wiley -

Special Issue: Abstracts for the Joint 34th European Immunogenetics and Histocompatibility and 31st British Society for Histocompatibility and Immunogenetics Conference Glasgow, Scotland, United Kingdom, April 26‐29, 2020 Oral presentation P163. International audience. 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

• Description
• Sujet/Public
• Outil
• Outil d'anticipation
• Mémoire et thèse
• Gestion