Assessment of the Purity of Isolated Cd3+ T Cell Populations by A New Molecular Biology Technology: Impact on the Quantification of Chimerism Monitoring After Hematopoietic Stem Cell Transplantation

of Purity of Isolated Cd3+ T Cell Populations Molecular Biology Technology: Impact on the Quantification of Chimerism Monitoring After Hematopoietic Stem Cell Transplantation. Abstract Chimerism is the key analysis of monitoring after Hematopoietic Stem Cell Transplantation (HSCT). When chimerism is performed on sorted cells, the EFI and ASHI standards recommend documenting the purity of sorted cells populations. The current reference method for analyzing purity of sorting is today the Flow Cytometry (FCM) method, which is relatively expensive and needs a specialized laboratory. In this context, new qPCR Non-T Genomic Kit (Accumol ® , Calgary, AB, Canada), based on the quantification by real-time PCR of genomic rearrangements of TCR genes, undergone by T cells during their differentiation seems very interesting for HLA laboratory. In our hands, the qPCR Non-T Genomic Kit is an efficient tool, allowing accurate and reproducible assessment of contamination levels in a CD3+ sorted cell sample. Routine use will help meet EFI/ASHI requirements and improve the follow-up of HSCT recipient at a cost equivalent to that of FCM.

actually controversial to appreciate early the risk of acute or chronic GVHD and the risk of rejection [3][4][5]. From the total collected blood, T lymphocytes are isolated through most often at a positive sorting from beads having anti-CD3 antibodies. When performed from sorted cells whatever the method, the European Federation for Immunogenetic (EFI) and American Society of Histocompatibility and Immunogenetic (ASHI) recommends that laboratories check the purity of cell sorting, i.e EFI Standards, Section I4.190 and ASHI Guidelines, Section D.5.3.4.1.8. The current reference method of cell purification is Flow Cytometry (FCM), but this technique requires a specific expertise which is not available in all histocompatibility laboratories. Also, molecular biology could be an alternative method, easier and certainly less expensive.
Purity assessment using PCR-based Non-T Genomic Detection Kit (Accumol) has been showed to correlate closely to the purity defined gated cells by FCM [6]. The qPCR Non T Genomic Detection kit (Accumol) has also been developed to detect non-T cell genomic DNA by real-time quantitative PCR (RQ-PCR) in a CD3 + sorted sample. During T cell differentiation, the T Cell Receptor (TCR) undergoes gene rearrangements, including excision of some noncoding domains. The kit makes it possible, on the one hand, to amplify an Ultra-Conserved Sequence (UCS) of the genome found in all cells, and on the other hand a DNA sequence specific for non-T cells, corresponding to the domains excised during the T cell selection in thymus. After amplification in the presence of the Sybrgreen intercalant, a melting curve is produced from the RQ-PCR instruments which makes it possible to identify two peaks corresponding to the two amplified sequences. The first peak at a temperature of 80°C corresponds to the amplification of the UCS and the second peak at a temperature of 83.5°C corresponds to the amplification of specific fragment of the non-T population. The percentage of contamination is then calculated by the ratio of the height of the non-T specific peak to the UCS peak, based on a revised calibration curve after each new lot of the kit.
The aim of this study is to assess the purity of isolated CD3+ T cell populations by this new molecular biology assay. The CD3 + T     Between the qPCR Non-T Genomic Kit (Accumol) and the chimerism in one experiment of contaminations range.  were included in the routine chimerism study. Informed consent was obtained from parents/guardians for all samples. The DNA was conserved at -80°C. Purity of CD3 sorting was previously analyzed by FCM in 8 samples. Of these 8 samples, 7 exceeded 95% purity, and 1 were 50% purity.
The results obtained on patient samples, are presented in Table   3. They show that the purity of CD3 sorting by FCM are comparable to those of qPCR Non-T Genomic kit. Furthermore, this kit makes it possible to determine purity even when FCM cannot, especially when number of lymphocytes < 0,500 G/L. Finally, it shows that the purity of the sorted cells by EasySep Human Whole Blood CD3 positive selection kit is reliable if the patient has at least 10% of CD3 + T lymphocytes among its total white blood because 5 from 7 samples with < 10% CD3/WB have a rate of contamination of non CD3 > 5%. In our hands, the qPCR non-T Genomic kit seems to be an alternative technology to FCM to determine the percentage of cell purification. The sensitivity of this technique is near to 2-5%, with a good reproducibility of results between 1% and 30% contaminations.
Thus, it is therefore an efficient tool, allowing accurate evaluation, even retrospectively, of non-T cell contamination in a sorted CD3 + T cell sample. The study of the patient samples also made it possible to test the kit in the pathological conditions of lymphopenia for which the chimerism results obtained must be interpreted with caution.
Finally, its routine use will therefore meet the requirements of the EFI and improve the follow-up of HSCT patients at a cost equivalent to that of FCM.