Follow-up of chimerism status after allogeneic HLA-mismatched stem cell transplantation by detection of non-shared HLA alleles

Background and Objectives. Chimerism studies after allogeneic transplantati on are usually performed using cytogenetic analysis, PCR-VNTR or PCR-STR. H ere, we report an alternative method for following the chimerism status aft er an HLA-mismatched stem cell transplantation (SCT), detecting the presenc e of non-shared HLA alleles by reference-strand mediated conformation analy sis (RSCA). Design and Methods. We tested this new approach on allogeneic related haplo identical SCT, unrelated cord blood transplantation, and HLA-mismatched unr elated donor SCT. The quantification of the chimerism was performed by lase r detection of fluorescent-labeled primers on an automated DNA sequencer. Results. In all cases this technique was able to detect mixed chimeras. The technique detected above 5% of residual cells when the analysis was based on HLA-class I and above 3% for HLA-class II. This sensitivity is similar t o that of the PCR-VNTR analysis. Interpretation and Conclusions. This method avoids the need to search for a n informative locus (which is essential for PCR-VNTR or -STR). Moreover, we did not find the phenomenon of preferential amplification that is observed with most VNTR, thus avoiding the need for construction of standard curves to quantify mixed chimeras. We conclude that the detection of the non-shar ed HLA alleles by RSCA is a useful approach for chimerism follow-up after H LA-mismatched SCT. (C)2000, Ferrata Storti Foundation.