Chimerism monitoring after allogeneic hematopoietic stem cell transplantation using quantitative real-time PCR of biallelic insertion/deletion polymorphisms.

An accurate and sensitive determination of chimerism status is mandatory after allogeneic hematopoietic stem cell transplantation. We evaluated the performance of the AlleleSEQR Chimerism Assay, which is based on quantitative real-time PCR of biallelic indel markers, using 79 recipient-donor pairs. When the informativeness of the screening panel composed of 34 markers was determined using 130 unrelated individuals, it presented a >99.9% probability of selecting at least one informative marker. The analytic sensitivity of the indel marker assay was estimated using a serially diluted DNA mixture. The detection limit of the quantitative assay approached 0.024% when 250 ng of DNA was used. We used 175 samples that had undergone hematopoietic stem cell transplantation to compare the quantitative real-time PCR assay with short tandem repeat quantitation. Results from the AlleleSEQR Chimerism Assay showed excellent correlation with those from the short tandem repeat method (r(2) = 0.931). Moreover, in some patients with relapsed leukemia, the AlleleSEQR Chimerism Assay was able to detect an early increase in recipient DNA levels that was undetectable using the short tandem repeat method. The indel marker-based AlleleSEQR Chimerism Assay can be a useful tool for following up patients after hematopoietic stem cell transplantation because of its high sensitivity compared with the conventional short tandem repeat method.