P070 A post-transplant chimerism study protocol that incoporated both STR and QPCR methods

Abstract

Aim To monitor post-stem cell transplant chimerism status, most clinical laboratories use commercial kits for short tandem repeat (STR) or quantitative real-time PCR (qPCR). We present a protocol that combines both systems to maximize monitoring performance. Methods PowerPlex Fusion STR kit (Promega) was used to genotype 22 autosomal STR markers. Donor (%) was assigned as an average from five informative loci (Types 1 and 2). Fragment analysis used 3130xl Genetic Analyzer (Applied Biosystems) and GeneMapper software (Thermo Fisher Scientific). KMRType kit (GenDx) was used for genotyping 29 insertion/deletion markers (4-color multiplex) on QuantStudio 12K Flex Real-Time PCR instrument (Thermo Fisher). Two informative loci (insertion in recipient -deletion in donor) selected were used to determine minor allele (%) (one-color) by KMRTrack kit (GenDx) on the Roche LightCycler480 Real-Time PCR instrument (KMRTrack). The average from the two loci was assigned as minor allele (%). Results The average number of STR informative loci for a patient-donor pair was 18.3 (range: 16–21) for unrelated (N = 30) and 9.0 (range: 6–16) for related (N = 30). The number of qPCR informative loci was 6.0 (range: 4–9) for unrelated (N = 28) and 3.5 (range: 3–4) for related (N = 2). In qPCR, sensitivity increases when more DNA is used per reaction and surpasses STR sensitivity of 3–5% when >2 ng DNA is used per reaction. We validated the qPCR method using 183 different donor-recipient-marker combinations and 14 ng whole blood DNA/reaction, which achieved sensitivity of 0.5%. Correlation coefficient (r) of intended % donor (mixed DNA) vs. assay-determined was 0.999 by STR (N = 12 for range: 5–95% donor) and 0.91 by qPCR (range: 0.5–10% minor allele). Average %CV was similar at low minor allele range; STR (29.6% for 5% donor detection) vs. qPCR (average 27.8% for detecting 0.5%–10% minor allele). At high minor allele range, %CV decreased for STR (CV Conclusions The combination of STR and qPCR can improve a post-transplant monitoring protocol. STR (higher precision) is used immediately after transplantation when high recipient (minor) alleles are present. Testing will be switched to qPCR (higher sensitivity) once complete chimerism is achieved.