Monitoring of Hematopoietic Chimerism by Real-Time Quantitative PCR of Single Nucleotide Polymorphisms In Samples with Low DNA Quantities.

Abstract 3491

Novel methods to quantify chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) based on single-nucleotide polymorphism (SNP) specific real-time quantitative polymerase chain reaction (qPCR) require high amounts of input DNA. The applicability of these methods in cases where DNA quantity is limiting, however, is currently unclear. Here, we demonstrate that SNP typing performed with just 5ng of input DNA still allowed for the distinction of positive (mean ct 28.05) and negative (ct > 36) signals. In addition, we confirm the high informative value of a set of 19 SNP markers, with 12 markers exceeding 20% informativity in our population (n=74). Of interest, a four-fold reduction of input DNA for qPCR standard curves did not alter PCR efficiencies. Most importantly, in 7 out of 16 allogeneic HSCT patients who experienced disease relapse, a retrospective analysis using the SNP-qPCR method revealed re-appearance of recipient chimerism earlier (mean 95 days) compared to previous results obtained by a short tandem repeat (STR) specific PCR. Taken together our data clearly demonstrate that SNP-qPCR is more sensitive compared to the widely used STR-PCR even with reduced amounts of input DNA. We therefore recommend that SNP-qPCR is preferable to STR-PCR for chimerism analysis in cases where DNA quantity is a limiting factor.