126-P

Abstract

Aim For leukemic stem cell transplant recipients, chimerism analysis can be used to detect recurrent disease. However, available methods lack the limit of detection and limit of quantification to detect recurrent disease below 1% recipient cells, where this test would be most useful. Therefore, we developed and validated a digital PCR method to detect and accurately quantify chimerism down to 0.01% recipient cells. Methods Small insertions and deletions (indels) were identified by searching the dbSNP database. Indels that have been shown to be polymorphic across multiple populations were chosen as potential informative markers. Primers and probes were designed to identify the predominant alleles and tested against a panel of DNA samples using the Droplet Digital PCR system (Bio-Rad). To assess the lower limit of detection and quantification, recipient DNA was diluted into donor DNA at low concentrations (0.1%, 0.05%, 0.01%, and 0%). The samples were tested multiple times by 2 technologists. Results Observed values correlated well to expected values [ Table 1 ]: Based on this data, the limit of quantification for a single well is 0.05%. By performing replicates and combining multiple wells to increase the total number of droplets, the lower limit of quantification can be improved to 0.01%. Conclusions Droplet Digital PCR allows accurate quantification of microchimerism down to 0.01%. The limit of detection with a single well (0.05%) is far better than what can be achieved with STR analysis and more accurate than what can be achieved with quantitative real-time PCR. In addition, by combining wells, a high degree of confidence can be achieved down to 0.01% recipient DNA.