127-P

Abstract

We have designed a system of RUO Quantitative PCR assays and software enabling highly sensitive, stem cell engraftment monitoring. Our approach allows for facile quantification of minute amounts of minor components in a mixture, while also reducing workflow and analysis burdens. This approach expands the linear dynamic range over which increasing mixed chimerism may be monitored, effectively increasing the probability of detecting adverse transplant events earlier. We utilize a panel of 34 Quantitative PCR research assays to bi-allelic indels across the genome. Our system affords rapid pre-transplant marker identification and quantification of recipient levels post-transplant. It enables marker identification and quantification to occur within a few hours and without the need for capillary electrophoresis. In an effort to decrease our current sample volume requirements, increase throughput and enable multiple donor screening scenarios, we evaluated a multiplexed assay approach for marker identification. The Quantitative PCR assays are multiplexed, with 2-3 assays per well. The presence of alleles is scored in relation to amplification of an endogenous control assay. The results of samples within a group are compared against each other to identify informative markers for each sample. Up to eight samples can be evaluated in a single 96-well plate, with a variable number and configuration of samples within a group. The described multiplexed Quantitative PCR approach yielded results which were comparable to those generated when using the assays individually. When the single assay algorithms were applied to the samples tested in the multiplexed format, the same genotyping calls were made and informative alleles identified. Use of this RUO, multiplexed Quantitative PCR system for screening of multiple specimens enabled rapid identification of informative markers for all samples without post-PCR processing, while dramatically decreasing sample volume requirements. Sigua: Celera/Quest Diagnostics: Employee. Wadia: Celera/Quest Diagnostics: Employee; Stockholder. Bost: Celera/Quest Diagnostics: Employee.