Platform Independent Multiplex qPCR Research Assay for Chimerism Analysis

Aim: Following Hematopoietic stem cell transplantation (HSCT), the accurate monitoring of donor-derived cells prospectively is critical for understanding sustained engraftment or whether therapeutic intervention is needed, in turn predicting the success or failure of HSCT. We designed a highly sensitive, quantitative PCR (qPCR) research assay specifically for engraftment monitoring following HSCT in a research setting. It can accurately detect recipient cells up to 0.01% while also reducing workflow and analysis burdens as compared to routine STR testings. To enable broader access to the AlleleSEQR qPCR Chimerism Assay RUO across more clinical research laboratories, we have assessed performance of the multiplexed screening assay across five different qPCR platforms from four manufacturers.

Method: A panel of 34 qPCR research assays to bi-allelic indels across the genome enables informative marker identification and quantification to occur within a few hours and without the need for capillary electrophoresis. To decrease our current sample volume requirements, increase throughput and enable multiple donor screening scenarios across multiple transplantation centers, we evaluated a multiplexed assay approach for marker identification utilizing five different qPCR platforms. The alleles are scored in relation to amplification of an endogenous control assay and 6-8 samples can be evaluated simultaneously across all platforms.

Results: Multiplexing the assays to 2-4 assays/well yielded identical results when compared to single well 34 assays. In addition, using five different qPCR platforms i.e. Applied Biosystems 7500 and ViiA7™ Real-Time PCR System, Qiagen Rotor-Gene Q, Focus 3M™ Integrated Cycler and Roche LightCycler™ 480 System, the described multiplexed qPCR approach yielded results with 100% concordance in identifying informative markers on six unique Coriell DNA across platforms.  

Conclusions: The results from this multiplexed qPCR research system for screening of informative markers were 100% concordant to results generated when using the assays individually. Performance of the multiplex qPCR assays were independent of the platform providing supportive results for possible future use in transplant centers.