*Purpose: Peripheral blood chimerism is associated with clinically relevant events post liver transplantation (LT). Accurate quantification of donor chimerism in LT recipients with ultra-sensitive chimerism test AlloHeme may provide a novel biomarker to assess the association of chimerism with graft and recipient outcomes such as GvHD, engraftment, and tolerance. The most common method for chimerism testing utilizes Short Tandem Repeats (STR). However, STR-based chimerism has low sensitivity and high variability. These limitations may inhibit consistent and accurate detection of chimerism, including micro chimerism (defined as <1% donor chimerism). We describe the validation of AlloHeme^TM , an ultra-sensitive Next Generation Sequencing (NGS) based chimerism surveillance solution and compare it to the STR-based chimerism method.

*Methods: To evaluate the chimerism assays, reference samples were prepared by mixing DNA samples to mimic post-transplant DNA ratio in the range from 0.01% to 75%, using unrelated and related donors. AlloHeme testing was performed at CareDx’s central laboratory in Brisbane, CA, using NGS to genotype 405 Single Nucleotide Polymorphisms (SNPs) distributed across all 22 pairs of human autosomes. The STR test was performed in a CLIA-certified/CAP-accredited clinical laboratory, with an established Limit of Detection (LOD) of 1%.

*Results: For unrelated donors, AlloHeme LOD was 0.035% and 0.044% when 100ng or 8ng of DNA was used respectively. For haplo-identical donors, LOD was 0.043% and 0.067% when 100ng or 8ng of DNA was used respectively. The assay was highly precise with coefficients of variation of 1.35% and 1.39% for repeatability and reproducibility respectively, when tested at 1% chimerism across 6 replicates. Accuracy was analyzed and compared across AlloHeme and STR close to their respective LODs at 0.06% and 1% respectively, using bias calculation [(observed-expected)/expected]. At 0.06% chimerism, AlloHeme was highly accurate with no bias, while STR showed 75% bias at 1% chimerism.

*Conclusions: These results demonstrate the high accuracy and sensitivity, at low DNA inputs, of AlloHeme in detecting micro chimerism compared to the commonly used approach using STR. AlloHeme may be suited for LT recipient surveillance to identify early GvHD or longer term engraftment and tolerance acquisition, allowing precise clinical diagnosis and potential intervention for improved patient outcomes. AlloHeme as an ultra sensitive chimerism detection tool, along with a suite of other post-LT relevant biomarkers, is being evaluated for clinical validation in the prospective, multi-center Molecular Assessment and Profiling of Liver transplant rEcipients (MAPLE) study (NCT 04793360).

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