Alloseq HCT: A Wide Range Chimerism Testing Solution with High Sensitivity, Accuracy and Precision for Routine Post Hematopoietic Cell Transplant Monitoring

Genetic chimerism testing in hematopoietic cell transplant (HCT) recipients is typically done using methods based on Short Tandem Repeats (STR), with lower limit of detection around 2%. More sensitive chimerism detection methods may aid in engraftment monitoring and potentially predict early relapse of the disease. We have developed and analytically validated a highly sensitive chimerism test based on Next Generation Sequencing that utilizes single-nucleotide polymorphisms located throughout the genome. The test combines a clinical laboratory compatible protocol with streamlined workflow and a fully automated analysis software to accurately quantify the level of chimerism. Twenty-two samples designed to mimic genomic DNA (gDNA) derived from HCT recipient samples were generated using four distinct gDNA samples mixed at fractions ranging between 0.1-30%. These samples were used to validate the entire AlloSeq HCT™ workflow across multiple DNA input amounts, operators, reagent lots and sequencing runs. Sequencing was performed on the Illumina MiSeq and chimerism quantification was done using the AlloSeq HCT software. The data obtained by the AlloSeq HCT was compared to the expected mixtures and a correlation of 0.99 was obtained. The assay was sensitive down to 0.1%. We next evaluated within-run and across-run variability, which were on average 4.7% and 4.1%, respectively. The within-run and across-run variability was calculated over a range of input gDNA between 3 ng and 100 ng, showing the robustness of the method at different test conditions. AlloSeq HCT test accurately and reproducibly quantified the level of chimerism across a wide dynamic range with higher sensitivity than current standard of care. Combined with automated data analysis, 24-hour workflow and minimal hands-on time, AlloSeq HCT is an optimal method with high sensitivity and precision for routine chimerism testing.