Comparison of an NGS and ddPCR based assay for the quantification of mixed chimerism post hematopoetic stem cell transplantation

Chimerism analysis is essential in the clinical follow-up of patients after allogeneic stem cell transplantation to monitor engraftment, graft failure, and predict relapse. Detecting low levels of mixed chimerism (<0.1%) is clinically relevant, allowing early adjustment of treatment strategies. Various molecular methods with different sensitivities for assessing the relative proportions of donor and recipient cells are commonly employed, such as STR, qPCR, dPCR, and NGS-based assays.

Until 2023, our laboratory used the Mentype® DIPscreen Kit (Biotype) to identify deletion/insertion polymorphisms (DIPs) in a 33-plex PCR, which occur individually in either the donor or recipient. Two recipient-specific markers were selected for digital droplet PCR (ddPCR) to detect chimerism with a high sensitivity of 0.06%. To reduce workflow complexity, we implemented the Devyser Chimerism NGS Kit (One Lambda). Informative markers are determined by performing a multiplex PCR on 24 biallelic Indels. In a second PCR, individual barcodes and sequencing adapters are introduced, and the amplicons are sequenced on the NovaSeq platform (Illumina). Data analysis is conducted using the Advyser software (One Lambda).

One hundred patients and EQA samples were analyzed with the Devyser Chimerism NGS Kit and compared to ddPCR results. For all samples, at least two informative markers (on average 8) were identified. With the defined limit of at least 4,000 reads per marker, a sensitivity of 0.025% is theoretically achievable. Out of 31 samples defined as complete chimerism by ddPCR, 29 had values below 0.02%, and two were below 0.06%. The remaining samples had mixed chimerism between 0.06% and 90%, in concordance with ddPCR results, with a mean coefficient of variation (CV) of 7%. Two samples showed a background of >0.06%, calculated from all homozygous non-informative markers, indicating sample contamination.

The Devyser Chimerism NGS Kit detects mixed chimerism with high sensitivity, comparable to ddPCR (0.06%). Below this threshold, distinguishing between background and real chimerism is challenging. The results demonstrate high inter-assay accuracy (CV <5%) and concordance with ddPCR. Evaluating 24 markers per sample, rather than two, allows for more reliable control of contamination. Among 189 patient/donor pairs analyzed so far, informative markers were unavailable in only two cases (1%).

no conflict of interest