Multi-centre analytical performance verification of an IVD assay to quantify donor-derived cell-free DNA in solid organ transplant recipients.

Donor-derived cell-free DNA (dd-cfDNA) has been widely studied as biomarker for non-invasive allograft rejection monitoring. Earlier rejection detection enables more prompt diagnosis and intervention, ultimately improving patient treatment and outcomes. This multi-centre study aims to verify analytical performance of a next-generation sequencing-based dd-cfDNA assay at end-user environments. Three independent laboratories received the same experimental design and 16 blinded samples to perform cfDNA extraction and the dd-cfDNA assay workflow. dd-cfDNA results were compared between sites and against manufacturer validation to evaluate concordance, reproducibility, repeatability and verify analytical performance. A total of 247 sample libraries were generated across 18 runs, with completion time of <24 h. A 96.0% first pass rate highlighted minimal failures. Overall observed versus expected dd-cfDNA results demonstrated good concordance and a strong positive correlation with linear least squares regression r2 = 0.9989, and high repeatability and reproducibility within and between sites, respectively (p > 0.05). Manufacturer validation established limit of blank 0.18%, limit of detection 0.23% and limit of quantification 0.23%, and results from independent sites verified those limits. Parallel analyses illustrated no significant difference (p = 0.951) between dd-cfDNA results with or without recipient genotype. The dd-cfDNA assay evaluated here has been verified as a reliable method for efficient, reproducible dd-cfDNA quantification in plasma from solid organ transplant recipients without requiring genotyping. Implementation of onsite dd-cfDNA testing at clinical laboratories could facilitate earlier detection of allograft injury, bearing great potential for patient care.

HLA variants and TCR diversity against SARS-CoV-2 in the pre-COVID-19 era.

HLA antigen presentation and T-cell mediated immunity are critical to control acute viral infection such as COVID-19 caused by SARS-CoV-2. Recent data suggest that both the depth of peptide presentation and the breadth of the T-cell repertoire are associated with disease outcome. It has also been shown that unexposed subjects can develop strong T-cell responses against SARS-CoV-2 due to heterologous immunity. In this study, we explored the anti-SARS-CoV-2 T-cell repertoire by analyzing previously published T-cell receptor (TCR) CDR3ß immunosequencing data in a cohort of 116 healthy donors and in the context of immune reconstitution after allogeneic hematopoietic stem cell transplantation in 116 recipients collected during the pre-COVID-19 era. For this, 143,310 publicly available SARS-CoV-2 specific T-cell sequences were investigated among the 3.5 million clonotypes in the cohort. We also performed HLA class I peptide binding predictions using the reference proteome of the virus and high resolution genotyping data in these patients. We could demonstrate that individuals are fully equipped at the genetic level to recognize SARS-CoV-2. This is evidenced by the 5% median cumulative frequency of clonotypes having their sequence matched to a SARS-CoV-2 specific T-cell. In addition, any combination of HLA class I variants in this cohort is associated with a broad capacity of presenting hundreds of SARS-CoV-2 derived peptides. These results could be explained by heterologous immunity and random somatic TCR recombination. We speculate that these observations could explain the efficacy of the specific immune response against SARS-CoV-2 in individuals without risk factors of immunodeficiency and infected prior to vaccination.