Droplet digital PCR-based testing for donor-derived cell-free DNA in transplanted patients as noninvasive marker of allograft health: Methodological aspects

Abstract

In solid organ transplantation, donor-derived cell-free DNA (dd-cfDNA) is a promising universal noninvasive biomarker for allograft health, where high levels of dd-cfDNA indicate organ damage. Using Droplet Digital PCR (ddPCR), we aimed to develop an assay setup for monitoring organ health. We aimed to identify the least distinguishable percentage-point increase in the fraction of minute amounts of cfDNA in a large cfDNA background by using assays targeting single nucleotide polymorphisms (SNPs). We mimicked a clinical sample from a recipient in a number of spike-in experiments, where cfDNA from healthy volunteers were mixed. A total of 40 assays were tested and approved by qPCR and ddPCR. Limit of detection (LOD) was demonstrated to be approximately 3 copies per reaction, observed at a fraction of 0.002%, and which would equal 6 copies per mL plasma. Limit of quantification (LOQ) was 35 copies per reaction, estimated to 0.038%. The lowest detectable increase in percentage point of dd-cfDNA was approximately 0.04%. Our results demonstrated that ddPCR has great sensitivity, high precision, and exceptional ability to quantify low levels of cfDNA. The ability to distinguish small differences in mimicking dd-cfDNA was far beyond the desired capability. While these methodological data are promising, further prospective studies are needed to determine the clinical utility of the proposed method.

A mathematical description of non-self for biallelic genetic systems in pregnancy, transfusion, and transplantation

“…Non-self-situations are the prerequisite for an alloimmune response, although several additional conditions must be fulfilled for an immune response to occur. Assays for determining chimerism in transplantation without prior knowledge of the genotypes of the involved individuals have been developed (Clausen et al, 2023). For instance, one group has chosen 24 indel markers using both homozygous and heterozygous informative marker genotypes (Pettersson et al, 2021).…”

“…Assays for determining chimerism in transplantation without prior knowledge of the genotypes of the involved individuals have been developed (Clausen et al, 2023). For instance, one group has chosen 24 indel markers using both homozygous and heterozygous informative marker genotypes (Pettersson et al, 2021).…”

Technical Advances in Circulating Cell-Free DNA Detection and Analysis for More Personalized Medicine in Patients’ Care

“…Most studies were performed using EDTA BD vacutainer [57,58], which does not preserve blood cells from apoptosis and release of genomic DNA, affecting the quantity and quality of cfDNA itself [16,59] if plasma is not rapidly separated from the corpuscular part [13]. To prevent cfDNA degradation and its dilution into genomic DNA, ad hoc collection tubes are available from different companies (Qiagen, Germany, Roche, Swi erland, and Streck, USA), which were successfully used in some studies [60,61]. Their main advantage is that tubes keep cfDNA stable and free from genomic contamination for up to 14 days, improving the performance of the following research studies, drug discovery, and assay development.…”

All That Glitters in cfDNA Analysis Is Not Gold or Its Utility Is Completely Established Due to Graft Damage: A Critical Review in the Field of Transplantation

“…One reason may be using relatively long amplicons (100–130 bp) in the employed test. According to Clausen et al [ 36 ], the recommended amplicon length is 85.4 bp (66–103 bp).…”