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Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients.

ABSTRACT: Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patients' plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA.

SUBMITTER: Jeric Kokelj B

PROVIDER: S-EPMC8531215 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients.

Jerič Kokelj Barbara B Štalekar Maja M Vencken Sebastian S Dobnik David D Kogovšek Polona P Stanonik Matjaž M Arnol Miha M Ravnikar Maja M

Frontiers in medicine 20211008

Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting sin ...[more]

PMID: 34692738

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Project description:BackgroundFebrile haematological patients are at high risk for potential bloodstream infections, the rapid and accurate identification of pathogens is crucial for clinical diagnosis and treatment. Droplet Digital PCR (ddPCR) is a novel and ultra-sensitively molecular technique for the rapid detection of pathogens. We evaluated the ability of ddPCR to identify infectious etiologies to discuss the applicability of ddPCR in the diagnosis and treatment of infections for febrile haematological patients.MethodsThis study enrolled and analyzed 89 ddPCR tests performed on 71 febrile haematological patients. We conducted a comparison between ddPCR results, blood culture (BC), and conventional microbiological testing (CMT). Additionally, we analyzed the correlation between ddPCR results and inflammatory factors, as well as their impact on antimicrobial therapy.ResultsDdPCR detected 113 pathogens in 72 plasma samples, while CMT identified 39 pathogens in 32 plasma samples. The detection rate of bacteria and viruses using ddPCR was significantly higher than that of CMT (p <0.0001). The turnaround time (TAT) for pathogenic diagnosis was significantly shorter with ddPCR compared to CMT (p <0.0001). When we used the CMT as reference standard, the sensitivity and specificity of ddPCR were 93.8%, 26.3%, respectively. We observed a positive correlation between the ddPCR results and CRP, PCT and IL-6, and ddPCR (AUC=0.771) has better diagnostic performance. The anti-infective treatment strategies were adjusted for 30 patients based on the positive ddPCR results, with 86.7% (26/30) of these cases demonstrating effectiveness in the anti-infective treatment.ConclusionDdPCR has the potential to enhance pathogen detection in febrile haematological patients by offering high sensitivity, rapid, precise results, it demonstrates better diagnostic performance compared to inflammatory factors and can contribute to the real-time clinical optimization of antimicrobial regimens, thereby enhancing the efficacy of anti-infective therapy.

Project description:The incidence rate of human papillomavirus-driven oropharyngeal cancer (HPV-OPC) is increasing in countries with high human development index. HPV cell-free DNA (cfDNA) isolated from 3 to 4 mL blood plasma has been successfully used for therapy surveillance. A highly discussed application of HPV-cfDNA is early detection of HPV-OPC. This requires sensitive and specific cfDNA detection as cfDNA levels can be very low. To study the predictive power of pre-diagnostic HPV-cfDNA, archived samples from epidemiological cohorts with limited plasma volume are an important source. To establish a cfDNA detection workflow for low plasma volumes, we compared cfDNA purification methods [MagNA Pure 96 (MP96) and QIAamp ccfDNA/RNA] and digital PCR systems (Biorad QX200 and QIAGEN QIAcuity One). Final assay validation included 65 low-volume plasma samples from oropharyngeal cancer (OPC) patients with defined HPV status stored for 2-9 years. MP96 yielded a 28% higher cfDNA isolation efficiency in comparison to QIAamp. Both digital PCR systems showed comparable analytical sensitivity (6-17 copies for HPV16 and HPV33), but QIAcuity detected both types in the same assay. In the validation set, the assay had 80% sensitivity (n = 28/35) for HPV16 and HPV33 and a specificity of 97% (n = 29/30). In samples with ≥750 µL plasma, the sensitivity was 85% (n = 17/20), while in samples with <750 µL plasma, it was 73% (n = 11/15). Despite the expected drop in sensitivity with decreased plasma volume, the assay is sensitive and highly specific even in low-volume samples and thus suited for studies exploring HPV-cfDNA as an early HPV-OPC detection marker in low-volume archival material.IMPORTANCEHPV-OPC has a favorable prognosis compared to HPV-negative OPC. However, the majority of tumors is diagnosed after regional spread, thus making intensive treatment necessary. This can cause lasting morbidity with a large impact on quality of life. One potential method to decrease treatment-related morbidity is early detection of the cancer. HPV cfDNA has been successfully used for therapy surveillance and has also been detected in pre-diagnostic samples of HPV-OPC patients. These pre-diagnostic samples are only commonly available from biobanks, which usually only have small volumes of blood plasma available. Hence, we have developed a workflow optimized for small-volume archival samples. With this method, a high sensitivity can be achieved despite sample limitations, making it suitable to conduct further large-scale biobank studies of HPV-cfDNA as a prognostic biomarker for HPV-OPC.

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Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients.

Publications

Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients.

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