Quantification Of Cell-free DNA Research Articles

Quantification of human papillomavirus cell-free DNA from low-volume blood plasma samples by digital PCR.

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.

(70) - PCR-Based Absolute Quantification of Donor-Derived Cell-Free DNA After Heart Transplantation

(70) - PCR-Based Absolute Quantification of Donor-Derived Cell-Free DNA After Heart Transplantation

Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

Clinical validation of a blood-based liquid biopsy test integrating cell-free DNA quantification and next-generation sequencing for cancer screening in dogs.

To validate the performance of a novel, integrated test for canine cancer screening that combines cell-free DNA quantification with next-generation sequencing (NGS) analysis. Retrospective data from a total of 1,947 cancer-diagnosed and presumably cancer-free dogs were used to validate test performance for the detection of 7 predefined cancer types (lymphoma, hemangiosarcoma, osteosarcoma, leukemia, histiocytic sarcoma, primary lung tumors, and urothelial carcinoma), using independent training and testing sets. Cell-free DNA quantification data from all samples were analyzed using a proprietary machine learning algorithm to determine a Cancer Probability Index (High, Moderate, or Low). High and Low Probability of Cancer were final result classifications. Moderate cases were additionally analyzed by NGS to arrive at a final classification of High Probability of Cancer (Cancer Signal Detected) or Low Probability of Cancer (Cancer Signal Not Detected). Of the 595 dogs in the testing set, 89% (n = 530) received a High or Low Probability result based on the machine learning algorithm; 11% (65) were Moderate Probability, and NGS results were used to assign a final classification. Overall, 87 of 122 dogs with the 7 predefined cancer types were classified as High Probability and 467 of 473 presumably cancer-free dogs were classified as Low Probability, corresponding to a sensitivity of 71.3% for the predefined cancer types at a specificity of 98.7%. This integrated test offers a novel option to screen for cancer types that may be difficult to detect by physical examination at a dog's wellness visit.

Diagnostic value of quantification of cell-free DNA for suspected gallbladder cancer.

An accurate preoperative diagnosis as the basis for deciding the most appropriate surgical procedure is essential for patients with suspected gallbladder cancer (GBC). The aim of this study was to investigate the usefulness of cell-free DNA (cfDNA) for the preoperative detection of ≥T2 invasion in patients with suspected GBC. Twenty-four patients who underwent resection for suspected GBC were enrolled. The concentration of cfDNA obtained from blood samples preoperatively was measured and evaluated in two distributions. The first peak (less than 200 base pairs) of cfDNA distribution was defined as the shorter fragment cfDNA, considered to originate mainly from apoptosis; and the second peak (200 base pairs or more) was defined as the longer fragment cfDNA, originating mainly from necrosis. Pathological analysis identified benign disease in 12 patients and GBC in 12 patients, of whom 6 patients had ≥pT2 GBC. Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 were significantly higher in the ≥pT2 GBC group than in the benign/<pT2 groups (2.1 [0.7-11.0] vs 4.5 [1.7-13.0], P = 0.033 and 14.0 [<2.0-401] vs 37.0 [26.0-141.0], P = 0.007, respectively). When limited to patients in the GBC group (n = 12), only cfDNA of longer fragments was significantly lower in the ≥pT2 group than the <pT2 groups (2.98 [1.88-4.61] vs 1.98 [1.42-2.42], P = 0.026) but cfDNA of shorter fragments showed no significant difference between above both comparisons. CfDNA might have potential use as a diagnostic factor for patients with suspected GBC.

Portable and visual quantification of urine cell-free DNA through smartphone-based colorimetric biosensor

The trace amount of cell-free DNA (cfDNA) in urine provides a promising diagnosis of urological disease, but its clinical applications are restricted by complicated operations for enrichment and detection. Herein, a portable and visual smartphone-based colorimetric platform is created for urine cfDNA quantification in quick, low-cost, and instrument-free manners. cfDNA in urine is enriched by the zinc ions-dipicolylamino complexes on glass slides and labeled by PicoGreen (PG) to form the DNA-PG complexes with photocatalysis for the generation of a visible output signal. The high sensitivity is related to the amplification of the optical signal of enriched cfDNA through the photocatalysis strategy, which yields greater color changes without interference from other ingredients. Notably, accurate quantitation of cfDNA is achieved with the aid of the smartphone App equipped with collecting and processing Color Grab. As a powerful alternative, such a robust colorimetric biosensor platform is employed for urine cfDNA quantification of clinical samples with excellent linearity compared with the two-stage commercial kits (R2 > 0.95), helping to distinguish the difference between healthy and urological disease. The comprehensive performance of our developed platform will be the first to enable visual quantification of urine cfDNA in terms of quick detection, low cost, and high portability.

Fluorometric Quantification of Total Cell-Free DNA as a Prognostic Biomarker in Non-Small-Cell Lung Cancer Patients Treated with Immune Checkpoint Blockade.

The present study aimed to investigate the potential of basal cell-free fluorometric DNA (cfDNA) quantification as a prognostic biomarker in advanced non-small cell lung cancer (NSCLC) patients treated with an Immune Checkpoint Blockade (ICB). A discovery and validation cohort of 61 and 31 advanced lung cancer patients treated with ICB were included in this study. Quantification of cfDNA concentration was performed before the start of the treatment and patients were followed up for a median of 34 (30-40) months. The prognostic predicted value of cfDNA was evaluated based on ROC, and Cox regression was conducted via univariate and multivariate analyses to estimate the hazard ratio. We observed that a cfDNA cut-off of 0.55 ng/µL before the ICB determines the overall survival of patients with a log rank p-value of 3.3 × 10-4. That represents median survivals of 3.8 vs. 17.5 months. Similar results were obtained in the validation cohort being the log rank p-value 3.8 × 10-2 with median survivals of 5.9 vs. 24.3. The univariate and multivariate analysis revealed that the cut-off of 0.55 ng/µL before ICB treatment was an independent predictive factor and was significantly associated with a better survival outcome. High cfDNA concentrations identify patients with advanced NSCLC who do not benefit from the ICB. The determination of cfDNA is a simple test that could select a group of patients in whom new therapeutic strategies are needed.

Circulating tumor DNA sequencing of pediatric solid and brain tumor patients: An institutional feasibility study

The potential of circulating tumor DNA (ctDNA) analysis to serve as a real-time “liquid biopsy” for children with central nervous system (CNS) and non-CNS solid tumors remains to be fully elucidated. We conducted a study to investigate the feasibility and potential clinical utility of ctDNA sequencing in pediatric patients enrolled on an institutional clinical genomics trial. A total of 240 patients had tumor DNA profiling performed during the study period. Plasma samples were collected at study enrollment from 217 patients and then longitudinally from a subset of patients. Successful cell-free DNA extraction and quantification occurred in 216 of 217 (99.5%) of these initial samples. Twenty-four patients were identified whose tumors harbored 30 unique variants that were potentially detectable on a commercially-available ctDNA panel. Twenty of these 30 mutations (67%) were successfully detected by next-generation sequencing in the ctDNA from at least one plasma sample. The rate of ctDNA mutation detection was higher in patients with non-CNS solid tumors (7/9, 78%) compared to those with CNS tumors (9/15, 60%). A higher ctDNA mutation detection rate was also observed in patients with metastatic disease (9/10, 90%) compared to non-metastatic disease (7/14, 50%), although tumor-specific variants were detected in a few patients in the absence of radiographic evidence of disease. This study illustrates the feasibility of incorporating longitudinal ctDNA analysis into the management of relapsed or refractory patients with childhood CNS or non-CNS solid tumors.

Graft-Derived Cell-Free DNA Quantification following Liver Transplantation Using Tissue-Specific DNA Methylation and Donor-Specific Genotyping Techniques: An Orthogonal Comparison Study.

Background: Graft-derived cell-free DNA (gdcfDNA) analysis has shown promise as a non-invasive tool for monitoring organ health following solid organ transplantation. A number of gdcfDNA analysis techniques have been described; however, the majority rely on sequencing or prior genotyping to detect donor-recipient mis-matched genetic polymorphisms. Differentially methylated regions of DNA can be used to identify the tissue-of-origin of cell-free DNA (cfDNA) fragments. In this study, we aimed to directly compare the performance of gdcfDNA monitoring using graft-specific DNA methylation analysis and donor-recipient genotyping techniques in a pilot cohort of clinical samples from patients post-liver transplantation. Results: 7 patients were recruited prior to LT, 3 developed early, biopsy-proven TCMR in the first 6 weeks post-LT. gdcfDNA was successfully quantified in all samples using both approaches. There was a high level of technical correlation between results using the two techniques (Spearman testing, rs = 0.87, p < 0.0001). gdcfDNA levels quantified using the genotyping approach were significantly greater across all timepoints in comparison to the tissue-specific DNA methylation-based approach: e.g., day 1 post-LT median 31,350 copies/mL (IQR 6731-64,058) vs. 4133 copies/mL (IQR 1100-8422), respectively. Qualitative trends in gdcfDNA levels for each patient were concordant between the two assays. Acute TCMR was preceded by significant elevations in gdcfDNA as quantified by both techniques. Elevations in gdcfDNA, using both techniques, were suggestive of TCMR in this pilot study with a 6- and 3-day lead-time prior to histological diagnosis in patients 1 and 2. Conclusions: Both the graft-specific methylation and genotyping techniques successfully quantified gdcfDNA in patients post-LT with statistically significant concordance. A direct comparison of these two techniques is not only important from a technical perspective for orthogonal validation, but significantly adds weight to the evidence that gdcfDNA monitoring reflects the underlying biology. Both techniques identified LT recipients who developed acute TCMR, with several days lead-time in comparison to conventional diagnostic workflows. Whilst the two assays performed comparably, gdcfDNA monitoring based on graft-specific DNA methylation patterns in cfDNA offers major practical advantages over the donor-recipient genotyping, and hence enhances the potential to translate this emerging technology into clinical practice.

Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation.

Many studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx. This prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF). Quantification of total cfDNA was not correlated with the patient's status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%). With the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries.

(996) Relationship Between Absolute Quantification of Donor-Derived Cell-Free DNA and Donor-Derived Cell-Free DNA Fraction for Detection of Allograft Rejection in Heart Transplant Patients

(996) Relationship Between Absolute Quantification of Donor-Derived Cell-Free DNA and Donor-Derived Cell-Free DNA Fraction for Detection of Allograft Rejection in Heart Transplant Patients

Qualification and quantification of plasma cell-free DNA after long-term storage conditions in patients with benign prostatic hyperplasia (BPH): a pilot study

Abstract Objectives Free DNA is used as a cancer biomarker due to its low cost, high applicability, and fast, reliable results compared to invasive methods. This study aimed to evaluate the quantification of plasma-free DNA after long-term storage conditions and perform qualification through single nucleotide polymorphism (SNP) screening based on this DNA. Methods Plasma-free DNA samples were quickly isolated from the peripheral blood of both the benign prostatic hyperplasia (BPH) and control group participants and then maintained at −80 °C for four years. Upon thawing, first, free DNA was purified and fluorometric measurements were taken to determine the amount of DNA. Subsequently, the rs6983267, rs12628, and rs1799939 SNPs were screened in the CCAT2, HRAS, and RET genes, respectively. Results Significant results were obtained from the fluorometric measurements in terms of single-stranded DNA (ssDNA) (p&lt;0.001). However, there was no significant difference in SNPs rs6983267, rs12628, and rs1799939 in the BPH group compared to the healthy individuals. Conclusions The data show that fluorometric ssDNA measurements are suitable for quantifying free DNA. The fact that SNP screening can be done successfully in both healthy people and BPH patients suggests that plasma-free DNA can be stored in the laboratory under appropriate conditions.

Quantification of Tumor-Associated Cell-Free HPV DNA in Surgical Drain Fluid Equates to Molecular Residual Disease and Correlates with High-Risk Pathology

<h3>Purpose/Objective(s)</h3> Multi-institutional efforts aimed at safely de-escalating treatment in human papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPC) patients rely on subjective pathology for risk stratification. While plasma HPV assays are emerging for post-treatment disease surveillance, they have poor sensitivity to detect postoperative molecular residual disease (MRD). Here we present a liquid biopsy assay of the serosanguinous fluid removed from surgical drains, proximal to the surgical site, to sensitively measure postoperative MRD. We hypothesize that HPV quantified in surgical drain fluid (SDF), 1) represents post-operative regional MRD, and 2) could improve patient risk-stratification to guide precision radiotherapy. <h3>Materials/Methods</h3> We collected SDF at 24-48 hours post-surgery for cell-free (cf) DNA extraction. Our 87-patient head and neck cancer cohort included 74 HPV(+) and 8 HPV(-) OPCs, and 5 benign tumors. Paired plasma and tumor samples were collected from approximately half of all patients. We used the HPV16 E6T2aE7 plasmid to compare our Taqman quantitative PCR (qPCR) to digital droplet PCR (ddPCR). We validated HPV(+) OPC PCR results using next-generation HPV sequencing (NGS) in 9 patients. Then we correlated SDF HPV copies to extranodal extension (ENE), AJCC 8^th edition pathological stage, smoking pack-years, recurrence-free survival (RFS), and composite risk scores derived from OPC de-escalation clinical trials. Statistical methods included Fisher's Exact test, Mann-Whitney (MW) test, Kruskal-Wallis (KW) test, and Kaplan-Meier (KM) analysis. <h3>Results</h3> Our HPV qPCR assay's limit of detection was 9.7 copies, similar to what we saw with ddPCR. HPV genotyping was concordant between NGS and PCR, and all SDF cfDNA samples with nondetectable (ND) HPV via PCR were ND via NGS. HPV was detected in 63 of 74 (85%) SDF samples compared to only 7 of 48 (15%) paired plasma samples. Median HPV copies in ENE+ patients' SDF was 15-fold higher than SDF from ENE- patients (P = 0.002). HPV copies also increased with increasing nodal (P = 0.01) but not primary tumor stage (P = 0.4), suggesting post-operative SDF HPV load reflects invasive nodal disease and is a proxy for regional MRD. When we stratified our cohort according to high-risk and low-risk scores drawn from four clinical trials (RTOG 9501, EORTC 22931, ECOG 3311, MC1273), SDF HPV load was consistently higher in patients with trial-defined high-risk pathology (mean AUC = 0.73). Lastly, KM RFS analysis of HPV-high and HPV-low patients, defined by an RTOG 9501-derived SDF classifier, showed HPV-high patients trended toward worse RFS (HR = 3.1; P = 0.1). <h3>Conclusion</h3> Liquid biopsy assessment of SDF, the biofluid most proximal to the nodal environment, enables postoperative regional MRD detection in HPV(+) OPC, and correlates with high-risk nodal pathology and worse RFS. If confirmed, SDF cfDNA analysis could aid traditional pathology and imaging to personalize adjuvant radiotherapy for surgically treated HPV(+) OPC.

Isolation and Quantification of Plasma Cell-Free DNA Using Different Manual and Automated Methods.

Plasma cell-free DNA (cfDNA) originates from various tissues and cell types and can enable minimally invasive diagnosis, treatment and monitoring of cancer and other diseases. Proper extraction of cfDNA is critical to obtain optimal yields and purity. The goal of this study was to compare the performance of six commercial cfDNA kits to extract pure, high-quality cfDNA from human plasma samples and evaluate the quantity and size profiles of cfDNA extracts—among them, two spin-column based, three magnetic bead-based and two automatic magnetic bead-based methods. Significant differences were observed in the yield of DNA among the different extraction kits (up to 4.3 times), as measured by the Qubit Fluorometer and Bioanalyzer. All kits isolated mostly small fragments corresponding to mono-nucleosomal sizes. The highest yield and reproducibility were obtained by the manual QIAamp Circulating Nucleic Acid Kit and automated MagNA Pure Total NA Isolation Kit. The results highlight the importance of standardizing preanalytical conditions depending on the requirements of the downstream applications.

Cell-free mitochondrial DNA deletions in idiopathic, but not LRRK2, Parkinson's disease

Mitochondrial dysfunction happens in both idiopathic (iPD) and LRRK2-related Parkinson's disease (LRRK2-PD). Nonetheless, previous studies suggested that a different type of mitochondrial pathology underlies the neurodegeneration in these two disorders. To further explore this hypothesis, we developed a novel multiplex digital PCR assay that allows the absolute quantification of cell-free mitochondrial DNA (cf-mtDNA) copy number and deletion ratio directly in cerebrospinal fluid (CSF) by simultaneously measuring two opposed regions of the mtDNA circular molecule, one of them in the commonly deleted major arc. The results confirmed that the content of cf-mtDNA in CSF was statistically significantly different between iPD and LRRK2-PD patients. Moreover, we found high cf-mtDNA deletion levels in CSF from patients with iPD, but not LRRK2-PD. The high cf-mtDNA deletion frequency in iPD was validated in an independent cohort. These results indicated that the content and deletion ratio of cf-mtDNA may differentiate iPD from LRRK2-PD, and provides further evidence of the different mitochondrial pathophysiology between these two forms of the disease.

A standardised methodology for the extraction and quantification of cell-free DNA in cerebrospinal fluid and application to evaluation of Alzheimer’s disease and brain cancers

Cerebrospinal fluid (CSF) is a source of diagnostic biomarkers for a range of neurological conditions. Cell-free DNA (cfDNA) is detected in CSF and differences in the concentration of cell-free mitochondrial DNA have been reported in studies of neurodegenerative disorders including Alzheimer’s disease (AD). However, the influence of pre-analytical steps has not been investigated for cfDNA in CSF and there is no standardised approach for quantification of total cfDNA (copies of nuclear genome or mitochondria-derived gene targets). In this study, the suitability of four extraction methods was evaluated: QIAamp Circulating Nucleic Acid (Qiagen), Quick-cfDNA Serum & Plasma (Zymo), NucleoSnap® DNA Plasma (Macherey-Nagel) and Plasma/Serum Circulating DNA Purification Mini (Norgen) kits, for cfDNA extraction from CSF of controls and AD dementia patients, utilising a spike-in control for extraction efficiency and fragment size. One of the optimal extraction methods was applied to a comparison of cfDNA concentrations in CSF from control subjects, AD dementia and primary and secondary brain tumour patients. Extraction efficiency based on spike-in recovery was similar in all three groups whilst both endogenous mitochondrial and nucleus-derived cfDNA was significantly higher in CSF from cancer patients compared to control and AD groups, which typically contained < 100 genome copies/mL. This study shows that it is feasible to measure low concentration nuclear and mitochondrial gene targets in CSF and that normalisation of extraction yield can help control pre-analytical variability influencing biomarker measurements.

Quantification of Postoperative Graft-Derived Cell-Free DNA to Evaluate the Risks of Impaired Allograft Function at Early Stage of Kidney Transplantation

Graft-derived cell-free DNA (GcfDNA) is a promising biomarker for comprehensive monitoring of allograft injury because it overcomes the limitations of traditional approaches. The aim of this study is to investigate the association between the outliers of GcfDNA at initial time post transplantation and short-term renal graft function. A total of 230 recipients who underwent primary kidney transplantation were recruited in the study. For each recipient, 10 mL of peripheral blood were collected at day 1 post transplantation. Both of the GcfDNA fraction (%) and GcfDNA concentration (cp/mL) were determined using droplet digital PCR. The study was conducted in accordance with the 1964 Helsinki Declaration and its later amendments. There were no values that fall outside of the lower extreme in both of the GcfDNA fraction and GcfDNA concentration, and the upper fence of GcfDNA fraction and GcfDNA concentration were 13.5% and 680 cp/mL, respectively. Recipients with GcfDNA concentration ≥680 cp/mL had a statistically significant higher serum creatinine at day 7 post-transplantation, when compared with the other group (P=.008). The receiver operating characteristic analysis obtained an area under the curve value of 0.869 when using GcfDNA concentration to predict the risk of serum creatinine ≥400 μmol/L, an optimal cut-off value was indicated at 975 cp/mL with high sensitivity (87.5%) and specificity (85%). Our results suggest that the quantification of GcfDNA at initial time after transplantation might be used as a novel strategy for predicting short-term risk of impaired kidney allograft function or delayed graft function.

Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status

Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status

Absolute quantification of plasma mitochondrial DNA by droplet digital PCR marks COVID-19 severity over time during intensive care unit admissions.

Increased plasma mitochondrial DNA concentrations are associated with poor outcomes in multiple critical illnesses, including COVID-19. However, current methods of cell-free mitochondrial DNA quantification in plasma are time-consuming and lack reproducibility. Here, we used next-generation sequencing to characterize the size and genome location of circulating mitochondrial DNA in critically ill subjects with COVID-19 to develop a facile and optimal method of quantification by droplet digital PCR. Sequencing revealed a large percentage of small mitochondrial DNA fragments in plasma with wide variability in coverage by genome location. We identified probes for the mitochondrial DNA genes, cytochrome B and NADH dehydrogenase 1, in regions of relatively high coverage that target small sequences potentially missed by other methods. Serial assessments of absolute mitochondrial DNA concentrations were then determined in plasma from 20 critically ill subjects with COVID-19 without a DNA isolation step. Mitochondrial DNA concentrations on the day of enrollment were increased significantly in patients with moderate or severe acute respiratory distress syndrome (ARDS) compared with those with no or mild ARDS. Comparisons of mitochondrial DNA concentrations over time between patients with no/mild ARDS who survived, patients with moderate/severe ARDS who survived, and nonsurvivors showed the highest concentrations in patients with more severe disease. Absolute mitochondrial DNA quantification by droplet digital PCR is time-efficient and reproducible; thus, we provide a valuable tool and rationale for future studies evaluating mitochondrial DNA as a real-time biomarker to guide clinical decision-making in critically ill subjects with COVID-19.

Assessment of donor cell engraftment after hematopoietic stem cell transplantation for sickle cell disease: A review of current and future methods.

Hematopoietic stem cell transplantation (HSCT) is the only established curative treatment for sickle cell disease (SCD), a debilitating red blood cell (RBC) disorder with significant prevalence worldwide. Accurate assessment of RBC engraftment following HSCT is essential to evaluate the status of the graft and can enable early intervention to treat or prevent graft rejection. Currently, chimerism measurement is performed on whole blood samples, which mainly reflect white blood cell (WBC) chimerism. This approach has limitations in assessing engraftment in patients with SCD because RBCs engraft non-linearly with WBCs. Direct measures of RBC chimerism exist but are not routinely used. In this review, we critically examine the current methodologies for assessing donor engraftment; highlight the limitations of these different methods, and present emerging and novel technologies with the potential to improve clinical monitoring of RBC engraftment post-HSCT for SCD. Promising alternative methodologies include RBC-specific flow cytometry, RBC-specific RNA analysis, and quantification of plasma cell-free DNA derived specifically from nucleated RBCs.