HLA diversity in ethnic populations can affect detection of donor-specific antibodies by single antigen beads.

Introduction: In solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins. Materials: Allele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes. Results: For the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors. Discussion: In conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection.

Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design.

Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one target is detected per optical channel and the total number of targets is limited by the number of optical channels on the platform. Higher-order multiplexing has the potential to dramatically increase the usefulness of dPCR, especially in scenarios with limited sample. Other potential benefits of multiplexing include lower cost, additional information generated by more probes, and higher throughput. To address this unmet need, we developed a novel melt-based hairpin probe design to provide a robust option for multiplexing digital PCR. A prototype multiplex digital PCR (mdPCR) assay using three melt-based hairpin probes per optical channel in a 16-well microfluidic digital PCR platform accurately distinguished and quantified 12 nucleic acid targets per well. For samples with 10,000 human genome equivalents, the probe-specific ranges for limit of blank were 0.00%-0.13%, and those for analytical limit of detection were 0.00%-0.20%. Inter-laboratory reproducibility was excellent (r 2 = 0.997). Importantly, this novel melt-based hairpin probe design has potential to achieve multiplexing beyond the 12 targets/well of this prototype assay. This easy-to-use mdPCR technology with excellent performance characteristics has the potential to revolutionize the use of digital PCR in research and diagnostic settings.

Cell-free DNA as a solid-organ transplant biomarker: technologies and approaches.

High-quality biomarkers that detect emergent graft damage and/or rejection after solid-organ transplantation offer new opportunities to improve post-transplant monitoring, allow early therapeutic intervention and facilitate personalized patient management. Donor-derived cell-free DNA (DD-cfDNA) is a particularly exciting minimally invasive biomarker because it has the potential to be quantitative, time-sensitive and cost-effective. Increased DD-cfDNA has been associated with graft damage and rejection episodes. Efforts are underway to further improve sensitivity and specificity. This review summarizes the procedures used to process and detect DD-cfDNA, measurement of DD-cfDNA in clinical transplantation, approaches for improving sensitivity and specificity and long-term prospects as a transplant biomarker to supplement traditional organ monitoring and invasive biopsies.

Association of higher erythropoiesis stimulating agent dose and mortality in children on dialysis.

BACKGROUND: Higher doses of erythropoiesis-stimulating agents (ESA) have been associated with an increased risk of adverse outcomes in adults with chronic kidney disease (CKD) and end-stage kidney disease (ESRD), but to our knowledge no trials have been performed in children. We examined the association between ESA dose and all-cause mortality in a prevalent pediatric dialysis population. METHODS: Retrospective cohort study utilizing national data on all prevalent dialysis patients aged <18 years from the Centers for Medicare and Medicaid Services' 2005 ESRD Clinical Performance Measures (CPM) project, linked to 18-month mortality records from the United States Renal Data System. Multivariate Cox proportional hazards regression was performed to determine the risk of mortality by mean weekly ESA dose. RESULTS: Eight-hundred and twenty-nine children were included in the analysis; 7 % died during follow-up. A higher proportion of patients receiving ESA doses in the highest category (erythropoietin ≥350 units/kg/week or darbepoetin ≥1.5 units/kg/week) died (50 % vs 28 %, p = 0.002), and also demonstrated a trend toward lower hemoglobin (11.0 vs 11.4 g/dL, p = 0.05). In multivariate analysis, patients receiving the highest dose of ESA demonstrated an increased risk of mortality (hazard ratio 3.37; p value <0.01). CONCLUSION: Higher ESA dose is independently associated with mortality in children on chronic dialysis.

Comparative clinical outcomes between pediatric and young adult dialysis patients.

Published data on the comparative achievement of The Kidney Disease Dialysis Outcome Quality Initiative (KDOQI) recommended clinical performance targets between children and young adults on dialysis are scarce. To characterize the achievement of KDOQI targets among children (<18 years) and young adults (18-24 years) with prevalent end stage renal disease (ESRD), we performed a cross-sectional analysis of data collected by the Mid-Atlantic Renal Coalition, in conjunction with the 2007 and 2008 ESRD Clinical Performance Measures Projects. Data on all enrolled pediatric dialysis patients, categorized into three age groups (0-8, 9-12, 13-17 years), and on a random sample of 5% of patients ≥ 18 years in ESRD Network 5 were examined for two study periods: hemodialysis (HD) data were collected from October to December 2006 and from October to December 2007 and peritoneal dialysis (PD) data were collected from October 2006 to March 2007 and from October 2007 to March 2008. In total, 114 unique patients were enrolled the study, of whom 41.2% (47/114) were on HD and 58.8% (67/114) on PD. Compared to the pediatric patients, young adults were less likely to achieve the KDOQI recommended serum phosphorus levels and serum calcium × phosphorus product values, with less than one-quarter demonstrating values at or below each goal. Multivariate analysis revealed that both young adults and 13- to 17-year-olds were less likely to achieve target values for phosphorus [young adults: odds ratio (OR) 0.04, 95% confidence interval (95% CI) 0.01-0.19, p < 0.001; 13- to 17-year-olds: OR 0.17, 95% CI 0.04-0.77, p = 0.02] and calcium × phosphorus product (young adults: OR 0.01, 95% CI 0.002-0.09, p < 0.001; 13- to 17-year-olds: OR 0.09, 95% CI 0.02-0.56, p = 0.01) than younger children. In summary, there are significant differences in clinical indices between pediatric and young adult ESRD patients.

No difference in meeting hemoglobin and albumin targets for dialyzed children with urologic disorders.

Urologic disorders are the most common cause of chronic kidney disease in children. To determine whether children with urologic etiology of end-stage renal disease (ESRD) fare better than children with ESRD from other causes while on dialysis, we conducted a cross-sectional study of children <18 years receiving peritoneal and hemodialysis in the United States using data from the Centers for Medicare & Medicaid Services 2005 ESRD CPM Project. We compared baseline demographics and the study groups. In multivariate logistic regression analysis of 1,286 subjects, we assessed whether children with urologic disorders had a higher odds of meeting adult KDOQI targets for hemoglobin levels ≥11 g/dl and albumin ≥3.5 BCG/3.2 BCP g/dl. We conducted a subset analysis of 1,136 patients to examine the impact of erythropoietin on hemoglobin targets. Our results did not reveal differences in achievement of adult hemoglobin targets (adjusted OR: 1.27; p value 0.09; CI: 0.97-1.66) or in the subset analysis with erythropoietin (adjusted OR: 1.32; p value 0.06; CI: 0.98-1.78) or albumin targets (adjusted OR: 1.22; p value 0.21; CI: 0.90-1.65) in adjusted analyses. Due to our study's limitations, it is difficult to determine whether this may result from treatment prior to dialysis initiation or treatment effect of dialysis rather than underlying diagnosis.