A new lateral flow assay to detect sIL-2R during T-cell mediated rejection after kidney transplantation

Kidney is the most frequently transplanted among all solid organs worldwide. Kidney transplant recipients (KTRs) undergo regular follow-up examinations for the early detection of acute rejections. The gold standard for proving a T-cell mediated rejection (TCMR) is a biopsy of the renal graft often occurring as indication biopsy, in parallel to an increased serum creatinine that may indicate deterioration of renal transplant function. The goal of the current work was to establish a lateral flow assay (LFA) for diagnosing acute TCMR to avoid harmful, invasive biopsies. Soluble interleukin-2 (IL-2) receptor (sIl-2R) is a potential biomarker representing the α-subunit of the IL-2 receptor produced by activated T-cells, e.g., after allogen contact. To explore the diagnostic potential of sIL-2R as a biomarker for TCMR and borderline TCMR, plasma and urine samples were collected from three independent KTR cohorts with various distinct histopathological diagnostic findings according to BANFF (containing 112 rsp. 71 rsp. 61 KTRs). Samples were analyzed by a Luminex-based multiplex technique and cut off-ranges were determined. An LFA was established with two specific sIL-2R-antibodies immobilized on a nitrocellulose membrane. A significant association between TCMR, borderline TCMR and sIL-2R in plasma and between TCMR and sIL-2R in urine of KTRs was confirmed using the Mann-Whitney U test. The LFA was tested with sIL-2R-spiked buffer samples establishing a detection limit of 25 pM. The performance of the new LFA was confirmed by analyzing urine samples of the 2nd and 3rd patient cohort with 35 KTRs with biopsy proven TCMRs, 3 KTRs diagnosed with borderline TCMR, 1 mixed AMR/TCMR rsp. AMR/borderline TCMR and 13 control patients with a rejection-free kidney graft proven by protocol biopsies. The new point-of-care assay showed a specificity of 84.6% and sensitivity of 87.5%, and a superior estimated glomerular filtration rate (eGFR) at the time point of biopsy (specificity 30.8%, sensitivity 85%)

A method for interoperable knowledge-based data quality assessment

Background!#!Assessing the quality of healthcare data is a complex task including the selection of suitable measurement methods (MM) and adequately assessing their results.!##!Objectives!#!To present an interoperable data quality (DQ) assessment method that formalizes MMs based on standardized data definitions and intends to support collaborative governance of DQ-assessment knowledge, e.g. which MMs to apply and how to assess their results in different situations.!##!Methods!#!We describe and explain central concepts of our method using the example of its first real world application in a study on predictive biomarkers for rejection and other injuries of kidney transplants. We applied our open source tool-openCQA-that implements our method utilizing the openEHR specifications. Means to support collaborative governance of DQ-assessment knowledge are the version-control system git and openEHR clinical information models.!##!Results!#!Applying the method on the study's dataset showed satisfactory practicability of the described concepts and produced useful results for DQ-assessment.!##!Conclusions!#!The main contribution of our work is to provide applicable concepts and a tested exemplary open source implementation for interoperable and knowledge-based DQ-assessment in healthcare that considers the need for flexible task and domain specific requirements

Urinary NGAL Ratio Is Not a Sensitive Biomarker for Monitoring Acute Tubular Injury in Kidney Transplant Patients: NGAL and ATI in Renal Transplant Patients

Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is known to predict the prolonged delayed graft function after kidney transplantation. We examined the relation of uNGAL with histological findings of acute tubular injury (ATI). Analyses were made in biopsies taken at 6 weeks, 3 months, and 6 months after kidney transplantation. uNGAL was measured in the spot urines, normalized to urinary creatinine excretion, and correlated to biopsy findings and clinical, laboratory, and demographic variables. Controls included healthy individuals, individuals after kidney donation and ICU patients with acute kidney failure. Renal transplant recipients without ATI did not display elevated uNGAL levels compared to the healthy controls. Transplant patients with ATI had a higher uNGAL excretion at 6 weeks than patients without ATI (27,435 versus 13,605 ng/g; P=0.031). This increase in uNGAL was minor compared to ICU patients with acute renal failure (2.05×106 ng/g). Patients with repeated findings of ATI or severe ATI did not have higher urinary NGAL levels compared to those with only one ATI finding or moderate ATI. Female recipient gender and urinary tract infection were identified as potential confounders. uNGAL has a relation with histological signs of acute tubular injury. The usability of this biomarker in renal allograft recipients is limited because of the low sensitivity

Fetuin, matrix-Gla protein and osteopontin in calcification of renal allografts.

BACKGROUND: Calcification of renal allografts is common in the first year after transplantation and is related to hyperparathyroidism. It is associated with an impaired long-term function of the graft (Am J Transplant 5∶1934-41, 2005). Aim of this study is to examine the role of the anti-calcifying/calcifying factors in the pathophysiology of renal allograft calcification. METHODS: We analyzed protocol allograft biopsies, blood and urine samples of 31 patients with and 27 patients without allograft calcification taken at 6 weeks, 3 and 6 months after transplantation. Patient demographical data, cold ischemia time, initial graft function and donor characteristics were comparable between the two groups. Biopsies were stained for osteopontin, fetuin, and matrix-gla-protein. Serum and urine electrolytes, matrix-gla-protein, fetuin, Vitamin D and intact parathyroid hormone in serum and osteopontin (OPN) in urine were examined. RESULTS: Serum levels of fetuin and matrix-Gla protein as well as urinary levels of OPN showed specific time dependent changes (6 weeks vs. 3 months vs. 6 months; all p<0.0001). In patients with calcifications, urinary levels of OPN were decreased by 55% at 6 weeks and increased thereafter, showing 54% higher levels at 6 months compared to patients without calcification (6 weeks: p<0.01, 6 months: p<0.05). Local protein expression of fetuin-A, matrix-Gla protein and OPN in the graft was specifically increased around calcified plaques, without differences in the mRNA tissue expression. CONCLUSION: Anticalcifying factors show significant changes in the early phase after renal transplantation, which may be important for the prevention of allograft calcification. The differences in OPN indicate an involvement of this factor in the regulation of calcification

Urinary phosphate levels in NC patients in comparison to control transplant patients without signs of calcification at the indicated time points are shown.

<p>Patients with calcification are depicted in red.</p

Levels of parathyroid hormone (A), serum calcium (B) and vitamin D (C) are shown in NC patients and controls at the indicated time points.

<p>Patients with calcification are depicted in red. Data are shown as mean ± standard error of the mean (SEM).</p

Urinary concentrations of OPN are shown in NC patients and control transplant patients without signs of calcification at the indicated time points.

<p>Patients with calcification are depicted in red.</p

Serum concentrations of Fetuin A (A) as well as MGP (B) are shown in NC patients (n = 31) in comparison to control transplant patients without signs of calcification (n = 27) at the indicated time points.

<p>Patients with calcification are depicted in red.</p

Demographics and transplant data of patients with and without (controls) calcification.

<p>Continuous data are given as mean±SD unless otherwise stated;</p>*<p>p = 0.011.</p