The role of biomarkers in early identification of acute kidney injury among non-critically ill patients.

BACKGROUND: Prediction and/or early identification of acute kidney injury (AKI) and individuals at greater risk remains of great interest in clinical medicine. Acute kidney injury continues to be a common complication among hospitalized patients, with an incidence ranging from 6 to 58%, depending on the setting. Aim of this study was to determine the performance of Insulin-like growth factor binding protein-7 (IGFBP7), tissue metallopeptidase inhibitor 2 (TIMP2), and urinary neutrophil gelatinase-associated lipocalin (uNGAL) in early detection of AKI among non-critically ill patients. METHODS: In this prospective observational study at Mayo Clinic Hospitals in Rochester, Minnesota, USA, non-critically ill patients admitted from the emergency department between October 31st, 2016 and May 1st, 2018, who had an acute kidney injury (AKI) probability of 5% or higher were included. Biomarkers were measured in residual urine samples collected in the emergency department. The primary outcome was biomarker performance in predicting AKI development within the first 72 h. RESULTS: Among 368 included patients, the mean age was 79 ± 12 years, and 160 (43%) were male. Acute kidney injury occurred in 62 (17%) patients; 11.5% stage 1, 2.5% stage 2, and 3% stage 3. Twelve patients (3%) died during hospitalization and 102 (28%) within nine months after admission. The median uNGAL and IGFBP7-TIMP2 were 57 [20-236 ng/ml], and 0.3 [0.1-0.8], respectively. The C-statistic of uNGAL and IGFBP7-TIMP2 of > 0.3 and > 2.0 for AKI prediction were 0.56, 0.54, and 0.53, respectively. In a model where one point is assigned to each marker of AKI (elevated serum creatinine, IGFBP7-TIMP2 > 0.3, and uNGAL), a higher score correlated with higher nine-month mortality [OR of 1.32 per point (95% CI 1.02-1.71)]. CONCLUSION: Among non-critically ill hospitalized patients, the performance of uNGAL and IGFBP7-TIMP2 for AKI prediction within 72 h of admission was modest. This suggests a limited role for these biomarkers in AKI risk stratification among non-critically ill patients. Key learning points What was known Acute kidney injury (AKI) is a common complication among hospitalized patients. It is associated with increased morbidity and mortality. Various clinical prediction models and biomarkers have been developed to identify patients in special populations (such as ICU and cardiac surgery) who are at risk of AKI and diagnose AKI early. This study adds The performance of the biomarkers uNGAL, TIMP-2, and IGFBP-7 in predicting AKI within 72 h of admission in non-critically ill patients was modest. However, these biomarkers were found to have a prognostic value for predicting 9-month mortality. One potential application of these biomarkers is identifying patients at higher AKI risk before exposing them to nephrotoxic agents. Potential impact This study provides evidence regarding the real-world performance of current FDA-approved biomarkers (uNGAL, TIMP-2, and IGFBP-7) for predicting acute kidney injury (AKI) within 72 h of hospital admission among noncritically ill patients. While the performance of these biomarkers for predicting short-term AKI was modest, they may have a prognostic value for predicting 9-month mortality.

Noninvasive Diagnosis of PLA2R-Associated Membranous Nephropathy: A Validation Study.

BACKGROUND AND OBJECTIVES: Kidney biopsy is the current gold standard to diagnose membranous nephropathy. Approximately 70%-80% of patients with primary membranous nephropathy have circulating anti-phospholipase A2 receptor antibodies. We previously demonstrated that in proteinuric patients with preserved eGFR and absence of associated conditions (e.g., autoimmunity, malignancy, infection, drugs, and paraproteinemia), a positive anti-phospholipase A2 receptor antibody test by ELISA and immunofluorescence assay confirms the diagnosis of membranous nephropathy noninvasively. These data have not been externally validated. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The clinical and pathologic characteristics of patients with a positive anti-phospholipase A2 receptor antibody test at the Mayo Clinic, the University Hospital Vall D'Hebron (Barcelona), and the Columbia University Medical Center (New York) were retrospectively reviewed. Biopsy findings and presence or absence of a potential associated condition were assessed. RESULTS: From a total of 276 patients with positive anti-phospholipase A2 receptor serology, previously reported patients (n=33), kidney transplant recipients (n=9), pediatric patients (n=2), and patients without kidney biopsy (n=69) were excluded. Among the 163 remaining patients, associated conditions were identified in 47 patients, and 15 patients had diabetes mellitus. All 101 patients of the final cohort had a primary diagnosis of membranous nephropathy on kidney biopsy. In the 79 patients with eGFR≥60 ml/min per 1.73 m2, none of the biopsy findings altered diagnosis or management. Among the 22 patients with decreased eGFR, additional findings included superimposed acute interstitial nephritis (n=1). CONCLUSIONS: In patients with preserved eGFR and absence of associated conditions or diabetes, a positive anti-phospholipase A2 receptor test by either ELISA >20 RU/ml or a positive immunofluorescence assay confirms the diagnosis of membranous nephropathy, precluding the requirement for a kidney biopsy.

A Target Antigen-Based Approach to the Classification of Membranous Nephropathy.

OBJECTIVE: To describe the clinical and pathological phenotype of membranous nephropathy (MN) associated with M-type-phospholipase-A2-receptor (PLA2R), thrombospondin-type-1-domain-containing-7A (THSD7A), semaphorin 3B (SEMA3B), neural-epidermal-growth-factor-like-1-protein (NELL-1), protocadherin 7 (PCDH7), exostosin 1/exostosin 2 (EXT1/EXT2) and neural cell adhesion molecule 1 (NCAM-1) as target antigens. METHODS: A retrospective cohort of 270 adult patients with biopsy-proven MN diagnosed between January 2015 and April 2020 was classified as PLA2R-, THSD7A-, SEMA3B-, NELL-1-, PCDH7-, EXT1/EXT2-, NCAM-1-associated or septuple-negative MN using serologic tests, immunostaining, and/or mass spectrometry. Clinical, biochemical, pathologic, and follow-up data were systematically abstracted from the medical records, including disease activity of conditions traditionally associated with MN and occurring within 5 years of MN diagnosis. RESULTS: Patients with PLA2R-associated MN were predominantly middle-aged white men without associated disease. The presence of associated disease did not affect the clinical and pathologic characteristics of PLA2R-associated MN, suggesting that they were coincidental rather than causally linked. THSD7A-, NELL-1-, PCDH7-, and NCAM-1-associated MN were rare and SEMA3B-associated MN was not discovered in our cohort. EXT1/EXT2-associated MN was primarily diagnosed in younger women with active systemic autoimmunity. A significant proportion of septuple-negative patients had associated malignancy or systemic autoimmunity. CONCLUSION: The widely used distinction between primary and secondary MN has limitations. We propose a refined terminology that combines the target antigen and associated disease to better classify MN and guide clinical decision making.

Serum levels of DNAJB9 are elevated in fibrillary glomerulonephritis patients.

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease. Kidney biopsy is required to establish the diagnosis. Recent studies have identified abundant glomerular deposition of DNAJB9 as a unique histological marker of FGN. We developed an immunoprecipitation-based multiple reaction monitoring method to measure serum levels of DNAJB9. We detected a 4-fold higher abundance of serum DNAJB9 in FGN patients when compared to controls, including patients with other glomerular diseases. Serum DNAJB9 levels were also negatively associated with estimated glomerular filtration rate in patients with FGN. Serum DNAJB9 levels accurately predicted FGN with moderate sensitivity (67%) and with high specificity (98%) and positive and negative predictive value (89% and 95%, respectively). A receiver operating curve analysis demonstrated an AUC of 0.958. These results suggest that serum levels of DNAJB9 could be a valuable marker to predict FGN, with the potential to complement kidney biopsy for the diagnosis of FGN.

Noninvasive diagnosis of primary membranous nephropathy using phospholipase A2 receptor antibodies.

Kidney biopsy is the gold standard to diagnose membranous nephropathy (MN). Approximately 70%-80% of patients with primary MN have anti-phospholipase A2 receptor (PLA2R) antibodies. We hypothesized that PLA2R antibody testing without kidney biopsy may be a valid strategy to make a non-invasive diagnosis of MN in patients with a negative work-up for secondary causes. The medical records of all Mayo Clinic patients in Minnesota, Florida, and Arizona with serum PLA2R antibody tests between January 2015 and June 2018 were reviewed. PLA2R antibody testing was performed in 838 unique patients, with 143 testing positive. In 132 of these patients, a native kidney biopsy was performed. The primary diagnosis in all biopsies was MN. Potential secondary causes were identified in 35 cases, with the most common being malignancy and autoimmunity. Ninety-seven patients had a negative work-up for secondary causes of MN. Sixty of those 97 patients had an estimated glomerular filtration rate (eGFR) >60 ml/min/1.73m2. In these patients, the kidney biopsy did not provide significant information that altered management; one patient had a superimposed diabetic nephropathy and a second patient had a superimposed focal segmental glomerulosclerosis (FSGS) lesion. Among the 37 patients with primary MN and eGFR <60 ml/min/1.73m2, additional findings included acute interstitial nephritis, diabetic nephropathy, and cellular crescents in one case each. Thus, among patients with preserved kidney function and no evidence of secondary causes, a positive PLA2R antibody test highly predicts a tissue diagnosis of PLA2R-associated MN. Further validation in a prospective study is warranted to determine whether PLA2R antibody testing be used as a non-invasive diagnostic test to guide therapy.

Cyanide-Nitroprusside Colorimetric Assay: A Rapid Colorimetric Screen for Urinary Cystine.

BACKGROUND: Cystinuria is an autosomal recessive disorder resulting in poor proximal tubule reabsorption of cystine in the nephron, increasing the risk of cystine stone formation. A fast, inexpensive assay to screen for urinary cystine is needed because cystine stones are difficult to noninvasively differentiate from more common calcium-containing ones. Tandem mass spectrometry (MS/MS) is sensitive and specific but is labor-intensive and costly. Alternatively, a colorimetric assay is fast and cost-effective; however, creatinine interference is an issue. METHODS: A published cyanide-nitroprusside colorimetric assay was modified for a high-throughput microplate format. Creatinine interference was reduced using 0.1 mol/L PBS and a standard reaction time of 60 s and was further corrected using a formula derived from the slope of multiple creatinine standard curves. RESULTS: The limit of blank was determined to be 2.6 mg/L, the limit of detection 11.9 mg/L, and the limit of quantitation 15.3 mg/L. The analytic measurement range was established as 15.3-100 mg/L cystine. Intraassay and interassay CV was calculated to be 9.6% and 8.0%, respectively, for a high-level cystine concentration (83.6 mg/L). Low-level cystine (36.4 mg/L) intraassay and interassay CV was determined to be 18.1% and 17.6%, respectively. Passing-Bablok regression analysis of colorimetric vs LC-MS/MS results revealed a slope of 1.10 and y intercept of -7.14 mg/L, with an overall bias of 2% by Bland-Altman plot analysis. CONCLUSIONS: We analytically validated a rapid colorimetric assay suitable to quantify urinary cystine. The effect of thiol drugs on this assay remains to be determined.

Fecal electrolyte testing for evaluation of unexplained diarrhea: Validation of body fluid test accuracy in the absence of a reference method.

BACKGROUND: Validation of tests performed on body fluids other than blood or urine can be challenging due to the lack of a reference method to confirm accuracy. The aim of this study was to evaluate alternate assessments of accuracy that laboratories can rely on to validate body fluid tests in the absence of a reference method using the example of sodium (Na(+)), potassium (K(+)), and magnesium (Mg(2+)) testing in stool fluid. METHODS: Validations of fecal Na(+), K(+), and Mg(2+) were performed on the Roche cobas 6000 c501 (Roche Diagnostics) using residual stool specimens submitted for clinical testing. Spiked recovery, mixing studies, and serial dilutions were performed and % recovery of each analyte was calculated to assess accuracy. Results were confirmed by comparison to a reference method (ICP-OES, PerkinElmer). RESULTS: Mean recoveries for fecal electrolytes were Na(+) upon spiking=92%, mixing=104%, and dilution=105%; K(+) upon spiking=94%, mixing=96%, and dilution=100%; and Mg(2+) upon spiking=93%, mixing=98%, and dilution=100%. When autoanalyzer results were compared to reference ICP-OES results, Na(+) had a slope=0.94, intercept=4.1, and R(2)=0.99; K(+) had a slope=0.99, intercept=0.7, and R(2)=0.99; and Mg(2+) had a slope=0.91, intercept=-4.6, and R(2)=0.91. Calculated osmotic gap using both methods were highly correlated with slope=0.95, intercept=4.5, and R(2)=0.97. Acid pretreatment increased magnesium recovery from a subset of clinical specimens. CONCLUSIONS: A combination of mixing, spiking, and dilution recovery experiments are an acceptable surrogate for assessing accuracy in body fluid validations in the absence of a reference method.

Performance of flow cytometry to screen urine for bacteria and white blood cells prior to urine culture.

BACKGROUND: The gold standard test for the diagnosis of urinary tract infection is bacterial culture. However, urine cultures are labor intensive and costly. Furthermore, since results take 1-2 days many patients are treated presumptively prior to culture results being known. METHODS: We evaluated the Sysmex UF-1000i for the quantification of bacteria and white blood cells (WBCs) in urine in order to determine if it could be used to predict positive culture in comparison to the use of gram stain as a screening tool. RESULTS: The UF-1000i demonstrated good linearity, within and between run precision for bacterial and WBC quantification. Using ROC analysis, the AUC for predicting a positive culture (>10(5)cfu/mL) was 0.95 and 0.90 for bacteria and WBCs, respectively, with optimum cutoffs of 288.9 bacteria/µL and 31.8WBCs/µL, respectively. At these cutoffs, sensitivity (SE) and specificity (SP) for culture positivity were 0.93 and 0.86, respectively, for bacterial counts and 0.89 and 0.79, respectively, for WBC counts. The use of gender specific bacterial cutoffs improved performance, especially in males. In comparison, SE and SP of urine Gram stain were 0.94 and 0.68, respectively. CONCLUSIONS: Quantification of bacteria in unspun urine samples by the Sysmex UF-1000i can be used to screen urine samples for those likely to grow >10(5)cfu/mL. The Sysmex UF-1000i demonstrated increased SP over urine Gram stain, and in this study population could reduce unnecessary reflex urine cultures by 55%.