Validation of Neutrophil gelatinase associated lipocalin (NGAL), a urinary biomarker for Acute Kidney Injury and the positive interference from high leukocyturia samples

Abstract

Abstract Acute kidney injury (AKI) is a sudden and serious kidney damage condition that affects more than 1.2 million people every year. KIDNEY DISEASE IMPROVING GLOBAL OUTCOMES (KDIGO) guidelines currently suggest measuring the increase of serum creatinine as a diagnostic marker for AKI. However, the elevation of serum creatinine levels can take up to seven days or when >50% kidney function is lost. Neutrophil gelatinase-associated lipocalin (NGAL) has been identified as an early biomarker for tubular injury, which can be detected in the urine of AKI patients in as early as three hours. In the current study, we evaluated the performance of BioPorto’s (Hellerup, Denmark) NGAL assay, which uses particle enhanced turbidimetry immunoassay (PETIA) technology on the Roche (Indianapolis, USA) cobas c502 chemistry analyzer. Linearity was determined using linearity material from BioPorto and also verified by serial dilutions of a high NGAL patient sample. The limit of quantitation was determined by running urine samples with values of 20 ng/mL, 25 ng/mL, and 35 ng/mL, respectively in duplicate for 10 days. Within-run precisions were determined by running urine two samples containing 100 ng/mL and 400 ng/mL NGAL 20 times and between–run precisions were determined by running the same samples 2 times a day for 10 days. To determine the accuracy of the NGAL method on cobas 502, 40 samples were used to compare results with those measured on Siemens Atellica using the BioPorto’s NGAL assay. Reference interval was verified in 40 urine samples collected from healthy donors. A urine dipstick test was performed to confirm the normal urine samples did not contain leukocyte esterase activity. Since NGAL is also present in neutrophils, we evaluated the effect of leukocytes in urine with the NGAL assay. Results showed the sample with 75 leukocytes/µL had an NGAL of 68 ng/mL and the sample with 500 leukocytes/µL has an NGAL of 222 ng/mL. The linearity range was determined to be 0 - 3000 ng/mL with a limit of quantification of 26 ng/mL. The within-run precision mean and CV% are 121 ng/mL, 4.3% for Level 1 and 401 ng/mL, 1.8% for Level 2. Between-run precisions mean and CV% are 106 ng/mL, 4.8% for Level 1 and 399 ng/mL, 2.6% Level 2. Method comparison had a slope of 0.90 and an intercept of 32.9. The reference interval was determined to be <50 ng/mL and Leukocytes in urine did cause false-positive NGAL results.