||Serological hyaluronic acid (HA) has been proposed as a noninvasive alternative to liver biopsy for assessing the extent of liver fibrosis. Liver biopsy correctly identifies hepatic disease in about 65 to 75% of cases, being strongly dependent on the length of the biopsy obtained. Furthermore, hepatic fibrosis is often not distributed homogeneously throughout the liver. In contrast, studies suggest HA to have better sensitivity and specificity with areas under receiver operating curves of 0.86 and 0.92, and negative predictive values of 93% and 99% for fibrosis and cirrhosis respectively. Moreover, the non-uniform distribution of hepatic fibrotic tissue does not affect HA results. In this study, we evaluated and validated a commercially available enzyme-linked binding protein assay for measuring HA. We also investigated serum/plasma HA concentrations in subjects having elevated levels of hepatitis C virus (HCV) RNA. The HA assay (Corgenix Inc., Westminster, CO) is a spectrophotometric sandwich protein binding assay in microplate format. The test utilizes a highly specific HA binding protein (HABP) coated to the microwell surface to capture HA. An enzyme conjugated version of HAPB is subsequently used to detect the HA in the sample. The assay uses six calibrators with final results expressed as ng HA/mL. Serum and/or plasma samples were stored and assayed according to the kit manufacturer's instructions. The assay's limit of detection was 8 ng/mL resulting in an analytical measurement range of 8 - 800 ng/mL. A linearity study spanning this range generated a slope of 1.008, intercept of 13.64 and R2 of 0.998 (n = 7). The within-run precision at three levels (n = 8) was determined to be 29 ? 0.8, 138 ? 3.4 and 546 ? 6.7 ng/mL with CVs of 2.7, 2.5 and 1.2% respectively. Between-run precision at three levels (n = 7) resulted in values of 44? 3.9, 87? 7.6 and 459? 29.3 ng/mL generating CVs of 8.8, 8.5 and 6.4% respectively. A correlation study using samples previously assayed at Corgenix produced a slope, intercept and R2 of 0.957 -0.56 and 0.993 respectively as analyzed by Deming Regression (n = 22, range 29 - 866 ng/mL). Utilizing donations from 122 healthy individuals, an upper 97.5% reference limit of 54 ng/mL was established. HA was found stable for 24 hours at room temperature, and a minimum of two weeks at 4?C. Deidentified serum or plasma samples from patients with HCV infection were assayed for HA. These patients had HCV RNA levels greater than log 2.3 IU/mL as previously measured by real-time polymerase chain reaction. Of 68 specimens, 53% (36) were found to have elevated HA, with concentrations greater than 37 ng/mL. HA concentration did not correlate with HCV RNA level. In conclusion, the Corgenix HA test kit has shown acceptable performance characteristics for quantifying HA. Although the stages of liver fibrosis for the HCV infected subjects in this study were unavailable, the large percentage having elevated HA supports previous studies suggesting the possible use of HA in assessing liver fibrosis and/or cirrhosis in lieu of liver biopsy.