Automated Measurement of Plasma Cell-Free Hemoglobin Using the Hemolysis Index Check Function

The Roche Cobas chemistry analyzer's hemolysis index (HI) check function can directly report hemoglobin (Hb) concentrations. We aimed to validate the HI check function for the measurement of plasma cell-free Hb.

Plasma samples (6 μl) were taken by the analyzer and diluted in normal saline to measure the absorbance for Hb at 570 and 600 nm. Hb concentrations were calculated based on the molar extinction coefficient. Imprecision, lower limit of quantification (LLOQ), and analytical measurement range (AMR) of the assay were evaluated. The accuracy was determined by comparing the results between the new method and an existing spectrophotometric method. We further studied interference of icterus and lipemia and carryover. The performance of the assay in proficiency testing was also evaluated. The reference range was transferred from the existing method.

Within-run and total CVs were 1.7%-4.2% and 2.1%-7.0%, respectively (n = 20). The LLOQ was 11 mg/dL (CV = 8.1%) with the upper limit of AMR of 506 mg/dL. The results of the new method correlated well with the existing reference assay: Y (new method) = 0.974 x (reference method) + 4.9, r = 0.9990, n = 52. Bilirubin with a concentration up to 60 mg/dL and lipemic index up to 389 did not show significant interference. No significant carryover was detected. The average standard deviation index in proficiency testing was 0.03 ± 0.29. The reference range was <22 mg/dL.

Plasma cell-free Hb measurement using the HI check function meets the analytical requirements of the plasma cell-free Hb assays. It is simple and cost-effective.