A second-derivate fitting algorithm for the quantification of free hemoglobin in human plasma

Assessment of hemolysis in vivo is becoming increasingly relevant in critical care. Current methods (Harboe, 1959) for quantifying the free hemoglobin (fHb) content produce unsatisfactory results in case of hyperbilirubinemia, a frequent condition in patients at risk for intravascular hemolysis. A novel evaluation method based on second-derivative fitting to quantify fHb content was developed. The method uses spectrophotometric data from 350 to 650 nm recorded with standard instruments as input. To evaluate the power of the new method, plasma of patients and non-icteric plasma of healthy volunteers were spiked with fHb concentrations up to 2000 mg/L and compared to methods described in the literature by Harboe, Noe and Fairbanks. All measurements were done in compliance with the bioanalytical method validation protocol from the European Medicines Agency. Both the second-derivative fitting algorithm as well as the methods of Harboe, Noe and Fairbanks quantified fHb accurately in non-icteric samples, with inaccuracy and imprecision below 10%. For icteric specimen, false high results were obtained with the established formulas for fHb concentrations below 700 mg/L. In contrast, no interference was found with the second-derivate fitting method for bilirubin concentrations up to 465 μmol/L. The lower limits of quantifications for the second-derivative fitting algorithm were specified in agreement with the EMA guideline with 25 mg/L fHb for both non-icteric and icteric specimens. A user-friendly, computer-based algorithm is reported that allows the accurate quantification of fHb concentrations in the presence of high bilirubin concentrations. The new method allows for uniform sample preparation with only a single dilution step and can be readily implemented in any laboratory on standard spectrophotometers using the provided supplementary Microsoft Excel macro.