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

Area of Science:

• Clinical Chemistry
• Spectrophotometry
• Biomarker Quantification

Background:

• Hemolysis assessment is crucial in critical care.
• Existing free hemoglobin (fHb) quantification methods fail in hyperbilirubinemia.
• Hyperbilirubinemia is common in patients at risk for intravascular hemolysis.

Purpose of the Study:

• Develop and validate a novel method for accurate fHb quantification.
• Address limitations of current methods in the presence of bilirubin.
• Provide a user-friendly tool for clinical laboratories.

Main Methods:

• Developed a second-derivative fitting algorithm for fHb quantification.
• Utilized spectrophotometric data (350-650 nm).
• Validated against established methods (Harboe, Noe, Fairbanks) using spiked patient and healthy volunteer plasma, adhering to EMA guidelines.

Main Results:

• The novel method accurately quantifies fHb in non-icteric samples (<10% inaccuracy/imprecision).
• Established methods yield false high fHb results in icteric specimens (<700 mg/L).
• The second-derivative fitting method shows no interference with bilirubin up to 465 μmol/L, with a lower limit of quantification of 25 mg/L.

Conclusions:

• A computer-based algorithm accurately quantifies fHb in the presence of high bilirubin.
• The method offers uniform sample preparation and is implementable on standard spectrophotometers.
• This facilitates reliable assessment of hemolysis in icteric critical care patients.