Spectrophotometric evaluation of hemolysis in plasma by quantification of free oxyhemoglobin, methemoglobin, and methemalbumin in presence of bilirubin

Severe intravascular hemolysis leads to the simultaneous presence of free heme pigments (oxyhemoglobin, methemoglobin, and methemalbumin) bilirubin in human plasma. Standard spectrophotometric methods used assess vivo inadequately address this complex analytical situation. Thus, we propose a novel quantification algorithm ensure highest specificity. A corresponding second-derivative fitting was validated according guideline bioanalytical method validation from European Medicines Agency using plasma specimens (n = 1759) spiked with different concentrations oxyhemoglobin methemoglobin. The results were compared standard described by Harboe, Noe, Fairbanks. Based on method, methemoglobin/methemalbumin samples total ≤4.9 mg/dL (83.8 μmol/L) provided robust (inaccuracy ≤20%, imprecision ≤16%). Analyzing UV/VIS spectra patients confirmed severe evidenced an underestimation up 33% for combined pigment content. employed allows automated highly specific content diluted plasma, which cannot be realized evaluation methods. An Excel-based tool readily applicable clinical datasets accompanies manuscript.