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

Abstract

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

Upconverting nanoparticle-containing erythrocyte-sized hemoglobin microgels that generate heat, oxygen and reactive oxygen species for suppressing hypoxic tumors

“…Representative UV-VIS spectra peaks for met-Hb and oxy-Hb were observed at ∼406 and ∼412 nm, respectively [ 60 , 61 ]. Similarly, our Hb 6 μGel and ascorbic acid-reduced Hb 6 μGel (presumably oxygenated Hb μGel) had UV-VIS spectral peaks at ∼408 and ∼413 nm, respectively ( Fig.…”

Optimized RT-qPCR and a novel normalization method for validating circulating miRNA biomarkers in ageing-related diseases

“…However, recent studies on haemolysis focus on developing and improving absorbance methods by measurement of few different wavelengths and additional mathematical algorithm for interference determination and elimination. Increasing number of reports on lysis quantification in blood sample have confirmed the effectiveness of using absorbance-based method improved by mathematical algorithm 22 , 23 , 33 – 35 . Interestingly, we have also shown that usage of the entire absorbance spectrum for the samples to evaluate haemolysis could be a viable alternative method that would not rely on a priori selection of important wavelengths (Supplementary Fig.…”

Carboxyhemoglobin and Methemoglobin Levels and Hemolysis in Children Undergoing Cardiac Surgery With Cardiopulmonary Bypass

“…One possible explanation for these findings is that hemolysis can elevate MetHb levels. In fact, studies reported that methemoglobinemia originated from intravascular hemolysis 18 and observed in-bag hemolysis of stored RBCs due to oxidative stress. 19,20 The difference in MetHb level between both groups was minimal, and the predictive ability for hemolysis was not excellent; however, MetHb elevation could be due to hemolysis during CPB.…”

“…During hemolysis, hemoglobin is released into the circulation as oxyhemoglobin, which is expected to be oxidized to MetHb. Studies have reported methemoglobinemia caused by intravascular hemolysis 18 and in-bag hemolysis of stored red blood cells (RBCs) due to oxidative stress. 19,20…”

“…During hemolysis, hemoglobin is released into the circulation as oxyhemoglobin, which is expected to be oxidized to MetHb. Studies have reported methemoglobinemia caused by intravascular hemolysis 18 and in-bag hemolysis of stored red blood cells (RBCs) due to oxidative stress. 19,20 Plasma-free hemoglobin (PFH) is released from RBCs during hemolysis, and measuring its level is the gold standard for monitoring intravascular hemolysis, as the Extracorporeal Life Support Organization recommends.…”