Detection and quantification of heme and hemoglobin for diagnosis of intravascular hemolysis

Introduction Hemolysis, caused by e.g. disorders like sickle cell disease, paroxysmal nocturnal hemoglobinuria and vascular trauma or by infections and injuries, is often characterized by serious conditions and consequences for the patients, such as kidney injury or vasoocclusion [1] [2] [3]. These clinical concerns are mainly attributed to elevated levels of hemoglobin and labile heme from premature rupture of red blood cells during intravascular hemolysis. Diagnosis of hemolysis is usually done based on elevated reticulocyte, lactate dehydrogenase (LDH), and bilirubin levels, as well as reduced haptoglobin plasma concentrations [4] [6]. Additionally, urine hemosiderin and urine/blood extracellular hemoglobin indicate intravascular hemolysis [1]. Although several methods are available for the diagnosis and monitoring of hemolytic disorders, only hemoglobin is currently considered as a diagnostic marker [5] [7]. However, due to the proven inflammation- and thrombosis-triggering effects of heme, quantification of this biomarker should also be considered in clinical settings to achieve a complete picture of the hemolytic state and the treatment strategies [7] [8].

Method The aim of our study was to evaluate the validity and applicability of the quantification methods for hemoglobin and heme levels as well as to differentiate between hemoglobin-bound heme and labile heme. Indirect and direct approaches, including e.g., chromatography, spectroscopy, and mass spectrometry as well as enzymatic test systems, were used to determine the concentration of heme and hemoglobin as well as mixtures thereof.

Results A clear distinction between hemoglobin-bound heme and labile heme with one method was, however, not possible, suggesting the use of a combined approach. With a specific spectroscopic approach and a newly established equation we were able to determine both analytes in human plasma samples of different hemolytic states.

Conclusion Our study gives a broader perspective by adding to the knowledge about hemoglobin and heme quantification in research and/or clinical diagnosis. The implementation of an amalgamated method is an easy-to use technique requiring low sample volumes and can thus enable fast detection via spectrophotometric tools. This would enable monitoring of heme as a biomarker to understand the molecular basis of hemolytic disorders in clinical diagnosis.

Conflict of Interest

The authors declare no competing interests.

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Publication History

Article published online:
26 February 2024