Determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay

Abstract

Transfusion effectiveness of red blood cells (RBCs) has been associated with duration of the storage period. Storage-dependent RBC alterations lead to hemolysis and release of toxic free heme, but the increase of free heme levels over time is largely unknown. In the current study, an apo-horseradish peroxidase (apoHRP)-based assay was applied to measure levels of free heme at regular intervals or periodically in supernatants of RBCs until a maximum storage period of 42 days. Free heme levels increased with linear time-dependent kinetics up to day 21 and accelerated disproportionally after day 28 until day 42, as determined with the apoHRP assay. Individual time courses of free heme in different RBC units exhibited high variability. Notably, levels of free hemoglobin, an established indicator of RBC damage, and those of total heme increased with continuous time-dependent linear kinetics over the entire 42 day storage period, respectively. Supernatants from RBC units with high levels of free heme led to inflammatory activation of human neutrophils. In conclusion, determining free heme in stored RBCs with the applied apoHRP assay may become feasible for testing of RBC storage quality in clinical transfusion medicine.

Alpha1-antitrypsin improves survival in murine abdominal sepsis model by decreasing inflammation and sequestration of free heme

“…Measurement of labile heme concentration in PL fluid samples was performed in 96-well plates as previously described ( 22 ). Briefly, 5 µL of an appropriately diluted PL fluid sample was added to 95 µL of HBSS buffer containing 0.75 µM Apo-Horseradish Peroxidase (Apo-HRP) (BBI Solutions, Gwent, UK) and incubated for 10 min at 4°C.…”

Hematin- and Hemin-Induced Spherization and Hemolysis of Human Erythrocytes Are Independent of Extracellular Calcium Concentration

“…Free heme, ferrous (Fe 2+ ) protoporphyrin (PP) IX, and heme derivatives are widely recognized pathological molecules in a number of hemolytic conditions, and their high concentrations (up to 500 µM) emerge in the human organism during malaria [ 1 , 2 ], hemorrhagic stroke [ 3 , 4 , 5 ], gastric ulcer [ 6 ], sickle cell disease, and beta-thalassemia [ 7 ]. Moreover, accumulation of degraded hemoglobin in stored RBCs can also lead to hematin/hemin formation, and the supernatant of such blood units was shown to trigger inflammatory activation of neutrophils, meaning that transfusion of such units might contribute to transfusion effectiveness decrease and in the worst cases to poor clinical outcomes [ 8 , 9 ]. Hematin (ferric (Fe 3+ ) PP IX liganded with OH − ) is formed predominantly during malaria RBC invasion as a by-product of the parasite’s digestion of host cell hemoglobin (Hb), while hemin (Fe 3+ PP IX liganded with Cl − ) is constantly produced in the human body during the reactions of Hb autooxidation either spontaneous or disease-triggered ( Supplementary Figure S1 ) [ 10 ].…”

Interaction of haemin with albumin-based macroporous cryogel: Adsorption isotherm and fluorescence quenching studies

“…In a functioning body, free haem uptake is facilitated by specific serum transport proteins (globin, albumin and haemopexin) and distributed according to their concentrations, affinities for haem, and rates of clearance from the circulation ( Morgan et al, 1976 ). However, in patients ( Fontanellas et al, 1994 ), blood transfusion bags ( Vijayan et al, 2022 ) or when designing haemoglobin-based oxygen carriers (HBOCs) ( Bialas et al, 2019 ), the free haem ions should be removed by other means and methods, such as filtration or dialysis ( Fontanellas et al, 1994 ; Coronel et al, 2003 ), to minimise their side effects. However, these methods have a few limitations, such as fouling and low efficiency when dealing with suspensions and complex mixtures.…”