On the source of the non-transferrin-bound iron which accumulates in packed red blood cell units during storage

Abstract

Background: Recent studies have shown large increases in non-transferrin-bound iron (NTBI) and biomarkers of oxidative stress in the extracellular medium of packed red blood cell units during storage. It has been further suggested that transfusion-mediated iron and oxidative load may contribute to transfusion-related morbidity in premature babies. The origin and nature of the NTBI is currently unclear, but the release of iron from oxidatively modified haemoglobin and haem has been suggested. The purpose of this study was to investigate whether this may be the case.

Materials and methods: The concentration of haem in the extracellular fluid of paediatric packed cell units stored from 3 to 35 days was measured using a commercial haem assay. In vitro studies were conducted using haem (haemin; ferriprotoporphyrin IX chloride) to determine whether the NTBI assay was able to react with and measure iron associated with haem in the presence and absence of oxidising agents.

Results: The level of haem in the extracellular fluid of paediatric packed cell units rose gradually from day 3 to day 21, then more rapidly to day 35. Very little NTBI was released from haem in the absence of oxidising agents, but the amount rose in a dose- and time-dependent manner in proportion to the oxidation of haem by incubation with H2O2.

Discussion: The results of the study imply that the NTBI measured in previous studies may derive from the oxidatively modified haem that builds up in the extracellular fluid of packed red blood cell units during storage. The potential influence of this on transfusion mediated morbidity is discussed.

Figure 1

The concentration of haem in the extracellular milieu of stored paediatric packed cell units as a function of storage age. Results are presented as the mean±SD of ten sets of paediatric packs. There were no significant differences between levels measured between days 3 and 14. Thereafter the levels increased significantly (p<0.05) with storage age. ANOVA with Duncan’s multiple comparison test.

Figure 2

The concentration of NTBI measured in haem solutions of 10 μmol/L (dark grey columns), 20 μmol/L (light grey columns) and 50 μmol/L (medium grey columns) following incubation for 30 minutes at room temperature without H₂O₂ (unoxidised), 1 mM H₂O₂ and 5 mM H₂O₂. Results are expressed as the mean±SD of five experiments. There was very little NTBI in the non-oxidised group, and no significant differences with regard to haem concentration in this group. In the oxidised groups the level of NTBI increased with haem concentration (p<0.05). ANOVA with Duncan’s multiple comparison test.

Figure 3

The amount of NTBI released from 50 μM haem as a function of H₂O₂ concentration. Haem was incubated for 30 minutes at room temperature at the different concentrations of H₂O₂. Results are expressed as the mean±SD of five experiments. NTBI concentration increased significantly (p<0.05) with H₂O₂ concentration beyond 1 mM. ANOVA with Duncan’s multiple comparison test.

Figure 4

The percentage of available haem iron present as NTBI in the extracellular milieu of paediatric packed cell units as a function of storage age. Results are expressed as the mean percentage±SD of ten sets of paediatric packed cell units. There were significant differences (p<0.05) between all time-points with the exception of between days 21 and 14, and days 21 and 28. ANOVA with Duncan’s multiple comparison test.