Iron-deficient erythropoiesis in blood donors and red blood cell recovery after transfusion: initial studies with a mouse model

Abstract

Background: Most frequent red cell (RBC) donors and many first-time donors are iron deficient, but meet haemoglobin standards. However, the effects of donation-induced iron deficiency on RBC storage quality are unknown. Thus, we used a mouse model to determine if donor iron deficiency reduced post-transfusion RBC recovery.

Methods: Weanling mice received a control diet or an iron-deficient diet. A third group receiving the iron-deficient diet was also phlebotomised weekly. This provided 3 groups of mice with different iron status: (1) iron replete, (2) mild iron deficiency with iron-deficient erythropoiesis, and (3) iron-deficiency anaemia. At ten weeks of age, blood was collected, leucoreduced, and stored at 4 ºC. After 12 days of storage, 24-hour (h) post-transfusion RBC recovery was quantified in recipients by flow cytometry.

Results: Before blood collection, mean haemoglobin concentrations in the iron-replete, iron-deficient, and iron-deficiency anaemia donor mice were 16.5±0.4, 11.5±0.4, and 7.0±1.4 [g/dL± 1 standard deviation (SD)], respectively (p<0.01 for all comparisons between groups). The 24-h post-transfusion RBC recoveries in recipients receiving transfusions from these three cohorts were 77.1±13.2, 66.5±10.9, and 46.7±15.9 (% ±1 SD), respectively (p<0.05 for all comparisons between groups).

Discussion: In summary, donor iron deficiency significantly reduced 24-h post-transfusion RBC recovery in recipient mice. RBCs from mice with mild iron deficiency and iron-deficient erythropoiesis, with haemoglobin levels similar to those used for human autologous blood donation, had intermediate post-transfusion RBC recovery, as compared to iron-replete donors and those with iron-deficiency anaemia. This suggests that, in addition to the effects of iron deficiency on donor health, frequent blood donation, leading to iron-deficient erythropoiesis, may also have adverse effects for transfusion recipients.

Figure 1

Red blood cell (RBC) zinc protoporphyrin progressively increases with increasing severity of iron deficiency. Iron deficiency progresses from reduced iron stores to iron depletion, in which there is normal erythropoiesis with normal zinc protoporphyrin levels, but decreased ferritin due to absent iron stores. The next stage is iron-deficient erythropoiesis, in which increased RBC levels of zinc protoporphyrin and very low serum ferritin levels reflect both absent iron stores and the lack of sufficient iron for normal haemoglobin production. As iron deficiency progresses further, frank iron deficiency anaemia develops with insufficient iron for maintaining adequate haemoglobin levels. Adapted from Hastka J et al. and Brittenham GM,.

Figure 2

Blood donor mice with varying iron status were generated by dietary manipulation. Three groups of weanling mice (N=15–20 per group) were fed either an iron-replete or iron-deficient diet (IDE) for 6 weeks. A third group was fed the iron deficient diet and was phlebotomised weekly to induce iron deficiency anaemia (IDA). After 6 weeks on the diet, the mice were euthanised and blood collected by exsanguination. (A) The average haemoglobin per group across 3 replicate experiments. (B) Liver iron, as a measure of total body iron, quantified in 5 representative mice per group from a single experiment. (C) Zinc protoporphyrin levels in the pooled blood from each group across two representative experiments (these measurements were not performed in one of the replicate experiments). *p<0.05, **p<0.01, ***p<0.001 by One-way ANOVA with Tukey's multiple comparison test. ***Without brackets in panel B represents significance compared to both other groups.

Figure 3

Additional laboratory parameters demonstrating the validity of the mouse model of variable iron status. Additional parameters were measured on the animals described in Figure 2. Three groups of weanling mice (N=15–20 per group) were fed either an iron-replete or iron-deficient diet (IDE) for 6 weeks. A third group was fed the iron deficient diet and was phlebotomised weekly to induce iron deficiency anaemia (IDA). After 6 weeks on the diet, blood was collected from the saphenous vein and pooled per cage in each group. The measurements are as labeled in the figure panels. *p<0.05, **p<0.01, ***p<0.001 by One-way ANOVA with Tukey's multiple comparison test. ***Without brackets in panel B represents significance compared to both other groups.

Figure 4

Donor RBCs from mice with iron deficiency exhibit decreased 24-hour post-transfusion recovery. Three groups of adult mice with varying iron status: iron replete, iron-deficient erythropoiesis with mild iron deficiency (IDE), and severe iron deficiency anaemia (IDA) were generated by modifying the iron-content of their diets with or without additional phlebotomy. At ~10 weeks of age, blood was collected from each cohort, pooled, leucoreduced, packed, and refrigerator-stored in CPDA-1 for 12 days. The 24-hour post-transfusion recoveries of the stored RBCs were quantified using flow cytometry in enhanced Green Fluorescent Protein-transgenic recipient mice. The experiment was repeated three times in its entirety (n=8 recipient mice per group per experiment; 24 mice per group total). Shown are the individual 24-hour post-transfusion recovery results for each recipient mouse, combined across all three replicate experiments. *p<0.05, ***p<0.001 by One-way ANOVA with Tukey's multiple comparison test.