Electron paramagnetic resonance oximetry as a novel approach to monitor the effectiveness and quality of red blood cell transfusions

Abstract

Background: The goal of red blood cell transfusion is to improve tissue oxygenation. Assessment of red blood cell quality and individualised therapeutic needs can be optimised using direct oxygen (O₂) measurements to guide treatment. Electron paramagnetic resonance oximetry is capable of accurate, repeatable and minimally invasive measurements of tissue pO₂. Here we present preclinical proof-of-concept of the utility of electron paramagnetic resonance oximetry in an experimental setting of acute blood loss, transfusion, and post-transfusion monitoring.

Materials and methods: Donor rat blood was collected, leucocyte-reduced, and stored at 4 °C in AS-3 for 1, 7 and 14 days. Red blood cell morphology, O₂ equilibrium, p50 and Hill numbers from O₂ binding and dissociation curves were evaluated in vitro. Recipient rats were bled and maintained at a mean arterial pressure of 30-40 mmHg and hind limb muscle (biceps femoris) pO₂ at 25-50% of baseline. Muscle pO₂ was monitored continuously over the course of experiments to assess the effectiveness of red blood cell preparations at different stages of blood loss and restoration.

Results: Red blood cell morphology, O₂ equilibrium and p50 values of intra-erythrocyte haemoglobin were significantly altered by refrigerated storage for both 7 and 14 days. Transfusion of red blood cells stored for 7 or 14 days demonstrated an equivalently impaired ability to restore hind limb muscle pO₂, consistent with in vitro observations and transfusion with albumin. Red blood cells refrigerated for 1 day demonstrated normal morphology, in vitro oxygenation and in vivo restoration of tissue pO₂.

Discussion: Electron paramagnetic resonance oximetry represents a useful approach to assessing the quality of red blood cells and subsequent transfusion effectiveness.

Figure 1

Red blood cell morphology. Scanning electron microscopy images of Lewis rat RBC before and after refrigerated storage. (A) Fresh (day 1) RBC, (B) RBC stored for 7 days, (C) RBC stored for 14 days, (D) zoomed in image of cells from the dashed box in (C) with arrows indicating pores on the RBC surface. Scale bars = 5 microns. RBC: red blood cells.

Figure 2

Oxygen equilibrium, ATP and 2,3-diphosphoglycerate (2,3-DPG) in red blood cells. (A) Oxygen binding curves for fresh (day 1) RBC, RBC stored for 7 days, and RBC stored for 14 days (n=4 storage units per time point). (B) Oxygen dissociation curves for fresh (day 1) RBC, RBC stored for 7 days, and RBC stored for 14 days (n=4 storage units per time point). Dashed lines indicate the p50 values for each RBC preparation. (C) Mean ± standard deviation (SD) p50 values derived from oxygen binding (●) and dissociation curves (■), p<0.0001 by one-way ANOVA with a multiple comparisons test. (D) Mean ± SD Hill numbers (n50) derived from oxygen binding (●) and dissociation Hill plots (■) showed no significant differences following a one-way ANOVA with a multiple comparisons test. (E) Individual data with mean ± SD for ATP concentrations at days 1, 7 and 14, p<0.0001 by one-way ANOVA with a multiple comparisons test. (F) Individual data with mean ± SD for 2,3-DPG concentrations at days 1, 7 and 14, p<0.0001 by one-way ANOVA with a multiple comparisons test. ATP: adenosine triphosphate; RBC: red blood cells.

Figure 3

Model parameters. (A) Diagram of the study design and EPR magnet/resonator with a dashed oval outline showing the location/positioning of the rat (arrow indicating close-up of resonator) OxyChip prior to implantation and hind limb with resonator over implanted chip (arrow indicating location of OxyChip). (B) Blood loss to induce and maintain, for 30 minutes, a mean arterial pressure (MAP) of 30-40 mmHg and a biceps femoris muscle pO₂ at 25-50% in the groups given fresh (day 1) RBC, RBC stored for 7 days, RBC stored for 14 days and albumin (n=7 in each group). (C) MAP values (mean ± SD) over 30 minutes at baseline and 30 minutes at blood loss (n=7), *p<0.0001 by a paired t-test comparing basal MAP and MAP after blood loss. (D) Biceps femoris pO₂ values (mean ± SD) over 30 minutes at baseline and 30 minutes at blood loss (n=6), *p<0.0001 by a paired t-test comparing basal MAP and MAP after blood loss. Four outlier animals had abnormally high basal pO₂ values and are shown, but not included in the statistical analysis.

Figure 4

Biceps femoris muscle tissue O₂ concentrations. Data are shown for animals transfused with RBC stored for 7 days and, separately, for those transfused with RBC stored for 14 days: in both cases comparisons were made with the groups given fresh (day 1) RBC and albumin. (A) Individual, mean ± standard deviation (SD) rat pO₂ values at baseline, after blood loss, during transfusion and after transfusion are shown for the 30-minute time frames for RBC stored in a refrigerator for 7 days (n=6). (B) Individual, mean ± standard deviation (SD) rat pO₂ values at baseline, after blood loss, during transfusion and after transfusion are shown for the 30-minute time frames for RBC stored in a refrigerator for 14 days (n=6). (C) Scans showing pO₂ values in three rats/group for 7-day refrigerator-stored RBC and (D) 14-day refrigerator-stored RBC. Data for animals given fresh (day 1) RBC or albumin are repeated in each plot. Values at baseline vs blood loss and at blood loss vs transfusion and post-transfusion were compared using a one-way ANOVA with a multiple comparisons test. For (A) and (B) all p values are shown on the plots for comparison. Four animals had abnormally high basal pO₂ values and these outlier values are represented by muted circles; data from these animals were not included in the statistical analysis.