Predicting storage-dependent damage to red blood cells using nitrite oxidation kinetics, peroxiredoxin-2 oxidation, and hemoglobin and free heme measurements

Abstract

Background: Storage-dependent damage to red blood cells (RBCs) varies significantly. Identifying RBC units that will undergo higher levels of hemolysis during storage may allow for more efficient inventory management decision-making. Oxidative-stress mediates storage-dependent damage to RBCs and will depend on the oxidant:antioxidant balance. We reasoned that this balance or redox tone will serve as a determinant of how a given RBC unit stores and that its assessment in "young" RBCs will predict storage-dependent hemolysis.

Study design and methods: RBCs were sampled from bags and segments stored for 7 to 42 days. Redox tone was assessed by nitrite oxidation kinetics and peroxiredoxin-2 (Prx-2) oxidation. In parallel, hemolysis was assessed by measuring cell-free hemoglobin (Hb) and free heme (hemin). Correlation analyses were performed to determine if Day 7 measurements predicted either the level of hemolysis at Day 35 or the increase in hemolysis during storage.

Results: Higher Day 7 Prx-2 oxidation was associated with higher Day 35 Prx-2 oxidation, suggesting that early assessment of this variable may identify RBCs that will incur the most oxidative damage during storage. RBCs that oxidized nitrite faster on Day 7 were associated with the greatest levels of storage-dependent hemolysis and increases in Prx-2 oxidation. An inverse relationship between storage-dependent changes in oxyhemoglobin and free heme was observed underscoring an unappreciated reciprocity between these molecular species. Moreover, free heme was higher in the bag compared to paired segments, with opposite trends observed for free Hb.

Conclusion: Measurement of Prx-2 oxidation and nitrite oxidation kinetics early during RBC storage may predict storage-dependent damage to RBC including hemolysis-dependent formation of free Hb and heme.

Figure 1. Effects of storage on hemolysate mediated nitrite oxidation kinetics

Panel A shows a representative tracing for nitrite (250μM)-mediated oxidation by d7 and d35 RBC hemolysates (25μM hemoglobin, (concentrations reflect heme) assessed by absorbance change at 570nm, at 25°C, in PBS + 100μM DTPA. Panel B-D show respectively, lag times, lag rates and propagation rates plotted as a function of nitrite added to d7 (○) and d35 (■) hemolysates. Data are mean ± SEM, n=10. *p<0.05 by 2-way ANOVA with Bonferroni post tests.

Figure 2. Effects of storage on hemolysis and Prx-2 oxidation

Shown are paired assessments in d7 and d35 supernatant fraction for cell-free oxyhemoglobin (panel A), cell-free methemoglobin (Panel B), cell-free heme (Panel C), RBC oxyhemoglobin (Panel D), RBC-methemoglobin (Panel E) and RBC-heme (Panel F) Each point (n=32) denotes sampling from an individual donated RBC unit. Panel G shows oxidized Prx-2 : total Prx-2 ratio determined by western blotting (n= 42). A representative western blot with 5 paired d7 and d35 samples are shown. Indicated P-values by paired t-test using *normal or ^#non-normal (Wilcoxon signed rank test) distribution.

Figure 3. Heterogeneity of storage dependent changes in hemolysis and Prx-2 oxidation

Storage dependent changes (d35 – d7) in cell-free oxyhemoglobin (Panel A), methemoglobin (Panel B), free heme (Panel C) and Prx-2 oxidation in RBC (Panel D) were calculated. Each symbol represents a unique RBC unit.

Figure 4. Basal Prx-2 oxidation dependent correlations

Panel A shows correlations between Prx- oxidation at d7 and d35. Panels B-D show respectively, d7 Prx-2 oxidation correlations with d7 lag times, d7 lag rates and d7 propagation rates for nitrite oxidation. P-values indicate significance by Pearson correlations; line show linear regression with 95% confidence intervals of fit.

Figure 5. Nitrite oxidation kinetics dependent correlations

Panel A-B show correlations between rate of nitrite oxidation in the lag phase from d7 RBC with d7 and d35 free heme levels, respectively. Panel C-D show correlations between rate of nitrite oxidation in the propagation phase from d7 RBCs with d7 and d35 extracellular free hemoglobin levels respectively. Panel E shows the correlation between rate of nitrite oxidation in the propagation phase from d7 RBCs with storage dependent increases in Prx-2 oxidation (d35 – d7). P-values indicate significance by *Pearson or ^#Spearman correlations; line show linear regression with 95% confidence intervals of fit.

Figure 6. Heme and hemoglobin dependent correlations

Panel A-B show correlations between d7 free heme levels and d35 free heme and d35 oxyhemoglobin, respectively. Panel C-D show correlations between d7 oxyhemoglobin and storage-dependent increases in free oxyhemoglobin and heme, respectively. Panel E shows the correlation between storage dependent changes in free oxyhemoglobin and free heme. P-values indicate significance by *Pearson or ^#Spearman correlations; line show linear regression with 95% confidence intervals of fit.

Figure 7. Effects of bag vs segment sampling

RBC collected from bags and paired segments after storage for 35 days or 42 days and nitrite oxidation kinetics measured by lag times (Panel A using 250μM nitrite), lag rates (Panel B using 500μM nitrite), propagation rates (Panel C using 250μM nitrite). Panels D-F show respectively levels of oxyhemoglobin, free heme and the total (oxyhemoglobin + free heme) in paired bags and segments. *P < 0.05 by paired t-test relative to respective bag or segment at d35. NS = not significant by paired t-test. Data show mean ± SEM, n=4-5.