Red blood cell washing, nitrite therapy, and antiheme therapies prevent stored red blood cell toxicity after trauma-hemorrhage

Abstract

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma-hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0-10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.

Keywords: Acute lung injury; Heme; Hemin; Hemoglobin; Nitric oxide; Resuscitation; Storage lesion.

Figure 1. Characterization of murine RBC storage

Panel A C57BL/6 blood was filtered by either Purecell NEO Neonatal High Efficiency Leukocyte Reduction Filter (Pall corporation) (NN) or by Sephadex G25: microcellulose column. (G25). Leukocyte content was determined by FACs and staining for the surface antigen CD45. * P< 0.05 compared to unfiltered (UF) by 1-way ANOVA with Tukey post test (n=3). Panel B: Storage time dependent hemolysis in control (non leukoreduced) RBC (□), leukoreduced (LR) RBC (●) or LR RBC after washing (▼), change indicated by dashed arrows *P<0.01 by 2-way ANOVA with Bonferroni post test (n = 3–9). ^#P<0.02 by paired t-test (n=3–6). Panel C: Storage time dependent formation of glycophorin positive microparticles in LR mRBC before and after washing. *P<0.01 by 1-way ANOVA with Tukey post test relative to day 0 unwashed RBC. ^#P<0.04 by paired t-test relative to respective unwashed RBC (n=3–6). Panel D Storage time dependent changes in P₅₀. *P<0.05 relative to day 0 (n=3) by 1-way ANOVA with Tukey post test. Panel E shows relative rate constants for NO-dioxygenation by intra-erythrocytic (K_RBC) hemoglobin vs. acellular hemoglobin (K_Hb) as a function of storage age. *P<0.05 relative to day 0 by 1-way ANOVA with Tukey post test (n=3–6). Panel F shows lag times for hemolysate mediated nitrite oxidation. *P<0.01 by t-test (n=5).

Figure 2

Standard spectra for oxyhemoglobin, methemoglobin, and hemin in Adsol buffer, pH 7.4 (Panel A). Solutions of hemin alone (0–50µM) or in combination with oxyHb (10µM) + metHb 10µM or with oxyHb 40µM + metHb (60µM) were prepared in Adsol at pH 7.4 and spectra measured (Panel B–D respectively). Spectra were deconvoluted and calculated hemin (Panel E) and oxyHb and metHb (panel F) plotted vs hemin added. Deconvolution analysis was performed using standard spectra shown in Figure 2. Data in panels E–F were fitted by linear regression and are mean ± STDEV (n=3). Gradients determined from regression fit are indicated on respective plots. Key: Panel E: ● = hemin alone group, □ = hemin + oxyHb (10µM) + metHb (10µM) group; ◊ = hemin + oxyHb (50µM) + metHb (50µM) group. Panel F: ▼ = oxyHb, Δ= metHb.

Figure 3. Cell-free heme increases with RBC storage

Panel A: Mouse RBC were stored for 0, 5, or 10d and cell-free heme measured by spectral deconvolution before and after washing. Data show mean ± SEM (n=3–8). *P<0.05 vs. all other groups by 1-way ANOVA with Tukey post test. Panel B,C,D show respectively levels of cell-free heme, cell-free oxyhemoglobin and cell-free methemoglobin measured in the same sample of human RBC collected from bag associated segments after 7d or 35d of storage. Each point represents distinct RBC sample. Indicated P-values calculated by unpaired t-test (N=42). Panel E–F shows residuals for fits of experimental spectra (d7 and d35 respectively) by deconvolution. Panel G shows percent of each species relative to total extracellular heme (free or present in hemoglobin). Data are mean ± SEM (n=42) *P<0.05 for hemin and oxyHb relative to day 7. Deconvolution analysis were performed with mouse (panel A) or human hemoglobin (panel B–E) standard spectra for respectively.

Figure 4. Effects of RBC storage on survival after trauma-hemorrhage

Shown is the percent of mice that survived or died during trauma-hemorrhage resuscitation. Lethality was only observed post resuscitation. Indicated are n-values per group. *P<0.05 or ^#P<0.01 by two-tailed N-1 two proportion test. (□ = dead; ▪ = live)

Figure 5. Effects of RBC storage and washing on acute lung injury after trauma-hemorrhage

Mice were exposed to trauma-hemorrhage and resuscitated with either saline or RBCs that were stored for either 0d or 5d and BAL levels of protein (Panel A) or inflammatory cell (Panel B), with differential analysis (Panel C–D) determined. As indicated, tested variables included transfusing with 1unit (1U) or 3 units (3U), and washing RBC prior to transfusion (grey bars). Black bars indicate pRBC transfusion without washing. *P<0.05 or **P<0.01 as indicated by 1-way ANOVA with Tukey post test. ^#P<0.01 relative to respective unwashed d0 group by 1-way ANOVA with Tukey post test. n= 3–12 per group.

Figure 6. Effects of nitrite therapy on stored RBC mediated acute lung injury after trauma-hemorrhage

Mice were exposed to trauma-hemorrhage and resuscitated with either saline or pRBC that were stored for either 0d or 5d and BAL levels of protein (Panel A) or inflammatory cell (Panel B), with differential analysis (Panel C–D) determined. Nitrite therapy was injected once immediately prior to transfusion at either 10µmol (□) or 100µmol (▪) injected amount per mouse. Note black bars are the same as data presented in Figure 5 and plotted here to improve ease of comparison of nitrite effects on pRBC-dependent acute lung injury test. ^#P<0.01 by 1-way ANOVA with Tukey post test. n= 3–12 per group.

Figure 7. Role of free-heme in stored RBC mediated acute lung injury after trauma-hemorrhage

Mice were exposed to trauma-hemorrhage and resuscitated with either saline or pRBC that were stored for either 0d or 5d, or hemin (15µM stock, 100µl transfusion) and BAL levels of protein (Panel A) or inflammatory cell accumulation (Panel B), with differential analysis (Panel C–D) determined. Hemopexin (Hx), intralipid (vehicle) or TAK-242 (TLR-4 inhibitor) therapy was administered once prior to hemorrhage (grey bars) or once 5–10 min prior to resuscitation (white bars). Note black bars are the same as data presented in Figure 5 and plotted here to allow comparison of TLR-4 inhibition and hemin effects to saline and RBC-dependent acute lung injury. *P<0.05 relative to respective vehicle by t-test. ^#P<0.05 by t-test relative to saline (N= 3–12 per group).

Figure 8. Effects of RBC storage on blood pressure post transfusion

Shown are MAP measured 30min post transfusion of saline or 1unit of RBC stored for 0d or 5d. Data are mean ± SEM (n=3–12 per group). *P<0.05 relative to saline by t-test. #P<0.02 relative to 5d RBC.