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 RBC are thought to underlie this association. In this study we determined the effects of RBC washing, nitrite and anti-heme 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 RBC stored for 0–10d was used. Tested variables included whether washing RBC to remove lower MWt 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 5d RBC increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5d RBC prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10d RBC elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5d RBC. Both washing and nitrite therapy significantly protected against 10d 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 RBC. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBC. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5d and 10d mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy and / or anti-heme and TLR4 strategies may prevent stored RBC toxicities.

Exposure of Stored Packed Erythrocytes to Nitric Oxide Prevents Transfusion-associated Pulmonary Hypertension

“…The increase in PAP after transfusion is more pronounced when endothelial dysfunction (caused by systemic diseases such as diabetes, or cardiopulmonary bypass) reduces nitric oxide bioavailability 11,13,27 . In addition, stored RBCs themselves may have inhibitory effects on nitric oxide-mediated vasodilation through nitric oxide scavenging 28-30 . Solomon and co-workers demonstrated in an exchange transfusion model in septic dogs that transfusion of stored blood produces persistent pulmonary hypertension that was linked to severe lung damage including necrosis, hemorrhage and thrombosis 26 .…”

Absorbance and redox based approaches for measuring free heme and free hemoglobin in biological matrices

“…Spectra were analyzed by deconvolution algorithms with multilinear regression fitting (Excel) as described [6], [33], [34].…”

Circulating Erythrocyte Microparticles and the Biochemical Extent of Myocardial Injury in ST Elevation Myocardial Infarction

“…These findings have been supported by studies investigating the role of ex vivo generated RBCm (i.e. in packed erythrocyte units) transfused in experimental models [10,14]. Moreover, there are data supporting the concept that after a longer duration of erythrocyte packing, the ‘associated' scavenging of nitric oxide by hemoglobin is faster [15].…”

“…Accordingly, Stapley et al [14] showed that preprocessing of erythrocyte packs (i.e. washing in order to remove potentially harmful components like RBCm) or employment of processes that prevent the ‘toxic' effects of hemoglobin (e.g.…”