The development of respiratory dysfunction compromises the recovery of severely ill patients and may contribute to their morbidity and death. While some patients may progress to either ALI or ARDS, the association with recent blood transfusion may be overlooked [20
]. Thus, many cases of ALI/ARDS may in fact represent TRALI, and the true scale of the risks posed by TRALI in the critical care setting are likely to be under-appreciated. Prospective studies have revealed an incidence of TRALI ranging from 5 to 8% in general critical care patients [23
] and up to 29% in ELSD critical care patients admitted with GI bleeding [25
]. This study provides additional evidence that both patient and blood product factors contribute to the development of TRALI, and that the type of blood product influences the severity of injury.
Patients with severe illness are hypothesised to be more likely to develop TRALI, thus critically ill patients may be particularly susceptible to the development of TRALI [14
]. In this study, TRALI only developed in "ill" sheep and did not develop in any of the "healthy" sheep, even following transfusion of "stored PRBC." This is consistent with previous TRALI models, in which both a clinical first event, either LPS-infusion or, in the case of mice, their exposure to a germ environment, and an appropriate second event (that is, stored blood or leucocyte antibody) were required for TRALI to develop [9
]. Thus, this study reaffirms the importance of patient factors in the development of TRALI.
The age of the transfused blood product was also found to be crucial to the development of TRALI, as it predominantly developed in LPS-primed sheep transfused with "stored PRBC" and not "fresh PRBC." This adds to findings from previous in vivo
models in which TRALI has been described subsequent to transfusion with supernatant from stored human PRBC in rats [12
] or stored human PLT in both rats [40
] and in sheep [10
]. During routine storage of PRBC and PLT, proteins and lipids (or their metabolites) are released by cells into the storage medium [28
]. These soluble factors are retained in the supernatant and are thought to contribute to the development of TRALI [1
], although some studies have also implicated the transfused cells [44
]. In this study, cytokine array and ELISA analyses were used to identify the soluble factors that may have contributed to the development of TRALI in the sheep. It was demonstrated that "stored PRBC" contained higher levels of EGF, ENA-78, GRO-α, IL-8, IL-16 and MCP-1 relative to "fresh PRBC", while levels of lactate and potassium increased and levels of sodium decreased. Since neutrophils are key effector cells in TRALI pathogenesis, the biological relevance of these changes was confirmed by the increased in vitro
neutrophil priming ability present in "stored PRBC" compared to "fresh PRBC".
Heat-inactivation of the human blood product supernatant used in this and in previous studies [9
] was necessary to prevent widespread thrombus formation and mortality due to non-specific actions of complement and fibrinogen [9
]; however, represents a limitation of these models. As was demonstrated for sCD40L, heat-inactivation may reduce the concentration of some protein BRMs; however, levels of EGF, ENA-78, GRO-α, IL-8, IL-16 and MCP-1 were all unaffected by heat-inactivation. It remains possible that heat-inactivation may have affected other parameters not investigated, and may have influenced the development of TRALI. The alternative approach of transfusing homologous ovine with PRBC rather than with heat-inactivated supernatant from human PRBC was not used in this study because of the limitations of this alternative approach. First, while the preparation of ovine PRBC is not technically difficult, this process requires standardisation and validation to ensure that the ovine PRBC provide a suitable model of human PRBC. Second, as has been demonstrated in small animal models [45
], there are likely to be differences between the storage lesions of ovine and human PRBC. Detailed comparative data comparing the storage lesions of ovine PRBC and human PRBC are, therefore, essential to validate an ovine model of homologous transfusion for the study of effects related to the age of blood. While future studies are planned to address these limitations of homologous transfusion models, it was felt that, at the present time, the transfusion of heat-inactivated supernatant from human blood products, provided a more relevant clinical model of TRALI.
PRBC that have not undergone pre-storage leucoreduction comprise a significant proportion of the PRBC used in the USA (approximately 20% of the approximately 17 million PRBC transfused in 2009) [50
]. Hence, the findings of this study are of particular clinical relevance to the USA and other countries in which universal pre-storage leucoreduction of blood products has not yet been implemented. Leucoreduction has been shown to reduce the concentration of leucocyte-derived factors in the storage lesion of cellular blood products [41
]; however, whether it also reduces the risk of TRALI remains a matter of conjecture based upon current evidence [7
]. Of note, analyses of 89 TRALI cases from two tertiary care medical centres in the USA [7
] and of 60 TRALI cases in The Netherlands [8
] failed to demonstrate any association between the length of storage of leucoreduced PRBC and TRALI, although these analyses may have been confounded by the presence of leucocyte antibodies in a proportion of leucoreduced PRBC. Hence, the importance of the present study, and the ovine model, as a historical marker allowing for further investigation of the effects of leucoreduction upon TRALI pathogenesis. Accordingly, follow-up studies using equivalent leucoreduced PRBC have been planned to identify common BRM and to elucidate the effects that transfusion of supernatant from stored leucoreduced human PRBC may have upon TRALI pathogenesis in the ovine model. These effects may then be compared to those reported in the present study.
The definition of TRALI used in this study included cases in which a sustained worsening of pre-existing hypoxaemia was evident following transfusion. This was justified because the control groups and detailed monitoring used in the experimental setting make it possible to clearly define such cases. Two separate analyses confirmed the robustness of these data. First, analyses of averaged data demonstrated that the LPS-stored group had lower PaO2
values post-transfusion compared to the LPS-control group (Figure ). Second, analyses using mixed modelling demonstrated that the LPS-stored group had lower PaO2
values post-transfusion compared to both the LPS-control and LPS-fresh groups (Table ). Thus, it was possible to conclude that the worsening hypoxaemia was related to the transfusion of "stored PRBC" rather than the continued effects of LPS-infusion. In contrast to the experimental setting, defining worsening hypoxaemia related to transfusion is problematic in the clinical setting. Therefore, more restrictive criteria, in which TRALI is only defined by the onset of new hypoxaemia, are used clinically [52
]. However, Koch et al
. have demonstrated that, regardless of transfusion history, over 60% of cardiac surgical patients were hypoxaemic upon admission to ICU, this highlighting the difficulty in applying current TRALI definitions in the critical care setting [54
The relative similarity in pulmonary anatomy and physiology between sheep and humans [55
] represents a significant advantage of this ovine model over existing small animal rodent models of TRALI. Another distinct advantage is the larger size of the sheep relative to the rats and mice used for other TRALI models. This enabled detailed monitoring of the respiratory and haemodynamic changes associated with TRALI. In sheep that developed TRALI, the observed reduction in Cstat
and decrement in oxygenation represents a physiological manifestation of the loss of the open alveolar structure evident upon post-mortem histological analysis. The continuous physiological monitoring also revealed that TRALI was associated with development of pulmonary arterial hypertension, further increasing the workload on the right heart, which may lead to poorer tissue oxygenation, with increased venous pressures and reduced cardiac output. Hence, TRALI worsens oxygenation at the arterio-alveolar interphase, as well as diminishing tissue oxygen delivery, due to the cardiovascular perturbations. In addition to predisposing the development of TRALI, it is possible that by worsening tissue dysoxia in other organs the transfusion of stored blood might also contribute to the development of multiple organ dysfunction syndrome (MODS) [21
], although further studies would be required to investigate this hypothesis.
This study provides further evidence that both recipient (first event) and blood product (second event) factors contribute to the development of TRALI. Such a two-event mechanism was first postulated for some instances of ARDS [61
], then was adapted for TRALI [15
], and recently has been re-stated as a threshold mechanism for TRALI [14
]. This proposes that the development of TRALI is associated with both the severity of underlying illness and the strength of blood product factors [14
]. This interaction may provide an explanation for both the unexpected lack of TRALI in a single LPS-infused sheep transfused with "stored PRBC" as well as the unexpected development of TRALI in a single LPS-infused sheep transfused with "fresh PRBC." In the former case, it is possible that recipient factors were protective against TRALI. Genetic factors have been implicated in the development of ALI [62
], and it is possible that they may also play a role in TRALI as only some patients transfused with stored PRBC go on to develop TRALI. In the latter case, post hoc
analyses revealed that abnormal baseline respiratory data were indicative of pre-existing lung injury (initial PaO2
was 277.5, which recovered to 452.5 at the start of the experiment). Therefore, we speculate that pre-existing injury in combination with LPS-infusion may have rendered this sheep more susceptible to the development of TRALI, such that a weaker second event stimulus ("fresh PRBC") was sufficient to induce TRALI. This would be consistent with the proposed threshold mechanism. Thus, critical care patients may be particularly susceptible to the development of TRALI because of the severity of their illness. In addition, the risk of developing TRALI may be further increased if they are transfused with stored blood products which have a higher BRM content [1
Finally, this study demonstrated that the injury profile of TRALI induced by "stored PRBC" was more severe than that previously described by "stored PLT" [10
]. Data re-modelling confirmed a reduction in MAP and CO as well as higher CVP and temperature in TRALI induced by "stored PRBC." The strength of the recipient factors was consistent, as the same dose of LPS was used in both studies [10
]. Therefore, the difference in symptoms may be attributable to a difference in blood product factors. This is supported by the higher concentrations of EGF, IL-8, IL-16, MCP-1, lactate and potassium measured in "stored PRBC" than in "stored PLT." The observation that these higher concentrations, present in the transfused blood product were associated with more severe symptoms is suggestive of a dose-response relationship; however, further research would be required to confirm this hypothesis. Also, the mechanism by which each of these potential BRM may act requires further elucidation. As no differences were observed in the in vitro
neutrophil priming ability of "stored PRBC" and "stored PLT," direct actions of these potential BRMs upon neutrophils are unlikely to contribute to the differences observed in vivo
. It is possible that other cells, such as platelets [38
], T-lymphocytes [63
] and endothelial cells [16
], which also contribute to the pathophysiology of TRALI, may have contributed to the observed haemodynamic differences and this warrants further investigation.