Heat-inactivation of the human blood product supernatant used in this and in previous studies [9,10,12,28,39] was necessary to prevent widespread thrombus formation and mortality due to non-specific actions of complement and fibrinogen [9,12,39]; however, represents a limitation of these models. As was demonstrated Imatinib Mesylate for sCD40L, heat-inactivation may reduce the concentration of some protein BRMs; however, levels of EGF, ENA-78, GRO-��, 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 [45,47-49], 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 [7,12,18,51].

Of note, analyses of 89 TRALI cases from two tertiary care medical centres in the USA [7] and AV-951 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.