Lyophilized Platelet Transfusion Does Not Constitute An Immunologic “Second Hit” In a Non-Human Primate Hemorrhagic Shock Model

Apheresed platelet concentrates accumulate biologic response modulators (BRMs) with prolonged storage. BRMs have been localized to the supernatant fraction of stored blood products and BRM levels correlate with length of storage. BRMs, and hence transfusion, have been demonstrated to function as a recipient immune “second hits”, in the construct of the two hit model of multiple organ dysfunction and failure. Furthermore, contemporary practices for apheresed platelet processing and storage restricts transfusibility to five days or less from time of collection. Platelet processing by lyophilization offers the potential for long term platelet storage and transfusibility. Lyophilized platelets (LP) are preserved in a metabolically inactive state until rehydrated in sterile water immediately prior to infusion and therefore, intuitively, of limited BRM activity. We hypothesized that LP transfusion will not constitute an immunologic “second hit”.

Rhesus Macaques were anesthetized and subjected to grade III hemorrhagic shock to induce a systemic inflammatory profile consistent with an immunological “first hit”. To determine the “second hit” capability of LP, 2 × 10¹⁰ reconstituted LP (a weight based equivalent dose of an average size human receiving 1 six pack of platelets) were infused at 15 minutes (T=15) following the initiation of shock. In parallel experiments, normal saline (NS) or 2 × 10¹⁰ fresh (day 1 – 3) apheresed human platelets (FAP) were infused at T=15 15 minutes following initiation of shock to serve as controls. Subsequently, volume resuscitation with additional normal saline, per ATLS guidelines, was performed and all animals survived for 480 minutes (T=480) post-shock initiation. Physiologic parameters were continually monitored, serum collected immediately preceding shock (T=0), T=240 and T=480 for TNF α, IL-1β, IL-6, and IL-8 analysis. Supernatants from the LP and FAP administered during the study were assayed for pro-inflammatory cytokines. All cytokine detection was performed on a Luminex IS 100. Analysis: paired sample t-test and one-way ANOVA; results reported as mean (SEM).

Grade III hemorrhagic shock, determined by a reduction in MAP (43% (4.5), p<0.05) and blood loss index (24% (3.9), p<0.05), resulted in a significant pro-inflammatory response determined by significant changes in the serum levels of IL-6 and IL-8 from T=0 to T=240 and T=480 (p<0.05) in NS controls. No statistically significant differences were detected in serum concentration of TNFα, IL-1β, IL-6, and IL-8 at T=240 and T=480 amongst the three groups (NS, LP, FAP). The supernatants from transfused LP and FAP contained undetectable levels of GMCSF, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-13, and IL-15. However, the FAP supernatant contained increased concentrations of in TNFα (255%), MCP1 (405%), IP-10 (817%) and IL-8 (864%) as compared to LP.

The model utilized in this study effectively constitutes an immunological first hit as determined by altered systemic levels of IL6 and IL8 in response to the hemorrhagic shock. As expected based upon the nature of LP processing and storage, the supernatant of the infused LPs was lacking in BRMs even though infused LP was > 6 months post-collection. The infusion of fresh apheresed platelets did not constitute an immunologic second hit in this study; given the age (day 1 – 3) of the FAP utilized this result is consistent with previous reports and is a sound negative control and excludes a xenogeneic pro-inflammatory response to the infusion of human platelets. The inflammatory profile of LP treated animals was not different than NS control or FAP treated animals, in conjunction with the above indicate that lyophilized platelet transfusion does not constitute an immunologic second hit in the setting of hemorrhagic shock.

No relevant conflicts of interest to declare.