Temporal Changes in Innate and Adaptive Immunity During Sepsis as Determined by ELISpot.

BACKGROUND: The inability to evaluate host immunity in a rapid quantitative manner in patients with sepsis has severely hampered development of novel immune therapies. The ELISpot assay is a functional bioassay that measures the number of cytokine-secreting cells and the relative amount of cytokine produced at the single-cell level. A key advantage of ELISpot is its excellent dynamic range enabling a more precise quantifiable assessment of host immunity. Herein, we tested the hypothesis that the ELISpot assay can detect dynamic changes in both innate and adaptive immunity as they often occur during sepsis. We also tested whether ELISpot could detect the effect of immune drug therapies to modulate innate and adaptive immunity. METHODS: Mice were made septic using sublethal cecal ligation and puncture (CLP). Blood and spleens were harvested serially and ex vivo IFN-γ and TNF-α production were compared by ELISpot and ELISA. The capability of ELISpot to detect changes in innate and adaptive immunity due to in vivo immune therapy with dexamethasone, IL-7, and arginine was also evaluated. RESULTS: ELISpot confirmed a decreased innate and adaptive immunity responsiveness during sepsis progression. More importantly, ELISpot was also able to detect changes in adaptive and innate immunity in response to immune-modulatory reagents, for example dexamethasone, arginine, and IL-7 in a readily quantifiable manner, as predicted by the reagents known mechanisms of action. ELISpot and ELISA results tended to parallel one another although some differences were noted. CONCLUSION: ELISpot offers a unique capability to assess the functional status of both adaptive and innate immunity over time. The results presented herein demonstrate that ELISpot can also be used to detect and follow the in vivo effects of drugs to ameliorate sepsis-induced immune dysfunction. This capability would be a major advance in guiding new immune therapies in sepsis.

Changes in massive transfusion over time: an early shift in the right direction?

BACKGROUND: Increasing evidence suggests that high fresh frozen plasma:packed red blood cell (FFP:PRBC) and platelet:PRBC (PLT:PRBC) transfusion ratios may prevent or reduce the morbidity associated with early coagulopathy which complicates massive transfusion (MT). We sought to characterize changes in resuscitation which have occurred over time in a cohort severely injured patients requiring MT. METHODS: Data were obtained from a multicenter prospective cohort study evaluating outcomes in blunt injured adults with hemorrhagic shock. MT was defined as requiring ≥10 units PRBCs within 24 hours postinjury. Mean PRBC, FFP, and PLT requirements (per unit; 6 hours, 12 hours, and 24 hours) were determined over time (2004-2009). Sub-MT, those patients just below the threshold for MT, were defined as requiring ≥7 and <10 units PRBCs in the initial 24 hours. The percent of resuscitation given at 6 hours relative to 24 hours total (6 of 24%) was determined and compared across "early" (admission until December 2007) and "recent" (after December 2007) periods for each component. RESULTS: Over the study time period (2004-2009) for the MT group (n = 526), initial base deficit and presenting international normalized ratio were unchanged, while Injury Severity Score was significantly higher. The percent of patients who required MT overall significantly decreased over time. No significant differences were found over time for six-hour, 12-hour, or 24-hour FFP:PRBC and PLT:PRBC transfusion ratios in MT patients. Sub-MT patients (n = 344) had significantly higher six-hour FFP:PRBC ratios and significantly higher six-hour,12-hour, and 24-hour PLT:PRBC ratios in the recent time period. The six h/24 h% total for FFP and PLT transfusion was significantly greater in the recent time period. (FFP: 54% vs.70%; p = 0.004 and PLT 46% vs. 61%; p = 0.048). CONCLUSION: In a severely injured cohort requiring MT, FFP:PRBC and PLT:PRBC ratios have not changed over time, whereas the rate of MT overall has significantly decreased. During the recent time period (after December 2007), significantly higher transfusion ratios and a greater percent of 6-hour/24-hour FFP and PLT were found in the sub-MT group, those patients just below the PRBC transfusion threshold definition of MT. These data suggest early, more aggressive attainment of high transfusions ratios may reduce the requirement for MT and may shift overall blood requirements below those which currently define MT. Further prospective evidence is required to verify these findings.