Michael Adam Meledeo

Background: Plasma has been used as an efficient resuscitation fluid to improve prehospital survival from hemorrhagic shock. The 25% albumin (25% ALB, w/v in aqueous diluent) is a hyperoncotic fluid, and we hypothesize that it can be used as a lower‐volume alternative to plasma in hypovolemic shock. Method: Isoflurane‐anesthetized Sprague‐Dawley rats were subjected to a lethal decompensated hemorrhagic shock via removing 65% estimated blood volume (EBV, calculated by 6% body weight (gram) + 0.77 mL) of blood within 20 min. At 20 min post‐hemorrhage, rats randomly received plasma (20% EBV), 25% ALB (4% EBV), or respective vehicles, followed by fresh whole blood (FWB, 20% EBV) at 1 h post‐hemorrhage. The outcome was also compared between plasma and 25% ALB without FWB resuscitation in a separate experiment. Results: 20% EBV of plasma or 4% EBV of 25% ALB led to 100% survival at 60 min from the start of hemorrhage, compared to zero survival without treatment. Survival was maintained at least 4 h following limited resuscitation, with or without FWB at 1 h post‐hemorrhage. The lack of fibrinogen in 25% ALB was notable and compromised the hemostatic outcome as compared to using plasma, which was partially restored by FWB resuscitation with the reduction in prothrombin time. Conclusion: The current study indicates that 25% ALB enhances survival and maintains hemodynamics at only one‐fifth the volume of plasma. The results also suggest that 25% ALB requires hemostatic agents or subsequent blood products to counteract the hemostatic deficiency caused by hemodilution.

Background: Cold‐stored platelets (CSPs) are used in active bleeding due to their extended shelf life and preserved hemostatic function. Although both CSPs and cold‐stored whole blood (CS‐WB) are used clinically, the comparative effects of cold storage on their hemostatic and immunological properties remain partially understood. This study evaluated longitudinal changes in platelet function and immune markers stored at 4°C for 21 days. Study Design and Methods: Eight healthy donors each provided an apheresis platelet (AP) unit in plasma and a whole blood (WB) unit, leukoreduced using a platelet‐sparing filter. Baseline samples were collected prior to cold‐storage and on days 2, 7, 14, and 21 of cold‐storage. Hemostatic, metabolic, and immunological parameters were assessed by aggregometry, clot retraction assays, and flow cytometry. Results: At baseline, CD62P, Annexin V binding, CD47 expression, and aggregation amplitude did not differ significantly between products. CD154 was higher in apheresis platelets. Over 21‐day cold storage, CS‐WB exhibited a more pronounced decline in aggregation and metabolic functions. Expression of activation markers (CD62P, Annexin V) increased in both products, with a stronger rise in CS‐WB. CD47 ⁺ platelets remained high in both products, whereas CD154 ⁺ platelets transiently increased in CS‐WB but remained low in CSPs. Discussion: Cold‐storage differentially affects platelet in vitro characteristics across product types. While CS‐WB exhibited greater functional and metabolic deterioration, both products retained viable platelet function through the end of 21‐day cold storage. Larger studies are required to determine whether the observed in vitro differences translate into clinical outcomes.

Background: The anticipated multidomain operations of future battlefields necessitates minimalistic medical strategies for treatment of combat wounded. Direct transfusion, involving the administration of blood directly from a donor to a recipient, may be a solution for treating hemorrhagic shock patients in an austere environment where the tools to execute clinical practice guidelines are unavailable.Study Design and Methods: Six participants were enrolled in this observational pilot study. Blood was drawn through medical tubing using a 60 mL syringe and pushed through a second medical tubing set with output to a waste container. Three participants were collected with untreated lines, and three collections were performed with heparin flushed lines. Visible clotting was assessed, and blood samples were collected from the output at baseline, after 240 mL of blood collected, and at study endpoint, which was 400 mL collection or termination due excessive clotting or draw difficulties. Coagulation, hematology, and chemistry testing were performed on the samples.Results: Visible clotting was observed at endpoint in the four procedures with the longest draw times. There were statistically significant differences in platelet count and fibrinogen concentration between baseline and endpoint samples of the non‐heparin group. Clot strength decreased over time in the non‐heparin group.Discussion: The association between longer procedure times and visible clotting indicates direct transfusions should be completed based on time constraints rather than target volumes. Maintenance of platelet count, fibrinogen concentration, and clot strength in the heparin group suggest that heparin coated lines may enhance the safety of direct transfusions.

Introduction: Hemorrhage is the leading cause of death after trauma. Blood transfusions are used to restore physiology but are stored in citrate preservative which can bind electrolytes, particularly calcium, leading to hypocalcemia. Few data exist on the changes that occur in humans because of whole blood donation/transfusion. We sought to determine the electrolyte changes that occur during whole blood donation/reinfusion.Methods: We conducted a prospective observational study of military personnel that served as donor/recipient for personnel performing autologous whole blood transfusion training. Trained research staff analyzed whole blood samples collected pre‐donation, post‐donation, and post‐reinfusion of one autologous unit of whole blood. We used the i‐STAT laboratory analyzer. Laboratory data are reported using means and standard deviations.Results: We prospectively enrolled 40 participants. The median age was 22 years (interquartile range [IQR] 20–26), and 95% were male. The median body mass index was 25.3 (IQR 23.0–29.1). There were four participants that reported use of dietary supplements: three reported taking creatine, and the other one reported taking fish oil, magnesium, multivitamin, creatine, caffeine, and beetroot powder. Mean iCa was 1.25 mmol/L (standard deviation [SD] 0.04) pre‐donation, 1.26 (SD 0.04) post‐donation, and 1.12 (SD 0.14) post‐reinfusion. Comparatively, the mean potassium values were 3.90 mEq/L (SD 0.36), 4.10 (SD 0.62), and 4.09 (SD 0.72). Hemoglobin levels decreased by 0.34 g/dL (p < .001) from post‐donation to post‐reinfusion. Other labs had no significant changes.Conclusions: We noted a decrease in ionized calcium measurements post‐reinfusion. Future studies should assess changes after larger volume transfusions and assess repletion methods.

Platelet transfusions are essential for managing bleeding and hemostatic dysfunction and could be expanded as a cell therapy due to the multifunctional role of platelets in various diseases. Creating these cell therapies will require modifying transfusable donor platelets to express therapeutic proteins. However, there are currently no appropriate methods for genetically modifying platelets collected from blood donors. Here, we describe an approach using platelet-optimized lipid nanoparticles containing mRNA (mRNA-LNP) to enable exogenous protein expression in human and rat platelets. Within the library of mRNA-LNP tested, exogenous protein expression did not require nor correlate with platelet activation. Transfected platelets retained hemostatic function and accumulated in regions of vascular damage after transfusion into rats with hemorrhagic shock. We expect this technology will expand the therapeutic potential of platelets.

BackgroundCitrate is the only anticoagulant currently Food and Drug Administration (FDA)-approved for the long-term storage of blood for transfusion. Citrate inhibits phosphofructokinase and may play a pro-inflammatory role, suggesting that there may be an advantage to using alternative anticoagulants. Here, we examine the use of pyrophosphate as an anticoagulant. Study design and methodsWhole blood samples from healthy donors were anticoagulated either with citrate-phosphate-adenine-dextrose (CPDA-1) or our novel anticoagulant mixture pyrophosphate-phosphate-adenine-dextrose (PPDA-1). Samples were assessed for coagulation capacity by thromboelastography immediately after anticoagulation (T0) with and without recalcification, as well as 5 hours after anticoagulation (T1) with recalcification. Complete blood counts were taken at both timepoints. Flow cytometry to evaluate platelet activation as well as blood smears to evaluate cellular morphology were performed at T1. ResultsNo clotting was detected in samples anticoagulated with either solution without recalcification. After recalcification, clotting function was restored in both groups. R-Time in recalcified PPDA-1 samples was shorter than in CPDA-1 samples. A reduction in platelet count at T1 compared to T0 was observed in both groups. No significant platelet activation was observed in either group at T1. Blood smear indicated platelet clumping in PPDA-1. ConclusionWe have shown initial proof of concept that pyrophosphate functions as an anticoagulant at the dose used in this study, though there is an associated loss of platelets over time that may limit its usefulness for blood storage. Further dose optimization of pyrophosphate may limit or reduce the loss of platelets.

BackgroundPlatelets stored at room temperature (22-24 degrees C) for transfusion purposes have a shelf life of 5-7 days, or 72 h when stored refrigerated (1-6 degrees C). The limited shelf life of platelet products severely compromises platelet inventory. We hypothesized that cold storage of platelets in 100% plasma using xenon gas under high pressure would extend shelf life to 14 days. Study Design and MethodsDouble apheresis platelet units were collected and split equally between two bags. One unit was placed in a hyperbaric chamber, pressurized to 4 bars with a xenon/oxygen gas mixture, and placed in a refrigerator for 14 days (Xe). The remaining unit was aliquoted into mini-bags (10 ml) for storage at room temperature (RTP) or in cold (CSP). Samples were assayed on days 5 (RTP) or 14 (Xe and CSP) for count, metabolism, clot strength, platelet aggregation, and activation markers. ResultsThe platelet count in Xe samples was lower than that of RTP but significantly higher than CSP. Despite similar levels of glucose and lactate, the pH of Xe samples was significantly lower than CSP. Glycoprotein expression was better preserved by Xe storage compared to CSP, but no differences in activation were observed. Thromboelastography and aggregometry results were comparable between all groups. DiscussionCold storage of platelets in plasma with hyperbaric xenon provides no significant improvement in platelet function over cold storage alone. The use of a hyperbaric chamber and the slow off-gassing of Xe-stored units complicate platelet storage and delivery logistics.

Background: Hemorrhagic shock remains a leading cause of death in both military and civilian trauma casualties. While standard of care involves blood product administration, maintaining normothermia, and restoring hemostatic function, alternative strategies to treat severe hemorrhage at or near the point of injury are needed. We reviewed adjunct solutions for managing severe hemorrhage in the prehospital environment.Methods: We performed a literature review by searching PubMed with a combination of several keywords. Additional pertinent studies were identified by crossreferencing primary articles. Clinical experience of each author was also considered.Results: We identified several promising antishock therapies that can be utilized in the prehospital setting: ethinyl estradiol sulfate (EES), polyethylene glycol 20,000 (PEG20K), C1 esterase inhibitors (e.g. Berinert, Cinryze), cyclosporin A, niacin, bortezomib, rosiglitazone, icatibant, diazoxide, and valproic acid (VPA).Conclusion: Several studies show promising adjunct treatment options in the management of severe prehospital hemorrhage. While some are rarely used, many others are readily available and commonly utilized for other indications. This suggests the potential for future use in resourcelimited settings. Human studies and case reports supporting their use are currently lacking.

Background: Never frozen liquid plasma (LP) has limited shelf life versus fresh frozen plasma (FFP) or plasma frozen within 24 h (PF24). Previous studies showed decreasing factor activities after Day (D)14 in thawed FFP but no differences between LP and FFP until D10. This study examined LP function through D40.Study design and methods: FFP and PF24 were stored at −20°C until assaying. LP was assayed on D5 then stored (4°C) for testing through D40. A clinical coagulation analyzer measured Factor (F)V, FVIII, fibrinogen, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thromboelastography (TEG) and thrombogram measured functional coagulation. Ristocetin cofactor assay quantified von Willebrand factor (vWF) activity. Residual platelets were counted.Results: FV/FVIII showed diminished activity over time in LP, while PT and aPTT both increased over time. LP vWF declined significantly by D7. Fibrinogen remained high through D40. Thrombin lagtime was delayed in LP but consistent to D40, while peak thrombin was significantly lower in LP but did not significantly decline over time. TEG R‐time and angle remained constant. LP and PF24 (with residual platelets) had initially higher TEG maximum amplitudes (MA), but by D14 LP was similar to FFP.Conclusion: Despite significant declines in some factors in D40 LP, fibrinogen concentration and TEG MA were stable suggesting stored LP provides fibrinogen similarly to frozen plasmas even at D40. LP is easier to store and prepare for prehospital transfusion, important benefits when the alternative is crystalloid.