Publications by authors named "James A Bynum"

41 Publications

Trends in Prehospital Blood, Crystalloid, and Colloid Administration in Accordance With Changes in Tactical Combat Casualty Care Guidelines.

Mil Med 2021 Dec 22. Epub 2021 Dec 22.

Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.

Introduction: Hemorrhage is the leading threat to the survival of battlefield casualties. This study aims to investigate the types of fluids and blood products administered in prehospital trauma encounters to discover the effectiveness of Tactical Combat Casualty Care (TCCC) recommendations.

Materials And Methods: This is a secondary analysis of a previously described dataset from the Department of Defense Trauma Registry with a focus on prehospital fluid and blood administration in conjunction with changes in the TCCC guidelines. We collected demographic information on each patient. We categorized receipt of each fluid type and blood product as a binary variable for each casualty and evaluated trends over 2007-2020 both unadjusted and controlling for injury severity and mechanism of injury.

Results: Our original dataset comprised 25,897 adult casualties from January 1, 2007 through March 17, 2020. Most (97.3%) of the casualties were male with a median age of 25. Most (95.5%) survived to hospital discharge, and 12.2% of the dataset received fluids of any kind. Medical personnel used crystalloids in 7.4% of encounters, packed red blood cells in 2.0%, and whole blood in 0.5% with very few receiving platelets or freeze-dried plasma. In the adjusted model, we noted significant year-to-year increases in intravenous fluid administration from 2014 to 2015 and 2018 to 2019, with significant decreases noted in 2008-2009, 2010-2012, and 2015-2016. We noted no significant increases in Hextend used, but we did note significant decreases in 2010-2012. For any blood product, we noted significant increases from 2016 to 2017, with decreases noted in 2009-2013, 2015-2016, and 2017-2018. Overall, we noted a general spike in all uses in 2011-2012 that rapidly dropped off 2012-2013. Crystalloids consistently outpaced the use of blood products. We noted a small upward trend in all blood products from 2017 to 2019.

Conclusions: Changes in TCCC guidelines did not immediately translate into changes in prehospital fluid administration practices. Crystalloid fluids continue to dominate as the most commonly administered fluid even after the 2014 TCCC guidelines changed to use of blood products over crystalloids. There should be future studies to investigate the reasons for delay in guideline implementation and efforts to improve adherence.
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http://dx.doi.org/10.1093/milmed/usab461DOI Listing
December 2021

Intravenous administration of mesenchymal stromal cells leads to a dose dependent coagulopathy and is unable to attenuate acute traumatic coagulopathy in rats.

J Trauma Acute Care Surg 2021 Nov 17. Epub 2021 Nov 17.

Blood and Shock Resuscitation, United States Army Institute of Surgical Research, Fort Sam Houston, TX 78234.

Background: Mesenchymal stromal cells (MSC) express surface tissue factor (TF), which may affect hemostasis and detract from therapeutic outcomes of MSCs if administered intravenously (IV). In this study, we determine a safe dose of MSCs for IV administration, and further demonstrate the impact of IV-MSC on acute traumatic coagulopathy (ATC) in rats.

Methods: TF expression of rat bone marrow derived MSC (BMSC) or adipose derived MSC (AMSC) was detected by immunohistochemistry and ELISA. The coagulation properties were measured in MSC-treated rat whole blood, and blood samples collected from rats after IV administration of MSCs. ATC-rats underwent polytrauma and 40% hemorrhage, followed by IV administration of 5 or 10million/kg BMSCs (BMSC-5, BMSC-10), or vehicle at 1 hr after trauma.

Results: Rat MSCs expressed TF, and incubation of rat BMSCs or AMSCs with whole blood in vitro led to a significantly shortened clotting time (CT). However, a dose-dependent prolongation of prothrombin time (PT) with reduction in platelet counts and fibrinogen was found in healthy rat treated with IV-MSCs. BMSCs at 5 million/kg or less led to minimal effect on hemostasis. MSCs were not found in circulation, but in the lungs after IV administration regardless of the dosage. ATC with prolonged PT was not significantly affected by 5 or 10million/kg BMSCs. BMSC-10 led to significantly lower fibrinogen and platelet counts; while significantly higher levels of lactate, wet/dry weight ratio and leukocyte infiltration in the lung were present compared to BMSC-5 or vehicle. No differences were seen in immune or inflammatory profiles with BMSC treatment in ATC-rats, at least in the acute timeframe.

Conclusions: Intravenous administration of MSCs leads to a risk of coagulopathy associated with a dose-dependent reduction in platelet counts and fibrinogen, and is incapable of restoring hemostasis of rats with ATC after polytrauma and hemorrhagic shock.

Level Of Evidence: Animal and laboratory studies; Study type: therapeutic.
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http://dx.doi.org/10.1097/TA.0000000000003476DOI Listing
November 2021

Coagulation function of never frozen liquid plasma stored for 40 days.

Transfusion 2021 07;61 Suppl 1:S111-S118

U.S. Army Institute of Surgical Research, JBSA-Fort Sam Houston, Texas, USA.

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.
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http://dx.doi.org/10.1111/trf.16526DOI Listing
July 2021

Evaluation of KP-1199: a novel acetaminophen analog for hemostatic function and antinociceptive effects.

Transfusion 2021 07;61 Suppl 1:S234-S242

United States Army Institute of Surgical Research, JBSA Fort Sam Houston, San Antonio, Texas, USA.

Background: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples.

Methods: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 μg/ml) and 10× (2140 μg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation.

Results: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested.

Conclusion: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.
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http://dx.doi.org/10.1111/trf.16497DOI Listing
July 2021

Cold-stored platelets have better preserved contractile function in comparison with room temperature-stored platelets over 21 days.

Transfusion 2021 07;61 Suppl 1:S68-S79

Blood and Coagulation Research Department, Combat Mortality Prevention Division, United States Army Institute of Surgical Research, JBSA Fort Sam Houston, San Antonio, Texas, USA.

Although it is well established that transfusion of platelets in cases of severe bleeding reduces mortality, the availability of platelets is hampered by harsh restrictions on shelf life due to elevated risks of microbial contamination and functional losses with room temperature-stored platelets (RTP) kept at 22°C. In contrast, many recent studies have shown that 4°C cold-stored platelets (CSP) are able to overcome these shortcomings leading to the recent Food and Drug Administration licensure for 14-day stored CSP when conventional platelets are unavailable. This work expands the evidence supporting superiority of CSP function by assaying the less explored platelet-mediated clot retraction of RTP and CSP in either autologous plasma (AP) or platelet additive solution (PAS) for up to 21 days. The results demonstrate that CSP have better preservation of contractile function, exhibiting retraction for up to 21 days in both AP and PAS and forming highly ordered fibrin scaffolds similar to those of fresh platelets. In contrast, RTP stored in AP showed impaired contractile function by Day 5 with no retraction after 10 days, whereas PAS-stored RTP retained contractile function for up to 21 days. Collectively, these findings support extended storage of CSP and suggest that storage in PAS can mitigate functional losses in RTP.
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http://dx.doi.org/10.1111/trf.16530DOI Listing
July 2021

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

J Trauma Acute Care Surg 2021 08;91(2S Suppl 2):S81-S88

From the Coagulation and Blood Research (Blood) (T.C.C., X.W., J.D.K., J.G.-M., C.L.S., B.L., A.P.C., J.A.B.), United States Army Institute of Surgical Research, San Antonio, Texas; Capricor Therapeutics Institute (J.J.M., K.A.P., L.R.-B., N.A.A., L.S.M.), Beverly Hills, California; Department of Biological Chemistry (S.J.G.), Johns Hopkins, Baltimore, Maryland; and Department of Biomedical Engineering (C.R.R.), The University of Texas at San Antonio, San Antonio, Texas.

Background: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined.

Methods: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock.

Results: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy.

Conclusion: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.
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http://dx.doi.org/10.1097/TA.0000000000003218DOI Listing
August 2021

Minimal Effects of Intravenous Administration of Xenogeneic Adipose Derived Stem Cells on Organ Function in a Porcine 40% TBSA Burn Model.

J Burn Care Res 2021 09;42(5):870-879

United States Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas, USA.

Adipose stem cells (ASCs) have shown therapeutic promise for various conditions, including burn injury. While ASCs have immunomodulatory properties, concerns exist over pro-coagulant activity after intravenous (IV) administration. In the present study, we examined IV human ASC delivery in terms of coagulation, organ function, and inflammation in a 40% total body surface area (TBSA) swine burn model. Anesthetized female Yorkshire swine were burned and randomized to receive 15 ml/kg Lactated Ringer's containing: no ASCs; a low dose (5 × 105 ASCs/kg); or a high dose (5 × 106 ASCs/kg). For biochemical analysis, blood was collected at baseline (BL), 3, 6, 12, and 24 h post-burn, while kidney and liver tissue was collected post-euthanasia. A significant, but transient, effect of ASCs was seen on prothrombin times and INR, wherein low doses revealed slight hypercoagulation. Burns increased partial thromboplastin time, fibrinogen, and d-dimer levels, which was unchanged with ASC administration. ASCs tended to exacerbate increases in bilirubin at 3 h, but this didn't reach statistical significance. A significant effect of ASCs on creatinine and BUN was seen, wherein low doses elevated levels at 24 h (creatinine, P = 0.0012; BUN, P = 0.0195). Hepatic and renal TUNEL staining were similar for all groups. A dose-dependent decrease in IL-8 was observed, while low doses significantly increased IL-1RA at 3h (P = 0.050), IL-12 at 12h (P = 0.021) and IL-6 at 24 h post-burn (P = 0.035). IV administration of xenogeneic ASCs slightly increased coagulation, but effects on burn-induced renal and hepatic dysfunction effects were minimal. Despite some significant immunomodulation, organ dysfunction effects were modest. Collectively, this study provides evidence to be skeptical about xenogeneic ASC administration in regards to burns.
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http://dx.doi.org/10.1093/jbcr/irab094DOI Listing
September 2021

Lessons Learned From the Battlefield and Applicability to Veterinary Medicine - Part 2: Transfusion Advances.

Front Vet Sci 2021 7;8:571370. Epub 2021 May 7.

U.S. Army Institute of Surgical Research, Joint Base San Antonio, San Antonio, TX, United States.

Since the inception of recent conflicts in Afghanistan and Iraq, transfusion practices in human military medicine have advanced considerably. Today, US military physicians recognize the need to replace the functionality of lost blood in traumatic hemorrhagic shock and whole blood is now the trauma resuscitation product of choice on the battlefield. Building on wartime experiences, military medicine is now one of the country's strongest advocates for the principle of hemostatic resuscitation using whole blood or balanced blood components as the primary means of resuscitation as early as possibly following severe trauma. Based on strong evidence to support this practice in human combat casualties and in civilian trauma care, military veterinarians strive to practice similar hemostatic resuscitation for injured Military Working Dogs. To this end, canine whole blood has become increasingly available in forward environments, and non-traditional storage options for canine blood and blood components are being explored for use in canine trauma. Blood products with improved shelf-life and ease of use are not only useful for military applications, but may also enable civilian general and specialty practices to more easily incorporate hemostatic resuscitation approaches to canine trauma care.
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http://dx.doi.org/10.3389/fvets.2021.571370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138582PMC
May 2021

The Use of Whole Blood Transfusion During Non-Traumatic Resuscitation.

Mil Med 2021 Apr 15. Epub 2021 Apr 15.

Department of Emergency Medicine, Brooke Army Medical Center, JBSA Fort Sam Houston, San Antonio, TX 78234, USA.

Background: Evidence from military populations showed that resuscitation using whole blood (WB), as opposed to component therapies, may provide additional survival benefits to traumatically injured patients. However, there is a paucity of data available for the use of WB in uninjured patients requiring transfusion. We sought to describe the use of WB in non-trauma patients at Brooke Army Medical Center (BAMC).

Materials And Methods: Between January and December 2019, the BAMC ClinComp electronic medical record system was reviewed for all patients admitted to the hospital who received at least one unit of WB during this time period. Patients were sorted based on their primary admission diagnosis. Patients with a primary trauma-based admission were excluded.

Results: One hundred patients were identified who received at least one unit of WB with a primary non-trauma admission diagnosis. Patients, on average, received 1,064 mL (750-2,458 mL) of WB but received higher volumes of component therapy. Obstetric/gynecologic (OBGYN) indications represented the largest percentage of non-trauma patients who received WB (23%), followed by hematologic/oncologic indications (16%).

Conclusion: In this retrospective study, WB was most commonly used for OBGYN-associated bleeding. As WB becomes more widespread across the USA for use in traumatically injured patients, it is likely that WB will be more commonly used for non-trauma patients. More outcome data are required to safely expand the indications for WB use beyond trauma.
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http://dx.doi.org/10.1093/milmed/usab128DOI Listing
April 2021

Interactions of human mesenchymal stromal cells with peripheral blood mononuclear cells in a Mitogenic proliferation assay.

J Immunol Methods 2021 05 18;492:113000. Epub 2021 Feb 18.

Blood and Coagulation Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX, United States of America. Electronic address:

Background: Immunomodulation by mesenchymal stromal cells (MSCs) is a potentially important therapeutic modality. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, suggesting a mechanism for suppressing inflammatory responses in vivo. This study details the interactions of PBMCs and MSCs.

Methods: Pooled human PBMCs and MSCs were co-cultured at different MSC:PBMC ratios and harvested from 0 to 120 h, with and without phytohaemagglutin A (PHA) stimulation. Proliferation of adherent MSCs and non-adherent PBMCs was assessed by quantitation of ATP levels. PBMC surface marker expression was analyzed by flow cytometry. Indoleamine 2,3-dioxygenase (IDO) activity was determined by kynurenine assay and IDO mRNA by RT-PCR. Cytokine release was measured by ELISA. Immunofluorescent microscopy detected MSC, PBMC, monocyte (CD14+) and apoptotic events.

Results: PBMC proliferation in response to PHA gave a robust ATP signal by 72 h, which was suppressed by co-culture with densely plated MSCs. Very low level MSC seeding densities relative to PBMC number reproducibly stimulated PBMC proliferation. The CD4+/CD3+ population significantly decreased over time while the CD8+/CD3+ population significantly increased. No change in CD4+/CD8+ ratio is seen with high density MSC co-culture; very low density MSCs augment the changes seen in PHA stimulated PBMCs alone. IDO activity in MSCs co-cultured with PBMCs correlated with PBMC suppression. MSCs increased the secretion of IL-10 and IL-6 from stimulated co-cultures and decreased TNF-α secretion. In stimulated co-culture, low density MSCs decreased in number; fluorescence immunomicroscopy detected association of PBMC with MSC and phosphatidyl serine externalization in both cell populations.

Conclusions: A bidirectional interaction between MSCs and PBMCs occurs during co-culture. High numbers of MSCs inhibit PHA-stimulated PBMC proliferation and the PBMC response to stimulation; low numbers of MSCs augment these responses. Low density MSCs are susceptible to attrition, apparently by PBMC-induced apoptosis. These results may have direct application when considering therapeutic dosing of patients; low MSC doses may have unintended detrimental consequences.
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http://dx.doi.org/10.1016/j.jim.2021.113000DOI Listing
May 2021

Mesenchymal stromal cell delivery via an ex vivo bioreactor preclinical test system attenuates clot formation for intravascular application.

Stem Cells Transl Med 2021 06 1;10(6):883-894. Epub 2021 Feb 1.

Sentien Biotechnologies, Inc, Lexington, Massachusetts, USA.

While mesenchymal stromal cells are an appealing therapeutic option for a range of clinical applications, their potential to induce clotting when used systemically remains a safety concern, particularly in hypercoagulable conditions, such as in patients with severe COVID-19, trauma, or cancers. Here, we tested a novel preclinical approach aimed at improving the safety of mesenchymal stromal cell (MSC) systemic administration by use of a bioreactor. In this system, MSCs are seeded on the exterior of a hollow-fiber filter, sequestering them behind a hemocompatible semipermeable membrane with defined pore-size and permeability to allow for a molecularly defined cross talk between the therapeutic cells and the whole blood environment, including blood cells and signaling molecules. The potential for these bioreactor MSCs to induce clots in coagulable plasma was compared against directly injected "free" MSCs, a model of systemic administration. Our results showed that restricting MSCs exposure to plasma via a bioreactor extends the time necessary for clot formation to occur when compared with "free" MSCs. Measurement of cell surface data indicates the presence of known clot inducing factors, namely tissue factor and phosphatidylserine. Results also showed that recovering cells and flushing the bioreactor prior to use further prolonged clot formation time. Furthermore, application of this technology in two in vivo models did not require additional heparin in fully anticoagulated experimental animals to maintain target activated clotting time levels relative to heparin anticoagulated controls. Taken together the clinical use of bioreactor housed MSCs could offer a novel method to control systemic MSC exposure and prolong clot formation time.
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http://dx.doi.org/10.1002/sctm.20-0454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133341PMC
June 2021

Neglected No More: Emerging Cellular Therapies in Traumatic Injury.

Stem Cell Rev Rep 2021 08 8;17(4):1194-1214. Epub 2021 Jan 8.

Blood & Coagulation Research Department, US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX, USA.

Traumatic injuries are a leading cause of death and disability in both military and civilian populations. Given the complexity and diversity of traumatic injuries, novel and individualized treatment strategies are required to optimize outcomes. Cellular therapies have potential benefit for the treatment of acute or chronic injuries, and various cell-based pharmaceuticals are currently being tested in preclinical studies or in clinical trials. Cellular therapeutics may have the ability to complement existing therapies, especially in restoring organ function lost due to tissue disruption, prolonged hypoxia or inflammatory damage. In this article we highlight the current status and discuss future directions of cellular therapies for the treatment of traumatic injury. Both published research and ongoing clinical trials are discussed here.
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http://dx.doi.org/10.1007/s12015-020-10086-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793620PMC
August 2021

Cold storage of platelets in platelet additive solution maintains mitochondrial integrity by limiting initiation of apoptosis-mediated pathways.

Transfusion 2021 01 8;61(1):178-190. Epub 2020 Dec 8.

US Army Institute of Surgical Research, JBSA-Fort Sam Houston, Sam Houston, Texas, USA.

Background: Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold-stored platelets (CSPs) and RTPs after 5-day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days.

Study Design And Methods: This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial-associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics.

Results: CSP preserved mitochondria-associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro-apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs.

Conclusion: Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well-documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients.
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http://dx.doi.org/10.1111/trf.16185DOI Listing
January 2021

A streamlined proliferation assay using mixed lymphocytes for evaluation of human mesenchymal stem cell immunomodulation activity.

J Immunol Methods 2021 01 16;488:112915. Epub 2020 Nov 16.

Blood and Coagulation Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, TX, United States of America.

Background: Mesenchymal stromal cells (MSCs) have been proposed for treatment of acute respiratory distress syndrome (ARDS), graft versus host disease (GVHD), wound healing and trauma. A consensus is building that immunomodulation by MSCs is important for therapeutic potential. MSCs suppress peripheral blood mononuclear cell (PBMC) proliferation in vitro, potentially reflecting an ability to suppress PBMC inflammatory responses in vivo. Current mixed lymphocyte reaction (MLR) assays commonly used to evaluate MSC potency generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated PBMCs in the presence of mitotically inactive MSCs. Proliferation of PBMCs is monitored by several methods, including incorporation of radiolabeled nucleotides, BRDU labeling and ELISA assay or flow cytometry of carboxyfluorescein labeled PBMCs. Here we present a streamlined assay using MSCs in a direct co-culture system with unmodified MSCs using a luminescent ATP assay to evaluate both PBMC and MSC proliferation/survival.

Methods: PBMCs were isolated from fresh anti-coagulated whole blood by centrifugation over Ficoll-Paque in LeucoSep tubes. Isolated PBMCs from 8 to 10 donors were pooled and cryopreserved at 1 × 10/ml in 50% RPMI medium,10% DMSO, 40% human AB serum. MSCs derived from bone marrow, adipose tissue or umbilical cord (BM-MSC, Ad-MSC, UC-MSC, respectively) were serially diluted starting at 50-60,000 cells/well and cultured in 96 well plates for 4-48 h in their respective medium. On Day 0, MSCs were washed, resuspended in PBMC media (RPMI with 10% FBS, 2 mM Glutamine, 10 mM HEPES, pH 7.4) and incubated with or without 150,000 freshly thawed pooled PBMCs/well, in the presence or absence of phytohemagglutinin A (PHA, 0-5 μg/ml). Proliferation of both MSCs (adherent) and PBMCs (non-adherent) was assessed by quantitation of ATP levels using the bioluminescent reagent Cell Titer-Glo (Promega). Culture supernatant contained PBMC, while washed adherent cells were primarily MSCs. Both cell types were incubated for 30 min with an equal volume of Cell Titer-Glo reagent and then assayed in white plates on a luminescence plate reader.

Results: PBMC proliferation in response to PHA stimulation resulted in a robust increase in ATP by 72 h, with >6 fold increase over unstimulated PBMCs, which showed no increase. MSC proliferation was decreased <20% at the highest PHA concentrations. Co-culture with MSCs suppressed PBMC proliferation dependent upon MSC passage number, source, and prior growth conditions. Total time to complete the ATP assay was under an hour including incubations. With minimal manipulations in the assay, intra- and inter- assay variations averaged 11.1 and 15.7% respectively.

Conclusions: Direct co-culture of live unmodified MSCs with freshly thawed pooled PBMCs gives a robust determination of immunosuppression by MSCs with unparalleled ease. Graded responses can be determined, allowing comparison of potency between MSC preparations as in comparisons between freshly thawed and cultured MSCs as well as interferon-γ licensed MSCs. With the 96 well plate assay, far fewer PBMCs are generally required than in a typical flow cytometry determination. This streamlined assay can be performed within 72 h, without irradiating cells and without specialized equipment.
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http://dx.doi.org/10.1016/j.jim.2020.112915DOI Listing
January 2021

Burn resuscitation strategy influences the gut microbiota-liver axis in swine.

Sci Rep 2020 09 24;10(1):15655. Epub 2020 Sep 24.

Blood Coagulation Research Department, USA Army Institute of Surgical Research, JBSA Ft. Sam Houston, San Antonio, TX, 78234, USA.

Fluid resuscitation improves clinical outcomes of burn patients; however, its execution in resource-poor environments may have to be amended with limited-volume strategies. Liver dysfunction is common in burn patients and gut dysbiosis is an understudied aspect of burn sequelae. Here, the swine gut microbiota and liver transcripts were investigated to determine the impact of standard-of-care modified Brooke (MB), limited-volume colloid (LV-Co), and limited-volume crystalloid (LV-Cr) resuscitation on the gut microbiota, and to evaluate its' potential relationship with liver dysfunction. Independent of resuscitation strategy, bacterial diversity was reduced 24 h post-injury, and remained perturbed at 48 h. Changes in community structure were most pronounced with LV-Co, and correlated with biomarkers of hepatocellular damage. Hierarchical clustering revealed a group of samples that was suggestive of dysbiosis, and LV-Co increased the risk of association with this group. Compared with MB, LV-Co and LV-Cr significantly altered cellular stress and ATP pathways, and gene expression of these perturbed pathways was correlated with major dysbiosis-associated bacteria. Taken together, LV-Co resuscitation exacerbated the loss of bacterial diversity and increased the risk of dysbiosis. Moreover, we present evidence of a linkage between liver (dys)function and the gut microbiota in the acute setting of burn injury.
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http://dx.doi.org/10.1038/s41598-020-72511-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515893PMC
September 2020

Use of multiple potency assays to evaluate human mesenchymal stromal cells.

J Trauma Acute Care Surg 2020 08;89(2S Suppl 2):S109-S117

From the Blood and Coagulation Research Department (B.A.C., M.C.H., C.P.D., I.A., C.C., C.S., S.L., L.G., K.J., R.M., A.P.C., J.A.B.), US Army Institute of Surgical Research, JBSA Fort Sam Houston, Houston; Department of Molecular Medicine (B.A.C.), UT Health San Antonio; Joint Interdisciplinary Program in Biomedical Engineering Program (A.P.C., J.A.B.), UT San Antonio and UT Health San Antonio; Department of Surgery (A.P.C.), UT Health San Antonio, San Antonio; and Institute of Biomedical Studies (J.A.B.), Baylor University, Waco, Texas.

Background: There is broad interest in the use of cell therapies and cell products for treatment of a variety of diseases and problems. Of interest to the military, cellular therapies have the potential to confer tremendous benefit for treatment of both acute and chronic injuries. Although many different cell therapy products are currently under investigation, mesenchymal stromal cells (MSCs) are good candidates, based on their ability to respond to inflammation, limit vascular permeability, and modulate immune responses to injury. Although a large number of clinical trials utilize MSCs or their products, there is no firm consensus defining the characteristics and activities of a good MSC product. Here, we test multiple human MSCs in several assays designed to test potency, to determine if functionally relevant differences between MSCs can be defined using in vitro assays, allowing identification of superior MSC products for preclinical or clinical testing.

Methods: Human MSCs derived from several tissue sources (adipose, bone marrow, umbilical cord) were evaluated for their ability to respond to inflammatory signaling by upregulating indoleamine-2,3-dioxygenase and TSG6, suppress lymphocyte proliferation, alter the polarization of macrophages, and affect tube formation by endothelial cells.

Results: All MSCs tested displayed activity in the functional assays utilized, but differences in potency were observed in each assay.

Conclusion: The indoleamine-2,3-dioxygenase enzyme activity assay represents a simple way to screen multiple samples. The mixed lymphocyte reaction and monocyte assays used to test interactions between MSCs and immune cells are more involved but give direct information on immunomodulation potential. The endothelial cell tube formation assay is relatively simple to perform but a large number of images must be generated and analyzed. However, it tests a functional activity other than immunomodulation and, therefore, adds another facet to MSC evaluation.
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http://dx.doi.org/10.1097/TA.0000000000002743DOI Listing
August 2020

Similar hemostatic responses to hypovolemia induced by hemorrhage and lower body negative pressure reveal a hyperfibrinolytic subset of non-human primates.

PLoS One 2020 24;15(6):e0234844. Epub 2020 Jun 24.

Coagulation and Blood Research, United States Army Institute of Surgical Research, Fort Sam Houston, Texas, United States of America.

Background: To study central hypovolemia in humans, lower body negative pressure (LBNP) is a recognized alternative to blood removal (HEM). While LBNP mimics the cardiovascular responses of HEM in baboons, similarities in hemostatic responses to LBNP and HEM remain unknown in this species.

Methods: Thirteen anesthetized baboons were exposed to progressive hypovolemia by HEM and, four weeks later, by LBNP. Hemostatic activity was evaluated by plasma markers, thromboelastography (TEG), flow cytometry, and platelet aggregometry at baseline (BL), during and after hypovolemia.

Results: BL values were indistinguishable for most parameters although platelet count, maximal clot strength (MA), protein C, thrombin anti-thrombin complex (TAT), thrombin activatable fibrinolysis inhibitor (TAFI) activity significantly differed between HEM and LBNP. Central hypovolemia induced by either method activated coagulation; TEG R-time decreased and MA increased during and after hypovolemia compared to BL. Platelets displayed activation by flow cytometry; platelet count and functional aggregometry were unchanged. TAFI activity and protein, Factors V and VIII, vWF, Proteins C and S all demonstrated hemodilution during HEM and hemoconcentration during LBNP, whereas tissue plasminogen activator (tPA), plasmin/anti-plasmin complex, and plasminogen activator inhibitor-1 did not. Fibrinolysis (TEG LY30) was unchanged by either method; however, at BL, fibrinolysis varied greatly. Post-hoc analysis separated baboons into low-lysis (LY30 <2%) or high-lysis (LY30 >2%) whose fibrinolytic state matched at both HEM and LBNP BL. In high-lysis, BL tPA and LY30 correlated strongly (r = 0.95; P<0.001), but this was absent in low-lysis. In low-lysis, BL TAFI activity and tPA correlated (r = 0.88; P<0.050), but this was absent in high-lysis.

Conclusions: Central hypovolemia induced by either LBNP or HEM resulted in activation of coagulation; thus, LBNP is an adjunct to study hemorrhage-induced pro-coagulation in baboons. Furthermore, this study revealed a subset of baboons with baseline hyperfibrinolysis, which was strongly coupled to tPA and uncoupled from TAFI activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234844PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314422PMC
September 2020

Use of Specialized Pro-Resolving Mediators to Alleviate Cold Platelet Storage Lesion.

Transfusion 2020 06 1;60 Suppl 3:S112-S118. Epub 2020 Jun 1.

Coagulation and Blood Research Program, U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas, USA.

Background: Cold-stored platelets are an attractive option for treatment of actively bleeding patients due to a reduced risk of septic complications and preserved hemostatic function compared to conventional room temperature-stored platelets. However, refrigeration causes increased platelet activation and aggregate formation. Specialized pro-resolving mediators (SPMs), cell signaling mediators biosynthesized from essential fatty acids, have been shown to modulate platelet function and activation. In this study, we sought to determine if SPMs could be used to inhibit cold-stored platelet activation.

Methods: Platelets were collected from healthy donors (n = 4-7) and treated with SPMs (resolvin E1 [RvE1], maresin 1 [MaR1], and resolvin D2 [RvD2]) or vehicle (VEH; 0.1% EtOH). Platelets were stored without agitation in the cold and assayed on Days 0 and 7 of storage for platelet activation levels using flow cytometry, platelet count, aggregation response using impedance aggregometry, and nucleotide content using mass spectrometry.

Results: Compared to VEH, SPM treatment inhibited GPIb shedding (all compounds), significantly reduced both PS exposure and activation of GPIIb/IIIa receptor (RvD2, MaR1), and preserved aggregation response to TRAP (RvD2, MaR1) after 7 days of storage. Similar to untreated cold-stored platelets, SPM-treated samples did not preserve platelet counts or block the release of P-Selectin. Nucleotide content was unaffected by SPM treatment in cold-stored platelets.

Conclusions: SPM treatment, particularly Mar1 and RvD2, led to reduced platelet activation and preserved platelet function after 7 days of storage in the cold. Future work is warranted to better elucidate the mechanism of action of SPMs on cold platelet function and activation.
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http://dx.doi.org/10.1111/trf.15750DOI Listing
June 2020

Field-expedient thawing of fresh-frozen plasma.

Transfusion 2020 06 1;60 Suppl 3:S87-S95. Epub 2020 Jun 1.

US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

Introduction: Frozen plasma is superior to crystalloids for hemorrhage resuscitation but remains logistically challenging in austere environments because of specialized clinical equipment for on-demand thawing. This research examines some ad hoc thawing techniques that have been implemented by military medical personnel.

Methods: Fresh-frozen plasma (FFP) units were thawed accordingly: using a slow cooker (three temperature settings) with preheated or room temperature water; affixing flameless ration heaters from meals ready-to-eat (MREs) to FFP and submerging in water; exposing FFP to electric kettle-boiled water; incubating with a sous vide immersion circulator; or using a clinical thawer (control). Hemostatic function, thrombin generation, factor activities, and essential chemistry were measured after thawing.

Results: Even at the highest temperatures, without preheated water the slow cooker doubled thawing time (62.5 min vs. control, 32.5 min; p < 0.0001), and the final temperature was 13.5°C versus 28.8°C in control (p < 0.01). When preheated, the slow cooker thawed in 31.3 minutes (p < 0.05), with a final temperature of 22.4°C. Kettle-boiled water thawed in 23.0 minutes with a final temperature of 25.1°C. The sous vide thawed in 28.1 minutes, with a final temperature of 20.2°C. MRE heaters were insufficient. Functional measures were similar in all conditions.

Discussion: In emergencies, protracted plasma thawing is unacceptable, and slower thawing methods also produced cryoprecipitate. Although no functional changes were observed with boiled water thawing, potential negative physiological impacts must be examined. Safe, controlled thawing can be obtained with the sous vide, although optimization requires further testing.
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http://dx.doi.org/10.1111/trf.15652DOI Listing
June 2020

Blood component separation of pathogen-reduced whole blood by the PRP method produces acceptable red cells but platelet yields and function are diminished.

Transfusion 2020 06 1;60 Suppl 3:S124-S133. Epub 2020 Jun 1.

Coagulation & Blood Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas, USA.

Background: This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined.

Study Design And Methods: PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity.

Results: Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC.

Conclusion: Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.
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http://dx.doi.org/10.1111/trf.15766DOI Listing
June 2020

Human mesenchymal stromal cell source and culture conditions influence extracellular vesicle angiogenic and metabolic effects on human endothelial cells in vitro.

J Trauma Acute Care Surg 2020 08;89(2S Suppl 2):S100-S108

From the Coagulation and Blood Research (T.C.C., M.C.H., B.A.C., C.D., A.P.C., J.A.B.), The United States Army Institute of Surgical Research, and Biomedical Engineering (T.C.C., C.R.R.), The University of Texas at San Antonio, San Antonio, Texas.

Background: Mesenchymal stem/stromal cell (MSC)-derived extracellular vesicles (EVs) are a possible cell-free alternative to MSCs because they retain the regenerative potential of MSCs, while still mitigating some of their limitations (such as the possible elicitation of host immune responses). The promotion and restoration of angiogenesis, however, is an important component in treating trauma-related injuries, and has not been fully explored with EVs. Herein, we describe the effects of monolayer adipose-derived EVs, spheroid adipose-derived EVs (SAd-EVs), monolayer bone marrow-derived EVs (MBM-EVs), and spheroid bone marrow-derived EVs (SBM-EVs) on human umbilical vein endothelial cell (HUVEC) tube formation and mitochondrial respiration.

Methods: The successful isolation of EVs derived from adipose MSCs or bone marrow MSCs in monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). The EV angiogenic potential was measured using a 24-hour HUVEC tube formation assay. The EV effects on HUVEC mitochondrial function were evaluated using the Seahorse respirometer machine.

Results: The number of junctions, branches, and the average length of branches formed at 24 hours of tube formation were significantly affected by cell and culture type; overall adipose-derived EVs outperformed bone marrow-derived EVs, and spheroid-derived EVs outperformed monolayer-derived EVs. Additionally, adipose-derived EVs resulted in significantly increased HUVEC mitochondrial maximal respiration and adenosine triphosphate (ATP) production, while only MBM-EVs negatively impacted HUVEC proton leak.

Conclusion: Adipose-derived EVs promoted HUVEC tube formation significantly more than bone marrow-derived EVs, while also maximizing HUVEC mitochondria function. Results demonstrate that, as with MSC therapies, it is possible to tailor EV culture and production to optimize therapeutic potential.

Level Of Evidence: Basic or Foundational Research.
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http://dx.doi.org/10.1097/TA.0000000000002661DOI Listing
August 2020

Freeze-dried plasma mitigates the dilution effects of a hemoglobin-based oxygen carrier (HBOC-201) in a model of resuscitation for hemorrhage and hemodilution.

J Trauma Acute Care Surg 2019 07;87(1S Suppl 1):S83-S90

From the United States Army Institute of Surgical Research, JBSA-Fort Sam Houston (G.C.P., C.S.M., A.S.T., J.A.B., A.E.P., A.P.C.)., Houston, Texas.

Background: Hemoglobin-based oxygen carriers (HBOCs) have proven useful for supplementing oxygen delivery when red cells are unavailable; however, HBOCs do not promote hemostasis. The need for prehospital bridges to blood transfusion informed this study which sought to determine the impact of HBOCs on coagulation, with or without cotransfusion of freeze-dried plasma (FDP).

Methods: Treatment was simulated in vitro by replacing whole blood volume (or whole blood prediluted with 25% plasmalyte A as a hemodilution model) with HBOC-201, FDP, or both at ratios of 10% to 50% of original volume. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, complete blood count, viscosity, thromboelastography (TEG), and platelet adhesion to collagen under flow were evaluated. Subsequently, tissue plasminogen activator was added to model hemorrhagic shock effects on fibrinolysis.

Results: Substituting blood with HBOC resulted in dose-dependent decreases in fibrinogen and cells, which lengthened PT (+61% at highest dose) and aPTT (+40% at highest dose) and produced TEG parameters consistent with dilutional coagulopathy. While substituting blood with FDP decreased cell counts accordingly, fibrinogen, PT, aPTT, and TEG parameters were not statistically changed. When HBOC and FDP were combined 1:1 for volume replacement, observed HBOC-only detriments were mitigated: PT and aPTT were increased by 17% and 11%, respectively, at the highest doses. In prediluted samples, similar trends were seen with exacerbated differences. Platelet adhesion to collagen was directly affected by hematocrit. Samples containing both HBOC and tissue plasminogen activator were highly susceptible to fibrinolysis.

Conclusion: A dose equivalent to 1 unit to 2 units each of HBOC-201 and FDP had a modest impact on functional coagulation measures and is reasonable to consider for clinical study as a part of early transfusion intervention. Higher doses may impart hemodilution risks similar to resuscitation with crystalloid or other colloids in coagulation-compromised patients. Further study of HBOC effects on fibrinolysis is also indicated.

Study Type: In vitro laboratory study.
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http://dx.doi.org/10.1097/TA.0000000000002317DOI Listing
July 2019

The effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles.

J Trauma Acute Care Surg 2019 07;87(1S Suppl 1):S74-S82

From the Blood and Coagulation Research Task Area, U.S. Army Institute of Surgical Research, (T.C.C., R.M.K., G.C.P., A.P.C., J.A.B.), Fort Sam Houston, San Antonio, Texas; Department of Biomedical Engineering, University of Texas at San Antonio, (T.C.C., C.R.R.) San Antonio, Texas.

Background: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have great potential as a cell-free therapy in wound healing applications. Because EV populations are not equivalent, rigorous characterization is needed before clinical use. Although there has been much focus on their RNA composition and regenerative capabilities, relatively less is known regarding the effects of MSC cell type (adipose tissue [Ad-MSCs] or bone marrow [BM-MSCs]) and culture condition (monolayer or spheroid) on MSC-EV performance, including characteristics related to their ability to promote coagulation, which could determine EV safety if administered intravenously.

Methods: The successful isolation of EVs derived from Ad-MSCs or BM-MSCs cultured in either monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using thromboelastography and a flow-based adhesion model simulating blood flow over a collagen-expressing surface.

Results: The MSC cell type and culture condition did not impact EV size distribution. Extracellular vesicles from all groups expressed phosphatidylserine and tissue factor on their surfaces were functionally thrombogenic and tended to increase clotting rates compared to the negative control of serum-free media without EVs. On average, EVs did not form significantly larger or stronger clots than the negative control, regardless of cell source or culture condition. Additionally, EVs interfered with platelet adhesion in an in vitro flow-based assay.

Conclusion: Adipose-derived EVs were more thrombogenic and expressed higher amounts of phosphatidylserine. Our findings suggest that, like intact MSCs, source variability among EVs is an important factor when considering EVs for potential therapeutic purposes.

Level Of Evidence: Therapeutic care management, level II.
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http://dx.doi.org/10.1097/TA.0000000000002225DOI Listing
July 2019

Optimizing whole blood storage: hemostatic function of 35-day stored product in CPD, CP2D, and CPDA-1 anticoagulants.

Transfusion 2019 04;59(S2):1549-1559

United States Army Institute of Surgical Research, JBSA-Fort Sam Houston, Texas.

Background: Transitioning from whole blood (WB) to components developed from efforts to maximize donor yield. Components are advantageous for specific derangements, but treating hemorrhage with components requires significantly more volume to provide similar effects to WB. Because storage lesion and waste remain problematic, this study examined hemostatic function of refrigerated WB stored for 35 days in anticoagulants citrate-phosphate-dextrose-adenosine (CPDA-1), citrate-phosphate-dextrose (CPD), or citrate-phosphate-double dextrose (CP2D).

Methods: Refrigerated WB units from healthy donors were sampled over 35 days. Global hemostatic parameters were measured by thromboelastometry, thrombogram, platelet aggregometry, and platelet adhesion to collagen under shear conditions. The effects of transfusion filtration and mixing 35-day stored product with fresh WB were evaluated.

Results: Countable platelets declined as aggregation clusters appeared in microscopy. While gross platelet agonist-induced aggregation declined over time, normalization revealed aggregation responses in remaining platelets. Peak thrombin generation increased over time. Clot strength diminished over storage in tissue factor-activated samples (normalized by filtration of aggregates). Functional fibrinogen responses remained consistent throughout. Filtration was necessary to maintain consistent platelet adhesion to collagen beyond collection day. Few differences were observed between anticoagulants, and stored/fresh mixing studies normalized coagulation parameters.

Conclusions: WB is easier to collect, store, and transfuse. WB provides platelets, an oft-neglected, critical resuscitation component, but their individual numbers decline as aggregates appear, resulting in diminished coagulation response. WB has better performance in these assays when examined at earlier time points, but expirations designated to specific anticoagulants appear arbitrary for hemostatic functionality, as little changes beyond 21 days regardless of anticoagulant.
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http://dx.doi.org/10.1111/trf.15164DOI Listing
April 2019

Human primary fibroblasts perform similarly to MSCs in assays used to evaluate MSC safety and potency.

Transfusion 2019 04;59(S2):1593-1600

Coagulation & Blood Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas.

Background: Cellular therapeutic agents may benefit trauma patients by modulating the immune response to injury, and by reducing inflammation and vascular leakage. Administration of allogeneic mesenchymal stromal cells (MSCs) shows some benefit in preclinical and clinical trials, but less testing has been performed with other cell types. Human primary fibroblasts (FBs) were compared to MSCs in assays designed to evaluate MSCs to determine if these assays actually evaluate properties unique to MSCs or whether related cell types perform similarly.

Study Design And Methods: MSC-related surface marker expression, tissue factor, and human leukocyte antigen-D related were evaluated by flow cytometry, and in vitro adipogenic and osteogenic differentiation potential were determined. Procoagulant activity was determined by thromboelastography. Two potency assays correlated with immunomodulation potential were utilized: the mixed lymphocyte reaction and indoleamine 2,3-dioxygenase enzyme activity assays.

Results: Human primary FBs performed similarly to MSCs in assays designed to evaluate MSC characteristics and potency. Although similar for MSC-positive cell surface marker expression, FBs did not show robust adipose differentiation and expressed some level of markers not expected on MSCs.

Conclusions: Human primary FBs are very similar to human MSCs, at least in assays currently used to evaluate MSC potency. Preclinical and clinical testing are required to determine if FBs show similar activity to MSCs in vivo. If FBs show inferior activity in vivo, development of new MSC-specific potency assays will be necessary to evaluate properties relevant to their unique clinical benefits.
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http://dx.doi.org/10.1111/trf.15187DOI Listing
April 2019

Evaluation of a lyophilized platelet-derived hemostatic product.

Transfusion 2019 04;59(S2):1490-1498

Coagulation and Blood Research Program, U.S. Army Institute of Surgical Research, Fort Sam Houston, Texas.

Background: Current limitations of platelet shelf life to 5 days have led to an increasingly greater demand for hemostatic agents with greater longevity. The objective of this study was to evaluate the function of a lyophilized platelet-derived hemostatic product (thrombosome [TS]) as a potential alternative to fresh platelets.

Methods: Platelets were collected from whole blood from healthy donors. TSs were reconstituted with water and added to various configurations of reassembled whole blood (platelets, plasma, and RBCs); measures included rotational thromboelastometry (ROTEM), optical aggregometry, mitochondrial function, calibrated automated thrombogram, collagen adhesion under flow (shear flow assay), and flow cytometry.

Results: In ROTEM, no differences were observed between maximum clot formation values for contact pathway activation thromboelastometry tests with TSs or platelet samples. Significantly decreased aggregation was observed in the TSs versus platelets (p < 0.001 for all agonists). Flow cytometry measures demonstrated significant decreases in glycoprotein Ib expression and increases in phosphatidylserine expression in the TS group (p < 0.01). The calibrated automated thrombogram assay was suggestive (lag time and peak thrombin) that the TSs might have some thrombogenic properties. Measurements of mitochondrial function revealed that TSs had no functional mitochondria.

Conclusion: In this study, TSs were shown to have nonfunctional mitochondria. ROTEM measures revealed that the TSs had no impact on clot strength. Likewise, compared to platelets, the TSs displayed minimal aggregation, had significantly more phosphatidylserine (measure of activation status), but had the ability to adhere to a collagen surface under flow conditions and contribute to clot formation and induced greater thrombin generation.
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http://dx.doi.org/10.1111/trf.15167DOI Listing
April 2019

Spray-dried plasma deficient in high-molecular-weight multimers of von Willebrand factor retains hemostatic properties.

Transfusion 2019 02 19;59(2):714-722. Epub 2018 Nov 19.

JBSA-Fort Sam Houston, United States Army Institute of Surgical Research, San Antonio, Texas.

Background: Dried plasmas can overcome logistical barriers that prevent fresh frozen plasma (FFP) usage in acute resuscitation, but processing of these products can detrimentally alter the composition. Spray-dried plasma (SpDP) from single units is deficient in high-molecular-weight multimers of von Willebrand factor (vWF), a critical facilitator of platelet adhesion and thrombus formation. We hypothesized that converting high-molecular-weight multimers to smaller-molecular-weight multimers would retain vWF's capacity to mediate platelet adhesion.

Study Design And Methods: SpDP obtained from untreated FFP was reconstituted with glycine-hydrochloric acid (HCl) and glycine (20 mM:50 mM) or pretreated with glycine-HCl (20 mM) or glycine-glycine-HCl (20 mM:50 mM) and reconstituted with water. In vitro hemostatic potential of SpDPs versus FFP or FFP spiked with 70 mM of glycine was evaluated, leading to a more detailed in vitro study of glycine-HCl-glycine (20 mM:50 mM) pretreated SpDP. Plasmas were combined with RBCs and platelets to observe global coagulation response.

Results: While vWF-ristocetin cofactor activity is significantly decreased (-41.13%; p < .0001) in SpDP, a model of vWF-mediated platelet adhesion to collagen under flow showed enhanced function (+13%; p < .01). Fewer microparticles, particularly of platelet origin, were observed in SpDP versus FFP (p < .0001). Small but significant differences in thromboelastography results were observed, although SpDP and FFP were within normal ranges.

Conclusion: Comparable coagulability was observed in FFP and SpDP. The apparent paradox between vWF-ristocetin cofactor assay and vWF-mediated platelet adhesion may be explained by the increase in smaller multimers of vWF in SpDP, producing different outcomes in these assays.
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http://dx.doi.org/10.1111/trf.15038DOI Listing
February 2019

Cold storage of platelets in platelet additive solution: an in vitro comparison of two Food and Drug Administration-approved collection and storage systems.

Transfusion 2018 07 30;58(7):1682-1688. Epub 2018 Mar 30.

Coagulation and Blood Research Program, US Army Institute of Surgical Research, Fort Sam Houston, Texas.

Background: Refrigeration of platelets (PLTs) in a PLT additive solution (PAS) reduces PLT activation compared to storage in plasma and preserves function for at least 15 days. Currently only two PASs are licensed by the Food and Drug Administration, each for use with only one apheresis platform. In this study, we compared the metabolic, functional, and activation status of PLTs collected on a Trima apheresis collection system and stored refrigerated in Isoplate (ISO) PAS to PLTs collected on an Amicus collection system and stored refrigerated in Intersol (INT) PAS.

Study Design And Methods: Apheresis PLTs (n = 4-7 donors) were collected on a Trima in ISO PAS or on an Amicus in INT PAS. PLTs were stored in a walk-in refrigerator (1-6°C) without agitation for long-term storage. Bags were assayed at Days 1, 5, 10, and 15 of storage. Measurements included PLT counts, pH, aggregation response, rotational thromboelastometry, and activation markers.

Results: Cold-stored Trima-collected PLTs in ISO were slightly more hemostatic than Amicus-collected PLTs in INT and displayed better adhesion to collagen under flow conditions. Amicus-collected PLTs in INT showed increased microaggregate formation on Days 5 and 10 and a significant decrease in PLT count over storage. Trima-collected PLTs in ISO displayed better clot strength than Amicus-collected PLTs in INT.

Conclusion: Compared to cold-stored Amicus PLTs in INT, Trima PLTs in ISO display superior in vitro function and may be better suited for treatment of bleeding patients. Clinical studies are warranted to confirm these findings.
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http://dx.doi.org/10.1111/trf.14603DOI Listing
July 2018

Functional stability of the TEG 6s hemostasis analyzer under stress.

J Trauma Acute Care Surg 2018 06;84(6S Suppl 1):S83-S88

From the United States Army Institute of Surgical Research (M.A.M., G.C.P., C.S.M., C.R.V., J.A.B., A.P.C.), Joint Base San Antonio, Fort Sam Houston, Texas.

Background: Viscoelastic measurements of coagulation provide much needed information, including guidance for triage and insight into bleeding disorders. The current clinical standards for these devices are the thromboelastogram (TEG) 5000 and the rotational thromboelastometer (ROTEM) delta, but a new product, the TEG 6s, has recently come to market, designed to simplify the user experience, reduce the required blood volume, and conduct multiple assays simultaneously. This study compares the performance of these three devices and examines the resiliency of the TEG 6s under various stresses.

Methods: The variances of coagulation metrics obtained by the TEG 6s (prototype and production models), TEG 5000, and ROTEM delta were compared using manufacturers' reagents and citrate-collected blood from healthy donors. Variability between devices was examined, and their performances under various motion and temperature stresses were compared by placing one unit on a linear or orbital shaker, in the cold, or in the heat while a counterpart remained stationary at room temperature.

Results: Although most comparable parameters had low degrees of variance, there were small but significantly increased variances found in some ROTEM delta and TEG 5000 parameters versus comparable TEG 6s parameters. Orbital rotation of the TEG 6s had no effect on means of any parameter but resulted in increased variance of 2 parameters, but linear motion with sudden striking had no observed impact on results. Similarly, 7-day exposure to heat (45°C) or cold (4°C) only resulted in minor deviations within normal ranges of the TEG 6s.

Discussion: The TEG 6s provides several improvements over other coagulation analyzers: it is easier to use and robustly resilient against motion and temperature stresses. These features suggest that it may be capable of deployment not only in the clinical laboratory but also to a variety of austere settings.

Level Of Evidence: Diagnostic test, level III.
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http://dx.doi.org/10.1097/TA.0000000000001810DOI Listing
June 2018

Procoagulant activity of human mesenchymal stem cells.

J Trauma Acute Care Surg 2017 07;83(1 Suppl 1):S164-S169

From the Coagulation & Blood Research (B.A.C., M.C.H., R.K.M., C.D., J.A.B., K.M.R., A.P.C.), US Army Institute of Surgical Research, JBSA Fort Sam Houston, Texas; Department of Molecular Medicine (B.A.C.), University of Texas Health Science Center at San Antonio, San Antonio, Texas, and Department of Surgery (A.P.C.), University of Texas Health Science Center at San Antonio, San Antonio, Texas.

Background: Allogeneic mesenchymal stem cells (MSCs) show great potential for the treatment of military and civilian trauma based on their reduced immunogenicity and ability to modulate inflammation and immune function in the recipient. Although generally considered to be safe, MSCs express tissue factor (TF), a potent activator of coagulation. In the current study, we evaluated multiple MSC populations for tissue factor expression and procoagulant activity to characterize safety considerations for systemic use of MSCs in trauma patients who may have altered coagulation homeostasis.

Methods: Multiple MSC populations derived from either human adipose tissue or bone marrow were expanded in the recommended stem cell media. Stem cell identity was confirmed using a well-characterized panel of positive and negative markers. Tissue factor expression on the cell surface was evaluated by flow cytometry with anti-CD142 antibody. Effects on blood coagulation were determined by thromboelastography and calibrated automated thrombogram assays using platelet-poor plasma or whole blood.

Results: Mesenchymal stem cells express tissue factor on their surfaces and are procoagulant in the presence of blood or plasma. The adipose-derived MSCs (Ad-MSC) evaluated were more procoagulant and expressed more tissue factor than bone marrow MSCs (BM-MSCs), which showed a greater variability in TF expression. Bone marrow MSCs were identified that exhibited low procoagulant activity, whereas all Ad-MSCs examined exhibited high procoagulant activity. The percentage of cells in a given population expressing surface tissue factor correlates roughly with functional procoagulant activity. Mesenchymal stem cell tissue factor expression and procoagulant activity change over time in culture.

Conclusions: All MSC populations are not equivalent; care should be taken to select cells for clinical use that minimize potential safety problems and maximize chance of patient benefit. Adipose-derived MSCs seem more consistently procoagulant than BM-MSCs, presenting a potential safety concern for systemic administration in coagulopathic patients. Donor variation exists between different cell populations, and culture handling conditions may also determine coagulation activity. Cells must be routinely monitored during preparation to ensure that they retain the desired characteristics before patient administration.
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http://dx.doi.org/10.1097/TA.0000000000001485DOI Listing
July 2017
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