Pyrophosphate as a novel anticoagulant for storage of whole blood: A proof-of-concept study.

BACKGROUND: Citrate 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 METHODS: Whole 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. RESULTS: No 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. CONCLUSION: We 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.

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

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.

Field-expedient thawing of fresh-frozen plasma.

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.

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

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.

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

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.

Macular hole: a rare complication of ocular bartonellosis.

A 37-year-old woman presented with an anterior optic neuropathy related to Bartonella henselae. Twenty-nine days after symptom onset, a partial thickness macular hole developed in the involved eye. Fundus photography and optical coherence tomography confirmed the conversion to a full-thickness macular hole in 2 months. Macular hole as a complication of cat scratch disease is a rare entity, with 2 prior reported cases in children. The development of a macular hole following cat scratch disease can appear without the clinical picture of multiple white chorioretinal lesions, macular star, or vitritis.

A Brief Primer on the Concept of the Neuroweapon for U.S. Military Medical Personnel.

The malevolent application of neuroscience is an emerging threat to the U.S. military. At present, U.S. military medical personnel are not capable of adequately diagnosing or treating the injuries and illnesses that may result from exposure to potential neuroweapons. This fact was illustrated in 2016 when U.S. diplomats serving in Havana, Cuba reported hearing strange noises accompanied by a constellation of unexplained health effects. Similar incidents have been reported in China and Russia. Although various hypotheses have been put forward to explain these symptoms, none of them have been verified. The reported symptoms were analogous to the physiological responses that have been produced in the laboratory by exposing volunteers to pulsed microwave energy. However, these incidents of undetermined origin demonstrate that widespread neurological illness can be disruptive to U.S. government operations and that it is currently not possible to identify the cause, determine the correct treatment, or ascribe attribution to potential neuroweapon use in an overseas setting. Since it is likely that Special Operations medical personnel will be among the first to respond to neuroweapon attacks in the deployed environment, it is essential that they be made aware of this emerging threat and that efforts be made to incorporate potential directed energy neuroweapons and other neuroweapon configurations into future Chemical, Biological, Radiological, Nuclear, and high yield Explosives (CBRN-E) training modules. The intention of this article is to introduce the concept of the neuroweapon to military medical personnel and to provide a brief review of the relevant literature.

Stability of SARS-CoV-2 on the Army Combat Uniform and Recommendations for Cleaning.

SARS-CoV-2 is the virus responsible for the disease that is known as COVID-19. While there have been numerous studies detailing the survival rates of SARS-CoV-2 on various materials, there are currently no published data regarding whether this virus is stable on standard military uniforms. Consequently, there are no standard operating procedures for washing uniforms once exposed to the virus. This study aimed to determine whether SARS-CoV-2 could be removed from Army combat uniform material by washing with a commercially available detergent and tap water. Washing the fabric with detergent followed by a rinse step with tap water effectively removes detectable viral particles. Importantly, it was found that washing with hot water alone was not effective. Therefore, it is recommended that military personnel wash their uniforms with detergent and water as soon as possible after exposure to SARS-CoV-2; hot water should not be used as a substitute for detergent.

An Introduction to the Processionary Caterpillar, An Underrecognized Threat to US Military Personnel in Australia.

Processionary caterpillars are well-described threats to human and animal health. They are found throughout Central Asia, Northern Africa, and Southern Europe. However, US military personnel may not be familiar with the threat that these organisms pose in Australia. The larval form of the bag-shelter moth (Ochrogaster lunifer) is a processionary caterpillar that has been found throughout inland and coastal Australia. These organisms are habitually associated with Acacia and Eucalyptus trees and they tend to form long chains known as "processions" as they travel between nesting and pupating sites. They are covered with numerous hairs that can detach, become airborne, and cause potentially life-threatening inflammatory reactions and ocular trauma in susceptible personnel. They can also cause severe inflammatory reactions in military working animals. It is important that military and preventive medical personnel become aware of the presence of processionary caterpillars in Australia, and that they can identify aerial or ground-based nests so that these dangerous organisms can be avoided by both humans and animals. Early identification is important so that prompt medical treatment can be rendered in the event of an accidental exposure.

Detection of Potential Pathogenic Bacteria on the Surfaces of Female Urinary Diversion Devices Following a Short Duration Military Training Exercise.

BACKGROUND: Female Servicemembers are increasingly being incorporated into the combat arms and Special Operations communities. Female urinary diversion devices (FUDDs) have been used to facilitate urination in the austere environments that are encountered by Servicemembers. Importantly, the potential for the bacterial contamination of these devices has not been evaluated. The goals of this study were to determine whether microorganisms adhere to the surfaces of FUDDs in the field environment and to demonstrate the presence of potential pathogens on the used devices. MATERIALS AND METHODS: A total of 15 devices that were used in a comprehensive 18-24-hour military field exercise were tested for the presence of microorganisms. Briefly, each device was swabbed, and the swabs were used to inoculate blood agar plates to encourage bacterial growth. The resulting bacterial colonies were identified, and the surface topography of the devices was investigated with electron microscopy. RESULTS: Although microscopy revealed few surface features capable of facilitating bacterial attachment, several species were recovered. Significantly, a biofilm-forming strain of Proteus mirabilis (P. mirabilis) was detected on two of the devices. P. mirabilis is a mobile urinary pathogen that can potentially migrate from the surface of the device into the urinary tract of the user. CONCLUSION: Commercial FUDDs can support bacterial growth and harbor potential pathogens. Care should be taken to ensure that Servicemembers are aware of the importance of the proper care and cleaning of these devices in the field environment. To this end, standard operating procedures should be developed and distributed.

Factors predictive for computed tomography use and abnormality in paediatric head injuries in Australia and New Zealand.

OBJECTIVES: To investigate patient-level factors predictive for computed tomography of the brain (CTB) use and abnormality in head injured children in Australia and New Zealand. METHODS: Retrospective data from tertiary, urban/suburban and regional/rural EDs including factors predictive for CTB use and abnormality. RESULTS: Of 3072 children at 31 EDs, 212 (6.9%) had a CTB scan, of which 66 (31%) were abnormal. Increasing age, serious mechanisms of injury and decreasing Glasgow Coma Score were predictive for ordering CTB. Decreasing age was predictive for CTB abnormalities. Other factors were not. CONCLUSION: Patient-level drivers of CTB use in children in Australia and New Zealand are consistent with international data.

Human Bocavirus as a Possible Contributor to Respiratory Disease in the Georgian Military Population.

The coronavirus disease 2019 (COVID-19) pandemic has demonstrated that new and devastating respiratory pathogens can emerge without warning. It is therefore imperative that Special Operations medical personnel be aware of the presence of emerging pathogens within their area of operation. Human bocavirus (HBoV) is a newly described member of a family of viruses known as the Parvovirinae that are often associated with acute respiratory illness. The presence of HBoV in the country of Georgia has not been previously reported. Nasal and throat swabs were collected from 95 symptomatic members of the Georgian military. HBoV was detected in 11 of them (12%). To our knowledge, this is the first report of HBoV infection in the country of Georgia. This finding may have a significant impact on members of the Special Operations community who train in Georgia as more data concerning the transmission, pathogenesis, and treatment of HBoV are accumulated and the role of HBoV in human disease is more clearly defined.

An Unusual Wound Infection Due to Acinetobacter junii on the Island of Oahu: A Case Report.

The genus Acinetobacter has long been associated with war wounds. Indeed, A baumannii was responsible for so many infected wounds during Operation Iraqi Freedom that it was given the nickname "Iraqibacter." Therefore, it is important to monitor the occurrence and spread of Acinetobacter species in military populations and to identify new or unusual sources of infection. A junii is an infrequently reported human pathogen. Here, we report a case of a slow-healing wound infection with A junii in a woman on the island of Oahu. This case highlights the pathogenic potential of this organism and the need for proper wound care when dealing with slow-healing wounds of unknown etiology. It also underscores the need for identifying species of Acinetobacter that are not A baumannii to better understand the epidemiology of slow-healing wound infections.

Lymphatic vessels identified in failed corneal transplants with neovascularisation.

BACKGROUND: Corneal transplant failure with neovascularisation is a leading indication for full-thickness grafts in patients. Lymphangiogenesis is implicated in the pathology of graft failure, and here we systematically evaluate failed human corneal transplants with neovascularisation for the presence of lymphatic vessels. METHODS: Nine failed grafts with neovascularisation, based on H&E staining with subsequent immunoperoxidase staining for CD31, a blood vessel marker, were selected. Lymphatics were investigated by immunohistochemical and immunofluorescence approaches using podoplanin as a lymphatic marker. In two of nine cases, fluorescence in situ hybridisation (FISH) was used for detection of lymphatic mRNAs including podoplanin, VEGFR-3 and LYVE-1. All immunofluorescence and FISH samples were compared with positive and negative controls and visualised by confocal microscopy. RESULTS: Corneal neovascularisation was established in all cases by H&E and further confirmed by CD31 immunoreactive profiles. Immunohistochemistry for the podoplanin antibody was positive in all cases and showed morphologies ranging from distinct luminal structures to elongated profiles. Simultaneous immunofluorescence using CD31 and podoplanin showed lymphatic vessels distinct from blood vessels. Podoplanin immunofluorescence was noted in seven of nine cases and revealed clear lumina of varying sizes, in addition to lumen-like and elongated profiles. The presence of lymphatic mRNA was confirmed by FISH studies using a combination of at least two of podoplanin, VEGFR-3 and LYVE-1 mRNAs. CONCLUSIONS: The consistent finding of lymphatic vessels in failed grafts with neovascularisation implicates them in the pathogenesis of corneal transplant failure, and points to the lymphatics as a potential new therapeutic target.

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

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.

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

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.

Arthropod Borne and Zoonotic Infections Among Military Personnel in Georgia.

Military personnel are at an increased risk for exposure to arthropod- borne and zoonotic pathogens. The prevalence of these pathogens has not been adequately described in the country of Georgia. As the Georgian military moves toward an increased level of capability and the adoption of European Union and North Atlantic Treaty Organization standards, international field exercises will become more frequent and will likely involve an increasing number of international partners. This study was undertaken with the goal of defining the arthropod-borne and zoonotic pathogen threat in Georgia so force health protection planning can proceed in a rational and data-driven manner. To estimate disease burden, blood was taken from 1,000 Georgian military recruits between October 2014 and February 2016 and screened for previous exposure to a set of bacterial and viral pathogens using a antibody-based, serologic procedure. The highest rate of exposure was to Salmonella enterica serovar Typhi, and the lowest rate of exposure was to Coxiella burnettii (the causative agent of Q fever). These data provide insight into the prevalence of arthropod-borne infections in Georgia, fill a critical knowledge gap, will help guide future surveillance efforts, and will inform force health protection planning.

The Hidden Complexity of Biological "Dirty Bombs": Implications for Special Operations Medical Personnel.

The recent capture of a terrorist in Belgium carrying explosives, fecal matter, and animal tissue may indicate a shift from conventional weapons to crude bacteriological preparations as instruments of terror. It is important to note that although such weapons lack technological sophistication, bacteria are inherently complex, unpredictable, and undetectable in the field. Therefore, it is important that Special Operations medical personnel understand the complications that such seemingly simple devices can add to the treatment of casualties in the field and subsequent evaluation in the clinic.