Extracellular vesicles in fresh frozen plasma and cryoprecipitate: Impact on in vitro endothelial cell viability.

BACKGROUND: Transfusion-related acute lung injury (TRALI) remains a major contributor to transfusion-associated mortality. While the pathogenesis of TRALI remains unclear, there is evidence of a role for blood components. We therefore investigated the potential effects of fresh frozen plasma (FFP), cryoprecipitate, and extracellular vesicles (EVs) derived from these blood components, on the viability of human lung microvascular endothelial cells (HLMVECs) in vitro. METHODS: EVs were isolated from FFP and cryoprecipitate using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. The potential effects of these blood components and their EVs on HLMVEC viability (determined by trypan blue exclusion) were examined in the presence and absence of neutrophils, either with or without prior treatment of HLMVECs with LPS. RESULTS: EVs isolated from FFP and cryoprecipitate displayed morphological and biochemical properties conforming to latest international criteria. While FFP, cryoprecipitate, and EVs derived from FFP, each reduced HLMVEC viability, no effect was observed for EVs derived from cryoprecipitate. CONCLUSION: Our findings demonstrate clear differences in the effects of FFP, cryoprecipitate, and their respective EVs on HLMVEC viability in vitro. Examination of the mechanisms underlying these differences may lead to an improved understanding of the factors that promote development of TRALI.

Investigation of the effect of pre-analytical factors on particle concentration and size in cryoprecipitate using nanoparticle tracking analysis.

BACKGROUND: Cryoprecipitate is used primarily to replenish fibrinogen levels in patients. Little is known about the presence of micro- or nano-sized particles in cryoprecipitate. Therefore, we aimed to quantify these particles and investigate some pre-analytical considerations. MATERIALS AND METHODS: Particle concentration and size distribution were determined in 10 cryoprecipitate units by nanoparticle tracking analysis (NTA). The effects of freeze-thawing cryoprecipitate and 0.45 µm filtration with either regenerated cellulose (RC) or polytetrafluoroethylene (PTFE) filters before sample analysis were examined. RESULTS: Neither the size nor concentration of particles were affected by two freeze/thaw cycles. PTFE filtration, but not RC filtration, significantly reduced particle mean and mode size compared to RC filtration and mode size compared to unfiltered cryoprecipitate. The 10 cryoprecipitate units had an average particle concentration of 2.50 × 1011 ± 1.10 × 1011 particles/mL, a mean particle size of 133.8 ± 7.5 nm and a mode particle size of 107.9 ± 11.1 nm. CONCLUSION: This study demonstrated that preanalytical filtration of cryoprecipitate units using RC filters was suitable for NTA. An additional freeze/thaw cycle did not impact NTA parameters, suggesting that aliquoting cryoprecipitate units prior to laboratory investigations is suitable for downstream analyses.

Using whole-genome sequencing to characterize clinically significant blood groups among healthy older Australians.

There have been no comprehensive studies of a full range of blood group polymorphisms within the Australian population. This problem is compounded by the absence of any databases carrying genomic information on chronically transfused patients and low frequency blood group antigens in Australia. Here, we use RBCeq, a web server-based blood group genotyping software, to identify unique blood group variants among Australians and compare the variation detected vs global data. Whole-genome sequencing data were analyzed for 2796 healthy older Australians from the Medical Genome Reference Bank and compared with data from 1000 Genomes phase 3 (1KGP3) databases comprising 661 African, 347 American, 503 European, 504 East Asian, and 489 South Asian participants. There were 661 rare variants detected in this Australian sample population, including 9 variants that had clinical associations. Notably, we identified 80 variants that were computationally predicted to be novel and deleterious. No clinically significant rare or novel variants were found associated with the genetically complex ABO blood group system. For the Rh blood group system, 2 novel and 15 rare variants were found. Our detailed blood group profiling results provide a starting point for the creation of an Australian blood group variant database.

RBCeq: A robust and scalable algorithm for accurate genetic blood typing.

BACKGROUND: While blood transfusion is an essential cornerstone of hematological care, patients requiring repetitive transfusion remain at persistent risk of alloimmunization due to the diversity of human blood group polymorphisms. Despite the promise, user friendly methods to accurately identify blood types from next-generation sequencing data are currently lacking. To address this unmet need, we have developed RBCeq, a novel genetic blood typing algorithm to accurately identify 36 blood group systems. METHODS: RBCeq can predict complex blood groups such as RH, and ABO that require identification of small indels and copy number variants. RBCeq also reports clinically significant, rare, and novel variants with potential clinical relevance that may lead to the identification of novel blood group alleles. FINDINGS: The RBCeq algorithm demonstrated 99·07% concordance when validated on 402 samples which included 29 antigens with serology and 9 antigens with SNP-array validation in 14 blood group systems and 59 antigens validation on manual predicted phenotype from variant call files. We have also developed a user-friendly web server that generates detailed blood typing reports with advanced visualization (https://www.rbceq.org/). INTERPRETATION: RBCeq will assist blood banks and immunohematology laboratories by overcoming existing methodological limitations like scalability, reproducibility, and accuracy when genotyping and phenotyping in multi-ethnic populations. This Amazon Web Services (AWS) cloud based platform has the potential to reduce pre-transfusion testing time and to increase sample processing throughput, ultimately improving quality of patient care. FUNDING: This work was supported in part by Advance Queensland Research Fellowship, MRFF Genomics Health Futures Mission (76,757), and the Australian Red Cross LifeBlood. The Australian governments fund the Australian Red Cross Lifeblood for the provision of blood, blood products and services to the Australian community.

Acute Effects of Nitrate-Rich Beetroot Juice on Blood Pressure, Hemostasis and Vascular Inflammation Markers in Healthy Older Adults: A Randomized, Placebo-Controlled Crossover Study.

Aging is associated with a vasoconstrictive, pro-coagulant, and pro-inflammatory profile of arteries and a decline in the bioavailability of the endothelium-derived molecule nitric oxide. Dietary nitrate elicits vasodilatory, anti-coagulant and anti-inflammatory effects in younger individuals, but little is known about whether these benefits are evident in older adults. We investigated the effects of 140 mL of nitrate-rich (HI-NI; containing 12.9 mmol nitrate) versus nitrate-depleted beetroot juice (LO-NI; containing ≤0.04 mmol nitrate) on blood pressure, blood coagulation, vascular inflammation markers, plasma nitrate and nitrite before, and 3 h and 6 h after ingestion in healthy older adults (five males, seven females, mean age: 64 years, age range: 57-71 years) in a randomized, placebo-controlled, crossover study. Plasma nitrate and nitrite increased 3 and 6 h after HI-NI ingestion (p < 0.05). Systolic, diastolic and mean arterial blood pressure decreased 3 h relative to baseline after HI-NI ingestion only (p < 0.05). The number of blood monocyte-platelet aggregates decreased 3 h after HI-NI intake (p < 0.05), indicating reduced platelet activation. The number of blood CD11b-expressing granulocytes decreased 3 h following HI-NI beetroot juice intake (p < 0.05), suggesting a shift toward an anti-adhesive granulocyte phenotype. Numbers of blood CD14++CD16⁺ intermediate monocyte subtypes slightly increased 6 h after HI-NI beetroot juice ingestion (p < 0.05), but the clinical implications of this response are currently unclear. These findings provide new evidence for the acute effects of nitrate-rich beetroot juice on circulating immune cells and platelets. Further long-term research is warranted to determine if these effects reduce the risk of developing hypertension and vascular inflammation with aging.

Evidence of altered haemostasis in an ovine model of venovenous extracorporeal membrane oxygenation support.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a life-saving modality used in the management of cardiopulmonary failure that is refractory to conventional medical and surgical therapies. The major problems clinicians face are bleeding and clotting, which can occur simultaneously. To discern the impact of pulmonary injury and ECMO on the host's haemostatic response, we developed an ovine model of smoke-induced acute lung injury (S-ALI) and ECMO. The aims of this study were to determine if the ECMO circuit itself altered haemostasis and if this was augmented in a host with pulmonary injury. METHODS: Twenty-seven South African meat merino/Border Leicester Cross ewes underwent instrumentation. Animals received either sham injury (n = 12) or S-ALI (n = 15). Control animal groups consisted of healthy controls (ventilation only for 24 h) (n = 4), ECMO controls (ECMO only for 24 h) (n = 8) and S-ALI controls (S-ALI but no ECMO for 24 h) (n = 7). The test group comprised S-ALI sheep placed on ECMO (S-ALI + ECMO for 24 h) (n = 8). Serial blood samples were taken for rotational thromboelastometry, platelet aggregometry and routine coagulation laboratory tests. Animals were continuously monitored for haemodynamic, fluid and electrolyte balances and temperature. Pressure-controlled intermittent mandatory ventilation was used, and mean arterial pressure was augmented by protocolised use of pressors, inotropes and balanced fluid resuscitation to maintain mean arterial pressure >65 mmHg. RESULTS: Rotational thromboelastometry, platelet aggregometry and routine coagulation laboratory tests demonstrated that S-ALI and ECMO independently induced changes to platelet function, delayed clot formation and reduced clot firmness. This effect was augmented with the combination of S-ALI and ECMO, with evidence of increased collagen-induced platelet aggregation as well as changes in factor VIII (FVIII), factor XII and fibrinogen levels. CONCLUSIONS: The introduction of an ECMO circuit itself increases collagen-induced platelet aggregation, decreases FVIII and von Willebrand factor, and induces a transient decrease in fibrinogen levels and function in the first 24 h. These changes to haemostasis are amplified when a host with a pre-existing pulmonary injury is placed on ECMO. Because patients are often on ECMO for extended periods, longer-duration studies are required to characterise ECMO-induced haemostatic changes over the long term. The utility of point-of-care tests for guiding haemostatic management during ECMO also warrants further exploration.

Procoagulant role of microparticles in routine storage of packed red blood cells: potential risk for prothrombotic post-transfusion complications.

During routine storage, packed red blood cells (PRBC) undergo biochemical and morphological changes including loss of red blood cell (RBC) membrane asymmetry and release of microparticles (MPs) bearing phosphatidylserine (PS), a procoagulant phospholipid. This study investigated the association between PRBC storage duration, MP profile and procoagulant activity. Leukodepleted PRBC-supernatant (PRBC-SN; n=13) was prepared at weekly intervals throughout storage. Phospholipid-dependent procoagulant activity, assessed using a factor X-activated clotting time (XACT) assay, decreased throughout storage (p<0.0001), corresponding with increased procoagulant phospholipid content. As determined by flow cytometry, total numbers of MPs and of PS-bearing MPs increased by Day 28 of storage (p<0.01 and p<0.05, respectively, versus D1), and these MPs were predominantly RBC-derived (CD235+). Depletion of MPs from stored (Day 42) PRBC-SN using 0.22 µm filters reduced the number of PS-bearing MPs (p<0.01) but did not increase XACT clotting times. Furthermore, the reduction in procoagulant activity when lactadherin was used to block PS was not altered pre- or post-filtration of PRBC-SN. In conclusion, routine PRBC storage was associated with accumulation of MPs (particularly RBC-derived PS-bearing MPs) and of procoagulant phospholipids; however, depletion of PS-bearing MPs by 0.22 µm filtration did not reduce phospholipid-dependent procoagulant activity.

Reversal of acute coagulopathy during hypotensive resuscitation using small-volume 7.5% NaCl adenocaine and Mg2+ in the rat model of severe hemorrhagic shock.

OBJECTIVE: Acute traumatic coagulopathy occurs early in hemorrhagic trauma and is a major contributor to mortality and morbidity. Our aim was to examine the effect of small-volume 7.5% NaCl adenocaine (adenosine and lidocaine, adenocaine) and Mg on hypotensive resuscitation and coagulopathy in the rat model of severe hemorrhagic shock. DESIGN: Prospective randomized laboratory investigation. SUBJECTS: A total of 68 male Sprague Dawley Rats. INTERVENTION: Post-hemorrhagic shock treatment for acute traumatic coagulopathy. MEASUREMENTS AND METHODS: Nonheparinized male Sprague-Dawley rats (300-450 g, n=68) were randomly assigned to either: 1) untreated; 2) 7.5% NaCl; 3) 7.5% NaCl adenocaine; 4) 7.5% NaCl Mg²âº; or 5) 7.5% NaCl adenocaine/Mg²âº. Hemorrhagic shock was induced by phlebotomy to mean arterial pressure of 35-40 mm Hg for 20 mins (~40% blood loss), and animals were left in shock for 60 mins. Bolus (0.3 mL) was injected into the femoral vein and hemodynamics monitored. Blood was collected in Na citrate (3.2%) tubes, centrifuged, and the plasma snap frozen in liquid N2 and stored at -80°C. Coagulation was assessed using activated partial thromboplastin times and prothrombin times. RESULTS: Small-volume 7.5% NaCl adenocaine and 7.5% NaCl adenocaine/Mg²âº were the only two groups that gradually increased mean arterial pressure 1.6-fold from 38-39 mm Hg to 52 and 64 mm Hg, respectively, at 60 mins (p<.05). Baseline plasma activated partial thromboplastin time was 17±0.5 secs and increased to 63±21 secs after bleeding time, and 217±32 secs after 60-min shock. At 60-min resuscitation, activated partial thromboplastin time values for untreated, 7.5% NaCl, 7.5% NaCl/Mg²âº, and 7.5% NaCl adenocaine rats were 269±31 secs, 262±38 secs, 150±43 secs, and 244±38 secs, respectively. In contrast, activated partial thromboplastin time for 7.5% NaCl adenocaine/Mg²âº was 24±2 secs (p<.05). Baseline prothrombin time was 28±0.8 secs (n=8) and followed a similar pattern of correction. CONCLUSIONS: Plasma activated partial thromboplastin time and prothrombin time increased over 10-fold during the bleed and shock periods prior to resuscitation, and a small-volume (~1 mL/kg) IV bolus of 7.5% NaCl AL/Mg²âº was the only treatment group that raised mean arterial pressure into the permissive range and returned activated partial thromboplastin time and prothrombin time clotting times to baseline at 60 mins.

Elevated plasma fibronectin levels associated with venous thromboembolism.

Elevated plasma fibronectin levels occur in various clinical states including arterial disease. Increasing evidence suggests that atherothrombosis and venous thromboembolism (VTE) share common risk factors. To assess the hypothesis that high plasma fibronectin levels are associated with VTE, we compared plasma fibronectin levels in the Scripps Venous Thrombosis Registry for 113 VTE cases vs. age and sex matched controls. VTE cases had significantly higher mean fibronectin concentration compared to controls (127% vs. 103%, p < 0.0001); the difference was greater for idiopathic VTE cases compared to secondary VTE cases (133% vs. 120%, respectively). Using a cut-off of >90% of the control values, the odds ratio (OR) for association of VTE for fibronectin plasma levels above the 90(th) percentile were 9.37 (95% CI 2.73-32.2; p < 0.001) and this OR remained significant after adjustment for sex, age, body mass index (BMI), factor V Leiden and prothrombin nt20210A (OR 7.60, 95% CI 2.14-27.0; p = 0.002). In particular, the OR was statistically significant for idiopathic VTE before and after these statistical adjustments. For the total male cohort, the OR was significant before and after statistical adjustments and was not significant for the total female cohort. In summary, our results suggest that elevated plasma fibronectin levels are associated with VTE especially in males, and extend the potential association between biomarkers and risk factors for arterial atherothrombosis and VTE.

High-density lipoprotein and the risk of recurrent venous thromboembolism.

BACKGROUND: High-density lipoprotein (HDL) protects against arterial atherothrombosis, but it is unknown whether it protects against recurrent venous thromboembolism. METHODS AND RESULTS: We studied 772 patients after a first spontaneous venous thromboembolism (average follow-up 48 months) and recorded the end point of symptomatic recurrent venous thromboembolism, which developed in 100 of the 772 patients. The relationship between plasma lipoprotein parameters and recurrence was evaluated. Plasma apolipoproteins AI and B were measured by immunoassays for all subjects. Compared with those without recurrence, patients with recurrence had lower mean (+/-SD) levels of apolipoprotein AI (1.12+/-0.22 versus 1.23+/-0.27 mg/mL, P<0.001) but similar apolipoprotein B levels. The relative risk of recurrence was 0.87 (95% CI, 0.80 to 0.94) for each increase of 0.1 mg/mL in plasma apolipoprotein AI. Compared with patients with apolipoprotein AI levels in the lowest tertile (<1.07 mg/mL), the relative risk of recurrence was 0.46 (95% CI, 0.27 to 0.77) for the highest-tertile patients (apolipoprotein AI >1.30 mg/mL) and 0.78 (95% CI, 0.50 to 1.22) for midtertile patients (apolipoprotein AI of 1.07 to 1.30 mg/mL). Using nuclear magnetic resonance, we determined the levels of 10 major lipoprotein subclasses and HDL cholesterol for 71 patients with recurrence and 142 matched patients without recurrence. We found a strong trend for association between recurrence and low levels of HDL particles and HDL cholesterol. CONCLUSIONS: Patients with high levels of apolipoprotein AI and HDL have a decreased risk of recurrent venous thromboembolism.

High-density lipoprotein deficiency and dyslipoproteinemia associated with venous thrombosis in men.

BACKGROUND: Although dyslipoproteinemia is associated with arterial atherothrombosis, little is known about plasma lipoproteins in venous thrombosis patients. METHODS AND RESULTS: We determined plasma lipoprotein subclass concentrations using nuclear magnetic resonance spectroscopy and antigenic levels of apolipoproteins AI and B in blood samples from 49 male venous thrombosis patients and matched controls aged <55 years. Venous thrombosis patients had significantly lower levels of HDL particles, large HDL particles, HDL cholesterol, and apolipoprotein AI and significantly higher levels of LDL particles and small LDL particles. The quartile-based odds ratios for decreased HDL particle and apolipoprotein AI levels in patients compared with controls were 6.5 and 6.0 (95% CI, 2.3 to 19 and 2.1 to 17), respectively. Odds ratios for apolipoprotein B/apolipoprotein AI ratio and LDL cholesterol/HDL cholesterol ratio were 6.3 and 2.7 (95% CI, 1.9 to 21 and 1.1 to 6.5), respectively. When polymorphisms in genes for hepatic lipase, endothelial lipase, and cholesteryl ester transfer protein were analyzed, patients differed significantly from controls in the allelic frequency for the TaqI B1/B2 polymorphism in cholesteryl ester transfer protein, consistent with the observed pattern of lower HDL and higher LDL. CONCLUSIONS: Venous thrombosis in men aged <55 years old is associated with dyslipoproteinemia involving lower levels of HDL particles, elevated levels of small LDL particles, and an elevated ratio of apolipoprotein B/apolipoprotein AI. This dyslipoproteinemia seems associated with a related cholesteryl ester transfer protein genotype difference.