Point-of-care blood coagulation assay enabled by printed circuit board-based digital microfluidics

The monitoring of coagulation function has great implications in many clinical settings. However, existing coagulation assays are simplex, sample-consuming, and slow in turnaround, making them less suitable for point-of-care testing....

Point-of-care detection and differentiation of anticoagulant therapy - development of thromboelastometry-guided decision-making support algorithms

Background DOAC detection is challenging in emergency situations. Here, we demonstrated recently, that modified thromboelastometric tests can reliably detect and differentiate dabigatran and rivaroxaban. However, whether all DOACs can be detected and differentiated to other coagulopathies is unclear. Therefore, we now tested the hypothesis that a decision tree-based thromboelastometry algorithm enables detection and differentiation of all direct Xa-inhibitors (DXaIs), the direct thrombin inhibitor (DTI) dabigatran, as well as vitamin K antagonists (VKA) and dilutional coagulopathy (DIL) with high accuracy. Methods Following ethics committee approval (No 17–525-4), and registration by the German clinical trials database we conducted a prospective observational trial including 50 anticoagulated patients (n = 10 of either DOAC/VKA) and 20 healthy volunteers. Blood was drawn independent of last intake of coagulation inhibitor. Healthy volunteers served as controls and their blood was diluted to simulate a 50% dilution in vitro. Standard (extrinsic coagulation assay, fibrinogen assay, etc.) and modified thromboelastometric tests (ecarin assay and extrinsic coagulation assay with low tissue factor) were performed. Statistical analyzes included a decision tree analyzes, with depiction of accuracy, sensitivity and specificity, as well as receiver-operating-characteristics (ROC) curve analysis including optimal cut-off values (Youden-Index). Results First, standard thromboelastometric tests allow a good differentiation between DOACs and VKA, DIL and controls, however they fail to differentiate DXaIs, DTIs and VKAs reliably resulting in an overall accuracy of 78%. Second, adding modified thromboelastometric tests, 9/10 DTI and 28/30 DXaI patients were detected, resulting in an overall accuracy of 94%. Complex decision trees even increased overall accuracy to 98%. ROC curve analyses confirm the decision-tree-based results showing high sensitivity and specificity for detection and differentiation of DTI, DXaIs, VKA, DIL, and controls. Conclusions Decision tree-based machine-learning algorithms using standard and modified thromboelastometric tests allow reliable detection of DTI and DXaIs, and differentiation to VKA, DIL and controls. Trial registration Clinical trial number: German clinical trials database ID: DRKS00015704.

The role of the calibrated automated thrombogram in neonates: describing mechanisms of neonatal haemostasis and evaluating haemostatic drugs

Premature infants are at high risk of haemorrhage and thrombosis. Our understanding of the differences between the neonatal and adult haemostatic system is evolving. There are several limitations to the standard coagulation tests used in clinical practice, and there is currently a lack of evidence to support many of the transfusion practices in neonatal medicine. The evaluation of haemostasis is particularly challenging in neonates due to their limited blood volume. The calibrated automated thrombogram (CAT) is a global coagulation assay, first described in 2002, which evaluates both pro- and anti-coagulant pathways in platelet-rich or platelet-poor plasma. In this review, the current applications and limitations of CAT in the neonatal population are discussed.Conclusion: CAT has successfully elucidated several differences between haemostatic mechanisms in premature and term neonates compared with adults. Moreover, it has been used to evaluate the effect of a number of haemostatic drugs in a pre-clinical model. However, the lack of evidence of CAT as an accurate predictor of neonatal bleeding, blood volume required and the absence of an evidence-based treatment algorithm for abnormal CAT results limit its current application as a bedside clinical tool for the evaluation of sick neonates. What is Known:• The Calibrated automated thrombogram (CAT) is a global coagulation assay which evaluates pro- and anti-coagulant pathways.• CAT provides greater information than standard clotting tests and has been used in adults to evaluate bleeding risk. What is New:• This review summarises the physiological differences in haemostasis between neonates and adults described using CAT.• The haemostatic effect of several drugs has been evaluated in neonatal plasma using CAT.

Compatible properties and behaviour of dually loaded electrospun polyurethane bone tissue scaffolds

In this study, composite based on polyurethane (PU) containing lemon grass oil (LG) and zinc nitrate (ZnNO3) was fabricated using electrospinning technique. Morphology study revealed the fabricated scaffolds PU/LG and PU/LG/ZnNO3 diameter was lower than polyurethane. LG and ZnNO3 form hydrogen bond with polyurethane as revealed in the infrared analysis. The developed PU/LG composite rendered hydrophobicity while PU/LG/ZnNO3 showed hydrophilic nature than PU. Atomic force microscopy (AFM) depicted the decrease in surface roughness of the nanocomposite compared to polyurethane. The addition of LG and ZnNO3 improved the mechanical strength of the pristine PU as indicated in tensile analysis. Coagulation assay measurements indicated a delay in the activation of clot and also exhibited reduced toxicity for the developed composites PU/LG and PU/LG/ZnNO3. Moreover, the deposition of calcium in the developed composites were found to be higher compared to the PU as noted in the bone mineralization testing. Hence, these developed nanocomposites with desirable properties will translate them as potential candidates for bone tissue engineering.

HEMOSTATIC POTENTIAL ASSESSMENT OF PATIENTS WITH LIVER CIRRHOSIS AND ATRIAL FIBRILLATION BY LOW-FREQUENCY PIEZOELECTRIC THROMBOELASTOGRAPHY

The aim: Our aim was to assess the hemostatic potential of patients with liver cirrhosis and atrial fibrillation by LPTEG global coagulation assay, to investigate changes in LPTEG parameters according to the stage of liver cirrhosis and compare results with liver cirrhosis group. Materials and methods: We performed a prospective cross-sectional study including 70 patients with liver cirrhosis and atrial fibrillation, 36 patients with liver cirrhosis and 20 healthy individuals. LPTEG parameters were measured using ARP-01M “Mednord” in order to assess coagulation abnormalities. Results: t1 and Intensity of contact coagulation didn't differ (p>0,05), Constant of thrombin activity was increased (47.53±0.8vs.34.51±1.88, p<0.001), t3 was reduced (5,0±0.1vs.6.7±0.36 p<0.001), Intensity of coagulation drive was increased (52.8±1.8vs.38.55±1.54, p = 0.001), Intensity of clot polymerization was increased (19.66±0.28vs.16.29±0.28, p<0.001), time t5 was reduced (32.94±0.36 vs. 36.8±1.30, p<0.01), Maximum amplitude was increased (655.7±9.19 vs. 547±19.38, p<0.001), Intensity of total coagulation was increased (19.41±0.34vs.15,09±0.56, p<0.001), Intensity of clot retraction and lysis was increased (4.1±0.07vs.3±0.15, p<0.001) and Coefficient of total anticoagulant activity was increased (2.81±0.05 vs. 2.48 ± 0.06, p<0.001) compared to liver cirrhosis. Conclusions: In patients with liver cirrhosis and atrial fibrillation the hemostatic potential is significantly shifted towards hypercoagulation with a gradual worsening of coagulation disorders, starting from the compensated stage of liver cirrhosis.

Rotational thrombelastometry (ROTEM) improves hemostasis assessment compared to conventional coagulation test in ACLF and Non-ACLF patients

Background Patients with liver cirrhosis typically exhibit abnormal coagulation parameters in conventional coagulation tests (CCTs). Rotational thromboelastometry (ROTEM) is a holistic blood coagulation assay. This method provides an insight into the global hemostatic capabilities and has been suggested to provide a better overview of the coagulation system in liver cirrhosis. Methods The goal of this study was to examine hemostasis in patients with stable liver cirrhosis (Non-ACLF) and in acute-on-chronic liver failure (ACLF) by CCT and ROTEM including agreement of both tests and the prospective assessment of test performance based on clinical outcomes in ACLF patients. Therefore, ACLF patients were additionally subgrouped by bleeding events. Fifty-five Non-ACLF patients and twenty-two patients with ACLF were analysed in this prospective cohort study. Results Coagulation parameters analysed by CCT were outside the normal range in Non-ACLF and ACLF patients, but were significantly more aberrant in ACLF patients. Non-ACLF patients analysed by ROTEM revealed parameters largely within the normal limits, while significantly more ROTEM parameters in ACLF patients were affected. Maximum clot firmness (MCF) was significantly divergent between both patient groups and correlated well with levels of fibrinogen and platelet count. Using Cohen’s Kappa coefficient κ, the strength of agreement between CCT and ROTEM analyses was determined to be fair for Non-ACLF patients and moderate for ACLF patients. Bleeding events occurred significantly more often in ACLF group with significantly reduced A10 and MCF. Conclusions For assessing hemostasis in Non-ACLF and ACLF patients the underlying dataset shows advantages of ROTEM over CCT. A10 and MCF represent suitable prognostic parameters in predicting bleeding events in ACLF group.

Varespladib Inhibits the Phospholipase A2 and Coagulopathic Activities of Venom Components from Hemotoxic Snakes

Phospholipase A2 (PLA2) enzymes are important toxins found in many snake venoms, and they can exhibit a variety of toxic activities including causing hemolysis and/or anticoagulation. In this study, the inhibiting effects of the small molecule PLA2 inhibitor varespladib on snake venom PLA2s was investigated by nanofractionation analytics, which combined chromatography, mass spectrometry (MS), and bioassays. The venoms of the medically important snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus, Echis ocellatus, and Oxyuranus scutellatus were separated by liquid chromatography (LC) followed by nanofractionation and interrogation of the fractions by a coagulation assay and a PLA2 assay. Next, we assessed the ability of varespladib to inhibit the activity of enzymatic PLA2s and the coagulopathic toxicities induced by fractionated snake venom toxins, and identified these bioactive venom toxins and those inhibited by varespladib by using parallel recorded LC-MS data and proteomics analysis. We demonstrated here that varespladib was not only capable of inhibiting the PLA2 activities of hemotoxic snake venoms, but can also effectively neutralize the coagulopathic toxicities (most profoundly anticoagulation) induced by venom toxins. While varespladib effectively inhibited PLA2 toxins responsible for anticoagulant effects, we also found some evidence that this inhibitory molecule can partially abrogate procoagulant venom effects caused by different toxin families. These findings further emphasize the potential clinical utility of varespladib in mitigating the toxic effects of certain snakebites.