Activated Partial Thromboplastin Time Assays Research Articles

Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-β2-glycoprotein I antibody titres for all three LA assays. TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

Differentiation of Acquired Hemophilia a from Lupus Anticoagulant Using Global Coagulation Assays and Magnesium-Dependent Activated Partial Thromboplastin Time Assay

CONCLUSION Rapid and reliable differentiation between bleeding and thrombotic tendency in cases with prolonged activated partial thromboplastin time (APTT) remains challenging in clinical practice, in particular, distinguishing cases positive for coagulation factor inhibitor or lupus anticoagulant (LA). To differentiate acquired hemophilia A (AHA) from LA using three global coagulation assays: rotational thromboelastometry (ROTEM), thrombin generation assay (TGA), and clot waveform analysis (CWA); also to validate the effectiveness of magnesium-dependent APTT (APTT-Mg) assay. This method uses APTT-Mg and normal APTT using Ca to screen for LA, and APTT-Mg/APTT ratio is smaller in LA. We included patients with prolonged APTT without congenital coagulation factor deficiency who were referred to our department between November 2019 and September 2021. APTT prolongation was defined as >35 s (normal range: 25-35 s) measured routinely with Coagpia APTT-N (Sekisui Medical). The AHA group had a confirmed diagnosis and underwent treatment of AHA. The LA group was divided into LA1 (possible LA) and LA2 (definite LA). LA1 was defined by any of the following conditions: (1) index of circulation anticoagulant ≥15 in the cross-mixing test performed with Coagpia APTT-N; (2) positive dilute Russell's viper venom time (DRVVT) phospholipid neutralization method using Gladipore LA (Medical & Biological Laboratories); (3) positive APTT phospholipid neutralization test using Hemosil SCT (Instrumentation Laboratory); (4) positive for anti-CL antibody; (5) positive for anti-CLβ2GPI antibody; or (6) positive for phosphatidylserine-dependent anti-PT antibody. LA2 was defined by index of circulation anticoagulant >12.4 in the cross-mixing test performed by Thrombocheck APTT-SLA (Sysmex Corporation), or item (2) or (3) above, in accordance with criteria of LA in the International Society of Thrombosis and Haemostasis. LA1 and LA2 did not overlap, because cases that did not meet the conditions of LA2 were treated as LA1, and global coagulation assays were performed in all LA cases. In the global coagulation assays, 12 healthy volunteer samples were tested and regarded as the normal goroup. For the APTT-Mg assay, equal volumes of 25 mmol MgCl 2 and 25 mmol CaCl 2 were used instead of the normally used CaCl 2, and Thrombocheck APTT-SLA was used to measure APTT-Mg, which was then divided by the normal APTT concentration measured with the same APTT reagent. Ten patients in the AHA, 12 in the LA1, and 32 in the LA2 groups were eligible. ROTEM showed that clotting time in the non-activated rotational thromboelastometry assay (NATEM) mode was >3600 s (minimum 1363 s) for AHA, 501 s (IQR: 430-632 s) for LA1, 533 s (IQR: 443-641 s) for LA2, and 500 s (IQR: 429-645 s) for the normal group. TGA showed that peak thrombin concentration was 16 nM (IQR: 13-29 nM) in the AHA group, 242 nM (IQR: 175-273 nM) in the LA1 group, 174 nM (IQR: 129-259 nM) in the LA2 group, and 190 nM (IQR: 125-288 nM) in the normal group. In the CWA, Ad|min1| was 2.1 (IQR: 1.7-3.2) in the AHA group, 8.7 (IQR: 8.0-9.5) in the LA1 group, 6.7 (IQR: 5.8-8.4) in the LA2 group, and 10.3 (IQR: 9.8-10.6) in the normal group. APTT-Mg/APTT was 1.23 (IQR: 1.18-1.29), 1.26 (IQR: 1.23-1.38), and 1.05 (IQR: 0.95-1.20) in the AHA, LA1, and LA2 groups, respectively. ROTEM, TGA, and CWA effectively differentiated between the LA1/2 and AHA groups. Clotting time in non-activated rotational thromboelastometry assay mode (ROTEM) and peak height (TGA) were better at distinguishing high-titer LA cases from LA-positive AHA cases. APTT-Mg/APTT could be suitable for distinguishing AHA from high-titer LA; however, it may not be useful for differentiating AHA from low-titer or suspected LA.

Isolation of the Anticoagulant and Procoagulant Fractions of the Venom of Iranian Endemic Echis carinatus.

The venom of Echis carinatus contains both procoagulant and anticoagulant components that can either promote or block the blood coagulation cascade, and some of these components affect platelet function in different ways. The present study focuses on setting up a procedure for the purification of crude venom and designing appropriate clotting tests in order to characterize the procoagulant and anticoagulant fractions of E. carinatus venom. Chromatographic methods, including gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (HPLC), were applied for purifying these fractions. Coagulant activity testing, prothrombin time (PT), and activated partial thromboplastin time (APTT) were used to determine procoagulant and anticoagulant properties. For measuring molecular weight, 15% SDS-PAGE electrophoresis with a molecular weight standard ranging from 6.5 to 200 kDa was used. We obtained five fractions named F1, F2, F3, F4, and F5. The F1 and F2 fractions showed procoagulant activity, and the F5 fraction had anticoagulant activity. The molecular weight of F2.4.2 from fraction F2 and F5.1 from fraction F5 were analyzed by SDS-PAGE electrophoresis under the reducing condition. These factors were identified as a single protein band at the end of purification. The molecular weights of these purified fractions were estimated to be 7.5 kDa and 38 kDa for F5.1(b) and F2.4.2(b), respectively. Our findings suggest an efficient and suitable procedure for the identification and purification of the procoagulant and anticoagulant factors of the venom of Iranian E. carinatus using the PT and APTT assays.

Harmonizing factor assay-related testing performed in a large laboratory network.

Coagulation factor testing is commonly performed within haemostasis laboratories, either to assess for bleeding disorders, such as haemophilia, or to investigate unexplained prolongation in routine coagulation assays. The aim of this evaluation was to harmonize procedures and normal reference ranges (NRRs) for investigation of coagulation factors on the ACL TOP 50 family of instruments in a large laboratory network. We employed comparative evaluations using newly installed ACL TOPs 550 and 750 and HemosIL reagents vs. existing 'reference' instrumentation and reagents, predominantly Stago and Siemens, as well as assessment of factor sensitivity in routine coagulation assays, prothrombin time (PT) and activated partial thromboplastin time (APTT). Also, establishment of coagulation factor NRRs using normal plasma samples. HemosIL factor assays showed good comparability with the existing reference methods ( R > 0.9). Factor sensitivity for PT and APTT assays were acceptable at around 30 U/dl. NRRs were established and harmonized across the laboratory network. This evaluation of factor testing on ACL TOP 50 Family instruments identified overall acceptable performance using Werfen reagents and enabled harmonization of coagulation factor testing in our large network.

A multi-laboratory assessment of lupus anticoagulant assays performed on the ACL TOP 50 family for harmonized testing in a large laboratory network.

IntroductionLupus anticoagulant (LA) testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new “single platform” of hemostasis instrumentation. Our aim was to evaluate these instruments and manufacturer reagents or alternatives for utility in LA testing.MethodsComparative evaluations of LA testing using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing “reference,” predominantly Stago, instrumentation, and reagents. Evaluations comprised both dilute Russell viper venom time (dRVVT) and activated partial thromboplastin time (APTT)‐based assays. Establishment of normal reference ranges (NRR).ResultsThe HemosIL dRVVT‐based assays showed good comparability with the existing Stago reference method (R > 0.9) and could be considered as verified as fit for purpose. A variety of APTT assays was additionally evaluated for LA utility, and we identified from the assessment good utility of a non‐Werfen solution in Hyphen BioMed Cephen reagents. NRR were established based on ≥120 normal individual plasma samples.ConclusionThis evaluation of LA reagents on ACL TOP 50 Family instruments identified overall acceptable performance of both dRVVT (Werfen solution) and APTT (non‐Werfen solution) to enable harmonization of LA testing in our large network.

An Analysis of the Sensitivity of the Activated Partial Thromboplastin Time (APTT) Assay, as Used in a Large Laboratory Network, to Coagulation Factor Deficiencies.

To advance knowledge in using the ex vivo method to identify factor sensitivity of the activated partial thromboplastin time (APTT), using data from a hemophilia and reference hemostasis laboratory; to evaluate application of inclusion and exclusion criteria to eliminate data outliers; and to discuss outcomes with reference to comparable studies. An ex vivo, retrospective analysis was performed on patient samples with conjointly ordered APTT and intrinsic pathway factors (VIII, IX, XI, XII) for application to a large network of laboratories. The relationship between factor levels and APTT, before and after application of exclusion criteria, is demonstrated. Curvilinear relationships were found between all factor levels and APTTs, which demonstrated both similarities and differences with available studies. Factor sensitivity data are presented. Study strengths include large sample size and use of real-world data. Limitations include inability to exclude all residual outliers and paucity of patient samples singularly deficient in factors other than FVIII. This ex vivo, retrospective analysis of the sensitivity of the APTT assay to intrinsic pathway factor deficiencies using real-world data from a hemophilia and reference hemostasis laboratory contains the largest sample size using this approach to date. The outcomes assist in informing practice in this area and can be used as a reference for further studies.

Preliminary Results on Heparin-Modified Double-Layered PCL and PLA-Based Scaffolds for Tissue Engineering of Small Blood Vessels.

Low-diameter blood vessels are challenging to replace with more traditional synthetic vascular grafts. Therefore, the obvious choice is to try to regenerate small veins and arteries through tissue-engineering approaches. However, the layered structure of native vessels and blood compatibility issues make this a very challenging task. The aim of this study is to create double-layered tubular scaffolds with enhanced anticoagulant properties for the tissue engineering of small blood vessels. The scaffolds were made of a polycaprolactone-based porous outer layer and a polylactide-based electrospun inner layer modified with heparin. The combination of thermally induced phase separation and electrospinning resulted in asymmetric scaffolds with improved mechanical properties. The release assay confirmed that heparin is released from the scaffolds. Additionally, anticoagulant activity was shown through APTT (activated partial thromboplastin time) assay. Interestingly, the endothelial cell culture test revealed that after 14 days of culture, HAECs (human aortic endothelial cell lines) tended to organize in chain-like structures, typical for early stages of vascular formation. In the longer culture, HAEC viability was higher for the heparin-modified scaffolds. The proposed scaffold design and composition have great potential for application in tissue engineering of small blood vessels.

Relative Interfering Effects of In Vivo Direct Oral Anticoagulants on Routine Coagulation Tests

Background:The interfering effects of DOACs on the screening coagulation tests, such as prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen assay, have been shown mainly by in vitro spiking experiments. However, the effects of DOACs on coagulation tests in real-world samples from anticoagulated patients are unknown because of the difficulty in selectively eliminating DOAC from blood samples already containing DOACs.Method:Citrated blood samples were drawn from patients on anticoagulation therapy (rivaroxaban and edoxaban). In addition, blood samples from patients not on anticoagulation were collected. PT INR and APTT were measured from those samples by coagulometers from two manufacturers (Roche t711, Swiss and ACL-TOP, USA). We also measured DOAC levels from the same samples by anti-FXa activity (Hyphen Biomed, France). Then, we compared the test results in relation to the DOAC levels.Results:The PT INR, APTT, and fibrinogen assay results from non-anticoagulated patients measured by the two coagulometers were comparable (PT INR: y = -3.353 + 1.029 x; APTT: y = -6.276 + 1.101x; fibrinogen: y = -3.353 + 1.029 x; Passing Bablok). We included blood samples from 61 patients on rivaroxaban and 75 patients on edoxaban. From the rivaroxaban samples we observed the regression line change for PT INR (y = 0.6303 + 0.3712x) and for APTT (y = -10.71+1.358x). The comparability of fibrinogen assay was not affected significantly (y = -17.39+1.01x). From the edoxaban samples we also observed the similar change of the regression line (PT INR: y = 0.4728 + 0.5661x; APTT: y = -133.07+2.014x). Fibrinogen levels were comparable (y = -28.95+1.082x).Conclusion:The susceptibility of screening coagulation tests to the interfering effects of in vivo DOAC is dependent on the reagents and coagulometers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Performance Characteristics of DOAC-Remove for Neutralization of the Effects of Apixaban and Rivaroxaban in Lupus Anticoagulant Assays.

This study established the performance characteristics of DOAC-Remove for neutralization of the effects of rivaroxaban and apixaban in lupus anticoagulant (LAC) testing. Normal donor, LAC control, and patient samples were spiked with rivaroxaban or apixaban to simulate their effects on the dilute Russell's viper venom time (dRVVT), activated partial thromboplastin time (APTT), and dilute prothrombin time (dPT). Anti-Xa activity was measured after spiking and after DOAC-Remove neutralization. Accuracy, complex precision, and reference interval verification were evaluated. DOAC-Remove neutralized rivaroxaban and apixaban concentrations as high as 415 ng/mL and 333 ng/mL, respectively. Percentage positive and negative agreement between the baseline and postneutralization interpretations were 75% or higher for the dRVVT and APTT methods but not for the dPT method. Coefficients of variation (CVs) were 10% or less for all assays except the Staclot-LA delta, which had a standard deviation of 2.5 seconds or CV of 25% or less depending on the level. The laboratory's reference intervals were verified for the dRVVT and APTT assays after DOAC-Remove treatment but not for the dPT assays. DOAC-Remove appears to have acceptable performance characteristics for neutralizing the effects of rivaroxaban and apixaban in the dRVVT and APTT methods but not in the dPT method.

Laboratory Assays to Address the Problem of Heparin-Protamine Balance Following Neonatal Open Heart Surgery

Introduction: Neonates are especially susceptible to bleeding during and after cardiopulmonary bypass (CPB) surgery seemingly by virtue of a proportionately larger foreign surface exposure area for blood, significant hemodilution, and a coagulation system that differs from older patients. During CPB, heparin is administered to prevent thrombin formation. After CPB, it is reversed with protamine sulfate. Although administered to reverse anticoagulation, high levels of protamine sulfate paradoxically inhibit blood coagulation. This study evaluated the effect of heparin anticoagulation and protamine sulfate reversal on the accuracy of diagnostic coagulation tests and bleeding outcomes in a neonatal population undergoing CPB for congenital heart defects in the first 30 days of life.Methods: Blood samples were obtained at baseline (Draw 1), on CPB (Draw 2), following protamine reversal (Draw 3) and upon admission to the cardiac intensive care unit (Draw 4). Thrombin time (±protamine), FXa activity, activated partial thromboplastin time (APTT), Rotational Thromboelastometry (ROTEM) and Calibrated Automated Thrombography (CAT) were performed. Excessive bleeding was defined by meeting one or more of the following criteria: ≥ 84 mL/kg total chest tube output (CTO) in the first 24 hours, ≥ 7 mL/kg/hr CTO for ≥2 consecutive hours in the first 12 hours, or re-exploration in the first 24 hours for bleeding or tamponade.Results: This prospective observational study enrolled 44 neonates. Of these, 16 (36%) had excessive bleeding. The thrombin time assay was sensitive to heparin but was only minimally impacted by protamine sulfate, unless heparin was in the sample. Therefore, it was selected as the “gold-standard” assay for measurement of heparin. Relative heparin levels, determined by rate of fibrin formation, inversely correlated with thrombin generation measured using CAT, particularly in comparison to the thrombin generation rate in Draw 4 samples (exponential fit R2= 0.333). Higher plasma heparin concentration was present in 85% of the patients at Draw 4 than at Draw 3, indicating postoperative heparin rebound. This was important because, although the plasma heparin concentration in bleeders varied greatly, those patients with the two highest heparin concentrations at Draw 4 had excessive bleeding. The FXa activity assay was altered by protamine and by heparin. Therefore, samples with elevated FXa activity but normal thrombin time likely had high protamine. There were 4 such samples and 2 were bleeders. Additional coagulation assays were compared to the thrombin time assay to assess their performance for detection of heparin. ROTEM and APTT assays also correlated weakly (R2= 0.0531 with P value 0.1527 and R2 = 0.1549 with P value 0.0099, respectively) with the thrombin time assay at Draw 4. However, no correlation was present at Draw 3.Conclusions: Bleeding remains a common complication in neonates undergoing CPB for repair of heart defects. While there are many possible causes for the bleeding, excess heparin or protamine may contribute to its severity in a subset of cases. The simple to perform and inexpensive thrombin time test readily and accurately detects excess heparin. Increased use of this test may be beneficial, particularly for detection of heparin rebound that is observed in approximately 85% of patients. Excess protamine may also contribute to bleeding in these patients, and can be assessed using a combination of thrombin time and factor Xa activity assays. DisclosuresMast:Novo Nordisk: Research Funding.

New Insights into the Biological Activity of Lichens: Bioavailable Secondary Metabolites of Umbilicaria decussata as Potential Anticoagulants.

This study reports the in vitro anticoagulation activity of acetonic extract (AE) of 42 lichen species and the identification of potential bioavailable anticoagulant compounds from Umbilicaria decussata as a competent anticoagulant lichen species. Lichens' AEs were evaluated for their anticoagulant activity by monitoring activated partial thromboplastin time (APTT) and prothrombin time (PT) assays. A strong, positive correlation was observed between total phenolics concentration (TPC) of species and blood coagulation parameters. U. decussata was the only species with the longest clotting time in both APTT and PT assays. The research was moved forward by performing in vivo assays using rats. The results corroborated the dose-dependent impact of U. decussata's AE on rats' clotting time. Major secondary metabolites of U. decussata and their plasma-related bioavailability were also investigated using LC-ESI-MS/MS. Atranol, orsellinic acid, D-mannitol, lecanoric acid, and evernic acid were detected as possible bioavailable anticoagulants of U. decussata. Our findings suggest that U. decussata might be a potential anticoagulant lichen species that can be used for the prevention or treatment of coagulation-related issues such as cardiovascular diseases (CVDs).

ANTI-COAGULANT PROPERTIES OF FLAVONOID COMPOUNDS: POTENTIAL STRUCTURE-FUNCTIONAL RELATIONSHIP

Objective: Flavonoids, naturally-occurring compounds in fruits and vegetables, possess anti-coagulant property. However, a very few studies wereattempted to understand how flavonoid structure influences its anti-coagulation property, such as clotting time. In this study, we investigatedstructurally similar flavonoid compounds which differ in the number of hydroxyl groups and compared their anti-coagulation properties.Methods: We selected and evaluated five flavonoid compounds, that is, chrysin, apigenin, luteolin, kaempferol, and quercetin, for their anti-coagulantproperties using in vitro prothrombin time (PT) assays and activated partial thromboplastin time (APTT) assay.Results: Our findings suggested that quercetin, kaempferol, and luteolin showed a significant anti-coagulant effect on APTT (p<0.05) in a dosedependentmanner. The dose of 500 μM quercetin showed potent prolong APTT with 37.43±1.60 s, followed by 500 μM of kaempferol and luteolin(34.63±1.29 s and 4.83±1.56 s, respectively). Furthermore, a combination of 500 μM of quercetin with 0.25 U/ml of heparin demonstrated prolongAPTT (52.16±5.18 s) when compared with individual effects of either 0.25 U/ml heparin (33.4±0.50 s) or 500 μM quercetin (37.43±1.62 s) alone.Conclusion: Our results demonstrated that numbers of the hydroxyl group on flavonoid compounds influence anti-coagulation properties. Inaddition, the prolonged APTT assay results suggested that quercetin, kaempferol, and luteolin could affect factors VIII, IX, XI, and XII of intrinsicpathway. Moreover, the synergistic effect of quercetin further enhances the heparin anti-coagulation effect. Based on our findings, we recommendthat the consumption of vegetables and fruits rich in quercetin, luteolin, and kaempferol could help prevent thrombotic stroke in high-risk patients.

IN VITRO ANTIPLATELET AND ANTICOAGULANT ACTIVITY OF INDIGENOUS VEGETABLES FROM SOUTHERN THAILAND

Objective: Epidemiological studies have indicated that diets rich in fruits and vegetables help reduce the risk of cardiovascular diseases (CVDs).However, data about the antithrombotic activity of local vegetables is rare. The objective of this study was to evaluate antiplatelet and anticoagulantactivity in indigenous vegetables with high phenolic compounds collected from Southern Thailand.Methods: Five selected indigenous vegetables were crudely extracted by distilled water and 80% methanol. The extracts were screened for in vitroantiplatelet and anticoagulant activity at a concentration of 10 μg/μl. The antiplatelet activity was measured by inhibition of platelet adhesion tocollagen and thrombin-induced platelet aggregation, while the anticoagulant activity was assessed by the prothrombin time (PT) and activated partialthromboplastin time (APTT) tests.Results: Among the selected vegetables, the extracts of mon-pu (Glochidion perakense Hook.f.) and young cashew leaves (Anacardium occidentale L.)showed high antithrombotic properties. The highest antithrombotic activity was observed in the methanolic extract of mon-pu, which showed92.79±0.78% of platelet adhesion inhibition, 102.9±1.53% of platelet aggregation inhibition, and a prolonged APTT assay (48.92±0.94 s). Theprolonged APTT but normal PT results suggested that the extract could affect factors VIII, IX, XI, and XII of the intrinsic coagulation pathway.Conclusion: Our findings demonstrated antiplatelet and anticoagulation properties of indigenous vegetables from Southern Thailand. The multipotentialeffects of mon-pu extracts on antithrombosis evidently suggest that mon-pu can be considered as an excellent nutraceutical option in theprevention of thrombosis-related CVDs caused by different mechanisms.

Revisiting the effects of spectral interfering substances in optical end‐point coagulation assays

Hemolysis, icterus, and lipemia (HIL) are common pre-analytical variables in the clinical laboratory. Understanding their effects on coagulation laboratory results is essential. HIL effects on the prothrombin time (PT), activated partial thromboplastin time (APTT), dilute Russell's viper venom time (DRVVT), thrombin time (TT), and protein C chromogenic activity (CFx) were evaluated on the ACL TOP 750 optical analyzer and STA-R Evolution mechanical analyzer (PT and APTT only) by spiking normal donor, patient, and commercial control samples with varying concentrations of hemolysate, bilirubin, or a lipid emulsion. The relative difference or bias compared to the original results was determined. Hemolysis (H) indices up to 900mg/dL did not affect the APTT, PT, DRVVT Confirm, TT, and CFx; however, H indices above approximately 200mg/dL resulted in a false-negative DRVVT screen and screen/confirm ratio in samples with a lupus anticoagulant. There was an artifactual prolongation of the PT and APTT when conjugated bilirubin was dissolved in aqueous solvents and not when it was dissolved in dimethyl sulfoxide. Icterus (I) indices up to 45mg/dL did not result in significant (>15%) bias for all assays evaluated. The PT and APTT assays failed to produce a robust clot curve when the lipemia (L) index exceeded 6000 milliabsorbance units (mAbs), and the TT and DRVVT assays failed when the L index exceeded 3000 mAbs; the CFx assay was unaffected by lipemia. Verification of the manufacturer's recommended interference thresholds is important since it may avoid inappropriate instrument flagging and/ or sample rejection.

Elevated anti-human factor Xa activity in rabbit and rodent plasma: Implications for preclinical assessment of human factor X in animal models of hemostasis

A wide variety of animal models on thrombosis and hemostasis are used in thrombosis and hemostasis research for the preclinical assessment of hemostatic agents. While the vertebrate coagulome is highly conserved, human and animal plasmas differ considerably when evaluated in coagulation assays such as prothrombin time (PT), activated partial thromboplastin time (APTT), and calibrated automated thrombography (CAT). Here, we have aimed to provide a reference framework for the evaluation of coagulation assays and inhibition of activated human FXa (hFXa) in various animal plasmas. To do so, a side-by-side evaluation of the extrinsic and intrinsic pathway of coagulation was performed by means of PT, APTT, and CAT measurements on (diluted) pooled plasmas from goats, pigs, rabbits, rats, mice, and humans. Plasma anti-FXa activity was assessed by determining the rate of recombinant hFXa inhibition through chromogenic activity analyses and immunoblotting. In general, rabbit, rat, and mouse plasmas exhibited robust clotting upon stimulation of both the extrinsic and intrinsic pathway, produced more thrombin during CAT upon plasma dilution, and displayed relatively high hFXa inhibitory activities. By comparison, goat, porcine, and human plasma displayed a similar profile in PT and APTT assays, produced less thrombin during CAT upon plasma dilution, and displayed comparable hFXa inhibitory activities. In conclusion, the observed differences in clotting parameters and anti-hFXa activity point to a higher anticoagulant threshold in plasma from rabbits, rats, and particularly in mice relative to human, goat, and porcine plasma. Finally, rat plasma was found to be more relevant to the preclinical assessment of human FX(a) in comparison to murine plasma.

Pharmacopeia Potency Equated Sulodexide Produces Comparable Anticoagulation as Studied in Whole Blood

IntroductionSulodexide is a widely used glycosaminoglycan derived drug for oral (antithrombotic) and parenteral (anticoagulant) indications and is composed of fast moving heparin (80%) and dermatan sulfate (20%). In view of the current shortage of porcine heparin supply sulodexide may be a potential substitute for heparin for surgical and interventional anticoagulation. The purpose of this study is to compare the anticoagulant activities of sulodexide with heparin in simulated studies to mimic surgical and interventional anticoagulation.Materials and MethodsPowder versions of sulodexide were obtained from Alfasigma (Bologna, Italy). Powder versions of porcine mucosal heparin were obtained from Medefil (Glendale Heights, IL, USA). Stock solution of both heparin and sulodexide were prepared in sterile saline at 10mg/ml. Sterile solutions were made at 1.0 and 0.1mg/ml. USP potency for both drugs were measured, cross referenced against US pharmacopiel standard and expressed as units per milligram. Whole blood activated clotting time was measured in healthy human volunteers at graded concentrations of both drugs. Plasma based anticoagulant assays such as activated partial thromboplastin time (APTT) and thrombin time (TT) were measured. Protamine neutralization studies were also carried out in the different assays used.ResultsThe USP potency of sulodexide range from 90 – 105 U/mg whereas the porcine heparin exhibited a potency range of 180 – 200 U/mg as measured by the anti‐Xa activity. In the ACT assays USP potency adjusted sulodexide and heparin produced concentration dependent prolongation of this test which were comparable to heparin. Both the sulodexide and heparin produce concentration dependent anticoagulant effects in the APTT and TT assays. Protamine sulfate produced effective neutralization of the anticoagulant effects of both the sulodexide and heparin.ConclusionsThese results suggest that at USP potency equated levels sulodexide is capable of producing similar anticoagulant effects as unfractionated heparin. The USP potency of sulodexide can be adjusted at a comparable potency to porcine heparin to obtain comparable anticoagulant responses in the in vitro setting. Additional in vivo studies are warranted to compare the potency adjusted sulodexide with heparin.

Anticoagulant Heparin Mimetics via RAFT Polymerization.

Heparin, a sulfated polysaccharide derived from animal sources, is the most commonly used parenteral anticoagulant drug, but it suffers from significant safety and supply issues. Herein, we describe the preparation of heparin mimetic homo- and copolymers via the reversible addition-fragmentation chain transfer (RAFT) polymerization in water of commercially available (non-carbohydrate) sulfonated and carboxylated monomers. The anticoagulant activities of the polymers were assessed by activated partial thromboplastin time (APTT), thrombin clotting time (TCT), and for the more promising polymers, thrombin generation, antifactor Xa, and antifactor IIa assays. Sulfonated homopolymers studied herein displayed low cytotoxicity and significant anticoagulant activity in APTT, TCT, and thrombin generation assays. In addition, copolymers of sodium styrenesulfonate and acrylic acid [poly(SSS-co-AA)] displayed unprecedented antifactor IIa activity. This study demonstrates the potential of RAFT polymers as alternative anticoagulants for biomedical applications.

The accuracy of a point of care measurement of activated partial thromboplastin time in intensive care patients

Point of care (POC) devices are increasingly being used in intensive care units to obtain faster results. Data are limited on the performance of these devices in critically ill patients, especially those on heparin infusion. The objective of this study was to assess the agreement between POC activated partial thromboplastin time (APTT) and laboratory APTT results in patients on heparin infusion and to determine its impact on the clinical decisions regarding heparin dosage. We screened all patients admitted to the intensive care unit (ICU) at St George Hospital, Sydney, over a 7-month period and enrolled those who were receiving intravenous heparin infusion. We measured APTT by two methods: bedside POC test (Hemochron Junior Signature Plus) and central laboratory method (STA analyser). We used the Bland-Altman method to test the statistical agreement between the two measurements and Cohen's kappa statistic to test the clinical agreement regarding heparin dosing decision. A total of 176 paired samples from 44 patients (mean age 63 years, 64% males, mean APACHE 18) were analysed. The mean turnaround time for the point of care APTT result was significantly shorter than the central laboratory result (5.0±0.2 min vs 64.6±2.7 min, p<0.0001). Despite the statistically significant correlation, the overall agreement tested by the Bland-Altman method was poor. The 95% limits of agreement were widest (-27.266 to 64.791) and mean percentage bias was highest (24%) for the comparison between POC APTT using citrated blood and laboratory APTT. When POC APTT results of less than 90 seconds using whole blood were compared to laboratory APTT results, the limits of the agreement became narrower (-23.243 to 28.419), and the mean percentage bias decreased to 5%. The agreement between clinical decisions regarding heparin dosage based on the two methods was poor for plain and citrated blood (kappa 0.35 and 0.11, respectively). The POC APTT results were not sufficiently accurate for use in patients on heparin infusion compared to laboratory APTT assay.

Anti-Factor Xa–Based Monitoring of Unfractionated Heparin: Clinical Outcomes in a Pediatric Cohort

To assess clinical outcomes in children treated with unfractionated heparin and monitored using an anti-factor Xa (Anti-FXa)-based nomogram. We also sought to assess the correlation between activated partial thromboplastin time (APTT) and Anti-FXa. This was a single-center, observational cohort study conducted over a 20-month period that included all pediatric patients (<21years) who received therapeutic unfractionated heparin and were monitored using an anti-FXa-based nomogram. In total, 95 patients met prespecified inclusion criteria, and 1098 pairs of APTT and Anti-FXa measurements were performed. The median unfractionated heparin dose required to reach therapeutic Anti-FXa goal was significantly greater in infants compared with older children (P<.0001). The median time to achieve therapeutic Anti-FXa was 10hours (range 2-96hours) and was significantly shorter in patients who received a bolus compared with those who did not (P = .03). Five (5.3%) major bleeding events were noted. Age, peak Anti-FXa, peak APTT, lowest platelet count, and fibrinogen were not predictive of major and clinically relevant nonmajor bleeds. Moderate correlation between the APTT and Anti-FXa (r=0.75; 95% CI 0.72-0.77) assays was appreciated. Using an anti-FXa-based nomogram to monitor unfractionated heparin in children is feasible. Although moderate correlation was observed between the APTT and Anti-FXa assays, the APTT frequently overestimated heparin activity. Safety and efficacy of an Anti-FXa nomogram needs further validation.

Toxicological Evaluation of Bioactive Sulfated Polysaccharides from the Edible Marine Algae Padina tetrastromatica and Ulva fasciata in Sprague-Dawley Rats

The bioactivities of edible brown algae Padina tetrastromatica and green algae Ulva fasciata are mainly attributed to the presence of sulfated polysaccharides (SPSs). The toxicological evaluation of a particular dose of a compound is necessary before practicing its pharmacological activities. In the absence of detailed in vivo toxicological studies of the sulfated polysaccharides from the above mentioned edible marine algae, the present study focused on the toxicological evaluation of these compounds in adult male Sprague-Dawley rats through oral administration at a dose of 50 mg/Kg Body weight for 45 days. The minimum effective dose was fixed from the preliminary dose fixation studies as per OECD test guideline 420. The compounds were initially identified and proved to be non-toxic using in vitro and in vivo studies. The compounds did not induce statistically significant toxicological changes with respect to relative organ weight, hematology, serum toxicity parameters, and histopathology. Both compounds significantly increased the antioxidant status of hepatic tissue and coagulation time in APTT (Activated partial thromboplastin time) assay with no significant change in prothrombin time (PT), indicating their heparin-like activities through the intrinsic pathway. Our study demonstrated that in this particular dose, both these natural compounds are non-toxic to the vital organs and can exhibit potent antioxidant and anticoagulant activities without producing life-threatening side effects.