aPTT Reagents Research Articles

Strategies for Performing Factor Assays in the Presence of Emicizumab or Other Novel/Emerging Hemostatic Agents.

For several decades, therapeutic options for inherited deficiencies of factor VIII or IX (hemophilia A or B, respectively) have largely been the replacement of the missing clotting factor with plasma-derived or recombinant products. Hemostasis laboratories use standard activated partial thromboplastin time (aPTT)-based clotting or chromogenic assays to monitor plasma factor levels to guide therapy. The emergence in the past 10 years of extended half-life replacement products and other novel therapies for hemophilia has led to a reappraisal of assay suitability, with studies of product measurement showing some existing assay types or reagents to be unsuitable for some products. The hemostasis laboratory must adapt to the changing landscape by adding new assays or modifying existing assays to ensure accurate results for product measurement. These strategies include switching from a chromogenic assay to a clotting assay, or vice versa, changing an aPTT reagent brand, or introducing product specific calibrators. This article evaluates the effects of some of the newer treatment options on the laboratory testing of factor levels and related assays.

Synthesis, Characterization, DFT Calculations and Anticoagulant Activity of Symmetrical and Unsymmetrical N-Aryl Thiourea Derivatives

The present study reports a methodology for the synthesis of symmetrical and unsymmetrical thiourea derivatives 1-9, which have been prepared in good overall yields from condensation reactions between phenylisothiocyanate and different primary amines. The structures of all products were confirmed by the spectroscopic analysis (UV, IR, and NMR). These derivatives were tested for anticoagulant activity using PT and APTT reagents, and those reagents are considered also a controller, the results showed that the theN-aryl thiourea derivatives have very high anticoagulant activity. Theoretical calculations based on DFT/B3LYP method have been performed in order to get insights into the molecular geometry and chemical reactivity of the studied compounds.

Impact of Anticoagulant Function By the Addition of Heparinoid in Vitro in the Plasmas from Patients with Hemophilia and Von Willebrand Disease

Background: Heparinoid (Hirudoid ®) is a local anticoagulant preparation as skin application that relieves pain and inflammation with addition to promoting healing in superficial thrombophlebitis and bruising. Heparinoid is mainly composed of chondroitin sulfate and has high moisturizing ability due to the large number of hydrophilic group. Thus, it is used as topical treatments for sebum deficiency in Japan. Since heparinoid has a structure similar to heparin, the package insert states the contraindication for hemorrhagic diseases such as hemophilia and von Willebrand disease (VWD) in Japan. Hemophilia A and B (HA/HB) are inherited bleeding disorders caused by a defect in factor (F) VIII and FIX coagulant protein, respectively. Based on its activity levels, the clinical severity is classified as severe (<1 IU/dL), moderate (1-5 IU/dL), and mild (>5-40 IU/dL). VWD is the common congenital bleeding disorders and is caused by a deficiency of von Willebrand factor (VWF). VWD is classified into partial deficiency (type 1), qualitative deficiency (type 2), and complete quantitative deficiency (type 3) of VWF, based on the VWF antigen (VWF:Ag) and ristocetin cofactor activity (VWF:RCo) level. In the clinical practice, there are cases of hemorrhagic patients using heparinoid at the discretion of their families. However, the influence of heparinoid on coagulant function in human subjects remains to be investigated, and it is unclear if the patients with hemophilia and VWD can use heparinoid safely. Aim: To examine the impact of anticoagulant function of heparinoid in vitro in the plasmas from patients with hemophilia and VWD. Methods: The maximum blood concentration from absorbed heparinoid was estimated to be 2.5 µg/mL, based on the previous report, when heparinoid was applied to the skin as stated in package insert. Heparinoid (0-6.5 µg/mL) or unfractionated heparin (0-0.6 IU/mL) were added to normal plasma (NP), followed by measuring activated partial thromboplastin time (aPTT) by one-stage clotting assay. A modified clot waveform analysis (CWA) using an aPTT reagent were performed on the CS-2400™ (Sysmex), followed by measuring clotting time (CT) and adjusted maximum coagulation velocity (Ad|min1|). Heparinoid was also added to the patients' plasmas with HA, HB, or VWD, followed by CWA assessment. The CT and Ad|min1| of 20 healthy individuals were 31.1±0.1 sec and 6.90±0.05 (median±SEM). Results: aPTT and CT values in NP by the spike of heparinoid were prolonged and Ad|min1| was decreased dose-dependently. The addition of 2.5 µg/mL heparinoid showed that aPTT (34.3 sec; 1.2-fold of control (no heparinoid)), CT (36.3 sec; 1.2-fold of control), and Ad|min1| (9.5; 0.96-fold of control) were comparable to the effect of ~0.2 IU/mL heparin, suggesting that heparinoid exhibited slight anticoagulant effect for NP. To examine the effect of heparinoid on HA/HB plasmas, recombinant (r) FVIII or rFIX (0-50 IU/dL) were added to FVIII- or FIX-deficient plasmas (3 lots each) together with heparinoid (0-6.3 µg/mL), respectively. The addition of heparinoid had prolonged the CT and decreased the Ad|min1| at each concentration of FVIII and FIX dose-dependently. The addition of 2.5 µg/mL heparinoid showed modest anticoagulant effect for HA and HB at each concentration of FVIII and FIX (CT; 1.3- and 1.5-fold of control, Ad|min1|; 0.82- and 0.87-fold of control). Next, the effect of heparinoid on the plasma from one VWD patient each with type 1 (VWF:Ag/VWF:RCo 28 IU/dL/26 IU/dL), type 2 (20 IU/dL/9 IU/dL), and type 3 (<2 IU/dL/<3 IU/dL) was assessed. The addition of 2.5 µg/mL heparinoid showed slight anticoagulant effect for each type of VWD (CT; ~1.3-fold of control, Ad|min1|; ~0.91-fold of control). Conclusion: The presence of heparinoid demonstrated mild anticoagulant effect in vitro in the plasmas of patients with hemophilia and VWD. It might be possible to apply the heparinoid-contained product as skin treatment for these patients.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N=35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.

Agreement between one stage and chromogenic assays in samples from patients receiving recombinant porcine FVIII (Obizur, Susoctocog-alfa).

Recombinant porcine FVIII (rpFVIII) (Obizur, Susoctcog-alfa, Takeda, Japan) is licensed for the treatment of bleeding in acquired Haemophilia A (AHA). The summary of product characteristics state that monitoring should be by one stage assay (OSA) rather than chromogenic assay (CSA). CSA have been shown to underestimate activity when rpFVIII is added to plasma in vitro. Samples from three AHA patients (n = 21) (pre- and post rpFVIII) were assessed using FVIII:C assays; OSA methods: Actin, Actin FS, Actin FSL and Pathromtin SL performed on CS5100i (Sysmex, Kobe, Japan); APTT-SP, SynthASil and SynthAFax performed on ACL TOP (Werfen, Barcelona, Spain). CSA methods on CS5100i: Siemens Chromogenic Assay, Biophen FVIII:C, Technochrom FVIII:C; on ACL TOP: Rox Factor VIII, Coamatic Factor VIII and CRYOcheck Factor VIII. OSA and CSA varied according to reagent used median OSA 61 IU/dL (range 41.5-81 IU/dL (ANOVA p < 0.0001)) median CSA 46.5 IU/dL (range of method specific medians 36.5-84 IU/dL (ANOVA p < 0.0001)). Amongst OSA, Actin FS was associated with the highest FVIII:C, APTT-SP was associated with the lowest. Variation in CSA results by different methods was also seen with highest FVIII:C levels obtained using the Technochrom FVIII:C and the lowest levels obtained with Siemens Assay. The relationship between OSA and CSA was not consistent between method or patient. Previously there has been reports of underestimation by CSA in in vitro spiked samples. Investigation into concentration of phospholipids in the APTT reagents may explain some of these variations.

A performance evaluation of sthemO 301 coagulation analyzer and associated reagents.

The study objective was to evaluate the performance of sthemO 301 system and to compare it with the analyzer used in our university hospital laboratory (STA R Max® 2), for a selection of hemostasis parameters. Method comparison (according to CLSI EP09-A3), carryover (according to CLSI H57-A), APTT sensitivity to heparin (according to CLSI H47-A2), HIL level assessment, and productivity were performed using leftover samples from our laboratory (n > 1000). Commercial quality control materials were used to evaluate precision (according to CLSI EP15-A3) and accuracy. The assays tested on sthemO 301 were: PT, APTT (silica and kaolin activators), fibrinogen (Fib), thrombin time (TT), chromogenic and clotting protein C (PC) activity, and von Willebrand factor antigen (VWF:Ag) levels. All intra-assay and inter-assay precision CVs were below the maximal precision limit proposed by the French Group for Hemostasis and Thrombosis (GFHT). Accuracy was verified with bias below GFHT criteria and most Z-scores were between -2 and +2. No clinically relevant carryover was detected. Silica APTT reagent sensitivity to unfractionated heparin was moderate, as expected. Productivity results were consistent over the 10 repeats performed. The overall agreement between the two systems was excellent for all assays, with Spearman rank correlation coefficient all above 0.9 and slopes of Passing-Bablok correlation near 1 and intercepts close to 0. For the methods tested, sthemO 301 system met all the criteria to implement a novel coagulation analyzer in the laboratory and result comparability with STA R Max® 2 was good.

FIBRINOLYTIC POTENTIAL INCREASING DURING ACTIVATION OF BLOOD COAGULATION IN THE COURSE OF PREGNANCY WITH PLACENTAL DYSFUNCTION

Aim. The study of molecular mechanisms of hemostasis balance is one of the most vivid tasks for clinical biochemistry. In present communication we aimed to underline the constant connection between blood coagulation and fibrinolysis. Methods. Blood coagulation activation was estimated by the soluble fibrin accumulation. For it determination we used the sandwich ELISA method. As the catch-antibody we used fibrin-specific mAb FnI-3C. As the tag-antibody we used another mAb (II-4d) that has an epitope in the NH 2 -terminal fragment of the γ-chain of the D-region of the fibrin(ogen) molecule. The rate of activation of fibrinolysis was estimated by measuring of Fibrinolytic Potential (FP). It was measured by turbidimetric method with recording the scattering of light by a fibrin clot at 405 nm on a microplate reader Multiscan (Finland). The clot was formed in the microplate wells in blood plasma activated by APTT reagent in the presence or without t-PA. Results. SF was found in blood plasma of 12 pregnant women with placenta dysfunction. Six of studied patients had SF less than 4 µg/ml that were assumed as the control meanings. We divided patients on two groups according to this parameter. It was shown that patients of the 1st group (SF ≤ 4) exhibited FP as 24 ou/s. In the same time patients of the 2nd group (SF ≥ 4) had much higher FP – 62 ou/s. The level of statistical significance was P = 0,05. Conclusions. Blood coagulation activation (estimated by SF measurement) was shown to be accompanied by fibrinolysis activity increasing (measured by FP evaluation) in pregnant women with placental dysfunctions. These findings can be evidence of constant balance between blood coagulation and fibrinolysis that stabilize hemostasis in pathological conditions for avoiding thrombosis or hemorrhages.

DETERMINATION OF THROMBIN AND PLASMIN ACTIVITY IN HUMAN BLOOD PLASMA USING THE TURBIDIMETRIC CURVE OF CLOT FORMATION AND DISSOLUTION

The aim of the study was to develop a method for determination the activity of thrombin, which is based on the turbidimetry curve of the formation and dissolution of a blood plasma clot. Methods. Donor blood samples were collected in 3.8% sodium citrate (1 part of sodium citrate and 9 parts of blood, pH 7.4). Plasma was separated from blood cells within 1 hour after blood collection by centrifugation the latter at 1200 g for 20 minutes. Aliquots of plasma were stored at -20 °C. Results. To determine the concentrations of thrombin and plasmin, TDCs of the formation and dissolution of blood plasma clots, initiated by the APTT reagent, were used. Based on the values of τ obtained, a calibration curve was constructed in the coordinates 1/τ – [Thr] (the rate of protofibrils formation in s-1 vs thrombin concentration in NIH units in 1 ml). Conclusion. The proposed methods to determine the activity of thrombin and plasmin made it possible to quantitatively calculate the rate of prothrombin activation in the lag period, the concentration and activity of thrombin based on the rate of fibrin and protofibrils formation as well as the activity and concentration of plasmin at the point of the complete clot dissolution,

POSTER ABSTRACTS

POSTER ABSTRACTS

The combination of emicizumab and recombinant factor VIII in plasma: Which assays can we use for accurate measurement?

The bispecific antibody, emicizumab, is a prophylactic therapy used for the treatment of haemophilia A (HA). Patients may require additional replacement factor VIII (FVIII) to ensure adequate haemostasis. This study investigated the laboratory measurement of severe HA (SHA) plasma spiked with 36 combinations of emicizumab plus recombinant (r) FVIII concentrates. FVIII assays were performed by one stage assay (OSA) using eight APTT reagents from three manufacturers and chromogenic assays (CSA) using seven kits. CSA kits comprised a range of bovine FX/FIXa, bovine FX/human FIXa or human FX/FIXa. Thrombin generation (TG) was assessed by CAT and ST-Genesia. Emicizumab-calibrated modified OSA and human FX CSA both overestimated rFVIII in the presence of emicizumab; median FVIII:C of up to 89% higher was observed in plasma spiked with both drugs compared to just rFVIII. In bovine FX CSA assays, there was a FVIII:C increase of up to 11% in plasmas spiked with both drugs compared to rFVIII alone. TG parameters were not all normalized by the presence of emicizumab however addition of rFVIII increased TG. ETP and peak thrombin were normalized at 50 μg/ml emicizumab using ST-Genesia but were still reduced at 75 μg/ml with CAT. Addition of rFVIII further normalized results. Modified OSA and human FX CSA could not distinguish between rFVIII or emicizumab. The presence of both emicizumab and rFVIII increased thrombin generation to normal levels compared to each drug alone. Bovine FX CSA can be used to accurately determine FVIII activity of rFVIII in plasma which also contains emicizumab.

Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments.

Accurate measurement of clotting factors VIII (FVIII) or IX (FIX) is vital for comprehensive diagnosis and management of patients with hemophilia A or B. The one-stage activated partial thromboplastin time (aPTT)-based clotting assay is the most commonly used method worldwide for testing FVIII or FIX activities. Alternatively, FVIII and FIX chromogenic substrate assays, which assess the activation of factor X, are available in some specialized laboratories. The choice of reagent or methodology can strongly influence the resulting activity. Variation between one-stage FVIII or FIX activities has been reported in the measurement of some standard and extended half-life factor replacement therapies and gene therapy for hemophilia B using different aPTT reagents. Discrepancy between one-stage and chromogenic reagents has been demonstrated in some patients with mild hemophilia A or B, the measurement of some standard and extended half-life factor replacement therapies, and the transgene expression of hemophilia A and B patients who have received gene therapy. Finally, the measurement of bispecific antibody therapy in patients with hemophilia A has highlighted differences between chromogenic assays. It is imperative that hemostasis laboratories evaluate how suitable their routine assays are for the accurate measurement of the various hemophilia treatment therapies.

New CMC Algorithm for Evaluation of Lupus Anticoagulant

Introduction: Lupus Anticoagulant (LA) is an autoantibody that in vitro inhibit phospholipid-dependent coagulation reactions in the absence of specific coagulation factor inhibition Primary laboratory criteria for LA detection include prolonged clotting time of a phospholipid dependent test, failure of correction on mixing study, shortening of the clotting time in the presence of high phospholipid concentrations & absence of other inhibitors. (1) According to guidelines,for diagnosing LA two tests based on different principles should be used- dRVVT(Dilute Russel's Viper Venom Time) should be the first test considered. Second test should be a sensitive aPTT (Activate Partial Thromboplastin Time).LA is a multistep procedure with screening, mixing and confirmation tests. Devreese et al recently concluded that integration of screening and confirmation in a single procedure and skipping the mixing tests result in a poor diagnostic efficacy for LA (2).LA is an in-vitro inhibitor and elevated Screening test without mixing studies does not prove inhibitor effect & it wastes time & resources to perform confirmatory tests skipping the mixing. Hence, we devised a new algorithm- Christian Medical College (CMC) Algorithm as shown in figure-1 which starts with DRVVT Screen Mix. To identify interferences due to anticoagulants like heparin and fibrin degradation products (FDP) thrombin time (TT) was also included. If Screen Mix is normal APTT Screen Mix was performed. Aim of our new algorithm was to reduce false positives and avoid interferences in LA Materials and methods: Four hundred and ninety-one (491) samples were analysed retrospectively for a period of 5 months from January 2022 to May 2022. The new algorithm was incorporated into STA R Max2 (Diagnostica Stago) analyser and automatic calculation of ratios and interpretation of results were done based on flow charts. LA testing was performed using DRVVT screen and confirm reagents (STA-Staclot DRVV Screen and STA-Staclot DRVV Confirm) and/or APTT reagent (STA-PTT Synthasil). LA test was considered positive when Screen/ Confirm(S/C) ratio was >1.2. We compared this result to the three hundred forty seven(347) samples, over a period of 4 months(September 2021 - December 2021) analyzed in old algorithm where DRVVT screen & APTT were done as the initial steps instead of DRVVT mixing studies as the 1st step. Results: In the new algorithm result were classified into 7 categories. Category 1 had normal DRVVT screen Mix and normal APTT; these represent true negative cases comprising 68.8 % of the cases. Category 2 had normal DRVVT screen Mix, prolonged APTT Screen Mix with normal TT. 1 case in this category had other Antiphosholipid Antibody (APLA) negative & repeat testing for LA was advised. Category 3 had normal DRVVT Screen mix, prolonged APTT Screen Mix with prolonged TT. There was 1 case in this category, who was on heparin, APLA antibody was negative. Repeat testing was advised at a later date. Category 4 had prolonged DRVVT Screen Mix, normal S/C ratio , with normal TT. There were 61 cases (12%) in this category among which 5 cases had normal DRVVT Screen with prolonged Mix Category 5 had Prolonged DRVVT Screen Mix, with normal S/C Ratio, prolonged TT and this category had 13 (2 %) cases. On further evaluation they were found to be on anticoagulants. Category 6 had prolonged S/C Ratio with normal TT, which are the true positive cases, n=67 (13.6%). There were 4 cases where Screen mix was more prolonged than Screen, and Screen fell below the cut off & could have been missed if mixing step was not done Category 7 had prolonged S/C ratio with prolonged TT. There were 10 cases (2%) in this category. On further evaluation 6 patients had history of anticoagulants and 2 had features of DIC. These 8 patients could represent false positive LA and would have been misclassified if TT was not done. Old algorithm had 20 % positive cases & 80 % negative cases. Since the algorithm were incorporated into STA R Max2 hands on time decreased from 240 minutes to 80 minutes. Cost of the reagents were similar by 2 algorithms. Conclusion: The new algorithm gives better clarity in classification of patients into true positive and true negative & detection of interference due to heparin as thrombin time is also done in parallel. Possible false positive cases could be identified at the onset even before repeat confirmatory lupus testing. This study concludes the importance of mixing studies in avoiding false negatives. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Discrepancy between the Results of One-Stage and Chromogenic Factor VIII Assays: Switch in Diagnosis from Mild to Moderate Hemophilia a in Three Cases

Introduction Factor VIII (FVIII) activity can be measured with different methods including one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Some persons with mild hemophilia A (HA) have a significant discrepancy between the assays which has been previously reported. Discrepancy between these two methods, albeit less frequent, can also lead to a switch in diagnosis from mild to moderate HA. Herein, we report 3 persons with HA (PwHA) whose diagnosis changed from mild to moderate HA after obtaining FVIII levels assessed by CSA. Materials and Methods Data were extracted from the medical records of the PwHA including demographics, bleeding rates, and factor levels. FVIII activity was measured using the OSA on an ACL Top 500 with SynthaSil aPTT reagent (Instrumentation Laboratories Bedford, MA, USA). Factor VIII activity was also determined photometrically via the Chromogenix Coatest® SP4 FVIII chromogenic assay kit (bovine chromogenic FVIII activity, Diapharma Group, West Chester, OH). Results Data were collected from 3 PwHA who were previously diagnosed with mild HA yet were noted to have a bleeding phenotype worse than expected for mild HA (Table-1). Case 1 is 26 years old and had been experiencing multiple joint bleeds. His OSA FVIII level was 6.8%. He was on clotting factor concentrate (CFC) on demand. After having severe hemophilic arthropathy affecting his social life, prophylaxis with CFC was commenced. Significant improvement was observed after the initiation of prophylaxis. His CSA FVIII level was 2.7%, consistent with his bleeding phenotype, and he has been on emicizumab prophylaxis for a year without any bleeding events. Case 2 is 22 years old and had been experiencing frequent muscle bleeds. His OSA FVIII level was 6.1% and CSA FVIII level was 2.7%. He remains on CFC on demand based on his preference despite the frequent bleeding. Case 3 is 15 years old and was diagnosed with mild HA at the age of 8 after recurrent joint bleeds. His OSA FVIII level was 13.8% and CSA FVIII level was 1.5%. He has been on emicizumab prophylaxis for 10 months without any bleeding event. Conclusion While discrepancies between assays in mild hemophilia have been widely reported, there are fewer reports on an assay discrepancy changing a diagnosis from mild to moderate HA. As such, we strongly suggest performing both OSA and CSA for the diagnosis and classification of HA especially if the PwHA has more bleeding episodes than expected. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Lupus anticoagulant associates with thrombosis in patients with COVID‐19 admitted to intensive care units: A retrospective cohort study

BackgroundThrombosis is a frequent and severe complication in patients with coronavirus disease 2019 (COVID‐19) admitted to the intensive care unit (ICU). Lupus anticoagulant (LA) is a strong acquired risk factor for thrombosis in various diseases and is frequently observed in patients with COVID‐19. Whether LA is associated with thrombosis in patients with severe COVID‐19 is currently unclear. ObjectiveTo investigate if LA is associated with thrombosis in critically ill patients with COVID‐19. Patients/MethodsThe presence of LA and other antiphospholipid antibodies was assessed in patients with COVID‐19 admitted to the ICU. LA was determined with dilute Russell's viper venom time (dRVVT) and LA‐sensitive activated partial thromboplastin time (aPTT) reagents. ResultsOf 169 patients with COVID‐19, 116 (69%) tested positive for at least one antiphospholipid antibody upon admission to the ICU. Forty (24%) patients tested positive for LA; of whom 29 (17%) tested positive with a dRVVT, 19 (11%) tested positive with an LA‐sensitive aPTT, and 8 (5%) tested positive on both tests. Fifty‐eight (34%) patients developed thrombosis after ICU admission. The odds ratio (OR) for thrombosis in patients with LA based on a dRVVT was 2.5 (95% confidence interval [CI], 1.1–5.7), which increased to 4.5 (95% CI, 1.4–14.3) in patients at or below the median age in this study (64 years). LA positivity based on a dRVVT or LA‐sensitive aPTT was only associated with thrombosis in patients aged less than 65 years (OR, 3.8; 95% CI, 1.3–11.4) and disappeared after adjustment for C‐reactive protein. ConclusionLupus anticoagulant on admission is strongly associated with thrombosis in critically ill patients with COVID‐19, especially in patients aged less than 65 years.

Novel salivary antihemostatic activities of long-form D7 proteins from the malaria vector Anopheles gambiae facilitate hematophagy

To successfully feed on blood, hematophagous arthropods must combat the host's natural hemostatic and inflammatory responses. Salivary proteins of blood-feeding insects such as mosquitoes contain compounds that inhibit these common host defenses against blood loss, including vasoconstriction, platelet aggregation, blood clotting, pain, and itching. The D7 proteins are some of the most abundantly expressed proteins in female mosquito salivary glands and have been implicated in inhibiting host hemostatic and inflammatory responses. Anopheles gambiae, the primary vector of malaria, expresses three D7 long-form and five D7 short-form proteins. Previous studies have characterized the AngaD7 short-forms, but the D7 long-form proteins have not yet been characterized in detail. Here, we characterized the A. gambiae D7 long-forms by first determining their binding kinetics to hemostatic agonists such as leukotrienes and serotonin, which are potent activators of vasoconstriction, edema formation, and postcapillary venule leakage, followed by ex vivo functional assays. We found that AngaD7L1 binds leukotriene C4 and thromboxane A2 analog U-46619; AngaD7L2 weakly binds leukotrienes B4 and D4; and AngaD7L3 binds serotonin. Subsequent functional assays confirmed AngaD7L1 inhibits U-46619-induced platelet aggregation and vasoconstriction, and AngaD7L3 inhibits serotonin-induced platelet aggregation and vasoconstriction. It is therefore possible that AngaD7L proteins counteract host hemostasis by scavenging these mediators. Finally, we demonstrate that AngaD7L2 had a dose-dependent anticoagulant effect via the intrinsic coagulation pathway by interacting with factors XII, XIIa, and XI. The uncovering of these interactions in the present study will be essential for comprehensive understanding of the vector-host biochemical interface.

Coagulation assay discrepancies in Japanese patients with non-severe hemophilia A.

Patients with non-severe hemophilia A often show discrepancies in factor VIII (FVIII) activity. However, information on variant-specific coagulation assay characteristics in Japanese patients is limited. Pathogenic variants were classified into three groups, thrombin-cleavage site (TC), A1-A2-A3 interface (IF), and non-discrepant, with reference to previous studies. Cutoff values for the one-stage assay (OSA)/chromogenic substrate assay (CSA) ratio, which is suitable for distinguishing discrepancies, were determined for all five aPTT reagents. TGA and CWA parameters and bleeding scores were compared between groups. Two of the 39 patients with non-severe hemophilia A (5%) were classified as TC, 10 (26%) as IF, and 27 (69%) as non-discrepant. The OSA/CSA cutoff values between the groups varied widely by aPTT reagent and tended to be relatively low compared to previous studies. As an indicator of bleeding tendency, TGA had a low correlation coefficient for the IF variant, but this was not significant and was comparable to FVIII activity and CWA. Moreover, various parameters and bleeding tendency differed among patients with the same variants. Thus, our findings suggest that it is difficult to adequately assess the bleeding tendency of individual patients, even with the various assessments currently available.

Comparison of the sensitivity of commercial aPTT tests in measurement of the pharmacodynamic response of milvexian, a novel FXIa inhibitor

Abstract Introduction The activated partial thromboplastin time (aPTT) assay is a test routinely used to evaluate abnormalities or deficiencies in coagulation factors of the intrinsic and common pathways. The composition of the surface activator and phospholipids in the aPTT reagent is known to influence the response of heparin and direct FXa or thrombin inhibitors. Milvexian (formerly referred to as BMS-986177/JNJ-70033093) is an investigational small-molecule Factor XIa (FXIa) inhibitor being studied for the prevention and treatment of major thrombotic conditions. Clinical pharmacology evaluation of milvexian includes aPTT as the primary pharmacodynamic assay, thus underscoring the need for a sensitive reagent to evaluate its anticoagulant activity. Purpose This study evaluated the sensitivity of six commercially available aPTT reagents in plasma from individual donors spiked with milvexian, at concentrations spanning the anticipated clinically relevant exposures. Methods Platelet-poor plasma (PPP) prepared from citrated whole blood collected from consenting, healthy adult volunteers (n=12) was spiked with vehicle (DMSO, 0.5% v/v) or 0.1–10 μM milvexian. The aPTT was measured using six commercially available aPTT reagent kits which included silica; kaolin, or ellagic acid as the contact activator, in combination with natural or synthetic phospholipids. The coagulation automated analysers used for testing were matched to the kit's manufacturer recommendations. All reagents and respective normal or abnormal controls were prepared as instructed by the manufacturer. Results Milvexian exhibited dose-dependent prolongation of aPTT with all reagents tested. Assays performed with aPTT reagents containing kaolin or ellagic acid demonstrated the highest sensitivity, as measured by the concentration that achieved a 2-fold aPTT prolongation (EC2x), and showed the widest dynamic range of response. Coefficient of variability of aPTT measurements in plasma from 12 individual donors was between 5.6–7.9% for Dade® Actin® FS, and 7.1–8% for STA®-C.K. Prest® 5. Conclusions Prolongation of aPTT in PPP spiked with milvexian exhibited a dose-dependent relationship, with statistically significant differences observed among reagents at milvexian concentrations above 0.3 μM. The highest sensitivity measured by changes in the ratio to baseline was obtained with aPTT reagents containing an activator/procoagulant phospholipid combination of ellagic acid + purified soy phosphatides or kaolin + cephalin, which performed similarly. Identification of the reagents with the best combination of sensitivity, precision, and dynamic range may help guide the selection of reagent for assessing milvexian activity. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Janssen Research &amp; Development, LLC

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317–Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317–Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317–Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Lack of Inter-Laboratory Agreement in the Monitoring of Unfractionated Heparin Using the Anti-Factor Xa Activity and the Anti-Factor Xa-Correlated Activated Partial Thromboplastin Time

Background: Unfractionated heparin (UFH) has been in clinical use for more than half a century. Due to the risk of hemorrhage in case of over dosage and the risk of thrombosis in case of under dosage, monitoring UFH treatments is mandatory. The activated partial thromboplastin time (aPTT) is the most widely used method as it is simple, cheap, and readily available. However, it is also poorly standardized and affected by many parameters, both analytic and pre-analytic, that are unrelated to the heparin effect. Particularly, the UFH-induced prolongation of aPTT is highly dependent on the reagent and analyzer used. In an effort to improve inter-laboratory agreement in the monitoring of UFH, the College of American Pathologists (CAP) recommends that the aPTT therapeutic range must be defined in each laboratory through correlation with a direct measure of heparin activity such as the factor Xa (FXa) inhibition assay. Such a strategy was endorsed years ago by the American College of Chest Physicians (ACCP) and the 9thACCP Consensus guideline recommends monitoring UFH treatments by using either the anti-FXa activity, with the therapeutic range between 0.30 and 0.70 IU/mL, or the anti-FXa-correlated aPTT. However, whether and to what extent this approach enhances the inter-laboratory agreement of UFH monitoring has been weakly evaluated, so far.Objectives: To compare the inter-laboratory agreement of the anti-FXa activity and the anti-FXa-correlated aPTT for monitoring therapeutic UFH treatment, we conducted a cross-validation study among 3 accredited coagulation laboratories that used different technical conditions.Patients and Methods: 125 inpatients treated with UFH were sampled in one of the centers. Blood was collected into evacuated polymer tubes containing 3.2% tri-Na citrate. Tubes were centrifuged twice within 2 h after collection, and plasma was stored frozen in aliquots at -70°C that were shipped in dry ice to the 2 other centers to be locally evaluated. Anti-FXa activity (in IU/mL) was evaluated using 4 chromogenic assays i.e. Biophen Heparin LRT (Hyphen BioMed), HemosIL Liquid Anti-Xa (Instrumentation Laboratory, IL), Innovance Heparin (Siemens), and STA-Liquid Anti-Xa (Stago). APTT (expressed as the patient-to-control ratio) was evaluated using 4 reagents i.e. HemosIL SynthASil and HemosIL APTT SP (IL), Pathromtin SL (Siemens), and STA-PTT Automate 5 (Stago). With some exceptions, participating centers used combinations of reagents and analyzers from the same manufacturer i.e. ACL TOP 700 (IL), CS-5100 (Siemens) and STAR (Stago).Results: In the studied population, anti-FXa test results were found to be highly variable depending of the assay used, with the median activity ranging from 0.57 IU/mL with one reagent to 0.37 IU/mL with another, the two other reagents giving intermediate results (Table). Test results obtained using the different reagents were well correlated (r>0.91 in all cases) even though some comparisons performed according to Bland-Altman demonstrated unacceptable bias. If 0.30 to 0.70 IU/mL was used as the therapeutic range of anti-FXa activity, such a discrepancy in test results led to a lack of agreement as to whether a sample was subtherapeutic, therapeutic or supratherapeutic in more than 25% of the patients.The median aPTT test result (ratio) ranged from 2.22 with the less sensitive to 2.93 with the most sensitive reagent. Using the anti-FXa-correlation method, aPTT therapeutic ranges were found to be highly different from one combination of reagent/analyzer to another (Table), and the same applied when correlations were made between one single aPTT reagent and different anti-FXa assays performed on the same analyzer. Consequently, agreement among all aPTT reagents was found in in less than 40% of the patient samples.Conclusions: The reported discrepancy between test results evaluated using commercially available anti-FXa assays are consistent with the results of various national and international EQA programs, and clearly suggests a lack of standardization of that assay. As the consequence, the anti-FXa-correlated aPTT therapeutic range could be significantly impacted. In addition, the anti-FXa-correlation method did not appear to enhance interlaboratory agreement in UFH monitoring as it produces considerable disparity in UFH dosing decisions among different centers, although the clinical impact of this disparity is not known. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pre-Analytical Stability of Coagulation Parameters in Plasma

Introduction:It is well known that haemostasis testing is influenced by many pre-analytical variables, such as storage time and temperature, that can affect the stability of coagulation factors and influence the results of coagulation assays. The stability of coagulation parameters may also depend on measurement principle and reagent used for analysis. The main aim of this study was to investigate the stability of haemostasis tests after storage of aliquoted plasma at room temperature (RT) for different time periods. Additionally, we investigated whether it is possible to accept a longer storage period at RT, compared to the current Clinical and Laboratory Standard Institute (CLSI) H21 A5 guidelines, without significant stability problems. Also, the influence of using different measurement principles and reagents on the stability of coagulation parameters was investigated by using two different fully automated haemostasis analysers with corresponding reagents.Methods:Blood samples from 20 healthy volunteers were obtained, immediately processed to platelet poor plasma (PPP) and aliquoted. Aliquots were stored at RT for 0h, 2h, 4h, 6h, 8h, 12h, 24h and 48h. Routine haemostasis parameters (PT, aPTT, fibrinogen and D-dimers) were performed on non-frozen plasma. Coagulation factors (FII, FV, FVII, FX, FVIII, FIX, FXI and FXII), von Willebrand factor antigen (vWF:Ag) and von Willebrand factor activity (vWF:Act) were performed batch-wise on frozen plasma aliquots.All these parameters were determined by two analysers, STA-R Max® (Diagnostica Stago®, S.A.S., France) using a viscosity-based principle and ACL-TOP® 350CTS (Instrumentation Laboratory, Werfen, Bedford USA) using a turbidimetric measurement principle. Also analysis of PT and aPTT were performed by multiple reagents. VWF:Ag and vWF:Act were determined by the AcuStar® (Instrumentation Laboratory, Werfen, Bedford USA) using a chemiluminescence technique.Statistically significant differences compared to the baseline results were defined by a Friedman repeated measures analysis of variance and a post hoc Wilcoxon signed rank test with bonferroni adjustment. A clinically relevant change in coagulation assay value compared to the initial measurement (analysis at 0h storage at RT) was denoted as a percentage change of > 10 % according to the 99% confidence interval (CI).Results:For both analysers, a decrease in factor activity of > 10% (according to the 99% CI) was observed for FV after 2h, FVIII after 4h and for FII and FX after 48h of storage at RT. A clinically significant decline of FVII activity, determined by ACL-TOP®, was observed at 24h storage at RT (99% CI: -13.3% - -0.7%). According to the 99% CI a clinically relevant decrease of FVII activity was seen after 48h of storage with mean percentage changes of 9.5% and 21.1% for STA-R Max® and ACL-TOP® respectively. A statistically significant and clinically relevant increase in FXII activity was seen after 48h storage of aliquoted plasma at RT.For aPTT, a statistically significant difference was seen after 6h storage and mean percentage changes of > 10% were observed after 48h of storage at RT for all three aPTT reagents used. Statistically significant differences, but no clinically relevant changes were observed after a storage time of 48h for PT, fibrinogen and FIX. For D-Dimers, FXI, vWF:Ag and vWF:Act no statistically significant nor clinically relevant differences were observed after a storage period of 48h.Conclusions:Some differences between measurements by STA-R Max® and ACL-TOP® could be explained by the difference in reagent used for analysis. Overall, compared to the stated time limits given by the current CLSI guidelines, for most coagulation parameters investigated in this study a longer storage period could be accepted. Time intervals for FVIII and FV dosage were shorter than recommended by the CLSI guidelines. For PT determination our findings were consistent with the current CLSI guidelines. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.