One-stage Clotting Research Articles

Guidance for establishing a factor VIII testing protocol for the myriad of factor VIII products.

Management of hemophilia A has changed significantly in the past few years with the expansion of new and/or modified products as treatment options. Unfortunately, many of the standard factor VIII assays do not always accurately measure all available treatment products; therefore, the laboratory must investigate various assay algorithms to ensure the reporting of the correct results. Requirements for factor testing, diagnosis and severity levels, product testing, factor VIII inhibitor detection and titers, are evaluated, and potential algorithms are created for optimal assessment of patients with hemophilia A. The potential for inaccurate result reporting for patients with hemophilia A or those being treated with the myriad of products has left many laboratories uncertain as to which assay algorithm to implement to ensure reporting the correct results for all products used in their hemophilia program. Algorithms for using either One-stage Clotting assays or Chromogenic assays or a combination of both types of assays are presented for each laboratory to implement based on their clinical situation. Several algorithms are considered based on the needs of the clinical providers and their patients. Each laboratory must select a testing algorithm that is cost-effective and within available resources, yet that encompasses the needs of their providers and patients. Laboratory personnel must consider all assay uses (factor VIII levels, different products, interfering products, and inhibitor titers) in determining the best algorithm for their laboratory. This paper is a starting guide for developing the best factor VIII testing assays and protocols for your laboratory.

Factor VIII Level Comparison in Patients with Severe Hemophilia a on Emicizumab with Inhibitors with One Stage, Bovine and Human Chromogenic Assays and the Factor VIII Equivalency of Emicizumab Using In Vivo Global Hemostasis Assays

IntroductionEmicizumab is a recombinant, humanized bispecific monoclonal antibody that mimics the function of factor VIII (FVIII) which results in a significant reduction in the annualized bleeding rate in patients with hemophilia A (HA), however, the degree with which emicizumab corrects the coagulation defect remains unclear. The objective of this study was to compare the current available laboratory methods in clinical practice; one-stage clotting factor assays (OSCA), bovine and human chromogenic FVIII activity (bovCHR and humCHR, respectively) and FVIII Equivalency of Emicizumab by Thrombin Generation (F8EmT).AimsThe aim of this study is to address the differences of FVIII activity with different techniques in patients with severe HA with inhibitors on emicizumab.Materials and MethodsFactor VIII levels are determined with an activated partial thromboplastin time (aPTT), OSCA using SynthASil on the ACL TOP 500 (Instrumentation Laboratory, Bedford, MA). Factor VIII activity is also determined photometrically via the Chromogenix Coatest® SP4 FVIII chromogenic assay kit (bovCHR, Diapharma Group, West Chester, OH) and the Biophen FVIII:C chromogenic assay kit (humCHR, Aniara Diagnostica, West Chester, OH). For F8EmT, linear regression was utilized to model the FVIII levels as a function of the endogenous thrombin potential (ETP) and peak thrombin values for patients with mild/moderate hemophilia. Then, we used the ETP and peak thrombin results of the severe HA patients on emicizumab with the calibration curve to calculate their F8EmT. Association between patient weight and their F8EmT were also examined and evaluated by linear regression.ResultsData is presented for eight patients with severe HA with inhibitors on emicizumab in the non-bleeding state (Table-1). All patients' FVIII levels measured with OSCA are in or above the normal range (94.0-289.1). Bovine chromogenic FVIII activity is in the severe hemophilia range for five out of eight patients, for the rest it is in the moderate hemophilia range. Human chromogenic FVIII activity ranged between 12.5-49.8%. Factor VIII Equivalency of Emicizumab by Thrombin Generation is either in the mild hemophilia or normal range in all participants of the study.ConclusionOne-stage clotting factor assays demonstrated falsely high results as expected since it is activated partial thromboplastin time based. Bovine chromogenic FVIII activity results were consistent with the severe HA range of the patients though a few had results slightly above that level. Previous literature has stated that the humCHR in patients on emicizumab results in FVIII levels of ∼30% when emicizumab is at its therapeutic concentration (∼50 mcg/ml). This study also demonstrated similar results with 5/8 patients having levels 30-50%. F8EMT levels were mostly consistent with the humCHR. In conclusion, understanding the degree to which emicizumab corrects the coagulation defect of is an important goal as it has clinical implications.Certainly, additional studies with higher participant numbers are needed to confirm these findings. [Display omitted] DisclosuresYoung: Apcintex, BioMarin, Genentech/Roche, Grifols, Novo Nordisk, Pfizer, Rani, Sanofi Genzyme, Spark, Takeda, and UniQure: Consultancy; Genentech/Roche, Grifols, and Takeda: Research Funding.

Monitoring of different factor VIII replacement products using a factor VIII one-stage clotting assay on cobas t 511/711 analysers.

IntroductionRecombinant coagulation factor VIII (FVIII) products are the standard of care for patients with haemophilia A. The development of modified FVIII products has provided benefit for patients but presented challenges for monitoring FVIII activity.AimThis single‐centre study evaluated the Roche FVIII one‐stage clotting assay (OSA) in measuring FVIII activity in plasma samples spiked with seven FVIII products at clinically relevant concentrations.MethodsFVIII‐deficient plasma samples were spiked with two batches of recombinant FVIII products (octocog alfa, moroctocog alfa, simoctocog alfa, efmoroctocog alfa, damoctocog alfa pegol, rurioctocog alfa pegol, lonoctocog alfa) at 1–120 IU/dL FVIII activity, according to their labelled potency. Measurement was conducted on the cobas t 511/711 analysers using the Roche FVIII OSA and the Technoclone TECHNOCHROM FVIII:C chromogenic substrate assay (CSA).ResultsUsing the OSA, FVIII activity was close to labelled potency for most analysed FVIII products including a recombinant FVIII Fc fusion protein. PEGylated FVIII product, damoctocog alfa pegol, was marginally above and single‐chain product, lonoctocog alfa, below the predefined acceptance criteria: for FVIII activity < 25 IU/dL: ± 5 IU/dL; for FVIII activity ≥ 25 IU/dL: ± 20% (relative). The different principles of OSA and CSA led to discrepancies in the estimation of all analysed FVIII products. Additionally, in vitro recovery was increased at lower levels of FVIII activity using the OSA, whereas recovery was more consistent using the CSA.ConclusionThese data allow the interpretation of FVIII activity results for different FVIII products using the Roche FVIII OSA on the cobas t 511/711 analysers.

Regulation of Factor VIII By Activated Protein C: Assessment In Vitro and In Vivo

Factor VIII (FVIII) plays an essential role in the activation of factor X (FX), acting as a factor IXa (FIXa) cofactor in the intrinsic Xase complex. Activated protein C (APC) is a serine protease that inactivates FV/Va. Resistance of FV/FVa to APC due to the Leiden mutation is the most common inherited thrombophilia. By cleaving FVIII/FVIIIa at Arg336 and Arg 562, APC can also inactivate FVIII; however, the role of this reaction, especially in vivo, is not well defined. Further, thrombophilic mutations at either of these sites have yet to be identified, calling into question the importance of this reaction. Understanding the role of APC in FVIII regulation is important as there are several novel therapeutics in development for hemophilia A (HA) that either bypass FVIII regulatory pathways or target inhibition of APC. Additionally, while data using a high-specific activity factor IX variant has been successful in a hemophilia B gene transfer trial, there is no such described hyperactive FVIII protein. In principle, manipulation of APC cleavage sites may result in a more procoagulant FVIII protein with enhanced hemostastic function that may serve as a novel therapeutic for HA in the setting of a recombinant protein treatment or gene therapy.We sought to further understand the role of APC regulation of FVIII and to evaluate the in vivo hemostatic effect of an APC-resistant FVIII as a potential treatment strategy for HA. We conducted site-directed mutagenesis to introduce missense arginine to glutamine mutations at FVIII APC cleavage sites (R336Q and R562Q) on a B-domain deleted FVIII (FVIII-WT) backbone. The resulting recombinant, purified FVIII variant protein (FVIII-QQ) was then compared to FVIII-WT. Following incubation with APC, SDS-PAGE analysis of FVIII-QQ demonstrated no apparent cleavage by APC. In contrast, FVIII-WT demonstrated expected cleavage at R336 and R562, thereby confirming FVIII-QQ is resistant to APC cleavage. Surprisingly, one-stage clotting assay determination of FVIII-QQ specific activity was approximately 30% higher than FVIII-WT; this was unexpected given the absence of thrombomodulin or APC. We speculate this may be related to the ability of FXa to cleave FVIIIa at Arg336 and Arg562 inactivating FVIIIa that is likely disrupted in the FVIII-QQ variant (Eaton D et al. Biochemistry 1986 and Plantier JL et al. J Thromb Haemost 2010). To test the role of APC regulation of FVIII, we performed plasma-based in vitro thrombin generation assays in the presence of APC. Human FVIII deficient plasma was reconstituted to 1 nM (normal plasma FVIII concentration) with FVIII-QQ or FVIII-WT wherein increasing concentrations of APC were then added and thrombin generation was measured. Consistent with the known role of APC being an anticoagulant, the amount of thrombin generation decreased with increasing APC concentrations. However, the amount of APC necessary to inhibit 50% peak thrombin generation with FVIII-WT was 5.2 nM ± 0.3 nM versus FVIII-QQ was 11.3 nM ± 0.8 nM, which again demonstrated FVIII-QQ is resistant to APC inhibition. We suspect the observed decrease in thrombin generation with increasing APC concentrations is due to the role of APC in FV/FVa inhibition. Overall, our data support a role for APC inactivation of FVIIIa in this in vitro system. Interestingly, FVIII-QQ reliably demonstrated an approximate 2-fold increase in peak thrombin in the thrombin generation assay. This is being explored, but we again speculate this may be related to the effects of FXa on FVIII-WT at R336 and R562.We next evaluated the in vivo hemostatic efficacy of FVIII-WT vs. FVIII-QQ in HA mice. HA mice were infused with FVIII-WT or FVIII-QQ protein prior to tail clip assay. Comparison of both proteins at a dose of 3 µg/kg demonstrated greater blood loss in HA mice treated with FVIII-WT vs. FVIII-QQ (p=0.006). Further, blood loss in HA mice treated with FVIII-WT was greater than wild type mice (p=0.007); in contrast, there was no difference between wild type and HA mice treated with 3 µg/kg of FVIII-QQ (p=0.26).Our data demonstrate FVIII-QQ has a higher specific activity, requires 2-fold greater APC concentrations to inhibit thrombin generation in vitro and an approximate 2-fold greater in vivo hemostastic efficacy. Our results support that APC has physiologic significance in FVIII/FVIIIa inactivation and provide rationale for the study of APC in FVIII/FVIIIa regulation to develop potential novel HA therapeutics. DisclosuresDavidson:Spark Therapeutics: Consultancy. Camire:Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Bayer: Consultancy, Research Funding. George:Pfizer: Consultancy; Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B.

Pharmacokinetic variability of factor VIII concentrates in Chinese pediatric patients with moderate or severe hemophilia A.

ABSTRACTImportanceThe use of factor VIII (FVIII) concentrates under pharmacokinetic (PK) guidance has become the main approach for treatment of hemophilia. However, limited PK research has been conducted in Chinese pediatric patients.ObjectiveTo investigate the PK parameters of various FVIII concentrates in Chinese pediatric patients.MethodsSeventy‐nine patients were enrolled (28 treated with Kogenate FS®, 23 treated with Advate ®, and 28 treated with GreenMono™). All enrolled patients participated in single‐dose PK analysis after at least a 3‐day washout period. Blood samples were collected predose, as well as at 1 h, 9 h, 24 h, and 48 h after infusion; FVIII levels were measured using a one‐stage clotting assay. von Willebrand Factor Antigen (VWF:Ag) levels and blood types were also determined. PK parameters were evaluated by WAPPS‐Hemo.ResultsMean values of terminal elimination half‐life time (t1/2) for the Kogenate FS®, Advate®, and GreenMono™ FVIII groups were 12.24 h, 10.18 h, and 9.62 h; median clearance values were 4.16, 6.23, and 5.11 mL·kg−1·h−1; and median in vivo recovery values were 1.97, 1.55, and 1.61 IU/dL per IU/kg. Longer t1/2, higher in vivo recovery, and lower clearance were observed in patients with higher VWF:Ag level who were treated with recombinant concentrates.InterpretationChinese pediatric patients with hemophilia had FVIII PK characteristics similar to those previously observed in non‐Chinese children, including large variation among individuals. VWF:Ag level and FVIII brand were associated with differences in FVIII PK. Thus, PK‐guided dosing should be used to optimize individualized therapy in Chinese children.

Pharmacodynamics of eftrenonacog-alfa (rFIX-Fc) in severe hemophilia B patients: A real-life study

Eftrenonacog-alfa is a recombinant factor IX-Fc fusion protein increasingly prescribed in hemophilia B patients. We aimed to assess its pharmacodynamics (PD) in real-life setting via FIX activity measurement and thrombin generation assay (TGA).Sixty samples from 15 severe hemophilia B treated patients were collected at different time points. FIX activity was measured using product-specific one-stage clotting assay (reference method) and two chromogenic assays (CSA) (Biophen FIX and Rox FIX). TGA was triggered with 1 pM tissue factor. Five parameters were analyzed: lag time (LT), time to peak (TTP), peak height (PH), endogenous thrombin potential (ETP), and velocity. PD models were built to characterize their relationships with FIX activity, using mixed effects models.Mean trough FIX level was estimated at 4.64 (±1.50) IU/dl with a recovery at 0.78 (±0.16) IU/dl per 1 IU/kg injected dose. FIX activity ranged between 1 and 86 IU/dl with 21.5 IU/dl median value. Biophen FIX and Rox FIX allowed reliable measurements except in samples with FIX <20 IU/dl in which values were underestimated (delta >30%). PD models revealed that velocity was the most sensitive TGA parameter to FIX activity followed by PH, ETP, TTP and finally LT. Following FIX activity peak after eftrenonacog-alfa injection, velocity decreased first, followed by PH then ETP.Both CSA failed to accurately measure FIX in severe hemophilia B patients receiving eftrenonacog-alfa throughout the measuring range. TGA could be an additional valuable tool to evaluate hemostasis balance in treated patients.

Activity of transgene-produced B-domain–deleted factor VIII in human plasma following AAV5 gene therapy

AbstractAdeno-associated virus (AAV)-based gene therapies can restore endogenous factor VIII (FVIII) expression in hemophilia A (HA). AAV vectors typically use a B-domain–deleted FVIII transgene, such as human FVIII-SQ in valoctocogene roxaparvovec (AAV5-FVIII-SQ). Surprisingly, the activity of transgene-produced FVIII-SQ was between 1.3 and 2.0 times higher in one-stage clot (OS) assays than in chromogenic-substrate (CS) assays, whereas recombinant FVIII-SQ products had lower OS than CS activity. Transgene-produced and recombinant FVIII-SQ showed comparable specific activity (international units per milligram) in the CS assay, demonstrating that the diverging activities arise in the OS assay. Higher OS activity for transgene-produced FVIII-SQ was observed across various assay kits and clinical laboratories, suggesting that intrinsic molecular features are potential root causes. Further experiments in 2 participants showed that transgene-produced FVIII-SQ accelerated early factor Xa and thrombin formation, which may explain the higher OS activity based on a kinetic bias between OS and CS assay readout times. Despite the faster onset of coagulation, global thrombin levels were unaffected. A correlation with joint bleeds suggested that both OS and CS assay remained clinically meaningful to distinguish hemophilic from nonhemophilic FVIII activity levels. During clinical development, the CS activity was chosen as a surrogate end point to conservatively assess hemostatic efficacy and enable comparison with recombinant FVIII-SQ products. Relevant trials are registered on clinicaltrials.gov as #NCT02576795 and #NCT03370913 and, respectively, on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database; https://eudract.ema.europa.eu) as #2014-003880-38 and #2017-003215-19.

ADAMTS13 activity, high VWF and FVIII levels in the pathogenesis of deep vein thrombosis

BackgroundDeep vein thrombosis (DVT) is a common multi-factorial disease with a partially understood aetiology. Although the roles of high factor (F)VIII and von Willebrand factor (VWF) levels are recognized, that of ADAMTS13 is still unclear. AimTo assess the association between ADAMTS13 activity levels, VWF antigen (VWF:Ag) and FVIII coagulant activity (FVIII:C) levels and DVT. Materials and methods365 Italian DVT patients and 292 age- and sex-matched controls were considered. Plasma ADAMTS13 activity was measured using FRETS-VWF73 assay. VWF:Ag and FVIII:C were measured using immunoassay and one-stage clotting assay (ACL TOP analyzer), respectively. Quartile analyses were performed to evaluate the individual association between ADAMTS13 activity, VWF:Ag, FVIII:C and DVT. The combined effect of high VWF levels (> 4th quartile) and low ADAMTS13 levels (< 1st quartile) was evaluated using binary variables. All models were age- and sex-adjusted. Estimated risks were reported as Odds ratio (OR) with 95% confidence intervals (CI). ResultsADAMTS13 activity was lower in DVT patients (94% vs. 98% of controls). Patients with an ADAMTS13 activity <1st quartile (86%) showed a 1.6-fold increased risk of DVT (95%CI, 1.05–2.55). The combination of low ADAMTS13 activity and high VWF:Ag levels was associated with a 15–fold increased risk (95%CI, 7.80–33.80). VWF:Ag and FVIII:C were associated to DVT with a dose-response relationship. ConclusionsADAMTS13 activity < 86% was associated with a moderate risk of DVT. The co-presence of low ADAMTS13 activity and high VWF levels resulted in a strong synergistic effect on DVT risk. The association of VWF:Ag and FVIII:C with DVT was confirmed.

Weight-Based Dosing in Hemophilia a: A Randomized, Controlled, Open-Label, Crossover Trial to Measure Factor VIII Recovery Following Factor VIII Concentrate Dosing Based on Total Body Weight, Ideal Body Weight, and Lean Body Mass

Introduction: The increasing prevalence of obesity has not spared the hemophilia population with rates similar to those in the general population. Obesity alters the pharmacokinetic properties of many drugs making it difficult to determine the appropriate dose when administering medications based on total body weight (TBW). Alternative descriptors of body weight, such as lean body mass (LBM) and ideal body weight (IBW) are sometimes used in these situations. Under-or overdosing of clotting factor concentrates in hemophilia may have catastrophic consequences. It has been demonstrated that when recombinant factor VIII (rFVIII) is dosed according to TBW in overweight and obese hemophiliacs, FVIII recovery values are greater than the expected 2.0 IU/dl per IU/kg. These findings suggest the need for alternative methods of rFVIII dosing in individuals whose morphometric characteristics differ from the ideal. Methods: We performed a single-center, randomized, controlled, open-label, crossover trial to determine if rFVIII dosing based on LBM and IBW achieves a targeted FVIII recovery with better precision than based on TBW in overweight and obese (body mass index ≥25 mg/m2), adult males (age ≥18) with hemophilia A (FVIII activity ≤0.40 IU/dl). Participants were randomized to 1 of 6 possible dosing sequence scenarios based on 3 different weight-based dosing regimens (TBW, LBM, and IBW). Recombinant FVIII dosing was calculated based on weight and a targeted FVIIII recovery of 2.0 IU/dl per IU/kg. Each subject administered his own rFVIII on three separate weeks following a washout period of at least 72 hrs. FVIII recovery was determined using the one-stage clotting assay obtained before and 30 min after infusion. Outcomes consisted of FVIII recovery and the proportion of participantsachieving a targeted FVIII recovery of 2.0 +/- 0.2 IU/dl per IU/kg 30 min after infusion. An intention-to-treat analysis was performed using mixed effects linear and logistic regression with random subject effect adjusted for week. Results: Between September 2015 and March 2019, 30 eligible patients were randomized to 1 of 6 rFVIII weight-based dosing scenarios consisting of TBW, LBM, and IBW. Nineteen participants completed the study. The mean age of participants was 34.6 +/- 11.3 years. The average body mass index (BMI) of participants was 29.2 +/- 3.5 kg/m2. Twenty-six participants had severe hemophilia A and 2 participants each had moderate and mild hemophilia A. Of those receiving at least 1 dose of rFVIII, Eloctate, 66.7%, Advate, 14.3%, and Novoeight, 9.5%, were the most commonly administered products. The mean FVIII recovery for TBW, LBM, and IBW-based dosing was 2.45 (95% CI: 2.27, 2.64), 2.08 (95% CI: 1.89, 2.26), and 2.17 (95% CI: 1.99, 2.35) IU/dl per IU/kg, respectively. The mean FVIII recovery was higher in TBW vs LBW and IBW-based dosing (Table 1).The proportion of participants with a targeted FVIII recovery of 2.0 +/- 0.2 IU/dl per IU/kg was 0.25 (95% CI; 0.09, 0.52), 0.39 (95% CI: 0.18, 0.64), and 0.54 (95% CI: 0.30, 0.77) for TBW, LBM, and IBW-based dosing, respectively. There was no difference in the proportion of participants with a targeted FVIII recovery of 2.0 +/- 0.2 IU/dl per IU/kg in TBW vs LBM and IBW-based dosing (Table 1). Discussion: In this study, rFVIII dosed according to TBW resulted in a higher FVIII recovery value than LBM and IBW-based dosing in overweight and obese, adult males with hemophilia A. There was no significant difference in the proportion of participants with a targeted FVIII recovery of 2.0 +/- 0.2 IU/dl per IU/kg in TBW vs LBM and IBW-based dosing; however, effect estimates favor alternative dosing strategies with LBM and IBW-based dosing 1.93 and 3.65 times, respectively, having greater odds of achieving targeted FVIII recovery. This may be due to limited statistical power as only 79.2% of the necessary number of participants completed the study. In addition, the rFVIII dose was erroneously calculated in 7 participants resulting in the administration of a smaller than intended amount. This primarily affected IBW and LBM-based dosing, which may have served to reduce the effect size above. In conclusion, based on these findings, overweight and obese patients with hemophilia A should undergo individualized pharmacokinetic studies using alternative descriptors of body weight to determine the most accurate, and cost-effective, method of achieving targeted FVIII recovery values. Disclosures Seaman: Takeda: Consultancy; Genentech: Consultancy; Spark Therapeutics: Consultancy; Bayer: Consultancy. Ragni:Alnylam Pharmaceuticals Inc., Baxalta/Takeda, BioMarin, Bioverativ, and Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Sangamo: Consultancy, Research Funding; Takeda: Research Funding; Alnylam/Sanofi, ATHN, BioMarin, Bioverativ, Sangamo, Spark: Research Funding; Bioverativ: Consultancy, Research Funding; Spark: Consultancy, Research Funding; BioMarin: Consultancy, Research Funding; Alnylam/Sanofi, BioMarin, Bioverativ, Spark: Consultancy; American Thrombosis Hemostasis Network: Other: Committee work; Baxalta/Takeda, CSL Behring, Genentech, a member of the Roche Group, OPKO Biologics, and Vascular Medicine Institute: Research Funding.

Extended Half-Life Factor VIII/Factor IX Products: Assay Discrepancies and Implications for Hemophilia Management.

Due to structural differences between extended half-life (EHL) factor VIII (FVIII) or FIX products and equivalent plasma wild-type molecules used for assay calibration, reagent-dependent discrepancies during monitoring of FVIII- and FIX-replacement therapies with EHL products have been described. To assess the performance of available one-stage clotting and chromogenic substrate assays on the Siemens Atellica COAG 360 analyzer, an in vitro study using spiked plasma samples was performed. The described results confirm previously described findings and allowed allocation of each EHL product to an appropriate assay. In addition, corresponding EHL product-specific analytes were defined within the order entry system of the University Hospital Bonn. The requirement of product-specific FVIII and FIX assays complicates patient monitoring and demonstrates the need for both continuous education and communication between treating physicians and the coagulation laboratory.

Cross-reacting recombinant porcine FVIII inhibitors in patients with acquired haemophilia A.

Acquired haemophilia A (AHA) is a rare bleeding disorder caused by the development of autoantibodies to endogenous human factor VIII (hFVIII). If treatment of bleeding is required, one option is recombinant porcine FVIII (rpFVIII). Cross-reactivity between factor VIII inhibitors and rpFVIII has previously been described. The aim of this study was to retrospectively assess the incidence of cross-reacting anti-porcine inhibitors in patients diagnosed with AHA in two UK centres. The plasma of fifty-one patients diagnosed with AHA via reduced FVIII:C and positive FVIII inhibitor titre as detected with a Nijmegen-Bethesda assay (NBA) was also tested by a porcine Bethesda assay (PBA). The NBA was modified by replacement of human FVIII with rpFVIII in the PBA, with determination of residual FVIII by one-stage clotting assay. The median FVIII inhibitor titre by NBA was 22.8 BU/mL (range: 0.8-1000 BU/mL). 37% of samples exhibited linear, type 1 kinetics in the NBA. Negative PBA was observed in 26 patients, and 25 were positive (median PBA: 3.5 BU/mL; range: 0.8-120 BU/mL). Type 1 kinetics were observed in 40% of PBA-positive patients. At NBA tires of greater than 100 BU/mL, the positive predictive value for the presence of porcine cross-reactivity was 100%. At NBA below 5 BU/mL, the negative predictive value for the presence of porcine cross-reactivity was 71%. Cross-reactivity between FVIII inhibitors and rpFVIII was observed in 49% of patients. The presence of inhibitors to rpFVIII may influence the treatment choice for patients with acquired haemophilia A.

Direct comparison of two extended half-life PEGylated recombinant FVIII products: a randomized, crossover pharmacokinetic study in patients with severe hemophilia A

An open-label, crossover randomized study was performed to compare the pharmacokinetics (PK) of damoctocog alfa pegol and rurioctocog alfa pegol, two recombinant factor VIII (FVIII) products indicated in patients with hemophilia A, both conjugated to polyethylene glycol to reduce clearance and extend time in circulation. Adult patients (N = 18) with severe hemophilia A (FVIII < 1 IU/dL), previously treated with any FVIII product for ≥ 150 exposure days, were randomized to receive a single 50 IU/kg infusion of damoctocog alfa pegol followed by rurioctocog alfa pegol, or vice versa, with ≥ 7-day washout between doses. FVIII activity was measured using the one-stage clotting assay. PK parameters, including area under the curve from time 0 to the last data point (AUC0–tlast, primary parameter), dose-normalized AUC (AUCnorm), and time to threshold, were calculated based on 11 time points between 0.25 and 120 h post-dose and evaluated using a noncompartmental model. Due to differences in batch-specific vial content used for the study, actual administered median doses were 54.3 IU/kg for damoctocog alfa pegol and 61.4 IU/kg for rurioctocog alfa pegol. Based on actual dosing, a significantly higher geometric mean (coefficient of variation [%CV]) AUCnorm was observed for damoctocog alfa pegol (43.8 h kg/dL [44.0]) versus rurioctocog alfa pegol (36.0 h kg/dL [40.1, P < 0.001]). Based on population PK modeling, median time to reach 1 IU/dL was 16 h longer for damoctocog alfa pegol compared with rurioctocog alfa pegol. No adverse events or any immunogenicity signals were observed. Overall, damoctocog alfa pegol had a superior PK profile versus rurioctocog alfa pegol. Trial registration number: NCT04015492 (ClinicalTrials.gov identifier). Date of registration: July 9, 2019

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Genetics and Hemostatic Potential in Persons with Mild to Moderate Hemophilia A with a Discrepancy between One-Stage and Chromogenic FVIII Assays.

Factor VIII (FVIII) activity (FVIII:C) can be measured by different methods including one-stage clotting assays (OSAs) and chromogenic assays (CSAs). Discrepancy between FVIII:C assays is known and associated with genetic variations causing mild and moderate hemophilia A (HA). We aimed to study the discrepancy phenomenon and to identify associated genetic alterations. Further, we investigated if hemostatic global assays could discriminate the group with discrepant FVIII:C from them. The study contained plasma samples from 45 patients with HA (PwHA) from Hemophilia Centers in Stockholm, Sweden, and Belgrade, Serbia. We measured FVIII:C with OSA and CSA, sequenced the F8 gene, and performed two global hemostatic assays; endogenous thrombin potential and overall hemostatic potential. Nineteen of 45 PwHA had a more than twofold higher FVIII:C using OSA compared to CSA and were considered discrepant. Thirty-four causal mutations were detected, where of five had not previously been associated with assay discrepancy. These novel mutations were p.Tyr25Cys, p.Phe698Leu, p.Met699Leu, p.Ile1698Thr, and Ala2070Val. We found no difference between discrepant and nondiscrepant cases with either of the global assays. There was a discrepancy between FVIII:C assays in almost half of the PwHA, which for some could lead to missed HA diagnoses or misclassification of severity. Genotyping confirmed that mutations associated with FVIII:C discrepancy cluster in the A domains of F8, and five mutations not previously associated with FVIII:C discrepancy was identified. Global hemostatic assays did not contribute to distinguish assay discrepancy in PwHA.

The traceability of commercial plasma calibrators to the plasma International Standards for factor VIII and factor IX.

Accurate measurement of coagulation factors is essential, especially for diagnosis of deficiency. Clinical laboratories use commercially available plasma calibrators, which should be traceable to the relevant plasma International Standard (IS). This study assessed the relationship between the plasma IS for factors IX (FIX) and VIII (FVIII) and some commonly used commercial calibrators. Calibrators from seven manufacturers were assayed for FIX and FVIII activity by one‐stage clotting assay (OSCA) using different activated partial thromboplastin time (APTT) reagents and deficient plasmas, or chromogenic assay (CA). Results were calculated relative to the 4th IS Factors II,VII,IX,X, Plasma or the 6th IS Factor VIII/VWF, Plasma. Results for each calibrator were similar across the APTT reagents and deficient plasmas used. All calibrators showed a recovery of 90%‐111% of the manufacturers’ values, except calibrator C, which had recovery of around 85%. CA gave similar results, with good recovery for all but calibrator C. Similar low recoveries for OSCA and CA were found for a different lot of calibrator C and for a different calibrator product from manufacturer C. When all calibrators from manufacturer C were assayed by OSCA using the manufacturer's own deficient plasmas and APTT reagents, the mean recovery was still below 90%. Overall, there was good traceability of the international unit between the IS and commercial calibrator plasmas. Calibrators from one manufacturer consistently yielded lower than expected values for FIX and FVIII. This could lead to an over‐estimation of the coagulation factor content in patient samples and demonstrates the importance of careful choice of calibrator.

Gene therapy for hemophilias: the end of phenotypic testing or the start of a new era?

: Hemophilia comprises two distinct genetic disorders caused by missing or defective clotting factor VIII (hemophilia A) or clotting factor IX (hemophilia B). The management of these conditions has been for long based on replacement therapies, but emerging evidence garnered from recent landmark studies suggests that a promising avenue toward routine use of gene therapy is clearly progressing forward, thus generating unavoidable consequences on laboratory hemostasis, especially as pertaining to phenotypic testing. Although it seems likely that widespread use of gene therapy will be associated with a relative decrease of hemostasis tests requests in this patient population due to the relatively stable effect of transgene delivery and persistent production of endogenous clotting factor, some important aspects persuade us that conventional laboratory diagnostics, especially encompassing activated partial thromboplastin time, as well as one-stage and two-stage clotting factor assays, will not be completely voided in the gene therapy era. In particular, phenotypic testing will remain essential for excluding acquired or sporadic cases of hemophilia, for identifying and titrating factor inhibitors, as well as for defining and monitoring the long-term therapeutic effectiveness of gene transfection in hemophiliacs.

Laboratory issues in gene therapy and emicizumab.

The treatment options for the haemostatic disorders, haemophilia A and haemophilia B, have progressed rapidly over the last decade. The introduction of extended half-life recombinant factor VIII (FVIII) and factor IX (FIX) concentrates to replace these missing clotting factors highlighted discordance between one-stage activated partial thromboplastin time (APTT)-based clotting factor assays and chromogenic factor assays with some products. This raised awareness of the importance of investigation of potential reagent or assay differences by pharmaceutical companies. In 2017, the FVIII mimetic, emicizumab, was approved as a prophylactic treatment for haemophilia A patients with anti-FVIII inhibitors. The mechanism of action of emicizumab causes interference with some commonly used haemostasis tests including the APTT and its associated one-stage APTT-based clotting assays. Chromogenic assays may also be affected but this is dependent on the particular constituents of the reagents. Chromogenic assays containing human factor IXa (FIXa) and factor X (FX) are sensitive to the presence of emicizumab but those containing bovine FIXa and FX are unaffected. Many haemostasis laboratories have been required to evaluate new assays to enable accurate monitoring of emicizumab in patient plasma. A number of gene therapy approaches have been trialled in both haemophilia A and haemophilia B but there are scant data published on the measurement of FVIII and FIX in these patients and whether there are discrepancies between reagents or assay methodologies.

Factor VIII and IX assays for post-infusion monitoring in hemophilia patients: Guidelines from the French BIMHO group (GFHT).

Replacement therapy with plasma-derived or recombinant FVIII and FIX (pdFVIII/pdFIX or rFVIII/rFIX) concentrates is the standard of treatment in patients with haemophilia A and B, respectively. Measurement of factor VIII (FVIII:C) or factor IX (FIX:C) levels can be done by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). The French study group on the Biology of Hemorrhagic Diseases (a collaborative group of the GFHT and MHEMO network) presents a literature review and proposals for the monitoring of FVIII:C and FIX:C levels in treated haemophilia A and B patients, respectively. The use of CSA is recommended for the monitoring of patients treated with pdFVIII or rFVIII including extended half-life (EHL) rFVIII. Except for rFVIII-Fc, great caution is required when measuring FVIII:C levels by OSA in patients substituted by EHL-rFVIII. The OSA is recommended for the monitoring of patients treated with pdFIX or rFIX. Large discordances in the FIX:C levels measured for extended half-life rFIX (EHL-rFIX), depending on the method and reagents used, must lead to great attention when OSA is used for measuring FIX:C levels in patients substituted by EHL-rFIX. Data of most of recent studies, obtained with spiked plasmas, deserve to be confirmed in plasma samples of treated patients.

Activity measurements of dalcinonacog alfa.

IntroductionMany recombinant and modified FIX products have been, and continue to be, developed with the aim of improving treatment for patients with haemophilia B. One such new product is dalcinonacog alfa, a recombinant FIX with modifications to provide improved features such as subcutaneous administration.AimIn view of previously observed assay discrepancies with modified FIX therapeutics, the aim of this study was to assess potential discrepancies in potency measurement of dalcinonacog alfa between and within different assay methods.MethodsPotency of dalcinonacog alfa was measured against the 5th International Standard (IS) for FIX Concentrate and the 4th IS for FIX Plasma by One‐Stage Clotting Assay, using 9 different APTT reagents and 2 commercially available FIX chromogenic kits. Plasma‐derived concentrate and recombinant FIX samples were also included for comparison in every assay.ResultsSubstantial discrepancies were observed when assaying dalcinonacog alfa using the one‐stage clotting assay against both standards. No statistically valid results were obtained when testing dalcinonacog alfa using either chromogenic kit. Increasing the incubation time with the activation reagent in both chromogenic kits resulted in valid assays and increased the potency to become more in line with potencies by one‐stage clotting assays. Increasing the incubation time in the chromogenic kits had no effect on the potencies of the plasma‐derived or recombinant samples. However, incubation time influenced in the one‐stage clotting assay using Dapttin.ConclusionsWithin and between assay method discrepancy was found when assaying dalcinonacog alfa. Methods for potency labelling and clinical monitoring should be given careful consideration.

Evaluation of Chromogenic Factor VIII Assay Compared with One-Stage Clotting Assay.

Congenital factor VIII (FVIII) deficiency causes hemophilia A due to different types of defects in the FVIII gene. Although the chromogenic measurement is the reference method and shows less variability, a one-stage assay is the most commonly preferred method for measurement of FVIII. In this study, we aimed to evaluate the analytical performances of chromogenic and one-stage assays, and compare the results prior to introduction of newly developed extended half-life recombinant FVIII products. Sixty-six blood samples from residual material of Istanbul Faculty of Medicine, Central Laboratory workflow comprised the study group. Samples were classified; plasma FVIII > 40 IU and FVIII < 40 IU. FVIII activities were measured using one-stage clotting and chromogenic assays on a CS-2500 analyzer. Analytical performances were determined through precision, linearity, carryover, and comparability studies. The within-run CV% of the one-stage assay on the CS-2500 had 1.6%, 2.6%, the between day CV% were 8.5%, 4.9 % for low and high controls, respectively. The within-run CV% of chromogenic method had 1.2% and 0.9%. Both methods demonstrated good linearity (R2 > 0.998), and the comparisons of both assays exhibited good agreement with minor bias for FVIII activity > 40 IU. However, a significant bias was obtained for FVIII activity < 40 IU. We obtained higher results using the one-stage assay compared with the chromogenic assay, and a significant bias was found for the samples lower than 40 IU. The discrepancy can explained by the presence of a weak agreement for samples lower than 10 IU due to the lower detection limit of the chromogenic assay used in this study (1.5%).