One-stage Assay Research Articles

Acquired Hemophilia A: A Narrative Review and Management Approach in the Emicizumab Era

Acquired Hemophilia A (AHA) is a rare bleeding disorder caused by inhibitory autoantibodies to factor VIII (FVIII). The goals of treatment are two-fold, namely immunosuppressive therapy (IST) to eradicate the inhibitor and hemostatic management to control bleeding. Emicizumab, a bispecific antibody that acts as a factor VIIIa (FVIIIa) mimetic, has seen growing use in AHA following its approval for congenital hemophilia A (cHA). This review provides an overview of the epidemiology, pathophysiology, diagnosis, and treatment of AHA. Registry, trial, and case series data are assimilated and summarized with an emphasis on a standardized approach that integrates the use of emicizumab. With recent registry data suggesting the need to focus on immunosuppression-related mortality in AHA, we provide treatment recommendations in an algorithmic format that have become the standard of care at our institution. These recommendations are intended to minimize hemostatic product usage and potential toxicity related to IST, while reducing morbidity and re-hospitalization rates for bleeding. The proposed treatment algorithm, which includes key interventions by phase of therapy, can be readily implemented at centers that have rapid access to plasma FVIII activity using a one-stage assay. A case is presented to illustrate the proposed diagnostic and management considerations.

Coagulation factors VIII and factors IX testing practices in China: Results of the 5-year external quality assessment program

Background and aimsTesting for coagulation factors VIII (FVIII) and IX (FIX) plays significant importance in the diagnostic and treatment of hemophilia A and B. External quality assessment (EQA) scheme aimed to assess the participants’ performance of testing for coagulation factors and identify shortcomings in clinical practice. This study aimed to investigate the performance trends of the participating laboratories in China national external quality assessment Scheme (China NEQAS) for FVIII and FIX over a five-year period (2019–2023). Materials and methodsA total of ten external quality assessment (EQA) rounds were conducted from 2019 to 2023 in the China NEQAS for FVIII and FIX. The distribution of method, reagent and instrument were calculated. The trends of method- specific inter-laboratory coefficient of variation (CV) and pass rates were analyzed over 5 years. The dilutions for coagulation factor testing were also investigated. ResultsAll laboratories use one-stage assays to detect FVIII and FIX activity. The inter-laboratory overall CV decreased year by year (10.9 % to 9.3 % for FVIII and 13.5 % to 10.2 % for FIX), and the laboratory pass rate steadily increased (88.0 % to 93.4 % for FVIII and 81.3 % to 92.7 % for FIX). The majority of laboratories employed a single dilution methodology for the assessment of FVIII and FIX activity. The interlaboratory CV was elevated for the Siemens reagent (Actin FSL) during analysis of moderately abnormal FIX concentrations of EQA samples in most batches. ConclusionsThe implementation of the external quality assessment has contributed to facilitate the enhancement of testing quality. Chromogenic assay is a supplement to accurate determination when necessary. Laboratories may choose to perform dilution tests or direct assays to identify the presence of inhibitors, particularly when they are suspected.

The Role of Clot Waveform Analysis and Related Parameters in the Diagnosis and Treatment of Hemophilia A.

Hemophilia A (HA) is an inherited bleeding disorder caused by a deficiency or defect in factor VIII (FVIII). We investigated the role of clot waveform analysis (CWA) of activated partial thromboplastin time in the diagnosis and therapeutic monitoring of HA. The changes in CWA parameters the maximum clotting velocity (|Min1|), maximum clotting acceleration (|Min2|), and maximum clotting deceleration (|Max2|) were detected among mild, moderate, and severe HA groups. As the severity of HA subtypes increased, the levels of |Min1|, |Min2|, and |Max2| progressively decreased (p < 0.05). Receiver operating characteristic curve analysis showed that |Max2| and |Min2| were more effective than |Min1| in distinguishing different types of HA patients, with higher diagnostic efficacy. The standard curves based on Actin FSL reagent for normal and low levels of FVIII:C-|Max2| were established, with R2 values of 0.98 and 0.99, respectively. These curves can be utilized for monitoring during replacement therapies involving full-length recombinant FVIII and B-domain-deleted FVIII. Thirty cases of HA patients utilized the FVIII-|Max2| standard curve to obtain individual pharmacokinetics characteristic parameters. The clearance, half-life (t1/2), time to FVIII:C of 1% above baseline (tt1%), and predicted dosage showed no statistically significant differences compared with one-stage assay (p > 0.05). CWA is an economical and practical tool, and its related parameters are associated with the severity of HA. It has promising clinical prospects in predicting FVIII:C levels and individualized treatment when HA patients undergo replacement therapy.

Canadian clinical experience on switching from standard half-life recombinant factor VIII (rFVIII), octocog alfa, to extended half-life rFVIII, damoctocog alfa pegol, in persons with haemophilia A ≥ 12 years followed in a Comprehensive Hemophilia Care Program in Canada.

Damoctocog alfa pegol (BAY 94-9027, Jivi®) is an extended half-life recombinant factor (F)VIII replacement, indicated for the treatment of haemophilia A in patients aged ≥12 years. Following introduction of damoctocog alfa pegol in Canada in 2020, there have been no reports on routine clinical effectiveness and satisfaction, when switching from a previous FVIII product in Canada. To report changes in pharmacokinetics, effectiveness, utilization and patient satisfaction when switching to damoctocog alfa pegol prophylaxis from previous standard half-life octocog alfa (BAY 81-8973, Kovaltry®) treatment. A single-centre, intra-patient comparison of pharmacokinetics and clinical outcomes was performed. Blood samples drawn once pre-dose and ≥2 times post-dose were measured by a one-stage assay to assess pharmacokinetic parameters including area under the curve (AUC, primary endpoint). Patient-reported outcomes data were collected using the Patient-Reported Outcomes, Burdens and Experiences questionnaire (PROBE). Clinical outcomes included annualized bleeding rate (ABR) and factor utilization. Dose-normalized AUC was significantly increased after switch to damoctocog alfa pegol from octocog alfa. Median (quartile [Q]1; Q3) annualized bleeding rates were 0.67 (0.00; 1.33) with damoctocog alfa pegol and 1.33 (0.00; 2.67) with octocog alfa. Half of the patients receiving damoctocog alfa pegol prophylaxis experienced zero bleeds (n=9, 50.0%) versus 38.9% (n=7) of patients treated with octocog alfa. Patients' good quality of life was maintained. This study provides routine clinical evidence supporting the benefits of switching from octocog alfa to damoctocog alfa pegol for patients with severe haemophilia A.

A Generative and Causal Pharmacokinetic Model for Factor VIII in Hemophilia A: A Machine Learning Framework for Continuous Model Refinement.

In rare diseases, such as hemophilia A, the development of accurate population pharmacokinetic (PK) models is often hindered by the limited availability of data. Most PK models are specific to a single recombinant factor VIII (rFVIII) concentrate or measurement assay, and are generally unsuited for answering counterfactual ("what-if") queries. Ideally, data from multiple hemophilia treatment centers are combined but this is generally difficult as patient data are kept private. In this work, we utilize causal inference techniques to produce a hybrid machine learning (ML) PK model that corrects for differences between rFVIII concentrates and measurement assays. Next, we augment this model with a generative model that can simulate realistic virtual patients as well as impute missing data. This model can be shared instead of actual patient data, resolving privacy issues. The hybrid ML-PK model was trained on chromogenic assay data of lonoctocog alfa and predictive performance was then evaluated on an external data set of patients who received octocog alfa with FVIII levels measured using the one-stage assay. The model presented higher accuracy compared with three previous PK models developed on data similar to the external data set (root mean squared error = 14.6 IU/dL vs. mean of 17.7 IU/dL). Finally, we show that the generative model can be used to accurately impute missing data (< 18% error). In conclusion, the proposed approach introduces interesting new possibilities for model development. In the context of rare disease, the introduction of generative models facilitates sharing of synthetic data, enabling the iterative improvement of population PK models.

The influence of dead space in blood sampling needle on FVIII level and pharmacokinetic profiles in children with hemophilia

ABSTRACT Objective To investigate the influence of the dead space in disposable blood sampling needle on activated partial thromboplastin time (APTT), FVIII level and pharmacokinetic (PK) profiles in children with hemophilia. Methods Children (<18 years) with severe hemophilia A were enrolled. After three days’ washout-period, blood samples were collected at pre-dose, 1 h, 3 h, 9 h, 24 h and 48 h post-infusion. At each timepoint, two 2 mL vacuum tubes with 3.2% trisodium citrate were used. The first tube was signed as ‘non-standard’ (NS) and the second tube was signed as ‘standard’ (S). FVIII activities were evaluated by one-stage assay. WAPPS-Hemo was used to generate PK profiles like half-life time (t1/2), clearance (CL), trough level and time to 1, 2 and 5IU/dL after a dose of 50 ± 10IU/dL. The FVIII activities at 9 h and 24 h post-infusion were put into WAPPS and thus brought four combinations by true or biased FVIII level that used. Result Compared with standard-collected blood samples, prolonged APTT results (P-values < 0.01) and decreased FVIII activity (P-values < 0.05) were revealed in those non-standard blood samples. The corresponding bias was in positive relation to both APTT-S (r = 0.44, P < 0.0001) and FVIII-S level(r = 0.68, P < 0.001). The FVIII bias percentage got larger as FVIII-S level reduced (r = −0.24, P < 0.01). During the four combinations of FVIII activity at 9 h and 24 h, statistically longer t1/2, lower CL and longer time to 1, 2 or 5IU/dL were observed in 9H-S&24H-S group and 9H-NS&24H-S group. Conclusion While using vacuum tubes for clotting indicators and PK profiles, the dead space of blood sampling needle should be eliminated in advance.

Bleeding Outcomes in Participants with Factor VIII Activity &amp;lt;5 IU/DL Post-Gene Transfer in GENEr8-1

Background: Valoctocogene roxaparvovec is expected to transfer a functional FVIII coding sequence that would eliminate the need for regular prophylaxis for people with severe hemophilia A (HA). At the same valoctocogene roxaparvovec dose, FVIII levels varied among participants in the phase 3 GENEr8-1 trial. The protective effect of low transgene-derived FVIII is unknown. Aim: To determine clinical outcomes for participants with low FVIII activity 3 years post-gene transfer in GENEr8-1. Methods: In the open-label, single-arm, phase 3 GENEr8-1 trial (NCT03370913), 134 adult males (intention-to-treat [ITT] population) with severe HA (FVIII activity &amp;lt;1 IU/dL) previously receiving regular FVIII prophylaxis and with no history of FVIII inhibitors received a single dose of 6x10 13 vg/kg valoctocogene roxaparvovec. Plasma FVIII activity was measured via chromogenic substrate assay (reported in text; lower limit of quantitation [LLOQ], 1.5 [previously 3.0] IU/dL) or one-stage assay (LLOQ, 1.0 IU/dL). For participants who resumed prophylaxis, the valid FVIII measurement closest to, but not after, return to prophylaxis (RTP) is reported. Presented FVIII activity at a visit week is the median value in the 4- or 6-week window around the target date. Week 156 FVIII activity was imputed as 0 for participants who discontinued; other missing FVIII measurements were imputed as the smaller of valid values immediately prior to or post week 156. Bleeds were self-reported during baseline and after cessation of regular FVIII prophylaxis (scheduled for week 4 post-infusion). RTP was defined per-protocol as usual FVIII prophylaxis administered ≥1 time/week for ≥4 consecutive weeks or ≥2 emicizumab injections/month. Outcomes are reported for up to 156 weeks. Results: Of 134 ITT participants, 131 completed the week 156 visit. Mean (standard deviation [SD]) FVIII activity at week 156 was 18.2 (30.6) IU/dL in the ITT population. At week 156, 46 of 134 (34.3%) ITT participants had median FVIII activity &amp;lt;5 IU/dL (range, 0 to 4.9 IU/dL). These 46 participants had annualized bleeding rate (ABR) for treated spontaneous and traumatic bleeds at baseline between 0 and 27.5 (mean, 4.8) bleeds/year and from post-prophylaxis to week 156 between 0 and 12.0 (mean, 1.7) bleeds/year. Of these 46 participants, 8 resumed prophylaxis before week 156 (range, 58-141 weeks; Figure 1) and 38 did not RTP before week 156 ( Figure 2). The 8 participants who resumed prophylaxis had FVIII activity between &amp;lt;1.5 and 4.5 IU/dL proximal to RTP; the 38 who did not RTP had median FVIII activity between 0 and 4.9 IU/dL at week 156. At week 156, 3 of 8 and 15 of 38 participants who did and did not RTP had FVIII of 0 IU/dL, respectively. For the 8 participants who resumed prophylaxis before week 156, baseline ABR for treated bleeds ranged from 0.0 to 22.0 (mean, 8.5) bleeds/year, compared with 0 to 27.5 (mean, 4.0) bleeds/year for the 38 participants who did not RTP before week 156. Treated ABR from post-prophylaxis to RTP ranged from 0.9 to 13.4 (mean, 4.8) bleeds/year for the 8 participants who resumed prophylaxis before week 156, compared with 0 to 12.0 (mean, 1.0) bleeds/year from post-prophylaxis to week 156 for the 38 participants who did not RTP before week 156. For 2 of 8 participants who resumed prophylaxis before week 156, treated ABR was higher during the post-prophylaxis period up to RTP than at baseline; treated ABR was higher post-prophylaxis to week 156 compared with baseline for 5 of 38 participants who did not RTP before week 156. Most of the 38 participants who did not RTP before week 156 had lower ABR for treated bleeds compared with baseline, low post-prophylaxis ABRs for treated bleeds, or no substantial treated spontaneous bleeds. Decisions to not RTP were multifactorial and based on participant-investigator shared decision making; narratives outlining determinants that may have influenced decisions around prophylaxis for participants who had week 156 FVIII activity &amp;lt;5 IU/dL will be presented. Conclusions: Most participants with low FVIII activity had low bleeding rates, suggesting that low endogenous FVIII expression may provide protective hemostatic benefits. Many participants who resumed prophylaxis had clinical presentation consistent with moderate hemophilia; the individual decision to RTP was multifactorial and influenced by FVIII activity, bleeding rates, desired physical activity levels, and personal preferences.

Immunogenicity of a Plasma-Derived Von Willebrand Factor/Factor VIII Concentrate during the Wil-31 Prophylaxis Study in Patients with Von Willebrand Disease

Background: Long-term prophylaxis with a von Willebrand factor (VWF) concentrate is recommended in people with von Willebrand disease (VWD) with a history of frequent and severe bleeds. The WIL-31 study demonstrated the efficacy of prophylaxis with a plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrate containing VWF and FVIII in a 1:1 activity ratio (wilate ®) in adults and children with VWD of all types. The primary endpoint was met, showing a decrease in mean total annualized bleeding rate (ABR) of 84% compared with prior on-demand treatment. Aim: To assess the immunogenicity of pdVWF/FVIII when used during the WIL-31 study in children and adults with VWD of all types. Methods: WIL-31 (NCT04052698) was a prospective, non-controlled, international, multicenter phase 3 trial that enrolled male/female patients aged ≥6 years with VWD type 1 (VWF: RCo &amp;lt;30 IU/dL), type 2 (except 2N) or type 3. Prior to entering the WIL-31 study, all patients had received on-demand treatment with a VWF-containing product during a 6-month, prospective, observational, run-in study (WIL-29); patients who experienced at least 6 bleeding episodes (BEs), excluding menstrual bleeds, of which ≥2 were treated with a VWF-containing product, were eligible to enter WIL-31. During WIL-31, patients received regular pdVWF/FVIII (containing VWF and FVIII in a 1:1 activity ratio) prophylaxis 2-3 times per week at a dose of 20-40 IU/kg for 12 months. FVIII (chromogenic and one-stage assays) and VWF (VWF:RCo assay) activities were assessed at baseline and throughout the study. Retention samples were collected at screening and baseline visits for possible later inhibitor testing (to be performed at a central laboratory using the Nijmegen-Bethesda assay for FVIII inhibitors and the mixing study based on VWF:RCo for VWF inhibitors). Inhibitor testing was also performed if inhibitor development was suspected. Results: Inhibitor testing was performed in 2 of 33 patients because of unusual pharmacokinetic results at the 6-month visits, even though neither patient had any clinical signs of an inhibitor. The VWF inhibitors were detected in samples from two brothers taken at their 6-month visits. However, when retained blood samples from the screening visits of both boys were tested, both samples tested inhibitor-positive, demonstrating that both boys had developed the inhibitors prior to starting prophylaxis during WIL-31. During and before the WIL-29 run-in study, both boys had been treated on demand with another pdVWF/FVIII concentrate. The boys had both been diagnosed with VWD shortly after birth, and it was suspected that they had type 2A based on family history and were 15 (patient A) and 11 years old (patient B) at the start of WIL-31. They had a family history of VWD (both parents were diagnosed with VWD), but no family history of inhibitors. After the completion of WIL-31, both patients were reclassified to type 3 VWD following further investigation including next-generation sequencing. Baseline VWF activity indicated low VWF:RCo levels (Patient A: 0.092 IU/mL and Patient B: 0.65 IU/mL; Figure 1). The boys received 38 (patient A) and 34 IU/kg (patient B) pdVWF/FVIII respectively, twice weekly during WIL-31, and their mean total ABRs decreased by 85% and 91%, respectively, during WIL-31 compared with WIL-29. All bleeding episodes during WIL-31 were spontaneous nose bleeds. Both boys continued the same prophylactic regimen until the end of WIL-31. In one patient the inhibitor titer was negative by the end of the study. Conclusion: VWF inhibitors were detected in 2 of the 33 patients on prophylaxis with pdVWF/FVIII in the WIL-31 study and subsequent investigation revealed that both patients were already positive for VWF inhibitors prior to the study start. In one of the patients, the inhibitor resolved during continued prophylaxis. Despite the inhibitors, prophylaxis was effective in both patients with an ≥85% reduction in ABR compared to on demand treatment.

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.

PB1112 UK National External Quality Assessment Scheme for Blood Coagulation (UK NEQAS BC) Emicizumab Programme, Survey July 2022 Results for Modified One-Stage Assay and Chromogenic Assay

PB1112 UK National External Quality Assessment Scheme for Blood Coagulation (UK NEQAS BC) Emicizumab Programme, Survey July 2022 Results for Modified One-Stage Assay and Chromogenic Assay

A-160 Factor VIII Chromogenic Low Activity Sample Analysis in a Low Calibration Curve Model

Abstract Background Adequate laboratorial diagnostics of hemophilia is essential and could be done by chromogenic and one-stage assays based on APTT methodologies. Adverse effects of medications became less severe recently, but it remains important to be aware of to ensure that treatment is effective and ensure the best possible conditions for affected individuals. Classification of hemophilia according to FVIII activity: mild 25%–5%; moderate, 5%–1% and severe &amp;lt;1%. For new therapies such as emicizumab and some drugs with an extended half-life, the World Federation of Hemophilia recommends the use of a chromogenic assay of FVIII containing bovine FX for monitoring factor FVIII activity. Objetive: evaluate the applicability of a low calibration curve (LCC) in the chromogenic factor VIII (FVIII-Chr) assay by standardizing the dilution of the standard (calibrator) and thus ensuring the accuracy of the analyses in the lower values like severe hemophilia range. Methods The samples were obtained from H&amp;H LAB SAS, which guarantees all requirements. Sysmex® CS 2100i Siemens Healthineers. Calibration was performed with Standard Human Plasma (SHP), together with FVIII-Chr. Quality Control (QC); the accuracy of the LCC was checked. Results Preparation of the LCC: SHP reconstitution, followed by 1:10 dilution between the SHP and the plasma deficient in FVIII. The curve was named FVIII-Chr Low. QC was performed through pathological control (PC) with 2 dilutions: 1) identical to the formation of the low curve, i.e., dilution 1:10 (2.3%–3.9%). 2) dilution 1:30 (0.2%–2%) to accommodate the range of activity comprising severe hemophilia. The values found confirms the curve’s quality. Discussion This study sought to develop a way to detect FVIII activities &amp;lt;1.5% in a reproductive manner, even with a reduced “N” number of samples for severe state. The chromogenic test presents a limit of detection for the optical density of D.O. 0.0080 reporting &amp;lt;0,3% FVIII-Chr activity, which is very close to the most diluted point of the LCC. Conclusion QC performance with PC 1/10 and 1/30 showed excellent reproducibility. With the protocol FVIII Low and LCC (SHP + PDF) it is possible to obtain results of FVIII-Chr &amp;lt;1.0% compatible patients with severe hemophilia clinical condition.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

High variability in Factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system.

Haemophilia B (HB) is an X-linked hereditary bleeding disorder characterized by coagulation factor IX (FIX) deficiency. To improve the quality of life of patients and adherence to treatment, recombinant factor concentrates modified to extend their half-life have been developed, called extended half-life factors (EHL: extended half-life). Nonacog beta pegol (N9-GP) is a glycopegylated recombinant human FIX molecule that has a half-life of 93 h with a single dose and has shown a higher recovery percentage than other molecules. To diagnose and monitor the treatment of haemophiliac patients, FIX activity is determined with the one-stage clotting assay (OSA) and/or the chromogenic assay. The objective of this work, carried out in three centres, was to measure the recovery of N9-PG with 10 different activated partial thromboplastin time (APTT) reagents on three platforms, in samples spiked in vitro with N9-GP, at four different concentration levels. It was measured the recovery of N9-GP with 10 different APTT reagents (polyphenol, ellagic acid, silice dioxide, colloidal silica as APTT activator on three platforms, in sample spiked in vitro with N9-GP. The results show heterogeneity in the activity of N9-GP measured by OSA with the different APTT reagents when the calibrations were performed with the specific calibrator of each coagulometer. A recovery percentage between 87% and 108% was obtained only with polyphenol and ellagic acid as activator in the three platforms evaluated. The other reagents studied overestimate or underestimate, with no clear profile. When a calibration curve was performed with a calibrator prepared from the N9-GP vial, all APTT reagents met the established recovery requirement. APTT reagents with polyphenol or ellagic acid as activator would be the only ones appropriate when using the commercially available OSA with specific calibrator to monitor patients treated with N9-GP.

Relationship between factor VIII levels and bleeding for rFVIII-SingleChain in severe hemophilia A: A repeated time-to-event analysis.

Publications on the exposure-effect relationships of factor concentrates for hemophilia treatment are limited, whereas such analyses give insight on treatment efficacy. Our objective was to examine the relationship between the dose, factor VIII (FVIII) levels and bleeding for rFVIII-SingleChain (lonoctocog alfa, Afstyla). Data from persons with severe hemophilia A on rFVIII-SingleChain prophylaxis from three clinical trials were combined. The published rFVIII-SingleChain population pharmacokinetic (PK) model was evaluated and expanded. The probability of bleeding was described with a parametric repeated time-to-event (RTTE) model. Data included 2080 bleeds, 2545 chromogenic stage assay, and 3052 one-stage assay FVIII levels from 241 persons (median age 19 years) followed for median 1090 days. The majority of the bleeds occurred in joints (65%) and the main bleeding reason was trauma (44%). The probability of bleeding decreased during follow-up and a FVIII level of 8.9IU/dL (95% confidence interval: 6.9-10.9) decreased the bleeding hazard by 50% compared to a situation without FVIII in plasma. Variability in bleeding hazard between persons with similar FVIII levels was large, and the pre-study annual bleeding rate explained part of this variability. When a FVIII trough level of 1 or 3IU/dL is targeted during prophylaxis, simulations predicted two (90% prediction interval [PI]: 0-17) or one (90% PI: 0-11) bleeds per year, respectively. In conclusion, the developed PK-RTTE model adequately described the relationship between dose, FVIII levels and bleeds for rFVIII-SingleChain. The obtained estimates were in agreement with those published for the FVIII concentrates BAY 81-8973 (octocog alfa) and BAY 94-9027 (damoctocog alfa pegol), indicating similar efficacy to reduce bleeding.

POSTER ABSTRACTS

POSTER ABSTRACTS

Treatment changes in hemophilia A with chromogenic factor VIII assay implementation

BackgroundThe one-stage assay (OSA) and the chromogenic assay (CSA) are 2 factor VIII (FVIII) assays used for the diagnosis and classification of hemophilia A. Discrepancies between the 2 assays exist in approximately one-third of patients with mild hemophilia A. ObjectivesThe objectives of this study were to report the proportion of patients with mild or moderate hemophilia A and OSA-CSA discrepancies and to report the observed changes in treatment approach prompted by the presence of assay discrepancy. The study aimed to identify OSA:CSA ratio associated with the highest sensitivity for identification of patients in whom modification of treatment approach may be recommended. MethodsThis is a retrospective cohort study including adult (>18-year-old) patients with mild or moderate hemophilia A who were followed up at the Adult British Columbia Hemophilia Program between January 2013 and March 2019. ResultsA total of 75 patients with mild and 23 with moderate hemophilia A based on baseline OSA were included. Overall, 52% of study patients had OSA-CSA discrepancies, and change in treatment approach was observed in 27% of patients with OSA-CSA discrepancy. The OSA:CSA ratio of 1.8 to 3.5 demonstrated the highest area under the receiver operating characteristics curve and sensitivity for identification of patients in which modification of treatment approach may be recommended (AUC 0.75; sensitivity 71%). ConclusionIn our population, OSA-CSA discrepancy was observed in 52% of patients with mild or moderate hemophilia A, and the treatment approach in 27% of these patients had to be modified.

Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency

BackgroundThe mixing test is useful to investigate the cause of unexpectedly prolonged activated partial thromboplastin time (APTT). Several indexes are available for distinguishing correction from non-correction (ie, factor deficiency from inhibitors), but their performance characteristics may differ because of their different formulas. Furthermore, it is unclear how each index performs under the coexistence of factor deficiency and inhibitors. ObjectivesThe objective of this study was to examine the differences in indexes, depending on factor VIII activity (FVIII:C) levels and lupus anticoagulant (LA) titers in test samples. MethodsAPTT was measured in spiked samples with various FVIII:C levels and LA titers, normal pooled plasma (NPP), and their 4:1, 1:1, and 1:4 mixtures. The following 5 indexes were calculated: index of circulating anticoagulant, mixing test normalized ratio, 4:1 and 1:1 percent corrections, and an APTT difference between the 1:1 mixture and NPP. The samples with LA, showing correction, were measured for FVIII:C in a one-stage assay to check parallelism. ResultsAll indexes showed correction under FVIII deficiency and non-correction under higher LA titers. However, under lower LA titers, some indexes showed non-correction but others showed correction because of dilution effects and variations in formulas and/or sample mix ratios. The differences among the indexes were more pronounced under coexistent FVIII deficiency and LA, even though LA titers were equal in the tested samples; samples with lower FVIII:C showed correction, whereas those with normal FVIII:C showed non-correction. The samples tested for FVIII:C showed non-parallelism. ConclusionEach index had different performance characteristics to LA samples, which were pronounced under low FVIII:C levels in test samples.

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.

The effect of a next generation factor VIII mimetic bispecific antibody (Mim8) on assays of factor VIII activity and thrombin generation

BackgroundMim8 is a next generation bispecific antibody developed for the prophylactic treatment of hemophilia A. The sensitivity of activated plasma thromboplastin time (APTT), assays measuring factor VIII activity (FVIII:C) and thrombin generation to plasma containing Mim8 was assessed. MethodsCongenital severe hemophilia A plasma was spiked with Mim8 at 0 μg/mL to 20 μg/mL. APTT was measured with 4 reagents, one-stage FVIII with 9 reagents, and chromogenic FVIII with 6 assays. Thrombin generation was assessed in a low tissue factor system. ResultsAt 1-μg/mL Mim8, the APTT was shortened to within normal limits and one-stage FVIII:C was >1.00 IU/mL with all APTT reagents. Modified one-stage assays calibrated with Mim8 reference material calibrated recovered within 20% of the target Mim8 concentration with most APTT reagents. Factor VIII:C of bovine only chromogenic assays was <0.04 IU/mL at all Mim8 concentrations. Factor VIII:C of human only chromogenic assay was >4.00 IU/mL at 20-μg/mL Mim8. Factor VIII:C of hybrid bovine FX, human FIXa chromogenic assays ranged from 0.076 to >3.00 IU/mL at 20-μg/mL Mim8. Normal thrombin generation was restored at 5-μg/mL Mim8. ConclusionsAPTT-based assays are sensitive to Mim8 and should not be performed in the presence of the drug. Chromogenic assays containing human proteins or hybrid human/bovine proteins demonstrated variable sensitivity to Mim8. Bovine only chromogenic assays were largely insensitive to the presence of Mim8. Thrombin generation normalized at increased Mim8 concentrations. Modified one-stage and chromogenic assays could be used to quantify the Mim8 concentration in plasma.

Bleeding Phenotype Does Not Appear to Correlate with Factor VIII Levels or Genetic Mutation in an Exclusive Pediatric Cohort of Hemophilia a Carriers

Introduction: Inherited Hemophilia A is defined as Factor VIII (FVIII) deficiency, with levels <0.40 IU/ml. Female carriers exhibit varying degrees of X chromosome inactivation, with resultant variable FVIII activity levels. Bleeding phenotype in Hemophilia A carriers has been thought to be due to low FVIII levels; however, adult studies have shown lack of correlation of FVIII levels and bleeding phenotype in some patients. Our study aimed to correlate FVIII levels with bleeding phenotype and genetic mutations in pediatric hemophilia A carriers. Methods: Retrospective medical record review of Hemophilia A carriers <18 years of age was undertaken at our center. Inclusion criteria were family history of Hemophilia A and FVIII mutation or FVIII level <0.40 IU/ml in patients without genetic testing done. Patients with other co-existing bleeding disorders were excluded. Statistical analysis was done by Chi Square/Exact tests. Results: Twenty-three pediatric Hemophilia A carriers were eligible, with median age of 12 years (range1-16). Fifteen were adolescents >10 years. Fourteen (60.9%) had FVIII level >0.40 IU/ml with 8 (57.1%) categorized as asymptomatic, and 6 (42.9%) symptomatic carriers in accordance with the new ISTH nomenclature. Nine (39.1%) patients had FVIII <0.40 IU/ml, with 8 (88.8%) categorized as mild (FVIII 0.05-0.40 IU/ml), 1 (11.1%) as moderate (FVIII 0.01-0.05 IU/ml), and none as severe (<0.01 IU/ml) Hemophilia A, as per the new nomenclature. Bleeding Scores (BS) were obtained in all patients using the ISTH Bleeding Assessment Tool (ISTH-BAT) with a median BS of 1 (range 0-7). A significant BS of >2 was present in 4/9 (44.4%) patients with FVIII <0.40 IU/ml, and 4/14 (28.6%) with FVIII >0.40 IU/ml, the difference was not statistically significant (p=0.40) (Table 1). Two symptomatic carriers with FVIII >0.4 IU/ml, had BS of 1 and 2. 5/15 adolescents (33.3%) had BS>2 (BS of 4 in 3; BS of 7 in 2). FVIII was measured with one stage assay (OSA) (N=17; 73.9%), chromogenic substrate assay (CSA) (N= 2; 8.7%) or both (N=4; 17.4%), with 1 patient's (4.3%) CSA discrepantly lower than OSA by 0.62 IU/ml and none with inverse discrepancy. Genetic testing was done in 20/23 (86.9%) patients, with FVIII missense mutations in 14 (70%), intron inversions in 4 (20%), and deletions in 2 (10%), their association with BS did not show statistically significant difference (p=0.6) (Table 2). Conclusion: Our study was done exclusively in pediatric Hemophilia A carriers, as it is important to identify children at risk of bleeding and institute preventive measures early. Unlike in adult women, higher number (one-third) of our patients experienced mucosal bleeding, and none reported joint or muscle bleeding. Our study showed lack of statistically significant association of FVIII levels with bleeding phenotype. According to the new ISTH nomenclature, 39.1% of our study patients have the diagnosis of Hemophilia A, of whom 55.6% did not have a significant BS. Of the 60.9% categorized as carriers, 28.6% had a significant BS. In addition, we found lack of statistically significant correlation of genetic mutation type with the bleeding phenotype, with none, 5% and 25% of the patients with deletion, intron inversion and missense mutation having a significant BS respectively. Follow-up/BS reassessment in our cohort is planned to monitor BS increase with age. Our study limitations include small cohort and retrospective analysis. Future pediatric studies are required to identify coagulation/genetic parameters that correlate better with bleeding phenotype, which may then help with early recognition and prompt institution of preventive therapy to pediatric Hemophilia A carriers at risk of bleeding, in order to avoid bleeding complications during adulthood. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal