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Issues complicating precision dosing for factor VIII prophylaxis

Open AccessPublished:July 15, 2018DOI:https://doi.org/10.1016/j.transci.2018.07.007

      Abstract

      We previously showed that personalizing prophylaxis on the basis of an individual’s pharmacokinetic (PK) response to factor VIII (FVIII) infusion reduces joint and other bleeding events in patients with hemophilia A. We theorized that the FVIII assay used, FVIII product selected, and interpatient differences impact PK assessment and the ability to precisely dose prophylaxis.
      A comprehensive search of the literature for articles published from January 2004 to September 2017 was performed to identify the variables associated with these three domains.
      Collectively, product- and patient-related assay discrepancies, variability among plasma-derived and unmodified and modified recombinant FVIII products, and interpatient differences in the response to FVIII infusions are obstacles to precision prophylactic dosing.
      Stringent laboratory quality assurance programs and proficiency testing to improve the accuracy of FVIII measurement, the widespread use of PK assessment to fine-tune FVIII dosing, and new research to identify patient characteristics and other contributors to bleeding risk and complication development are essential to optimizing outcomes for patients with hemophilia A receiving FVIII prophylaxis.

      Keywords

      1. Introduction

      Prophylaxis is recommended as optimal care for children and adults with severe hemophilia A [
      • National Hemophilia Foundation Medical and Scientific Advisory Council
      MASAC recommendation concerning prophylaxis (regular administration of clotting factor concentrate to prevent bleeding). MASAC document 170.
      ,
      • Srivastava A.
      • Brewer A.K.
      • Mauser-Bunschoten E.P.
      • Key N.S.
      • Kitchen S.
      • Llinas A.
      • et al.
      Guidelines for the management of hemophilia.
      ], yet a generic approach is not ideal, possibly leading to inefficient or unnecessarily costly dosing. Using an individual’s pharmacokinetic (PK) response to factor VIII (FVIII) infusions to calculate the dose and dosing frequency is one strategy for personalizing the prophylactic regimen [
      • Bjorkman S.
      Prophylactic dosing of factor VIII and factor IX from a clinical pharmacokinetic perspective.
      ,
      • Bjorkman S.
      Limited blood sampling for pharmacokinetic dose tailoring of FVIII in the prophylactic treatment of haemophilia A.
      ,
      • Valentino L.A.
      Considerations in individualizing prophylaxis in patients with haemophilia A.
      ]. We previously showed that PK-guided prophylaxis is as effective as standard thrice-weekly prophylaxis in preventing joint and other bleeding events and may allow the dosing interval to be extended [
      • Valentino L.A.
      • Mamonov V.
      • Hellmann A.
      • Quon D.V.
      • Chybicka A.
      • Schroth P.
      • et al.
      A randomized comparison of two prophylaxis regimens and a paired comparison of on-demand and prophylaxis treatments in hemophilia A management.
      ]. Yet PK is not immutable, and the FVIII assay used, FVIII product selected, and interpatient differences all have the potential to impact the PK assessment and, thus, the ability to precisely dose prophylaxis.
      Here, we consider the influence of these three domains on FVIII PK and examine how they may complicate efforts at precision prophylactic dosing for patients with hemophilia A. In addition, we speculate on opportunities to reduce the influence of these factors with the intent of determining a more precise dosing strategy.

      2. Methods

      We conducted a search of the Medline database from January 2004 to September 2017 for English-language articles pertaining to variability in FVIII assays, FVIII products, and patients with hemophilia A. To remedy the lack of systematic studies investigating the influence of von Willebrand factor (VWF) on assays of FVIII-deficient plasma, we performed a one-stage clotting assay according to a pharmacopoeia method [
      • Turecek P.L.
      • Mitterer A.
      • Matthiessen H.P.
      • Gritsch H.
      • Varadi K.
      • Siekmann J.
      • et al.
      Development of a plasma- and albumin-free recombinant von Willebrand factor.
      ]. The assay was run on a coagulation analyzer and was based on the activated partial thromboplastin time (aPTT) clotting assay. Equal volumes of activator reagent, FVIII-deficient plasma, and diluted test sample were incubated at 37 °C for 4 min until a complex was formed, after which calcium chloride was added. Time to clot formation was recorded, and a reference curve constructed from different dilutions of the World Health Organization (WHO) 8th International Standard FVIII Concentrate was used to assign FVIII activity from unknown samples. A commercially available FVIII-deficient plasma devoid of VWF was supplemented with increasing amounts of a highly pure recombinant VWF (rVWF) preparation. This plasma was used for preparation of the predilution to 1 international unit (IU) FVIII/mL for both the standard and test sample. A commercial FVIII-deficient plasma with a high VWF concentration was used as the control.

      3. Results

      3.1 Selection of an FVIII assay

      FVIII primarily functions as a cofactor for activated factor IX (FIXa) in the activation of factor X (FXa). This activity can be measured downstream of the coagulation cascade by monitoring clot generation, or upstream by directly measuring FXa enzymatic activity following activation by the FVIIIa-FIXa (tenase) complex [
      • Lippi G.
      • Franchini M.
      • Favaloro E.J.
      One-stage clotting versus chromogenic assays for assessing recombinant factor VIII: two faces of a haemostasis coin.
      ]. The accurate assessment of FVIII coagulant activity (FVIII:C) is essential for: 1) establishing the diagnosis of hemophilia A; 2) determining the severity of the bleeding phenotype; 3) identifying the post-infusion bioavailability and PK of FVIII concentrates; 4) monitoring FVIII replacement therapy (including perioperative) and prophylaxis; and 5) assigning potency to FVIII concentrates, which defines the quantity of active ingredient in a vial of drug product, guides dosing, determines pricing, and is required by regulatory authorities for product release [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ,
      • Peyvandi F.
      • Oldenburg J.
      • Friedman K.D.
      A critical appraisal of one-stage and chromogenic assays of factor VIII activity.
      ].

      3.1.1 Types of assays

      Three assays are currently available for measuring FVIII in biologic samples: the one-stage (OSA) and two-stage (TSA) clotting assays and the chromogenic substrate assay (CSA) (Fig. 1A-C). All measure FVIII:C in generating FXa in the presence of calcium ions and phospholipids [
      • Fay P.J.
      Factor VIII structure and function.
      ,
      • Lenting P.J.
      • van Mourik J.A.
      • Mertens K.
      The life cycle of coagulation factor VIII in view of its structure and function.
      ]. Because the TSA is more difficult to automate than the OSA, and since the principle and results for the TSA are similar to the CSA, the OSA and CSA are preferentially used [
      • Peyvandi F.
      • Oldenburg J.
      • Friedman K.D.
      A critical appraisal of one-stage and chromogenic assays of factor VIII activity.
      ,
      • Rodgers S.E.
      • Duncan E.M.
      • Barbulescu D.M.
      • Quinn D.M.
      • Lloyd J.V.
      In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results.
      ].
      The OSA (Fig. 1A) is based on correction of the prolonged aPTT by dilution of the patient’s plasma into substrate plasma from a FVIII-deficient individual or normal plasma made FVIII deficient via immunochemical absorption, both of which are phenotypically variable [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ]. This assay relies on the intrinsic coagulation pathway, in which the reaction is initiated by a contact activator (ie, ellagic acid, kaolin, silica, Celite®, polyphenols), is propagated by natural or synthetic phospholipids, and ends with fibrinogen clotting [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ,
      • Peyvandi F.
      • Oldenburg J.
      • Friedman K.D.
      A critical appraisal of one-stage and chromogenic assays of factor VIII activity.
      ,
      • Moser K.A.
      • Adcock Funk D.M.
      Chromogenic factor VIII activity assay.
      ]. Owing to its simplicity, the availability of reagents, ease of automation, and lower cost relative to the CSA, the OSA is used by the vast majority of clinical laboratories worldwide [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ,
      • Potgieter J.J.
      • Damgaard M.
      • Hillarp A.
      One-stage vs. chromogenic assays in haemophilia A.
      ,
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ] and is required by the US Food and Drug Administration for the assignment of FVIII concentrate potency [
      • Makris M.
      • Peyvandi F.
      Assaying FVIII activity: one method is not enough, and never was.
      ]. Accurate potency assignment is necessary to: 1) define the quantity of active substance in a vial, thereby ensuring safety and efficacy and allowing comparison among products; 2) uphold the integrity of the IU; and 3) guide dosing [
      • Hubbard A.R.
      • Dodt J.
      • Lee T.
      • Mertens K.
      • Seitz R.
      • Srivastava A.
      • et al.
      Recommendations on the potency labelling of factor VIII and factor IX concentrates.
      ].
      The CSA (Fig. 1C) is based upon quantitation of FXa as a measure of FVIIIa cofactor activity on FIXa [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ]. Two sequential reactions initially form FXa, after which a chromogenic substrate specific for FXa is enzymatically cleaved and photometrically quantified (Fig. 1B) [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ,
      • Peyvandi F.
      • Oldenburg J.
      • Friedman K.D.
      A critical appraisal of one-stage and chromogenic assays of factor VIII activity.
      ,
      • Seghatchian M.J.
      A new approach to the determination of coagulation factors using chromogenic substrates.
      ]. The CSA is the reference method for FVIII potency designation required by the European Pharmacopoeia and used by the International Society on Thrombosis and Haemostasis for this purpose [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ,
      • Makris M.
      • Peyvandi F.
      Assaying FVIII activity: one method is not enough, and never was.
      ].

      3.1.2 Assay limitations

      The OSA and CSA are compared in Fig. 2. A major challenge to the use of the FVIII OSA is marked variability among laboratories [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ,
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ,
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ,
      • Kitchen S.
      • Beckmann H.
      • Katterle Y.
      • Bruns S.
      • Tseneklidou-Stoeter D.
      • Maas Enriquez M.
      BAY 81-8973, a full-length recombinant factor VIII: results from an International comparative laboratory field study.
      ,
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ], which is related to differences in methodology (eg, source of aPTT reagents and phospholipid composition), instrumentation, calibration standards, and selected factor-deficient substrate plasma [
      • Barrowcliffe T.W.
      Standardization of FVIII & FIX assays.
      ,
      • Kitchen S.
      • Jennings I.
      • Preston F.E.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Interlaboratory variation in factor VIII:C inhibitor assay results is sufficient to influence patient management: data from the UK national quality external assessment scheme for blood coagulation.
      ,
      • Meijer P.
      • Verbruggen B.
      The between-laboratory variation of factor VIII inhibitor testing: the experience of the external quality assessment program of the ECAT foundation.
      ,
      • Barrowcliffe T.W.
      • Cattaneo M.
      • Podda G.M.
      • Bucciarelli P.
      • Lussana F.
      • Lecchi A.
      • et al.
      New approaches for measuring coagulation.
      ]. Additionally, aPTT-based assays are inherently subject to interference from lipids and small amounts of heparin contained in samples and are sensitive to the extent of FVIII preactivation.
      Fig. 2
      Fig. 2Comparison of the one-stage clotting assay (OSA) and the chromogenic substrate assay (CSA).
      The CSA shows no interference by heparins, direct thrombin inhibitors, or lupus anticoagulants [
      • Chandler W.L.
      • Ferrell C.
      • Lee J.
      • Tun T.
      • Kha H.
      Comparison of three methods for measuring factor VIII levels in plasma.
      ] and is thought to be more precise than the OSA [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]. Nonetheless, according to a recently published report, inter-laboratory variation is also a problem with the CSA [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ] and may be comparable to that observed with the OSA [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]. Because the CSA is more expensive and labor intensive to perform than the OSA, the availability of the CSA in clinical laboratories is limited. Batch testing and aliquoting of chromogenic kit reagents may improve CSA cost efficiency, thereby facilitating wider adoption of this method for assaying FVIII [
      • Kitchen S.
      • Blakemore J.
      • Friedman K.D.
      • Hart D.P.
      • Ko R.H.
      • Perry D.
      • et al.
      A computer-based model to assess costs associated with the use of factor VIII and factor IX one-stage and chromogenic activity assays.
      ].

      3.1.3 Influence of VWF

      VWF is the natural chaperone protein for FVIII, stabilizing it both in the circulation and in vitro. Sufficient amounts of VWF must be present in assay set-ups to ensure FVIII stability and guarantee valid test results [
      • Barrowcliffe T.W.
      Standardization of FVIII & FIX assays.
      ]. One source of VWF is the test sample itself, since human plasma, including that from patients with hemophilia A, typically contains a normal range of VWF levels (50–200%) [
      • Ng C.
      • Motto D.G.
      • Di Paola J.
      Diagnostic approach to von Willebrand disease.
      ], as an unidentified codeficiency of FVIII and VWF is highly unlikely [
      • Lindsay H.
      • Bergstrom K.
      • Srivaths L.
      Co-inheritance of mild hemophilia A and heterozygosity for type 2N von Willebrand disease: a diagnostic and therapeutic challenge.
      ]. Depending on whether the OSA or CSA is used, plasma samples are diluted to different concentrations of FVIII. As a result, the assay mixture may contain VWF levels below the critical concentration needed for FVIII stabilization.
      While dilutions in bioanalytical tests are usually performed using aqueous buffers, for FVIII determination, artificially depleted FVIII-deficient plasma is used [
      • Mazurier C.
      • Parquet-Gernez A.
      • Goudemand M.
      Validation of a procedure for potency assessing of a high purity factor VIII concentrate--comparison of different factor VIII coagulant assays and effect of prediluent.
      ,
      • Cinotti S.
      • Paladino E.
      • Morfini M.
      Accuracy of FVIII: C assay by one-stage method can be improved using hemophilic plasma as diluent.
      ]. The use of artificially depleted FVIII plasma preparations circumvents the need for plasma from patients with severe hemophilia A, the availability of which is limited by the number of affected patients and their increasing treatment with prophylactic regimens (meaning some circulating FVIII is always present). Methods for artificially depleting FVIII (eg, immunoadsorption) also deplete VWF, rendering the plasma preparation deficient in both FVIII and VWF [
      • Rothschild C.
      • Amiral J.
      • Adam M.
      • Meyer D.
      Preparation of factor-VIII-depleted plasma with antibodies and its use for the assay of factor VIII.
      ,
      • Barrowcliffe T.W.
      • Tubbs J.E.
      • Wong M.Y.
      Evaluation of factor VIII deficient plasmas.
      ]—information that may not be included in the label of FVIII-deficient plasma.
      The influence of VWF content in FVIII-deficient plasma on OSA results was studied using a commercially available FVIII-deficient plasma devoid of VWF and supplemented with increasing amounts of a highly purified rVWF preparation [
      • Turecek P.L.
      • Mitterer A.
      • Matthiessen H.P.
      • Gritsch H.
      • Varadi K.
      • Siekmann J.
      • et al.
      Development of a plasma- and albumin-free recombinant von Willebrand factor.
      ]. This plasma was used to dilute the standard and test samples to 1 IU FVIII/mL (Fig. 3). As the amount of rVWF in the FVIII-deficient plasma was increased, it produced a corresponding rise in FVIII potency at the same FVIII concentration, such that a relatively low concentration of VWF/mL (0.01 IU, the equivalent of 1% of normal human plasma) was sufficient to stabilize FVIII and achieve the expected FVIII concentration. These findings underscore the importance of using FVIII-deficient plasma containing adequate amounts of VWF [
      • Barrowcliffe T.W.
      Standardization of FVIII & FIX assays.
      ].
      Fig. 3
      Fig. 3Influence of von Willebrand factor (VWF) concentration on FVIII recovery in the OSA [
      • Turecek P.L.
      • Mitterer A.
      • Matthiessen H.P.
      • Gritsch H.
      • Varadi K.
      • Siekmann J.
      • et al.
      Development of a plasma- and albumin-free recombinant von Willebrand factor.
      ].

      3.1.4 Product-related assay discrepancies

      While both the OSA and CSA yield comparable results in patients treated with plasma-derived (pd) FVIII concentrates [
      • Pouplard C.
      • Caron C.
      • Aillaud M.F.
      • Ternisien C.
      • Desconclois C.
      • Dubanchet A.
      • et al.
      The use of the new ReFacto AF laboratory standard allows reliable measurement of FVIII:C levels in ReFacto AF mock plasma samples by a one-stage clotting assay.
      ], discrepancies have been observed following infusions of recombinant FVIII (rFVIII) [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ,
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ,
      • Mikaelsson M.
      • Oswaldsson U.
      Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products.
      ]. The level of discordance between the two methods does not typically exceed 20% in patients receiving full-length rFVIII products [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ]. Field studies of Advate (Shire), for example, show an OSA:CSA ratio of 1:1 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ] and in vivo recovery of approximately 100% [
      • Di Paola J.
      • Smith M.P.
      • Klamroth R.
      • Mannucci P.M.
      • Kollmer C.
      • Feingold J.
      • et al.
      ReFacto and Advate: a single-dose, randomized, two-period crossover pharmacokinetics study in subjects with haemophilia A.
      ], indicative of good agreement between the two assays. Discordance increases to up to 50%, however, when B-domain-deleted (BDD) rFVIII concentrate ReFacto (Pfizer) is infused (OSA:CSA ratio = 0.82 ± 0.12 for FVIII > 25 U/dL and 1.42 ± 0.99 for FVIII < 25 U/dL [
      • Morfini M.
      • Cinotti S.
      • Bellatreccia A.
      • Paladino E.
      • Gringeri A.
      • Mannucci P.M.
      • et al.
      A multicenter pharmacokinetic study of the B-domain deleted recombinant factor VIII concentrate using different assays and standards.
      ]) [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ,
      • Mikaelsson M.
      • Oswaldsson U.
      • Jankowski M.A.
      Measurement of factor VIII activity of B-domain deleted recombinant factor VIII.
      ]. This discrepancy can be attenuated through the use of a product-specific reference standard rather than a plasma reference standard [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ,
      • Morfini M.
      • Cinotti S.
      • Bellatreccia A.
      • Paladino E.
      • Gringeri A.
      • Mannucci P.M.
      • et al.
      A multicenter pharmacokinetic study of the B-domain deleted recombinant factor VIII concentrate using different assays and standards.
      ,
      • Ingerslev J.
      • Jankowski M.A.
      • Weston S.B.
      • Charles L.A.
      • ReFacto Field Study Participants
      Field Study Collaborative field study on the utility of a BDD factor VIII concentrate standard in the estimation of BDDr factor VIII:C activity in hemophilic plasma using one-stage clotting assays.
      ] and does not appear to be common to all FVIII products not encoded by the full FVIII sequence [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]. For example, N8 (Novo Nordisk), a new B-domain-deleted truncated product, can be adequately measured using both OSA and CSA systems [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ].

      3.1.5 Patient-related assay discrepancies

      Individuals with severe hemophilia A who share a similar genetic defect(s) may have heterogeneous clinical phenotypes [
      • Gissel M.
      • Whelihan M.F.
      • Ferris L.A.
      • Mann K.G.
      • Rivard G.E.
      • Brummel-Ziedins K.E.
      The influence of prophylactic factor VIII in severe haemophilia A.
      ,
      • Barnes C.
      • Blanchette V.
      • Lillicrap D.
      • Mann K.
      • Stain A.M.
      • Leggo J.
      • et al.
      Different clinical phenotype in triplets with haemophilia A.
      ]. Such diversity may be explained by differing levels of prothrombin and antithrombin concurrent with the aggregate influence of normal range variation in factors other than FVIII that impact thrombin-generating capacity [
      • Gissel M.
      • Whelihan M.F.
      • Ferris L.A.
      • Mann K.G.
      • Rivard G.E.
      • Brummel-Ziedins K.E.
      The influence of prophylactic factor VIII in severe haemophilia A.
      ].
      Interindividual differences in basal plasma concentrations of clotting factors are well recognized. For example, among healthy adults, FX levels range from 90% to 149% of normal and protein C from 78% to 148% of normal, with age and physiologic development contributing to this heterogeneity [
      • Appel I.M.
      • Grimminck B.
      • Geerts J.
      • Stigter R.
      • Cnossen M.H.
      • Beishuizen A.
      Age dependency of coagulation parameters during childhood and puberty.
      ]. Such differences may also contribute to FVIII (or FIX) PK discrepancies among patients with hemophilia. One study found that patients with severe hemophilia A or B had basal FVIIa levels that were 40% and 90% lower, respectively, than normal [
      • Wildgoose P.
      • Nemerson Y.
      • Hansen L.L.
      • Nielsen F.E.
      • Glazer S.
      • Hedner U.
      Measurement of basal levels of factor VIIa in hemophilia A and B patients.
      ]. The contributions of other clotting factors to phenotypic variability and PK require additional study.
      In 10% to nearly 50% of persons with mild hemophilia A (baseline FVIII > 5% to < 40% of normal [
      • White G.C.
      • Rosendaal F.
      • Aledort L.M.
      • Lusher J.M.
      • Rothschild C.
      • Ingerslev J.
      Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis.
      ]), FVIII:C levels are higher with the OSA compared with the CSA [
      • Duncan E.M.
      • Rodgers S.E.
      • McRae S.J.
      Diagnostic testing for mild hemophilia A in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays.
      ,
      • Bowyer A.E.
      • Van Veen J.J.
      • Goodeve A.C.
      • Kitchen S.
      • Makris M.
      Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A.
      ,
      • Cid A.R.
      • Calabuig M.
      • Cortina V.
      • Casana P.
      • Haya S.
      • Moret A.
      • et al.
      One-stage and chromogenic FVIII:C assay discrepancy in mild haemophilia A and the relationship with the mutation and bleeding phenotype.
      ,
      • Pavlova A.
      • Delev D.
      • Pezeshkpoor B.
      • Muller J.
      • Oldenburg J.
      Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays.
      ,
      • Poulsen A.L.
      • Pedersen L.H.
      • Hvas A.M.
      • Poulsen L.H.
      • Thykjaer H.
      • Ingerslev J.
      Assay discrepancy in mild haemophilia A: entire population study in a National haemophilia Centre.
      ,
      • Trossaert M.
      • Boisseau P.
      • Quemener A.
      • Sigaud M.
      • Fouassier M.
      • Ternisien C.
      • et al.
      Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity.
      ,
      • Goodeve A.C.
      • Peake I.R.
      The molecular basis of hemophilia A: genotype-phenotype relationships and inhibitor development.
      ], which can result in missed diagnoses or under-estimation of bleeding risk [
      • Potgieter J.J.
      • Damgaard M.
      • Hillarp A.
      One-stage vs. chromogenic assays in haemophilia A.
      ]. The discordance between the two assays is attributable to genetic mutations clustered in the A1, A2, and A3 domains of the F8 molecule that impair FVIII stability [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ]. Less common mutations observed in patients with non-severe hemophilia primarily affect thrombin activation and FVIII binding to FIXa in the assembly of the tenase complex. These alterations are partially responsible for the inverse discrepancy, where FVIII:C levels are higher with the CSA than with the OSA [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ].

      3.2 Variability in FVIII products

      The FVIII molecule is synthesized as a 2329 single-chain amino acid protein composed of six domains: A1-A2-B-A3-C1-C2 [
      • Fay P.J.
      Factor VIII structure and function.
      ]. It is processed extensively during secretion and transport to provide a heterogeneous group of molecules from which portions of the B-domain are removed [
      • Venkateswarlu D.
      Structural investigation of zymogenic and activated forms of human blood coagulation factor VIII: a computational molecular dynamics study.
      ]. Owing to this extensive protein processing, mature FVIII, unlike other proteins, does not have a single molecular weight, a characteristic that influences its pharmacologic properties. Ideally, FVIII formulation would be based on the number of moles of FVIII infused and the activity of FVIII per mole, thereby allowing infusions to be calculated according the moles or mass infused, as is used for other drugs. FVIII potency assignment is based on units and is standardized and controlled through international scientific committees and the WHO with the aim of standardizing the FVIII content of a pharmaceutical’s formulation in relation to internationally calibrated reference preparations.
      The total mass of FVIII molecules and their activities may change during circulation, the result of transport-related fragmentation and alterations in binding affinity [
      • Lenting P.J.
      • van Mourik J.A.
      • Mertens K.
      The life cycle of coagulation factor VIII in view of its structure and function.
      ]. The natural breakdown processes affecting these molecules include selective limited proteolysis, resulting in degradation and spontaneous dissociation of FVIIIa. Both processes are complex, governed by enzymatic reactions and subunit separation and the involvement of FIXa in FVIII stabilization and inactivation. Enzymatic cleavage in the FVIIIa heavy chain by FIXa, FXa, and activated protein C (APC) limits the availability of FVIIIa—both in terms of mass and time—to participate in the tenase complex. In addition, the FVIIIa molecule is intrinsically unstable because of the weak interaction between the A2 domain and the A1-A3-C1-C2 dimer that favors the inactive, dissociated state.
      Plasma-derived (pd) and recombinant FVIII concentrates are considered to have virtually indistinguishable PK [
      • Carcao M.
      Changing paradigm of prophylaxis with longer acting factor concentrates.
      ]. Nonetheless, the specific activity (SA) (FVIII:C/FVIII protein in molar concentration) of these concentrates varies not only from product to product but also among lots of a single product [
      • Butenas S.
      • Parhami-Seren B.
      • Gissel M.T.
      • Gomperts E.D.
      • Fass D.N.
      • Mann K.G.
      Potency and mass of factor VIII in FVIII products.
      ,
      • Orfeo T.
      • Elsman R.
      • Gissel M.
      • Mann K.G.
      • Butenas S.
      Activation, activity and inactivation of factor VIII in factor VIII products.
      ]. Butenas and colleagues used a calibrated immunoassay to study three separate lots of five different FVIII concentrates (two plasma-derived, three recombinant; 15 total lots) to determine FVIII molar concentration (mass) [
      • Butenas S.
      • Parhami-Seren B.
      • Gissel M.T.
      • Gomperts E.D.
      • Fass D.N.
      • Mann K.G.
      Potency and mass of factor VIII in FVIII products.
      ]. For three of the products, the inter-lot concentration (standard deviation) of FVIII protein varied by 2% to 6%, for the fourth product by 12%, and the fifth product by nearly 40%. Similarly, inter-product SA varied widely among the five products and was consistent with the results of a synthetic thrombin generation assay. When tested at an equimolar FVIII protein concentration (0.7 nM), those products with higher specific activity generated thrombin more quickly and robustly.

      3.3 Interpatient differences in response to FVIII

      Interindividual differences in the response to FVIII are well recognized. The usual circulating half-life of unmodified FVIII infusions is approximately 8–12 h [
      • Srivastava A.
      • Brewer A.K.
      • Mauser-Bunschoten E.P.
      • Key N.S.
      • Kitchen S.
      • Llinas A.
      • et al.
      Guidelines for the management of hemophilia.
      ]. However, this number can vary more than twofold in patients with severe hemophilia A [
      • Collins P.W.
      • Fischer K.
      • Morfini M.
      • Blanchette V.S.
      • Bjorkman S.
      Implications of coagulation factor VIII and IX pharmacokinetics in the prophylactic treatment of haemophilia.
      ], especially in those who develop non-neutralizing antibodies to FVIII [
      • Hofbauer C.J.
      • Whelan S.F.
      • Hirschler M.
      • Allacher P.
      • Horling F.M.
      • Lawo J.P.
      • et al.
      Affinity of FVIII-specific antibodies reveals major differences between neutralizing and nonneutralizing antibodies in humans.
      ], profoundly impacting PK. Inheritance partially explains this variation in drug response [
      • Vesell E.S.
      • Page J.G.
      Genetic control of drug levels in man: antipyrine.
      ], with environment, age, ethnicity, and physical activity level all contributing factors [
      • Kaddurah-Daouk R.
      • Weinshilboum R.M.
      Pharmacometabolomics Research Network. Pharmacometabolomics: implications for clinical pharmacology and systems pharmacology.
      ]. The impact of other factors on the response to FVIII, such as the microbiome, the collection of genomes of microbes harbored primarily in the gut [
      • Ursell L.K.
      • Metcalf J.L.
      • Parfrey L.W.
      • Knight R.
      Defining the human microbiome.
      ], are less certain [
      • Li H.
      • He J.
      • Jia W.
      The influence of gut microbiota on drug metabolism and toxicity.
      ].

      4. Discussion

      The ability to precisely dose FVIII prophylaxis, key to preventing bleeding episodes, is complicated by the variability in laboratory assays, FVIII products, and interpatient differences.

      4.1 Clinical implications of assay variability

      Ideally, potency labeling correlates with assay methods used by clinical laboratories [
      • Hubbard A.R.
      • Dodt J.
      • Lee T.
      • Mertens K.
      • Seitz R.
      • Srivastava A.
      • et al.
      Recommendations on the potency labelling of factor VIII and factor IX concentrates.
      ,
      • Kitchen S.
      • Kershaw G.
      • Tiefenbacher S.
      Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.
      ]. Incongruity resulting from the use of two different assays—one for product labeling, the other for clinical assessment—may result in missed diagnoses and/or inaccurate assessment of the severity of the bleeding phenotype, as can occur in patients with mild hemophilia A [
      • Duncan E.M.
      • Rodgers S.E.
      • McRae S.J.
      Diagnostic testing for mild hemophilia A in patients with discrepant one-stage, two-stage, and chromogenic factor VIII:C assays.
      ,
      • Bowyer A.E.
      • Van Veen J.J.
      • Goodeve A.C.
      • Kitchen S.
      • Makris M.
      Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A.
      ,
      • Cid A.R.
      • Calabuig M.
      • Cortina V.
      • Casana P.
      • Haya S.
      • Moret A.
      • et al.
      One-stage and chromogenic FVIII:C assay discrepancy in mild haemophilia A and the relationship with the mutation and bleeding phenotype.
      ,
      • Pavlova A.
      • Delev D.
      • Pezeshkpoor B.
      • Muller J.
      • Oldenburg J.
      Haemophilia A mutations in patients with non-severe phenotype associated with a discrepancy between one-stage and chromogenic factor VIII activity assays.
      ,
      • Poulsen A.L.
      • Pedersen L.H.
      • Hvas A.M.
      • Poulsen L.H.
      • Thykjaer H.
      • Ingerslev J.
      Assay discrepancy in mild haemophilia A: entire population study in a National haemophilia Centre.
      ,
      • Trossaert M.
      • Boisseau P.
      • Quemener A.
      • Sigaud M.
      • Fouassier M.
      • Ternisien C.
      • et al.
      Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity.
      ,
      • Goodeve A.C.
      • Peake I.R.
      The molecular basis of hemophilia A: genotype-phenotype relationships and inhibitor development.
      ]. Assay discordance may also adversely impact PK assessment, potentially resulting in under-treatment or over-treatment [
      • Spencer Chapman M.
      • Batty P.
      • Platton S.
      • Hart D.
      Accuracy of published FVIII/FIX:C assay result times: implications for pharmacokinetic (PK) guided dose predictions: a UK-PK study pilot.
      ] and leading to a heightened risk for hemorrhage and breakthrough bleeding during prophylaxis [
      • Reininger A.J.
      • Chehadeh H.E.
      The principles of PK-tailored prophylaxis.
      ], or serious adverse events and higher-than-necessary treatment costs. Finally, discrepancies between the two types of assay may translate to the need to have both the OSA and CSA available to hemophilia treatment centers. In fact, the current consensus is to use both assays for the evaluation of patients with a personal or family history consistent with mild hemophilia A [
      • Oldenburg J.
      • Pavlova A.
      Discrepancy between one-stage and chromogenic factor VIII activity assay results can lead to misdiagnosis of haemophilia A phenotype.
      ,
      • Poulsen A.L.
      • Pedersen L.H.
      • Hvas A.M.
      • Poulsen L.H.
      • Thykjaer H.
      • Ingerslev J.
      Assay discrepancy in mild haemophilia A: entire population study in a National haemophilia Centre.
      ,
      • Kitchen S.
      • Hayward C.
      • Negrier C.
      • Dargaud Y.
      New developments in laboratory diagnosis and monitoring.
      ]. Such a practice is costly in terms of equipment and personnel.

      4.2 Potency disparity and bleeding

      The administration of an equal FVIII potency in units means the administration of different amounts of FVIII protein. Given that potency designation is used to calculate dosage [
      • Butenas S.
      • Parhami-Seren B.
      • Gissel M.T.
      • Gomperts E.D.
      • Fass D.N.
      • Mann K.G.
      Potency and mass of factor VIII in FVIII products.
      ,
      • Orfeo T.
      • Elsman R.
      • Gissel M.
      • Mann K.G.
      • Butenas S.
      Activation, activity and inactivation of factor VIII in factor VIII products.
      ], these disparities may underlie some of the variability observed in PK determinations and provide an explanation for breakthrough bleeding despite optimal dosing and adherence to the prophylactic regimen.

      4.3 Challenges with extended half-life rFVIII products

      All new, unmodified FVIII products, as well as single-chain and extended half-life (EHL) concentrates with molecular modification of Fc fusion or site-specific polyethylene glycol (PEG), can be labeled (assigned potency) and monitored using the CSA [
      • Kitchen S.
      • Kershaw G.
      • Tiefenbacher S.
      Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.
      ]. However, clinical laboratories predominantly use the OSA, and that assay is entirely unsuitable for monitoring some of these new FVIII concentrates (Table 1), and certain modified rFVIII molecules have shown discordant results when measured using the OSA and CSA [
      • Barrowcliffe T.W.
      • Raut S.
      • Sands D.
      • Hubbard A.R.
      Coagulation and chromogenic assays of factor VIII activity: general aspects, standardization, and recommendations.
      ,
      • Mikaelsson M.
      • Oswaldsson U.
      • Jankowski M.A.
      Measurement of factor VIII activity of B-domain deleted recombinant factor VIII.
      ]. In field studies, OSA accuracy with BAX 855 (Adynovate, Shire), a full-length, PEGylated, EHL rFVIII [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ], and Eloctate (Bioverativ), an rFVIII fusion protein product [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ], was comparable to that with Advate, although slightly higher CSA values were observed for Eloctate [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]. In contrast, and as is true for standard BDD rFVIII concentrates, the OSA potency assessment of BDD or truncated EHL products (eg, BAY 94-9027; Bayer), may be highly dependent on the choice of aPTT reagent [
      • Hubbard A.R.
      • Dodt J.
      • Lee T.
      • Mertens K.
      • Seitz R.
      • Srivastava A.
      • et al.
      Recommendations on the potency labelling of factor VIII and factor IX concentrates.
      ,
      • Gu J.M.
      • Ramsey P.
      • Evans V.
      • Tang L.
      • Apeler H.
      • Leong L.
      • et al.
      Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.
      ], potentially resulting in significant incongruence between the OSA and CSA [
      • Gu J.M.
      • Ramsey P.
      • Evans V.
      • Tang L.
      • Apeler H.
      • Leong L.
      • et al.
      Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.
      ]. Finally, rFVIII-Fc yields marginally higher results with the CSA than the OSA, although a product-specific rFVIII-Fc standard is unnecessary [
      • Gu J.M.
      • Ramsey P.
      • Evans V.
      • Tang L.
      • Apeler H.
      • Leong L.
      • et al.
      Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.
      ].
      Table 1Assays used to assess new and/or extended half-life products.
      ProductModificationAssay used for potency labelingLabel activity for OSA and CSA (IU/mL)OSA mean recovery (%)CSA mean recovery (%)
      rFVIII

      (Advate; Shire)
      Used as the reference.
      NoneOSA.8 [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]

      .2

      .05

      .8 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]

      .2

      .05

      .9 [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]

      .2

      .03

      .865 [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ]

      .375

      .043

      .6 [
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ]

      .3
      96.7 [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]

      110.2

      118.1

      114.0 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]

      129.8

      138.3

      154 [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]

      120

      93

      97 [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ]

      106

      119

      127 [
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ]

      138
      108.1 [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]

      112.4

      101.8

      129.0 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]

      127.4

      92.4

      108 [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]

      106

      102

      116 [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ]

      114

      115

      143 [
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ]

      119
      N8 (NovoEight; Novo Nordisk)NoneOSA.9 [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]

      .2

      .03
      94 [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]

      116

      157
      118 [
      • Viuff D.
      • Barrowcliffe T.
      • Saugstrup T.
      • Ezban M.
      • Lillicrap D.
      International comparative field study of N8 evaluating factor VIII assay performance.
      ]

      116

      104
      BAY 81-8973 (Kovaltry; Bayer)NoneCSA.043 [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ]

      .375

      .865
      106 [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ]

      93

      88
      102 [
      • Kitchen S.
      • Jennings I.
      • Makris M.
      • Kitchen D.P.
      • Woods T.A.
      • Walker I.D.
      Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.
      ]

      98

      98
      Human-cl rhFVIII (Nuwiq; Octapharma)NoneCSA800 [
      • NUWIQ
      Prescribing information.
      ]

      400

      200

      100
      81-98 [
      • Sandberg H.
      • Kannicht C.
      • Stenlund P.
      • Dadaian M.
      • Oswaldsson U.
      • Cordula C.
      • et al.
      Functional characteristics of the novel, human-derived recombinant FVIII protein product, human-cl rhFVIII.
      ]
      NA
      rFVIII-Fc (Eloctate/Elocta; Bioverativ)Fusion to Fc domain of IgG1CSA.87 [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]

      .22

      .054
      94.6 [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]

      106.0

      115.7
      119.0 [
      • Sommer J.M.
      • Moore N.
      • McGuffie-Valentine B.
      • Bardan S.
      • Buyue Y.
      • Kamphaus G.D.
      • et al.
      Comparative field study evaluating the activity of recombinant factor VIII Fc fusion protein in plasma samples at clinical haemostasis laboratories.
      ]

      133.4

      120.2
      BAX 855 (Adynovate; Shire)20 kDa branched PEGOSA.8 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]

      .2

      .05
      101 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]

      112.9

      124.3
      124 [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]

      123.7

      95.4
      N8-GP (Novo Nordisk)40 kDa at O-linked glycanNA30-130
      Highly dependent on aPTT reagent.
      [
      • Hillarp A.
      • Bowyer A.
      • Ezban M.
      • Persson P.
      • Kitchen S.
      Measuring FVIII activity of glycopegylated recombinant factor VIII, N8-GP, with commercially available one-stage clotting and chromogenic assay kits: a two-centre study.
      ]
      108-140 [
      • Hillarp A.
      • Bowyer A.
      • Ezban M.
      • Persson P.
      • Kitchen S.
      Measuring FVIII activity of glycopegylated recombinant factor VIII, N8-GP, with commercially available one-stage clotting and chromogenic assay kits: a two-centre study.
      ]
      BAY 94-9027 (Bayer)BDD, 60 kDA PEG at CysNA0-236
      Highly dependent on aPTT reagent and coagulation analyzer.
      Values are estimated from an abstract and poster presented by Leong L et al at the International Society of Thrombosis and Haemostasis 2011 Congress.
      80-110 [
      • Gu J.M.
      • Ramsey P.
      • Evans V.
      • Tang L.
      • Apeler H.
      • Leong L.
      • et al.
      Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.
      ]
      CSL 627 (Afstyla; CSL Behring)Single-chain rFVIII with truncated B-domainCSA.6 [
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ]

      .3
      61 [
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ]

      61
      106 [
      • St. Ledger K.
      • Fuessner A.
      • Kalina U.
      • Metzner H.
      • Horn C.
      • Stowers A.
      • et al.
      Characteristics of rFVIII-single chain in the one-stage and the chromogenic substrate assay: results of an international field study.
      ]

      119
      aPTT, activated partial thromboplastin time; BDD, B-domain-deleted; CSA, chromogenic substrate assay; NA = information not available; OSA, one-stage assay; PEG, polyethylene glycol.
      a Used as the reference.
      b Highly dependent on aPTT reagent.
      c Highly dependent on aPTT reagent and coagulation analyzer.
      d Values are estimated from an abstract and poster presented by Leong L et al at the International Society of Thrombosis and Haemostasis 2011 Congress.

      4.4 Impact of joint inflammation on FVIII PK

      Host response to disease, including inflammation, impacts the PK response to treatment [
      • Schmith V.D.
      • Foss J.F.
      Effects of inflammation on pharmacokinetics/pharmacodynamics: increasing recognition of its contribution to variability in response.
      ]. The expression of various drug-metabolizing enzymes and transporters is affected by cytokines produced during the inflammatory state associated with cancer and end-stage renal disease [
      • Naldini A.
      • Morena E.
      • Belotti D.
      • Carraro F.
      • Allavena P.
      • Giavazzi R.
      Identification of thrombin-like activity in ovarian cancer associated ascites and modulation of multiple cytokine networks.
      ]. Whether such changes are operative and relevant in hemophilia and play a role in determining individual FVIII PK remains to be determined. However, inflammation is a common sequela of hemophilia-related bleeding, associated with both acute hemarthroses and chronic synovitis resulting from recurrent hemorrhages into the same joint [
      • Rodriguez-Merchan E.C.
      Pathogenesis, early diagnosis, and prophylaxis for chronic hemophilic synovitis.
      ]. Furthermore, the connection between inflammation and coagulation has long been recognized, linked in part to the regulation of APC, a proteolytic enzyme that controls FVIII inactivation [
      • Dahlback B.
      • Villoutreix B.O.
      Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition.
      ] and, thus, may influence FVIII bioavailability and PK. Among other potential mechanisms are cytokines such as interleukin-1 beta and tumor necrosis factor alpha, which downregulate thrombomodulin, and the endothelial protein C receptor, which reduces the ability to generate APC [
      • Esmon C.T.
      The interactions between inflammation and coagulation.
      ].

      4.5 Impact of intense physical activity on FVIII PK

      Physical activity and muscular exercise, like inflammation, elicit an acute-phase response leading to an increase in VWF and other coagulation factors, including those in the fibrinolytic pathway [
      • Smith J.E.
      Effects of strenuous exercise on haemostasis.
      ]. In patients with hemophilia, moderate-intensity aquatic exercise reduces prothrombin time consistent with a rise in the heart rate but has minimal effects on FVIII and fibrinogen levels. Nonetheless, physical activity has the benefits of accelerating the metabolic rate; burning fat; enhancing muscle strength, endurance, and proprioception; helping maintain joint range of motion; and improving psychosocial well-being. All of these effects may upregulate or downregulate metabolic pathways, thereby impacting coagulation or cofactor function and indirectly influencing FVIII activity and stability in the circulation [
      • Saroglakis G.
      • Kritikos A.
      • Arzoglou P.
      • Kokaridas D.
      • Patsiaouras A.
      The effect of aerobic and weight lifting exercise on blood clotting.
      ].

      4.5.1 Solutions related to FVIII product

      The use of standard techniques to measure FVIII levels following infusions of FVIII concentrate containing modified proteins that extend half-life may not be reliable. Performing a PK assessment at the tail end of the curve to predict terminal half-life may be more helpful than measuring FVIII activity at a single time point (eg, during the elimination phase).

      4.5.2 Solutions related to patient selection

      Selecting an FVIII concentrate on the basis of patient characteristics is not possible at this time. Previously, Viel et al speculated that FVIII haplotype may influence the risk for the development anti-FVIII alloantibodies (inhibitors), particularly when patients receive mismatched FVIII replacement therapy [
      • Viel K.R.
      • Ameri A.
      • Abshire T.C.
      • Iyer R.V.
      • Watts R.G.
      • Lutcher C.
      • et al.
      Inhibitors of factor VIII in black patients with hemophilia.
      ]. Although data from the Hemophilia Inhibitor Genetics Study (HIGS) combined cohort failed to support this hypothesis [
      • Schwarz J.
      • Astermark J.
      • Menius E.D.
      • Carrington M.
      • Donfield S.M.
      • Gomperts E.D.
      • et al.
      F8 haplotype and inhibitor risk: results from the Hemophilia Inhibitor Genetics Study (HIGS) combined cohort.
      ], matching product to patient genotype may become a reality in the future, as the factors regulating FVIII PK are deciphered.

      4.5.3 Solutions related to FVIII assays

      The simplest way to avoid FVIII assay discrepancies is for all laboratories to exclusively use the CSA, as recommended by the Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation [
      • National Hemophilia Foundation Medical and Scientific Advisory Council
      MASAC statement regarding use of various clotting factor assays to monitor factor replacement therapy. MASAC document 228.
      ]. However, as previously discussed, this solution is expensive, labor intensive, and may have even higher variability than the OSA [
      • Turecek P.L.
      • Romeder-Finger S.
      • Apostol C.
      • Bauer A.
      • Crocker-Buque A.
      • Burger D.A.
      • et al.
      A world-wide survey and field study in clinical haemostasis laboratories to evaluate FVIII:C activity assay variability of ADYNOVATE and OBIZUR in comparison with ADVATE.
      ]. Consequently, the CSA is unlikely to be adopted. Given that OSAs will continue to be used for the diagnosis, severity assessment, and monitoring of hemophilia A treatment, these assays must be performed in a way that closely approximates the labeled potency for a given product [
      • Kitchen S.
      • Kershaw G.
      • Tiefenbacher S.
      Recombinant to modified factor VIII and factor IX - chromogenic and one-stage assays issues.
      ]. Ultimately, this can be ensured by creating an international normalized ratio for the OSA to allow direct comparison of results across different laboratories despite the use of a variety of reagent and instrument systems [
      • Pi D.W.
      • Raboud J.M.
      • Filby C.
      • Carter C.J.
      Effect of thromboplastin and coagulometer interaction on the precision of the International Normalised Ratio.
      ]. Until that occurs, some temporizing solutions may help to improve OSA accuracy.
      With regard to methodology, it is essential that manufacturers of clotting factor concentrates perform studies to elucidate variability related to specific reagents. For example, the use of silica reagents when assessing FVIII:C following infusion of N8-GP or BAY 94-9027 results in under-estimation of FVIII activity [
      • Gu J.M.
      • Ramsey P.
      • Evans V.
      • Tang L.
      • Apeler H.
      • Leong L.
      • et al.
      Evaluation of the activated partial thromboplastin time assay for clinical monitoring of PEGylated recombinant factor VIII (BAY 94-9027) for haemophilia A.
      ,
      • Tiefenbacher S.
      • Robinson M.
      • Ross E.A.
      • Williams P.
      • Cogswell C.
      • Wham T.
      • et al.
      FVIII OS/CS ratios for select novel recombinant FVIII replacement products measured in four FDA approved FVIII one-stage clot assay sytems and one chromogenic assay.
      ].
      Concerning instrumentation, automated rather than manual techniques should be used whenever possible to minimize dilution and other errors. In addition, normal reference ranges for a particular instrument must be determined and validated by external surveys and proficiency testing.
      Regarding calibration, the reference plasma used must be calibrated in IU against an appropriate WHO international standard and should allow adequate residual FVIII activity to remain despite multiple dilutions [
      • Over J.
      Methodology of the one-stage assay of factor VIII (VIII:C).
      ].
      With regard to the plasma source, while either FVIII immunodepleted plasma from healthy individuals or plasma from patients with severe hemophilia A can be used, it is essential that adequate amounts of VWF are present, particularly when using immunodepleted plasma [
      • Turecek P.L.
      • Mitterer A.
      • Matthiessen H.P.
      • Gritsch H.
      • Varadi K.
      • Siekmann J.
      • et al.
      Development of a plasma- and albumin-free recombinant von Willebrand factor.
      ,
      • Barrowcliffe T.W.
      Standardization of FVIII & FIX assays.
      ].
      Finally, per MASAC recommendations, laboratories that routinely perform factor assays should participate in regular proficiency testing and field surveys done by manufacturers of newer clotting factor concentrates [
      • National Hemophilia Foundation Medical and Scientific Advisory Council
      MASAC statement regarding use of various clotting factor assays to monitor factor replacement therapy. MASAC document 228.
      ].

      5. Conclusion

      The ability to precisely dose FVIII according to an individual’s PK is key to optimizing prophylaxis for hemophilia A. Assay limitations and discrepancies, variability in FVIII products, and interpatient differences in the response to FVIII impede attempts at precision dosing. Overcoming these impediments will require rigorous laboratory quality assurance programs and proficiency testing to ensure the accuracy and fidelity of FVIII measurements, more generalized adoption of PK as a tool for fine-tuning FVIII dosing, and finally, novel research to better understand patient characteristics and elucidate the factors that determine the risk for bleeding and bleeding-related complications, including arthropathy and intracranial hemorrhage.

      Funding

      This work was funded by Shire. Michele Grygotis, an independent consultant, provided medical writing services that were funded by Shire.

      Conflicts of interest

      LAV was an employee and stockholder of Shire at the time of manuscript preparation. He is now an employee and stockholder of Spark Therapeutics and retains a faculty appointment as professor of pediatrics at Rush University, Chicago, IL.
      PLT is a full-time employee and stockholder of Shire and has no conflicts of interest to disclose.
      HG is a full-time employee and stockholder of Shire and has no conflicts of interest to disclose.
      SB is a consultant for Diagnostica Stago.
      KGM is a consultant to Alnylam, Baxalta, Bayer, Novo Nordisk, and Stago; a stockholder of Alnylam, Novo Nordisk, and Shire; and chairman of the board of Haematologic Technologies.

      Authorship

      All of the authors contributed to writing and editing the article, and have approved the final version.

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