Extended half-life (EHL) testing for FVIII and FIX therapies

Posted on: November 13, 2024

David McGlasson

The complexities of testing for hemophilia A and B are vast. This Clot Club post serves as an overview of what the laboratory must consider when testing for new EHL products.  

When comparing different methods used to determine FVIII and FIX activity, the testing laboratory should evaluate the calibrators used, individual kit manufacturer reagents, instrumentation (mechanical or optical) FVIII or FIX deficient plasma products used in the individual EHL products.

Following is a summary of therapies with information from the drug manufacturer’s labeling.

AFSTYLA: (CSL Behring GmbH) AFSTYLA, Antihemophilic Factor (Recombinant), single chain is a recombinant, antihemophilic factor indicated in adults and children with hemophilia A (congenital Factor VIII deficiency) for:

• On-demand treatment and control of bleeding episodes

• Routine prophylaxis to reduce the frequency of bleeding episodes,

 • Perioperative management of bleeding. Limitation of Use of AFSTYLA is not indicated for the treatment of von Willebrand disease.

Monitoring Laboratory Tests

• Monitor plasma Factor VIII activity in patients receiving AFSTYLA using either the chromogenic assay (CA)or the one-stage clotting assay (OSA) routinely used in US clinical laboratories.

  • The chromogenic assay result most accurately reflects the clinical hemostatic potential of AFSTYLA. The OSA result underestimates the Factor VIII activity level compared to the CA result by approximately one-half.
  • If the OSA is used, multiply the result by a conversion factor of 2 to determine the patient’s Factor VIII activity level. Incorrect interpretation of the Factor VIII activity obtained by the OSA could lead to unnecessary additional dosing, higher chronic dosing, or investigations for an inhibitor.

• Monitor for the development of Factor VIII inhibitors: Perform a Bethesda inhibitor assay if expected plasma Factor VIII activity levels are not attained or if bleeding is not controlled with the expected dose of AFSTYLA. Use Bethesda Units (BU) to report inhibitor levels.

https://www.fda.gov/media/98080/download

ADYNOVATE: (Takeda Pharmaceutical company)

 Antihemophilic factor, Recombinant PEGylated. Used for treating congenital FVIII deficiency.

Monitoring Laboratory Tests

Use a validated (OSA). Use Bethesda inhibitor assay to determine if FVIII inhibitor is present.  

Not indicated for treatment of Von Willebrand’s disease.

Use vendor recommended aPTT reagents for OSA testing.

https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/adynovate

ALTUVIIIO: (Sanofi) [antihemophilic factor (recombinant), Fc-VWF-XTEN fusion protein-extended half-life, lyophilized powder for solution, for intravenous use Initial U.S. Approval 203

  • If assessment of plasma Factor VIII activity is needed, it is recommended to use a validated one stage clotting assay. The ALTUVIIIO Factor VIII activity level is overestimated by the chromogenic assay and a specific ellagic acid- based APTT reagent in one-stage clotting assay by approximately 2.5-fold. If those assays are used, divide the result by 2.5 to approximate the patient’s ALTUVIIIO Factor VIII activity level.
  •  Use of a reference laboratory is recommended when a qualified one-stage clotting assay or chromogenic assay is not available locally.

 Monitor for the development of Factor VIII inhibitors. When bleeding is not controlled with ALTUVIIIO and the expected factor VIII activity plasma levels are not attained, perform an assay to determine if Factor VIII inhibitors are present (use Bethesda Units to titer inhibitors).

https://www.fda.gov/vaccines-blood-biologics/altuviiio

ELOCATE: (Biogen Idec, Inc.) ELOCTATE, Antihemophilic Factor (Recombinant), Fc Fusion Protein, is a recombinant DNA derived, antihemophilic factor indicated in adults and children with Hemophilia A (congenital Factor VIII deficiency).

  •  Monitor plasma Factor VIII activity by performing a validated test (e.g., one stage clotting assay) to confirm that adequate Factor VIII levels have been achieved and maintained
  • Monitor for the development of Factor VIII inhibitors. Perform a Bethesda inhibitor assay if expected Factor VIII plasma levels are not attained, or if bleeding is not controlled with the expected dose of ELOCTATE. Use Bethesda Units (BU) to report inhibitor levels.

https://www.fda.gov/media/88746/download?attachment

ESPEROCT: (Novo Nordisk, Inc.) Antihemophilic factor (recombinant), glycopegylated-exei is a coagulation Factor VIII concentrate indicated for use in adults and children with hemophilia A for:

• On-demand treatment and control of bleeding episodes

• Perioperative management of bleeding

• Routine prophylaxis to reduce the frequency of bleeding episodes Limitation of Use ESPEROCT is not indicated for the treatment of von Willebrand disease. If monitoring of a Factor VIII is performed, using a chromogenic or one-stage clotting assay appropriate for use with ESPEROCT].

  • Factor VIII activity levels may be affected by the type of activated partial thromboplastin time (aPTT) reagent used in the assay. Some silica-based APTT reagents can underestimate the activity of ESPEROCT by up to 60%; other reagents may overestimate the activity by 20%.  APTT-SP, APTT-A were the reagents that underestimated the FVIII activity. The CA tests used were reliable.
  •  If an appropriate one-stage clotting or chromogenic assay is not available locally, then use a reference laboratory. If bleeding is not controlled with the recommended dose of ESPEROCT, or if the expected Factor VIII activity levels in plasma are not attained, then perform a Bethesda assay to determine if Factor VIII inhibitors are present.

https://www.fda.gov/media/120351/download?attachment

HEMLIBRA: (Genentech, Inc.) HEMLIBRA is a bispecific factor IXa- and factor X-directed antibody indicated for routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adult and pediatric patients with hemophilia A (congenital factor VIII deficiency) with factor VIII inhibitors.

Coagulation Test Results Affected and Unaffected by HEMLIBRA Results:

  • Affected by HEMLIBRA; Activated partial thromboplastin time (aPTT) Bethesda assays (clotting-based) for FVIII inhibitor titers One-stage, aPTT-based, single-factor assays APTT-based Activated Protein C Resistance (APC-R) Activated clotting time (ACT)
  • Unaffected by HEMLIBRA: Bethesda assays (bovine chromogenic) for FVIII inhibitor titers Thrombin time (TT) One-stage, prothrombin time (PT)-based, single factor assays, Chromogenic-based single-factor assays other than FVIII* Immuno-based assays (i.e., ELISA, turbidimetric methods). Genetic tests of coagulation factors (e.g., Factor V Leiden, Prothrombin 20210).

https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/761083s000lbl.pdf

HUMATE-P: (CSL Behring GmbH)Laboratory Tests Antihemophilic Factor/von Willebrand Factor (Human), Dried, Pasteurized, Humate-P®, contains blood group isoagglutinins (anti-A and anti-B). When large or frequently repeated doses are needed, as when inhibitors are present or when pre- and post-surgical care is involved, patients of blood groups A, B and AB should be monitored for signs of intravascular hemolysis and decreasing hematocrit values and be treated appropriately, as required.

CSL Behring Antihemophilic Factor/ von Willebrand Factor Complex (Human), ® US Package Insert Revised: 10/2007 Dried, Pasteurized, Humate-P Page 12 The Factor VIII levels of VWD patients receiving  Humate-P® may be monitored using standard coagulation tests, especially in cases of surgery. Strong consideration should also be given to monitoring VWF:RCo levels in VWD patients receiving Humate-P® for the prevention of excessive bleeding during and after surgery.

  • It is advisable to monitor trough levels of VWF:RCo and FVIII:C levels at least once daily in order to adjust the dosage of Humate-P® as needed to avoid excessive accumulation of coagulation factors.

JIVI: (Bayer) JIVI, antihemophilic factor (recombinant), PEGylated-aucl, is a recombinant DNA-derived, Factor VIII concentrate indicated for use in previously treated adults and adolescents (12 years of age and older) with hemophilia A (congenital Factor VIII deficiency. If monitoring of Factor VIII activity is performed, use a validated chromogenic assay or a selected validated one stage clotting assay.

• Laboratories intending to measure the Factor VIII activity of JIVI should check their procedures for accuracy. For JIVI, select silica-based one-stage assays may underestimate the Factor VIII activity of JIVI in plasma samples; some reagents, e.g., with kaolin-based activators, have the potential for overestimation. Therefore, the suitability of the assay must be determined. If a validated one-stage clotting or chromogenic assay is not available locally, then use of a reference laboratory is recommended.

• Monitor for development of Factor VIII inhibitors. Perform a Bethesda inhibitor assay if expected Factor VIII plasma levels are not attained, or if bleeding is not controlled with the expected dose of JIVI. Use Bethesda Units (BU) to report inhibitor titers.

https://www.fda.gov/vaccines-blood-biologics/jivi

KOVALTRY: (Bayer) Antihemophilic Factor (Recombinant), is a recombinant, human DNA sequence derived, full length Factor VIII concentrate indicated for use in adults and children with hemophilia A (congenital Factor VIII deficiency). Monitor plasma Factor VIII activity levels using a validated test to confirm that adequate Factor VIII levels have been achieved and maintained.

• Monitor for development of Factor VIII inhibitors. Perform a Bethesda inhibitor assay if expected Factor VIII plasma levels are not attained or if bleeding is not controlled with the expected dose of KOVALTRY. Use Bethesda Units (BU) to report inhibitor titers.

https://www.fda.gov/media/96215/download

NUWIQ: (Octapharma) NUWIQ is a recombinant antihemophilic factor [blood coagulation factor VIII (Factor VIII)] indicated in adults and children with Hemophilia A for:

 • On-demand treatment and control of bleeding episodes

• Perioperative management of bleeding

• Routine prophylaxis to reduce the frequency of bleeding episodes NUWIQ is not indicated for the treatment of von Willebrand Disease. Monitor plasma Factor VIII activity by performing a validated test (e.g., one stage clotting assay), to confirm that adequate Factor VIII levels have been achieved and maintained.

  • Monitor for the development of Factor VIII inhibitors. Perform a Bethesda inhibitor assay if expected Factor VIII plasma levels are not accomplished, or if bleeding is not controlled with the expected dose of NUWIQ. Use Bethesda Units (BU) to report inhibitor levels.

https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/nuwiq

OBIZUR: (TAKEDA): OBIZUR, Antihemophilic Factor (Recombinant), Porcine Sequence, is an antihemophilic factor indicated for the on-demand treatment and control of bleeding episodes in adults with acquired hemophilia A.

Limitations of Use:  Safety and efficacy of OBIZUR has not been established in patients with a baseline anti-porcine factor VIII inhibitor titer of greater than 20 BU. 

· OBIZUR is not indicated for the treatment of von Willebrand disease.

Perform one-stage clotting assay to confirm that adequate factor VIII levels have been achieved and maintained.  Monitor factor VIII activity 30 minutes and 3 hours after initial dose. Monitor factor VIII activity 30 minutes after subsequent doses.

· Monitor the development of inhibitory antibodies to OBIZUR. Perform a Nijmegen Bethesda inhibitor assay with recombinant porcine factor VIII as substrate for recombinant porcine factor VIII antibodies if expected plasma factor VIII activity levels are not attained or if bleeding is not controlled with the expected dose of OBIZUR. Use Bethesda Units (BU) to report inhibitor levels.

https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/obizur

WILATE: (Octapharma) Pharmazeutika Produktionsges.m.b.H. WILATE is indicated in children and adults with von Willebrand disease for:

• On-demand treatment and control of bleeding episodes.  Perioperative management of bleeding.  Routine prophylaxis to reduce the frequency of bleeding episodesWILATE is indicated for routine prophylaxis in children 6 years of age and older and adults with von Willebrand disease. WILATE is indicated in adolescents and adults with hemophilia A for: Routine prophylaxis to reduce the frequency of bleeding episodes. On-demand treatment and control of bleeding episodes.

  •  Monitor plasma Factor VIII activity by performing a validated test (e.g., one stage clotting assay), to confirm that adequate Factor VIII levels have been achieved and maintained.
  • Monitor for the development of Factor VIII inhibitors. Perform a Bethesda inhibitor assay if expected Factor VIII plasma levels are not attained, or if bleeding is not controlled with the expected dose of WILATE. Use Bethesda Units (BU) to report inhibitor levels.
  • Monitor plasma levels of VWF:RCo and FVIII activities in patients receiving WILATE to avoid sustained excessive VWF and FVIII activity levels, which may increase the risk of thromboembolism, particularly in patients with known clinical or laboratory risk factors.Monitor for development of VWF and FVIII inhibitors. Perform assays to determine whether VWF and/or FVIII inhibitor(s) is present if bleeding is not controlled with the expected dose of WILATE.

https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/wilate

XYNTHA:(Wyeth Pharmaceuicals, LLC) XYNTHA is a recombinant antihemophilic factor indicated in adults and children with hemophilia A for on-demand treatment and control of bleeding episodes, for perioperative management, and for routine prophylaxis to reduce the frequency of bleeding episodes. XYNTHA is not indicated in patients with von Willebrand’s disease. Use individual factor VIII values for recovery and, if clinically indicated, other pharmacokinetic characteristics to guide dosing and administration.

 • Monitor plasma factor VIII activity levels by the one-stage clotting assay to confirm that adequate factor VIII levels have been achieved and are maintained, when clinically indicated

 • Monitor for development of factor VIII inhibitors. Perform assay to determine if factor VIII inhibitor is present when expected factor VIII activity plasma levels are not attained, or when bleeding is not controlled with the expected dose of XYNTHA. Use Bethesda Units (BU) to titer inhibitors.

The detection of antibody formation is highly dependent on the sensitivity and specificity of the assay. Additionally, the observed incidence of antibody, including neutralizing antibody, positivity in an assay may be influenced by several factors, including assay methodology, sample handling, timing of sample collection, concomitant medications, and underlying disease. For these reasons, comparisons of the incidence of antibodies to XYNTHA with the incidence of antibodies to other products may be misleading.

https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/xyntha

FACTOR IX EHL CONCENTRATES

ALPROLIX: (Bioverative Therapeutics, Inc.)

ALPROLIX, Coagulation Factor IX (Recombinant), Fc Fusion Protein, is a recombinant DNA derived coagulation Factor IX concentrate indicated in adults and children with hemophilia B for:

• On-demand treatment and control of bleeding episodes,

 • Perioperative management of bleeding,

 • Routine prophylaxis to reduce the frequency of bleeding episodes. Limitation of Use: ALPROLIX is not indicated for induction of immune tolerance in patients with hemophilia B

Monitoring Laboratory Tests

  • To confirm adequate Factor IX levels have been achieved and maintained, monitor patient plasma Factor IX levels by performing a validated one-stage clotting assay.
  • There are now chromogenic FIX assays that are FDA cleared that can be used if the presence of an inhibitor interferes with the one-stage clotting method used in the laboratory.
  • Factor IX results can be affected by the type of aPTT reagent used. Measurement with a one-stage clotting assay using a kaolin-based aPTT reagent has been shown to result in an underestimation of Factor IX levels.

• Monitor for the development of Factor IX inhibitors if the expected Factor IX levels in patient plasma are not attained, or if bleeding is not controlled with the recommended dose of ALPROLIX. Perform a Bethesda assay to determine if Factor IX inhibitors are present.

https://www.fda.gov/media/88119/download

BENEFIX: (Pfizer Hemophilia)

BeneFIX is a recombinant human blood coagulation factor IX indicated for the treatment of:

· Adults and children with hemophilia B (congenital factor IX deficiency or Christmas disease) for: On-demand treatment and control of bleeding episodes.

 · Patients 16 years of age and older with hemophilia B (congenital factor IX deficiency or Christmas disease) for: Routine prophylaxis to reduce the frequency of bleeding episodes. Limitations of Use:  BeneFIX is not indicated for induction of immune tolerance in patients with hemophilia B.

Monitoring Laboratory Tests

· Monitor patients for factor IX activity levels by the one-stage clotting assay to confirm that adequate factor IX levels have been achieved and maintained, when clinically indicated.

· Monitor patients for the development of inhibitors if expected plasma factor IX activity levels are not attained, or if bleeding is not controlled with the recommended dose of BeneFIX. Determine plasma factor IX inhibitor levels in Bethesda Units (BUs).

https://www.fda.gov/media/73556/download

REFERENCES FOR BENEFIX:

1. Roberts HR, Eberst ME. Current management of hemophilia B. Hematol Oncol Clin North Am. 1993;7(6):1269-1280.

2. Shapiro AD, Ragni MV, Lusher JM, et al. Safety and efficacy of monoclonal antibody purified factor IX concentrate in previously untreated patients with hemophilia B. Thromb Haemost. 1996;75(1):30-35.

IDELVION: (CSL Behring LLC)

IDELVION, Coagulation Factor IX (Recombinant), Albumin Fusion Protein (rIX-FP), a recombinant DNA-derived coagulation Factor IX concentrate, is indicated in children and adults with Hemophilia B (congenital Factor IX deficiency) for:

 • On-demand treatment and control of bleeding episodes

 • Perioperative management of bleeding

 • Routine prophylaxis to reduce the frequency of bleeding episodes Limitations of Use: IDELVION is not indicated for immune tolerance induction in patients with Hemophilia B.

Monitoring Laboratory Tests

• Monitor Factor IX plasma levels by a one-stage clotting assay to confirm that adequate Factor IX levels have been achieved and maintained. Factor IX activity assay results may vary with the type of activated partial thromboplastin time (aPTT) reagent used in the assay system.1 For example, kaolin based aPTT reagents along with other reagents designed to exhibit low responsiveness to lupus anticoagulant have been shown to result in approximately 50% lower than expected recovery based on labeled potency.

• Consistent with similar findings for other recombinant Factor IX products, overestimation of Factor IX activity in spiked samples of IDELVION (mean overestimation 32%) occurred at low Factor IX levels with commonly used aPTT reagents.

 • Monitor patients for the development of inhibitors if expected Factor IX activity plasma levels are not attained, or if bleeding is not controlled with the recommended dose of IDELVION. Assays used to determine if a Factor IX inhibitor is present should be titered in Bethesda Units (BUs).

https://www.fda.gov/media/96526/download

REFERENCES for Idelvion:

 1. Wilmot HV, Hogwood J, Gray E. Recombinant factor IX: discrepancies between onestage clotting and chromogenic assays. Haemophilia 2014, 1-7.

2. Fritsma GA, Dembitzer FR, Randhawa A, et al. Recommendations for Appropriate Activated Partial Thromboplastin Time Reagent Selection and Utilization. Am J Clin Pathol 2012;137:904-908.

 3. Sommer JM, Buyue Y, Bardan S, Peters RT, Jiang H, Kamphaus G, Gray E, Pierce GF. Comparative field study: impact of laboratory assay variability on the assessment of recombinant factor IX Fc fusion protein (rFIXFc) activity. Thrombosis and Haemostasis 2014, 112(5):932-4

REBINYN: (Novo Nordisk)

REBINYN, Coagulation Factor IX (Recombinant), GlycoPEGylated, is a recombinant DNA-derived coagulation Factor IX concentrate indicated for use in adults and children with hemophilia B for:

• On-demand treatment and control of bleeding episodes

• Perioperative management of bleeding Limitations of Use: REBINYN is not indicated for routine prophylaxis in the treatment of patients with hemophilia B. REBINYN is not indicated for immune tolerance induction in patients with hemophilia B.

Monitoring Laboratory Tests

If monitoring of Factor IX activity is performed, use a chromogenic assay or selected one-stage clotting assay validated for use with REBINYN.  The OSA results can be significantly affected by the type of  time (aPTT) reagent used, which can result in over- or under-estimation of Factor IX activity.

  • Avoid the use of silica-based reagents, as some may overestimate the activity of REBINYN.
  • If a validated OSA or CA is not available locally, then use of a reference laboratory is recommended.
  • If bleeding is not controlled with the recommended dose of REBINYN, or if the expected Factor IX activity levels in plasma are not attained, then perform a Bethesda assay to determine if Factor IX inhibitors are present.

REFERENCES for Rebinyn:

Duboscq C, Sueldo E, Rosa C. et al: High variablilty in Factor IX one-stage assay in samples spiked with nonacog beta pegol among different pairs of reagent/detection system. Int J lab Hematol 2024 46(1): 128-134.

Robinson MM, George LA, Carr ME. Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua. J Thromb Haemos. 2021;19:1212-1218.

https://www.fda.gov/vaccines-blood-biologics/approved-blood-products/rebinyn

SUGGESTIONS FOR WORLD SOLUTIONS IN MONITORING FVIII AND FIX EXTENDED HALF LIFE CONCENTRATES:

  • Use product inserts and literature that the manufacturer used when validating the potency of the product produced.  Duplicate the reagents (calibrators, OA and CA like methods.)
  • Use product specific calibrators.
  • Have the ability to perform one-stage factor assays and chromogenic factor assays.
  • Use correction factors when necessary. (See manufacturers package insert)
  • Communicate with physicians and pharmacists for collaboration when using these products for patient care.
  • Be prepared to have a reference laboratory with the ability to perform these assays when these EHL products need to be measured.

RECOMMENDED REFERENCES:

  1. Robinson MM, George LA, Carr ME et al: Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua. J Thromb Haemost. 2021;19:1212-1218.
  2. Marlar RA, Strandberg K, Shima M, Adcock DM. Clinical utility and impact of the use of the chromogenic vs one-stage factor activity assays in haemophilia A and B. Eur J Haematol. 2020;104:3-14.
  3. Bowyer AE, Gosselin RC. Factor VIII and Factor IX Activity Measurements for Hemophilia Diagnosis and Related Treatments. Semin Thromb Hemost. 2023;49:609-620.
  4. Schmidt DE, Truedsson A, Stralfors A et al: Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B. Thromb Hemost 2024;124:32-39.
  5. Van Moort I, Meijer P, Priem-Visser D, van Gammeren AJ et al: Analytical variation in factor VIII one-stage and chromogenic assays: Experiences from the ECAT  external quality assessment programme. Haemophilia. 2019;25:162-169.
  6. Van Moort I, Meijer P, Priem-Visser D, van Gammeren AJ et al: Analytical variation in factor VIII one-stage and chromogenic assays: Experiences from the ECAT  external quality assessment programme. Haemophilia. 2019;25:162-169.

I would like to thank Mr. George Fritsma MS, MLS(ASCP) (Fritsma Factor) and Dr. Russell Higgins, MD, Clinical Professor, Department of Pathology and Laboratory Services University of Texas Health Science Center San Antonio,TX, Medical Director, University Hospital System Pathology Services for their advice and contribution of literature.