Protocol - Emicizumab Therapy: Individual Pharmacokinetic Study of Extended Half-life Factor VIII Products: Chromogenic Substrate Assay with Bovine Reagents
This protocol provides instructions and guidance for collecting and processing samples for coagulation testing, performing an individual pharmacokinetic study using the chromogenic substrate assay, and interpreting pharmacokinetic results in response to infusion of extended half-life Factor VIII products. Because there are many comparable assays for performing the chromogenic assay, the protocol also provides basic guidelines to aid comparability among different studies.
The PhenX Hemophilia Inhibitor Research Working Group (WG) notes that these measures are intended for use in observational and interventional trials but are not sufficient to define hemophilia phenotypes when used in isolation.
The WG recommends that when measuring inhibitor recovery in non-severe patients, endogenous factor should be measured by the same assay that was optimized prior to inhibitor development.
The WG recommends that Factor VIII and IX assays, either by one-stage clotting factor or chromogenic substrate methodology, should be performed by a laboratory that is College American Pathologists (CAP) accredited or Clinical Laboratory Improvement Amendments of 1998 (CLIA) certified. For multi-center clinical trials, the use of a central laboratory is strongly encouraged.
Emicizumab is a bispecific antibody that mimics the cofactor function of activated factor VIII (FVIIIa) by bridging activated factor IX (FIXa) and factor X (FX). Factor VIII (FVIII) levels and inhibitors can be measured in the presence of emicizumab using chromogenic assays with bovine reagents. One-stage clot-based assays cannot be used for determination of FVIII levels and FVIII inhibitors in the presence of emicizumab because results will show artifactually elevated FVIII levels. For more information see the Medical and Scientific Advisory Committee (MASAC) of the National Hemophilia Foundations MASAC Update on the Approval and Availability of the New Treatment: Emicizumab (Hemlibra), for Persons with Hemophilia A with Inhibitors to Factor VIII: Interim Guidance on Acute Bleed Management and Use of Laboratory Assays (https://www.hemophilia.org/sites/default/files/MASAC-Update-on-the-Approval-and-Availability-of-the-New-Treatment.pdf).
The WG notes the activity of FVIII extended half-life products in plasma determined by the chromogenic clotting factor assay can vary according to the reagents and instrumentation used (Kitchen et al., 2017). Investigators should select an assay that aligns with the one used to determine the potency of the product. Additionally, investigators should record the make and manufacturer of equipment, the repeatability and coefficients of variation for the assay, and the reagents used.
The WG expects that a pharmacokinetic study of standard and extended half-life FVIII products in the presence of emicizumab will primarily be used to guide prophylaxis for surgery and for episodic prophylaxis around peak activity.
The WG recommends that investigators wait at least 6 months from the patients last dose of emicizumab before using assays with regular (non-bovine) reagents.
Additionally, the WG notes that in the HAVEN 2 study, one patient developed an antibody to emicizumab which was associated with increased bleeding, decreased chromogenic factor VIII activity, and a prolonged activated partial thromboplastin time (aPTT).
If a washout is not performed, comparison of pharmacokinetic data between studies should be conducted under steady-state conditions. Steady-state is defined as a patient-specific condition during which a pharmacokinetic assessment remains valid over a clinically useful period of time. Examples of non-steady state conditions include:
- Bleeding state
- Peri- and immediate post-surgical states in which patients are receiving continuous or regular high (non-routine prophylactic) doses of FVIII
- Immune tolerance induction during which inhibiter titers are in flux
- Children in whom age- and weight-based clearance is still developing toward adult physiologic states.
Emicizumab Therapy: Individual Pharmacokinetic Study of Extended Half-life Factor VIII Products: Chromogenic Substrate Assay with Bovine Reagents
The PhenX Hemophilia Inhibitors Working Group (WG) recommends that investigators follow the sample collection procedures outlined in Lippi et al. (2012) to ensure quality specimens for coagulation testing. These recommendations include basic criteria for venipuncture (e.g., proper patient identification, use of correct techniques, appropriate devices and needles) as well as additional guidance for critical parameters which can affect the outcome of clot-based tests. These critical parameters include prevention of prolonged venous stasis, collection of nonhemolyzed samples, order of blood draw, and appropriate filling and mixing of collection tubes.
Additionally, the WG highlights that blood should be collected by direct venipuncture into 3.2% sodium citrate tubes and filled within 11% of fill line. A second tube should be collected. A discard tube should be drawn if using a winged butterfly collection system.
The WG recommends that investigators follow the sample collection procedures outlined in Adcock Fuck et al. (2012). The procedures include that:
- unprocessed or processed sodium citrate samples remain capped and at room temperature until testing,
- samples should not be refrigerated or stored on ice or in an ice bath,
- samples should be transported vertically, and
- processed samples should not be agitated during transportation to avoid remixing of components.
Additionally, samples can be transported and stored as:
- unprocessed sodium citrate whole blood samples,
- whole blood samples centrifuged and maintained in sodium citrate tubes, or
- plasma processed by centrifugation and aliquoted into a second tube.
Ideally, whole blood samples should be processed to platelet poor plasma (PPP) within 1 hour of collection and assayed within 4 hours of collection.
If centrifuging samples, the centrifuge should be validated so that post-centrifuged samples contain less than 10,000 platelets/microliter. Centrifuged samples should be frozen and can be stored at -20o C for 2 weeks. Samples should be transferred to < -70o C for longer storage, including shipment.
Emicizumab Therapy: Chromogenic Substrate Assay with Bovine Reagents for Determining Factor VIII Activity in Plasma
In patients being treated with emicizumab, Factor VIII inhibitors can only be measured using a chromogenic assay with bovine reagents. Investigators should select an assay that aligns with the one used to determine the potency of the product. Once an assay is chosen for a particular study, the WG recommends that no changes in the protocol be made over the course of the study. Because results can vary with the instrumentation and reagents, the WG recommends that the investigator record the make and manufacturer of equipment, the repeatability and coefficients of variation for the assay, and the reagents used. Use of a central laboratory for multi-center clinical trials is strongly encouraged.
Extended Half-life Factor VIII Products: Individual Pharmacokinetic Study
Prior to pharmacokinetic evaluations, participants should undergo a treatment-free washout period lasting longer than five half-lives. Samples should be taken before infusion (baseline), 10-15 minutes after infusion, and then up to eight other timepoints that cover 2.5 half-lives of the extended half-life FVIII products. The WG notes that the list of timepoints necessary for the pharmacokinetic study to cover 2.5 half-lives will depend on the specific extended half-life FVIII product being used and can be identified in relevant pre-licensure studies. Investigators should report which timepoints were used.
Extended Half-life Factor VIII Products: Interpretation of Individual Pharmacokinetic Study Results
The half-life of extended half-life FVIII products is highly variable and the level at which an inhibitor is suggested has not been established.
Personnel and Training Required
Equipment NeedsLaboratory with the ability to perform the chromogenic substrate assay with bovine reagents.
|Specialized requirements for biospecimen collection||Yes|
|Average time of greater than 15 minutes in an unaffected individual||Yes|
Mode of Administration
Toddler, Child, Adolescent, Adult
The PhenX Hemophilia Inhibitors Working Group selected the recommendations from Lippi et al. (2012) and Adcock Funk et al. (2012) as the best standardized methodology for collecting and processing samples for coagulation testing. The International Society on Thrombosis and Haemostasis (Lee et al., 2001) provides standard timepoints for consistent implementation of the pharmacokinetic study.
|caDSR Common Data Elements (CDE)||Individual Pharmacokinetic Study Coagulation Factor VIII Extended Half Life Chromogenic Substrate Clotting Factor Assay||6712203||CDE Browser|
The results of this protocol can be combined with the results of Emicizumab Therapy: Determination of Factor VIII Inhibitors Using the Bethesda Assay with Nijmegen Modification: Chromogenic Substrate Assay with Bovine Reagents to document:
Presence of an Inhibitor
The presence of an inhibitor is indicated by one or more of the following:
- lack of clinical response (cessation of bleeding) to factor infusion for treatment of bleeding;
- less than expected (< 66%) recovery of Factor VIII level immediately after infusion (Response to Factor VIII Infusion in the Presence of Emicizumab - Individual Pharmacokinetic Study);
- positive inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity in the Presence of Emicizumab);
- Reduced FVIII half-life below 6 hours.
Evolution of an Inhibitor
Inhibitor evolution is indicated by:
- change in inhibitor titer over time (Quantitative Measure of Factor VIII Inhibitor Activity in the Presence of Emicizumab), with or without immune tolerance induction;
- change in clinical response (i.e., bleeding) to FVIII infusion;
- change in FVIII activity after factor infusion (Response to Factor VIII Infusion in the Presence of Emicizumab - Individual Pharmacokinetic Study).
Resolution of an Inhibitor
Inhibitor resolution is indicated by the following:
- for patients receiving immune tolerance therapy for eradication of an FVIII inhibitor, success is defined as a negative inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity in the Presence of Emicizumab) and a normal recovery (>= 66% of expected) and half-life >= 6 hours of infused FVIII concentrate (Response to Factor VIII Infusion in the Presence of Emicizumab - Individual Pharmacokinetic Study).
Persistence of an Inhibitor
A persistent inhibitor is indicated by a decreased response to FVIII concentrate infusion (Response to Factor VIII Infusion in the Presence of Emicizumab - Individual Pharmacokinetic Study) with or without a persistently positive inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity in the Presence of Emicizumab).
Blanchette, V. S., Key, N. S., Ljung, L. R., Manco-Johnson, M. J., van den Berg, H. M., & Srivastava, A.; Subcommittee on Factor VIII, Factor IX and Rare Coagulation Disorders of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis. (2014). Definitions in hemophilia: Communication from the SSC of the ISTH. Journal of Thrombosis and Haemostasis, 12(11), 1935-1939.
Process and Review
Protocol Name from Source
Medical and Scientific Advisory Committee (MASAC) of the National Hemophilia Foundation (NHF), 2017
Adcock Funk, D. M., Lippi, G., & Favaloro, E. J. (2012). Quality standards for sample processing, transportation, and storage in hemostasis testing. Seminars in Thrombosis and Hemostasis, 38(6), 576-585.
Lippi, G., Salvagno, G. L., Montagnana, M., Lima-Oliveira, G., Guidi, G. C., & Favaloro, E. J. (2012). Quality standards for sample collection in coagulation testing. Seminars in Thrombosis and Hemostasis, 38(6), 565-575.
Medical and Scientific Advisory Committee (MASAC) of the National Hemophilia Foundation (NHF). (2017). MASAC update on the approval and availability of the new treatment: Emicizumab (hemlibra), for persons with hemophilia A with inhibitors to factor VIII: Interim guidance on acute bleed management and use of laboratory assays. https://www.hemophilia.org/sites/default/files/MASAC-Update-on-the-Approval-and-Availability-of-the-New-Treatment.pdf
Blanchette, V. S., Key, N. S., Ljung, L. R., Manco-Johnson, M. J., van den Berg, H. M., & Srivastava, A.; Subcommittee on Factor VIII, Factor VIII and Rare Coagulation Disorders of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis. (2014). Definitions in hemophilia: Communication from the SSC of the ISTH. Journal of Thrombosis and Haemostasis, 12(11), 1935-1939.
Hay, C. R., & DiMichele, D. M.; International Immune Tolerance Study. (2012). The principal results of the International Immune Tolerance Study: A randomized dose comparison. Blood, 119(6), 1335-1344.
Iorio, A., Edginton, A.N., Blanchette, V., Blatny, J., Boban, A., Cnossen, M., Collins, P., Croteau, S.E., Fischer, K., Hart, D.P., Ito, S., Korth-Bradley, J., Lethagen, S., Lillicrap, D., Makris, M., Math
Kitchen, S., Kershaw, G., & Tiefenbacher, S. (2016). Recombinant to modified factor VIII and factor VIII Chromogenic and one-stage assays issues. Haemophilia, 22(Suppl. 5), 72-77.
Kitchen, S., Tiefenbacher, S., & Gosselin, R. (2017). Factor activity assays for monitoring extended half-life FVIII and factor IX replacement therapies. Seminars in Thrombosis and Haemostasis, 43(3), 331-337.
Lee, M., Morfini, M., Schulman, S., & Ingerslev, J.; Factor VIII/Factor VIII Scientific and Standardization Committee of the International Society for Thrombosis and Haemostasis. (2001). Scientific and Standardization Committee communication. The design and analysis of pharmacokinetic studies of coagulation factors. https://www.isth.org/members/group_content_view.asp?group=100348&id=159244. Retrieved August 30, 2018.
Mahlangu, J., Oldenburg, J., Paz-Priel, I., Negrier, C., Niggli, M., Mancuso, M. E., Schmitt, C., Jim
Morfini, M., Lee, M., Messori, A. and the Factor VIII/Factor IX Scientific and Standardization Committee of the International Society for Thrombosis and Haemostasis. (1991). The design and analysis of half-life and recovery studies for factor VIII and factor IX. Thrombosis and Haemostasis, 66(3), 384-386.
Oldenburg, J., Mahlangu, J. N., Kim, B., Schmitt, C., Callaghan, M. U., Young, G., Santagostino, E., Kruse-Jarres, R., Negrier, C., Kessler, C., Valente, N., Asikanius, E., Levy, G. G., Windyga, J., & Shima, M. (2017). Emicizumab prophylaxis in hemophilia A with inhibitors. The New England Journal of Medicine, 377(9), 809-818.
Peyvandi, F., Oldenburg, J., & Friedman, K. D. (2006). A critical appraisal of one-stage and chromogenic assays of factor VIII activity. Journal of Thrombosis and Haemostasis, 14(2), 248-261.
Shima, M., Hanabusa, H., Taki, M., Matsushita, T., Sato, T., Fukutake, K., Fukazawa, N., Yoneyama, K., Yoshida, H., & Nogami, K. (2016). Factor VIII-mimetic function of humanized bispecific antibody in hemophilia A. New England Journal of Medicine, 374(21), 2044-2053.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX911402040500||Was Factor VIII activity tested 1 hour after more||N/A|
|PX911402040900||Was Factor VIII activity tested 24 hours more||N/A|
|PX911402041000||Was Factor VIII activity tested 28 hours more||N/A|
|PX911402040600||Was Factor VIII activity tested 3 hours more||N/A|
|PX911402041100||Was Factor VIII activity tested 32 hours more||N/A|
|PX911402040700||Was Factor VIII activity tested 6 hours more||N/A|
|PX911402040800||Was Factor VIII activity tested 9 hours more||N/A|
|PX911402040300||Was Factor VIII activity tested 10-15 more||N/A|
|PX911402040400||Was Factor VIII activity tested 30 minutes more||N/A|
|PX911402040200||Was Factor VIII activity tested before more||N/A|
|PX911402050000||Was the Factor VIII half-life (t) less than more||N/A|
|PX911402040100||Have partipants undergone a treatment-free more||N/A|
|PX911402010100||Were the sample collection procedures more||N/A|
|PX911402010400||Was the order of blood draw recorded?||N/A|
|PX911402010300||Were nonhemolyzed samples collected?||N/A|
|PX911402010500||Were collection tubes filled and mixed as more||N/A|
|PX911402011000||Was a discard tube drawn?||N/A|
|PX911402010700||Were the tubes filled within 11% of the fill line?||N/A|
|PX911402010800||Was a second tube collected?||N/A|
|PX911402010900||Was a winged butterfly collection system used?||N/A|
|PX911402010600||Was blood collected by direct venipuncture more||N/A|
|PX911402010200||Were steps taken to prevent prolonged venous more||N/A|
|PX911402020100||Were the sample collection and processing more||N/A|
|PX911402020500||Were samples agitated during transportation?||N/A|
|PX911402021000||Were samples assayed within 4 hours of collection?||N/A|
|PX911402021100||Was the centrifuge validated so that process more||N/A|
|PX911402021300||Was the sample transferred to <= -70 C, more||N/A|
|PX911402021200||Was the sample frozen immediately and stored more||N/A|
|PX911402020900||Were samples processed to platelet poor more||N/A|
|PX911402020300||Were samples refrigerated or stored on ice more||N/A|
|PX911402020200||Did unprocessed or processed sodium citrate more||N/A|
|PX911402020800||Were samples stored as plasma processed by more||N/A|
|PX911402020600||Were samples stored as unprocessed sodium more||N/A|
|PX911402020700||Were samples stored as whole blood samples more||N/A|
|PX911402020400||Were samples transported vertically?||N/A|
|PX911402030100||Were any changes made in the protocol over more||N/A|
|PX911402030202||Is this study a part of multi-center more||N/A|
|PX911402030300||Is a central laboratory being used?||N/A|
|PX911402030201||Were the make and manufacturer of equipment, more||N/A|
Response to Factor VIII Infusion in the Presence of Emicizumab - Individual Pharmacokinetic Study
May 7, 2019
A series of plasma Factor VIII activity determinations in blood samples are obtained immediately prior to, and at timepoints after, infusion of FVIII concentrate.
The results of an individual pharmacokinetic study (i.e., initial recovery and half-life) of infused Factor VIII concentrate can characterize an individual?s response to a new drug or can confirm the success of immune tolerance induction (ITI).
Hemophilia inhibitors, Factor VIII, FVIII, hemophilia A, inhibitors, pharmacokinetic study, immune tolerance induction, prophylaxis, recovery, half-life
|Protocol ID||Protocol Name|
|911401||Emicizumab Therapy: Individual Pharmacokinetic Study of Standard Half-life Factor VIII Products: Chromogenic Substrate Assay with Bovine Reagents|
|911402||Emicizumab Therapy: Individual Pharmacokinetic Study of Extended Half-life Factor VIII Products: Chromogenic Substrate Assay with Bovine Reagents|
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