Protocol - Emicizumab Therapy: Determination of Factor VIII Inhibitors Using the Bethesda Assay with Nijmegen Modification: Chromogenic Substrate Assay with Bovine Reagents

Description

This protocol provides instructions and guidance for collecting and processing samples for coagulation testing, performing the chromogenic assay using bovine reagents, and interpreting results. Because there are many comparable assays for performing Bethesda Assay with Nijmegen Modification Using the Chromogenic Substrate Assay, the protocol also provides basic guidelines to aid comparability among different studies.

Specific Instructions

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. Factor VIII 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 recommends that investigators wait at least 6 months from the patients last dose of emicizumab before using assays with regular (non-bovine) reagents.

Availability

Available

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Protocol

Emicizumab Therapy: Determination of Factor VIII Inhibitors Using the Bethesda Assay with Nijmegen Modification: Chromogenic Substrate Assay with Bovine Reagents

Sample Collection

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.

Sample Processing

The WG recommends that investigators follow the sample collection procedures outlined in Adcock Funk 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 immediately and can be stored at -20^o C for 2 weeks. Samples should be transferred to < -70^o C for longer storage, including shipment.

Emicizumab Therapy: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate Assay with Bovine Reagents

In patients being treated with emicizumab, Factor VIIII inhibitors can only be measured using a chromogenic assay with bovine reagents. The WG notes that there are a number of different assays and instruments that are appropriate to perform the Bethesda assay with Nijmegen modification using the chromogenic substrate assay with bovine reagents. 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.

The WG notes that the sample should be heated prior to testing to eliminate exogenous Factor VIII (Miller et al., 2012).

Emicizumab Therapy: Interpretation of Bethesda Assay with Nijmegen Modification Results

The International Society on Thrombosis and Haemostasis (Blanchette et al., 2014) consensus definition for a relevant Factor VIII inhibitor is a result of >= 0.6 Bethesda Units (BU) per mL on two or more separate assays over a 1-4 week period.

Personnel and Training Required

Phlebotomist

Equipment Needs

Laboratory with the ability to perform the Bethesda assay with Nijmegen modification using the chromogenic substrate assay with bovine reagents.

Requirements

Requirement Category	Required
Major equipment	No
Specialized training	No
Specialized requirements for biospecimen collection	Yes
Average time of greater than 15 minutes in an unaffected individual	No

Mode of Administration

Bioassay

Lifestage

Toddler, Child, Adolescent, Adult, Senior

Participants

Toddler, child, adolescent, adult

Selection Rationale

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 (Blanchette et al., 2014) provides consensus definitions for the consistent interpretation of results.

Language

English

Standards

Standard	Name	ID	Source

Derived Variables

The results of this protocol can be combined with the results of Emicizumab Therapy: Individual Pharmacokinetic Study of Standard Half-life Factor VIII Products: Chromogenic Substrate Assay with Bovine Reagents or Emicizumab Therapy: Individual Pharmacokinetic Study of Extended Half-life Factor VIII Products: 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), and
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); and
change in FVIII half-life.

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 hour 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 to Monitor Immune Tolerance Induction in Presence of Emicizumab) 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

Not applicable.

Protocol Name from Source

Medical and Scientific Advisory Committee (MASAC) of the National Hemophilia Foundation (NHF), 2017

Source

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

General References

Kasper, C. K., Aledort, L. M., Couns, R. B., Edson, J. R., Frantantoni, J., Green, D., Hampton, J. W., Higartner, M. W., Lazerson, J., Levine, P. H., McMillan, C. W., Shapiro, S. S., Schulman, N. R., & van Eys, J. (1975). A more uniform measurement of factor VIII inhibitors. Thrombosis et Diathesis Haemorrhagica, 34, 869-872.

Kitchen, S., Kershaw, G., & Tiefenbacher, S. (2016). Recombinant to modified factor VIII and factor IX Chromogenic and one-stage assays issues. Haemophilia, 22(Suppl. 5), 72-77.

Mahlangu, J., Oldenburg, J., Paz-Priel, I., Negrier, C., Niggli, M., Mancuso, M. E., Schmitt, C., Jimnez-Yuste, V., Kempton, C., Dhalluin, C., Callaghan, M. U., Bujan, W., Shima, M., Adamkewicz, J. I., Asikanius, E., Levy, G. G., & Kruse-Jarres, R. (2018). Emicizumab prophylaxis in patients who have hemophilia A without inhibitors. New England Journal of Medicine, 379(9), 811-822.

Miller, C. H., Platt, S. J., Rice, A. S., Kelly, F., Soucie, & J. M.; Hemophilia Inhibitor Research Study Investigators. (2012). Validation of Nijmegen-Bethesda assay modifications to allow inhibitor measurement during replacement therapy and facilitate inhibitor surveillance. Journal of Thrombosis and Haemostasis, 10(6), 1055-1061.

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. 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, 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.

Verbruggen, B., Novakova, I., Wessels, H., Boezeman, J., van den Berg, M., & Mauser-Bunschoten, E. (1995). The Nijmegen modification of the Bethesda assay for factor VIII:C inhibitors: Improved specificity and reliability. Thrombosis and Haemostasis, 73(2), 247-251.

Protocol ID

911301

Variables

Export Variables

Variable Name	Variable ID	Variable Description	dbGaP Mapping
PX911301_Factor_Eight_Emicizumab_Sample_Collection
	PX911301010100	Were the sample collection procedures more outlined in Lippi et al. (2012) followed? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Blood_Draw
	PX911301010400	Was the order of blood draw recorded?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Nonhemolyzed
	PX911301010300	Were nonhemolyzed samples collected?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Tubes
	PX911301010500	Were collection tubes filled and mixed as more outlined in Lippi et al. (2012)? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Tubes_Discard
	PX911301011000	Was a discard tube drawn?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Tubes_Filled
	PX911301010700	Were the tubes filled within 11% of the fill line?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Tubes_Second
	PX911301010800	Was a second tube collected?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Tubes_Winged
	PX911301010900	Was a winged butterfly collection system used?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Venipuncture
	PX911301010600	Was blood collected by direct venipuncture more into 3.2% sodium citrate tubes? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Collection_Venous_Statsis
	PX911301010200	Were steps taken to prevent prolonged venous more stasis? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing
	PX911301020100	Were the sample collection and processing more procedures outlined in Adcock Funk et al. (2012) followed? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Agitation
	PX911301020500	Were samples agitated during transportation?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Assayed
	PX911301021000	Were samples assayed within 4 hours of collection?	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Centrifuge_Validated
	PX911301021100	Was the centrifuge validated so that process more results in less than 10,000 platelets/microliter show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Deep_Freeze
	PX911301021300	Was the sample transferred to <= -70 C, more including shipment? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Frozen
	PX911301021200	Was the sample frozen immediately and stored more at -20 C for at most 2 weeks? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Platelet_Poor_Plasma
	PX911301020900	Were samples processed to platelet poor more plasma (PPP) within 1 hour of collection? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Refrigerated
	PX911301020300	Were samples refrigerated or stored on ice more or in an ice bath? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Sodium_Citrate
	PX911301020200	Did unprocessed or processed sodium citrate more samples remain capped and at room temperature until testing? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Storage_Plasma
	PX911301020800	Were samples stored as plasma processed by more centrifugation and aliquoting into a second tube? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Storage_Unprocessed
	PX911301020600	Were samples stored as unprocessed sodium more citrate whole blood samples? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Storage_Whole_Blood
	PX911301020700	Were samples stored as whole blood samples more centrifuged and maintained in sodium citrate tubes? show less	N/A
PX911301_Factor_Eight_Emicizumab_Sample_Processing_Transportation
	PX911301020400	Were samples transported vertically?	N/A
PX911301_Factor_Eight_Emicizumab_Therapy_Bethesda_Assay_Nijmegen_Modification_Chromogenic_Substrate_Assay
	PX911301030100	Were any changes made in the protocol over more the course of the study? show less	N/A
PX911301_Factor_Eight_Emicizumab_Therapy_Bethesda_Assay_Nijmegen_Modification_Chromogenic_Substrate_Assay_Guidelines
	PX911301030400	Were the parameters outlined by the more International Society on Thrombosis and Haemostasis Subcommittee on Factor VIII, Factor VIII and Rare Coagulation Disorders (Iorio et al., 2017) followed? show less	N/A
PX911301_Factor_Eight_Emicizumab_Therapy_Bethesda_Assay_Nijmegen_Modification_Chromogenic_Substrate_Assay_Interpretation_Results
	PX911301040000	Was a result of of >= 0.6 Bethesda Units more (BU) per mL on two or more separate assays over a 1-4 week period achieved? show less	N/A
PX911301_Factor_Eight_Emicizumab_Therapy_Bethesda_Assay_Nijmegen_Modification_Chromogenic_Substrate_Assay_Record
	PX911301030200	Were the make and manufacturer of equipment, more repeatability and coefficients of variation for the assay, and the reagents used recorded? show less	N/A
PX911301_Factor_Eight_Emicizumab_Therapy_Bethesda_Assay_Nijmegen_Modification_Chromogenic_Substrate_Assay_Samples
	PX911301030300	Were the samples heated prior to testing to more eliminate exogenous Factor VIII? show less	N/A

Hemophilia Inhibitor

Measure Name

Quantitative Measure of Factor VIII Inhibitor Activity in the Presence of Emicizumab

Release Date

May 7, 2019

Definition

In hemophilia A, inhibitors are antibodies developed against Factor VIII in response to therapeutic infusion of clotting factors. Inhibitors neutralize the ability of infused FVIII concentrates to prevent or stop bleeding.

Purpose

This measure can be used to determine the activity of a Factor VIII inhibitor when treating with emicizumab. The results can be used to guide treatment for breakthrough bleeds and can also be combined with the results of the Response to Factor VIII Infusion in the Presence of Emicizumab measure to document the evolution, resolution, or persistence of inhibitors.

Keywords

Hemophilia inhibitors, hemophilia, hemophilia A, Factor VIII, FVIII

Measure Protocols

Protocol ID	Protocol Name
911301	Emicizumab Therapy: Determination of Factor VIII Inhibitors Using the Bethesda Assay with Nijmegen Modification: Chromogenic Substrate Assay with Bovine Reagents

Publications

There are no publications listed for this protocol.