Protocol - Determination of Factor VIII Inhibitors: Bethesda Assay with Nijmegen Modification Using One-Stage Clotting Factor Assay

Description

This protocol provides instructions and guidance for collecting and processing samples for coagulation testing, performing the one-stage clotting factor assay, and interpreting results. Because there are many comparable assays for performing Bethesda assay with Nijmegen modification using the one-stage clotting factor test, 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.

The WG notes the one-stage clotting factor assay for determining Factor VIII Activity in Plasma can vary by reagent or instrument used, particularly for modified recombinant Factor VIII products, and does not correlate with bleeding risk in the setting of emicizumab. 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.The WG notes that samples should be heat inactivated to enable quantification of inhibitors in patients with circulating exogenous Factor VIII.

Availability

Available

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Protocol

Determination of Factor VIII Inhibitors: Bethesda Assay with Nijmegen Modification Using One-Stage Clotting Factor Assay

Sample Collection

The 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
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 aliquoting into a second tube.

Ideally, whole blood samples should be processed to platelet poor plasma within 1 hour of collection and assayed within 4 hours of collection.

If centrifuging samples, the centrifuge should be validated so that process results in less than 10,000 platelets/microliter. Centrifuged and processed plasma can be stored at -20^o C for 2 weeks and should be transferred to <= -70^o C for longer storage, including shipment.

Bethesda Assay with Nijmegen Modification Using One-Stage Clotting Factor Assay

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 one-stage clotting factor assay. 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.

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

Interpretation of 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 one-stage clotting factor assay.

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

Participants

Any age

Selection Rationale

The 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 Individual Pharmacokinetic Study Using One-stage Clotting Factor Assay - Standard Half-life Factor VIII Products, Individual Pharmacokinetic Study Using One-stage Clotting Factor Assay - Extended Half-life Factor VIII Products, Individual Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor VIII Products, or Individual Pharmacokinetic Study Using Chromogenic Substrate Assay - Extended Half-life Factor VIII Products 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- Individual Pharmacokinetic Study), and
positive inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity)
Reduced Factor VIII 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), with or without immune tolerance induction;
change in clinical response (i.e., bleeding) to Factor VIII infusion;
change in Factor VIII activity after factor infusion (Response to Factor VIII Infusion - Individual Pharmacokinetic Study); and
change in Factor VIII half-life.

Resolution of an Inhibitor

Inhibitor resolution is indicated by the following:

for patients receiving immune tolerance therapy for eradication of an Factor VIII inhibitor, success is defined as a negative inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity) and a normal recovery (>= 66% of expected) and half-life >= 6 hours of infused Factor VIII concentrate (Response to Factor VIII Infusion - Individual Pharmacokinetic Study).

Persistence of an Inhibitor

A persistent inhibitor is indicated by a decreased response to Factor VIII concentrate infusion (Response to Factor VIII Infusion - Individual Pharmacokinetic Study) measured by recovery and/or half-life with or without a persistently positive inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity).

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

Blanchette et al. Definitions in hemophilia: communication from the SSC of the ISTH. J Thromb Haemost, 2014

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.

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<97>Chromogenic and one-stage assays issues. Haemophilia, 22(Suppl. 5), 72-77.

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.

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.

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.

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

910502

Variables

Export Variables

Variable Name	Variable ID	Variable Description	dbGaP Mapping
PX910502_Quantitive_Factor_Eight_Clotting_Bethesda_Assay_Nijmegen_Modification_One
	PX910502030000	Were appropriate assays and instruments to more perform the Bethesda assay with Nijmegen modification using the one-stage clotting factor assay chosen? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Individual_Centrifuge_Validated
	PX910502021003	Was the centrifuge validated so that process more results in less than 10,000 platelets/microliter? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Individual_Frozen_Immediately
	PX910502021004	Were centrifuged samples frozen immedietely more and stored at -20C for 2 weeks? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Individual_Transferred_Storage
	PX910502021005	Were samples transferred to <-70C for longer more storage, including shipment? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Individual_Whole_Blood_Assayed
	PX910502021002	Were samples assayed within 4 hours of collection?	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Individual_Whole_Blood_PPP
	PX910502021001	Were whole blood samples processed to more platelet poor plasma (PPP) within 1 hour of collection? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Interpretation_Results
	PX910502040000	Was a result of >= 0.6 Bethesda Units (BU) more per mL on two or more separate assays over a 1-4 week period achieved? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Collection
	PX910502010100	Were the sample collection procedures more outlined in Lippi et al. (2012) to ensure quality specimens for coagulation testing followed? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing
	PX910502020100	Were the sample collection procedures more outlined in Adcock Funk et al. (2012) followed? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Capped_Room_Temp
	PX910502020200	Did unprocessed or processed sodium citrate more samples remain capped and at room temperature until testing? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Not_Agitated
	PX910502020500	Were samples agitated during transportation more to avoid remixing of components? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Not_Refrigerated_Stored
	PX910502020300	Were samples refrigerated or stored on ice more or in an ice bath? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Not_Transported_Vertically
	PX910502020400	Were samples transported vertically?	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Plasma_Processed_Centrifugation
	PX910502020900	Were samples transported and stored as more plasma processed by centrifugation and aliquoting into a second tube? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Transported_Stored
	PX910502020600	Were procedures for samples transportation more and storage followed? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Unprocessed_Sodium_Citrate
	PX910502020700	Were samples transported and stored as more unprocessed sodium citrate whole blood samples? show less	N/A
PX910502_Quantitive_Factor_Eight_Clotting_Sample_Processing_Whole_Blood_Centrifuged
	PX910502020800	Were samples transported and stored as whole more blood samples centrifuged and maintained in sodium citrate tubes? show less	N/A
PX910502_Quantitive_Factor_Eight_Individual_Discard_Tube
	PX910502010202	Was a discard tube drawn?	N/A
PX910502_Quantitive_Factor_Eight_Individual_Winged_Butterfly
	PX910502010201	Was a winged butterfly collection system used?	N/A

Hemophilia Inhibitor

Measure Name

Quantitative Measure of Factor VIII Inhibitor Activity

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 Factor VIII concentrates to prevent or stop bleeding.

Purpose

This measure can be used to screen for and quantitate the concentration of a Factor VIII inhibitor. The results can also be combined with the Response to Factor VIII Infusion measure to determine the presence of an inhibitor and to document the evolution, resolution, or persistence of the inhibitor.

Keywords

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

Measure Protocols

Protocol ID	Protocol Name
910501	Determination of Factor VIII Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate Assay
910502	Determination of Factor VIII Inhibitors: Bethesda Assay with Nijmegen Modification Using One-Stage Clotting Factor Assay

Publications

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