Protocol - Determination of Factor IX Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate 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 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.

The WG notes that samples should be heat inactivated to enable quantification of inhibitors in patients with circulating exogenous Factor IX .

The WG also notes that while the chromogenic substrate assay is used for the determination of FIX activity, it has not been validated, and there are no U.S. Food and Drug Administration (FDA)-approved assays as of June 2018.

Availability

Available

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Protocol

Determination of Factor IX Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate 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 ^oC for 2 weeks and should be transferred to <= -70^o C for longer storage, including shipment.

Determination of Factor IX Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate 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 chromogenic substrate 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 IX (Miller et al., 2012).

Interpretation of Results

The International Society on Thrombosis and Haemostasis (Blanchette et al., 2014) consensus definition for a relevant Factor IX 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.

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

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 IX Products, Individual Pharmacokinetic Study using One-stage Clotting Factor Assay - Extended Half-life Factor IX Products, Individual Pharmacokinetic Study using Chromogenic Assay - Extended Half-life Factor IX Products, or Individual Pharmacokinetic Study Using Chromogenic Assay - Standard Half-life Factor IX 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 IX infusion for treatment of bleeding,
less than expected (< 66%) recovery of Factor IX levels immediately after infusion (Response to Factor IX Infusion - Individual Pharmacokinetic Study) and positive inhibitor titer (Quantitative Measure of Factor IX Inhibitor Activity) in routine surveillance.

Evolution of an Inhibitor

Inhibitor evolution is indicated by:

change in inhibitor titer over time (Quantitative Measure of Factor IX Inhibitor Activity), with or without immune tolerance induction,
change in clinical response (i.e., bleeding) to Factor IX infusion,
change in Factor IX activity after factor infusion (Response to Factor IX Infusion - Individual Pharmacokinetic Study),
change in Factor IX half-life.

Resolution of an Inhibitor

Inhibitor resolution is indicated by the following:

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

Persistence of an Inhibitor

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

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.

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.

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.

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

910401

Variables

Export Variables

Variable Name	Variable ID	Variable Description	dbGaP Mapping
PX910401_Quantitive_Factor_Nine_Chromogenic_Bethesda_Assay_Nijmegen_Modification_One
	PX910401040000	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
PX910401_Quantitive_Factor_Nine_Chromogenic_Interpretation_Results
	PX910401050000	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
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Collection
	PX910401010000	Were the sample collection procedures more outlined in Lippi et al. (2012) followed to ensure quality specimens for coagulation testing? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing
	PX910401020100	Were the sample collection procedures more outlined in Adcock Funk et al. (2012) followed? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Capped_Room_Temp
	PX910401020200	Did unprocessed or processed sodium citrate more samples remain capped and at room temperature until testing? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Not_Agitated
	PX910401020500	Were samples agitated during transportation more to avoid remixing of components? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Not_Refrigerated_Stored
	PX910401020300	Were samples refrigerated or stored on ice more or in an ice bath? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Not_Transported_Vertically
	PX910401020400	Were samples transported vertically?	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Plasma_Processed_Centrifugation
	PX910401030400	Were samples transported and stored as more plasma processed by centrifugation and aliquoting into a second tube? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Transported_Stored
	PX910401030100	Were procedures for samples transportation more and storage followed? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Unprocessed_Sodium_Citrate
	PX910401030200	Were samples transported and stored as more unprocessed sodium citrate whole blood samples? show less	N/A
PX910401_Quantitive_Factor_Nine_Chromogenic_Sample_Processing_Whole_Blood_Centrifuged
	PX910401030300	Were samples transported and stored as whole more blood samples centrifuged and maintained in sodium citrate tubes? show less	N/A

Hemophilia Inhibitor

Measure Name

Quantitative Measure of Factor IX Inhibitor Activity

Release Date

May 7, 2019

Definition

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

Purpose

This measure can be used to screen for and quantitate the concentration of a Factor IX inhibitor. The results can also be combined with Response to Factor IX 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 B, Factor IX, FIX

Measure Protocols

Protocol ID	Protocol Name
910401	Determination of Factor IX Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate Assay
910402	Determination of Factor IX Inhibitors: Bethesda Assay with or without Nijmegen Modification Using One-Stage Clotting Factor Assay

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