Protocol - Factor VIII Activity in Plasma Using the Chromogenic Substrate Assay

Description

This protocol provides instructions and guidance for collecting and processing samples for coagulation testing and for interpreting results. Because there are many comparable assays for performing 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 in comparison to the one-stage clotting factor assay, the chromogenic substrate assay can produce more uniform results, thus enabling cross-laboratory comparisons. It is not influenced by emicizumab when using bovine reagents.

Availability

Available

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Protocol

Factor VIII Activity in Plasma Using the 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^o C for 2 weeks and should be transferred to <= -70^o C for longer storage, including shipment.

Chromogenic Substrate Assay

The WG notes that there are a number of different assays and instruments that are appropriate to perform 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.

Interpretation of Results

The International Society on Thrombosis and Haemostasis (Blanchette et al., 2014) provides the following consensus definitions for the severity of hemophilia A based on plasma levels of Factor VIII activity:

severe hemophilia A if < 1% of normal,
moderate hemophilia A if > 1% and < 5% of normal,
mild hemophilia A if > 5% of normal.

Personnel and Training Required

Phlebotomist

Equipment Needs

Laboratory with the ability to perform 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

Participants

Any age

Selection Rationale

The Hemophilia Inhibitors Working Group (WG) 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

None

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

Bowyer, A. E., Duncan, E. M., & Antovic, J.P. (2018). Role of chromogenic assays in haemophilia A and B diagnosis. Haemophilia, 24(4), 578-583.

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.

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(2), 248-261.

Teichman, J., Chaudhry, H. R., & Sholzberg, M. (2018). Novel assays in the coagulation laboratory: A clinical and laboratory perspective. Transfusion and Apheresis Science, 57(4), 480-484.

Protocol ID

910301

Variables

Export Variables

Variable Name	Variable ID	Variable Description	dbGaP Mapping
PX910301_Factor_Eight_Plasma_Chromogenic_Interpretation_Results_Mild
	PX910301050300	Was a result > 5% of normal?	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Interpretation_Results_Moderate
	PX910301050200	Was a result > 1% and < 5% of normal?	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Interpretation_Results_Severe
	PX910301050100	Was a result < 1% of normal?	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Collection
	PX910301010000	Were the sample collection procedures more outlined in Lippi et al. (2012) followed to ensure quality specimens for coagulation testing? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing
	PX910301020100	Were the sample collection procedures more outlined in Adcock Funk et al. (2012) followed? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Capped_Room_Temp
	PX910301020200	Did unprocessed or processed sodium citrate more samples remain capped and at room temperature until testing? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Not_Agitated
	PX910301020500	Were samples agitated during transportation more to avoid remixing of components? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Not_Refrigerated_Stored
	PX910301020300	Were samples refrigerated or stored on ice more or in an ice bath? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Not_Transported_Vertically
	PX910301020400	Were samples transported vertically?	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Plasma_Processed_Centrifugation
	PX910301030400	Were samples transported and stored as more plasma processed by centrifugation and aliquoting into a second tube? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Transported_Stored
	PX910301030100	Were procedures for samples transportation more and storage followed? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Unprocessed_Sodium_Citrate
	PX910301030200	Were samples transported and stored as more unprocessed sodium citrate whole blood samples? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Sample_Processing_Whole_Blood_Centrifuged
	PX910301030300	Were samples transported and stored as whole more blood samples centrifuged and maintained in sodium citrate tubes? show less	N/A
PX910301_Factor_Eight_Plasma_Chromogenic_Substrate_Assay
	PX910301040000	Were appropriate assays and instruments to more perform the chromogenic substrate assay chosen? show less	N/A

Hemophilia Inhibitor

Measure Name

Factor VIII Activity in Plasma

Release Date

May 7, 2019

Definition

Factor VIII is an essential blood-clotting protein.

Purpose

Factor VIII activity measured in plasma can determine the presence and severity of hemophilia A. Values that are less than anticipated may reflect the presence of a neutralizing antibody (inhibitor).

Keywords

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

Measure Protocols

Protocol ID	Protocol Name
910301	Factor VIII Activity in Plasma Using the Chromogenic Substrate Assay
910302	Factor VIII Activity in Plasma Using the One-Stage Clotting Factor Assay

Publications

There are no publications listed for this protocol.