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

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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 Hemophilia Inhibitor Research Working Group (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.

The WG also notes that while the chromogenic substrate assay is available for the determination of Factor IX activity, it is currently a research use-only assay because it has not been validated and there are no U.S. Food and Drug Administration (FDA)-approved assays as of June 2018.

Availability

Available

Protocol

Factor IX 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 -20o C for 2 weeks and should be transferred to <= -70o 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 B based on plasma levels of Factor IX activity:

  • severe hemophilia B if < 1% of normal,
  • moderate hemophilia B if > 1% and <= 5% of normal,
  • mild hemophilia B if > 5% of normal.
Personnel and Training Required

Phlebotomist

Equipment Needs
Laboratory with the ability to perform the chromogenic substrate assay.
Requirements
Requirement CategoryRequired
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
StandardNameIDSource
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.

Kershaw, G. W., Dissanayake, K., Chen, V. M., & Khoo, T. L. (2018). Evaluation of chromogenic factor IX assays by automated protocols. Haemophilia, 24(3), 492-501.

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.

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

910201

Variables
Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX910201_Factor_Nine_Plasma_Chromogenic_Interpretation_Results_Mild
PX910201050300 Was a result > 5% of normal? N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Interpretation_Results_Moderate
PX910201050200 Was a result > 1% and < 5% of normal? N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Interpretation_Results_Severe
PX910201050100 Was a result < 1% of normal? N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing
PX910201020100 Were the sample collection procedures more
outlined in Adcock Funk et al. (2012) followed? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Capped_Room_Temp
PX910201020200 Did unprocessed or processed sodium citrate more
samples remain capped and at room temperature until testing? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Not_Agitated
PX910201020500 Were samples agitated during transportation more
to avoid remixing of components? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Not_Refrigerated_Stored
PX910201020300 Were samples refrigerated or stored on ice more
or in an ice bath? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Not_Transported_Vertically
PX910201020400 Were samples transported vertically? N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Plasma_Processed_Centrifugation
PX910201030500 Were samples transported and stored as more
plasma processed by centrifugation and aliquoting into a second tube? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Transported_Stored
PX910201030100 Were procedures for samples transportation more
and storage followed? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Unprocessed_Sodium_Citrate
PX910201030200 Were samples transported and stored as more
unprocessed sodium citrate whole blood samples? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Whole_Blood_Centrifuged
PX910201030300 Were samples transported and stored as whole more
blood samples centrifuged and maintained in sodium citrate tubes? show less
N/A
PX910201_Factor_Nine_Plasma_Chromogenic_Substrate_Assay
PX910201040000 Were appropriate assays and instruments to more
perform the chromogenic substrate assay chosen? show less
N/A
PX910201_Factor_Nine_Plasma_Clotting_Sample_Collection
PX910201010100 Were the sample collection procedures more
outlined in Lippi et al. (2012) followed to ensure quality specimens for coagulation testing? show less
N/A
Hemophilia Inhibitor
Measure Name

Factor IX Activity in Plasma

Release Date

May 7, 2019

Definition

Factor IX is an essential blood-clotting protein.

Purpose

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

Keywords

Hemophilia inhibitors, hemophilia, hemophilia B, Factor IX, FIX

Measure Protocols
Protocol ID Protocol Name
910201 Factor IX Activity in Plasma Using the Chromogenic Substrate Assay
910202 Factor IX Activity in Plasma Using the One-Stage Clotting Factor Assay
Publications

There are no publications listed for this protocol.