Protocol - Factor IX Activity in Plasma Using the Chromogenic Substrate Assay
- Determination of Factor IX Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate Assay
- Determination of Factor IX Inhibitors: Bethesda Assay with or without Nijmegen Modification Using One-Stage Clotting Factor Assay
- Individual Pharmacokinetic Study Using Chromogenic Assay - Extended Half-life Factor IX Products
- Individual Pharmacokinetic Study Using Chromogenic 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 One-stage Clotting Factor Assay - Standard Half-life Factor IX Products
- Population-based Pharmacokinetic Study Using Chromogenic Substrate Assay - Extended Half-life Factor IX Products
- Population-based Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor IX Products
- Population-based Pharmacokinetic Study Using One-stage Clotting Factor Assay - Extended Half-life Factor IX Products
- Population-based Pharmacokinetic Study Using One-stage Clotting Factor Assay - Standard Half-life Factor IX Products
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
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 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
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 ID | Variable Description | dbGaP 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 | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Capped_Room_Temp | ||||
| PX910201020200 | Did unprocessed or processed sodium citrate more | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Not_Agitated | ||||
| PX910201020500 | Were samples agitated during transportation more | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Not_Refrigerated_Stored | ||||
| PX910201020300 | Were samples refrigerated or stored on ice more | 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 | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Transported_Stored | ||||
| PX910201030100 | Were procedures for samples transportation more | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Unprocessed_Sodium_Citrate | ||||
| PX910201030200 | Were samples transported and stored as more | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Sample_Processing_Whole_Blood_Centrifuged | ||||
| PX910201030300 | Were samples transported and stored as whole more | N/A | ||
| PX910201_Factor_Nine_Plasma_Chromogenic_Substrate_Assay | ||||
| PX910201040000 | Were appropriate assays and instruments to more | N/A | ||
| PX910201_Factor_Nine_Plasma_Clotting_Sample_Collection | ||||
| PX910201010100 | Were the sample collection procedures more | N/A | ||
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.