Protocol - Population-based Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor IX Products
This protocol provides instructions and guidance for collecting and processing samples for coagulation testing, performing a population-based pharmacokinetic study using the chromogenic substrate assay, and interpreting pharmacokinetic results in response to infusion of extended half-life Factor IX products. Because there are many comparable assays for performing the chromogenic substrate assay, the protocol also provides basic guidelines to aid comparability among different studies.
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 demonstrates less variation by reagents when testing different modified Factor IX (FIX) products, thus enabling cross-laboratory comparisons.
The WG also notes that while the chromogenic substrate assay is widely used for the determination of FIX activity in research studies, there are no U.S. Food and Drug Administration (FDA)-approved assays (as of August 2018).
Standard Half-life Factor IX Products: Population-based Pharmacokinetic Study Using Chromogenic Substrate Assay
The PhenX Hemophilia Inhibitors 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.
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
- processed 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 aliquoted into a second tube.
Ideally, whole blood samples should be processed to platelet-poor plasma (PPP) within 1 hour of collection and assayed within 4 hours of collection.
If centrifuging samples, the centrifuge should be validated so that post-centrifuged samples contain less than 10,000 platelets/microliter. Centrifuged samples should be frozen immediately and can be stored at -20o C for 2 weeks. Samples should be transferred to < -70o C for longer storage, including shipment.
Standard Half-life Factor IX Products: 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.
Standard Half-life Factor IX Products: Population-based Pharmacokinetic Study
The WG recommends that investigators follow parameters outlined by the International Society on Thrombosis and Haemostasis Subcommittee on Factor VIII, Factor IX and Rare Coagulation Disorders (Iorio et al., 2017). These parameters include taking two measurements at least 24 hours apart after a routine dose of the extended half-life product (i.e., no washout period and no standardized dose) at the following timepoints:
- 24-36 hours after infusion; and
- 48-60 hours after infusion.
Standard Half-life Factor IX Products: Population-based Pharmacokinetics Model
Investigators should use a "robust" population pharmacokinetics model, such as WAPPS-Hemo or PKFit.
Personnel and Training Required
Equipment NeedsLaboratory with the ability to perform the chromogenic substrate assay.
|Specialized requirements for biospecimen collection||Yes|
|Average time of greater than 15 minutes in an unaffected individual||Yes|
Mode of Administration
Toddler, Child, Adolescent, Adult
The PhenX 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 (Iorio et al., 2017) provides recommended parameters for consistent implementation of the pharmacokinetic study.
Process and Review
Protocol Name from Source
Iorio et al. Estimating and interpreting the pharmacokinetic profiles of individual patients with hemophilia A or B using a population pharmacokinetic approach: Communication from the SSC of the ISTH. J Thromb Haemost, 2017
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.
Iorio, A., Blanchette, V., Blatny, J., Collins, P., Fischer, K., & Neufeld, E. (2017). Estimating and interpreting the pharmacokinetic profiles of individual patients with hemophilia A or B using a population pharmacokinetic approach: Communication from the SSC of the ISTH. Journal of Thrombosis and Haemostasis, 15(12), 2461-2465.
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.
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.
Hay, C. R., & DiMichele, D. M.; International Immune Tolerance Study. (2012). The principal results of the International Immune Tolerance Study: A randomized dose comparison. Blood, 119(6), 1335-1344.
Iorio, A., Keepanasseril, A., Foster, G., Navarro-Ruan, T., McEneny-King, A., Edginton, A. N., & Thabane, L.; WAPPS-Hemo Co-investigator Network. (2016). Development of a Web-Accessible Population Pharmacokinetic Service-Hemophilia (WAPPS-Hemo): Study protocol. JMIR Research Protocols, 5(4), e239.
Iorio, A., Edginton, A.N., Blanchette, V., Blatny, J., Boban, A., Cnossen, M., Collins, P., Croteau, S.E., Fischer, K., Hart, D.P., Ito, S., Korth-Bradley, J., Lethagen, S., Lillicrap, D., Makris, M., Math
Kitchen, S., Kershaw, G., & Tiefenbacher, S. (2016). Recombinant to modified factor VIII and factor IX Chromogenic and one-stage assays issues. Haemophilia, 22(Suppl. 5), 72-77.
Lee, M., Morfini, M., Schulman, S., & Ingerslev, J.; Factor VIII/Factor IX Scientific and Standardization Committee of the International Society for Thrombosis and Haemostasis (2001). Scientific and Standardization Committee communication. The design and analysis of pharmacokinetic studies of coagulation factors. https://www.isth.org/members/group_content_view.asp?group=100348&id=159244. Retrieved August 30, 2018.
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.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX911003030100||Were any changes made in the protocol over more||N/A|
|PX911003030200||Were the make and manufacturer of equipment, more||N/A|
|PX911003040400||Was a robust population pharmacokinetics more||N/A|
|PX911003040100||Were the parameters outlined by the more||N/A|
|PX911003040200||Was a measurement taken 24-36 hours after more||N/A|
|PX911003040300||Was a measurement taken 48-60 hours after more||N/A|
|PX911003010100||Were the sample collection procedures more||N/A|
|PX911003010400||Was the order of blood draw recorded?||N/A|
|PX911003010300||Were nonhemolyzed samples collected?||N/A|
|PX911003010500||Were collection tubes filled and mixed?||N/A|
|PX911003011000||Was a discard tube drawn?||N/A|
|PX911003010700||Were the tubes filled within 11% of the fill line?||N/A|
|PX911003010800||Was a second tube collected?||N/A|
|PX911003010900||Was a winged butterfly collection system used?||N/A|
|PX911003010600||Was blood collected by direct venipuncture more||N/A|
|PX911003010200||Were steps taken to prevent prolonged venous more||N/A|
|PX911003020100||Were the sample collection and processing more||N/A|
|PX911003020500||Were samples agitated during transportation?||N/A|
|PX911003021000||Were samples assayed within 4 hours of collection?||N/A|
|PX911003021100||Was the centrifuge validated so that process more||N/A|
|PX911003021300||Was the sample transferred to <= -70 C, more||N/A|
|PX911003021200||Was the sample frozen immediately and stored more||N/A|
|PX911003020900||Were samples processed to platelet poor more||N/A|
|PX911003020300||Were samples refrigerated or stored on ice more||N/A|
|PX911003020200||Did unprocessed or processed sodium citrate more||N/A|
|PX911003020800||Were samples stored as plasma processed by more||N/A|
|PX911003020600||Were samples stored as unprocessed sodium more||N/A|
|PX911003020700||Were samples stored as whole blood samples more||N/A|
|PX911003020400||Were samples transported vertically?||N/A|
Response to Factor IX Infusion - Population-based Pharmacokinetic Study
May 7, 2019
A series of plasma Factor IX activity determinations in blood samples are obtained immediately prior to, and at timepoints after, infusion of Factor IX concentrate.
The results of the population-based pharmacokinetic study (i.e., initial recovery and half-life) can be used to guide prophylactic dosage of standard and extended half-life Factor IX products in hemophiliacs who do not have FIX inhibitors.
Hemophilia inhibitors, Factor IX, FIX, hemophilia B, pharmacokinetic study, population pharmacokinetic study, popPK, prophylaxis, half-life, recovery
|Protocol ID||Protocol Name|
|911001||Population-based Pharmacokinetic Study Using One-stage Clotting Factor Assay - Standard Half-life Factor IX Products|
|911002||Population-based Pharmacokinetic Study Using One-stage Clotting Factor Assay - Extended Half-life Factor IX Products|
|911003||Population-based Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor IX Products|
|911004||Population-based Pharmacokinetic Study Using Chromogenic Substrate Assay - Extended Half-life Factor IX Products|
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