Protocol - Individual Pharmacokinetic Study using One-stage Clotting Factor Assay - Standard Half-life Factor IX Products
This protocol provides instructions and guidance for collecting and processing samples for coagulation testing, performing an individual pharmacokinetic study using the one-stage clotting factor assays, and interpreting pharmacokinetic results in response to factor infusion. Because there are many comparable assays for performing the one-stage clotting factor test, 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 the one-stage clotting factor assay for determining Factor IX Activity in Plasma can vary by reagent and instrument used and recommends that the investigator record the make and manufacturer of equipment, the repeatability and coefficients of variation for the assay, and the reagents used.
If a washout is not performed, comparison of pharmacokinetic data between studies should be conducted under steady-state conditions. Steady-state is defined as a patient-specific condition during which a pharmacokinetic assessment remains valid over a clinically useful period of time. Examples of non-steady state conditions include:
- Bleeding state
- Peri- and immediate post-surgical states in which patients are receiving continuous or regular high (non-routine prophylactic) doses of FVIII
- Immune tolerance induction during which inhibiter titers are in flux
- Children in whom age- and weight-based clearance is still developing toward adult physiologic states.
Standard Half-life Factor IX Products: Individual Pharmacokinetic Study Using One-stage Clotting Factor 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: One-stage Clotting Factor Assay
The WG notes that there are a number of different assays and instruments that are appropriate to perform the one-stage clotting factor 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. Use of a central laboratory for multi-center clinical trials is strongly encouraged.
Standard Half-life Factor IX Products: Individual Pharmacokinetic Study
The WG recommends that the pharmacokinetic evaluations using the one-stage clotting factor assay be performed according to the parameters outlined by Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (Lee et al., 2001). This includes infusing a 100% correction dose of Factor IX concentrate after a treatment-free washout period lasting longer than 5 half-lives. Factor IX activity is then tested at 10 timepoints:
- before infusion (baseline),
- 10-15 minutes after infusion,
- 30 minutes after infusion,
- 1 hour after infusion,
- 3 hours after infusion,
- 6 hours after infusion,
- 9 hours after infusion,
- 24 hours after infusion,
- 48 hours after infusion, and
- 50 hours after infusion.
A sample taken at 72 hours after infusion is optional if the patient received > 75 u/kg of standard half-life Factor IX product.
Standard Half-life Factor IX Products: Interpretation of Individual Pharmacokinetic Study Results
The half-life of Factor IX therapies is highly variable (see [Iorio, 2017]), and the level at which an inhibitor is suggested has not been established.
Personnel and Training Required
Equipment NeedsLaboratory with the ability to perform the one-stage clotting factor 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 (Lee et al., 2001) provides standard timepoints for consistent implementation of a pharmacokinetic study.
|caDSR Common Data Elements (CDE)||Individual Pharmacokinetic Study Coagulation Factor IX Standard Half Life One-stage Clotting Factor Assay||6706629||CDE Browser|
The results of this protocol can be combined with the results of Determination of Factor IX Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate Assay or Determination of Factor IX Inhibitors: Bethesda Assay with or without Nijmegen Modification Using One-Stage Clotting Factor Assay 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
Protocol Name from Source
Lee et al., Scientific and Standardization Committee Communication: The Design and Analysis of Pharmacokinetic Studies of Coagulation Factors. ISTH, 2001.
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.
Lee, M., Morfini, M., Schulman, S., & Ingerslev, J.; Factor VIII/Factor VIII 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.
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., 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
Iorio, A. (2017). Using pharmacokinetics to individualize hemophilia therapy. Hematology, 2017(1), 595-604.
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.
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.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX910901040300||Was Factor IX activity tested 10-15 minutes more||N/A|
|PX910901040500||Was Factor IX activity tested 1 hour after more||N/A|
|PX910901040900||Was Factor IX activity tested 24 hours after more||N/A|
|PX910901040600||Was Factor IX activity tested 3 hours after more||N/A|
|PX910901041000||Was Factor IX activity tested 48 hours after more||N/A|
|PX910901040700||Was Factor IX activity tested 6 hours after more||N/A|
|PX910901041100||Was Factor IX activity tested 50 hours after more||N/A|
|PX910901040800||Was Factor IX activity tested 9 hours after more||N/A|
|PX910901040400||Was Factor IX activity tested 30 minutes more||N/A|
|PX910901040200||Was Factor IX activity tested before more||N/A|
|PX910901020903||Was the centrifuge validated so that process more||N/A|
|PX910901010201||Was a discard tube drawn?||N/A|
|PX910901020904||Were centrifuged samples frozen immedietely more||N/A|
|PX910901040100||Was a one-stage clotting factor assay more||N/A|
|PX910901050000||Were results interpreted according to: The more||N/A|
|PX910901030000||Were appropriate assays and instruments to more||N/A|
|PX910901010100||Were the sample collection procedures more||N/A|
|PX910901020100||Were the sample collection procedures more||N/A|
|PX910901020200||Did unprocessed or processed sodium citrate more||N/A|
|PX910901020500||Were samples not agitated during more||N/A|
|PX910901020300||Were samples not refrigerated or stored on more||N/A|
|PX910901020400||Were samples not transported vertically?||N/A|
|PX910901020900||Were samples transported and stored as more||N/A|
|PX910901020600||Were procedures for samples transportation more||N/A|
|PX910901020700||Were samples transported and stored as more||N/A|
|PX910901020800||Were samples transported and stored as whole more||N/A|
|PX910901020905||Were samples transferred to <-70C for longer more||N/A|
|PX910901020902||Were samples assayed within 4 hours of collection?||N/A|
|PX910901020901||Were whole blood samples processed to more||N/A|
|PX910901010200||Was a winged butterfly collection system used?||N/A|
Response to Factor IX Infusion - Individual 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 FIX concentrate.
The results of an individual pharmacokinetic study (i.e., initial recovery and half-life) of infused Factor IX concentrate can characterize an individual’s response to a new drug or can confirm the success of immune tolerance induction (ITI).
Hemophilia inhibitors, Factor IX, FIX, hemophilia B, inhibitors, pharmacokinetic study, immune tolerance induction, prophylaxis, half-life, recovery
|Protocol ID||Protocol Name|
|910901||Individual Pharmacokinetic Study using One-stage Clotting Factor Assay - Standard Half-life Factor IX Products|
|910902||Individual Pharmacokinetic Study using One-stage Clotting Factor Assay - Extended Half-life Factor IX Products|
|910903||Individual Pharmacokinetic Study Using Chromogenic Assay - Standard Half-life Factor IX Products|
|910904||Individual Pharmacokinetic Study Using Chromogenic Assay - Extended Half-life Factor IX Products|
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