Protocol - Individual Pharmacokinetic Study Using Chromogenic Assay - Extended 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 chromogenic substrate assay, and interpreting pharmacokinetic results in response to factor infusion. Because there are many comparable assays for performing the chromogenic 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).
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.
Extended Half-life Factor IX Products: Individual 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
- 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 (PPP) 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 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.
Extended 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.
Extended Half-life Factor IX Products: Individual Pharmacokinetic Study
Prior to pharmacokinetic evaluations, participants should undergo a treatment-free washout period lasting longer than five half-lives. Samples should be taken before infusion (baseline), 10-15 minutes after infusion, and then up to eight other time points that cover 2.5 half-lives of the extended half-life Factor IX products. The WG notes that the list of timepoints necessary for the pharmacokinetic evaluations to cover 2.5 half-lives will depend on the specific extended half-life Factor IX product being used and can be identified in relevant pre-licensure studies. Investigators should report which timepoints were used.
Extended 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 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 (Lee et al., 2001) provides standard timepoints for consistent implementation of a pharmacokinetic study.
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%) 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
Adcock Funk et al. Quality standards for sample processing, transportation, and storage in hemostasis testing. Semin Thromb Hemost, 2012
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.
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. (2017). Using pharmacokinetics to individualize hemophilia therapy. Hematology, 2017(1), 595-604.
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
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 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.
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.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX910904020903||Was the centrifuge validated so that process more||N/A|
|PX910904010201||Was a discard tube drawn?||N/A|
|PX910904030000||Were assays and instruments that are more||N/A|
|PX910904040000||Prior to pharmacokinetic evaluations, did more||N/A|
|PX910904050000||Were results interpreted according to? The more||N/A|
|PX910904010100||Were the sample collection procedures more||N/A|
|PX910904020100||Were the sample collection procedures more||N/A|
|PX910904020200||Did unprocessed or processed sodium citrate more||N/A|
|PX910904020500||Were samples not agitated during more||N/A|
|PX910904020300||Were samples not refrigerated or stored on more||N/A|
|PX910904020400||Were samples not transported vertically?||N/A|
|PX910904020900||Were samples transported and stored as more||N/A|
|PX910904020600||Were procedures for samples transportation more||N/A|
|PX910904020700||Were samples transported and stored as more||N/A|
|PX910904020800||Were samples transported and stored as whole more||N/A|
|PX910904020904||Were centrifuged samples frozen immedietely more||N/A|
|PX910904020905||Were samples transferred to <-70C for longer more||N/A|
|PX910904020902||Were samples assayed within 4 hours of collection?||N/A|
|PX910904020901||Were whole blood samples processed to more||N/A|
|PX910904010200||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).
individual pharmacokinetic study using chromogenic assay - extended half-life factor ix products, 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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