Protocol - Individual Pharmacokinetic Study Using One-stage Clotting Factor Assay - Extended Half-life Factor VIII 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 assay, and interpreting pharmacokinetic results in response to infusion of extended half-life Factor VIII products. Because there are many comparable assays for performing the one-stage clotting factor 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 certain one-stage assays are not suitable to monitor specific extended half-life products (see "General References"). Investigators should select an assay that aligns with the one used to determine the potency of the extended half-life product. Additionally, investigators should 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 Factor VIII
- 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.
Individual Pharmacokinetic Study Using One-stage Clotting Factor Assay - Extended Half-life Factor VIII Products
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.
Extended Half-life Factor VIII Products: One-stage Clotting Factor Assay
There are a number of different assays and instruments that are available to perform the one-stage clotting factor assay. However, the WG notes the activity of Factor VIII extended half-life products measured by the one-stage clotting factor assay can vary according to the reagents and instrumentation. Some one-stage assays are not suitable to monitor specific extended half-life products (see "General References"). Therefore, Investigators should select an assay that aligns with the one used to determine the potency of the extended half-life product.
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 VIII 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 VIII products. The Hemophilia Inhibitors WG notes that the timepoints necessary for the pharmacokinetic study to cover 2.5 half-lives will depend on the specific extended half-life Factor VIII product being used and can be identified in relevant pre-licensure studies. Investigators should report which timepoints were used.
Extended Half-life Factor VIII Products: Interpretation of Individual Pharmacokinetic Study Results
The half-life of extended half-life Factor VIII products is highly variable 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 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.
|caDSR Common Data Elements (CDE)||Population Pharmacokinetic Study Coagulation Factor IX Extended Half Life One-stage Clotting Factor Assay Ê||6706642||CDE Browser|
The results of this protocol can be combined with the results of Determination of Factor VIII Inhibitors: Bethesda Assay with Nijmegen Modification Using Chromogenic Substrate Assay or Determination of Factor VIII Inhibitors: Bethesda Assay with Nijmegen Modification Using One-Stage Clotting Factor Assay to document:
Presence of an Inhibitor
The presence of an inhibitor is indicated by:
- lack of response (cessation of bleeding) to factor infusion for treatment of bleeding in a clinical setting,
- less than expected (< 66%) of Factor VIII levels after infusion (Response to Factor VIII Infusion - Individual Pharmacokinetic Study) and positive inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity) in routine surveillance.
Evolution of an Inhibitor
Inhibitor evolution is indicated by:
- change in inhibitor titer over time (Quantitative Measure of Factor VIII Inhibitor Activity), with or without immune tolerance induction;
- change in clinical response (i.e., bleeding) to Factor VIII infusion;
- change in Factor VIII activity after factor infusion (Response to Factor VIII Infusion - Individual Pharmacokinetic Study).
Resolution of an Inhibitor
Inhibitor resolution is indicated by the following:
- for patients receiving immune tolerance therapy for eradication of an Factor VIII inhibitor, success is defined as a negative inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity), a normal recovery (>= 66% of expected) and a normal half-life of infused Factor VIII concentrate (Response to Factor VIII Infusion- Individual Pharmacokinetic Study).
Persistence of an Inhibitor
A persistent inhibitor is indicated by a decrease response to Factor VIII concentrate infusion (Response to Factor VIII Infusion - Individual Pharmacokinetic Study) with or without a persistently positive inhibitor titer (Quantitative Measure of Factor VIII 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 VIII 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
Kitchen, S., Kershaw, G., & Tiefenbacher, S. (2016). Recombinant to modified factor VIII and factor VIII Chromogenic and one-stage assays issues. Haemophilia, 22(Suppl. 5), 72-77.
Kitchen, S., Tiefenbacher, S., Gosselin, R. (2017). Factor activity assays for monitoring extended half-life FVIII and Factor IX replacement therapies. Seminars in Thrombosis and Haemostasis, 43(3), 331-337.
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.
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|
|PX911102030200||Were any changes made in the protocol over more||N/A|
|PX911102030300||Were the make and manufacturer of equipment, more||N/A|
|PX911102030100||Does the assay selected align with the one more||N/A|
|PX911102050100||During interpretations of the results, was more||N/A|
|PX911102040100||Were the parameters outlined by the more||N/A|
|PX911102040400||Was a sample collected 10-15 minutes after more||N/A|
|PX911102040500||Were samples collected at up to eight other more||N/A|
|PX911102040300||Was a sample collected before infusion (baseline)?||N/A|
|PX911102040600||Was it taken into account that the more||N/A|
|PX911102040700||Were sample collection timepoints recorded?||N/A|
|PX911102040200||Did the patient undergo a treatment-free more||N/A|
|PX911102010100||Were the sample collection procedures more||N/A|
|PX911102010400||Was the order of blood draw recorded?||N/A|
|PX911102010300||Were nonhemolyzed samples collected?||N/A|
|PX911102010500||Were collection tubes filled and mixed?||N/A|
|PX911102011000||Was a discard tube drawn?||N/A|
|PX911102010700||Were the tubes filled within 11% of the fill line?||N/A|
|PX911102010800||Was a second tube collected?||N/A|
|PX911102010900||Was a winged butterfly collection system used?||N/A|
|PX911102010600||Was blood collected by direct venipuncture more||N/A|
|PX911102010200||Were steps taken to prevent prolonged venous more||N/A|
|PX911102020100||Were the sample collection and processing more||N/A|
|PX911102020500||Were samples agitated during transportation?||N/A|
|PX911102021000||Were samples assayed within 4 hours of collection?||N/A|
|PX911102021100||Was the centrifuge validated so that process more||N/A|
|PX911102021300||Was the sample transferred to <= -70 C, more||N/A|
|PX911102021200||Was the sample frozen immediately and stored more||N/A|
|PX911102020900||Were samples processed to platelet poor more||N/A|
|PX911102020300||Were samples refrigerated or stored on ice more||N/A|
|PX911102020200||Did unprocessed or processed sodium citrate more||N/A|
|PX911102020800||Were samples stored as plasma processed by more||N/A|
|PX911102020600||Were samples stored as unprocessed sodium more||N/A|
|PX911102020700||Were samples stored as whole blood samples more||N/A|
|PX911102020400||Were samples transported vertically?||N/A|
Response to Factor VIII Infusion - Individual Pharmacokinetic Study
May 7, 2019
A series of plasma Factor VIII activity determinations in blood samples are obtained immediately prior to, and at timepoints after, infusion of Factor VIII concentrate.
The results of an individual pharmacokinetic study (i.e., initial recovery and half-life) of infused Factor VIII concentrate can characterize an individual?s response to a new drug or can confirm the success of immune tolerance induction (ITI).
Hemophilia inhibitors, Factor VIII, FVIII, hemophilia A, inhibitors, pharmacokinetic study, immune tolerance induction, prophylaxis, half-life, recovery
|Protocol ID||Protocol Name|
|911101||Individual Pharmacokinetic Study Using One-stage Clotting Factor Assay - Standard Half-life Factor VIII Products|
|911102||Individual Pharmacokinetic Study Using One-stage Clotting Factor Assay - Extended Half-life Factor VIII Products|
|911103||Individual Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor VIII Products|
|911104||Individual Pharmacokinetic Study Using Chromogenic Substrate Assay - Extended Half-life Factor VIII Products|
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