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Protocol - Individual Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor VIII Products

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Description:

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 infusion of standard half-life Factor VIII 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.

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 WG notes that in comparison to the one-stage clotting factor assay, the chromogenic substrate assay demonstrates less variation by reagents, thus enabling cross-laboratory comparisons.

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.
Protocol:

Individual Pharmacokinetic Study Using Chromogenic Substrate Assay - Standard Half-life Factor VIII Products

Sample Collection

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.

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
  • 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 and processed plasma can be stored at -20o C for 2 weeks and should be transferred to < -70o C for longer storage, including shipment.

Standard Half-life Factor VIII 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 VIII Products: Individual Pharmacokinetic Study

The WG recommends that the pharmacokinetic evaluation using the chromogenic substrate assay be performed according to the parameters outlined by Subcommittee on Factor VIII and Factor VIII 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 VIII concentration after a treatment-free washout period lasting longer than five half-lives. Factor VIII 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;
  • 28 hours after infusion; and
  • 32 hours after infusion.

A sample taken at 48 hours after infusion is optional.

Standard Half-life Factor VIII Products: Interpretation of Individual Pharmacokinetic Study Results

A Factor VIII half-life (t) less than 6 hours is considered abnormal and evidence of an inhibitor.

Protocol Name from Source:

N/A; see source.

Availability:

Publicly available

Personnel and Training Required

Phlebotomist

Equipment Needs
Laboratory with the ability to perform the chromogenic substrate assay.
Requirements
Requirement CategoryRequired
Major equipment No
Specialized training No
Specialized requirements for biospecimen collection Yes
Average time of greater than 15 minutes in an unaffected individual Yes
Mode of Administration

Bioassay

Life Stage:

Toddler, Child, Adolescent, Adult

Participants:

Any age

Selection Rationale

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 the pharmacokinetic study.

Language

English

Standards
StandardNameIDSource
Derived Variables

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 the following:

  • lack of response (cessation of bleeding) to factor infusion for treatment of bleeding in a clinical setting, and
  • 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 FVIII infusion, and
  • change in FVIII 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 a Factor VIII inhibitor, success is defined as a negative inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity) and a normal recovery (>= 66% of expected) and half-life >= 6 hours of infused FVIII concentrate (Response to Factor VIII Infusion - Individual Pharmacokinetic Study).

Persistence of an Inhibitor

A persistent inhibitor is indicated by a decrease response to FVIII concentrate infusion (Response to Factor VIII Infusion) with or without a persistently positive inhibitor titer (Quantitative Measure of Factor VIII Inhibitor Activity - Individual Pharmacokinetic Study).

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

Not applicable.

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.

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.

General References

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., Matht, R., Morfini, M., Neufeld, E.J., Spears, J. (2018). Performing and interpreting individual pharmacokinetic profiles in patients with Hemophilia A or B: Rationale and general considerations. Research and Practice in Thrombosis and Haemostasis, 2(3), 535-548.

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.

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.

Protocol ID:

911103

Variables:
Export Variables
Variable NameVariable IDVariable DescriptionVersiondbGaP Mapping
PX911103_Factor_Eight_Individual_Chromogenic_Standard_One_Stage_Clotting_Factor_Assay_Changes PX911103030100 Were any changes made in the protocol over more
the course of the study? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_One_Stage_Clotting_Factor_Assay_Record PX911103030200 Were the make and manufacturer of equipment, more
repeatability and coefficients of variation for the assay, and the reagents used recorded? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Interpretation PX911103050100 Was the Factor VIII half-life (t) less than more
6 hours? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study PX911103040100 Were the parameters outlined by the more
Subcommittee on Factor VIII and Factor VIII of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (Lee et al., 2001) followed? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_1 PX911103040600 Was Factor VIII activity tested 1 hour after more
infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_24 PX911103041000 Was Factor VIII activity tested 24 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_28 PX911103041100 Was Factor VIII activity tested 28 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_3 PX911103040700 Was Factor VIII activity tested 3 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_32 PX911103041200 Was Factor VIII activity tested 32 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_48 PX911103041300 Was Factor VIII activity tested 48 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_6 PX911103040800 Was Factor VIII activity tested 6 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Hours_9 PX911103040900 Was Factor VIII activity tested 9 hours more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Minutes_10 PX911103040400 Was Factor VIII activity tested 10-15 more
minutes after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_After_Infusion_Minutes_30 PX911103040500 Was Factor VIII activity tested 30 minutes more
after infusion? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_Dose PX911103040200 Was the patient infused with a 100% more
correction dose of Factor VIII concentration after a treatment-free washout period lasting longer than 5 half-lives? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Pharmacokinetic_Study_Infusion_Baseline PX911103040300 Was Factor VIII activity tested before more
infusion (baseline)? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection PX911103010100 Were the sample collection procedures more
outlined in Lippi et al. (2012) followed? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Blood_Draw PX911103010400 Was the order of blood draw recorded? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Nonhemolyzed PX911103010300 Were nonhemolyzed samples collected? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Tubes PX911103010500 Were collection tubes filled and mixed as more
outlined in Lippi et al. (2012)? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Tubes_Discard PX911103011000 Was a discard tube drawn? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Tubes_Filled PX911103010700 Were the tubes filled within 11% of the fill line? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Tubes_Second PX911103010800 Was a second tube collected? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Tubes_Winged PX911103010900 Was a winged butterfly collection system used? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Venipuncture PX911103010600 Was blood collected by direct venipuncture more
into 3.2% sodium citrate tubes? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Collection_Venous_Statsis PX911103010200 Was steps taken to prevent prolonged venous more
stasis? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing PX911103020100 Were the sample collection and processing more
procedures outlined in Adcock Funk et al. (2012) followed? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Agitation PX911103020500 Were samples agitated during transportation? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Assayed PX911103021000 Were samples assayed within 4 hours of collection? N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Centrifuge_Validated PX911103021100 Was the centrifuge validated so that process more
results in less than 10,000 platelets/microliter show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Deep_Freeze PX911103021300 Was the sample transferred to <= -70 C, more
including shipment? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Frozen PX911103021200 Was the sample frozen immediately and stored more
at -20 C for at most 2 weeks? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Platelet_Poor_Plasma PX911103020900 Were samples processed to platelet poor more
plasma (PPP) within 1 hour of colleciton? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Refrigerated PX911103020300 Were samples refrigerated or stored on ice more
or in an ice bath? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Sodium_Citrate PX911103020200 Did unprocessed or processed sodium citrate more
samples remain capped and at room temperature until testing? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Storage_Plasma PX911103020800 Were samples stored as plasma processed by more
centrifugation and aliquoting into a second tube? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Storage_Unprocessed PX911103020600 Were samples stored as unprocessed sodium more
citrate whole blood samples? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Storage_Whole_Blood PX911103020700 Were samples stored as whole blood samples more
centrifuged and maintained in sodium citrate tubes? show less
N/A
PX911103_Factor_Eight_Individual_Chromogenic_Standard_Sample_Processing_Transportation PX911103020400 Were samples transported vertically? N/A
Hemophilia Inhibitor Research
Measure Name:

Response to Factor VIII Infusion - Individual Pharmacokinetic Study

Release Date:

May 7, 2019

Definition

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.

Purpose

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).

Keywords

Hemophilia inhibitors, Factor VIII, FVIII, hemophilia A, inhibitors, pharmacokinetic study, immune tolerance induction, prophylaxis, half-life, recovery