Evaluation of a Standardized Protocol for Thrombin Generation Assay

Brief Title

Evaluation of a Standardized Protocol for Thrombin Generation Assay

Official Title

Calibrated Automated Thrombogram: A Scandinavian Multicenter Study

Brief Summary

      This study is aims at determining the inter laboratory variation when using the thrombin
      generation assay calibrated automated thrombogram (TGA CAT). It is thus, not a clinical trial
      in its usual meaning. However, to achieve relevant test samples one patient will be treated
      with two different study drugs as part of the trial and therefore, approval by
      Läkemedelsverket is needed. Test plasma samples will be sent out to five participating
      centers in the Scandinavian countries (Gothenburg and Stockholm, Sweden, Århus Denmark, Oslo
      Norway and Helsinki Finland) and coefficients of variance (CV) and level of agreement will be
      analyzed. To obtain representative plasma samples with a wide range of thrombin generation
      capacity (TGC), blood samples will be collected from research persons that has given informed
      consent to participate in the study. To obtain plasma with low TGC, blood samples will be
      drawn from patients with severe hemophilia (n=4)(study group 1), to obtain plasma with normal
      TGC, blood samples will be drawn from healthy volunteers (n=3)(study group 2) and to obtain
      plasma with high TGC, plasma will be collected from healthy volunteers (n=3)(study group 3)
      that at previous measurements have been shown to have a TGC>2SD of the median of the control
      population. Moreover, one patient with severe hemophilia A (HA) will be treated with two
      factor FVIII concentrates, one with standard half- life (Advate™) and one with a pro-longed
      half-life (Adynovate™) at two separate occasions (Treated HA person). By taking repeated
      blood samples after administration, samples with a wide range of FVIII levels and TGC:s will
      be obtained. Moreover, the effect of using plasmas with low, normal and high TGC for
      normalization will be investigated. Plasma samples will be collected as soon as approval from
      the Swedish medical agency (SMA) has been obtained, we count on sending them to participating
      centers March 2017. All laboratory measurements, data analysis and report writing will be
      concluded before December 31 2017.
    

Detailed Description

      Main goal The aim is to further improve the inter-laboratory variability for the TGA-CAT
      (Thrombin generation assay-calibrated automated thrombogram) method by using an elaborate
      standardization protocol and in extension making the use of large prospective multi-center
      studies a reality.

      Goals of the project are:

        -  To determine the inter-laboratory variability by evaluating an enhanced standardized
           TGA-CAT protocol.

        -  To evaluate if the inter-laboratory variability of the TGA-CAT method is effected by
           administration of a recombinant FVIII product (Advate) in a person with severe
           hemophilia A

        -  To evaluate if the inter-laboratory variability of the TGA-CAT method is effected by
           administration of a long acting FVIII product (Adynovate) in a person with severe
           hemophilia A

        -  To evaluate three reference plasma´s ability, through normalization of data, to improve
           the lab-to-lab variability.

      Introduction The TGA CAT method has proved its usefulness for multiple purposes including
      diagnosis and management of bleeding disorders (1, 2), detecting hyper coagulability (3, 4),
      and monitoring and characterization of oral anticoagulant drugs (5, 6). Most of these studies
      are single-center studies and large prospective multi-center studies are needed to further
      validate the results. These multi-center studies have proved to be hard to produce due to
      lack of standardization of the method. In the last couple of years several articles have
      addressed the problem with large inter-laboratory variability for the TGA-CAT method by
      presenting thoroughly worked-out standardization protocols. Focus has been put both on the
      method itself and its pre-analytical factors as well as the usage of reference plasmas (RP)
      for normalization of results. One article showed that the choice of RP for the normalization
      of results could greatly reduce the inter-lab variability and that certain RP have a better
      ability to do so (7).

      Lately a number of recombinant Factor VIII (rFVIII) and IX (rFIX) drugs with prolonged
      half-life have been developed and are about to or even have reached the market. The length of
      the pro-longed activity of the drug differs between patients and a need for individual
      tailoring of the patient´s drug administration is obvious. Thrombin generation could prove to
      be an excellent tool in tailoring the optimal drug administration and monitoring the effect
      of the drug for each individual.

      Methods and materials Thrombin generation Thrombin generation will be measured according to
      the method described by Hemker et al.(8): calibrated automated thrombogram (CAT,
      Thrombinoscope BV, Maastricht, the Netherlands).

      Reagents PPP-reagent LOW (1 pM), PPP-reagent (5 pM), Thrombin Calibrator and FluCa-kit
      (TS31.00, TS30.00, TS20.00 and TS50.00, Thrombinoscope BV, Maastricht, The Netherlands) will
      be used in the test.

      Samples Three different plasmas will be tested, normal plasma, hypo- and hypercoagulable
      plasma. Normal and hypercoagulable plasma will be pooled from 3 donors and hypocoagulable
      plasma will be collected from 4 donors. Hypocoagulable plasma will be collected from five
      haemophilia A (HA) patients regularly treated at the Malmö Coagulation Unit by taking 10 4.5
      mL tubes of citrated blood at one occasion. Normal plasma will be collected from 3 co-workers
      at the same unit, each person donating 13 tubes of 4.5 mL citrated blood. Hypercoaguable
      plasma will be prepared from 3 healthy volunteers, shown to have TGA CAT parameter values >2
      SD of median by taking 13 tubes of 4.5 mL citrated blood. Plasma from one patient with severe
      HA patient that have been given FVIII-concentrates, Advate™ respectively the long-acting
      Adynovate™ at two separate occasions will also be tested. After informed consent the patient
      will be asked not to take his regular profylaxis dose for 72 hours. The patient will be given
      Advate™ and Adynovate™ respectively at 30 U/kg. The monitoring samples will be taken 1h, 8h,
      24h, 36h and 48h (Advate™) and 1h, 24h, 36h, 48h and 72h (Adynovate™) after injection. At
      each time point 3 tubes of 4.5 mL blood will be taken.

      Reference plasmas Siemens Control Plasma P diluted 1:5 will be used as hypocoaguable RP,
      HemosIL calibration plasma (Instrumentation Laboratory, Bedford, Ma, USA) will be used as
      Standard RP and as hypercoagable plasma PS-DP (protein S-deficiency plasma)(Enzyme research
      laboratories, South Bend, In, USA) will be used.

      Statistical analysis Raw data will be collected and analyzed at the Coagulation Unit, Skåne
      University Hospital, Malmö. Each participating center will be matched with Malmö Coagulation
      unit in a test of agreement where Bland-Altman bias plot will be used. The mean, standard
      deviation (SD) and coefficient of variation (CV%) will be calculated for each center with and
      without normalization and displayed in a table. The Advate/ Adynovate elimination profiles
      will be plotted and multivariate repeated measurements analyses of variance including a
      general test for parallel curves, will be applied to evaluate differences between Centers in
      the elimination profiles.

      Study design The study aims to evaluate the inter-laboratory variability of TGA-CAT performed
      TGT with a highly standardized protocol, how the use of RFs for normalization of data impact
      on variability of results and to evaluate the usefulness of TGA-CAT in monitoring prolonged
      FVIII drug administration. All Fluorometers of the participating center´s should be serviced
      and have temperature calibrated to 37 C prior start of the study. The latest software version
      must be installed (Thromboscope BV, Maastricht, The Netherlands, version V5.0.0.742). All
      laboratory equipment, pipettes, water baths, water purifiers, etc., needs to be calibrated.
      All plasmas, TGA-CAT reagents needed for the study will be administered and distributed to
      all participating centers by the Coagulation Unit, Malmö. An USB-memory stick containing a
      plate set-up scheme file and a video file covering critical pre-analytical and analytical
      steps will also be provided alongside the samples and reagents. Three pooled plasmas (normal,
      hypo- and hypercoaguable), three RF, five Adynovate and five Advate monitoring samples will
      be run five times on five different days by each participating center, following a scheme
      provided in the USB-memory stick, with two different triggers, PPP-reagent LOW (1 pM) and
      PPP-reagent (5 pM). Two test runs are scheduled each day to maximize RF and reagent usage.
      The reason for testing three different types of RF is the fact an earlier study results
      implied a better reduction of variability when using, for an example a hypocoaguable RF to
      hypocoaguable plasmas (9).
    

Study Type

Observational

Primary Outcome

Inter lab variability

Secondary Outcome

 Inter lab variability

Condition

Hemophilia A

Intervention

Thrombin Generation Assay

Study Arms / Comparison Groups

 Study group 1

Description:  Patients with severe hemophilia A

Publications

* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.

Recruitment Status

Diagnostic Test

Estimated Enrollment

9

Start Date

April 15, 2017

Completion Date

January 28, 2019

Primary Completion Date

January 28, 2019

Eligibility Criteria

        Inclusion Criteria:

          -  For patients with severe hemophilia A (study group 1)(n=4) that has not received any
             factor concentrate within 72 hours: Diagnosis of severe hemophilia, with regular
             check-ups at the hemophilia center at SUS, Malmö and willingness to participate

          -  For healthy controls (n=3) (study group 2): Willingness to participate

          -  For healthy controls with a documented high TGC (study group 3): Willingness to
             participate

        Exclusion Criteria

          -  For patients with severe hemophilia A (study group 1)(n=4) that has not received any
             factor concentrate within 72 hours: Intake of any other pharmaceutical product known
             to have an effect on the coagulation system the last 14 days, to the judgement of the
             including investigator

          -  For healthy controls (n=3) (study group 2): Any disorder known to affect the
             coagulation system and intake of any drug known to affect the coagulation system the
             last 14 days before blood sampling , to the judgement of the including investigator.

          -  For healthy controls with a documented high TGC (study group 3): Any disorder known to
             affect the coagulation system and intake of any drug known to affect the coagulation
             system within the last 14 days before blood sampling, to the judgement of the
             including investigator.
      

Gender

Male

Ages

18 Years - 65 Years

Accepts Healthy Volunteers

Accepts Healthy Volunteers

Contacts

Jan Astermark, MD PhD, , 

Location Countries

Sweden

Location Countries

Sweden

NCT ID

NCT03313531

Organization ID

TGA1

Responsible Party

Sponsor

Study Sponsor

Lund University

Collaborators

 Shire

Study Sponsor

Jan Astermark, MD PhD, Study Director, Department of hematology, Skane University hospital

Verification Date

January 2018