Optimizing thrombelastography (TEG) assay conditions to monitor rFVIIa (NovoSeven^®) therapy in haemophilia a patients

Abstract 

Introduction

There is no established laboratory method that can predict the most optimal dose of bypassing agents for treatment of haemophilia A. The objectives of the study was to develop an assay that can a) differentiate between the haemostatic capacity in blood from healthy individuals and severe and moderate haemophilia patients; b) show a dose-response correlation to rFVIIa addition; and c) show dose response differences of rFVIIa addition to plasma samples from non-inhibitor patients of different severity.

Materials and Methods

Citrated whole blood from 25 haemophilia A patients was used in four thrombelastography (TEG) assays initiated with: 1) kaolin, 2) Tissue Factor (TF, Innovin 1:42,500), 3) TF 1:42,500+1.2nM tPA (tissue plasminogen activator) or 4) TF 1:200,000. rFVIIa was added to give a final concentration in the range of 0.02-4.8µg/ml.

Results

The TEG assays showed large differences in clot formation demonstrated by prolonged clotting time (R-time), decreased maximum thrombus generation (MTG) between severe and moderate haemophilia A patients and between haemophilia patients and healthy males. The maximal amplitudes (MA) of the clot and resistance against fibrinolysis were only compromised when TF with tPA was added.

Conclusion

In vitro addition of rFVIIa improved all TEG profiles significantly in a dose-dependent manner; but only the TEG assay containing kaolin could differentiate between the rFVIIa doses, showing that blood from severe patients need higher doses of rFVIIa to normalize the clot formation profile compared to blood from moderate patients. Kaolin seems to be the most useful TEG assay for monitoring rFVIIa treatment.

Abbreviations: TEG, Thromboelastography, rFVIIa, recombinant Factor VIIa, FVIII, Factor VIII, FIX, Factor IX, FX, Factor X, tPA, tissue plaminogen activator, TF, tissue factor, R-time, Reaction time, MTG, maximum thrombus generation, MA, maximum amplitude, pd-aPCC, activated plasma derived prothrombin complex concentrate, EDTA, ethylenediaminetetraacetic acid, FVIII:C, Factor VIII concentration, HEPES, (2-hydroxyethyl)-1-piperazineethanesulfonic acid ), NaCl, Sodium chloride, BSA, Bovine serum albumin, sTF, soluble tissue factor, Vmax, Maximum velocity, ANOVA, analysis of variance, CRP, c-reactive protein, INR, international normalized ratio, aPTT, activated partial thromboplastin time, CV%, Coefficient percentage

Keywords: Haemophilia A, Monitoring, rFVIIa (NovoSeven), TEG, Thrombelastogram