Thrombin Generation Test (TGT) & Rotational Thromboelastography (ROTEM™) and its use in assessing Severe Haemophilia A phenotype The classification and diagnosis of Haemophilia A (HA) is commonly based on one stage clotting assays of factor VIII (FVIII:C). Patients with severe HA have a FVIII:C of less than 1IU/dL. Based on clinical observations, 10-15% of severe HA patients demonstrate a mild clinical phenotype 1. Recent published studies indicate that the measurement of prothrombotic markers and global parameters of haemostasis such as thrombin generation test (TGT) and thromboelastography may be useful predictors of clinical phenotype in severe HA 2,3. TGT is an in-vitro laboratory test that is performed on platelet poor plasma (PPP) or platelet rich plasma (PRP) using either a chromogenic or fluorogenic substrate to measure the generation of thrombin after the addition of re-lipidated tissue factor and CaCl 2. 4 Rotational Thromboelastography (ROTEM™) is a global real time measurement of the formation of a fibrin clot in a whole blood sample over a period of time 3. The data generated by TGT and ROTEM™ parameters suggest a wide range of variation in the patient group compared with normal individuals. The majority of patients in this study had received no prophylaxis as children making it difficult to score the true clinical phenotype and thus correlate with the TGT and ROTEM™ parameters. The information gained from ROTEM™ and TGT compared to conventional laboratory monitoring suggests a need for more comprehensive evaluation of these tools in the context of physiological and interpersonal variation of a thrombin generation. 1.Aledort, L.M., R.H. Haschmeyer, and H. Pettersson, A longitudinal study of orthopaedic outcomes for severe factor-VIII-deficient haemophiliacs. The Orthopaedic Outcome Study Group. J Intern Med, (4): p Beltran-Miranda, C.P., et al., Thrombin generation and phenotypic correlation in haemophilia A. Haemophilia, (4): p Sorenson B, Johansen P, Christiansen K, Woelke M, Ingerslav J. Whole blood coagulation thromboelastographic profiles employing minimal tissue factor activation. J. Thromb. Haemost. 1 (2003), pp. 551– Hemker H C, Beguin S. Phenotyping the clotting system. Thromb Haemost 2000; 84: TGT 80  L of PPP was added to 20mL initiation reagent (containing tissue factor (TF) (Innovin, DadeBehring, Germany), and phospholipid vesicles (PS/PC/PE) to obtain a final concentration 1pM TF and 4  M PS/PC/PE). For PRP, 80  L of PRP was added to 20mL initiation reagent (containing TF with final concentration of 0.5pM) After incubating at 37C o,  l starting reagent (containg100 mM CaCl 2 and 2.5mM Z-Gly- Gly-Arg-AMC.HCL fluorogenic substrate (Bachem, UK)) was added to each well. Relative fluorescent unit (RFU) were measured at 30 second intervals for one hour using a SpectraMax Gemini XS plate reader (Molecular Devices, UK) at excitation wavelength of 390nm and reading wavelength of 460nm. The data was exported into an excel™ file and the amount of thrombin generated was calculated according to the method by Hemker & Beguin, 1999 and Hemker et al 2000 to determine the amount of free thrombin in a continuous TGT with a fluorogenic substrate. ROTEM™ Citrated whole blood (CWB) was allowed to stand for 30 minutes before testing. 300  L of CWB was pipetted into a cup,  l TF (final concentration 0.35pM) was added and recalcified with 20  L 0.2MCaCl 2 as described by Sorenson. Prothrombothic Markers We have also measured intrinsic and extrinsic plasma clotting proteins, prothrombotic factors including: Protein C (Pro Clot, Instrumentation Laboratory (IL), Milan; Italy), protein S (HemosIL) and anti-thrombin (In-house assay); Activated protein C ratio (APCr), factor V Lieden (Arg506Glu) and prothrombin gene (G20210A ) mutation analysis (Third Wave™ Invader ®). Haemophilia A patients generate less thrombin than normal individuals in a rate dependent (on FVIII) manor. This is because of their inability to form the intrinsic Xase complex. It is apparent that very small amounts of FVIII, below those detectable in conventional FVIII assays, can result in significant amounts of thrombin generation. Hence, some factors, which may cause thrombophilic status, might modulate the bleeding tendency of severe haemophiliacs. Clinical presentation can be regarded as a function of the altered thrombo-haemoragic balance due to the action of many intervening factors. Therefore, it is conceivable that the presence of an abnormality i.e. prothrombotic facrors, may mitigate the clinical phenotype. High plasma concentration of coagulation proteins, such FIX or FXI, is shown to increase the risk of thrombosis in normal individuals. Presence other thrombotic factors such as factor V Leiden and prothrombin G202010A, on the other hand, may also lessen the severity of bleeding tendency in some haemophiliacs. Acknowledgements – Katharine Dormandy Haemophilia Centre & Dr Sanj Raut (NIBSC, Potters Bar, UK for the EXCELL™ spreadsheet to calculate the TGT curves TGT (parameters were expressed as percentage of normal) CTI containing PPP: AUC=12%-67%, mean=41%; Peak Height (PH)=6%-47%, mean=22%; time to peak (TP) expressed in minutes=11-56min, mean=22 CTI containing PRP: AUC=7%-68%, mean=35%; PH=7%-47%, mean=28%; TP=37-61min, mean=50min There was no correlation between TGT parameters in PPP and PRP (r=0.055) S Aghighi, A Riddell, P Chowdary, P Lilly, A Griffioen, E G Tuddenham. The Royal Free NHS Hospital, Pond Street, London NW3 2QG, U.K. Introduction Results Aim The aim of this study was to adapt the use of global assays –flurogenic thrombin generation tests and thromboelastography using ROTEM™, as a diagnostic tool to predict heterogeneity amongst these individuals Subjects and Samples subjects Thirty four patients with severe HA aged years with no inhibitor. Eighteen normal individuals. Ethical approval from the Royal Free Hospital Ethics committee. Informed consent obtained from patients and normal individuals. Samples Blood sample was drawn into citrate Monovettes (Sarstedt; 0.106M trisodium citrate) with and without corn trypsin inhibitor (CTI) giving final concentration of 20  g/ml. PRP was obtained by centrifuging samples for 12min at 150g. PPP was obtained by centrifuging samples for 15min at 2500g. After separating upper two third of plasma; samples centrifuged for further 15min at 2500g. PRP platelet count was adjusted by PPP at 150 x 10 9 /l Methods ROTEM™ CWB with CTI: AUC range 42-67, mean = 52, MaxVel (mm/min) range from , mean 3.8; tMaxVel (min)=10-46, mean=24. CWB without CTI: MaxVel=3-7.3mm/min, mean=4.9; tMaxVel= min mean=17.4 There was correlation between ROTEM parameters of WCB with CTI and WCB (r=0.7) The standard deviation in peak height thrombin generation was 21.2% with the range between % and coefficient of variation of 47%. The time to peak shows a range of min, standard deviation of 12.7%, and coefficient of variation of 55%. ROTEM parameters show similar variation between the group of severe haemophiliacs we investigated tested. FVIII<1 IU Figure 2: TGT; Peak Height in PPP with and without CTI in severe Haemophilia A. Figure 1: TGT parameters in severe Haemophilia A ; PPP and PRP with CTI expressed as percentage of normal Figure 3: ROTEM™; MaxVel(mm/min) in severe Haemophilia A CWB with and without CTI. Prothrombotic Results Two individuals (5.9%) had heterozygous FV Leiden mutation. Two individuals (5.9%) had heterozygous FII (G20210A ) mutation. Four individuals (11.8%) had below normal protein S, three low AT, and two low protein C. Twelve individuals (35%) had low FXI (52-69 IU) normal range IU. One individual (3%) had low FX. Results Discussion Conclusion References Severe HA PPP+CTI Range PRP+CTI Range AUC (%) Peak Height (%) Time to Peak (min) PH Severe HA ROTEM™ CWB+CTI Range CWB Range AUC (mm) Maximum Velocity(mm/min) Time to MaxVel (min)