This study aimed to use global assays like thrombin generation tests (TGT) and rotational thromboelastography (ROTEMTM) to assess heterogeneity in patients with severe haemophilia A (HA). TGT and ROTEMTM parameters were measured on plasma and whole blood samples from 34 severe HA patients. The results showed wide variation between patients, with TGT peak height ranging from 6-47% of normal and ROTEMTM MaxVel ranging from 1.7-6.7 mm/min. Some patients also had prothrombotic factors like factor V Leiden mutation or low natural anticoagulants. The study suggests TGT and ROTEMTM may help predict clinical phenotype in severe HA

1. 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
phenotype1
.
• 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 HA2,3
.
• 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 CaCl2.4
• 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 time3
.
•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, 1994. 236(4): p. 391-
2. Beltran-Miranda, C.P., et al., Thrombin generation and phenotypic correlation in haemophilia A. Haemophilia, 2005. 11(4): p. 326-34.
3. 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. 551–558.
4. Hemker H C, Beguin S. Phenotyping the clotting system. Thromb Haemost 2000; 84: 747-51
TGT
• 80 µL 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 4µM PS/PC/PE). For PRP, 80 µL of PRP was added to 20mL
initiation reagent (containing TF with final concentration of 0.5pM)
• After incubating at 37Co
, 20µl starting reagent (containg100 mM CaCl2 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. 300µL of
CWB was pipetted into a cup, 20µl TF (final concentration 0.35pM) was added and
recalcified with 20µL 0.2MCaCl2 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 20-70 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 20µg/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 109
/l
Methods
ROTEM™
• CWB with CTI: AUC range 42-67, mean = 52, MaxVel (mm/min) range from 1.7-6.7, mean 3.8;
tMaxVel (min)=10-46, mean=24.
• CWB without CTI: MaxVel=3-7.3mm/min, mean=4.9; tMaxVel=8.7-42 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 6.4-
47% and coefficient of variation of 47%. The time to peak shows a range of 11-50 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 70-150 IU.
• One individual (3%) had low FX.
Results
Discussion
Conclusion
ReferencesSevere HA
PPP+CTI
Range
PRP+CTI
Range
AUC (%) 12-67 7-68
Peak Height (%) 6-47 7-47
Time to Peak (min) 11-56 37-60
PH
Severe HA
ROTEM™
CWB+CTI
Range
CWB
Range
AUC (mm) 40-68 42-67
Maximum
Velocity(mm/min)
1.7-6.7 3-7.3
Time to MaxVel
(min)
10-46 8.7-42