1Elona Rrapo, 2Enriqueta Coll, 2Alane Drexler, 2John Francis 1University of Central Florida College of Medicine; 2Center for Thrombosis Research, Florida Hospital;

Orlando, FL, USA

INTRODUCTION

1. The TGA in WB and plasma appears to better

distinguish between the clinical grades of

cirrhosis in the patients with liver disease.

TGA in WB showed superior performance.

The TGA parameters, PTh and ETP, clearly

show a difference between each group (figure

3A and 3B). On the other hand, the INR (figure

3C) was not able to distinguish between the

patients without cirrhosis and those with mild

cirrhosis, or between patients with moderate

and advanced cirrhosis. Thus, the TGA might

be a better parameter to include in the MELD

score than the INR.

2. Decreased amount of thrombin generation as

reflected by TGA parameters, peak thrombin

and ETP, correlated with decreased levels of

coagulation factors II, V, VII and X, and

prolongations of the PT and INR in patients

with liver disease.

3. The MELD score directly correlated with PT

and INR and inversely correlated with

coagulation factors V, VII and X and the TGA

parameter, peak thrombin.

4. TGA in whole blood is the most physiological

approach and may be a better prognostic

clinical tool in patients with hepatic disease.

TGA correlates closely with conventional

methods of hemostatic assessment and may

correlate more closely than these methods

with the clinical stage of liver cirrhosis.

5. Caution should be used when interpreting our

results due to the relatively small numbers of

patients studied. More studies, especially

with patients with the more severe grades of

hepatic dysfunction, are needed to fully

evaluate the potential benefits of this new

assay.

The current tests, including PT/INR used to assess bleeding problems in patients

with hepatic disease do not reflect overall thrombin generation and therefore are

insensitive to hypercoagulable states.

The Thrombin Generation Assay (TGA), which measures the amount of thrombin

generated, performed in whole blood (WB) is theoretically the most physiological,

but to date, has been technically challenging.

Florida Hospital Center for Thrombosis Research has developed a technique for

measuring thrombin generation in whole blood. This test has not yet been studied

clinically, but is anticipated to be applicable to the study of hemostatic

abnormalities.

The primary purpose of this study was to compare TGA with the coagulation

parameters conventionally used to evaluate the hemostatic status and hepatic

function of patients with mild, moderate and advanced liver cirrhosis. The MELD

score (routinely calculated for all patients with cirrhosis) was used to classify the

patients with liver cirrhosis. A MELD score in the range of 6-9 indicates mild

cirrhosis, a score in the range of 10-14 indicates moderate cirrhosis, and a score

above 15 classifies the patients as advanced cirrhosis. We analyzed blood

samples from 16 patients with cirrhosis and 5 patients with non cirrhotic liver

disease.

RESULTS

Figure 1. (A) To measure the thrombin generation capacity of whole blood (WB), 20 μl of

buffer (HEPES saline buffer pH 7.35, containing 6 g/l bovine serum albumin) and 80 μl of

blood were pipetted in quintuplicate in transparent round-bottom 96-well microtiter plates.

Each sample had its own calibrator, comprised of 20 μl thrombin and 80 μl of PFP or WB

calibrator in duplicate. WB calibrator was prepared by mixing heat-inactivated PPP and

autologous washed red blood cells (in PBS) to the hematocrit of the original blood

sample. CaCl2 was added to trigger coagulation. The thrombin generated specifically

cleaves the fluorogenic substrate (Z-GGA-AMC HCl, Bachem BioSciences, PA) included

in each reaction mixture. The reaction was followed for 60 minutes. (B) The results are

obtained in fluorescence units (left) and transformed into a curve known as a

thrombogram (right). Lag time (LT), peak thrombin (PTh), time to peak (TTP) and

endogenous thrombin potential (ETP) were measured.

Patients versus control group test results

Table 1. (A) Patient demographics, clinical and routine test results.

(B) Coagulation Variables. (C) TGA variables

CONCLUSIONS

Variation of TGA, PT, INR and coagulation factors with MELD score

ACKNOWLEDGMENTS

Special thanks to Dr. Nikolaos Prysopoulos, Dr. Ali

Amirkhosravi, Liza Robles, Megan Hatfield, Kristin

Rathmann, Eduardo Reyes and my faculty advisor

Dr. Stephen Lambert

Figure 2. (A) A statistically significant negative correlation was found between PT

and INR with peak thrombin; and between PT and INR with ETP (p < 0.05)

indicating that, as expected, longer PT/INR values are associated with a decrease

in thrombin generation. (B) Coagulation factors, II, V, and VII were positively

correlated with peak thrombin and ETP (p < 0.05). Thus, decreases in the

procoagulant clotting factors are paralleled by a decrease in thrombin generation

potential.

TGA in whole blood is capable of detecting variation in

patients’ coagulability

Demographics and clinical classification of patients

with liver disease

The use of thrombin generation assays (TGA) as prognostic tools in patients with hepatic disease

METHODS

REFERENCES

Coll E, Amirkhosravi A, Francis JL. Detection of tissue factor activity in whole blood by a

fluorogenic thrombin generation assay. International Society for Thrombosis and

Hemostasis, Boston July 11-6, 2009.

Hemker H.C., et al. Pathophysiology of Haemostasis and Thrombosis, 2003, 33:4-15

Al Dieri R, Hemker C.H., British Journal of Haematology, 2008, 141:895-908

C

ETP (nM/min) in patients with liver disease

INR values in patients with liver disease

A A

B

B

Variable Patients Controls and normal ranges

Mean SD or Median (25-75% percentile)

Age (years) 53.8 9.73 43.9 10.35

Gender

Male

Female

10 (47.6%)

11 (52.4%)

6 (33.3%)

12 (66.7%)

Etiology on non-cirrhosis liver disease

Abnormal liver enzymes

Small hemangioma

NASH

HCV positive antibody

HCV and NASH

1 (4.8%)

1 (4.8%)

1 (4.8%)

1 (4.8%)

1 (4.8%)

N/A

Etiology of cirrhosis

Hepatitis C virus(HCV)

Nonalcoholic steatohepatitis (NASH)

Autoimmune hepatitis (AIH)

Alcoholic liver disease (ALD)

Nonalcoholic fatty liver disease (NAFLD)

Primary biliary cirrhosis (PBC) and AIH

8 (38.1%)

3 (14.3%)

2 (9.5%)

1 (4.8%)

1 (4.8%)

1 (4.8%)

N/A

Hematocrit (%) 34.60 4.63 35.70 3.01

Platelets (/µl) 174,000 (64,000 – 258,000) 225,400 55,540

Bilirubin (mg/dL) 1.30 1.03 < 1.2

Albumin (g/dL) 3.24 1.22 3.5 – 5.2

Creatinine (mg/dL) 0.74 0.20 0.6 – 1.0

MELD score 7.70 6.31 N/A

Variable Patients Controls

Prothombin time (PT) (sec) 14.9 (13.4 - 16.6) 13.5 0.57

International Normalized Ratio (INR) 1.18 (1.03 - 1.35) 1.00 0.06

FII (%) 73.0 (52.8 - 99.3) 100.1 14.60

FV (%) 83.3 23.73 97.6 11.37

VII (%) 108.4 47.90 116.4 30.70

FX (%) 93.7 28.73 104.0 (97 – 112)

Variable Result Controls and normal ranges

Lag time (min) 9.0 (8.4 - 10.1) 9.6 2.39

Peak thrombin (nM) 97.8 (68.4 - 132.6) 80.8 (64.3 - 107.5)

ETP (nM*min) 934 (754 – 1257) 950.3 349.32

2 (a) Results in whole blood

ANOVA Ht Platelets PTh ETP PT INR FII FV FVII FX

C vs Patients NS NS NS NS <0.05 <0.05 <0.05 NS NS <0.05

C vs Cirrhosis NS <0.05 NS NS <0.05 <0.05 <0.05 NS NS <0.05

C vs Non

Cirrhosis NS NS NS NS NS NS NS NS NS NS

Cirrhosis vs Non

Cirrhosis NS <0.05 NS NS NS NS NS NS NS <0.05

2 (b) Results in plasma

ANOVA Ht Platelets PTh ETP PT INR FII FV FVII FX

C vs Cirrhosis NS NS NS NS <0.05 <0.05 <0.05 <0.05 <0.05 <0.05

C vs Non

Cirrhosis NS NS NS NS NS NS NS NS NS NS

Cirrhosis vs Non

Cirrhosis NS NS NS NS NS NS NS NS NS <0.05

* NS= not significant; C = control; Ht= hematocrit; PTh = Peak Thrombin; ETP = endogenous thrombin potential; PT = prothrombin time; INR = international normalized ratio; F = factor

Peak Thrombin (nM) in patients with liver disease

Pe

ak T

hro

mb

in (

nM

)

Patients with liver disease categories according to MELD score

Peak Thrombin (nM)

Lag time (min) Time to peak (min)

ETP (nM*min)

Area under the curve

Time

Th

rom

bin

(N

m)

Time

Flu

ore

sc

en

ce

un

its

B

C

Table 2. A statistically significant difference among control and patient groups

was found for PT and INR, and coagulation factors II and X. PT and INR were

significantly higher in patients with liver disease than in controls (P = 0.001) while

factors II and X were significantly lower. Patients with cirrhosis had significantly

lower platelet numbers (p < 0.05). TGA showed no significant difference among

patients with liver disease and the control group even though the range of ETP

values was broader in the patients with liver disease. (A) whole blood; (B)

plasma

A B

Figure 3. Results

shown for TGA in whole

blood. The TGA

parameters: peak

thrombin (A) and ETP

(B) showed negative

correlation to MELD

score (p < 0.05). MELD

positively correlated

with PT and INR (p <

0.05) (C).

When the groups of

patients with non-

cirrhosis, mild,

moderate and

advanced cirrhosis

were compared with

each other, there were

statistically significant

differences in INR and

PT values of moderate

versus mild cirrhosis,

and moderate versus

non-cirrhosis (p < 0.01).

Even though the TGA

parameters were not

significantly different

among the patient

groups (most likely

because of the

relatively low numbers

of patients), there was

a very clear trend

towards diminishing

thrombin generation

potential with

increasing hepatic

dysfunction (A and B),

and this was mirrored

(albeit less clearly) by

the INR (C).

Figure 4.

Thrombogram in the

4 groups of patients

with liver disease:

non-cirrhosis, mild,

moderate and

advanced cirrhosis.

The maximum

amount of thrombin

produced and total

amount of thrombin

generated is highest

in the group of

patients with non-

cirrhotic liver

disease. PTh and

the total amount of

thrombin generated

are lowest in

patients with

advanced cirrhosis,

indicating a potential

for bleeding.

Patients with liver disease categories according to MELD score

Patients with liver disease categories according to MELD score INR

E

TP

(n

M/m

in)

Thrombogram (whole blood) in patients with

liver disease

time

Flu

ore

sc

en

ce

un

its

A

Measure fluorescent signal

Thrombin generated

Whole blood

(+) buffer

cleaves the fluorogenic substrate (Z-GGA-AMC HCl)

CaCl2

(+)

in transparent round-bottom

96-well microtiter plates

60 minutes

Platelet free plasma (PFP)

(+) Tissue factor (1 pM) / Phospholipids (4 µM)

90minutes