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1. ORIGINAL ARTICLE
Antithrombin III assay using thrombin in
disseminated intravascular coagulation
(DIC), other thromboembolic disorders and
hepatic diseases.
Saxena V, Mishra DK, Subramanya H, Satyanarayana S, Sharma A
Departments of Pathology, Medicine & Clinical Hematology, Armed
Forces Medical College, Pune.
Indian J Pathol Microbiol 2004; 47,(2):210-212

2. INTRODUCTION
• Blood coagulation.
• Natural anticoagulants.
• Antithrombin III- inhibitor of
thrombin.
• Assays.
• Low AT III levels were correlated
with DIC markers.

3. MATERIALS AND METHODS
• Fifty cases were evaluated, using clotting
assay.
• Eleven DIC, sixteen venous thrombosis, and
twenty three hepatic diseases.
• All cases were subjected to hematological
investigations.
• Twelve samples were evaluated, using synthetic
chromogenic assay.
• Twenty controls were also analysed to find out
the reference value for the techniques.

4. MATERIALS AND METHODS
• Citrated plasma was collected and stored at
minus 70C in 2 ml aliquots, and thawed before
the assay.
• No anticoagulant therapy at collection time.
• Plasma was defibrinated.
• Serum was incubated with excess thrombin and
any residual thrombin remaining at the end of
the incubation period reflected the
concentration of AT III in the serum.

5. MATERIALS AND METHODS
• Thrombin was added to the diluted serum,
and the mixture was incubated.
• An aliquot of the mixture was withdrawn
and added to a prewarmed fibrinogen
solution.
• The resulting clotting time of the
fibrinogen solution is a measure of the
active thrombin remaining, that is, after
it has been exposed and partially
neutralised by AT III.

6. MATERIALS AND METHODS
• When the CT was plotted against the
concentration of AT III, a straight line
was obtained.
• Normal range: 75-125 % by clotting
assay and 80-100 % by synthetic
chromogenic assay.
• FDP and D-dimer levels were retrieved
from the clinical records.

7. OBSERVATIONS
TABLE 1
ANTITHROMBIN PROFILE IN 50 CASES
Disease
category
Total
No.of
Cases
Range of
Age(Yrs)
Range of
AT
III (%)
% of
Cases
with low
AT III
Average
AT III
(%)
DIC 11 34-41 40-85 81.80 51.90
Hepatic
diseases
23 40-60 40-105 69.50 61.26
Venous
thrombosis
16 35-50 50-102 62.50 72.25
Significant difference between the AT III levels of the
three disease categories.

8. OBSERVATIONS
TABLE 2
Value of AT III by clotting and chromogenic
assay
Assay Range of AT III (%) Average AT
III (%)
Clotting 40-102 64.33
Chromogenic 45-104 62.16
No significant difference between the average AT
III levels measured by both the clotting and
synthetic chromogenic assay.

9. DISCUSSION
• Changes in AT III levels appear to be
of significance in some diseases.
• The AT III status was measured using
the clotting assay, and reconfirmed the
fact that a correlation between
abnormally low AT III levels and certain
diseases exists.
• AT III levels decrease more with
increasing severity of the disease.

10. DISCUSSION
• The plasma was defibrinated using reptilase R.
• Some workers proposed that chromogenic assays
are preferable, but the present study found no
significant difference between clotting and
chromogenic assay results.
• Many workers reported low AT III levels in DIC,
venous thrombosis, and hepatic diseases, which are
comparable with the results of the present study.

11. CONCLUSIONS
• Low AT III levels in all three disease
categories.
• Significant difference between levels in
all three disease categories.
• Lowest AT III levels in DIC, which
correlated well with FDP and D-dimer
levels.
• No significant difference between average
AT III levels, measured by both assays.

12. CONCLUSIONS
• Clotting assay- 6.5 US $ versus
Chromogenic assay- 12 US $.
• This study thus brings out the
possibility of using AT III as a
diagnostic tool in DIC, employing the
clotting assay, which if performed
under rigid quality control can be a
simple, and a relatively inexpensive
routine procedure.

13. ACKNOWLEDGEMENTS
• General Ramji Rai
• General H Subramanya
• Col DK Mishra
• Dr Shilpika Saxena
• Armed Forces Medical College.

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15. Thank You