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

ORIGINAL ARTICLE

INTRODUCTION

• Blood coagulation.

• Natural anticoagulants.

• Antithrombin III- inhibitor of thrombin.

• Assays.

• Low AT III levels were correlated with DIC markers.

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.

MATERIALS AND METHODS

• Citrated plasma was collected and stored at minus 70C 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.

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• An aliquot of the mixture was withdrawn and added to a prewarmed fibrinogen solution.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.

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.

OBSERVATIONS

TABLE 1ANTITHROMBIN PROFILE IN 50 CASES

Disease category

Total

No.ofCases

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.

OBSERVATIONS

TABLE 2Value 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.

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.

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.

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-dimerlevels.

• No significant difference between average AT III levels, measured by both assays.

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.

ACKNOWLEDGEMENTS

• General Ramji Rai

• General H Subramanya

• Col DK Mishra

• Dr Shilpika Saxena

• Armed Forces Medical College.

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