Determination of antithrombin activity in plasma with Chromogenic Substrate S-2238™
Measurement Principle
The antithrombin activity in plasma is measured after addition of an excess of heparin, to form an AT•Heparin complex. An excess of thrombin is then added and allowed to react quantitatively in a 1:1 stoichiometric relationship with the AT Heparin complex present. The residual thrombin splits off p-nitroaniline (pNA) from the chromogenic substrate H-D-Phe-Pip-Arg-pNA (S-2238). The rate at which pNA is released is measured photometrically at 405 nm. This can be followed on a recorder (initial rate method) or read after stopping the hydrolysis with acid (acid stopped method). The correlation between the change in absorbance per minute (DA/min) or absorbance (A) and the AT activity is linear and inversely proportional in the 5-125% range of normal plasma.
AT + Heparin (excess) → [AT · Heparin]
[AT · Heparin] + Thrombin (excess) → [AT · Heparin · Thrombin] + Thrombin (residual)
Thrombin (residual)
H-D-Phe-Pip-Arg-pNA + H2O → H-D-Phe-Pip-Arg-OH + pNA
Reagents
- Chromogenic Substrate S-2238, 25 mg Art. No. 82 03 24
Reconstitute the substrate S-2238 (MW: 625.6) in 53 ml of distilled water. Note: Polybrene ® can be added to the chromogenic substrate solution at a final concentration of 0.33 mg/ml. - Thrombin, 53 nkat Art. No. 82 07 12
Reconstitute with 1.5 ml sterile water. The solution is stable for 4 weeks at 2-8°C. - Buffer – Tris/Heparin, pH 8.4 (25°C)
- Tris 6.1 g (50 mmol/l)
NaCl 10.2 g (175 mmol/l)
Na2EDTA-2H2O 2.8 g (7.5 mmol/l)
Distilled water 800 ml
Adjust the pH to 8.4 at 25°C by adding an appropriate amount (approx. 22 ml) of 1 mol/l HCl. Add 3000 IU of heparin. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, will remain stable for two months at 2-8°C. - Acetic acid 20%
Acetic acid is used in the acid-stopped method.
Specimen collection
Nine parts of freshly drawn venous blood are collected into one part trisodium citrate.
Centrifugation: 2000 x g for 10-20 min at 20-25°C.
Standard curve
Normal plasma has an antithrombin activity of 100%. Two standards (e.g. 25% and 100%) made up fresh should be included in each test run. Check whether ΔA/min or A for the two standards correspond with the stored standard curve. The tolerance limit is ± 0.1 Absorbance units. Prepare the standards according to the table below:
| Antithrombin % | Normal plasma ml | Tris/Heparin buffer ml |
|---|---|---|
| 0 | – | 400 |
| 25 | 100 | 300 |
| 50 | 200 | 200 |
| 75 | 300 | 100 |
| 100 | 400 | – |
Method
Dilute samples and standards as follows:
Tris/Heparin Buffer: 3000 µl
Test plasma or standard: 50 µl
Initial rate method
| Diluted test plasma or standard | 400 µl |
| Incubate at 37°C | 3-6 min |
| Thrombin (20-25°C) | 100 µl |
| Mix and incubate at 37°C | 30 sec |
| Substrate | 300 µl |
Transfer immediately to a 1 cm semi-microcuvette (preheated to 37°C) for measurement of the absorbance change in a photometer at 405 nm and at 37°C, calculate ΔA/min.
Acid stopped method
| Diluted test plasma or standard | 400 µl |
| Incubate at 37°C | 3-6 min |
| Thrombin (20-25°C) | 100 µl |
| Mix and incubate at 37°C | 30 sec |
| Substrate | 300 µl |
| Incubate at 37°C | 30 sec |
| Acetic acid 20% | 300 µl |
Read the absorbance (A) of the sample against distilled water at 405 nm within 4 hours.
Limitations of the procedure
In some pathological states (DIC, sepsis) plasma alone may hydrolyze the chromogenic substrate S-2238. This interfering reaction may be determined by assay of a test sample in the absence of added thrombin. This activity rarely corresponds to more than 1% of that of the added thrombin. To improve the validity of the assay the value obtained in the absence of added thrombin can be subtracted from the sample value. Bilirubin, hemoglobin and plasma from hyperlipemic patients interfere in absorbance reading. Patients plasma blanks are necessary in these instances for the acid stopped method only. At concentrations below 25% AT it is recommended to double the plasma concentration (100 µl plasma + 3 ml buffer). The result is then divided by two.
Calculation
Plot A or ΔA/min for the standards against their known antithrombin activity.
Percent of normal AT activity is determined by plotting the A or ΔA/min for the test sample on the standard curve and reading the corresponding AT value.
Bibliography
- Odegard OR et al. Heparin cofactor activity measured with an amydolytic method. Thromb Res 6, 287-294 (1975).
- Odegard OR et al. Evaluation of an amidolytic heparin cofactor method. Thromb Res 7, 351-360 (1975).
- Abildgaard U et al. Antithrombin (heparin cofactor) assay with new chromogenic substrates. Thromb Res 11, 549-553 (1977).
- Kahlé LH et al. Antithrombin III, Evaluation of an automated antithrombin III method. Thromb Res 12, 1003-1014 (1975).