Determination of heparin in plasma with Chromogenic Substrate S-2238™
Measurement Principle
Heparin is analyzed as a complex with antithrombin (AT) present in the sample. The concentration of this complex is dependent on the availability of AT. In order to obtain a more constant concentration of AT, purified AT is added to the test plasma. Thrombin in excess is neutralized in proportion to the amount of heparin, which determines the amount of heparin-AT complex. The remaining amount of thrombin hydrolyses the chromogenic substrate H-D-Phe-Pip-Arg-pNA (Chromogenic Substrate S-2238) thus liberating the chromophoric group, pNA. The color is then read photometrically at 405 nm.
Heparin + AT → [Heparin · AT]
[Heparin · AT] + Thrombin (excess) → [Heparin · AT · 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) with 40 ml of distilled water. - Thrombin
Human thrombin or bovine thrombin can be used in 0.15 mol/l NaCl solution.
The activity of the solution should be 14 nkat/ml (about 6 NIH-U/ml).
If bovine thrombin 53 nkat from Chromogenix (Art. No. 82 07 12) is used, dissolve the content of one vial with 3.8 ml saline. - Antithrombin 10 IU Art No. 82 07 20
Reconstitute with 5 ml water to obtain a concentration of 2 IU/ml. - Tris Buffer, pH 8.4 (25°C)
Tris 6.1 g (50 mmol/l)
NaCl 10.2 g (175 mmol/l)
Na2EDTA 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. Fill up to 1 liter. - Normal plasma
Blood should be taken from normal donors. 10-30 ml of citrated blood (9 vol blood and 1 vol 0.1 mol/l sodium citrate) are taken from each donor. The first ml of blood is discarded and the tube is kept in an ice bath. Plasma is prepared by centrifugation at 2000 x g for 20 minutes at 4°C. Equal amounts of plasma from the donors are mixed and dispensed in small volumes. The normal plasma is stable for 3 months at -20°C or below. Thaw at 37°C and then keep on ice. - Acetic acid 20%
Acetic acid is used in the acid-stopped method.
Specimen collection
Blood (9 vol) is mixed with sodium citrate (1 vol) cooled to 0°C with ice and centrifuged at 2000 x g for 20 min at 4°C.
Dilute plasma 1:5 with Tris Buffer pH 8.4.
Standard curve
The same heparin as is used for the patient is diluted to 1 IU/ml with saline 0.9%. Then 100 µl dilution is further diluted with 1.9 ml buffer to obtain a concentration of 0.05 IU/ml.
| Standard IU/ml | Buffer µl | AT µl | Plasma dil 1:5µl | Heparin 0.05 IU/ml µl |
|---|---|---|---|---|
| 0.00 | 800 | 100 | 100 | 0 |
| 0.25 | 700 | 100 | 100 | 100 |
| 0.50 | 600 | 100 | 100 | 200 |
| 0.75 | 500 | 100 | 100 | 300 |
| 0.10 | 400 | 100 | 100 | 400 |
Method
| Initial rate method | Tube No. 1 |
|---|---|
| Buffer | 800 µl |
| AT | 100 µl |
| Test plasma | 100 µl |
| Mix | |
| Tube No. 2 | |
| Standard or tube No. 1 | 200 µl |
| Incubate at 37°C | 3-4 min |
| Thrombin | 100 µl |
| Incubate at 37°C | 30 sec |
| Substrate (37°C) | 200 µl |
| Mix |
Transfer sample immediately to a 1 cm micro-cuvette (preheated at 37°C) for measurement of the absorbance change at 405 nm. Calculate ΔA/min. Read the absorbance against a normal plasma blank in a photometer at 405 nm.
| Acid stopped method | Tube No. 1 |
|---|---|
|
Buffer |
800 µl |
| AT | 100 µl |
| Test plasma | 100 µl |
| Mix | |
| Tube No. 2 | |
| Standard or tube No. 1 | 200 µl |
| Incubate at 37°C | 3-4 min |
| Thrombin | 100 µl |
| Incubate at 37°C | 30 sec |
| Substrate (37°C) | 200 µl |
| Incubate at 37°C | 60 sec |
| Acetic acid 20% | 300 µl |
| Blanks for acid stopped method | Normal plasma blank | Test plasma blank |
|---|---|---|
| Standard 0 IU/ml | 200 µl | – |
| Sample from tube No. 1 | – | 200 µl |
| Acetic acid | 300 µl | 300 µl |
| Mix | ||
| Distilled water | 300 µl | 300 µl |
| Mix |
Note: As a rule a normal plasma blank or even water is used as a blank. If bilirubin exceeds 100 mmol/l or the test plasma is opaque, read the test plasma sample against its own blank.
Calculation
Plot A or ΔA/min for the standards against their known heparin concentration.
Heparin concentration is determined by plotting the A or ΔA/min for the test sample on the standard curve and read the corresponding heparin value.
Bibliography
- Bhargava AS et al. Characterization of a new potent heparin. 2nd communication: chemical analysis of the carbohydrate content and determination of the biological activity of a new potent heparin preparation in vitro, using protamine nutralization and amidolytic methods for factor Xa and thrombin. Arzneimittelforschung 30, 1071-1074 (1980).
- Sache E et al. Studies on a highly active anticoagulant fraction of high molecular weight isolated from porcine sodium heparin. Thromb Res 25, 443-458 (1982).
- Van Putten J et al. Determination of low molecular weight heparin in clinical laboratory. Haemostasis 14, 205-210 (1984).
- Van Putten J et al. Automated spectrophotometric heparin assays. Comparison of methods. Haemostasis 14, 195-204 (1984).
- Berry CN et al. Effects of the synthetic thrombin inhibitor argatroban on fibrin- or clot-incorporated thrombin: comparison with heparin and recombinant hirudin. Thromb Haemost 72, 381-386 (1994).
- Byun Y et al. Effect of fibronectin on the binding of antithrombin III to immobilized heparin. J Biomed Mater Res 30, 95-100 (1996).