Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES | METHODS | HEPARIN (Anti-FIIa)


Determination of heparin in plasma with Chromogenic Substrate S-2238

Measurement Principle
Heparin is analysed 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)
H-D-Phe-Pip-Arg-pNA + H2O Thrombin (residual)
H-D-Phe-Pip-Arg-OH + pNA

Reagents

  1. 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.
  2. 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.
  3. Antithrombin 10 IU Art No. 82 07 20
    Reconstitute with 5 ml water to obtain a concentration of 2 IU/ml.
  4. Tris Buffer, pH 8.4 (25C)
    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 25C by adding an appropriate amount (approx. 22 ml) of 1 mol/l HCl. Fill up to 1 liter.

  1. 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 4C. Equal amounts of plasma from the donors are mixed and dispensed in small volumes. The normal plasma is stable for 3 months at -20C or below. Thaw at 37C and then keep on ice.
  2. 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 0C with ice and centrifuged at 2000 x g for 20 min at 4C.

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 l dilution is further diluted with 1.9 ml buffer to obtain a concentration of 0.05 IU/ml.

Standard

IU/
ml
Buffer

ml
AT

ml
Plasma dil 1:5

ml
Heparin 0.05 IU/ml
ml
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 ml
AT 100 ml
Test plasma 100 ml
Mix   

 

Tube No. 2

Standard or tube No. 1 200 ml
Incubate at 37C 3-4 min
Thrombin 100 ml
Incubate at 37C 30 sec
Substrate (37C) 200 ml
Mix  

Transfer sample immediately to a 1 cm micro-cuvette (preheated at 37C) for measurement of the absorbance change at 405 nm. Calculate DA/min. Read the absorbance against a normal plasma blank in a photometer at 405 nm.

Acid stopped method

Tube No. 1

Buffer 800 ml
AT 100 ml
Test plasma 100 ml
Mix   

 

Tube No. 2

Standard or tube No. 1 200 ml
Incubate at 37C 3-4 min
Thrombin 100 ml
Incubate at 37C 30 sec
Substrate (37C) 200 ml
Incubate at 37C 60 sec
Acetic acid 20% 300 ml

 

Blanks for acid stopped
method

Normal
plasma blank

Test
plasma blank
Standard 0 IU/ml 200 ml -
Sample from tube No. 1 - 200 ml
Acetic acid 300 ml 300 ml
Mix    
Distilled water 300 ml 300 ml
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 DA/min for the standards against their known heparin concentration.
Heparin concentration is determined by plotting the A or
DA/min for the test sample on the standard curve and read the corresponding heparin value.

Bibliography

  1. 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).
  2. 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).
  3. Van Putten J et al. Determination of low molecular weight heparin in clinical laboratory. Haemostasis 14, 205-210 (1984).
  4. Van Putten J et al. Automated spectrophotometric heparin assays. Comparison of methods. Haemostasis 14, 195-204 (1984).
  5. 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).
  6. Byun Y et al. Effect of fibronectin on the binding of antithrombin III to immobilized heparin. J Biomed Mater Res 30, 95-100 (1996).
 

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