Chromogenix S-2238™ is a chromogenic substrate for thrombin. The substrate has been used for the determination of:

  • Prothrombin in plasma
  • Antithrombin in plasma
  • Platelet factor 3 in plasma
  • Heparin in plasma

Each vial contains chromogenic substrate S-2238™ 25 mg and mannitol 120 mg as a bulking agent. Stability: Substance: Stable until expiry date if stored at 2-8°C. Avoid exposure to light. The substance is hygroscopic and should be stored dry. Solution: 1 mmol/L in H2O is stable for more than 6 months at 2-8°C.

Chemical name: H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline dihydrochloride.

Formula: H-D-Phe-Pip-Arg-pNA·2 HCl

Mol. wt.: 625.6

chromogenix chromogenic substrate assay test kit

e316 nm: 1.27 . 104 mol-1 . L . cm-1

Solubility: > 10 mmol/L in H2O

Suitable stock solution: 1-2 mmol/L in H2O.

 

The current International Standard for thrombin is the Human a-thrombin 89/588 available from NIBSC. This is a high purity preparation of a-thrombin prepared from Cohn fraction III and assayed by a clotting time method against the first International Standard for thrombin, 75/157.

The National Institute of Health standard (Lot J) is also commonly used for calibration and a study conducted by Gaffney PJ et al. was focused on the relationship between the two standards, and between the International Units and the NIH Units. As a result of this study, based both on a clotting and a chromogenic assay (with the chromogenic substrate S-2238™), 1 NIH-U corresponds to 1.15 IU.

In an article it was shown that bovine thrombin has a higher amidolytic activity than human thrombin when the same NIH-U are compared. It was also underlined that the influence of b and g-forms, that were probably contaminating the bovine enzyme, might be the reason for this discrepancy.

In the same article it was concluded that 1 NIH-U bovine thrombin was equivalent to 3.4 nkat chromogenic substrate S-2238™, and that 1 NIH-U of human thrombin was equivalent to 2.7 nkat chromogenic substrate S-2238™.

From an earlier publication 1 NIH-U of human thrombin corresponded to 2.5 nkat chromogenic substrate S-2238™. The correspondence between NIH-U or IU of thrombin and the enzyme activity expressed in nkat, depends on the substrate, the enzyme preparation (content of a-, b- and g-thrombin) and the assay conditions.

From the article of Friberger , 1 µg thrombin corresponds to 2.2 NIH-U or 5.5 nkat chromogenic substrate S-2238™ or to 0.02 plasma equivalent units. In another study , 1 µg thrombin corresponds to 3.1 NIH-U.

In the experiments done by Chromogenix  1 µg thrombin was equivalent to 3 nkat chromogenic substrate S-2238™ (human) or 4.4 nkat chromogenic substrate S-2238™ (bovine). It might also be added that if all prothrombin is activated in 1 ml of human plasma, about 1.5 nanomoles or 17.5 NIH-U of thrombin are formed.

Determination of Thrombin in Plasma with Chromogenic Substrate S-2238


Reagents

  1. Chromogenic Substrate S-2238™: (0.56 mM) 25 mg Art. No. S820324. Disolve 25 mg lyophlized substrate in 7.14 ml sterile water = 5.6 mM stock solution.
  2. Buffer: 50 nM Tris, | 0.15, pH 8.3 with 0.2% BSA (10 ml buffer + 100 µl 20% BSA)

Assay

Dilute to a suitable sample concentration (0.5 – 1.0 nkat / ml after dilution with buffer)

Sample of buffer 100 µl
Incubate at 37°C 4 min
S-2238™ 100 µl
Incubate at 37°C 3 min
Acetic Acid, 20% 50 µl

Use buffer as a blank and subtract from sample absorbances

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 (ΔA/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)
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. S820324
    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.
  2. Thrombin, 53 nkat, Art. No. DPGBT-10
    Reconstitute with 1.5 ml sterile water. The solution is stable for 4 weeks at 2-8°C.
  3. Buffer – Tris/Heparin, pH 8.4 (25°C)
  4. 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.
  5. 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 hydrolyse 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, haemoglobin and plasma from hyperlipaemic 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

  1. Odegard OR et al. Heparin cofactor activity measured with an amydolytic method. Thromb Res 6, 287-294 (1975).
  2. Odegard OR et al. Evaluation of an amidolytic heparin cofactor method. Thromb Res 7, 351-360 (1975).
  3. Abildgaard U et al. Antithrombin (heparin cofactor) assay with new chromogenic substrates. Thromb Res 11, 549-553 (1977).
  4. Kahlé LH et al. Antithrombin III, Evaluation of an automated antithrombin III method. Thromb Res 12, 1003-1014 (1975).

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. S820324
    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 Diapharma (Art. No. DPGBT-10) is used, dissolve the content of one vial with 3.8 ml saline.
  3. Antithrombin 10 IU Art No. B820720
    Reconstitute with 5 ml water to obtain a concentration of 2 IU/ml.
  4. 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.
  5. 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.
  6. 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
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 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 100 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

  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).

Determination of prothombin activity in plasma with Chromogenix S-2238™


 

Background

A number of studies during the last few years support the notion that venous thromboembolism (VTE) is a multifactorial disease most often triggered by circumstantial risk factors (trauma, surgery, pregnancy, oral contraceptives, immobilization and age) in combination with one or more genetic or acquired coagulation disorders (see ref. 1 of a review).
Elevated activity of prothrombin in the absence of a known underlying genetic disorder is also associated with an increased thrombotic risk2.

A mutation G → A in the untranslated 3’-region of the prothrombin gene at nucleotide position 20210 constitutes a risk factor for VTE with an odds ratio of 3-52-10. About 90% of the carriers of this mutation have elevated levels (>115%) of prothrombin activity2,7,8. Levels above the upper limit of the normal range (75 – 130%) are commonly hetero- and homozygotes2,7-9.

So far, there is no explanation why a comparatively mild increase of prothrombin activity constitutes a risk factor for thrombosis and this is therefore an area of active clinical and biochemical research. Chromogenic methods for accurate determination of elevated activities of prothrombin and other coagulation factors, such as factor VIII11,12 are important tools for assessing the risk for VTE in patients and family members.


 

Measurement Principle

Prothrombin is activated to meizothrombin by the snake venom enzyme Ecarin from Echis Carinatus.

After a certain incubation time, the amount of meizothrombin formed is measured with the thrombin selective substrate Chromogenix S-2238™, which also is cleaved by meizothrombin.

The absorbance recorded at 405nm is proportional to the prothrombin activity in the sample.

Prothrombin Ecarin
Meizothrombin
S-2238™ Meizothrombin
 pNA + Peptide

 


 

Reagents

  1. Tris BSA Buffer (catalog# TB031-20)Buffer for sample dilution, containing 0.5mol/l Tris HCl pH 7.3, I = 2.0 with NaCl and 2% bovine serum albumin. Before use, dilute the stock solution 1+9 with sterile water to obtain a buffer working solution. The buffer working solution is prepared and used within the same day.
  2. Ecarin Diluent (catalog# ED0413-20)Buffer for dilution of Ecarin, containing 0.05mol/l Tris HCl pH 7.6, I=0.15 with NaCl, bovine serum albumin, polyethylene glycol and a fibrin polymerization inhibitor.
  3. Ecarin (catalog# ECARIN50B)Reconstitute with sterile water according to the Ecarin package insert. Freeze in suitable aliquots at -20°C or at -70°C. Stable for 3 months at both storage temperatures. Before use, dilute with Prothrombin Activator Diluent to obtain a concentration of 2.4U/ml. Stable for 8 hours at 20-25°C and for 1 week at 2-8°C.Note: Echis Carinatus crude venom can also be used. A suitable final concentration of this reagent is approximately 5µg/ml; however, this may vary between different sources. 10-20% loss of activity may occur upon freezing at -20°C.
  4. S-2238™ (catalog# S820324)Reconstitute with 13ml of sterile water to obtain a 3mmol/l solution.

 

Specimen Collection

Blood (9 volumes) is mixed with 0.1mol/l sodium citrate (1 volume) and centrifuged at 2000 x g for 20 min at 20-25°C. Separate plasma carefully from blood cells. Perform the analysis within 24 hours when plasma is stored at 2-25°C. Alternatively, freeze aliquots ≤ 1ml at -20°C or below. Perform the analysis of frozen samples within two months when stored at -20°C or within one year when stored at -70°C or below. No significant loss of prothrombin activity occurs upon freezing once, provided freezing is made in small aliquots (< 1 ml) and thawing is performed in a water bath or in an electric heater at 25-37°C.


 

Sample and Standard Dilutions

Standards
Calibrated normal plasma is diluted 1:23 – 1:160 to provide standard concentrations of 25-175%. The following table provides a suggestion of standard dilutions.

Standard Dilution Prothrombin Activity
1:18 167%
1:22 136%
1:30 100%
1:60 50%
1:120 25%

 

Samples
Plasma samples are diluted 1:40 in Tris BSA Buffer working solution for application on microplate and diluted 1:80 for application on ACL (see below).


 

Microplate Assay Procedure

Standard/Sample dilution 50μl
Incubate at 37°C 2-4min
Ecarin or Echis Carinatus (37°C) 50μl
Incubate at 37°C 3min
Substrate (37°C) 50μl
Read kinetically or incubate at 37°C 2min
Acetic acid, 20% 50μl

Determine the absorbance difference A405nm-490nm for the standard dilution and the samples. Draw a standard curve from the absorbances obtained for the standard dilutions. Read the prothrombin activity for the samples from the standard curve.

 

chromogenic assay test kit method

Fig. 1. Standard curve with the microplate method.

 


Application on ACL

Use the plasminogen channel program. Prepare a standard dilution 1:40, which corresponds to a nominal prothrombin activity of 100% (see above regarding calibration). Standard dilutions corresponding to 25% and 50% are then automatically prepared by the instrument. In order to allow determination of prothrombin activity up to 200%, sample plasma should be diluted 1:80 and the obtained result should be multiplied with two.

chromogenic assay test kit method

Fig. 2. Standard curve with the ACL method

 


 

Expected values13

The normal range is 75 – 130% (mean 102% 2 SD) as determined from analysis in microplate and on the ACL 300 of 101 healthy individuals (49 men and 52 women; age range 20 – 68 years). Analysis of plasma from 42 carriers of the G20210A mutation, who were not on oral anticoagulant treatment at the time of blood sampling, resulted in an activity range of 94 – 164% (mean 128% 2SD).


 

Interference and Limitations

No influence in the assay is obtained from variation of antithrombin activity in the range 50 – 150% of normal. Since meizothrombin is formed and measured, no influence in the assay is obtained from heparin levels ≤ 1 IU/ml plasma. Since Ecarin also activates decarboxyprothrombin, which is produced during oral anticoagulant therapy with anti-vitamin K drugs, plasma from patients undergoing such treatment should not be analysed with this method.


 

Repeatability

The imprecision, expressed as CV, within and between series (7 series, 5 replicates in each series) is ≤4% at 50% and 100% prothrombin activity.


 

Bibliography

  1. Lane DA, Mannucci PM, Bauer KA, Bertina RM, Bochkov NP, Boulyjenkov V, Chandy M, Dahlbäck B, Ginter EK, Miletich Jp, Roosendaal FR, Selingsohn U.
    Inherited Thrombophilia: part 1.
    Thromb Haemost 76, 651-662 (1996)
  2. Poort SR, Roosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3’-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.
    Blood 88, 3698-3703 (1996)
  3. Hillarp A, Zöller B, Svensson PJ, Dahlbäck B.
    The 20210 allele of the prothrombin gene is a common risk factor among Swedish out-patients with verified deep venous thrombosis.
    Thromb Haemost 78, 990-992 (1987)
  4. Cumming AM, Keeney S, Salden A, Bhavnani M, Shwe RH, Hay CRM. The prothrombin gene G20210A variant: prevalence in a UK anticoagulant clinic population.
    Br J Haematol 98, 353-355 (1997)
  5. Brown K, Luddington R, Williamson D, Baker P, Baglin T.
    Risk of venous thromboembolism associated with a G to A transition at position 20210 in the 3’-untranslated region of the prothrombin gene.
    Br J Haematol 98, 907-909 (1997)
  6. Makris M, Preston FE, Beuchamp NJ, Hampton KK, Daly ME, Cooper P, Bayliss P, Peake IR. Co-inheritance of the 20210A allele of the prothrombin gene increases the thrombotic risk in subjects with familial thrombophilia.
    Thromb Haemost 78, Suppl., 165 (1997)
  7. Ferraresi P, Marchetti G, Legnani C, Cavallari E, Castoldi E, Mascoli F, Ardissino D, Palareti G, Bernardi F. The heterozygous 20210G/A prothrombin genotype is associated with early venous thrombosis in inherited thrombophilias and is not increased in frequency in artery disease.
    Arterioscl Thromb Vasc Biol 17, 2418-2422 (1997)
  8. Kapur RK, Mills LA, Spitzer SG, Hultin MB. A prothrombin gene mutation is significantly associated with venous thrombosis.
    Arterioscl Thromb Vasc Biol 17, 2875-2879 (1997)
  9. Howard TE, Marusa M, Channel C, Duncan A. A patient homozygous for a mutation in the prothrombin gene 3’-untranslated region associated with massive thrombosis.
    Blood Coag Fibrinol 8, 316-319 (1997)
  10. Martinelli I, Sacchi E, Landi G, Taioli E, Duca F, Mannucci PM. High risk of cerebral-vein thrombosis in carriers of a prothrombin-gene mutation and in users of oral contraceptives.
    New Engl J Med 338, 1793-1797 (1998)
  11. Koster T, Blann AD, Briët E, Vanenbroucke JP, Roosendaal FR. Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis.
    The Lancet 345, 151-155 (1995)
  12. D’Onnel J, Tuddenham EGD, Manning R, Kemball-Cook G, Johnson D, Laffan M. High prevalence of elevated factor VIII levels in patients referred for thrombophilia screening: role of increased synthesis and relationship to the acute phase reaction.
    Thromb Haemost 77, 825-828 (1997)
  13. Rosén S, Andersson M, Ghosh R. A new chromogenic prothombin method providing accurate determination of elevated prothombin activity in plasma samples.
    ISTH 1999, Abstract 269