CHROMOGENIC SUBSTRATES UNIVERSITY | METHODS | PROTHROMBIN
Determination of prothrombin activity in plasma with Chromogenic Substrate S-2238
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, immobilisation 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 risk (2). A mutation G to 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 3-5 (2-10). About 90% of the carriers of this mutation have elevated levels (>115%) of prothrombin activity (2,7,8) .
Levels above the upper limit of the normal range (75 – 130%) are commonly hetero- and homozygotes (2,7-9). 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 VIII (11,12) are important tools for assessing the risk for VTE in patients and family members.
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 S-2238, which also is cleaved by meizothrombin. The absorbance recorded at 405 nm is proportional to the prothrombin activity in the sample.
||pNA + Peptide|
Blood (9 volumes) is mixed with 0.1 mol/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
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
|Standard Dilution||Prothrombin Activity|
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||50 ml|
|Incubate at 37ºC||2-4 min|
|Ecarin or Echis Carinatus (37ºC)||50 ml|
|Incubate at 37ºC||3 min|
|Substrate (37ºC)||50 ml|
|Read kinetically or incubate at 37ºC||2 min|
|Acetic acid, 20%||50 ml|
Determine the absorbance difference A405nm-490 nm 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.
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.
Standard curve with the ACL method.
Expected values (13)
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.
The imprecision, expressed as CV, within and between series (7 series, 5 replicates in each series) is < 4% at 50% and 100% prothrombin activity.
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