A Novel Chromogenic Assay Equivalent to the One Stage Prothrombin Time (PT)

1979 ◽  
Author(s):  
D. J. Baughman ◽  
A. Lytwyn
Keyword(s):  
Plasma Concentration ◽  
Prothrombin Time ◽  
Thrombin Generation ◽  
Specific Factor ◽  
Exponential Rate ◽  
Factor X ◽  
Chromogenic Assay ◽  
Initial Appearance ◽  
Tissue Thromboplastin ◽  
The One

A chromogenic assay equivalent to the PT has been developed which, it is expected, will allow the standardization of therapeutic limits. Assays were performed by combining 0.25 ml S-2238 (2mM), 0.40 ml tissue thromboplastin, and 0.050 ml plasma in 1.40 ml TRIS buffer (pH=8.50, I=0.15). The rate of thrombin generation was obtained by automatically computing the slope of In (A405nm/min. ) vs time. The results indicated that thromboplastin does not interfere with S-2238 hydrolysis. Thrombin production (A/min.) occurs just before clotting, rises rapidly to a maximum and falls. During its rise, the thrombin concentration increases as a positive exponential, exp(bt). The exponential rate, called b, is constant from the initial appearance to the maximum thrombin concentration, b is proportional to plasma concentration and to log Factor (X, VII, V) concentration for dilutions with deficient plasma. Log 1/b correlates with log PT using dicoumarolized plasmas. Thrombin appearance is quite delayed (12 min.) in diluted, deficient plasma. Thus, the assay appears sensitive to the extrinsic system and can be used to monitor dicoumarolized plasmas. Unlike the standard PT, this assay directly measures the rate of thrombin production and could be standardized by comparing the exponential rate to specific Factor concentrations.

Thromboplastin as a Reagent

1970 ◽  
Vol 23 (03) ◽  
pp. 585-592 ◽  
Author(s):  
A .J Quick
Keyword(s):  
Prothrombin Time ◽  
Oral Anticoagulant Therapy ◽  
Quantitative Measure ◽  
Rabbit Brain ◽  
Bacterial Action ◽  
Normal Human ◽  
Tissue Thromboplastin ◽  
Time Test ◽  
The One ◽  
Evacuated Tube

SummaryTissue thromboplastin is the key reagent in the one-stage prothrombin time test. To obtain reliable results, a potent thromboplastin with constant activity and stability is required. This need is met by acetone-dehydrated rabbit brain. This reagent, when protected against oxidation by sealing in an evacuated tube, retains its full activity indefinitely. The basic prothrombin time serves as a screening test for depletion of prothrombin, factors V, VII and X. Thromboplastin is slowly inactivated by oxidation and also by bacterial action. Phenol prevents the latter reaction and therefore a phenolized extract of either human brain or acetone-dehydrated rabbit brain serves as a satisfactory reagent for the prothrombin time when employed to control oral anticoagulant therapy.The constant 12 sec prothrombin time of fresh normal human plasma is fixed by the concentration of active prothrombin. By modifying the basic prothrombin time by adding excess of factors V, VII or X, the test is made a specific quantitative measure of each of these factors.

Biological Properties of the Thromboplastins and Plasmas Included in the ICTH/ICSH Collaborative Study on Prothrombin Time Standardization

1979 ◽  
Vol 42 (04) ◽  
pp. 1115-1127 ◽  
Author(s):  
E A Loeliger ◽  
L P van Halem-Visser
Keyword(s):  
Prothrombin Time ◽  
Collaborative Study ◽  
Biological Properties ◽  
Bovine Brain ◽  
Factor Vii ◽  
Factor X ◽  
One Stage ◽  
Activation Products ◽  
Position Sensitivity ◽  
The One

SummaryThe fourteen types of thromboplastin included in the ICTH/ICSH Collaborative Study on Prothrombin Time Standardization were investigated with respect to their sensitivity for factor VII, activation products, and PIVKAs. All of these thromboplastins displayed sufficient factor VII sensitivity, and all were sensitive to activation products. After correction for the overall sensitivity slope, rabbit plain thromboplastins were the most sensitive preparations and human as well as rabbit diluted the least sensitive; bovine brain thromboplastin lost its exceptional position. Sensitivity to activation products explains why factors II, VII, and X present in artificially prepared abnormal plasmas may be difficult to assess accurately. Also, all thromboplastins appear to be sensitive for both PIVKA VII and PIVKA X. PIVKA VII acts as a procoagulant which increases the amount of factor VII measured by the one-stage assay technique. PIVKA X inhibits and thus reduces the activity of factor X. The procoagulant activity of PIVKA VII is particulary pronounced for the two lung-brain rabbit thromboplastins (Lyoplastin and Simplastin), whereas the anticoagulant action of PIVKA X is strongest with bovine brain thromboplastin (Thrombotest). Considered as a group, rabbit thromboplastins appear to be less sensitive for PIVKA X and more sensitive for PIVKA VII.As judged from thromboplastin calibration results, all three types of lyophilized reference plasmas are to some degree distinguishable from fresh patient plasmas. Primary calibration of a thromboplastin must therefore still be performed with freshly prepared normal as well as patient plasmas.

An Assessment Of An Amidolytic Assay For Factor VII In The Laboratory Control Of Oral Anticoagulants

1981 ◽  
Author(s):  
A Bodzenta ◽  
Jean M Thomson ◽  
Z S Latallo
Keyword(s):  
Prothrombin Time ◽  
Oral Anticoagulant ◽  
Oral Anticoagulants ◽  
Factor Vii ◽  
Limiting Factor ◽  
Factor X ◽  
Present Evidence ◽  
Chromogenic Assay ◽  
Better Than

An amidolytic assay for factor VII, modified from the method of Seligsohn et al (1978), has been compared with the results of the prothrombin time using British Comparative Thromboplastin, Thronbotest and a clotting assay for factor VII. In ‘long-term’ oral anticoagulant administration agreement with the conventional methods was good and better than in our previous study when amidolytic assays for factors II and X respectively were studied (Latallo et al 1981). The method appeared to be reasonably specific for factor VII.On the present evidence the chromogenic assay for factor VII offers a limited but apparently dependable guide to dosage but it is elaborate to perform and difficult to standardise. The main limiting factor for its routine application is the need to prepare a purified factor X extract.

Mechanized amidolytic technique for determination of factor X and factor-X antigen, and its application to patients being treated with oral anticoagulants.

1980 ◽  
Vol 26 (7) ◽  
pp. 885-890 ◽  
Author(s):  
E M van Wijk ◽  
L H Kahlé ◽  
J W ten Cate
Keyword(s):  
Oral Anticoagulant ◽  
Oral Anticoagulants ◽  
Neutralization Assay ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Oral Anticoagulant Treatment ◽  
Chromogenic Assay ◽  
Russell’S Viper ◽  
The One

Abstract We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

ACTIVATION OF FACTOR VII BY ASBESTOS IN BEEF PLASMA AND SERUM

1958 ◽  
Vol 36 (9) ◽  
pp. 953-958 ◽  
Author(s):  
L. G. Israels ◽  
E. Friesen ◽  
C. Sinclair
Keyword(s):  
Prothrombin Time ◽  
Human Plasma ◽  
Factor Vii ◽  
One Stage ◽  
Tissue Thromboplastin ◽  
The One

The adsorption of beef plasma on asbestos markedly shortens the one-stage prothrombin time of the plasma when tissue thromboplastin is used as the throm boplastic agent. This adsorption on asbestos does not affect the Russell viper venom time of the plasma. The adsorbed plasma exhibits increased factor VII activity over that of the original plasma. An eluate can be prepared from the asbestos which prolongs the one-stage prothrombin time sof beef and human plasma.

Endotoxins And Procqagulant Activities Of Ascitic Fluid

1981 ◽  
Author(s):  
H Murr ◽  
P Schüssler ◽  
G E Rindfleisch ◽  
J Eisenburg ◽  
E Hiller
Keyword(s):  
Ascitic Fluid ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Cirrhosis Of The Liver ◽  
Coagulation System ◽  
Factor X ◽  
Monocytes And Macrophages ◽  
Tissue Thromboplastin ◽  
Generation Test ◽  
Limulus Test

Procoagulant activities in ascitic fluid of patients with cirrhosis of the liver have been reported repeatedly. It has been suggested that they cause DIC after reinfusicn of ascitic fluid, however, the nature of these factors is unknown. Since endotoxins have been found in ascitic fluid and it is known that they can greatly augment the procoagulant activities of monocytes and macrophages, we assayed for the presence of endotoxin and compared the procoagulant activities in each of 15 specimens of ascitic fluid caused either by cirrhosis of the liver or by metastasizing tumors.Endotoxins were assayed by the limulus test. Procoagulant activities were determined by thrcmbelastography, thrombin generation test, the presence of soluble fibrin monomer complexes ( SFMC ) and the ability to activate prothrombin complex and factor X.Endotoxins were detected in 12 of 15 specimens of ascitic fluid caused by cirrhosis of the liver. In 11 of the endotoxin positive and in 2 of the 3 endotoxin negative samples procoagulant activity was present ( i.e. shortened reaction time, increased thrombin generation, activation of prothrombin complex ). In contrast, in only one out of the 15 samples of tumerous ascitic fluid the limulus test was positive. Nevertheless, in 9 of these 15 specimens procoagulant activity was present. In all specimens increased amounts of SFMC were found.The precise mechanism for the activation of the coagulation system following ascitic fluid reinfusion remains to be established. In addition to stimulation of macrophages by endotoxin other factors like tissue thromboplastin release from tumor cells or injured tissues may account for the procoagulant activity of ascitic fluid.

Mechanized amidolytic technique for determination of factor X and factor-X antigen, and its application to patients being treated with oral anticoagulants.

1980 ◽  
Vol 26 (7) ◽  
pp. 885-890
Author(s):  
E M van Wijk ◽  
L H Kahlé ◽  
J W ten Cate
Keyword(s):  
Oral Anticoagulant ◽  
Oral Anticoagulants ◽  
Neutralization Assay ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Oral Anticoagulant Treatment ◽  
Chromogenic Assay ◽  
Russell’S Viper ◽  
The One

Abstract We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

ACTIVATION OF FACTOR VII BY ASBESTOS IN BEEF PLASMA AND SERUM

1958 ◽  
Vol 36 (1) ◽  
pp. 953-958
Author(s):  
L. G. Israels ◽  
E. Friesen ◽  
C. Sinclair
Keyword(s):  
Prothrombin Time ◽  
Human Plasma ◽  
Factor Vii ◽  
One Stage ◽  
Tissue Thromboplastin ◽  
The One

The adsorption of beef plasma on asbestos markedly shortens the one-stage prothrombin time of the plasma when tissue thromboplastin is used as the throm boplastic agent. This adsorption on asbestos does not affect the Russell viper venom time of the plasma. The adsorbed plasma exhibits increased factor VII activity over that of the original plasma. An eluate can be prepared from the asbestos which prolongs the one-stage prothrombin time sof beef and human plasma.

INFLUENCE OF HEPARIN ON FACTOR VIII (FVIII) ASSAY

1987 ◽  
Author(s):  
T H Tran ◽  
U Zuhlke ◽  
J Hauert ◽  
F Duckert ◽  
G A Marbet ◽  
...  
Keyword(s):  
Von Willebrand Disease ◽  
Factor Ix ◽  
Bleeding Tendency ◽  
Factor X ◽  
Heparin Infusion ◽  
Control Value ◽  
Chromogenic Assay ◽  
Before And After ◽  
The One

Thrombin-activated FVIII accelerates the conversion of factor X to activated factor X (FXa) by activated factor IX, phospholipids and calcium ions. In plasma, FVIII is activated by initial traces of thrombin, which, in the presence of heparin, is rapidly inhibited by binding to anti thrombin III and heparin cofactor II. To avoid the effect, we have experienced with increasing amounts of exogenous thrombin. We were able to match the heparin cofactors concentration in diluted plasma with thrombin, so that the presence of heparin did not affect the formation of FXa, whose activity was assessed with a chromogenic substrate. Indeed, addition of heparin at any concentration to citrated plasma showed no significant deviation from the FVIII control value. Levels of FVIII in heparinized plasmas similar to those in citrated plasmas further confirmed the finding. Patients plasmas showed comparable FVIII levels before and after heparin infusion, though plasma PTT was clearly prolonged after in vivo heparinization. FVIII chromogenic assay was correlated with the one-stage clotting assay by measuring FVIII levels in 60 hemophiliacs A and carriers, in patients with von Willebrand disease (27) and other congenital deficiencies (4), high risk of thrombosis (15), bleeding tendency (20), disseminated intravascular coagulation (4) and circulating anticoagulants (2), and commercial concentrates. There was a highly significant correlation between both techniques (N=127, r=0.97, Y=0.91X + 4, range 1-380%). Three severe hemophiliacs with were detected with both methods. Data obtained from both techniques were also in good agreement in the range of 1-20% FVIII.Thrombin was added both to activate instantaneously FVIII and to neutralize heparin cofactors in samples. It thus abolishes the incubation time needed to generate in situ traces of thrombin and the influence of heparin on our FVIII assay. An eventual fibrin formation does not affect the FXa formation and the reading. The technique is also suited for automation.

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