Recombinant Tissue Factor as Substitute for Conventional Thromboplastin in the Prothrombin Time Test

1992 ◽  
Vol 67 (01) ◽  
pp. 042-045 ◽  
Author(s):  
Armando Tripodi ◽  
Arnaldo Arbini ◽  
Veena Chantarangkul ◽  
Pier Mannuccio Mannucci
Keyword(s):  
Prothrombin Time ◽  
Tissue Factor ◽  
Oral Anticoagulant Therapy ◽  
Clotting Factor ◽  
Rabbit Brain ◽  
Sensitivity Index ◽  
Clotting Factors ◽  
Wide Range ◽  
Reference Preparation ◽  
Time Test

SummaryRelipidated recombinant tissue factor (r-TF) has been assessed in comparison with conventional rabbit brain thromboplastin (Manchester Reagent) for its suitability for measurement of prothrombin time (PT). The International Sensitivity Index (ISI) of r-TF calibrated against the International Reference Preparation BCT/253 (human plain) was found to be 0.96 and 1.12 with instrumental and manual techniques. Our study of plasmas from patients with congenital deficiencies of clotting factors covering a wide range of severity demonstrates that r-TF is able to detect even minor deficiencies of factors involved in the extrinsic and common coagulation pathways. Patients with liver diseases were correctly diagnosed with a prevalence of abnormal results comparable for both reagents. Between-assay reproducibility expressed as coefficient of variation was 2.3 % and 3.9 % at normal and abnormal PT levels.In conclusion, our evaluation shows that relipidated r-TF possesses the necessary requisites of sensitivity, diagnostic accuracy and reproducibility which make it a suitable candidate for PT determination both for monitoring oral anticoagulant therapy and diagnosing congenital and acquired clotting factor deficiencies. Moreover, being a highly defined reagent it may constitute a step forward in the standardization of PT testing.

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.

French Multicentric Evaluation of Recombinant Tissue Factor (Recombiplastin) for Determination of Prothrombin Time

1994 ◽  
Vol 72 (05) ◽  
pp. 698-704 ◽  
Author(s):  
J Roussi ◽  
L Drouet ◽  
M Samama ◽  
P Sié ◽  
C Bal ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Oral Anticoagulant Therapy ◽  
The Other ◽  
University Hospital ◽  
Factor Vii ◽  
Sensitivity Index ◽  
Diagnostic Systems ◽  
Multicentric Study

SummaryRecombiplastin, a recombinant a human tissue factor, elaborated by Ortho Diagnostic Systems, produced by Baculovirus and relipidated with highly purified phospholipids, was tested as a new reagent for determining prothrombin time (PT) in a French multicentric study. Its intralaboratory- performances, including sensitivity, repeatability, reproducibility and stability, were explored to establish whether its use would reduce the interlaboratory dispersion of PT values, and therefore improve the standardization of oral anticoagulant treatment.The 9 university hospital hematology laboratories involved in this study used the same type of instrument (KC 10). For 10 consecutive days, they determined PTS on a normal plasma pool, plasma dilutions of 1/2, 1/3 and 1/8, 3 identical lyophilized calibrated plasmas, as well as plasmas from 20 normal subjects, 50 patients on oral anticoagulant therapy with Recombiplastin which has an International Sensitivity Index (ISI) of 1, and 2 commercial thromboplastin extracts (ISI #1 or 2). In the patients on anticoagulants, factors VII, X and V were measured when results were conflicting.The intra and interlaboratory reproducibilities of Recombiplastin, calculated on the basis of either PTS expressed in seconds, or of the International Normalized Ratio (INR), were good, with coefficients of variation (CV) similar to those observed with the 5 other reagents used by the different laboratories (2% <CV <8%).The stability of Recombiplastin was excellent, with no variation in PT after 72 h of incubation at 37° C.A normal PT of 12 s was obtained with Recombiplastin, similar to the values found for the reagents with ISI #2. In the patients on anticoagulants, Recombiplastin gave the longest coagulation times (PTRecombipiastin = 64.2 s vs PTNeoPlastin = 32.8 s, and PTThromborel = 54.4 s). These results suggest that Recombiplastin is highly sensitive to the changes in coagulation induced by anticoagulants. Recombiplastin was more sensitive to factor VII deficiency than any of the other reagents, even those with ISI #1.The coefficients of correlation between the INRS calculated on the basis of the PTS obtained with Recombiplastin and the INRS based on the PTS for other thromboplastins, were satisfactory (0.85 <R <0.95) but a breakpoint in the slope of the regression curves was observed when INR >4. This observation requires further investigation, particularly in connection with the exact ISI values for Recombiplastin and the other thromboplastins used in this study.In conclusion, Recombiplastin is stable and sensitive and gives accurate reproducible results. However, the behavior of Recombiplastin is slightly different from that of the commercial reagents whether their ISI is 1 or 2, and its use did not reduce the interlaboratory dispersion of PT values.

The Calibration of the Second Primary International Reference Preparation for Thromboplastin (Thromboplastin, Human, Plain, Coded BCT/253)

1984 ◽  
Vol 52 (03) ◽  
pp. 336-342 ◽  
Author(s):  
Jean M Thomson ◽  
J A Tomenson ◽  
L Poller
Keyword(s):  
Statistical Analysis ◽  
Prothrombin Time ◽  
International System ◽  
Second Primary ◽  
Sensitivity Index ◽  
Calibration Model ◽  
International Reference ◽  
Reference Preparation ◽  
Time Test ◽  
International Sensitivity Index

SummaryAn international collaborative exercise has been undertaken to calibrate a replacement for the first WHO primary international reference preparation (IRP) for thromboplastin. The replacement preparation is a lyophilised batch of British Comparative Thromboplastin (BCT/253, human plain) for use in the Quick prothrombin time test. Seventeen centres participated. The experimental design, calibration model and statistical analysis were based on the recommended WHO procedure. As a result of this calibration exercise an International Sensitivity Index (ISI) of 1.1 has been assigned to the preparation by WHO and it has been officially recognised as the second primary IRP for thromboplastin.The calibration of BCT/253 is an essential link in a new hierarchical structure for the standardisation of the prothrombin time. The aim is to provide a uniform international system of reporting prothrombin time results using International Normalised Ratios (INR) derived from the ISI of individual thromboplastins.

APPLICATIONS OF INTERNATIONAL RECOMMENDATIONS ON THE STANDARDIZATION OF PROTHROMBIN TIME IN ORAL ANTICOAGULANT CONTROL

1987 ◽  
Author(s):  
F Dati ◽  
U Becker ◽  
N Heimburger
Keyword(s):  
Prothrombin Time ◽  
Oral Anticoagulant ◽  
Calibration Procedure ◽  
Sensitivity Index ◽  
Chromogenic Substrate ◽  
Clotting Factors ◽  
Reference Preparation ◽  
Broad Variation ◽  
The Mean

The determination of prothrombin time (PT) in oral anticoagulant control is affected by a broad variation. The responsible factors are: type of thromboplastin incorporated in the PT reagents, procedure for use, clotting factors or heparin inhibitors added to the reagent, method of expression of PT results. Recently, joint recommendations have been issued by International Committees (ICSH/ICTH) taking into account the system of International Thromboplastins and the statistical model for thromboplastin calibration established by WHO. The aim is a standardization of commercial thromboplastins for PT tests in order to allow the use of the international scale of oral anticoagulant intensity (INR: Intern. Normalized Ratio). Following such recommendations we have standardized two new PT tests, based on coagulometric and photometric methods which rely on the same sensitive human placental thromboplastin. The coagulometric PT test (Thromborel®S) is performed with conventional coagulometers. The photometric PT assay (Chromoquick®) uses a new chromogenic substrate specific for thrombin. This method is based on the measurement of the time necessary to reach a fixed increase of absorbance (0.1 A) using a special microprocessor-controlled photometer.The two PT reagents were calibrated either directly against a reference preparation (BCT) or via an intermediate standard thromboplastin in two multicentric studies. The calibration procedure by the WHO method allows to assign the corresponding ISI (Intern. Sensitivity Index) to the PT reagent used and the transformation of the obtained prothrombin ratio (PR) into INR by the equation INR = PRISI. The calculated ISI values were 1.08 for the coagulometric PT reagent (n = 330) and 1.07 for the photometric reagent (n = 365), respectively.The reproducibility of the ISI value for the new human placental thromboplastin for 64 different batches amounts to 3.6 %, the mean ISI value being 1.12.Comparison with the reference thromboplastins in PR values gave a good correlation.A) Coagul. PT assay (x): r = 0.964; y = 1.03x ™ 0.1;B) Photom. PT assay (x): r = 0.940; y = 1.02x ™ 0.1.

Use of Different Tissue Thromboplastins in the Control of Anticoagulant-Therapy

1958 ◽  
Vol 02 (05/06) ◽  
pp. 462-480 ◽  
Author(s):  
Marc Verstraete ◽  
Patricia A. Clark ◽  
Irving S. Wright
Keyword(s):  
Prothrombin Time ◽  
Anticoagulant Therapy ◽  
Factor Vii ◽  
Rabbit Brain ◽  
Minor Role ◽  
Rabbit Lung ◽  
Coagulation Mechanism ◽  
Time Test ◽  
The One ◽  
A Minor

SummaryAn analysis of the results of prothrombin time tests with different types of thromboplastins sheds some light on the problem why the administration of coumarin is difficult to standardize in different centers. Our present ideas on the subject, based on experimental data may be summarized as follows.Several factors of the clotting mechanism are influenced by coumarin derivatives. The action of some of these factors is by-passed in the 1-stage prothrombin time test. The decrease of the prothrombin and factor VII levels may be evaluated in the 1-stage prothrombin time determination (Quick-test). The prolongation of the prothrombin times are, however, predominantly due to the decrease of factor VII activity, the prothrombin content remaining around 50 per cent of normal during an adequate anticoagulant therapy. It is unlikely that this degree of depression of prothrombin is of major significance in interfering with the coagulation mechanism in the protection against thromboembolism. It may, however, play a minor role, which has yet to be evaluated quantitatively. An exact evaluation of factor VII is, therefore, important for the guidance of anticoagulant therapy and the method of choice is the one which is most sensitive to changes in factor VII concentration. The 1-stage prothrombin time test with a rabbit lung thromboplastin seems the most suitable method because rabbit brain preparations exhibit a factor VII-like activity that is not present in rabbit lung preparations.

THE RELIABILITY OF THE INTERNATIONAL NORMALISED RATIO (INR) DURING SHORT TERM ORAL ANTICOAGULANT TREATMENT

1987 ◽  
Author(s):  
A McKernan ◽  
J M Thomson ◽  
L Poller
Keyword(s):  
Induction Phase ◽  
Clotting Factors ◽  
Oral Anticoagulant Treatment ◽  
Sensitivity Indices ◽  
Freeze Dried ◽  
Wide Range ◽  
Reference Preparation ◽  
The Mean ◽  
The Individual ◽  
A Prospective Study

A prospective study has been performed to assess INR values obtained with a variety of thromboplastins during the early days of coumarin treatment. The reagents were BCT/253 (the primary International Reference Preparation), Diagen Activated, Diagen Freeze Dried, Manchester Reagent and Dade Thromboplastin FS. Prothrombin times were performed before the start of treatment and at regular intervals on fifteen patients who were given a slow induction regime. In theory INR should be the same irrespective of the thromboplastin. A wide range of values was however observed with the different thromboplastins on the same plasma samples. The mean deviations of the individual reagents from the mean INR obtained with the primary IRP were: Diagen Freeze-Dried 26%, Diagen Activated 13%, Dade FS 17%, Manchester Reagent 3%.There are two possible explanations for the discrepant findings. 1) In the induction phase Vitamin K dependent clotting factors are depressed at varying rates and thromboplastins differ in their sensitivity to the depression of these factors. The International Sensitivity Indices from which INR are derived are based on results from long-term stabilised patients. 2) The manufacturers' calibrations may be incorrect as demonstrated by the consistent differences of the results with some reagents from the IRP. The findings indicate therefore that INR values may not be dependable in the early days of oral anticoagulation with some thromboplastin reagents and that manufacturers' calibrations require independent assessment preferably by national control laboratories.

In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

2010 ◽  
Vol 104 (09) ◽  
pp. 514-522 ◽  
Author(s):  
Thomas Lecompte ◽  
Agnès Tournier ◽  
Lise Morlon ◽  
Monique Marchand-Arvier ◽  
Claude Vigneron ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Time Course ◽  
Anticoagulant Activity ◽  
Cathepsin G ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Factor V ◽  
Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

scholarly journals Reduction of salivary tissue factor (thromboplastin) activity by warfarin therapy

Blood ◽  
1979 ◽  
Vol 53 (3) ◽  
pp. 366-374 ◽  
Author(s):  
LR Zacharski ◽  
R Rosenstein
Keyword(s):  
Tissue Factor ◽  
Vitamin K ◽  
Coagulation Factors ◽  
Human Saliva ◽  
Factor Vii ◽  
Clotting Factor ◽  
Activate Factor ◽  
Total Protein Content ◽  
Factor X ◽  
Clotting Factors

Abstract The coagulant of normal human saliva has been identified as tissue factor (thromboplastin, TF) by virtue of its ability to cause rapid coagulation in plasmas deficient in first-stage coagulation factors and to activate factor x in the presence of factor VII and by virtue of the fact that its activity is expressed only in the presence of factor VII and is inhibited by an antibody to TF. The TF is related to cells and cell fragments in saliva. Salivary TF activity has been found to be significantly reduced in patients taking warfarin. The decline in TF activity during induction of warfarin anticoagulation occurs during the warfarin-induced decline in vitamin-K-dependent clotting factor activity, as judged by the prothrombin time. The decrease in TF activity is not related to a reduction in salivary cell count or total protein content or to a direct effect of warfarin on the assay. It is hypothesized that the mechanism by which warfarin inhibits TF activity may be related to the mechanism by which it inhibits expression of the activity of the vitamin-K-dependent clotting factors. Inhibition of the TF activity may be involved in the antithrombotic effect of warfarin.

Thrombin Generation Is Normal in Most Patients with Cirrhosis despite a Prolonged INR.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1826-1826
Author(s):  
Alexander Gatt ◽  
Anne Riddell ◽  
Vincenza Calvaruso ◽  
Michael Makris ◽  
Edward Tuddenham ◽  
...  
Keyword(s):  
Liver Disease ◽  
Tissue Factor ◽  
Patient Group ◽  
Protein C ◽  
Thrombin Generation ◽  
Control Sample ◽  
Normal Subjects ◽  
Sensitivity Index ◽  
Clotting Factors ◽  
The Mean

Abstract Introduction: Advanced liver disease is associated with prolongation of the prothrombin time (PT). In order to decrease the inter-laboratory variability of PT measurement, the international normalised ratio (INR) calculated as the ratio of patient’s PT to a normal (control) sample, raised to the power of the international sensitivity index (ISI) of the particular thromboplastin used was developed. However, the ISI is derived from PT results of patients on warfarin and results cannot be extrapolated to liver patients. Despite this, the INR is still commonly performed to assess bleeding risk in patients with liver disease worldwide. Furthermore the INR is only affected by factors I, II, V, VII, and X and is not influenced by other factors such as factor VIII which is usually raised in hepatic cirrhosis. Recently it has been reportd that thrombomodulin addition (to take into account the protein C pathway) normalises thrombin generation (TG)1 despite these patients having a low TG if thrombomodulin is not used. Aim: We speculated that TG, which is a global assay of coagulation and sensitive to all coagulation factors, when triggered by a low tissue factor (TF) concentration might not correlate with the INR in patients with liver disease and that contact inhibition with corn trypsin inhibitor (CTI) might better reflect the coagulation potential in this patient group. Results: 73 unselected patients with liver cirrhosis due to various diseases and 25 normal subjects were studied. INR and TG using the calibrated automated thrombogram (CAT) at 1pM tissue factor (TF) with CTI, 5pM without CTI and with and without Protac (a Protein C activator) were performed using platelet poor plasma (PPP). The INR range was 0.8–4.0 (mean 1.6). At 5pM TF without Protac, the patient group had a significantly lower endogenous thrombin potential (ETP) than the controls (mean ETP difference 752nM/min; P &lt;0.0001). With Protac, no significant differences could be detected between the 2 groups. However, if the ETP without Protac was divided by the ETP with Protac x 100, the liver group showed more resistance to PC activation (mean % difference 25.4; P 0.0002). At 1pM TF, the mean ETP in the cirrhosis cohort was slightly lower than the normal group (difference between means 216nM/min; P 0.03). However, only 7 (9.6%) patients had ETP values less than the normal range (mean±2SD). No correlation was found between the ETP at 1pM and the INR. The mean FVIII:C was raised at 185.6 (78–420U/dl). Conclusion: TG measured at low TF with CTI is normal in the majority of patients with cirrhosis. These patients are also more resistant to PC activation and have supranormal FVIII:C. Thus most patients have a normal or high thrombin potential despite an abnormal INR. These findings have important implications as in the absence of bleeding, “prophylactic” plasma and clotting factors are unnecessary.

Multicentric Evaluation of a New PT Reagent Based on Recombinant Human Tissue Factor and Synthetic Phospholipids

1994 ◽  
Vol 71 (03) ◽  
pp. 292-299 ◽  
Author(s):  
R Bader ◽  
P M M Mannucci ◽  
A Tripodi ◽  
J Hirsh ◽  
F Keller ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Human Tissue ◽  
Regression Line ◽  
Coagulation Factor ◽  
Phosphatidyl Serine ◽  
Factor Vii ◽  
Rabbit Brain ◽  
Sensitivity Index ◽  
Mean Values ◽  
Automated Instrument

SummaryA new PT reagent based on recombinant human tissue factor and synthetic phospholipids (phosphatidyl choline and phosphatidyl serine) with defined fatty acid side chains was calibrated against BCT/253 and CRM 149R. A small but consistent bias in the International Sensitivity Index (ISI) value was obtained using either the human or rabbit brain reference material. ISI values were around 1.0 or slightly lower depending on the respective instrument. Mixing studies with factor deficient plasmas showed a high factor sensitivity especially for factor VII as compared to commercial rabbit brain or human placenta thromboplastin. In an international field trial the reagent was tested using fully or semi automated Electra™ coagulometers in 4 different laboratories. Results with normal samples were in excellent agreement among the different laboratories. Mean values were 10.9, 10.9, 11.0, 11,7 s with a range of 9.5 to 12.5 s. Results of males and females were not different. In patients with liver disease very similar PT activities were found as compared to sensitive rabbit brain or human placental thromboplastins. In normals and patients with oral anticoagulation INR values correlated very well against BCT (r = 0.98, regression line y =-0.07 + 0.9 x). The distribution of samples was linear over the whole range. In the comparison against sensitive rabbit brain thromboplastin or human placental thromboplastin similar correlations were found. In a few cases higher INR values were observed for the recombinant reagent especially in patients with intensive treatment. Factor assays in those patients showed at least the strong reduction of one vitamin Independent coagulation factor. Over all the linearity was better against the rabbit brain reagent than against the human placental reagent which is slightly less factor VII sensitive as shown in mixing studies with normal and factor VII deficient plasma. Precision studies in the 4 laboratories showed excellent reproducibility of lyophilised controls or local patient plasma pools for all reagents with a better performance of the recombinant reagent. C. V. values from day to day ranged from 1.3% to 5% for normal and abnormal controls.These results show that the recombinant PT reagent, especially in conjunction with a precise automated instrument, may improve the results of PT testing and thus may lead to better patient care.

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