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PubMed1994

Effect of concentration of trisodium citrate anticoagulant on calculation of the International Normalised Ratio and the International Sensitivity Index of thromboplastin.

The aim of this study was to determine whether the concentration of trisodium citrate used to anticoagulate blood has an effect on the INR of the sample and the ISI of the thromboplastin. Five thromboplastins including and Australian reference material were used to measure the prothrombin time of normal and patient samples collected into two concentrations of trisodium citrate--109 mM and 129 mM. There was no effect of citrate concentration on the INRs determined with the reference material. However for the other four thromboplastins there was a significant difference between INRs for the two citrate groups. The prothrombin times of the samples collected into 129 mM were longer than those collected into 109 mM. This difference was only slight in normal plasma but more marked in patients receiving oral anticoagulants, causing the INRs for patient plasmas collected into 129 mM citrate to be higher then the corresponding samples collected into 109 mM citrate. From orthogonal regression of log prothrombin times by the reference method against each thromboplastin, we found that the ISI for each thromboplastin was approximately 10% lower when determined with samples collected into 129 mM citrate than with samples collected into 109 mM. These results suggest that the concentration of trisodium citrate used for collection of blood samples can affect the calculation of the INR and the calibration of the ISI of thromboplastin. This was found both for commercial thromboplastins prepared by tissue extraction and for a recombinant tissue factor.

Anticoagulants↗
PubMed1994

Implications of use of low international sensitivity index thromboplastins in prothrombin time testing.

The recent introduction of thromboplastin reagents with low international sensitivity index (ISI) values into the US market for the purpose of generating a more precise international normalized ratio than high ISI thromboplastins could has necessitated an evaluation of the impact of the low ISI reagents on prothrombin time (PT) testing in general. In this study, PT testing with three thromboplastin reagents, one of which (presently used in our laboratory) has an ISI of 2.10 and the other two ISI values of 0.92 and 1.06, respectively, was performed on normal individuals, on quality control reference plasma specimens and single-factor-deficient plasma specimens, and on patients with liver disease, intravascular coagulation, and receiving oral anticoagulant therapy. We found that PTs of normal individuals determined by all three thromboplastins were virtually identical. The thromboplastins with a low ISI generated much longer PTs on abnormal reference plasma specimens than did the high ISI product. Low ISI reagents also produced longer PTs in all three groups of patients. However, the degree of prolongation was far greater for patients receiving warfarin than for the other two groups of patients. Conversion of the PT to an international normalized ratio minimized the discrepancy seen in the PT ratio in patients receiving oral anticoagulants. The two low ISI thromboplastins did not produce near-identical values of PT, PT ratio, or international normalized ratio on plasma specimens obtained from patients who received warfarin therapy. The critical value set for PT with a high ISI thromboplastin would not be adequate if the reagent is to be replaced with a low ISI product.

Administration, Oral↗
PubMed1993

Multi-center study of thromboplastin calibration precision--influence of reagent species, composition, and International Sensitivity Index (ISI).

Four thromboplastin reagents were tested by 18 laboratories in Europe, North-America, and Australasia, according to a detailed protocol. One thromboplastin was the International Reference Preparation for ox brain thromboplastin combined with adsorbed bovine plasma (coded OBT/79), and the second was a certified reference material for rabbit brain thromboplastin, plain (coded CRM 149R). The other two thromboplastin reagents were another rabbit plain brain thromboplastin (RP) with a lower ISI than CRM 149R and a rabbit brain thromboplastin combined with adsorbed bovine plasma (RC). Calibration of the latter two reagents was performed according to methods recommended by the World Health Organization (W.H.O.). The purpose of this study was to answer the following questions: 1) Is the calibration of the RC reagent more precise against the bovine/combined (OBT/79) than against the rabbit/plain reagent (CRM 149R)? 2) Is the precision of calibration influenced by the magnitude of the International Sensitivity Index (ISI)? The lowest inter-laboratory variation of ISI was observed in the calibration of the rabbit/plain reagent (RP) against the other rabbit/plain reagent (CRM 149R) (CV 1.6%). The highest inter-laboratory variation was obtained in the calibration of rabbit/plain (RP) against bovine/combined (OBT/79) (CV 5.1%). In the calibration of the rabbit/combined (RC) reagent, there was no difference in precision between OBT/79 (CV 4.3%) and CRM 149R (CV 4.2%). Furthermore, there was no significant difference in the precision of the ISI of RC obtained with CRM 149R (ISI = 1.343) and the rabbit/plain (RP) reagent with ISI = 1.14. In conclusion, the calibration of RC could be performed with similar precision with either OBT/79 or CRM 149R, or RP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

[Comparative analysis of 2 thromboplastins].

BACKGROUND: This study was performed to evaluate the prothrombin time in normal healthy people (102 subjects) by means of two thromboplastins. METHODS: Dade Thromboplastin IS (rabbit brain thromboplastin) and Dade Innovin (recombinant tissue factor) were used. Derived fibrinogen, Claus fibrinogen and in vitro sensibility of these thromboplastins to known amount of heparin were also measured. RESULTS: A different behaviour of prothrombin time measurement linked to different thromboplastin sensibility connected to the age was observed. A different fibrinogen (Claus and derived) behaviour connected to the age that may help to explain thromboplastin sensibility difference with the age was also observed. Finally different sensibility of these two thromboplastins to heparin in vitro was observed. CONCLUSIONS: This result should be considered when anticoagulation is started with oral anti coagulant drug and heparin together.

Adult↗
PubMed1991

Effect of lipoprotein-associated coagulation inhibitor (LACI) on thromboplastin-induced coagulation of normal and hemophiliac plasmas.

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits the tissue factor/factor VIIa/calcium/phospholipid complex in a factor Xa-dependent manner. Recombinant LACI (rLACI) added exogenously to plasma prolonged the activated partial thromboplastin time (APTT) and prothrombin time (PT) as a function of rLACI concentration in linear and curvilinear manners, respectively. Under these standard assay conditions, the amounts of rLACI required to double the APTT and PT were approximately 350- and 90-fold the plasma concentration of LACI, respectively. Likewise, addition of antibodies against LACI to pooled normal, factor VIII-deficient, or factor IX-deficient plasma had no effect on their respective APTTs and PTs, demonstrating the insensitivity of these assays to endogenous LACI. The prothrombin time assay was modified by using dilute thromboplastin. Unlike the standard prothrombin time assay, the clotting times were prolonged for factors VIII- or IX-deficient plasma relative to pooled normal plasma in this modified PT assay. Additionally, the degree of factor deficiency, as determined by the APTT assay, was correlated with that determined by the modified PT assay using dilute thromboplastin. When antibodies against LACI were added to pooled normal, factor VIII-deficient, or factor IX-deficient plasma and the prothrombin time assay initiated using dilute thromboplastin, the clotting times for antibody-treated plasma were shorter than for the corresponding plasma in the absence of antibodies. Moreover, the clotting times for factors VIII- and IX-deficient plasmas treated with antibodies raised against LACI were at least as fast as pooled normal plasma in the absence of LACI antibodies when dilute thromboplastin was used to initiate clotting. These results suggest that the prothrombin time assay using dilute thromboplastin may more accurately reflect what occurs in vivo and that LACI may play an important role in the prolonged bleeding of those with hemophilia A or B.

Antibodies, Monoclonal↗
PubMed1990

Antiphospholipid antibodies and recurrent pregnancy loss: correlation between the activated partial thromboplastin time and antibodies against phosphatidylserine and cardiolipin.

Concordance was determined among the presence of the lupus anticoagulant measured by prolongation of the activated partial thromboplastin time and IgG and IgM antibodies against phosphatidylserine and cardiolipin in 47 patients selected for study because of histories of recurrent spontaneous pregnancy loss and a positive test for at least one antiphospholipid antibody. Forty-five of 47 patients (96%) had a prolonged activated partial thromboplastin time, ranging from 46 to 150 seconds. Elevated levels of IgG antiphosphatidylserine antibodies and IgM antiphosphatidylserine antibodies were present in 41 (87%) and in 19 (40%) of samples, respectively. Antibodies against cardiolipin were less commonly observed; IgG anticardiolipin antibodies in only 32 (68%) of 47 samples and IgM anticardiolipin antibodies in 15 (36%) of 42 samples. Neither the level of IgG antiphosphatidylserine antibodies nor the level of IgG anticardiolipin antibodies correlated well with the degree of prolongation of coagulation in the activated partial thromboplastin time (R = 0.312, p = 0.032 for IgG antiphosphatidylserine antibodies versus activated partial thromboplastin time; R = 0.251, p = 0.088 for IgG anticardiolipin antibodies versus activated partial thromboplastin time). Concordance with the activated partial thromboplastin time, however, was observed in 41 (87%) samples for IgG antiphosphatidylserine antibodies and in only 32 (68%) samples for IgG anticardiolipin antibodies. Our conclusion is that the antiphosphatidylserine assay correlates best, although not totally, with the presence of lupus anticoagulant and that the antiphosphatidylserine assay is more sensitive than testing for anticardiolipin.

Abortion, Habitual↗
PubMed1992

Heparin-induced prolongation of partial thromboplastin time after thrombolysis: relation to coronary artery patency. HART Investigators.

Having previously shown in the Heparin Aspirin Reperfusion Trial that the empiric use of early intravenous heparin after recombinant tissue-type plasminogen activator (rt-PA) is an important component in the overall treatment strategy, we examine in this report the specific relation between the degree of prolongation of activated partial thromboplastin time and coronary artery patency. To evaluate the hypothesis that arterial patency after administration of rt-PA for acute myocardial infarction is sustained by effective anticoagulation, activated partial thromboplastin time of heparin recipients was determined 8 and 12 h after the start of thrombolysis. Mean activated partial thromboplastin time was higher among patients with an open infarct-related artery than in those with a closed artery (81 +/- 4 vs. 54 +/- 9 s, p less than 0.02). Only 45% of patients with values less than 45 s at both 8 and 12 h had Thrombolysis in Myocardial Infarction (TIMI) flow grade 2 or 3 in the infarct-related artery at 18 h. In contrast, 88% of patients with activated partial thromboplastin time greater than 45 s and 95% of those with values greater than 60 s had an open infarct-related artery at 18 h (p = 0.003 and 0.0006, respectively). Among patients with an initially patent infarct-related artery who underwent repeat angiography at 7 days, activated partial thromboplastin time was similar in those with a persistently patent artery and those with late reocclusion. Excessive anticoagulation did not appear to increase hemorrhagic risk except that access site-related hemorrhage was more common in patients with activated partial thromboplastin time greater than 100 s at 8 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin↗
PubMed2001

Laboratory control of oral anticoagulant treatment by the INR system in patients with the antiphospholipid syndrome and lupus anticoagulant. Results of a collaborative study involving nine commercial thromboplastins.

Because of the variable responsiveness of thromboplastins to lupus anticoagulants (LA), concerns have been raised about the validity of the prothrombin time-International Normalized Ratio (PT-INR) in monitoring oral anticoagulant treatment in patients with the antiphospholipid syndrome (APS) and LA. To date, few studies have been performed, numbers of patients investigated are relatively small and results are conflicting. We report on a multicentre study organized to investigate further this clinically relevant issue. Each of nine thrombosis centres was asked to collect plasma samples from patients with APS who were on oral anticoagulants (cases) and patients without APS who were on oral anticoagulants (controls). Nine thromboplastins (three human recombinant, one from human placenta and five from rabbit brain) were calibrated at the co-ordinating centre according to World Health Organization guidelines. Measurements of the INR and factor X amidolytic activity for all frozen plasmas were performed centrally. The numbers of patients investigated were 58 cases and 57 controls. Between-reagent variability of the INR was higher in cases [coefficient of variation (CV) = 12.4%] than in controls (CV = 6.7%), but this was because of one of the thromboplastins only (Thromborel R, human recombinant), which measured considerably higher INR values than the others in cases but not in controls. In conclusion, our data indicate that LA interference on the PT-INR measured with the majority of commercial thromboplastins is not enough to cause concern if insensitive thromboplastins, properly calibrated to assign them an instrument-specific International Sensitivity Index are used. New thromboplastins, especially those made of relipidated tissue factor, should be checked for their responsiveness to LA before they are used to monitor oral anticoagulant treatment in patients with APS.

Animals↗
PubMed1992

The preparation of a sensitive partial thromboplastin reagent from bovine brain.

Commercial partial thromboplastin reagents markedly differ in their sensitivity to factor deficiency, heparin, or the lupus anticoagulant. These differences may be partly due to the variable phospholipid content of different commercially available reagents. For over 15 years, we have routinely used a partial thromboplastin prepared from human brain. In the past four years, we have been using a similarly prepared bovine partial thromboplastin reagent. This report describes the preparation of our partial thromboplastin reagent, as well as an analysis of the phospholipid composition of both the human and bovine thromboplastin reagents. Four separate brain preparations produced consistent percentages of the anionic phospholipids, phosphatidylserine, and phosphatidylinositol. The bovine reagent was also compared with commercial partial thromboplastin reagents in the detection of coagulation factor deficiency, heparin, and the lupus anticoagulant.

Animals↗
PubMed2003

Prolonged partial thromboplastin times in children with fever and petechiae without bacteremia or sepsis.

OBJECTIVES: In a prior uncontrolled study, 23% of children with fever and petechiae without bacteremia or sepsis had a prolonged partial thromboplastin time (PTT). We attempted to validate this finding by comparing the PTTs of children with fever and petechiae who were neither septic nor bacteremic with those of children without fever and petechiae. METHODS: Design. Secondary analysis of a prospective cohort with a newly identified retrospective control cohort. Setting. Emergency department of an urban pediatric teaching hospital from December 1993 to June 1995. Study group. All patients 1 month to 18 years old from a previously identified cohort with (1) fever (temperature >or=38.0); (2) petechiae; (3) prothrombin time and partial thromboplastin time performed in the emergency department (n = 273). Control group. Age-matched patients 1 month to 18 years old who presented to the emergency department during the same time period as the study patients with (1) an injury or other potentially surgical diagnosis; (2) neither fever nor petechiae; (3) prothrombin time and partial thromboplastin time performed in the emergency department. Main outcome measures. partial thromboplastin time and prothrombin time. RESULTS: 117 control patients were identified. Partial thromboplastin time was prolonged in 23% of study patients, but in only 6% of control patients (P < 0.001). Prothrombin time was prolonged in 9% of patients with fever and petechiae compared with only 4% of control patients (P = 0.09). CONCLUSION: Children with fever and petechiae without bacteremia or sepsis are more likely than controls to have prolonged partial thromboplastin time.

Adolescent↗
PubMed2004

Properties of recombinant human thromboplastin that determine the International Sensitivity Index (ISI).

Prothrombin Time (PT) clotting tests are widely used to monitor oral anticoagulation therapy and to screen for clotting factor deficiencies. The active ingredient in PT reagents (thromboplastins) is tissue factor, the integral membrane protein that triggers the clotting cascade through the extrinsic pathway. Several years ago, a system for calibrating and using thromboplastin reagents, known as the International Sensitivity Index (ISI) and the International Normalized Ratio (INR), was developed to standardize monitoring of oral anticoagulant therapy. The ISI/INR method, while revolutionizing the monitoring of coumarin therapy, has been criticized for a number of perceived shortcomings. We have undertaken a series of studies aimed at achieving a detailed understanding of which parameters influence the ISI values of thromboplastin reagents, with an ultimate goal of creating 'designer thromboplastins' whose sensitivities to the various clotting factors can be individually tailored. In this study, we demonstrate that ISI values of thromboplastin reagents based on relipidated, recombinant human tissue factor can be controlled by a combination of changes in the phospholipid content (in particular, the levels of phosphatidylserine and phosphatidylethanolamine) and ionic strength. The sensitivity of a given thromboplastin reagent can be increased (i.e. its ISI value decreased) by decreasing the content of phosphatidylserine and/or increasing the ionic strength. The molar ratio of phospholipid to tissue factor, on the other hand, had essentially no impact on ISI value.

Humans↗
PubMed1981

Alternative pathways of thromboplastin-dependent activation of human factor X in plasma.

To determine the interrelationships of the major coagulation pathways, the activation of 3H-labeled factor X in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin and calcium were added to plasma samples containing 3H-factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin, the rate of factor X activation in plasmas deficient in factor VIII or factor IX was 10% of the activation rate of normal plasma or of factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, reconstituted normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these plasma experiments, it is inferred that the dilute thromboplastin-dependent activation of factor X requires factors VII, IX, and VIII. An alternative extrinsic pathway that involves factors IX and VIII may be the physiologic extrinsic pathway and hence help to explain the consistent clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.

Animals↗
PubMed1982

Effect of purified protein derivative and sonicates of Mycobacterium leprae and Mycobacterium bovis BCG on thromboplastin response in human monocytes in vitro.

Human monocytes isolated from peripheral blood responded with increased thromboplastin expression upon stimulation in vitro with three mycobacterial antigens: tuberculin purified protein derivative and sonicates of Mycobacterium boviS BCG and Mycobacterium leprae. The stimulating principle of mycobacteria is probably a cell wall constituent since crude extracts of cell walls were 2.5 to 25 times more potent in stimulating thromboplastin synthesis than were whole sonicates. This thromboplastin response was inhibited by inhibitors of RNA and protein synthesis, dexamethasone, and agents that caused elevation of intracellular cyclic AMP. The presence of lymphocytes did not enhance the monocyte thromboplastin response significantly during the first 24 h of incubation. For M. bovis BCG and M. leprae sonicates, the thromboplastin response correlated with general activating effects measured by determining the release of lysozyme and beta-glucuronidase. The role of thromboplastin in chronic inflammatory reactions is discussed.

Antigens, Bacterial↗
PubMed1987

Differential effect of alpha-interferon and gamma-interferon on thromboplastin response in monocytes and endothelial cells.

Recombinant gamma-interferon (r gamma-IFN) has contrasting effects on thromboplastin (TPL) synthesis induced in monocytes (M) and endothelial cells by bacterial lipopolysaccharide (LPS), phorbol ester (TPA), and phytohaemagglutinin (PHA). In human umbilical vein endothelial cells (HUVEC) the induced thromboplastin response was significantly augmented by r gamma-IFN whereas the monocyte response was inhibited. Recombinant alpha-interferon (r alpha-IFN) had no effect on thromboplastin induction in endothelial cells but had a significant inhibitory effect on the TPL response in monocytes when LPS or LPS and cyclosporin A (CS) were used as inducing agents. Cyclosporin A, previously shown to enhance thromboplastin synthesis induced in monocytes, also contributes to a higher level of thromboplastin activity in endothelial cells. Its effect on monocytes was in most cases fully inhibited by r gamma-IFN (and also by r alpha-IFN when tested). gamma-Interferon and alpha-interferon alone had a weak stimulatory effect or none on thromboplastin synthesis in both cell types.

Cyclosporins↗
PubMed1978

Ultrastructural changes of the tissue thromboplastin after intravenous injection in rabbits.

Attempts were made to demonstrate ultrastructural changes of the tissue thromboplastin after intravenous injection, as a model experiment on the pulmonary microthrombi formation induced by the tissue thromboplastin circulating from venous return. Concentrically arranged membrane structures of the injected thromboplastin disappeared in extremely short time after the injection of the thromboplastin in rabbits. The long sheet membrane of the injected thromboplastin was frequently seen as adhered to the vascular endothelium or to the surface of blood corpuscles. Furthermore, fibrin fibres were formed in contact with the long sheet membrane of the thromboplastin. Membrane structures were not found anywhere in the control rabbits.

Animals↗
PubMed1993

A clinical study monitoring low-intensity anticoagulant therapy with a standard-sensitivity thromboplastin.

The ability of the prothrombin time to measure the anticoagulant effect of warfarin sodium varies depending on the particular tissue thromboplastin used in performing the test. Based on studies using sensitive thromboplastins, lower therapeutic ranges of anticoagulation are recommended. The adequacy of monitoring therapy in this lower range with the relatively insensitive thromboplastins commonly used in North America is unestablished. This 16-month prospective study used a standard North American thromboplastin to monitor 157 anticoagulated patients treated in a low therapeutic range. Of the 1734 prothrombin times generated, 876 (56%) were therapeutic, with 400 (23%) below and 458 (26%) above the therapeutic range. These results are comparable with those published in trials in which more sensitive thromboplastins were used in a similar therapeutic range. We conclude that standard North American thromboplastins are adequately suited to monitor therapy in this lower range.

Aged↗
PubMed2001

The role of factor XI in a dilute thromboplastin assay of extrinsic coagulation pathway.

Blood coagulation has been thought to be composed of both intrinsic and extrinsic pathways. Recent evidence strongly supports the critical role of the extrinsic pathway in the initiation of blood coagulation. This investigation established an assay that examines the role of FXI in the thromboplastin-initiated (extrinsic) coagulation based on this new concept. Plasma clotting times were measured at different concentrations of thromboplastin with activated FXII inhibited (FXIIa-inhibited Diluted Thromboplastin Time, FXIIaiDTT). Only at low concentrations of thromboplastin was FXIIaiDTT of FXI-deficient plasma significantly prolonged than that of normal plasma. Depletion of FXI from normal plasma prolonged its FXIIaiDTT and replenishment of FXI shortened it. FXIIaiDTTs of both FVIII-deficient and FIX-deficient plasma were remarkably prolonged, and addition of normal plasma dose-dependently shortened it. Furthermore, earlier alpha-thrombin inhibition was directly correlated with decreasing FXa generation. The amount of FXa production was: platelet-rich plasma > platelet-poor plasma > FXI-deficient plasma. Therefore, our findings from the FXIIaiDTT assays not only support the critical role of extrinsic pathway in blood coagulation initiation, but also demonstrate the importance of FXI as an amplifier of thrombin generation in thromboplastin-initiated coagulation.

Blood Coagulation↗
PubMed1994

Prognostic value of the activated partial thromboplastin time after orthotopic liver transplantation. A prospective study.

In this prospective study, we evaluated the predictive value of activated partial thromboplastin time on day 8 post transplantation for event-free survival in patients who had had orthotopic liver transplants; both death and retransplantation within 6 months were the events considered. In a 4-year period, 109 patients had orthotopic liver transplants in our hospital, and 104 were eligible for the study since they survived and were not given new transplants within 8 days. The activated partial thromboplastin time was significantly longer in patients who survived event-free for less than 6 months than in those with longer event-free survivals. Kaplan-Meier curves showed that patients with normal activated partial thromboplastin times were nine times more likely to survive more than 6 months without events than patients with prolonged values. The positive predictive value of activated partial thromboplastin time for event-free survival was 88% and the negative predictive value was 54%, indicating that the test is useful for predicting patient outcome. We suggest that activated partial thromboplastin time be performed on day 8 post transplantation to predict the medium-term event-free survival.

Adolescent↗