Dilute Russell Viper Venom Time Research Articles

A Hexagonal (II) Phase Phospholipid Neutralization Assay for Lupus Anticoagulant Identification

Lupus anticoagulants (LAs) are immunoglobulins (IgG, IgM, or both) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e. g. APTT, dilute PT, dilute Russell Viper Venom Time). These antibodies may be identified in a wide variety of clinical settings. With the exception of heparinized patient samples, the presence of LAs is often the most common cause of an unexplained APTT in a routine clinical laboratory. The diagnosis of LAs is difficult due to variable screening reagent sensitivity and intrinsic heterogeneity of LAs. Recently, Rauch and colleagues have shown human monoclonal hybridoma LAs were inhibited by hexagonal (II) phase PLs. In contrast, lamellar phase PLs had no effect. We have evaluated a new assay system, Staclot LA, which utilizes a hexagonal (II) phase PL (egg phosphatidylethanolamine [EPE]) as a confirmatory test for LAs. Plasma samples from the following patient populations were studied: LA positive, heparinized, oral anticoagulated, hemophilia A and B, and specific factor inhibitors (factors V, VIII, IX). Unlike previous studies, the LA positive patients were a mixed population including: autoimmune diseases, drug-induced, and post-infection. Our findings confirm the specificity of hexagonal (II) phase PL neutralization of LAs.

The Textarin/Ecarin Ratio: A Confirmatory Test for Lupus Anticoagulants

Lupus anticoagulants (LA) are immunoglobulins (IgG, IgM, IgA or a mixture) which interfere with in vitro phospholipid (PL) dependent tests of coagulation (e.g. APTT, KCT, dilute Russell Viper Venom Time). LA are heterogeneous; consequently, the laboratory diagnosis is difficult and relies on multiple tests. We have developed a sensitive and relatively specific confirmatory test system based on fractions of two snake venoms. Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern brown snake), activates prothrombin in the presence of PL, factor V and calcium ions. Ecarin, a protein fraction of Echis carinatus venom, will activate prothrombin in the absence of any cofactors. The activation of prothrombin by Textarin yields thrombin while Ecarin yields meizothrombin. In the presence of LA, the Textarin time is prolonged and the Ecarin time is unaffected. The test results are reported as a ratio of Textarin/Ecarin times (abnormal greater than 1.3). We have evaluated this test system in the following patient populations: LA positive, therapeutically heparinized, stable oral anticoagulated, liver disease, routine preoperative, anticardiolipin antibody positive LA negative, hemophilia A, various other hereditary factor deficiencies or dysfunctional proteins, and specific inhibitors of factor V and factor VIII. The LA positive patients represented a mixed population of autoimmune disease, drug-induced and post-infectious states. Our findings indicate the sensitivity of the Textarin/Ecarin system in the confirmation of LA. In order to use the test system most effectively, it is recommended to incorporate polybrene with Textarin when evaluating heparinized samples. Factor V deficiency and specific inhibitors of factor V yielded, in some instances, false positive results.

Antiphospholipid antibodies: proposed mechanisms of action.

Antiphospholipid antibodies (APA) are a family of immunoglobulins that react with anionic phospholipids, or anionic phospholipids-protein complexes. Recent evidence would support the latter definition. Lupus anticoagulants (LA) inhibit in vitro phospholipid dependent coagulation tests [e.g., activated partial thromboplastin time (APTT), prothrombin time (PT), and dilute Russell viper venom time (dRVVT)]. This inhibition appears to be specific for reagent phospholipids. The addition of freeze-thawed platelets or activated platelets will result in correction of the LA-induced abnormality. Anticardiolipin antibodies (ACA) are related to LA but appear to be distinct. ACA are detected by solid phase assays (ELISA, RIA) and require a plasma cofactor: beta 2 Glycoprotein-I (beta 2 GPI). ACA and LA activities can be separated in individual patient plasmas by affinity chromatography. In some instances they are of differing isotypes. Preliminary evaluation of beta 2 GPI in coagulation assays suggests it may function as a cofactor for LA activity. Recent work also suggests human prothrombin may represent a necessary cofactor for in vitro LA activity. Paradoxically, patients with LA/ACA may sustain thromboembolic events involving both venous and arterial sites. The prothrombotic properties of LA/ACA have not been satisfactorily characterized. A number of proposals have been reported, including inhibition of prostacyclin (PGI2) generation by endothelial cells, decreased activity of the protein C system, impaired fibrinolysis, and inhibition of beta 2GPI. Among these various hypotheses, down regulation of the protein C system appears most plausible. Also, LA/ACA may interfere with the phospholipase A2-phospholipid substrate complex involved in the generation of arachidonic acid from membrane phospholipids.

Lupus anticoagulant activity of autoimmune antiphospholipid antibodies is dependent upon beta 2-glycoprotein I.

It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself.

Laboratory heterogeneity of the lupus anticoagulant : A multicentre study using different clotting assays on a panel of 78 samples

The laboratory heterogeneity of the lupus anticoagulant (LA) was investigated in a multicentre study using a panel of 78 plasma samples diagnosed as containing a LA. Consecutive samples were collected by 12 participants using various screening tests, and sent to 7 laboratories which performed one or more clotting assays among the following : activated partial thromboplastin time (APTT), dilute Russell viper venom time, kaolin clotting time (KCT), dilute tissue thromboplastin time (dTTI) and a platelet neutralization test. For APTT and dTTI, 10 versions of these tests including standard and mixing procedures were carried out. They varied by reagents, phospholipid concentration or methodology. Cut-off times were determined for each test by comparing the results of the panel to those of a control population. When the data of all clotting assays were pooled, 70 of the 78 selected plasmas were considered to contain LA, 15 of them having a low-titer inhibitor. Sensitivity, defined as the proportion of positive results among LA-containing plasmas, varied from 62 to 100% and was positively related to responsiveness (defined as the mean ratio of clotting time to cut-off time). Laboratory heterogeneity of LA-containing plasma was illustrated by a star symbol plot analysis. Different populations of samples, with LA preferentially recognized by one assay (or group of assays) irrespective of the overall sensitivity of this assay, were identified. Multiple component analysis demonstrated the heterogeneity of low-titer inhibitors, which complicates their recognition in routine laboratory investigation.

Prospective study of fluctuations of lupus anticoagulant activity and anticardiolipin antibody titre in patients with systemic lupus erythematosus.

Fluctuations of lupus anticoagulant activity and anticardiolipin antibody titres were studied in 53 patients with systemic lupus erythematosus (SLE). The median study time was 26 months with a median number of 12 samples. Lupus anticoagulant was measured by the kaolin clotting time (KCT) and dilute Russell viper venom time (dRVVT) assays; anticardiolipin antibodies were assayed by an enzyme linked immunosorbent assay (ELISA). Normal and increased KCTs or dRVVTs were seen during follow up in 13 and 12 patients, respectively. IgG anticardiolipin antibodies changed from negative to positive or positive to negative in 26 patients and IgM anticardiolipin antibodies in 16 patients. Disease activity and treatment with prednisone could account for these fluctuations in the kaolin clotting time (KCT) in 7 of 13 patients and in the dRVVT in 2 of 12 patients. Whole group analysis showed that the KCT, dRVVT, and IgM anticardiolipin antibodies were not associated with disease activity, in contrast with IgG anticardiolipin antibodies. During treatment with prednisone normal KCT and dRVVT results were obtained more easily than normal anticardiolipin antibody levels. It is recommended that lupus patients should not be classified as antiphospholipid antibody positive or negative on the basis of only one sample.

Comparison Between a One-Point Dilute Phospholipid APTT and the Dilute Russell Viper Venom Time for Verification of Lupus Anticoagulants

In the present study, the dilute Russell viper venom time (RVVT) and the dilute phospholipid activated partial thromboplastin time (PL-APTT), which are two assays used for the verification of lupus anticoagulants (LA), were modified to increase standardization. The modified assays were then compared with respect to sensitivity and specificity in detecting LA in plasmas from 72 patients with a prolonged APTT. Modifications included utilizing a single dilution of phospholipid that was either bovine brain thromboplastin (Thrombofax) or liposomes comprised of phosphatidylcholine/phosphatidylserine, and expressing the results as a ratio of the clotting times of the mixture of patient and normal plasma/clotting time of normal plasma. In the RVVT, the correlation coefficient between assay results for liposomes and Thrombofax was 0.88 and in the PL-APTT, the correlation was 0.68. A positive test for LA was defined as a ratio of greater than or equal to 1.3 for the PL-APTT with liposomes and greater than or equal to 1.2 for the PL-APTT with Thrombofax and the RVVT with Thrombofax or liposomes. Regardless of the phospholipid source in the test system, the PL-APTT demonstrated higher sensitivity and the RVVT showed greater specificity in detecting patient plasmas that contained antiphospholipid antibodies.

A review of 100 consecutive patients with spontaneous or recurrent thrombosis

One hundred consecutive patients referred to the Royal Melbourne Hospital, Diagnostic Haematology Department for investigation of a possible hypercoagulable state were analysed 1) to determine the prevalence of congenital deficiencies of the naturally occurring anticoagulant protein C, protein S, antithrombin III and plasminogen, 2) the prevalence of acquired disorders, eg: antiphospholipid syndrome and abnormal fibrinolytic activity, and 3) whether there were clinical features associated with the inherited disorders that would identify high risk patients to allow more effective application of these tests. All patients referred were screened by the following tests: FBE, PT, APTT, thrombin time, fibrinogen, D-dimer, filtered APTT, lupus anticoagulant screening by dilute thromboplastin time and dilute Russell Viper venom time, protein C functional assay, protein S free and bound, anthithrombin III functional assay and plasminogen. Anticardiolipin assays were also performed. The age range of patients was 12-93 years, median 44 years. Both venous and arterial thrombi were studied, ratio 6:1. Abnormalities of the fibrinolytic system occurred in 35%, lupus anticoagulants in 13%, deficiencies of antithrombin III in 2%, protein C in 2% and plasminogen 2%. Neither age, documented family history, nor gender were useful in identifying patients with a detectable abnormality. The incidence of deficiencies of naturally occurring anticoagulants were similar to those previously reported. Protein S deficiency was not detected. The antiphospholipid syndrome and reduction in post stress fibrinolysis were the most frequently detected abnormalities. This suggests the need for greater understanding of the role of the fibrinolytic system in recurrent thrombosis.

Antiphospholipid antibody syndrome and recurrent fetal loss

The association between circulating antiphospholipid autoantibodies, anticardiolipin antibody and/or lupus anticoagulant with venous or arterial thrombosis, neural disorders, thrombocytopenia and fetal loss is well established (the antiphospholipid antibody syndrome). The mechanisms responsible for first trimester and later fetal losses may be different; the presence of placental infarction suggests mechanisms related to the syndrome. Since successful treatment of fetal loss is now possible, using antiplatelet therapy with low dose aspirin, with or without added immunosuppressive corticosteroids, identification of women with the syndrome is important. Screening is by the demonstration of anticardiolipin antibodies using radio-immunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) and detecting lupus anticoagulant by prolongation of phospholipid dependent coagulation tests, e.g. activated partial thromboplastin time (APTT), kaolin clotting time (KCT), or dilute Russell Viper Venom Time (dRVVT). Women may have no clinical or serological manifestations of autoimmune disorders apart from recurrent fetal loss; the screening tests may also be negative between pregnancies and early in pregnancy. Diagnosis of the antiphospholipid antibody syndrome identifies an important treatable cause of fetal loss and also indicates the potential for thrombotic and neurological problems that require ongoing active therapeutic and preventive measures.

Use of a simplified dilute Russell??s viper venom time (DRWT) confirms heterogeneity among 'lupus anticoagulants'

A simplified dilute Russell's viper venom time (DRVVT) test--in which the venom, trace phospholipid and calcium were combined into a single reagent--was evaluated for the detection of lupus anticoagulants (LA) in 28 plasma samples containing non-specific circulating anticoagulants. In agreement with previous studies, the DRVVT was found to be insensitive to defects in contact and haemophilic factors and was only marginally affected by antibodies directed against factor VIII. Thus, the use of a DRVVT test in investigations of anticoagulants reduces the risk of confusing a haemorrhagic inhibitor of factor VIII with a non-haemorrhagic LA. In comparisons of sensitivity against activated partial thromboplastin time tests (APTT-Actin FSL and Organon-Teknika reagents) the simplified DRVVT was prolonged slightly more than the APTT in most of the test plasmas containing various non-specific circulating anticoagulants. Three anticoagulants affecting APTTs more than the DRVVT were found to be associated with anticardiolipin IgMs. APTT-prolonging anticoagulants, whether prolonging DRVVT tests or not, showed similar 'correction' of their APTTs by the addition of platelets or phospholipid. Thus, phospholipid-dependent or LA show heterogeneity. Those affecting only the APTT and not DRVVT should perhaps be classified differently.

Comparison of Various Screening and Confirmatory Tests for the Detection of the Lupus Anticoagulant

Several assay systems have been proposed for detection of the lupus anticoagulant (LA). We compared several screening and confirmatory tests used for the detection of LA in 108 patients. LA was detected in 52 plasmas. The activated partial thromboplastin time (APTT) and the dilute Russell viper venom time (DRVVT) were the most sensitive screening tests as compared to the kaolin clotting time (p less than 0.001). The platelet neutralization procedure in both the APTT and DRVVT systems was superior to APTT performed with high phospholipid concentration (p less than 0.01) and tissue thromboplastin inhibition (p less than 0.001) as confirmatory tests. There was an association between the presence of LA and antiphospholipid antibodies detected by the enzyme-linked immunosorbent assay (p less than 0.001). In summary, our results show that APTT may be a sensitive test for the detection of LA when an appropriate reagent is employed, and that freeze-thawed platelets are more effective than phospholipids to neutralize LA activity.

Comparison of four laboratory tests for lupus anticoagulant

The lupus anticoagulant (LA) phenomenon created world-wide interest recently. Various tests have been devised to identify LA in plasma, but none of these methods have been universally accepted. In this study 16 cases labelled LA positive by a prior kaolin clotting time (KCT) test were reassessed by four other methods, namely delta KCT (delta kaolin clotting time), APTT (activated partial thromboplastin time), PNP (platelet neutralization procedure), and DRVVT (dilute Russell viper venom time). Anti-cardiolipin antibodies (ACL) were also looked for. In our hands the delta KCT proved to be a simple, sensitive test, not influenced by oral anticoagulant therapy, and we recommend it as a screening test. Where the presence of LA is strongly suspected on clinical and other groups, more than one method may be necessary for the diagnosis.

Screening for the lupus anticoagulant

The lupus anticoagulant may be defined as an immunoglobulin (IgG, IgM or both) which interferes with one or more of the in vitro phospholipid-dependent tests of coagulation. For many years, lupus anticoagulants were regarded as a laboratory nuisance; consequently, reagents were often selected on the basis of insensitivity to lupus anticoagulants. Recently, lupus anticoagulants have been associated with a variety of clinical conditions including recurrent thromboembolic events (both arterial and venous), obstetrical complications including fetal death and spontaneous abortion, and a variety of hematologic and neurologic complications. As a result, many laboratories are now being asked to identify the presence of lupus anticoagulants in selected patient populations. In addition to assays for lupus anticoagulants, there are immunologic assays designed to detect phospholipid antibodies using solid phase systems (RIA or ELISA). A variety of screening tests have been designed to enhance sensitivity to lupus anticoagulants. Test systems with decreased amounts of phospholipid (phosphatidylserine) appear to be most sensitive to lupus anticoagulants. Of the various tests used, the activated partial thromboplastin time (APTT) appears to be most sensitive. The sensitivity of any screening test system is inversely proportional to the residual platelets in the patient sample. APTT reagents differ widely in their sensitivity to lupus anticoagulants. The dilute Russell viper venom time is also highly dependent on the choice and concentration of phospholipid with respect to its sensitivity. Once an abnormality of a screening test has been identified, it is necessary to prove the abnormal result is due to the presence of an inhibitor. This step in the diagnosis may utilize either mixing studies or plasma agarose gels. The final step in the diagnosis of lupus anticoagulants is the demonstration of phospholipid specificity of the inhibitor. Two approaches have been utilized: 1. test systems designed to enhance anticoagulant effect (phospholipid-depleted), and 2. test systems with increased or altered phospholipids which will bypass or neutralize the anticoagulant.

Comparison of laboratory tests used for identification of the lupus anticoagulant

A comparison of the sensitivities of the ten most commonly used tests for the identification of the lupus anticoagulant (LA) and the lupus cofactor phenomenon was undertaken on 18 patients. All investigations, except the cardiolipin-antibody ELISA assay, were carried out using patient's plasma alone followed by a 1:1 mix with control plasma. Dilution studies (1:3, 1:6, 1:9--patient:control) were also carried out. The kaolin clotting time (KCT) was the only test positive in all patients at all dilutions, while the dilute activated partial thromboplastin time with kaolin (Dil-APTT) registered 17 of 18 positive at all dilutions. Both the dilute Russell viper venom time (Dil-RVVT) and the tissue thromboplastin inhibition time (TTI) (1/500 thromboplastin) identified the LA in 17 of 18 patients on initial testing but were less sensitive in the dilution studies. The KCT is not a suitable test for routine laboratory use, as it requires an individual filtration step. Therefore a combination of either the Dil-APTT or Dil-RVVT together with the TTI (1/500 dilution thromboplastin) is recommended for routine LA screening, as all patients with LA in this study were identified using these easily automated tests. The lupus cofactor phenomenon was most frequently demonstrated using the Dil-APTT.

Relative Sensitivity of Different Tests in the Detection of Low Titer Lupus Anticoagulants

Lupus anticoagulants (LA) and anticardiolipin antibodies have been strongly associated with recurrent abortion and fetal death. Because steroids have been reported to improve the fetal outcome of LA associated pregnancies, presumably by decreasing the levels of LA, it becomes desirable to have a simple and reliable test to monitor the levels of the putative antibody. To this effect, we assessed the capacity of the following coagulation tests to detect the presence of LA in serial dilutions of patient plasma with pooled normal plasma: kaolin clotting time (KCT), tissue thromboplastin inhibition test (TTIT), dilute Russell Viper venom time (DRVVT) and activated partial thromboplastin time with standard and high concentrations of phospholipids (SC and HCAPTT). All samples were also evaluated for the presence of anticardiolipin antibodies with an ELISA. The KCT was able to detect LA at a much greater dilution in normal plasma than any of the other clotting assays. The ELISA was comparable to KCT in its ability to detect high dilutions of LA.

The Use of the Dilute Russell Viper Venom Time for the Diagnosis of Lupus Anticoagulants

We describe here a test for lupus anticoagulants based on a modified Russell viper venom time (RVVT), using limiting amounts of phospholipid and venom. We have studied 29 patients with a prolonged dilute RVVT. Five of the 29 had a normal activated partial thromboplastin time and three of 14 tested by the tissue thromboplastin inhibition test were normal. In 17 of 19 patients tested, the dilute RVVT was completely normal when ionophore-treated platelets were substituted for phospholipid; the remaining two patients, both with very long phospholipid-dependent dilute RVVT's, were nearly completely normalized. The dilute RVVT is not prolonged in the presence of antibodies to factors VIII, IX, or XI. Thus, the dilute RVVT appears to be a simple, reproducible, sensitive, and relatively specific method for the detection of lupus anticoagulants.

The use of the dilute Russell viper venom time for the diagnosis of lupus anticoagulants

We describe here a test for lupus anticoagulants based on a modified Russell viper venom time (RVVT), using limiting amounts of phospholipid and venom. We have studied 29 patients with a prolonged dilute RVVT. Five of the 29 had a normal activated partial thromboplastin time and three of 14 tested by the tissue thromboplastin inhibition test were normal. In 17 of 19 patients tested, the dilute RVVT was completely normal when ionophore-treated platelets were substituted for phospholipid; the remaining two patients, both with very long phospholipid-dependent dilute RVVT's, were nearly completely normalized. The dilute RVVT is not prolonged in the presence of antibodies to factors VIII, IX, or XI. Thus, the dilute RVVT appears to be a simple, reproducible, sensitive, and relatively specific method for the detection of lupus anticoagulants.