In ECAT veritas?

The laboratory criteria of the antiphospholipid syndrome include one coagulation assay (lupus anticoagulant [LA]) and two solid phase assays (anticardiolipin [aCL] and anti-ß(2)glycoprotein I antibodies [aß(2)GPI]). External quality control (EQC) surveys show that negative and clearly positive LA samples are classified correctly by about 95% of laboratories. For 'weak' LA there is a wide variability in samples' classification. Furthermore, when a weak LA sample is used in two different EQC surveys more than 50% of laboratories classify it differently. In some surveys weak LA samples were found to be positive for aCL and for aß(2)GPI by a majority of laboratories; the main reason for laboratories which classified these samples as LA negative was a negative result in the mixing test. It is likely that, depending on the sensitivity of the assay, a weak LA cannot be detected anymore after 1:1 dilution of the sample with normal plasma. Therefore, we recommend the use of integrated assays, such as screen/confirm ratios, for the detection of weak LA samples.

Spacelab-2 plasma depletion experiments for ionospheric and radio astronomical studies.

The Spacelab-2 Plasma Depletion Experiments were a series of studies to examine shuttle-induced perturbations in the ionosphere and their application to ground-based radio astronomy. The space shuttle Challenger fired its orbital maneuvering subsystem engines on 30 July and 5 August 1985, releasing large amounts of exhaust molecules (water, hydrogen, and carbon dioxide) that caused the electrons and ions in Earth's upper atmosphere to chemically recombine, thereby creating so-called "ionospheric holes." Two burns conducted over New England produced ionospheric peak depletions ranging from 25 to 50 percent, affected the ionosphere over a 200-kilometer altitude range, and covered 1 degrees to 2 degrees of latitude. Optical emissions associated with the hole spanned an area of several hundred thousand square kilometers. A third burn was conducted over a low-frequency radio observatory in Hobart, Australia, to create an "artificial window" for ground-based observations at frequencies normally below the natural ionospheric cutoff (penetration) frequency. The Hobart experiment succeeded in making high-resolution observations at 1.7 megahertz through the induced ionospheric hole.

Biological effects of aspirin and clopidogrel in a randomized cross-over study in 96 healthy volunteers.

BACKGROUND: Some data suggest that biological 'resistance' to aspirin or clopidogrel may influence clinical outcome. OBJECTIVE: The aim of this study was to evaluate the relationship between aspirin and clopidogrel responsiveness in healthy subjects. METHODS: Ninety-six healthy subjects were randomly assigned to receive a 1-week course of aspirin 100 mg day(-1) followed by a 1-week course of clopidogrel (300 mg on day 1, then 75 mg day(-1)), or the reverse sequence, separated by a 2-week wash-out period. The drug effects were assessed by means of serum TxB2 assay, platelet aggregation tests, and the PFA -100 and Ultegra RPFA -Verify Now methods. RESULTS: Only one subject had true aspirin resistance, defined as a serum TxB2 level > 80 pg microL(-1) at the end of aspirin administration and confirmed by platelet incubation with aspirin. PFA-100 values were normal in 29% of the subjects after aspirin intake, despite a drastic reduction in TxB2 production; these subjects were considered to have aspirin pseudo-resistance. Clopidogrel responsiveness was not related to aspirin pseudo-resistance. Selected polymorphisms of platelet receptor genes were not associated with either aspirin or clopidogrel responsiveness. CONCLUSIONS: In healthy subjects, true aspirin resistance is rare and aspirin pseudo-resistance is not related to clopidogrel responsiveness.

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

New clinical, laboratory and experimental insights, since the 1999 publication of the Sapporo preliminary classification criteria for antiphospholipid syndrome (APS), had been addressed at a workshop in Sydney, Australia, before the Eleventh International Congress on antiphospholipid antibodies. In this document, we appraise the existing evidence on clinical and laboratory features of APS addressed during the forum. Based on this, we propose amendments to the Sapporo criteria. We also provide definitions on features of APS that were not included in the updated criteria.

D-dimer levels during delivery and the postpartum.

BACKGROUND: D-dimer (DD) measurement has proved to be very useful to exclude venous thromboembolism (VTE) in outpatients. However, during pregnancy, the progressive increase as well as the interindividual variations of DD means that in this instance they are of poor value to rule out VTE. Only a few studies have reported measurements of DD levels in the postpartum. OBJECTIVES: To measure DD sequentially in the puerperium in order to determine when DD levels return to values obtained in non-pregnant women and can again be used in the exclusion of VTE. PATIENTS AND METHODS: After uncomplicated pregnancies, 150 women delivering at term either vaginally (n = 100) or by cesarean section (n = 50) were included. DD levels were measured immediately following delivery and next at days 1, 3, 10, 30 and 45. RESULTS: There was a marked elevation of DD at delivery, especially when instrumental. All DD measurements were above 500 ng mL(-1) at delivery, at day 1 and at day 3 postpartum. A sharp decrease in DD was observed between day 1 and day 3, followed by a slight increase at day 10. At day 30 and day 45, respectively, 79% and 93% of women in the vaginal delivery group and 70% and 83% in the cesarean group had levels below 500 ng mL(-1). Bleeding, breastfeeding and heparin prophylaxis did not modify DD levels significantly. CONCLUSION: Using the Vidas DD new assay, our study provides reference intervals for DD in the postpartum period. Using a cut-off at 500 ng mL(-1), DD measurement for ruling out VTE was found to be useful again 4 weeks after delivery.

Determination of anti-cardiolipin and other antibodies in HIV-1-infected patients.

Anti-cardiolipin antibodies (ACA) were determined by an ELISA assay in 116 HIV-1-infected patients. A positive test was found in 27 patients (23.3%) with a predominance of IgG ACA isotype. No significant difference in ACA positivity was observed between homosexuals (22.2%) and intravenous drug users (25.8%). The presence of different immunological markers was compared in ACA-positive and ACA-negative patients: ACA-positive patients had higher IgG levels (p less than 0.05) and a tendency to higher frequencies of anti-ss DNA, anti-ds DNA, anti-i antibodies, as well as circulating immune complexes. When patients were classified according to CDC criteria, no significant difference was observed for the prevalence of ACA in class II (21.2%), in class III (25%), and in class IV (21%). Our results indicate that ACA antibodies occur with other immunological alterations in HIV-1-infected patients, but do not confirm that ACA is a useful prognostic marker for development of AIDS.

Effects of statins on adhesion molecule expression in endothelial cells.

BACKGROUND: Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. OBJECTIVES: We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. METHODS: Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4-6 h activation with tumor necrosis factor (TNF)-alpha or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). RESULTS: Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-alpha and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-alpha-mediated overexpression of E-selectin. CONCLUSIONS: Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-alpha or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

Aspirin inhibits endothelial cell activation induced by antiphospholipid antibodies.

BACKGROUND: Antiphospholipid antibodies (APLA) have been shown to activate endothelial cells (EC) in vitro, as documented by an increased expression of tissue factor as well as leukocyte adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin. Currently, treatment of patients with the antiphospholipid syndrome includes aspirin, particularly for women with recurrent fetal loss. OBJECTIVE: The present study was undertaken to investigate whether aspirin interferes with EC activation induced by APLA in vitro. METHODS: IgG from 14 patients with APLA, and suffering from thrombotic complications and/or pregnancy morbidity, and control IgG were tested for their ability to modify the expression of VCAM-1 in human umbilical vein endothelial cells. VCAM-1 antigen was measured by flow cytometry and its mRNA by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Incubation of EC with IgG from most of the patients led to a higher VCAM-1 expression compared with incubation with control IgG. The effect of aspirin was studied for the eight IgG samples that induced a more than 50% increase in VCAM-1. Aspirin (10 mm) treatment of the cells significantly reduced the VCAM-1 response to these APLA. CONCLUSIONS: Our results indicate that besides its antiplatelet properties, aspirin exerts a protective effect towards APLA at the EC level by decreasing leukocyte adhesion molecule expression at the cell surface.

D-dimer determination to exclude pulmonary embolism: a two-step approach using latex assay as a screening tool.

D-dimer (DD), when measured by a quantitative enzyme-linked immunosorbent assay (ELISA), is a valuable test to exclude venous thromboembolism (VTE). However, DD ELISA technique is not appropriate for emergency use and the available agglutination latex assays are not sensitive enough to be used as an alternative to rule out the diagnosis of VTE. Latex assays could still be used as screening tests. We tested this hypothesis by comparing DD levels measured by ELISA and latex assays in 334 patients suspected of pulmonary embolism. All but one patient with a positive (DD > or = 500 ng/ml) latex assay had DD levels higher than 500 ng/ml with the ELISA assay. Accordingly, ELISA technique could be restricted to patients with a negative result in latex assay. This two-step approach would have spared about 50% of ELISA in our cohort. In conclusion, our data indicate that a latex test can be used as a first diagnostic step to rule out pulmonary embolism provided a negative result is confirmed by ELISA and the performance of the latex assay used has been assessed properly.

Anticoagulant properties of three mucopolysaccharides used in rheumatology.

Three mucopolysaccharides (MPS) used in the treatment of degenerative joint disease were compared to heparin to establish their relative potencies on 3 coagulation tests, the aPTT, the antifactor Xa activity and the dilute thrombin time. One of the compounds, Arteparon, was one fourth as potent as heparin on the aPTT, but had little or no influence on the 2 other tests. Further in vitro studies suggested that Arteparon acted at a higher level than factor Xa generation in the intrinsic amplification system and that its effect was independent of antithrombin III. In vivo administration of Arteparon confirmed its anticoagulant properties, which raises the question of the clinical use of this MPS.

Performances of the fibrin monomer test for the exclusion of pulmonary embolism in symptomatic outpatients.

Many studies have shown that D-dimer determinations can be used for the exclusion of venous thromboembolism in symptomatic outpatients, depending however on the method of D-dimer measurement. Another related assay, the Fibrin Monomer test which measures soluble fibrin levels in plasma by ELISA, is now available. We have evaluated the performances of this assay for the exclusion of pulmonary embolism (PE) in 426 consecutive outpatients presenting at the emergency ward of our institution. Diagnosis of PE was made by D-dimer measurement, compression ultrasonography, lung scintigraphy, venography and pulmonary angiography. With a cut-off of 3 microg/ml. the sensitivity and the negative predictive value were both 100% (95% CI: 97.1-100 and 96.3-100 respectively) and the specificity 33% (95 % CI: 25.7-38.1). With 4 microg/ml, the corresponding figures were 98.4 (95% CI: 94.4-99.8), 98.3 (95% CI: 94.1-99.8) and 39% (95% CI: 33.6-44.7) respectively. The prevalence of PE was 30%, the exclusion rates were 23 and 27% for either cut-off. When compared with a reference D-dimer assay (Asserachrom D-Di), a good correlation was observed. In conclusion, this is the first study suggesting the interest of this Fibrin Monomer test to rule out PE; these results, however, need to be confirmed by other studies.

Thrombomodulin levels during normal pregnancy, at delivery and in the postpartum: comparison with tissue-type plasminogen activator and plasminogen activator inhibitor-1.

Some studies suggest that soluble thrombomodulin (TM) could be used as a marker of preeclampsia or eclampsia. However little is known about the sequential changes of TM during the course of normal pregnancy. Levels of TM were determined in 100 women with uneventful pregnancies. Samples (n = 394) were divided into five study intervals, three during pregnancy, one at delivery and one three days postpartum. As compared with TM levels (median 34.3 ng/ml, range 17.6-61) of a control group of 60 healthy non-pregnant women, TM levels were shown to increase throughout pregnancy, median (and range) values being respectively 38.5 (17.6-72.7) from 11 to 20 weeks, 45.2 (22.6-75.2) from 21 to 30 weeks and 54.3 (25.1-114.5) ng/ml from 31st week to delivery. One hour after delivery TM levels were still elevated and dropped three days postpartum to 40.5 (20.9-79.4) ng/ml. The increase of TM levels was correlated with those of tissue-type plasminogen activator and plasminogen activator inhibitor-1 antigens. The large overlap in TM levels between the study periods seems to preclude a clinical use of TM based on reference values from a control group. Our data suggest that it would be more appropriate to take into account TM baseline values in a given woman to examine her TM increase during pregnancy.

Plasma measurement of D-dimer as diagnostic aid in suspected venous thromboembolism: an overview.

This paper reviews the published experience with plasma measurement of D-dimer (DD), a specific degradation product of crosslinked fibrin, in the diagnostic approach of venous thromboembolism (VTE). Pooling 11 studies (with weighting of the figures according to sample size) with a total of 1337 patients clinically suspected of deep venous thrombosis (DVT) (prevalence of DVT 35%) disclosed an average weighted sensitivity of 96.8% (95% CI: 95.2-98.4) and specificity of 35.2% (95% CI: 32.0-38.4) for the presence of DVT when the ELISA technique was used. In 908 patients suspected of pulmonary embolism (PE) from 9 trials (prevalence of PE 38%), the ELISA technique was associated with a weighted sensitivity of 96.8% (95% CI: 95.0-98.6) and specificity of 45.1% (95% CI: 40.8-49.4) for the disease. Figures obtained with latex assays were definitely lower, precluding their use in the diagnostic approach of VTE. These results show that a low concentration of plasma DD measured by the ELISA technique (usually less than 500 micrograms/l) might be used to rule out VTE in clinically suspected patients. Increased plasma concentrations are of no utility because of the low specificity of this test result. The clinical usefulness of the DD ELISA test should now be assessed in management trials under routine conditions, in the frame of clinical decision-making diagnostic processes. Lastly, the promising data obtained in a small number of asymptomatic, postoperative patients at risk of VTE deserve confirmation before the test can be recommended for initial screening in thrombo-prophylactic trials.

Antithrombin III Geneva: a hereditary abnormal AT III with defective heparin cofactor activity.

A 43-year-old man presented a pulmonary embolism. The unusual circumstances of apparition, the age and the increased heparin requirements suggested an antithrombin III (AT III) deficiency. AT III activity was low in the propositus and seven other members of his family (mean 55%), but immunologic levels were normal (mean 110%). Crossed immunoelectrophoresis in absence of heparin showed a normal pattern, but in presence of heparin showed an abnormal peak as compared with controls. Kinetics experiments showed a normal inhibition of thrombin and Xa in absence of heparin, but abnormal in presence of heparin. Affinity chromatography on heparin-Sepharose revealed two populations of AT III, one of which was devoid of heparin cofactor activity. The toponym AT III Geneva is proposed for this new familial abnormal AT III with defective heparin cofactor activity.

Value of liquid crystal contact thermography and plasma level of D-dimer for screening of deep venous thrombosis following general abdominal surgery.

Because the use of radioactive fibrinogen uptake test (FUT) has become questionable both for ethical (risk of virus transmission) and technical (lack of sensitivity) reasons, we investigated the potential value of two alternative methods for screening of asymptomatic deep venous thrombosis following elective digestive surgery: liquid crystal contact thermography (LCCT) and measurement of plasma concentration of D-dimer (DD), as compared with bilateral ascending phlebography. Out of 194 patients, 185 underwent phlebography on the 8th (0-19, median and range) postoperative day. Despite prophylaxis with low-molecular-weight heparin and elastic stockings, DVT was detected on phlebography in 58 legs of 45 patients. Sensitivity of LCCT with respect to the presence of DVT was 55% (n = 184 patients) or 28% (n = 368 legs) with a specificity of 67% and 82%, respectively. These poor performances were obtained despite a good interobserver agreement for the LCCT assessments (overall kappa coefficient of 0.66 between three experts). The most accurate cut-off of DD for discriminating patients with or without DVT was 3,000 micrograms/l, as determined by ROC curve analysis. Sensitivity of a DD level of more than 3,000 micrograms/l for the presence of phlebographically documented DVT on the 8th postoperative day was 89% for a specificity of 48%. Thus, LCCT cannot be used for screening of postoperative, mainly asymptomatic DVT following general surgery. On the other hand, measurement of plasma DD may be useful for initial screening, a negative result (level less than 3,000 micrograms/l) allowing to exclude DVT (negative predictive value of 93%) and a positive result (positive predictive value of 35%) requiring confirmation by phlebography.(ABSTRACT TRUNCATED AT 250 WORDS)

Multicenter evaluation of nine commercial kits for the quantitation of anticardiolipin antibodies. The Working Group on Methodologies in Haemostasis from the GEHT (Groupe d'Etudes sur l'Hémostase et la Thrombose).

The performances of nine commercial kits and an in-house method (HM) for the quantitation of anticardiolipin antibodies (ACA) have been evaluated in a multicenter study. Ninety control and patient samples and six standards from Louisville University were run with kits and with the HM. Marked differences in positivity rate between kits were observed, ranging from 31 to 60% for IgG and 6 to 50% for IgM. Concordance between kits occurred in 59 and 51% of samples for IgG and IgM respectively. Concordance coefficients (kappa) ranged from 0.13 to 0.92. Slopes of regression lines between the declared units of Louisville standards and the units measured from the calibrators of the kits showed great diversity and ranged from 0.159 to 0.931 for IgG and from 0.236 to 0.836 for IgM. The beta 2-glycoprotein I (beta 2-GPI) content of the dilution buffers and the wells supplied with the kits revealed noticeable differences. However samples containing anti-beta 2-GPI antibodies were classified similarly by all but one kit. In contrast the ability to measure samples devoid of anti-beta 2-GPI antibodies differed markedly between the kits. This study shows that differences in positivity rates between the commercial kits may contribute to the differences in ACA prevalence rate found in the literature. The choice of cut-off levels may partly explain the moderate concordance between the kits. In addition some samples behave very differently depending on the kits. In spite of the expression of results in PL units, standardization of ACA assays has not been achieved.

Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants.

Some lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect anti-prothrombin autoantibodies in patient serum or plasma, no or very low signal was observed using human prothrombin immobilized on plain polystyrene plates. In contrast, the same LA-positive samples bound specifically to prothrombin coated on gamma-irradiated plates, depending on the radiation dose, in the absence of added calcium and phospholipid. Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was ascertained by competition using liposome-bound prothrombin, since fluid-phase prothrombin competed poorly. Seventy-seven of 139 patients (55.4%) with LA related to a variety of underlying diseases possessed anti-prothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either isolated or more often associated with anti-beta 2 glycoprotein I (beta 2GPI) antibodies. These included 67-71% of the patients with systemic lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19-20% of those with infection or malignancy (p < 0.001). As previously shown for anti-beta 2GPI antibodies, IgG2 was the predominant IgG subclass reactive with prothrombin. Thus, autoimmune patients with LA have a high incidence of antibodies to beta 2GPI and prothrombin, the binding of which could similarly require high antigen density and/or exposure of cryptic epitopes resulting from protein interaction with an irradiated (i.e. more anionic) polystyrene surface.

Contribution of a new, rapid, individual and quantitative automated D-dimer ELISA to exclude pulmonary embolism.

The performance of a new automated ELISA for a rapid, individual and quantitative measurement of plasma D-dimer (VIDAS D-dimer) has been evaluated. First, a study of 100 patients was performed in order to choose the best couple of antibodies in comparison with an already clinically validated ELISA. Then the results were certified in a prospective study including 195 consecutive patients suspected of pulmonary embolism (PE). For a cut-off level of 500 ng/ml VIDAS D-dimer showed a sensitivity of 100% (95% confidence interval 92-100), a specificity of 37.6%, a negative predictive value of 100% (95% CI 93.3-100) and a positive predictive value of 33.1%. During a 6 months' follow-up no patient (95% CI 0-6.4) with D-dimer < 500 ng/ml presented a new suspicion of venous thromboembolic disease. These results suggest that this rapid and single-dose ELISA provides a very useful tool for the clinician to exclude on a day-to-day basis the diagnosis of PE.

Superficial vein thrombosis of lower limbs: influence of factor V Leiden, factor II G20210A and overweight.

Superficial vein thrombosis (SVT) has been reported in patients with thrombophilia. In the present unmatched case-control study, the two most common thrombophilic abnormalities (factor V Leiden and factor II G20210A) were searched for in 112 consecutive patients with SVT of lower limbs and in 180 healthy donors. FV Leiden was present in 16/112 (14.3%) SVT patients and 11/180 (6.1%) controls (odds ratio 2.51, 95% CI 1.04-6.24) and FII G20210A in 4/112 (3.6%) patients and 2/180 (1.1%) controls (OR 3.28, 95% CI 0.46-36.84). In addition, body mass index (BMI) > or =28 kg/m2 was also associated with SVT (OR 2.81, 95% CI 1.60-5.00). After adjustment for BMI > or =28 kg/m2, the association between FV Leiden and SVT remained strong though no longer statistically significant. Among patients with SVT, the presence of FV Leiden was independently associated with the absence of varicose veins (OR 4.62, 95% CI 1.25-18.0) and with a BMI > or =28 kg/m2 (OR 3.74, 95% CI 1.05-15.1). In conclusion, both FV Leiden and overweight seem to predispose to SVT, a finding that should be confirmed in larger studies.

Lack of association between antiphospholipid antibodies and migraine.

We prospectively studied the prevalence of lupus anticoagulant, anticardiolipin antibodies (aCL) and various haemostatic parameters in 71 patients with migraine and compared the results with a control group of 32 subjects with back pain never having experienced migraine. The patients with migraine were divided into two groups: group I with migraine without (n = 18) and with aura lasting less than 60 min (n = 24) and group II with migraine with prolonged aura or migrainous infarction (complicated migraine, n = 29). The following results were obtained: a) no difference in aCL positivity was noted between migrainous patients and controls and between common migraine and complicated migraine patients and b) no statistically significant difference in haemostatic parameters (except for thrombin-antithrombin III complexes) was found between the two groups of migraine and between aCL positive and negative migrainous patients. These data suggest that anticardiolipin antibodies are not involved in the pathogenesis of migraine complications.