The contribution of antiphosphatidylethanolamine antibodies in the diagnosis of the antiphospholipid syndrome.

The diagnosis of seronegative antiphospholipid syndrome (APS) has been proposed for patients with well-defined clinical APS but persistently negative for the routinely tested antiphospholipid antibodies (aPLs): antibodies to cardiolipin (aCL) and to ß(2) glycoprotein I (aß(2)GPI) and lupus anticoagulant (LA). Antibodies directed to phosphatidylethanolamine (aPE) have been described as the sole aPLs in some patients with clinical manifestations of APS. Here, we briefly summarize the available data on the clinical associations of aPEs and propose their investigation in patients with a clinical profile highly suggestive of seronegative APS.

'Non-criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010.

Abstract: Current classification criteria for definite APS recommend the use of one or more of three positive standardized laboratory assays, including anticardiolipin antibodies (aCL), lupus anticoagulant (LA), and antibodies directed to ß(2)glycoprotein I (anti-ß(2)GPI) to detect antiphospholipid antibodies (aPL) in the presence of at least one of the two major clinical manifestations (i.e., thrombosis or pregnancy morbidity) of the syndrome. Several other autoantibodies shown to be directed to phospholipids and/or their complexes with phospholipids and/or to proteins of the coagulation cascade, as well as a mechanistic test for resistance to annexin A5 anticoagulant activity, have been proposed to be relevant to APS. A task force of worldwide scientists in the field discussed and analyzed critical questions related to 'non-criteria' aPL tests in an evidence-based manner during the 13th International Congress on Antiphospholipid Antibodies (APLA 2010, 13-16 April 2010, Galveston, Texas, USA). This report summarizes the findings, conclusions, and recommendations of this task force.

In vitro generation of endothelial microparticles and possible prothrombotic activity in patients with lupus anticoagulant.

Microparticles (MPs) resulting from vesiculation of platelets and other blood cells have been extensively documented in vitro and have been found in increased numbers in several vascular diseases, but little is known about MPs of endothelial origin. The aim of this study was to analyze morphological, immunological, and functional characteristics of MPs derived from human umbilical vein endothelial cells (HUVECs) stimulated by TNF, and to investigate whether these MPs are detectable in healthy individuals and in patients with a prothrombotic coagulation abnormality. Electron microscopy evidenced bleb formation on the membrane of TNF-stimulated HUVECs, leading to increased numbers of MPs released in the supernatant. These endothelial microparticles (EMPs) expressed the same antigenic determinants as the corresponding cell surface, both in resting and activated conditions. MPs derived from TNF-stimulated cells induced coagulation in vitro, via a tissue factor/factor VII-dependent pathway. The expression of E-selectin, ICAM-1, alphavbeta3, and PECAM-1 suggests that MPs have an adhesion potential in addition to their procoagulant activity. In patients, labeling with alphavbeta3 was selected to discriminate EMPs from those of other origins. We provide evidence that endothelial-derived MPs are detectable in normal human blood and are increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant. Thus, MPs can be induced by TNF in vitro, and may participate in vivo in the dissemination of proadhesive and procoagulant activities in thrombotic disorders.

Antigenic profile, prevalence, and clinical significance of antiphospholipid antibodies in women referred for in vitro fertilization.

The aim of this prospective study was to assess the prevalence of antiphospholipid antibodies (aPL) in women who had undergone in vitro fertilization (IVF) and the relationship between aPL and IVF outcome. A total of 101 infertile women with at least three unsuccessful IVF attempts were consecutively included in this study. Samples were collected in the follicular phase of a spontaneous ovarian cycle 2 months after the last ovulation induction treatment. Age-matched healthy fertile women (n = 160) were included as controls. All were evaluated for the presence of lupus anticoagulant (LA), antibodies (IgG, IgA, IgM) to cardiolipin (aCL), beta2-glycoprotein I (abeta2GPI), and phosphatidylethanolamine (aPE). Out of the 101 infertile women, 40 were persistently positive for aPL, showing a prevalence significantly higher than in controls (39.6% versus 5%, P < 0.0001). Among aPL, aPE were found with a significantly higher prevalence compared with LA, aCL, and aP2GPI (67.5% versus 0%, 15%, and 40%, respectively). Interestingly, aPE were found in 70% of the cases in the absence of the other aPL. The predominant isotype of aPL was IgA, in particular for abeta2GPI. Finally, no significant association was found between the presence of aPL and IVF outcome. This prospective study shows aPE as the most prevalent aPL in infertile women and IgA as more common than IgG and IgM. However, our results do not support an association between aPL and IVF outcome.

Can cryoglobulins interfere with the measurement of IgM antiphosphatidylethanolamine antibodies by ELISA?

Clinical manifestations of the antiphospholipid antibody syndrome (APS) have been recently related to the presence of phosphatidylethanolamine antibodies (aPE). However, it is well known that some molecules such as cryoglobulins, immunoglobulins that undergo a reversible precipitation at low temperatures, may interfere with biological assays. With this in view, we report the case of a patient with APS who was positive for both IgM aPE and type III cryoglobulinemia. Moreover, we show for this patient a potential implication of aPE in the cryoprecipitate formation. To further analyze the potential association between cryoglobulins and aPE, and also the possible consequences for aPE assay, we selected 55 patients according to positivity for both IgM aPE and cryoglobulinemia. Determination of IgM aPE levels was made before and after removal of cryoprecipitate from the serum. Of the 55 selected patients, 52 (95%) presented no significant difference for IgM aPE levels before and after cryoprecipitation. These results were ascertained whatever the aPE levels and clinical status of the patient. Taken together, our results indicate that cryoprecipitation does not interfere in most cases (95%) with the dosage of IgM aPE. Thus, IgM aPE do not appear to be involved in the formation of the cryoprecipitate.

Diagnostic value of anti-F-actin antibodies in a French multicenter study.

According to international criteria, autoimmune hepatitis (AIH) type 1 is characterized by the presence of antinuclear or anti-smooth muscle antibodies (SMA) with F-actin specificity. SMA have been found in 85% of AIH patients, but are not specific to this disease, and anti-F-actin specificity is not always verified when SMA are detected. The objective of this study was to determine the diagnostic value of anti-F-actin antibodies in a large population. A multicenter study involving 12 clinical centers was performed. Patients were selected on the basis of the presence of F-actin SMA detected by indirect immunofluorescence (IIF) on rat liver-kidney-stomach sections and was confirmed by IIF on Hep2 cells treated with colchicine, or F-actin dot-blot. The clinical status of patients was determined from their medical records. One hundred sixty-eight patients were included: 76% women, 24% men; mean age of 45 years (range, 2-88 years), with a bimodal age distribution. Sixty percent had AIH type 1, and 40% had another disease. In the group of women younger than 25 years, 90% had AIH type 1. Other pathologies associated with antiactin were other liver diseases (19%), including viral hepatitis C (7%), and non-liver diseases (21%), including connective tissue diseases (12%). Antibody titers were higher in AIH than in other diseases. Antiactin antibodies are of major diagnostic value in AIH, especially in young women; they may be found in other disease settings, but mostly at low levels.

Computerized edge tracking and lesion measurement in coronary angiograms. A pilot study comparing smokers with non-smokers.

Comparison of coronary atherosclerosis change in a pilot angiographic study of retrospectively matched smokers and controls indicates more rapid progression in smokers. The findings indicate the feasibility of small-scale angiographic trials of treatment designed to ameliorate arterial damage in atherosclerotic smokers who cannot quit.

Spatial reconstruction of human femoral atheromas showing regression.

Reports of atherosclerotic changes in human subjects previously described have been based on evaluation of arteriographic edge contours. They imply unchanged roentgenographic and physiologic conditions, including identical patient positioning and vascular tone which cannot always be obtained in sequential studies. We have previously described the development of quantitative angiographic densitometry which permits measurement of vascular cross-sectional chord length distributions and areas, independent of rotational changes in vascular position. In this paper we report on application of the method to sequential femoral angiograms in two patients in whom there were significant increases in vascular cross-sectional area after a program of exercise and risk reduction. The method excludes interpretive errors due to circumferential changes in vascular tone.

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.

Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations--a cooperative project of the European Antiphospholipid Forum.

Despite the widely recognized practical importance of anticardiolipin (aCL) ELISA, the reliability of this test has been recently discussed. In order to investigate this area on European scale, we sent to 30 experienced centers a questionnaire focusing on the diagnostic procedures applied to patients with antiphospholipid syndrome (APS) and on the detailed protocols used to perform aCL. Anticardiolipin ELISA was found to be the most frequently performed test in patients with suspected APS, but significant difference was shown among the various protocols. The cross-laboratory multiple examination of ten serum samples evaluated independently by the 24 centers pointed out the difficulty in getting comparable results. Therefore a "consensus" protocol was derived from the aCL methods giving the best performance. The materials and reagents necessary to perform the "consensus" method, including, as putative standards, one IgG and one IgM monoclonal antibody (HCAL and EY2C9) were distributed to 19 Centers. The results of one IgG and one IgM aCL high positive sera measured in serial dilutions were compared. A progressive decrease in the variability of the values obtained for a given sample appeared evident when all the laboratories used the same standard, in their own in-house ELISA and even more in the "consensus" ELISA. Our data show that aCL ELISA standardization is necessary in order to obtain comparable results in different laboratories.

Antibodies to phosphatidylethanolamine as the only antiphospholipid antibodies found in patients with unexplained thromboses.

The objective of this study was to assess the interest of antiphosphatidylethanolamine antibodies (aPE) in unexplained thrombosis (UT) defined as thrombotic episode without any of the main autoimmune and hereditary thrombophilic defects. Results from 98 UT were compared to those of (I) 142 patients with thrombophilia: 67 antiphospholipid syndrome (APS) and 75 hereditary hemostatic defects (HHD); (II) 110 patients without thrombosis: 60 with systemic lupus erythematosus (SLE) and 50 with infectious diseases (ID). As compared to controls (100 blood donors), aPE prevalence was significantly higher in both autoimmune contexts (APS: 43%; SLE: 40%, p<0.0001) and among non-autoimmune pathologies, only in UT (18%, p = 0.001) conversely to HHD (8%) or ID (10%). aPE prevalence in UT was not statistically different from that found in Primary APS (32%, p = 0.076) but lower than in Secondary APS (65%, p <0.005). In UT, aPE were mainly of IgM isotype like in Primary APS and they were found alone whereas in SLE they were always associated with classical antiphospholipid antibodies. No significant association was found between any isotype of aPE and a site of thrombosis in UT as well as in APS. In conclusion, this study demonstrates an increase of the prevalence of aPE in patients with unexplained thrombosis. Thus, aPE investigation appears to be of interest in UT and their persistent presence could define a biological variant of APS.

Antiphospholipid and antiprotein syndromes in non-thrombotic, non-autoimmune women with unexplained recurrent primary early foetal loss. The Nîmes Obstetricians and Haematologists Study--NOHA.

Various antiphospholipid and/or antiprotein antibodies have been suspected to be associated with recurrent early foetal loss in absence of any habitual aetiology. We conducted a hospital-based case control study on women with no antecedent of thromboembolic or autoimmune disease. We studied 3 groups of 518 women: patients with unexplained primary recurrent early foetal loss, patients with explained episodes and mothers with no previous obstetrical accident. Matching the 3 groups was carried out on the basis of age, number or pregnancies and time elapsed since the end of the last pregnancy. Significant biological markers were then prospectively tested. The various antibodies were shown to be dependent on parity and on the presence of previous foetal loss: cut-off values were thus calculated using data obtained from the group of explained accidents, and adjusted for parity. Only anti-phosphatidylethanolamine IgM [odds ratio: 6.0, 95% confidence interval (2.3-15.7), p = 0.0003], anti-beta2-glycoprotein I IgG [4.4, (1.6-11.7), p = 0.0035] anti-annexin V IgG antibodies [3.2 (1.2-8.1), p = 0.015] and lupus anticoagulant [3.0, (1.3-6.8), p = 0.009], were found to be independent retrospective risk factors for unexplained early foetal loss. These four markers were subsequently found to be, during the following pregnancy, associated with a significant risk of foetal loss despite a low-dose aspirin treatment. In non-thrombotic, non-auto-immune women with unexplained primary recurrent early foetal loss, subgroups of patients with positive anti-phosphatidylethanolamine IgM antibodies, or positive anti-beta2-glycoprotein-I IgG antibodies, or positive anti-annexin V IgG antibodies or lupus anticoagulant must be particularised. This should allow therapeutic trials to be carried in well-defined patients.

Studies of IgG-class anticardiolipin antibodies in myasthenia gravis.

IgG-class anticardiolipin antibodies (IgG-ACA) were found in 25% of patients with myasthenia gravis. The prevalence and the level distribution were significantly different from those of a normal donor population (p < 0.001). In myastenic patients, IgG-ACA bound negatively charged, but not zwitterionic, phospholipids. They were significantly associated with the thymic abnormalities, thymoma and thymic hyperplasia, but not with various factors such as age, sex, antinuclear antibodies, severity of the disease and clinical thrombosis. The IgG-ACA levels did not correlate with titers of anti-acetylcholine receptor antibodies. Thus in Myasthenia Gravis, asymptomatic IgG-ACA could reflect an immune dysregulation under the influence of thymic alterations.

Autoantibody formation after bone marrow transplantation: a comparison between autologous and allogeneic grafts.

Various autoantibodies were screened in 11 allogeneic, 14 autologous bone marrow transplanted patients and 11 healthy controls. Except anti-mitochondria antibodies, autoantibodies were found with similar frequencies after allogeneic and autologous transplantation. Moreover, 50% of autoantibodies were already present before transplantation. These results suggest that factors other than allogeneic reaction could be responsible for autoantibody formation after bone marrow transplantation, in particular, pre-graft conditioning treatment and/or viral and bacterial infection. Nevertheless, the number of patients with 2 or more autoantibodies was significantly higher after allogeneic than after autologous grafting (respectively, 82% and 36.7%; p less than 0.05). Forty percent of allografted but none of autografted patients were still positive at 12 months post graft. So, allogeneic reaction could play a role in intensity of humoral response and its maintenance for a long period.

Electrocardiographic and vectorcardiographic diagnosis of posterior wall myocardial infarction. Significance of the T wave.

The electrocardiographic diagnosis of posterior wall myocardial infarction remains elusive. To determine discriminating criteria a group of 27 patients with posterior infarction proven by biplane angiocardiography were compared to 97 controls. All patients had single-vessel obstruction of the circumflex artery or one of its major branches (greater than or equal to 75 percent area stenosis) without occlusive disease in the other coronary arteries. High-frequency, high-gain electrocardiograms and Cube and McFee vectorcardiograms were analyzed. Pathologic Q waves in the inferior leads were present in only 22 percent (six) of the patients; increased R-wave amplitude or duration in the right precordial leads was found in 17 to 26 percent, and an R/S ratio greater than or equal to I in lead V1 or greater than or equal to 1.5 in lead V2 was present in 22 percent (six) of patients. Vectorcardiographic criteria which improved the diagnostic yield were: (1) the presence of a QRS loop mostly anterior to the E point, and (2) the presence of an abnormally anterior T wave. This abnormal T-wave shift was present in over 70 percent of the patients with posterior infarctions and was clearly discernible from the 12-lead ECG, as manifested by tall T waves in lead V2 and flat T waves in lead V6. To approximate the T-wave angle in the 12-lead scalar ECG, an index was calculated by subtracting the amplitude of the T wave in lead V6 from its amplitude in lead V2 (T2-T6 index). An index of 0.38 mV or more yielded a sensitivity of 81 percent and a specificity of 75 percent; however, this was not as discriminating as the vectorcardiogram where a T angle of 60 degrees or more in the horizontal plane yielded a sensitivity of 70 percent and a specificity of 97 percent.

Gender differences in coronary angiographic findings from 1972 through 1981 in Los Angeles, California.

The angiographic findings of 930 men and 257 women who were referred to a Los Angeles County hospital from 1972 through 1981 are compared. Chi square test results indicated triple-vessel disease was more common in men (37.1%) compared with women (18.7%). The prevalence of normal angiographic findings was higher in women (23.3%) than in men (8.1%). However, for men and women with evidence of coronary artery disease, virtually identical percentages of subjects had left main, single-, double-, and triple-vessel disease. Although the incidence of coronary artery disease decreased during this time for the Los Angeles population as a whole, no differences in patterns of coronary vessel lesions were evidenced.

[Antiphospholipid antibodies: clinical significance and biological diagnosis]. / Les anticorps "antiphospholipides": intérêt clinique et diagnostic biologique.

The term "antiphospholipids" (aPLs) refers to an heterogeneous family of antibodies diagnosed either by clotting tests: the lupus anticoagulants or by Elisa: anticardiolipin (aCL) and anti-beta2-glycoprotein I (anti-beta2GP1) especially. aPLS recognize phospholipids, alone or bound to plasma protein cofactor(s), or the cofactors themselves. aPLs have long been described in autoimmune diseases such as SLE, but may also be found in other clinical settings including infections, malignancies and drug administration. Their persistent presence can be associated with venous and/or arterial thrombotic complications and/or recurrent miscarriage, thus defining the "antiphospholipid syndrome" (APS). The heterogeneity of aPLs makes a comprehensive approach to laboratory investigation essential. Detection of lupus anticoagulants relies on increased clotting times in phospholipid-dependent tests. Their 4 step diagnosis includes: 1) screening (by at least two different tests); 2) demonstration of an inhibitory activity; 3) evidence of its phospholipid dependence; 4) exclusion of an associated coagulopathy. Among the aPLs detected by Elisa, IgG aCL are the most frequently investigated. However, other antibodies may represent useful biological tools. Among them, anti-beta2GP1 are thought to be more closely associated with a history of thrombosis than aCL and testing for anti-beta2 GP1 should now be systematically included in the biological diagnosis of APS. The Elisa used for aCL and anti-beta2GP1 are not fully standardized, and a number of methodological parameters may account for the interlaboratory discrepancies often observed. The clinical importance of other antibodies such as antiphosphatidylethanolamine, antiprothrombin or antiannexin V is being evaluated. An appropriate laboratory investigation of APS should, in all cases, combine the use of clotting and immunological assays, and assess the persistence of autoantibodies over time.

[Auto-antibodies against acetylcholine receptors in myasthenia gravis]. / Auto-anticorps anti-récepteur de l'acétylcholine au cours de la myasthénie.

USEFULLNESS OF DETECTION OF ANTIBODIES AGAINST THE ACETYLCHOLINE RECEPTOR IN THE DIAGNOSIS OF MYASTHENIA GRAVIS (MG): MG is an autoimmune disorder including a fatigability of skeletal muscles and the presence of antibodies against the acetylcholine receptor (AChRAbs). These Abs are pathogenic by blocking the acetylcholine receptor within the neuromuscular junction. A transient neonatal MG occurs in 12% of babies born to myasthenic mothers. AChRAbs have been found in 77 to 89% of systemic MG and in 47 to 60% in ocular form of MG. These Abs are generally directed against the "Main Immunogenic Region" (MIR) within the a subunit of the receptor; others are directed against beta, gamma and epsilon subunits. Eleven to 23% of the MG are negative for the Abs directed against the a subunit reinforcing the interest to detect the other AChRAbs, specially the anti-epsilon subunit Abs and the Abs directed against the muscle-specific receptor tyrosine kinase. MULTICENTER EVALUATION: In order to compare the results of AChRAbs for a same patient from various laboratories, a multicenter study was performed on 26 sera with different titers of AChRAbs using radioimmunoassays. Six french laboratories have participated to this study. The antigen was obtained from TE671 cells for 5 laboratories (3 used a commercial test) or from human muscle (1 laboratory). At the end of the study, the qualitative analysis of the results showed a concordance of 92.3%. The interpretation of AChRAbs assays can thus be done by clinicians whatever the test is used. A same quality control is nowadays used by the six laboratories in order to estimate quantitative results according to the assay performed. USEFULNESS OF THE OTHER ABS: The Abs directed against the striated muscle are good markers of thymoma in MG but are not specific for MG. The Abs directed against titin which is the major target of the anti-striated muscle Abs, are also good biological markers of thymoma specially in MG adults younger than 60 years. The detection of anti-myoid cells and thymic reticuloepithelial cells Abs is no more useful due to a lower sensitivity compared to the Abs directed against the striated muscle.