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OBJECTIVE Immunoblot (IB) methods are widely used to detect myositis-specific autoantibodies (MSAs); however, false-positive results are common. In this study, we aimed to determine whether associating the anti-nuclear antibody (ANA) IIF pattern may help to improve the specificity of MSA detection by IB in patients with idiopathic inflammatory myositis (IIM). METHODS Serum samples from 104 patients presenting with muscle weakness/myalgia and positive to at least one MSA by IB (MYOS12 Diver and MIOS7 Diver, D-tek) were tested for ANAs on HEp-2000 cells (Immuno Concepts). The chi-square test was used to analyse the concordance of the MSA result and its corresponding pattern by ANA testing between patients with and without IIM. RESULTS Eighty-three of the 104 patients had a diagnosis of definite IIM, while in 21 cases, patients were affected by other autoimmune diseases or various non-systemic diseases. Forty nine of 83 (59%) patients in the IIM group and 4/21 (19%) in the non-IIM group showed a concordance between ANA pattern and MSAs by IB (P < 0.001). MSA monopositivity was significantly associated with IIM (91.6%) compared with 61.9% in the non-IIM group (P = 0.0005). CONCLUSIONS Considering both the MSA result and its corresponding pattern by ANA testing may help to improve the specificity of MSA detection by IB and to confirm the diagnosis of MSA-associated IIM. The monopositivity of MSAs is an important additional tool to validate IB results.

Serum anti-mitochondrial antibodies (AMA) are the serological hallmark of primary biliary cirrhosis (PBC), yet up to 15% of PBC sera are AMA negative at routine indirect immunofluorescence (IIF) while being referred to as “probable” cases. The diagnostic role of PBC-specific antinuclear antibodies (ANA) remains to be determined. We will report herein data on the accuracy of new laboratory tools for AMA and PBC-specific ANA in a large series of PBC sera that were AMA-negative at IIF. We will also provide a discussion of the history and current status of AMA detection methods. We included IIF AMA-negative PBC sera (n = 100) and sera from patients with other chronic liver diseases (n = 104) that had been independently tested for IIF AMA and ANA; sera were blindly tested with an ELISA PBC screening test including two ANA (gp210, sp100) and a triple (pMIT3) AMA recombinant antigens. Among IIF AMA-negative sera, 43/100 (43%) manifested reactivity using the PBC screening test. The same test was positive for 6/104 (5.8%) control sera. IIF AMA-negative/PBC screen-positive sera reacted against pMIT3 (11/43), gp210 (8/43), Sp100 (17/43), both pMIT3 and gp210 (1/43), or both pMIT3 and Sp100 (6/43). Concordance rates between the ANA pattern on HEp-2 cells and specific Sp100 and gp210 ELISA results in AMA-negative subjects were 92% for nuclear dots and Sp100 and 99% for nuclear rim and gp210. Our data confirm the hypothesis that a substantial part of IIF AMA-negative (formerly coined “probable”) PBC cases manifest disease-specific autoantibodies when tested using newly available tools and thus overcome the previously suggested diagnostic classification. As suggested by the recent literature, we are convinced that the proportion of AMA-negative PBC cases will be significantly minimized by the use of new laboratory methods and recombinant antigens.

Clinical studies have estimated a 10- to 20-fold increased risk for celiac disease (CD) in patients with selective IgA deficiency (SIgAD). For this reason, screening for CD is mandatory in SIgAD patients, but it represents a special challenge since the specific IgA class antibodies against gliadin (AGA), endomysium (EMA), and tissue-transglutaminase (tTG) are not produced in patients with CD. IgG class counterparts of these antibodies may be informative; in particular IgG EMA has been demonstrated to be a valid marker for diagnosing CD in SIgAD cases, but it is not used much in clinical laboratories, because it is cumbersome and involves some technical difficulties. Even if it was widely used in clinical laboratories, the measuring of IgG AGA has shown a less-than-optimum diagnostic accuracy, so that now it tends to be substituted by tests for anti-tTG IgG, for which the few available studies have shown diagnostic performances superior to AGA. Since it is not known whether various available methods for measuring IgG anti-tTG antibodies offer similar diagnostic performances, we have compared the results obtained from nine second-generation commercial methods (D-tek, Phadia, Immco, Orgentec, Radim, Euroimmun, Inova, Aesku, Generic Assays), measuring IgG anti-tTG antibodies in 20 patients with CD and SIgAD and in 113 controls (9 patients with SIgAD without CD, 54 patients with chronic liver disease, and 50 healthy individuals). Diagnostic sensitivity, calculated by means of ROC plot analysis, ranged between 75% and 95%, and specificity ranged from 94% to 100%. In the same population, the diagnostic sensitivity and specificity of AGA IgG were 40% and 87%, respectively. Even though they perform differently, all IgG anti-tTG methods evaluated are reliable serological assays for the diagnosis of CD in SIgAD patients, with diagnostic accuracy superior to the AGA IgG method. The methods that use a mix of tTG and gliadin peptides as the antigenic preparation have a specificity slightly lower than that of the methods that use only tTG.

The aim of this work was to evaluate the diagnostic accuracy of three different analytical methods for the detection of antineuronal antibodies and outline how they might be used to diagnose Paraneoplastic Neurological Syndromes (PNS) in a more effectively and rationally way. One hundred and four patients with neurological diseases were studied: 38 with paraneoplastic neurological disorder, 44 with other neurological diseases, and 22 with systemic autoimmune diseases and neurological disorders. 20 healthy subjects and 18 subjects with tumour without neurological disorders were also studied. Antineuronal antibodies were tested using three methods: Western blot (WB); Line-blot (LB); and indirect immunofluorescence (IIF) on primate cerebellum. The diagnostic sensitivity of the IIF, WB and LB methods was 28.9%, 26.3% and 36.8%, respectively, and their specificity was 95.2%, 97.1% and 98.1% respectively. The combined use of the three methods brought the sensitivity to 39.4%. The results of this study show that the methods used in clinical laboratories for the detection of antineuronal antibodies have good specificity. Among the three methods assessed, LB showed the highest diagnostic accuracy and also allowed for recognition of fine antibody specificities. According to these results we can suggest that LB should be used as the method of choice to search for paraneoplastic antibodies.

In this study we evaluated the diagnostic accuracy for antiphospholipid syndrome (APS) of new fully automated immunoassays for anticardiolipin (aCL) and anti-beta2 glycoprotein I (anti-beta2-GPI) auto-antibody detection (EliA-Phadia), and compared the results with those obtained with Orgentec and Inova ELISA methods. Sixty-two APS patients and 123 controls (20 syphilis, 33 Lyme disease, 30 HCV infection and cryoglobulinemia, 40 healthy subjects) were studied. Using the 99(th) percentile cutoff, the sensitivity and specificity of EliA aCL IgG, aCL IgM, anti-beta2-GPI IgG, and anti-beta2-GPI IgM were 69.4% and 81.9%, 64.5% and 86.7%, 64.5% and 98.8%, and 53.2% and 92.8%, respectively. Using the Sydney criteria cutoff (>40 GPL/MPL units), sensitivity and specificity of EliA aCL IgG and aCL IgM were 45.2% and 98.8%, and 35.5% and 97.5%, respectively. The best diagnostic efficiency was obtained combining the aCL tests (>40 GPL/MPL units) with the anti-beta2-GPI tests (sensitivity 85.7%, specificity 90.4%). The area under the ROC curves for EliA, Orgentec, and Inova methods were 0.870, 0.940, and 0.850 for aCL IgG; 0.820, 0.820, and 0.820 for aCL IgM; 0.910, 0.960, and 0.920 for anti-beta2-GPI IgG; 0.840, 0.840, and 0.820 for anti-beta2-GPI IgM, respectively. Finally, the overall agreement between EliA assays and the other two ELISA methods ranged from moderate (anti-beta2-GPI IgG EliA versus Orgentec: Cohen's k = 0.426) to good (anti-beta2-GPI IgM EliA vs. Inova: k = 0.841). In conclusion, newly developed EliA methods for antiphospholipid antibody detection perform similarly to other ELISA assays and represent a useful tool for APS laboratory diagnosis in daily practice.

Previous studies have demonstrated that serum anti-actin antibodies are a reliable marker of intestinal damage severity in coeliac disease.To validate in a multicentre study the clinical usefulness of serum IgA anti-actin antibody ELISA and its possible use in monitoring intestinal mucosa lesions during gluten-free diet.Four centres recruited 205 newly diagnosed coeliac disease patients with villous atrophy, 80 healthy controls and 81 "disease" controls. Twelve coeliac disease patients on gluten-free diet but with persistent symptoms underwent serum IgA anti-actin antibody assay and intestinal histology evaluation. IgA anti-actin antibody ELISA was performed with a commercial kit. All coeliac disease patients underwent intestinal histology study.IgA anti-actin antibodies showed a sensitivity of 80% and a specificity of 85% in the diagnosis of coeliac disease patients with villous atrophy. The area under the receiving operator curve for anti-actin antibodies was 0.873 [95% C.I. 0.805-0.899]. Serum anti-actin antibodies values were significantly higher in coeliac disease patients than in healthy or "disease" controls (P0.0001). Serum anti-actin antibodies were positive in 41 of the 60 coeliac disease patients with mild intestinal histology lesions (69%) and in 123 of the 145 with severe lesions (85.3%) (P0.05). There was a significant inverse correlation between anti-actin antibody values and the villi/crypts ratio (r=-0.423; P0.0001). In the 12 coeliac disease patients on gluten-free diet who underwent re-evaluation as they were persistently symptomatic, intestinal histology showed three cases with persistent villous atrophy: all of these were positive for serum anti-actin antibodies ELISA, whereas both serum anti-tTG and EmAs were negative. The other nine patients showed normal intestinal villi and were negative for serum anti-actin antibodies.Anti-actin antibodies are a reliable marker of severe intestinal mucosa damage in coeliac disease patients and a simple ELISA technique offers an accurate method for their determination. These antibodies seem to be a very reliable marker of persistent intestinal damage in coeliac disease patients.

In a previous study we characterized cancer procoagulant (CP), a 68 kd cysteine proteinase which directly activates coagulation factor X in various subtypes (from M1 to M5) of acute non-lymphoblastic leukemia (ANLL). The aim of this study was to determine whether CP is also expressed by acute lymphoblastic leukemia (ALL) cells. Blasts from 25 ALL patients were extracted and tested for their procoagulant properties. 16 samples (64%) shortened the recalcification time of normal human plasma, and 9 (36%) did not. 8 of the 16 active samples showed properties compatible with CP, i.e. independence from factor VII in triggering blood coagulation and sensitivity to cysteine proteinase inhibitors. Selected samples also cross-reacted with a polyclonal antibody raised against purified CP. The specific activity of CP in ALL extracts was significantly lower than in most ANLL types previously studied (all but M4). These finding indicate that CP can be a property of the lymphoid phenotype although its expression may be lower than in the myeloid phenotype.