Elio Tonutti

Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Diseases

The Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases has generated a series of guidelines for the laboratory diagnosis and monitoring of systemic autoimmune rheumatic diseases intendedfor the use of clinical pathologists and laboratory physicians. These guidelines are based on a systematic review of published works and expert panel discussion and consist of 13 recommendations for antinuclear antibodies, anti-double-stranded native DNA, and antinuclear specific antibodies. To improve analytic performances and help select the most appropriate test for specific autoantibodies, as well as provide education and guidance in the use of these tests, special emphasis is placed on laboratory methods.

Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems.

BACKGROUND Indirect immunofluorescence (IIF) plays an important role in immunological assays for detecting and measuring autoantibodies. However, the method is burdened by some unfavorable features: the need for expert morphologists, the subjectivity of interpretation, and a low degree of standardization and automation. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of anti-nuclear antibodies (ANA), the biomedical industry has developed technological solutions which might significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. METHODS We collected 104 ANA-positive sera from patients with a confirmed clinical diagnosis of autoimmune disease and 40 ANA-negative sera from healthy blood donors. One aliquot of each serum, without information about pattern and titer, was sent to six laboratories of our group, where the sera were tested with the IIF manual method provided by each of the six manufacturers of automatic systems. Assignment of result (pos/neg), of pattern and titer was made by consensus at a meeting attended by all members of the research team. Result was assigned if consensus for pos/neg was reached by at least four of six certifiers, while for the pattern and for the titer, the value observed with higher frequency (mode) was adopted. Seventeen ANA-positive sera and six ANA-negative sera were excluded. Therefore, the study with the following automatic instrumentation was conducted on 92 ANA-positive sera and on 34 ANA-negative sera: Aklides, EUROPattern, G-Sight (I-Sight-IFA), Helios, Image Navigator, and Nova View. Analytical imprecision was measured in five aliquots of the same serum, randomly added to the sample series. RESULTS Overall sensitivity of the six automated systems was 96.7% and overall specificity was 89.2%. Most false negatives were recorded for cytoplasmic patterns, whereas among nuclear patterns those with a low level of fluorescence (i.e., multiple nuclear dots, midbody, nuclear rim) were sometimes missed. The intensity values of the light signal of various instruments showed a good correlation with the titer obtained by manual reading (Spearmans rho between 0.672 and 0.839; P<0.0001 for all the systems). Imprecision ranged from 1.99% to 25.2% and, for all the systems, it was lower than that obtained by the manual IIF test (39.1%). The accuracy of pattern recognition, which is for now restricted to the most typical patterns (homogeneous, speckled, nucleolar, centromere, multiple nuclear dots and cytoplasmic) was limited, ranging from 52% to 79%. CONCLUSIONS This study, which is the first to compare the diagnostic accuracy of six systems for automated ANA-IIF reading on the same series of sera, showed that all systems are able to perform very well the task for which they were created. Indeed, cumulative automatic discrimination between positive and negative samples had 95% accuracy. All the manufacturers are actively continuing the development of new and more sophisticated software for a better definition in automatic recognition of patterns and light signal conversion in end-point titer. In the future, this may avert the need for serum dilution for titration, which will be a great advantage in economic terms and time-saving.

Diagnosis and classification of celiac disease and gluten sensitivity

Celiac disease is a complex disorder, the development of which is controlled by a combination of genetic (HLA alleles) and environmental (gluten ingestion) factors. New diagnostic guidelines developed by ESPGHAN emphasize the crucial role of serological tests in the diagnostic process of symptomatic subjects, and of the detection of HLA DQ2/DQ8 alleles in defining a diagnosis in asymptomatic subjects belonging to at-risk groups. The serological diagnosis of CD is based on the detection of class IgA anti-tissue transglutaminase (anti-tTG) and anti-endomysial antibodies. In patients with IgA deficiency, anti-tTG or anti-deamidated gliadin peptide antibody assays of the IgG class are used. When anti-tTG antibody levels are very high, antibody specificity is absolute and CD can be diagnosed without performing a duodenum biopsy. Non-celiac gluten sensitivity is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out. Diagnostic criteria include the presence of symptoms similar to those of celiac or allergic patients; negative allergological tests and absence of anti-tTG and EMA antibodies; normal duodenal histology; evidence of disappearance of the symptoms with a gluten-free diet; relapse of the symptoms when gluten is reintroduced.

IgG Antibodies against Deamidated Gliadin Peptides for Diagnosis of Celiac Disease in Patients with IgA Deficiency

BACKGROUND Assays for IgG antibodies against deamidated gliadin (IgG-anti-dGli) are comparable in performance with tests detecting IgA antibodies against tissue transglutaminase (IgA-anti-tTG) in diagnosing celiac disease (CD). IgA-anti-tTG are absent in IgA deficiency, a condition often associated with CD. In IgA deficiency, IgG-anti-tTG, which have a lower overall diagnostic accuracy, are routinely measured. We examined whether IgG-anti-dGli would be useful for diagnosing CD in patients with IgA deficiency. METHODS We studied 34 IgA-deficient CD patients, 185 IgA-competent newly diagnosed children with CD, 316 children without CD, 400 adult blood donors, and 6 control IgA-deficient individuals without CD. Anti-dGli and anti-tTG were measured by ELISA, and endomysium antibodies (EmA) were measured by immunofluorescence on monkey esophagus (IgA as well as IgG class for all antibodies). We calculated diagnostic sensitivity (percentage of patients above cutoff with 95% CIs) according to age-specific cutoffs for 95% diagnostic specificity and according to cutoffs proposed by the manufacturer of the assays. RESULTS No IgA-deficient CD patients were positive for any IgA-based antibody assay. Diagnostic sensitivity of IgG-anti-tTG was 91.2% (95% CI 76.3%-97.7%) according to age-specific cutoffs and 82.4% (66.1%-92.0%) according to manufacturer cutoffs. The diagnostic sensitivity of IgG-EmA was 75.8% (58.8%-87.4%) and the sensitivity of IgG-anti-dGli was 88.2% (72.8%-95.9%) according to both cutoffs. CONCLUSIONS IgG-anti-dGli and IgG-anti-tTG have comparable diagnostic sensitivities for IgA-deficient celiac patients. IgG-anti-dGli may be useful for diagnosing CD in IgA-deficient patients.

Cutting-Edge Issues in Celiac Disease and in Gluten Intolerance

Celiac disease (CD) is a gluten-dependent immune-mediated disease with a prevalence in the general population estimated between 0.3% and 1.2%. Large-scale epidemiological studies have shown that only 10–20% of cases of CD are identified on the basis of clinical findings and that laboratory tests are crucial to identify subjects with subtle or atypical symptoms. The correct choice and clinical use of these diagnostic tools may enable accurate diagnosis and early recognition of silent CD cases. In this review, we have considered some relevant aspects related to the laboratory diagnosis of CD and, more extensively, of gluten intolerance, such as the best combination of tests for early and accurate diagnosis, the diagnostic role of new tests for detecting antibodies against neoepitopes produced by the transglutaminase–gliadin complex, the forms of non-celiac gluten intolerance (gluten sensitivity), and the use and significance of measuring cytokines in CD.

Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: a 15-year longitudinal study.

Summary.  Objective: To evaluate the role of anti‐prothrombin (anti‐PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE). Methods: An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 ± 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15‐year follow‐up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti‐PT, anti‐cardiolipin (aCL) and anti‐β2glycoprotein I (β2GPI) antibodies were measured by enzyme‐linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell’s viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti‐PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan–Meier method. Results: In the 15‐year follow‐up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti‐PT, anti‐β2GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis‐free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti‐PT (P = 0.001), anti‐β2GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence. Conclusions: This longitudinal study shows that IgG anti‐PT antibodies are predictors of thrombosis in SLE patients.

Prevalence and Clinical Correlation of Anti-Phospholipid-Binding Protein Antibodies in Anticardiolipin-Negative Patients With Systemic Lupus Erythematosus and Women With Unexplained Recurrent Miscarriages

CONTEXT Anti-phospholipid antibodies (aPL) are a heterogeneous group of autoantibodies, the presence of which is associated with thrombotic events and miscarriage. OBJECTIVE To establish whether antibodies directed against phospholipid-binding plasma proteins such as beta(2)-glycoprotein I (beta(2)GPI), prothrombin (PT), and annexin V (Anx V) constitute a risk factor for thromboembolism in patients with systemic lupus erythematosus (SLE) and for miscarriage in women with recurrent pregnancy loss (RPL), independently of the presence of the classic anticardiolipin (aCL) antibodies, and whether their determination together with that of aCL would help to increase the diagnostic sensitivity of aPL tests. DESIGN The prevalence of various antibodies directed toward phospholipids (CL and other anionic phospholipids [APL]) and phospholipid-binding proteins (beta(2)GPI, PT, and Anx V) was determined by immunoenzymatic methods in 311 serum samples. PATIENTS Twenty-five patients with aCL-positive primary anti-phospholipid syndrome (pAPS); 89 patients with SLE, 23 of whom had thrombotic complications (SLE/APS) and 66 of whom had no thrombosis; and 77 women with unexplained recurrent pregnancy loss comprised our study group. One hundred twenty healthy subjects matched for age and sex were studied as the control group. RESULTS Immunoglobulin (Ig) G and/or IgM aAPL, anti-beta(2)GPI, anti-PT, and IgG anti-Anx V antibodies were detected in 25 (100%), 20 (80%), 15 (60%), and 6 (24%), respectively, of the 25 aCL-positive pAPS patients; IgG and/or IgM aCL, aAPL, anti-beta(2)GPI, anti-PT, and IgG anti-Anx V antibodies were detected in 33 (37%), 42 (47%), 31 (35%), 40 (45%), and 12 (13%) of the 89 SLE patients, respectively. Of the 56 SLE patients who proved to be aCL negative, anti-beta(2)GPI was present in 3 patients (5%), anti-PT in 13 (23%) patients, and anti-Anx V in 5 (9%) patients. In the subset of 23 SLE/APS patients, IgG anti-PT prevalence was higher than that of the other autoantibodies (87% vs 70% aCL, 66% aAPL, 57% anti-beta(2)GPI, and 4% anti-Anx V), and in 26% of cases, IgG anti-PT was the only antibody present. Anti-PT had a slightly lower specificity than aCL (46% vs 49%); however, the occurrence of both antibodies brought the specificity to 92.4%. The highest risk for thrombosis in SLE patients was associated with the presence of IgG anti-PT antibody (odds ratio [OR] 15.3, P < .001, vs 6.5 aCL, 3.5 aAPL, 3.4 anti-beta(2)GPI, 0.2 anti-Anx V). Fifty-one of the 77 women with recurrent pregnancy loss were negative for all antibodies investigated; the prevalence of IgG and/or IgM aCL, aAPL, anti-beta(2)GPI, anti-PT, and IgG anti-Anx V antibodies was 6% (5), 12% (9), 6% (5), 16% (12), and 17% (13), respectively. Of the 67 aCL-negative women, none had anti-beta(2)GPI antibodies, 7 (11%) were anti-PT positive, and 13 (19%) were anti-Anx V positive. In the subgroup of 26 recurrent pregnancy loss patients who had at least one antibody, anti-Anx V was present in 50% of cases (in 42% as the sole antibody) and was the only antibody significantly associated with miscarriage (P = .02). CONCLUSIONS The results of this study indicate that it is useful to measure anti-PT antibodies in addition to the more widely used aCL and anti-beta(2)GPI antibodies in the prognostic evaluation of SLE patients for the risk of thrombosis, and the results also confirm that anti-Anx V antibodies may play an important role in recurrent pregnancy loss.

Infectious serologies and autoantibodies in inflammatory bowel disease: insinuations at a true pathogenic role.

The aim of this study was to reevaluate the role of infection in inflammatory bowel disease (IBD). Sera from 119 patients with IBD [80 with Crohns disease (CD); 39 with ulcerative colitis] and 98 healthy controls were assessed using the Bio‐Rad BioPlex 2200 for the presence of Toxoplasma gondii, cytomegalovirus, Epstein–Barr virus, Treponema pallidum, and Saccharomyces cerevisiae. Hepatitis B virus, hepatitis C virus (HCV), and anti‐Helicobacter pylori antibodies were assessed by ELISA. In addition, sera were tested for a panel of antibodies associated with thrombophilia as well as various autoantibodies. Titers of antibodies toward HCV and T. gondii, and S. cerevisiae were higher in IBD patients than in controls, while the H. pylori autoantibodies were less prevalent among the patient population. Several thrombophilia‐associated antibodies were more common in CD patients, and a single patient had a thromboembolic event. Our results show an excess of anti‐HCV and anti‐T. gondii antibodies among patients with IBD compared to healthy controls. Whereas the former may be the result of immunosuppression from the inflammatory disease itself or from the medications used to treat it, the latter association suggests that T. gondii is involved in the etiopathogenesis of IBD, and especially CD, in humans, as has been shown in the murine model. However, our findings also reiterate the positive association between CD and anti‐S. cerevisiae antibodies as well as the negative association with H. pylori infections. These, in turn, lend indirect support to the “hygiene hypothesis” in IBD as well as the newly proposed role of commensal bacteria in the initiation of the disease process.

Diagnostic accuracy of ELISA methods as an alternative screening test to indirect immunofluorescence for the detection of antinuclear antibodies. Evaluation of five commercial kits.

Detection of antinuclear antibodies (ANA) is a fundamental laboratory test for diagnosing systemic autoimmune diseases. Currently, the method of choice is indirect immunofluorescence (IIF) on a HEp-2 cell substrate. The goal of this study was to evaluate the diagnostic accuracy of five commercially available enzyme immunoassay (EIA) kits for ANA detection and to verify the possibility of using them as an alternative to the IIF method. The study involved 1513 patients, 315 of whom were diagnosed with a systemic autoimmune disease and 1198 in whom an autoimmune disorder was excluded. For all sera, ANA detection was performed via IIF and with five different EIA kits. The results were evaluated in relation to clinical diagnosis and the presence of possible specific autoantibodies (anti-ENA or anti-dsDNA); lastly, they were compared with the results obtained using ANA-IIF as the method of reference. The positive rate of the ANA-IIF test in subjects with systemic autoimmune diseases was 92%, whereas in the five ANA-EIA kits there was broad diversity in terms of response, with positive rates ranging from 74 to 94%. All the EIA kits correctly detected the presence of antibodies (anti-dsDNA, anti-RNP, anti-Ro/SSA) responsible for homogeneous and speckled fluorescence pattern, but at the same time they showed substantial inaccuracy with the nucleolar pattern, with a mean sensitivity of approximately 50% in this case. Instead, there was a large kit-to-kit difference in terms of identification of anti-Scl70 and centromere patterns, for which sensitivities ranged between 45 and 91%, and between 49 and 100%, respectively. The results of the study demonstrate that the commercially available ANA-EIA kits show different levels of sensitivity and specificity. Some of them have a diagnostic accuracy that is comparable and, in some cases, even higher than the IIF method. Consequently, these could be used as an alternative screening test to IIF. However, others do not ensure acceptable results. Therefore, careful evaluation of the various kits on the market is advisable before including any of these methods in the clinical and diagnostic testing.

Overcoming a "Probable" diagnosis in antimitochondrial antibody negative primary biliary cirrhosis: Study of 100 sera and review of the literature

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.