Adult-onset Still's disease: analysis of a monocentric cohort of patients.

OBJECTIVE: Adult-onset Still's disease (AOSD) is a multigenic autoinflammatory disease with a severe systemic involvement. Because of the rarity of the disease, most published cohorts are multicentric. The aim of this report is to describe a monocentric cohort of AOSD patients, reporting clinical features and response to therapy in a long follow-up. METHOD: Thirty-eight patients, attending the Clinical Immunology Unit and fulfilling Yamaguchi, Fautrel, or Daghor-Abbaci classification criteria for AOSD, were recruited for this study. In all patients, clinical and serological data were collected at diagnosis and every 6 months thereafter. The Pouchot score was calculated at every visit. RESULTS: Fever, arthromyalgia, and skin rash were the most frequent manifestations, followed by lymphadenopathy, sore throat, arthritis, splenomegaly, hepatic involvement, pleuropericarditis, and weight loss. As far as the disease course is concerned, 25% presented a monocyclic and 35% a polycyclic pattern, and 40% developed chronic articular involvement. Severe complications were observed at disease onset in 21% of the patients. All of the patients were treated with steroids; 74% also received conventional synthetic disease-modifying anti-rheumatic drugs (methotrexate in most cases) and 71% biological disease-modifying anti-rheumatic drugs (interleukin-1 inhibitors in most cases). Therapeutic switching for lack/loss of efficacy or adverse drug reactions was necessary in 66%. CONCLUSION: The analysis of this cohort confirms that AOSD is a complex, severe, and heterogeneous disease. However, despite long-term treatment and comorbidities, therapies are effective and well tolerated. The therapeutic armamentarium now available allows long-lasting remission with low immunosuppression to be achieved in most patients.

Cytokines and soluble receptors of the interleukin-1 family in Schnitzler syndrome.

OBJECTIVES: Schnitzler syndrome (SchS) is an autoinflammatory disorder characterized by chronic urticaria, fever, and monoclonal gammopathy. The success of interleukin-1 (IL-1) blocking therapies suggests a crucial role for IL-1 in disease induction. The aim of this study is to perform a comprehensive analysis of IL-1 family cytokines and soluble receptors in a group of SchS patients. METHOD: Three patients fulfilling the criteria for the diagnosis of SchS were recruited; 80 blood donors formed the control group. IL-1 family cytokines (IL-1α, IL-1ß, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, sIL-1R4), and antagonists [IL-1Ra, IL-18 binding protein (IL-18BP)] were measured by a multiarray enzyme-linked immunosorbent assay. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Cytokine levels were compared by the Mann-Whitney test. RESULTS: IL-18 and free IL-18 were increased in patients compared with controls (p = 0.005 and p = 0.0082, respectively), while IL-18BP levels were not different. IL-1α, IL-1ß, and IL-33 were undetectable in both patients and controls. The soluble receptors sIL-1R1, sIL-1R2, and ST2/sIL-1R4, and the IL-1 antagonist IL-1Ra were all within normal ranges; sIL-1R3 was significantly lower in patients than in controls (p = 0.039). CONCLUSIONS: The data indicate that SchS is characterized by increased circulating levels of free IL-18, possibly leading to a higher activation of innate/inflammatory effector cells. At variance with other inflammatory diseases, the lack of increase in sIL-1R1 and sIL-1R2 and the decreased levels of sIL-R3 imply a failure in the counterbalancing mechanism aimed at inhibiting excessive IL-1ß in tissues.

Association between anti-citrullinated alpha enolase antibodies and clinical features in a cohort of patients with rheumatoid arthritis: a pilot study.

In recent years several antibodies against citrullinated peptides (ACPAs) have been identified in patients with rheumatoid arthritis (RA) and their pathogenic, diagnostic and prognostic significance is under intense investigation. Among ACPAs, those targeting citrullinated alpha enolase (anti-CEP1) have been identified in RA but data about their ability to predict the development of erosive disease are conflicting. Furthermore, no data are currently available concerning their possible association with extra-articular manifestations (EAMs) in RA. The aim of this study was to investigate the prevalence and significance of anti-CEP1 from a prognostic point of view. In this pilot study we confirmed that anti-CEP1 Abs are associated with higher prevalence of bone erosions, but we also provided the first evidence of an association between anti-CEP1 Abs and RA interstitial lung disease (ILD). These results provide the basis to investigate the association between anti-CEP1 Abs and EAMs in larger cohorts of RA patients to possibly confirm its role as biomarker for RA-ILD.

Label-free detection of immune complexes with myeloid cells.

The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.

Fingerprinting of anti-citrullinated protein antibodies (ACPA): specificity, isotypes and subclasses.

Anti-citrullinated protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific autoantibodies that recognize the amino acid citrulline, resulting from the post-translational modification of arginine. Peptidyl arginine deiminase, the enzyme responsible for citrullination, is present in humans in different isoforms with different tissue distribution, enzymatic activity and target specificity; nonetheless, the number of proteins citrullinated in physiological or pathological conditions is wide, but not every citrullinated protein is a target for antibodies. In pre-RA patients the immune response to citrullinated antigens is initially restricted, expands with time and, after the onset of the disease, is relatively stable. ACPA are heterogeneous in terms of not only fine specificity but also isotype and IgG subclasses usage. This heterogeneity may be relevant for the immunopathogenesis of RA, conditioning the interaction of antibodies with complement and Fc receptors.

Increased production of inflammatory cytokines by circulating monocytes in mesial temporal lobe epilepsy: A possible role in drug resistance.

We analyzed peripheral blood mononuclear cells (PBMCs) and serum inflammatory biomarkers in patients with mesial temporal lobe epilepsy (drug-resistant - DR, vs. drug-sensitive - DS). Patients with epilepsy showed higher levels of serum CCL2, CCL3, IL-8 and AOPP, and lower levels of FRAP and thiols compared to healthy controls (HC). Although none of the serum biomarkers distinguished DR from DS patients, when analysing intracellular cytokines after in vitro stimulation, DR patients presented higher percentages of IL-1ß and IL-6 positive monocytes compared to DS patients and HC. Circulating innate immune cells might be implicated in DR epilepsy and constitute potential new targets for treatments.

Free IL-18 and IL-33 cytokines in chronic spontaneous urticaria.

Overproduction of IL-18 has been described in chronic urticaria. To evaluate free IL-18 and IL-33 in chronic spontaneous urticaria (CSU). IL-18, its inhibitor IL-18BP, IL-33 and its soluble receptor ST2 (sST2) were measured (ELISA) in the sera of 73 CSU patients. Free IL-18 was calculated (law of mass action). Autologous serum skin test (ASST) was performed in all patients. Total IL-18, IL-18BP and free IL-18 serum levels were significantly higher in CSU than in controls. IL-18 and IL-18BP increased significantly in both ASST-positive and negative subgroups. Free IL-18 resulted significantly higher in the ASST-negative, but not in the ASST-positive subgroup. No differences in IL-33/sST2 levels were detected between CSU and controls. Increased levels of free IL-18 and IL-18BP, but not IL-33, was detected in CSU. Whether IL-18 up-regulation is a consequence of inflammation or one of the causes of the pathology needs to be addressed.

Cell surface expression of activating receptors and co-receptors on peripheral blood NK cells in systemic autoimmune diseases.

OBJECTIVES: A defined role for natural killer (NK) cells and their activating receptors in autoimmunity has not been clearly established. The aim of this study was to evaluate the levels of the CD3-CD56+ NK cells and their expression of receptors and co-receptors in the peripheral blood of patients with systemic autoimmune disorders. METHODS: Thirty-four subjects with systemic sclerosis (SSc), 14 with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), 14 with systemic lupus erythematosus (SLE), and 14 healthy donors were studied. The activating receptors NKp46, NKp44, NKp30, NKG2D, and DNAM-1 and the co-receptors NTB-A and 2B4 were analysed by flow cytometry on peripheral blood NK cells. RESULTS: In SSc, AAV, and SLE we detected a significant decrease in the percentage of CD3-CD56+ NK cells compared to healthy controls. No differences in the expression of NKp46, NKp44, and NKp30 were identified. On the contrary, NKG2D and DNAM-1 expression was decreased in SLE, but not in SSc and AAV, NTB-A was decreased in SLE, and 2B4 in both SLE and SSc. No differences were detected between active and inactive SLE patients. In SSc, only patients affected by pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) had a low expression of DNAM-1, 2B4, and NKp30. CONCLUSIONS: These data demonstrate that patients with different systemic autoimmune diseases differ in the expression of activating receptors and co-receptors on CD3-CD56+ NK cells. The down-regulation of receptors and co-receptors in SSc with lung involvement suggests their possible role in this manifestation of the disease.

Antibodies to a new viral citrullinated peptide, VCP2: fine specificity and correlation with anti-cyclic citrullinated peptide (CCP) and anti-VCP1 antibodies.

Anti-citrullinated protein/peptide antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and can be measured using different citrullinated substrates. In this paper we describe a new viral citrullinated peptide - VCP2 - derived from the Epstein-Barr virus-encoded protein EBNA-2 and analyse its potential as substrate for ACPA detection. Analysing sera from 100 RA patients and 306 controls, anti-VCP2 immunoglobulin (Ig)G were found in 66% of RA sera, IgM in 46% and IgA in 39%, compared with less than 3% of control sera. Anti-VCP2 IgG was associated with erosive arthritis, the presence of rheumatoid factor and anti-VCP1 and anti-cyclic citrullinated peptide (CCP) antibodies. Anti-VCP2 antibodies were detected in 1% and anti-VCP1 antibodies in 4% of CCP-negative RA sera; conversely, 3% of the VCP-negative sera were CCP-positive. Taken together, these data suggest that VCP2 could offer a valuable tool for ACPA detection. Inhibition assays showed that two non-overlapping epitopes - a citrulline-glycine stretch shared between VCP1 and VCP2 and the N-terminal portion of the VCP2 sequence - were targeted by anti-VCP2 antibodies. Moreover, in some RA sera that tested positive in CCP and VCP2 assays, preincubation with VCP2 inhibited binding to CCP, whereas in other sera the binding was unaffected. Thus, the reactivity with more than one ACPA substrate might be due in some RA patients to antibody populations with different specificities, and in others to cross-reactive antibody populations. Finally, affinity-purified anti-VCP2 antibodies immunoprecipitated deiminated Epstein-Barr virus nuclear antigen (EBNA-2) from an EBNA-2-transfected cell line, suggesting that viral sequences may be involved in the generation of the ACPA response.

Anti-DNA antibodies bind to DNase I.

Polyspecificity is a well-known property of the anti-DNA antibodies produced by autoimmune animals. In our search for antigen targets of anti-DNA antibodies within tissue extracts, we identified a 32-kD polypeptide that was recognized by a large panel of anti-DNA antibodies. Direct sequencing of this protein disclosed its identity with DNase I. 22 monoclonal anti-DNA antibodies bound to DNase I in direct and competitive immunoassays; out of 15 autoantibodies that did not bind DNA, none had the ability to bind DNase I. The ability of anti-DNA antibodies to interfere with DNase I enzymatic activity was evaluated in an assay based on the enzyme digestion of phage double strand DNA. Six monoclonal anti-single strand DNA antibodies that did not bind double strand DNA were tested in this assay. Three out of six inhibited DNase I-mediated digestion of phage DNA. The interaction of anti-DNA antibodies with DNase I was further investigated by testing their ability to bind a synthetic peptide that corresponds to the catalytic site of the molecule. 4 out of 22 anti-DNA antibodies bound the active site peptide; two of these had been shown to inhibit DNase I enzymatic activity. This report show that anti-DNA antibodies recognize both DNA and its natural ligand DNase I. Some anti-DNA antibodies inhibit DNase I enzymatic activity, thus displaying the potential to modulate DNA catabolism. The dual specificity of anti-DNA antibodies offers a clue for understanding the mechanisms that lead to anti-DNA antibody production in autoimmune animals.

Parallel sets of autoantibodies in MRL-lpr/lpr mice. An anti-DNA, anti-SmRNP, anti-gp70 network.

The public idiotype Id-H130 occurs in MRL-lpr/lpr serum both on a high proportion of anti-DNA autoantibodies as well as on antibodies that do not bind to DNA. To define members of the latter population, we prepared hybridomas and selected Id-H130+ mAbs that did not bind to DNA. One such antibody, mAb 28/12, was found to be an anti-SmRNP antibody. To determine whether mAb 28/12 had rheumatoid factor activity, we tested its ability to bind, in a solid-phase assay, to 16 mouse IgM mAbs. mAb 28/12 bound to only four of the panel, two anti-DNA antibodies (mAbs 512 and 319) and two anti-gp70 antibodies (mAbs 514 and 1417). In a liquid-phase competition assay with a panel of 32 monoclonal IgM and IgG antibodies, including allotype-matched Igs, mAb 28/12 reacted only with mAbs 512, 319, 514, and 1417. The binding of mAb 28/12 to mAbs 512 and 319 was displaced by DNA, but not by RNA, indicating that the idiotype it defines (Id-28/12) is in the antigen-binding region of the two anti-DNA antibodies. In the two anti-gp70 antibodies (mAbs 514 and 1417), Id-28/12 seems to occur in the framework region. To determine if all four Id-28/12+ antibodies shared a common antigen-binding property, they were tested for their ability to react with DNA and gp70. The two anti-gp70 antibodies did not bind to DNA. However, the two anti-DNA antibodies were found to immunoprecipitate viral proteins from retrovirus-infected cells. mAb 512 reacted with gp70, both in cell membrane lysates and in purified form; mAb 319 reacted with gp85, which contains both gp70 and the retroviral protein p15. Antibodies with properties similar to those of mAb 28/12 were found in MRL-lpr/lpr serum. It was possible, by affinity chromatography on an anti-gp70 antibody column, to isolate from serum those anti-(anti-gp70) antibodies with anti-SmRNP activity. These results show that parallel sets of autoantibodies, which share a common idiotype, but which bind to different autoantigens, occur in MRL-lpr/lpr mice. Some populations of anti-DNA, anti-SmRNP, and anti-gp70 antibodies appear to constitute a network of autoantibodies in that strain. We speculate that part of the anti-SmRNP population of autoantibodies can arise by mutation of germline-encoded anti-DNA antibodies.

An immunoglobulin light chain from a lupus-prone mouse induces autoantibodies in normal mice.

Autoantibodies against the 70-kD U1 RNP nucleoprotein autoantigen and DNA were elicited in normal BALB/c mice with a purified Ig light chain. This light chain, derived from a lupus-prone MRL-lpr/lpr mouse, has two distinctive properties: it contains an idiotypic marker recognized by a monoclonal MRL-lpr/lpr anti-snRNP autoantibody, and the amino acid sequence of its third hypervariable region (CDR3) is homologous to a sequence in an antigenic region of the 70-kD U1 RNP polypeptide. The results demonstrate that an Ig idiotype that mimics an autoantigen can induce autoimmunization.

Autoantibodies of various specificities encoded by genes from the VH J558 family bind to foreign antigens and share idiotopes of antibodies specific for self and foreign antigens.

We examined the binding to foreign antigens and the expression of crossreactive idiotypes by a panel of 20 murine monoclonal autoantibodies encoded by V genes from the VH J558 family. 9 of 20 antibodies bound to foreign antigens such as bacterial polysaccharides, poly(Glu50, Tyr50), poly(Glu54,Lys37,Phe9), arsonate, and lysozyme, known to interact with antibodies encoded by genes from the VH J558 family. A high proportion of our panel of autoantibodies expressed crossreactive idiotypes originally borne by monoclonal rheumatoid factors, anti-Sm, and anti-DNA antibodies, all encoded by V genes from the VH J558 family. Some of these VH J558+ autoantibodies shared crossreactive idiotypes with VH J558+ antibodies directed against foreign antigens such as influenza virus hemagglutinin, poly(Glu60,Ala30,Tyr10), arsonate, and dextran. The implications of these findings are discussed with respect to the process of activation of self-reactive clones.

Mortality rate and outcome factors in mixed cryoglobulinaemia: the impact of hepatitis C virus.

OBJECTIVES: Mixed cryoglobulinaemia (MC) is a chronic small-vessel vasculitis. Shortly after the discovery of hepatitis C virus (HCV) in 1989, an association between HCV infection and MC was being increasingly reported, suggesting the potential pathogenetic implication of HCV in most of the cases that had been previously diagnosed as essential MC. A number of studies have pointed out prognostic factors linked to mortality in this disorder. None of them, however, have clarified the impact of HCV discovery on the natural history of the disease. The aim of the present study was to evaluate mortality in MC after the discovery of HCV infection. METHODS: We retrospectively collected clinical and serological data in 70 unselected HCV-positive patients being followed up at our unit from 1990. Clinical and prognostic factors linked to poor outcome were evaluated. RESULTS: Chronic hepatitis, renal involvement, and intestinal vasculitis were the most frequent causes of death. CONCLUSION: Compared to other series, the outcome in our MC seemed to be better. Factors linked to a poor outcome were renal involvement, widespread vasculitis, male sex, and cryocrit.

Replication of the tumor necrosis factor receptor-associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study.

OBJECTIVE: We recently showed, using a candidate gene approach in a case-control association study, that a 65-kb block encompassing tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 is strongly associated with rheumatoid arthritis (RA). Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure and are not likely to be hampered by variation in population allele frequencies, as is seen for many genetic polymorphisms, including the TRAF1/C5 locus. The aim of this study was to confirm this association in populations of European origin by using a family-based approach. METHODS: A total of 1,356 western European white individuals from 452 "trio" families were genotyped for the rs10818488 polymorphism, using the TaqMan allelic discrimination assay. RESULTS: We observed evidence for association, demonstrating departure from Mendel's law, with an overtransmission of the rs10818488 A allele (A = 55%; P = 0.036). By taking into consideration parental phenotypes, we also observed an increased A allele frequency in affected versus unaffected parents (A = 64%; combined P = 0.015). Individuals carrying the A allele had a 1.2-fold increased risk of developing RA (allelic odds ratio 1.24, 95% confidence interval 1.04-1.50). CONCLUSION: Using a family-based study that is robust against population stratification, we provide evidence for the association of the TRAF1/C5 rs10818488 A allele and RA in populations of European descent, further substantiating our previous findings. Future functional studies should yield insight into the biologic relevance of this locus to the pathways involved in RA.

Evaluation of a novel particle-based assay for detection of autoantibodies in idiopathic inflammatory myopathies.

BACKGROUND: Myositis specific antibodies (MSA) represent not only important diagnostic tools for idiopathic inflammatory myopathies (IIM), but also help to stratify patients into subsets with particular clinical features, treatment responses, and disease outcome. Consequently, standardization of MSA is of high importance. Although many laboratories rely on protein immunoprecipitation (IP) for the detection of MSA, IP standardization is challenging and therefore reliable alternatives are mandatory. Recently, we identified significant variation between IP and line immunoassay (LIA) for the detection of MSA and myositis associated antibodies. In this study we aimed to compare the results from our previous study to the results obtained with a novel fully automated particle-based technology for the detection of MSA and MAA. METHODS: A total of 54 sera from patients with idiopathic inflammatory myopathy (IIM) were tested using three methods: IP, LIA (Euroimmun, Germany) and a novel particle-based multi-analyte technology (PMAT, Inova Diagnostics, US, research use only). The analysis focused on antibodies to EJ, SRP, Jo-1, NXP-2, MDA5, TIF1-γ, and Mi-2. RESULTS: Significant variations were observed among all methods. Overall, the novel PMAT assays showed slightly better correlation with IP, but the kappa agreement was strongly dependent on the antibody tested. When the results obtained from IP were used as reference for receiver operating characteristic (ROC) curve analysis, good discrimination and a high area under the curve (AUC) value were found for PMAT (AUC = 0.83, 95% confidence interval, CI 0.70-0.95) which was significantly higher (p = .0332) than the LIA method (AUC = 0.70, 95% CI 0.56-0.84). CONCLUSION: The novel PMAT used to detect a spectrum of MSA in IIM represents a potential alternative to IP and other diagnostic assays. Additional studies based on larger cohorts are needed to fully assess the performance of the novel PMAT system for the detection of autoantibodies in myositis.

Anti-C1q antibodies in pregnant patients with systemic lupus erythematosus.

OBJECTIVE: To study anti-C1q antibodies in pregnant patients with systemic lupus erythematosus (SLE) and to evaluate their prognostic significance for the occurrence of disease flares or pregnancy complications. METHODS: Twenty-one pregnancies in 19 SLE patients prospectively followed were analyzed. Disease activity was evaluated on the basis of the physician's intention to treat and a modified version of the ECLAM index. Anti-C1q and anti-dsDNA antibodies were detected in the sera by an ELISA assay. Antinuclear antibodies, anti-ENA antibodies, anticardiolipin antibodies and lupus anticoagulant were also performed. RESULTS: In all the patients the disease was inactive at the beginning of the pregnancy. Four flares of disease activity were observed in 4 pregnancies (19%) and obstetric complications were encountered in 7 pregnancies (43%). Anti-C1q antibodies were positive in 4 (19%) pregnancies and anti-dsDNA antibodies in 8 (38%). The presence of anti-phospholipid antibodies at the first assessment was correlated with the occurrence of obstetric complications (p<0.05). The presence of anti-C1q and anti-dsDNA antibodies at the first assessment had no prognostic significance for the occurrence of flares or obstetric complications during the course of pregnancy. Although the small number of patients studied did not allow for statistically significant analysis, flares appeared to be more likely to occur in patients presenting with anti-dsDNA or anti-C1q antibodies during pregnancy compared to patients with no changes in these antibody titers (43% vs 8% respectively). CONCLUSIONS: The presence of anti-C1q and anti-dsDNA antibodies does not seem to be prognostic for the occurrence of flares during pregnancy. Further studies are warranted to explore this possibility.

Antiprothrombin antibodies: a comparative analysis of homemade and commercial methods. A collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni (FIRMA).

OBJECTIVE: Prothrombin (PT) is a target for antibodies with lupus anticoagulant (LA) activity, suggesting the possible application of anti-prothrombin antibody (aPT) assays in patients with antiphospholipid syndrome (APS). Different methods - both homemade and commercial - for the detection of aPT are available, but they seem to produce conflicting results. The purpose of this study was to compare the performance of different assays on a set of well-characterized serum samples. PATIENTS AND METHODS: Sera were gathered from 4 FIRMA institutions, and distributed to 15 participating centres. Forty-five samples were from patients positive for LA and/or anticardiolipin antibodies (aCL) with or without APS, and 15 were from rheumatoid arthritis (RA) patients negative for antiphospholipid antibodies. The samples were evaluated for IgG and IgM antibodies using a homemade direct aPT assay (method 1), a homemade phosphatidylserine-dependent aPT assay (aPS/PT, method 2), and two different commercial kits (methods 3 and 4). In addition, a commercial kit for the detection of IgG-A-M aPT (method 5) was used. RESULTS: Inter-laboratory results for the 5 methods were not always comparable when different methods were used. Good inter-assay concordance was found for IgG antibodies evaluated using methods 1, 3, and 4 (Cohen k > 0.4), while the IgM results were discordant between assays. In patients with thrombosis and pregnancy losses, method 5 performed better than the others. CONCLUSION: While aPT and aPS/PT assays could be of interest from a clinical perspective, their routine performance cannot yet be recommended because of problems connected with the reproducibility and interpretation of the results.