Improved accuracy in DFS pattern interpretation using a novel HEp-2 ELITE system.

INTRODUCTION/OBJECTIVES: Accurate interpretation of DFS70 (dense fine speckled 70) and mixed antinuclear antibodies (ANAs) patterns can be challenging using conventional HEp-2 immunofluorescence (IIF) method. We evaluated a novel HEp-2 IIF substrate (HEp-2 ELITE/DFS70-KO) composed of a mixture of engineered HEp-2 devoid of the DFS70 autoantigen and conventional HEp-2 cells. The study assessed the utility of the new substrate in ANA screening and its advantages. METHOD: One thousand and five consecutive routine samples sent for ANA screening were tested on both standard HEp-2 and the HEp-2 ELITE DFS70 KO substrates (ImmuGlo ANA HEp-2 and HEp-2 ELITE/DFS70-KO, Trinity Biotech, Buffalo, NY). Anti-DFS70 antibody specificity was additionally determined by immunoblot (IB). Clinical and serological data were included in the analysis of the overall impact of the novel HEp-2 substrate on DFS pattern interpretation. RESULTS: Of the 22 cases suspected as positive for DFS pattern alone or in combination with homogeneous or speckled patterns on conventional HEp-2 cells, 17 were interpreted with a higher accuracy using the new HEp-2 ELITE method as positive for DFS70 (monospecific DFS70 (10), mixed DFS70 (7)), speckled (3), and DFS (2) patterns. CONCLUSIONS: The new substrate was not only useful in deciphering unclear mixed ANA patterns but also highly sensitive in detecting DFS70 pattern in comparison to the DFS70 positivity obtained using IB.

Decrease in 14-3-3η protein levels is correlated with improvement in disease activity in patients with rheumatoid arthritis treated with Tofacitinib.

14-3-3η protein is a proinflammatory mediator that may represent a novel diagnostic and prognostic biomarker for rheumatoid arthritis (RA). We assessed the correlation between changes in serum 14-3-3η levels and changes in clinical disease activity measures in RA patients treated with Tofacitinib (TOF). Paired serum samples from 35 patients with RA were obtained at baseline and 5 months after the initiation of treatment with TOF. The levels of 14-3-3η were measured by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). The cut-off was defined as 0.19 ng/ml. 14-3-3η positivity was found in 57% of the patients at baseline and in 37% of the patients after 5 months of treatment. Mean ± SD baseline 14-3-3η levels [4.92 ± 8.86 ng/ml] were significantly higher (p < 0.005) than 14-3-3η levels following treatment [1.97 ± 4.59 ng/ml]. A statistically significant improvement (p < 0.001) of CDAI, SDAI, DAS4ESR and DAS4CRP was achieved after 5 month of treatment. Decrease in 14-3-3η protein levels was highly correlated with improvement in DAS4ESR (r = 0.50, p < 0.01), DAS4CRP (r = 0.46, p < 0.01) and ESR (r = 0.36, p = 0.03) and moderately correlated with improvement in CDAI (r = 0.32, p = 0.065) and SDAI (r = 0.33, p = 0.051). The correlation between decrease in 14-3-3η levels and improvement in DAS4ESR remained significant in a partial correlation analysis controlling for ESR (r = 0.39, p = 0.02). This study demonstrates that in RA patients who were treated with TOF, decrease in 14-3-3η levels is correlated with improvement in clinical disease activity parameters. The 14-3-3η protein may serve as an objective biomarker for monitoring of TOF therapy response.

A better definition of the anti-DFS70 antibody screening by IIF methods.

BACKGROUND: Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies. METHODS: Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco). RESULTS: The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22). CONCLUSION: The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.

The diagnostic value of 14-3-3η protein levels in patients with rheumatoid arthritis.

14-3-3η may represent a useful diagnostic biomarker for rheumatoid arthritis (RA). We assessed the prevalence and serum levels of 14-3-3η in patients with RA and in patients with other rheumatic diseases. Serum levels of 14-3-3η were measured in 96 patients with RA, in 101 patients with other rheumatic diseases, and in 66 healthy subjects. All of the sera samples were evaluated by JOINT stat 14-3-3η ELISA test kits (Augurex Life Sciences Corp.). Median (IQR) 14-3-3η levels were significantly higher in the early RA group [0.25 ng/ml (0.075-3.11)] and in patients with established RA [0.15 ng/ml (0.08-1.26)] than in healthy subjects [0 ng/ml (0-0)] and disease controls: SLE [0.01 ng/ml (0-0.055)], AS [0.05 ng/ml (0-0.255)], and PsA [0.01 ng/ml (0-0.065)]. The prevalence of 14-3-3η positivity in patients with early RA was 58%, significantly higher than that in disease controls and healthy subjects (p < 0.001). In patients with established RA, this prevalence was 43%, and it was significantly higher than that in patients with other rheumatic diseases and healthy subjects (p < 0.05), excluding the AS group (p = 0.054). In the early RA cohort, the positivity for 14-3-3η, RF, and anti-CCP was 58%, 67%, and 71%, respectively. Eighty-two percent of the patients in this cohort were positive for at least one of these biomarkers. The concentration of 14-3-3η protein may be used to distinguish between patients with early RA and patients with other rheumatic diseases.

Anti-HMGCR antibodies demonstrate high diagnostic value in the diagnosis of immune-mediated necrotizing myopathy following statin exposure.

Anti-HMGCR antibodies represent a characteristic serological feature of statin-exposed and statin-unexposed patients with immune-mediated necrotizing myopathy (IMNM). We assessed anti-HMGCR antibodies in patients with suspected IMNM following statin exposure and patients with other autoimmune rheumatic diseases. We evaluated the presence of anti-HMGCR autoantibodies in sera samples from 13 statin-exposed patients who were suspected of having IMNM, 38 patients with different inflammatory and autoimmune rheumatic diseases and 29 healthy subjects. The autoantibodies were evaluated by two assays: a new chemiluminescence QUANTA Flash HMGCR kit utilizing BIO-FLASH system and QUANTA Lite® HMGCR ELISA kit. Twelve samples from patients with suspicion for IMNM were found positive for anti-HMGCR antibodies by both assays. Only one of the 13 samples that were found positive by ELISA was negative by CIA. A very good qualitative correlation (κ = 0.95; 95 % CI 0.85-1.0) and quantitative agreement (Spearman's rho 0.87; P value < 0.0001; 95 % CI 0.62-0.96) were found between these two assays. All samples from healthy subjects and from the disease-controlled patient cohort were negative for anti-HMGCR antibodies. In comparison with ELISA results, the CIA exhibited high sensitivity and specificity values of 92.3 and 100 %, respectively. Receiver operating characteristic analysis for CIA and ELISA yielded area under the curve values of 0.99. The presence of anti-HMGCR antibodies may be a useful biomarker of IMNM in statin-exposed patients. There is a good correlation between the two anti-HMGCR antibody assays evaluated in the present study.

A fully automated IIF system for the detection of antinuclear antibodies and antineutrophil cytoplasmic antibodies.

Indirect immunofluorescence (IIF) is the main technique for the detection of antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA). The fully automated IIF processor HELIOS(®) is the first IIF processor that is able to automatically prepare slides and perform automatic reading. The objective of the present study was to determine the diagnostic performance of this system for ANA and ANCA IIF interpretation, in comparison with visual IIF. ANA detection by visual IIF or HELIOS(®) was performed on 425 sera samples including: 218 consecutive samples submitted to a reference laboratory for routine ANA testing, 137 samples from healthy subjects and 70 ANA/ENA positive samples. For ANCA determination, 170 sera samples were collected: 40 samples for routine testing, 90 samples from healthy blood donors and 40 anti-PR3/anti-MPO positive subjects. Good correlation was found for the visual and automated ANA IIF approach regarding positive/negative discrimination of these samples (kappa = 0.633 for ANA positive samples and kappa = 0.657 for ANA negative samples, respectively). Positive/negative IIF ANCA discrimination by HELIOS(®) and visual IIF revealed a complete agreement of 100% in sera from healthy patients and PR3/MPO positive samples (kappa = 1.00). There was 95% agreement between the ANCA IIF performed by automated and visual IIF on the investigation of routine samples. Based on these results, HELIOS(®) demonstrated a high diagnostic performance for the automated ANA and ANCA IIF interpretation that was similar to a visual reading in all groups of samples.

Coincidence of granulomatosis and polyangiitis with atypical clinical manifestation and antiphospholipid syndrome.

Granulomatosis and angiitis (GPA) is a multisystemic disease characterized by a granulomatous inflammation, tissue necrosis, and vasculitis of small and medium-sized blood vessels. Although the disease has a predilection for the upper respiratory tract, lungs, and kidneys, any organ system may be affected. Here, we present a case of generalized GPA manifested initially by necrotizing isolated parotitis and later by pulmonary-renal syndrome. Simultaneously with pulmonary hemorrhage, our patient developed an antiphospholipid syndrome (APS) presenting with deep vein thrombosis and strongly positive lupus anticoagulant. To the best of our knowledge the coincidence of parotitis and pulmonary-renal syndrome due to GPA and APS has never been reported previously. Concomitant venous thromboembolism may be life-threatening in a patient with GPA. Early diagnosis and institution of the proper therapy are critical in order to prevent organ damage.

Novel insights into associations of antibodies against cardiolipin and beta2-glycoprotein I with clinical features of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a clinical autoimmune disorder characterized by arterial and/or venous thrombosis and/or pregnancy morbidity, associated with the persistence of lupus anticoagulant or anticardiolipin (aCL) antibodies. Accumulating evidence indicates that phospholipid binding protein, beta2-glycoprotein I (beta2GPI) represents the major target antigen for antiphospholipid (aPL) antibodies and plays a role in the pathogenesis of APS. It is widely accepted that aPL antibodies detected by conventional solid phase assays in patients with APS are mainly directed against a complex of aCL and anti-beta2GPI, although antibodies against beta2GPI protein can now also be detected by specific ELISA using purified proteins in solid phase. Despite the fact that these antibodies are not listed in the new diagnostic criteria, a high specificity of anti-beta2GPI assay for the clinical features of APS was established. During the last decade, numerous studies have investigated the clinical link between aCL and/or anti-beta2GPI antibodies and diverse features of APS. This manuscript reviews the current studies published recently in this field and discusses the relationship between the existence of aCL and anti-beta2GPI antibodies and the main and unusual manifestations of APS.

Probiotic supplementation with Lactobacillus casei (Actimel) induces a Th1 response in an animal model of antiphospholipid syndrome.

Probiotic fermented milk products have the capacity to modulate many immunological mechanisms. Several attempts have been made to alter the progression of various atopic and inflammatory disorders in which the immune system plays a major role. We studied this issue in an animal model of the antiphospholipid syndrome (APS) by supplementing the animals' daily intake with a probiotic mixture. We studied the effects of nutritional supplementation of a commercial product that consists of 10(8)/ml Lactobacillus casei (Actimel) on Balb/c mice that were immunized with beta-2- glycoprotein (beta2GPI) in order to induce a familiar murine model of APS. As controls, we used similar animals that were fed with either yogurt or sham solution as a supplement. We analyzed the effect of Actimel on the concentrations of interleukin (IL)-10 interferon gamma (IFNgamma) as well as the extent of the primary T cell response to beta2GPI, and the levels of autoantibodies to beta2GPI determined by ELISA. Two weeks after priming (in the hind footpad) of Balb/c mice with beta2GPI, we analyzed the cytokine profile of the animals by measuring the concentration of IL-10 and IFNgamma in the supernatants of lymphocytes that were extracted from the popliteal lymph nodes. Following stimulation with 10 microg/mL of beta2GPI, we noticed significant (P < 0.05) suppression of IL-10 production by the stimulated lymphocytes in the animals fed with Actimel and yogurt in comparison to sham solution (73.42 +/- 29.4, 84.7 +/- 8, 196 +/- 41.62 pg/mL, respectively). Both dairy products enhanced the secretion of IFNgamma from 657 +/- 47.09 pg/mL to 896 +/- 78.1, and 933 +/- 76.7 (P < 0.01), respectively; similarly they also accelerated by a mild degree the level of the T cell primary response to beta2GPI measured by [3H]thymidine incorporation. The level of autoantibodies to beta2GPI was suppressed in mice fed with actimel and yogurt in a significant manner (P < 0.05). Actimel as well as yogurt confer an immunological impact on Balb/c mice immunized with beta2GPI. Actimel was able not only to enhance IFNgamma secretion but also to inhibit IL-10 production.

Thickened carotid artery intima-media in rheumatoid arthritis is associated with elevated anticardiolipin antibodies.

Antiphospholipid and anti-oxidized LDL (anti-oxLDL) antibodies are associated with thrombosis and atherosclerosis. Rheumatoid arthritis (RA) is characterized by excess atherosclerosis and cardiovascular diseases. Our aim was to determine whether antiphospholipid and anti-oxLDL antibodies are associated with early atherosclerotic changes in RA. The levels of IgG and IgM anticardiolipin, IgG and IgM anti-beta-2-glycoprotein-I and anti-oxLDL autoantibodies have been evaluated in 82 patients having RA. Carotid artery intima-media thickness (IMT) was measured in the carotid arteries in the common carotid, bifurcation and internal carotid arteries. Elevated levels of IgG anticardiolipin antibodies were detected in 17 of 82 (21%) RA patients, including 7 with medium-to-high levels considered being clinically relevant. These patients had significantly elevated mean carotid and carotid bifurcation IMT compared with RA patients without elevated anticardiolipin. No such association was found regarding other autoantibodies tested. Anticardiolipin antibodies are prevalent in RA and are associated with early atherosclerotic changes, supporting a rational for measuring them in RA, and upon detection treat the patients in order to decrease chances of atherosclerosis progression and thrombosis.

Increased prevalence of anti-third generation cyclic citrullinated peptide antibodies in patients with rheumatoid arthritis and CREST syndrome.

To investigate the prevalence of anti-third generation cyclic citrullinated peptide antibodies (anti-CCP3) in patients with systemic connective tissue diseases, we assembled a training set consisting of 115 patients with rheumatoid arthritis (RA), 52 with Calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasia (CREST) syndrome, 21 with scleroderma, 20 with ankylosing spondylitis, 18 with reactive arthritis, 25 with juvenile rheumatoid arthritis (RA), 51 with osteoarthritis, 26 with mixed connective tissue disease, 23 with primary Sjogren's syndrome, 74 with systemic lupus erythematosus, 49 with Polymyalgia rheumatica, and 39 with polymyositis/dermatomyositis. The commercial enzyme-linked immunosorbent assay (ELISA) was used to detect anti-CCP antibodies, including anti-CCP2 (regular, second generation of CCP antigen) and anti-CCP3 (third generation of CCP antigen) in disease-related specimens and normal controls. These serum samples were also evaluated for anti-centomere antibodies by anti-centromere ELISA kit. The higher frequencies of anti-CCP3 and anti-CCP2 were detected in 75.6 and 70.4% patients with RA, respectively. At the same time, anti-CCP3 (not anti-CCP2) was significantly increased in samples isolated from patients with CREST syndrome. The clinical sensitivity of IgG anti-CCP3 for the patients with CREST syndrome was 29% (15 of 52) and the specificity was 96% (384 of 397), with the exception of the RA group. The anti-centromere antibodies were significantly higher in patients with CREST only. The results of our study suggest that compared to anti-CCP2 assay, the new anti-CCP3 assay can enhance the clinical sensitivity for diagnosis of RA and, as an associate marker combined with anticentromere, can distinguish CREST syndrome from other systemic connective tissue diseases, especially RA. The clinical specificity of anti-CCP3 was lower than anti-CCP2 assay in diagnosis of RA because of the crossreaction to the patients with CREST syndrome.

Application of a static fluorescence-based cytometer: the CellScan in clinical immunology.

The CellScan system is a laser scanning cytometer which enables repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. By monitoring FP changes in peripheral blood lymphocytes (PBL) following exposure to antigenic stimuli, the CellScan may have a role in the diagnosis of autoimmune diseases. Monitoring changes in FI and FP in PBLs from patients with atherosclerosis following exposure to various stimuli, has illustrated the role of the immune system in the atherosclerotic process. The CellScan has also been evaluated as a diagnostic tool for drug-induced allergy, based on FP reduction in PBLs following incubation with the suspected drugs. FI and FP changes in cancer cells have been found to correlate with the cytotoxic effect of different anti-neoplastic drugs, illustrating the potential role of the CellScan system in clinical oncology. In conclusion, the CellScan is a promising new tool with a variety of applications in cell biology, immunology, cancer research and clinical pharmacology.

Restricted specificity of anti-ribosomal P antibodies to SLE patients in Israel.

OBJECTIVE: Anti-ribosomal P antibodies (aRib-P Ab) are highly specific for systemic lupus erythematosus (SLE), but their correlatation with disease activity and manifestations including renal, hepatic and central nervous system (CNS) involvement is still controversial. The aim of our study was to evaluate the prevalence of aRib-P Ab and their correlation with clinical manifestations and anti-dsDNA antibodies in SLE patients from Israel. METHODS: Elevated titers of aRib-P Ab utilizing the ELISA method were analyzed in 141 sera samples from 44 SLE patients, 20 Familial Mediterranean Fever (FMF) patients, 22 primary antiphospholipid syndrome (PAPS) patients, 12 patients with infections, and 43 healthy individuals. The SLEDAI score was utilized for assessing SLE disease activity. RESULTS: Elevated titers of aRib-P Ab were present in 11% of SLE patients (n = 6). The mean SLEDAI was 7 (range: 3-10). No statistically significant association was observed between the presence of aRib-P Ab and disease manifestations present in the SLEDAI. The 6 SLE patients had renal disease (n = 1), leucopenia (n = 1), rash (n = 3), and CNS involvement manifested as psychosis (n = 1) or depression (n = 1). Elevated titers of anti-dsDNA antibodies were found in 50% of patients with elevated titers of aRib- P Ab. Patients with PAPS, FMF, infections or healthy controls did not harbor elevated titers of aRib-P Ab. CONCLUSION: Elevated titers of aRib-P Ab were restricted to SLE patients. We confirm previously reported associations of aRib-P Ab reactivity with disease activity and elevated anti-dsDNA Ab titers. No significant correlation with a specific manifestation described on the SLEDAI score was established in this small cohort of patients.

The diagnostic utility of anti-cyclic citrullinated peptide antibodies, matrix metalloproteinase-3, rheumatoid factor, erythrocyte sedimentation rate, and C-reactive protein in patients with erosive and non-erosive rheumatoid arthritis.

OBJECTIVE: To compare the diagnostic utility of laboratory variables, including matrix metalloproteinase-3 (MMP-3), anticyclic citrullinated peptide (CCP) antibodies, rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) in patients with erosive and non-erosive rheumatoid arthritis (RA). METHODS: We assembled a training set, consisting of 60 patients with RA, all fulfilling the revised criteria of the American College of Rheumatology. A commercial enzyme linked immunosorbent assay (ELISA) was used both to test for anti-CCP antibodies (second generation ELISA kit) and MMP; RF were detected by latex-enhanced immunonephelometric assay. CRP was measured by latex turbidimetric immunoassay. RESULTS: The levels of anti-CCP antibody titers and ESR were significantly higher in patients with erosive disease than those in non-erosive RA patients (p < 0.001 and 0.0341) respectively. Moreover, a higher frequency of elevated titers of anti-CCP antibodies was found in RA patients with erosions compared to patients with non-erosive RA (78.3% vs. 43.2% respectively). The ROC curves of anti-CCP passed closer to the upper left corner than those other markers and area under the curve (AUC) of anti-CCP was significantly larger than AUC of other markers (0.755 for anti-CCP, 0.660 for ESR, 0.611 for CRP, 0.577 for RF, and 0.484 for MMP-3 female). A positive predictive value was higher for anti-CCP antibodies in comparison to other markers. We did not find significant statistical correlation between anti-CCP antibody titers and inflammatory markers such as ESR or CRP. However, we confirmed the correlation of elevated titers of anti-CCP antibodies and RF in both groups of patients whereas the degree of correlation was more significant in non-erosive patients. CONCLUSION: The results of our study suggest that the presence of elevated anti-CCP antibody titers have better diagnostic performance than MMP-3, RF, CRP and ESR in patients with erosive RA.

Anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies as predictors of inflammatory bowel disease.

BACKGROUND AND AIMS: Several antibodies have been reported in the sera of patients with Crohn's disease (CD) and ulcerative colitis (UC). The most commonly described are anti-Saccharomyces cerevisiae mannan antibodies (ASCA) in CD and perinuclear antineutrophil cytoplasm antibodies (pANCA) in UC. Familial clustering of these antibodies has been described, suggesting they might be genetic markers. Our aim was to investigate the presence of these antibodies before the emergence of overt clinical manifestations. METHODS: Since 1980, the Israeli Defense Force (IDF) Medical Corps Serum Repository has stored serum samples obtained systematically from 5% of all recruits on enlistment, and from the same population on discharge from compulsory military service. We evaluated serum samples obtained from 32 subjects with CD and eight with UC before they were clinically diagnosed, along with samples from matched controls. RESULTS: ASCA were present in 10/32 (31.3%) CD patients before clinical diagnosis compared with 0/95 (0%) controls (p<0.001). None of the eight patients with serum samples available before diagnosis of UC were ASCA positive. ASCA was positive in 54.5% of patients after diagnosis of CD. The mean interval between ASCA detection and diagnosis was 38 months. In 90% of patients, antibodies were detected in the first available serum sample; therefore, measurements of the average time from the presence of ASCA to diagnosis may be even longer. pANCA were present in 2/8 (25%) patients with available sera before the diagnosis of UC. None of their 24 matched controls were positive (p = 0.014). CONCLUSIONS: ASCA and pANCA may predict development of inflammatory bowel disease years before the disease is clinically diagnosed.

Evaluation of the BioPlex 2200 ANA screen: analysis of 510 healthy subjects: incidence of natural/predictive autoantibodies.

The BioPlex 2200 ANA Screen is a fully automated system that determines levels for 13 different autoimmune antibodies of established clinical significance. The objective of this study was to determine the specificity of the BioPlex 2200 ANA Screen assay and to analyze the antibody profile samples collected from healthy subjects against comparative ELISA and IIF screening methods. A total of 510 specimens were randomly selected from a cohort of apparently healthy blood bank donors. Samples were distributed to five age brackets. All samples were tested using Bio-Rad's ANA Screen kit. Specificity was compared to IIF and ELISA results. Most of the samples were found negative in all ANA screening systems (84.5% by IIF, 92.5% by BioPlex 2200 ANA Screen kit, and 94.5% by ELISA). The frequency of positive results was highest (15.5%) using IIF, in comparison to almost similar results (5.5% vs. 7.5%) achieved by ANA ELISA and BioPlex 2200 ANA Screen kits. The positive rate of autoantibodies was significantly reduced when analyzed by different combinations of ANA screen assays (from 2.35% using IIF + BioPlex ANA Screen tests to 0.98% by using all three tests). Using the BioPlex 2200 ANA Screen system, we were able to identify samples with high levels of individual antibodies: anti-dsDNA at 20-63 IU/mL, antichromatin at 4-8 AI, anti-SmRNP at 2-6 AI, and anti-RNPA at 2-4.5 AI. Importantly, from 7 IIF and ELISA positive sera, 5 of these were also BioPlex 2200 positive, suggesting that the BioPlex is seeing the samples that are of the greatest interest, using the established techniques. The specificity of the BioPlex 2200 ANA Screen analysis of 13 different analytes (dsDNA, centromere B, chromatin, Jo1, ribosomal P, RNP 68, RNP A, Scl-70, Sm, SmPNP, SS-A52, SS-A60, SS-B) is comparable (P < 0.252) to the ELISA ANA screening test. Like the ELISA, the BioPlex 2200 has a lower (P < 0.001) positive rate than IIF for the autoantibody screening.

The influence of different cultivating conditions on polymorphonuclear leukocyte apoptotic process in vitro, II: ultrastructural characteristics of PMN populations incubated with proteinase 3 anti-neutrophil autoantibodies.

The present study shows the effects of proteinase 3 anti-neutrophil cytoplasmic autoantibodies (PR3 ANCA) on polymorphonuclear leukocytes (PMN) apoptotic processes in vitro. The results are part of a generalized morphological analysis of 3 identical experiments on the influence of different cultivating conditions on the apoptotic processes. As controls, the authors use the results on spontaneous PMN apoptosis (Guejes L, Zurgil N, Deutsch M, Gilburd B, Shoenfeld Y. Ultrastruct Pathol. 2003;27: 23-32) and PMN populations incubated with normal human IgG. Interaction of PR3 ANCA with the target antigen proteinase 3 (PR3) is one of the crucial pathogenic factors in Wegener granulomatosis (systemic autoimmune vasculitis). Following 40min and 12h incubation, PMN populations were evaluated by light microscopy, transmission electron microscopy, and immunogold electron microscopy. Twelve-hour cultures, either control or incubated with PR3 ANCA, contained different cell forms ranging from normal cells to cells at the final stages of apoptosis. Neutrophils at the state of complete manifestation of apoptotic phenotype were analyzed and compared. Three morphologically distinct apoptotic cell lines were characteristic for all PMN populations studied, regardless of cultivating conditions. As in spontaneous apoptosis, these cell lines are code-named "first," "second," and "third." The present study has shown, firstly, that in the presence of PR3 ANCA, all 3 apoptotic lines were modified or altered. Secondly, the modifications or alterations of apoptotic cell lines effected by PR3 ANCA are specific for each cell line: the "first" line is characterized by intensification and modification of activation; the "second" by vacuolized cell forms; and the "third" by pronounced lytic alterations of the nuclei, while the cytoplasm is fully identical to that of control cell lines.

Multiplexed AtheNA multi-lyte immunoassay for ANA screening in autoimmune diseases.

BACKGROUND: Multiplexed assays using fluorescence microspheres is an exciting technology with multiple applications including the detection of antinuclear autoantibodies (ANA) and autoantibody profiles. It is a rapid, sensitive and automatic method for simultaneous quantitative detection of several autoantibodies. The aim of our study was to determinate ANA and other autoantibodies to the nine extractable nuclear antigens by the AtheNA Multi-Lyte ANA system and compare the results achieved by this method to the routinely used enzyme immunoassay. METHODS: Four hundred eighteen serum samples were tested utililizing the multiplexed method: 96 healthy donors, 86 requested ANA specimens obtained from routine lab, and 236 samples from patients with known autoimmune diseases (43-scleroderma, 113-systemic lupus erythematosus, 38-Sjogren's syndrome, and 42 rheumatoid arthritis). The ANA and antibodies to nine different analytes (SS/A, SS/B, Sm, RNP, Jo-1, Scl-70, dsDNA, Centromere B and Histone) were tested. RESULTS: ANA screening by AtheNA system revealed high concordance of 99 and 97.7% with the enzyme immunoassay test in samples obtained from healthy donors and ANA requested samples, respectively. Evaluation of autoimmune disease-related samples for ANA by AtheNA technology also confirmed a high rate of concordance of 92-97.7% and correlated with the enzyme immunoassay. Positive discrepant results were found for Scl-70 specificity in 12.7% of SLE specimens by AtheNA technology, while all tested sera were negative for this antibody by enzyme immunoassay. Negative discrepant results were observed by the AtheNA system for anti-dsDNA. The sera (15 randomly obtained samples from SLE patients) were positive for anti-dsDNA in 50% of samples in Farr assay and 55% in enzyme immunoassay, respectively. CONCLUSION: We suggest that the AtheNA technology may be a useful diagnostic tool for ANA screening. Additional investigations are required to compare an analytic performance between AtheNA and routine methods in determination of the individual autoantibody profile.

Evaluation of Bacillus anthracis extractable antigen for testing anthrax immunity.

Three extractable Bacillus anthracis cell-wall-associated antigens were evaluated for potential use as skin testing agents, and as possible candidates for in-vitro diagnosis of anthrax immunity. Anthraxin and a partially purified extractable antigen (EAP) were produced from avirulent B. anthracis strain 34F2 (Sterne). The thermoextractable antigen used for the Ascoli reaction was obtained commercially. Guinea-pigs were immunised and boosted several times subcutaneously with the Sterne live veterinary anthrax vaccine. Four weeks after the last booster dose, animals were skin-tested with the three antigens. Serum antibody levels were also determined by ELISA, and the in-vitro T-cell response was evaluated by [3H]-thymidine incorporation. EAP was the most active antigen in both the serological and cellular reactions. EAP also elicited a distinct positive skin reaction in animals immunised with B. anthracis. The data obtained in this preliminary study indicated that extractable cell-wall antigens obtained from the vegetative form of B. anthracis may be used for skin tests and in-vitro testing of specific humoral and cell-mediated anthrax immunity.

Antineutrophil cytoplasmic autoantibodies penetrate into human polymorphonuclear leukocytes and modify their apoptosis.

OBJECTIVE: The interaction of extracellular anti-neutrophil cytoplasmic autoantibodies (ANCA) with neutrophilic granules may play an important role in the pathogenesis of ANCA-related disorders. It has been confirmed that apoptosis is an essential trigger associated with translocation of the cytoplasmic granules to the cell surface, and with the expression of ANCA antigens. Since cell penetration by autoantibodies and apoptosis may be associated processes, we tested the hypothesis that penetration of ANCA-autoantibodies into polymorphonuclear leukocytes (PMNs) has an effect on apoptosis and thereby can influence surface antigen expression. METHODS: PMNs were isolated from the blood of healthy volunteers and incubated in the presence of anti-proteinase3 (PR3) enriched IgG or normal human IgG. For each period of incubation (40 minutes or 12 hours) we evaluated: 1) PMN morphology by light microscopy (LM) and transmission electron microscopy (TEM) for general estimation of the apoptotic process, and 2) ANCA binding to the target antigen by immunogold electron microscopy (IgEM). RESULTS: Both normal and anti-PR3 IgG penetrate PMNs. The labeled PR3-ANCA were localized on PR3 granules, regardless of the granules' location within the cell, and in the sites where the PMN destruction processes were most expressed. The destructive processes showed extensive apoptotic characteristics, in contrast to PMNs penetrated by normal IgG. CONCLUSION: PR3 ANCA penetrate PMNs and, via the interaction between PR3-ANCA and PR3-containing granule components, initiate a modification of the apoptotic process.