Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.     

New genetically engineered DFS70 knock-out HEp-2 cells enable rapid and specific recognition of anti-DFS70 antibodies

Background: The correct identification of anti-dense fine speckled-70 (DFS70) antibodies represents an important issue in the detection of anti-nuclear antibodies (ANAs) as performed by the indirect immunofluorescence (IIF) test on HEp-2 substrates. In this study, we have evaluated a new method for anti-DFS70 antibody detection employing HEp-2 cells knocked-out for the DFS70 antigen.

Methods: We studied 148 sera with a DFS70-like pattern (91 positive and 57 negative when tested for anti-DFS70 antibodies by a specific chemoluminescence [CLIA] method); 116 sera with infectious disease; 100 healthy donors (HDs), 139 samples from patients with a defined diagnosis of autoimmune rheumatic disease (ARD), and 242 consecutive unselected samples screened for ANA during the routine work-up.

Results: The HEp2 DFS70-Ko substrate recognized anti-DFS70 antibodies in 86/91 (94.5%) of the DFS70 CLIA-positive sera and in 9/57 (15.8%) of the DFS70 CLIA-negative samples. None of the 116 infectious diseases were positive for DFS70 using the engineered IIF substrate. Two samples (2%) were positive among HDs and were then confirmed by CLIA. The 139 ANA-positive sera from patients with ARD displaying a defined antibody specificity showed their expected patterns also on DFS70-Ko HEp-2 substrate. Five of the 242 (2.1%) consecutive samples tested in the routine ANA-screening were identified as DFS70-positive using the HEp2 Ko-substrate and were then confirmed by CLIA.

Conclusions: The use of DFS70 HEp-2 Ko cells may offer the unique possibility of simultaneously identifying and confirming the presence of anti-DFS70 antibodies during the standard ANA evaluation, while keeping the expression of other autoantibody markers intact.     

Cytoplasmic staining pattern characteristic for anti Jo-1 antibody in HEp-2 cells]

We wished to confirm the staining pattern of HEp-2 cells by the anti Jo-1 antibody, because we found antibodies in serum with positive anti Jo-1 antibody which showed either a fibrilar cytoplasmic staining or a nuclear speckled staining pattern in indirect immuno fluorescence+ examinations using HEp-2 cells. Sera available from eight patients with PM DM (polymyositis and/or dermatomyositis) showed positive anti Jo-1 antibody in the double immuno-diffusion technique but had various staining patterns of HEp-2 cells in the immunofluorescent examination. We examined these eight sera with the immuno-blotting method utilizing whole cell extract of HeLa cells, and found the 50 kDa band from all sera tested, to which Jo-1 antigen had been reported to move. We eluted the antibody which formed the 50 kDa band from the nitrocellulose membrane and applied it on HEp-2 cells. This maneuver gave us the fine granular cytoplasmic staining of anti Jo-1 antibody on HEp-2 cells. We therefore concluded that the anti Jo-1 antibody should have cytoplasmic staining on HEp-2 cells although observers might miss it due to other types of associated antinuclear antibodies.     

Human autoantibodies to diacyl-phosphatidylethanolamine recognize a specific set of discrete cytoplasmic domains

The aim of this study was to characterize a novel human autoantibody-autoantigen system represented as cytoplasmic discrete speckles (CDS) in indirect immunofluorescence (IIF). A distinct CDS IIF pattern represented by 3-20 discrete speckles dispersed throughout the cytoplasm was identified among other cytoplasmic speckled IIF patterns. The cytoplasmic domains labelled by human anti-CDS-1 antibodies did not co-localize with endosome/lysosome markers EEA1 and LAMP-2, but showed partial co-localization with glycine-tryptophan bodies (GWB). CDS-1 sera did not react with several cellular extracts in immunoblotting and did not immunoprecipitate recombinant GW182 or EEA1 proteins. The typical CDS-1 IIF labelling pattern was abolished after delipidation of HEp-2 cells. Moreover, CDS-1 sera reacted strongly with a lipid component co-migrating with phosphatidylethanolamine (PE) in high performance thin-layer chromatography (HPTLC)-immunostaining of HEp-2 cell total lipid extracts. The CDS-1 major molecular targets were established by electrospray ionization-mass spectrometry (ESI-MS), HPTLC-immunostaining and chemiluminescent enzyme-linked immunosorbent assay as diacyl-PE species, containing preferentially a cis-C18 : 1 fatty acid chain at C-2 of the glycerol moiety, namely 1,2-cis-C18 : 1-PE and 1-C16 : 0-2-cis-C18 : 1-PE. The clinical association of CDS-1 sera included a variety of systemic and organ-specific autoimmune diseases but they were also observed in patients with no evidence of autoimmune disease.     

Characteristic nucleolar-reticular immunofluorescence staining pattern on touch prints of hamster liver: a marker for Scl-70 antibody

Sera from 48 patients with systemic sclerosis (SSc) and from 62 patients with other connective tissue diseases were studied for nuclear staining patterns by indirect immunofluorescence (IIF) using touch prints of hamster liver, HEp-2 and cryostat sections of monkey esophagus as substrates. Immunodiffusion studies performed in all cases disclosed Scl-70 antibody in 36 SSc patients. The IIF pattern of Scl-70 antibody is characterized by diffuse granular and nucleolar staining on HEp-2, a combined nucleolar and diffuse reticular staining on imprints of hamster liver and large speckled (nucleolar) fluorescence on cryostat sections of monkey esophagus. Touch prints of hamster liver revealed a partially nucleolar fluorescence as a dominant sign of the combined staining. Hamster liver substrate which is easily available might replace the expensive HEp-2 cells for detecting Scl-70 antibody.     

A comparative study on the reliability of an automated system for the evaluation of cell-based indirect immunofluorescence

Background

Automated interpretations systems for anti-nuclear antibody (ANA), anti-double stranded DNA antibody (dsDNAab), and anti-neutrophil cytoplasmic antibody (ANCA) assessment by indirect immunofluorescence (IIF) have been recently introduced. The aim of this study was to compare the diagnostic performance of the automated IIF reading system AKLIDES with both traditional visual interpretation of IIF by laboratory experts and confirmatory tests.

Methods

Visual and automated autoantibody interpretations of IIF findings using AKLIDES pattern recognition algorithms were performed for ANA on HEp-2 cells (n = 182), dsDNAab on Crithidia luciliae (n = 44) and ANCA on human neutrophils (n = 46). All serum samples tested by IIF for ANCA and dsDNAab were also assessed with the corresponding enzyme-linked immunosorbent assays (ELISAs). Out of the 182 sera tested for ANA by IIF, 116 were also assessed for antibodies to extractable nuclear antigens (ENA) by ELISA and dot immunoassay (DIA).

Results

ANA testing showed an excellent agreement between visual and AKLIDES reading (98.9%). The overall agreement of dsDNAab testing on C. luciliae substrate slides was 91.0%, whereas ANCA showed a concordance of 89.1%. There was a remarkable agreement of AKLIDES findings for dsDNAab with confirmatory tests.

Conclusion

Visual and automated interpretations of IIF findings for ANA, ANCA, and dsDNAab demonstrated a good agreement when assessing patients with suspected autoimmune diseases. Automated interpretation systems such AKLIDES may improve laboratory efficiency and support standardization of IIF in clinical laboratories.     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术（IIF）抗核抗体（ANA）检测法的底物HEp-2细胞，建立抗60kDRo60／SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA，克隆入真核表达载体pEGFP-C1，并转染HEp-2细胞。通过荧光显微镜、免疫印迹法（IBT）及IIF鉴定转染细胞（HEp-Ro60）中Ro60-绿色荧光蛋白（GFP）融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro／SSA对流免疫电泳（CIE）检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达，融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6．7倍（P〈0．01），而且2例HEp-2细胞上IIF检测为阴性的血清在HEI-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体，并增加了ANA检出的敏感性。     

Immunofluorescent patterns produced by antineutrophil cytoplasmic antibodies (ANCA) vary depending on neutrophil substrate and conjugate

BACKGROUND: The "International consensus statement on testing and reporting antineutrophil cytoplasmic antibodies (ANCA)" advocates screening by indirect immunofluorescence (IIF), but external quality assessment programmes often demonstrate different IIF patterns for a single serum. AIM: To determine whether the variation in IIF patterns can be attributed solely to errors in interpretation. METHODS: This study compared the IIF patterns produced by four sera (two with cytoplasmic or C-ANCA; one with perinuclear or P-ANCA with myeloperoxidase (MPO) specificity; and one P-ANCA without MPO specificity) that were tested in 11 different laboratories. The sera were examined according to individual laboratory protocols at dilutions of 1/10 to 1/40 using P1 (n = 4), P2 (n = 2), P3 (n = 2), or in house (n=3) neutrophil preparations and conjugates from manufacturers C1 (n = 3), C2 (n = 1), C3 (n = 2), C4 (n = 1), C5 (n = 2), and C6 (n = 2). The IIF patterns were noted in each laboratory, the testing repeated, and the fluorescent patterns photographed and subsequently discussed at a meeting of the Australian ANCA study group. RESULTS: All IIF patterns described in individual laboratories were confirmed on retesting and by the ANCA study group. Neutrophil substrates produced commercially or in house varied in their ability to demonstrate cytoplasmic granularity and interlobular accentuation, which distinguish between "C-ANCA" and "C-ANCA (atypical)". All commercial and in house neutrophil substrates demonstrated neutrophil nuclear extension of P-ANCA fluorescence, which correlates with MPO specificity. However, eight assays (eight of 43) from eight laboratories resulted in IIF patterns different from those usually seen. One of these produced a C-ANCA (atypical) rather than a C-ANCA pattern. The other seven resulted in at least some cytoplasmic fluorescence when the consensus pattern was P-ANCA with (n = 4) or without (n = 3) MPO specificity. These assays used three different commercial and one in house neutrophil substrate, and six different conjugates, with anti-IgG, anti-(Fab)'(2), anti-Ig (heavy and light chain), and anti-G, A, and M activity. Four of the seven assays tested on commercial substrates had used the manufacturer's conjugates. CONCLUSIONS: This study indicates that the variation in IIF patterns seen with ANCA positive sera tested in different laboratories does not necessarily result from errors in the interpretation of patterns and cannot be attributed solely to the use of a particular neutrophil substrate or conjugate, or to the use of substrate from one manufacturer and conjugate from another.     

Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases

The presence of anti-nuclear antibodies (ANA) is a hallmark of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and was recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of serum samples from healthy individuals (HI) have been reported to have a positive ANA test, the majority of which are directed to the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis of SARD, the reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test, significantly reduces the specificity and the positive likelihood of the ANA test. This has significant implications for diagnostic algorithms involving the detection of ANA. We summarize the current knowledge of anti-DFS70 antibodies and their impact on ANA testing. We also suggest a test algorithm which considers the DFS pattern and the presence of anti-DFS70 antibodies. In addition, we describe a novel method based on immunoadsorption of anti-DFS70 antibodies, which increases the specificity of the ANA HEp-2 test for SARD and which has the potential to overcome a significant limitation of the ANA HEp-2 assay.     

Ro60/SSA基因转染HEp-2细胞作为间接免疫荧光检测底物及其应用

本研究拟改造间接免疫荧光技术(IIF)抗核抗体(ANA)检测法的底物HEp-2细胞,建立抗60kDRo60/SSA抗体免疫荧光检测法。采用PCR扩增人源Ro60 cDNA,克隆入真核表达载体pEGFP-C1,并转染HEp-2细胞。通过荧光显微镜、免疫印迹法(IBT)及IIF鉴定转染细胞(HEp-Ro60)中Ro60-绿色荧光蛋白(GFP)融合蛋白的表达和抗原性。分别以HEp-Ro60以及HEp-2为底物的IIF检测10份抗Ro/SSA对流免疫电泳(CIE)检测阳性、其他抗体阴性血清以及对照血清。获得的转染细胞传十几代后仍具有较强的Ro60-GFP表达,融合蛋白保持Ro60的抗原性。IIF检测中HEp-Ro60的滴度比HEp-2增加了6.7倍(P<0.01),而且2例HEp-2细胞上IIF检测为阴性的血清在HEp-Ro60上为阳性。10例阳性血清中有8例出现了特征性荧光模式。结论是HEp-Ro60可用于IIF检测抗Ro抗体,并增加了ANA检出的敏感性。     

ANA和ENA检测在自身免疫性疾病中的应用评价

探讨抗核抗体和抗可溶性核抗原抗体的不同检测方法对自身免疫性疾病诊断的指导意义。回顾性分析2007年1月至2009年10月间在长征医院就诊的患者血清自身抗体的检测结果,549位患者,其中自身免疫病患者224例,非自身免疫病325例,所有患者血清同时检测ANA和ENA。以Hep-2细胞/肝组织为基质的间接免疫荧光法检测ANA,免疫印迹法检测ENA。两种检测方法产生4种检出模式:ANA+/ENA+、ANA-/ENA-、ANA+/ENA-和ANA-/ENA+。前两种模式共占检测的62.84%。ANA和ENA在自身免疫病患者中的阳性率(63.4%和58.5%)显著高于非自免病患者(16.9%和25.2%),ANA和ENA在自身免疫病组和非自身免疫病组间阳性率比较,差异有统计学意义(P<0.01)。两检测结果仅在MCTD和SLE患者中存在相关性(P<0.01),在其他观察组中不存在相关性(P>0.05)。间接免疫荧光法相对费时,而且需要操作者具备一定的经验,作为初筛实验,ANA的检测较ENA有更高的灵敏度,两者联合检测则将有利于提高检测的灵敏度和可靠性。     

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

BackgroundIndirect 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.MethodsWe 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.ResultsOverall 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 (Spearman's 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%.ConclusionsThis 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.     

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.     

Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA)

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjögren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P= 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P= 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P< 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.     

The association of solid-phase assays to immunofluorescence increases the diagnostic accuracy for ANA screening in patients with autoimmune rheumatic diseases

Aim

This study aimed to investigate whether it is useful both for diagnostic purposes and for cost savings to associate a solid-phase monotest called CTD screen to the immunofluorescence (IIF) test, to detect anti-nuclear and anti-cytoplasmic antibodies (ANA).

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.     

Evaluation of antinuclear antibodies by indirect immunofluorescence and line immunoassay methods′: four years′ data from Turkey

The presence of antinuclear antibodies (ANAs), directed against intracellular antigens, is a hallmark of systemic autoimmune rheumatic diseases. The indirect immunofluorescence (IIF) assay is among the most commonly used routine methods for ANA detection as the screening test. The objective of the study was to evaluate ANA patterns in a 4‐year period retrospectively. All 19 996 serum samples that were sent to the Laboratory of Medical Microbiology of the tertiary Hospital by any hospital department between 1 January 2009 and 1 January 2013 with a request to test for ANA, anti‐ENA or both were included in the study. Of these samples, 4375 (21.9%) were ANA‐IIF‐positive and 15621 (78.1%) were ANA‐IIF‐negative. The presented ANA‐positive samples consisted of 2392 (54.67%) homogenous, 818 (18.70%) speckled, 396 (9.05%) centromere, 242 (5.53%) nucleolar, 213 (4.87%) nuclear dots, 178 (4.07%) cytoplasmic (except for actin and golgi), 24 (0.55%) actin, 9 (0.21%) golgi, 53 (1.21%) nuclear membrane and 50 (1.14%) mixed pattern. Totally 7800 samples were examined by LIA. Of these samples, 3440 were positive and 4307 were negative with IIF and LIA. In addition, 22 samples were detected as IIF‐positive but LIA‐negative, whereas the rest 31 samples were IIF‐negative but LIA‐positive. ANA patterns in 22 IIF‐positive samples were homogenous (9), speckled (5), golgi (4), cytoplasmic (3) and nucleolar (1). SSA/Ro‐52, SSB/La and Scl‐70 positivity were detected in 31 IIF‐negative/LIA‐positive samples by LIA. The present study comes forward with its overall scope, which covers 4‐year data obtained in tertiary hospital located in the western part of Turkey.     

Anti-Granulocyte Antibodies (C-ANCA, P-ANCA, GS-ANA) Studied by Confocal Scanning Laser Fluorescence Microscopy, ELISA, and Chemiluminescence Techniques

Fifty-nine patient sera with antibodies against human polymorphonuclear neutrophil granulocyte (PMN) antigens, as determined primarily by indirect immunofluorescence microscopy (IIF) screening, were further analysed by enzyme-linked immunosorbent assays (ELISA). The antibodies were primarily characterized by their immunomorphological staining patterns on ethanol-fixed PMN as judged by conventional IIF microscopy, i.e. anti-neutrophil cytoplasmic antibodies (ANCA) giving a pancytoplasmic granular staining pattern (C-ANCA) or a diffuse perinuclear cytoplasmic pattern (P-ANCA), or granulocyte-specific anti-nuclear antibodies (GS-ANA) producing a homogeneous or peripheral nuclear staining pattern. The three distinct patterns were confirmed by confocal scanning laser IIF microscopy. As antigen substrates in the ELISA tests we used an extract from azurophil PMN granules, myeloperoxidase (MPO), and lactoferrin. As expected, most (but not all) of the C-ANCA positive sera turned out positive in the alpha-ELISA assay. Both P-ANCA and GS-ANA positive sera had high frequencies of antibodies against MPO. Occasional P-ANCA positive sera contained antilactoferrin antibodies. Although P-ANCA and GS-ANA in general probably represent the same type of auto-antibodies, we regard it appropriate to make a distinction between the two patterns, until the existence of 'true' granulocyte-specific ANAs has been ruled out. All sera were analysed for their ability to activate PMN in vitro as judged by the generation of a chemiluminescence (CL) response. Sera containing C-ANCA, as well as sera containing P-ANCA or GS-ANA, showed high frequencies of positive CL tests using 'resting' isolated PMN. The reactions were diminished, but not always abolished, by heat-treatment of the sera.