Multiple Nuclear Dots Research Articles

VI Brazilian consensus guidelines for detection of anti-cell autoantibodies on HEp-2 cells

BackgroundThe VI Brazilian Consensus on Autoantibodies against HEp-2 cells for determination of autoantibodies against cellular constituents on HEp-2 cells was held on September, 2019, in Fortaleza (CE, Brazil). The guidelines in this edition were formulated by the group of Brazilian experts discussing the classification of complex patterns, the classification of the nuclear discrete dots (few and multiple), the identification of the discrete fine speckled pattern (AC-4a) and improvements on the ANA report.MainbodySixteen Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of Brazil participated in the meeting. Four main topics were discussed: (1) How to classify patterns with fluorescence in more than one cell compartment considering three relevant categoris: composite patterns, mixed patterns and multiple patterns; (2) The splitting of the discrete nuclear dots pattern into the multiple discrete nuclear dots (AC-6) and few discrete nuclear dots (AC-7) patterns, respectively; (3) Inclusion of a novel nuclear pattern characterized by discrete fine speckled pattern highly associated with antibodies to SS-A/Ro60, classified as AC-4a. In addition, adjustments on the Brazilian Consensus nomenclature were implemented aiming to harmonize the designation of some patterns with the International Consensus on ANA Patterns (ICAP). Furthermore, the designations of the PCNA-like pattern (AC-13), CENP-F-like pattern (AC-14) and Topo I-like pattern (AC-29) were adjusted in accordance to ICAP. Finally, there was a recommendation for adjustment in the test report in order to address the status of nuclear envelope staining. For all topics, the aim was to establish specific guidelines for laboratories and clinicians. All recommendations were based on consensus among participants. All recommendations from the V Consensus were maintained and there was relevant progress in the BCA/HEp-2 guidelines and further harmonization with ICAP.ConclusionThe VI BCA/HEp-2 edition was successful in establishing important recommendations regarding the classification of complex patterns, in supporting the identification of a novel pattern within the AC-4 group and in the harmonization process with the ICAP terminology.

Presence of Serum Antinuclear Antibodies Does Not Impact Outcomes in HBV-Related Acute-on-Chronic Liver Failure.

Background The aim of this study was to provide new insights into the prevalence of positive antinuclear antibody (ANA) in patients with HBV-related acute-on-chronic liver failure (ACLF) and its impact on clinical outcomes. Methods A total of 116 patients with HBV-related ACLF treated at three clinical centers were retrospectively recruited. Serum concentrations of ANA were detected using the enzyme-linked immunosorbent assay kit. Multiple nuclear dots, rim-like, and centromere patterns of ANA were detected using indirect immunofluorescence assay on HEp-2 cells. Results Among the 116 patients with HBV-related ACLF, 17 (14.66%) were ANA positive. Most patients in both ANA positive and negative groups were males (88.2% and 83.8%). Patients with negative ANA had a higher international normalized ratio, model for end-stage liver disease (MELD), and MELD-sodium scores than those with positive ANA (all P < 0.05). Multiple nuclear dot pattern was detected in half of the patients (8/17, 47.06%), rim-like/membranous pattern was found in six patients, and centromere pattern was detected in the last three patients. For patients with ANA (+), IgM was lower, and it was positively correlated with IgG. For patients with ANA (-), C3 was positively correlated with C4, and both C3 and C4 were negatively correlated with INR and MELD (all P < 0.05). In addition, TBIL, INR, WBC, and PLT, but not ANA, resulted as independent risk factors associated with 90-day mortality. Conclusion Positive ANA is frequent in HBV-related ACLF, and it does not seem to be associated with poor outcomes, but the pathogenesis of ACLF may be different between ANA (+) and ANA (−) groups.

Investigation of the Contribution of Autoantibodies to Clinical Diagnosis in Liver Pathologies and the Identification of Accompanying Autoimmune Diseases

Autoantibodies are frequently detected in the presence of autoimmune liver diseases (ALD) [autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC)] and are widely used to classify the disease clinically. The aim of this study was to investigate the contribution of autoantibodies for the diagnosis of ALD and the identification of other accompanying systemic autoimmune rheumatic diseases (SARD). In addition, it was aimed to compare the results of indirect immunofluorescence (IIF) antinuclear antibody (ANA) patterns and extractable nuclear antigen (ENA) antibodies. A total of 593 patients, including 544 patients with high liver function tests from general surgery/gastroenterology clinics and 49 patients referred from internal medicine/rheumatology clinics to investigate ALD, were included in the study. HBsAg and anti-HCV test results of the patients were found to be negative. ANA, anti-mitochondrial antibody (AMA)/anti-liver-kidney microsomal antibody (LKM), anti-smooth muscle antibody (ASMA), antineutrophil cytoplasmic antibody (ANCA) assays were performed by indirect immunofluorescence method (IIF) (Euroimmune AG, Luebeck, Germany). Extractable nuclear antigen (ENA) (nRNP/Sm, Sm, SS-A, SS-B, Scl-70, Jo-1, dsDNA, nucleosome, histone, ribosomal P-protein, AMA-M2, Ro-52, PM-Scl, CENP-B, PCNA, DFS70) and liver profiles [soluble liver antigen\liver pancreas antigen (SLA/LP), liver cytosolic antigen1(LC-1), LKM-1, anti-mitochondrial antibody M2(AMA-M2)] (Euroimmune AG, Luebeck, Germany) were detected by immunoblot (IB) method. Demographic characteristics, clinical data, presence of systemic autoimmune rheumatic diseases (SARD), radiological and laboratory findings were determined from the medical records. Autoantibody tests were found to be negative in 461 (77.7%) of 593 patients (mean age= 53.3 ± 15.6, age range= 18-90), and were positive in 132 (22.3%) (86.4% female) of the patients. Of the patients with positive autoantibodies, 60.6% (80/132) were diagnosed as PBS and 1.5% (2/132) were diagnosed as AIH (positive anti-LC-1 and anti-LKM1 antibodies). Fourteen of the patients (10.6%) with centromere, nuclear membrane (NM), multiple nuclear dot (MND) staining patterns and elevated liver enzymes could not be diagnosed as a specific disease and were followed up. PBS (13/30) was detected in approximately half of the patients diagnosed with SARD. The most common accompanying SARD in PBC patients was Sjögren's syndrome (SS) (7.5%, 6/80), followed by rheumatoid arthritis (RA) (5.0%, 4/80), scleroderma (2.5%, 2/80), and systemic lupus erythematosus (SLE) (1.3%, 1/80) respectively. The most common pattern was the AMA staining pattern (34.8%, 46/132) among the autoantibody positive patients. AMA and ANA staining patterns were detected together in 31.1% (41/132) of the patients. In the ENA profile results of these patients, the most common profile detected was anti-Ro-52 (65.9%, 27/41), followed by anti-SSA (34.1%, 14/41), anti-SSB (22.0%, 9/41) and anti-CENP-B (12.2%, 5/41) autoantibodies , respectively. ANA patterns were detected in 32.6% (43/132) of the patients (NM 9.1%, centromere 9.1%, MND 6.8%, respectively). In our study, 87.5% (70/80) of the patients diagnosed as PBS were found to have AMA positivity and 12.5% (10/80) of them had ANA positivity (such as NM, CNN, centromere). The characteristics, laboratory and radiological findings of the patients with isolated AMA positivity alone (Group 1) and patients with multiple patterns/autoantibodies (Group 2) were compared. In patients with multiple patterns/autoantibodies (Group 2), the presence of cirrhosis and liver heterogeneity were found to be higher than Group 1 (p= 0.049). ALD associated autoantibodies can be detected before years from the clinical disease. ALD may be associated with various SARD. Detection of ALD-related autoantibodies in patients diagnosed with SARD can provide early diagnosis of these patients. These autoantibodies guide both diagnosis and prognosis as in PBC. Collaboration between the laboratory specialist and the clinician is critical in the diagnosis, management and early recognition of these patients before clinical disease.

Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern.

IntroductionThe indirect immunofluorescence assay on HEp-2 cells (HEp-2/IFA) is used worldwide for screening for autoantibodies to cellular antigens. Cell culture and fixation methods influence the cell distribution of autoantigens and the preservation of epitopes. Therefore, discrepancy of results obtained using different HEp-2/IFA kits (interkit nonreproducibility) is a common phenomenon in the clinical laboratory routine.ObjectiveThis study evaluated the interkit nonreproducibility of HEp-2/IFA results using samples from patients with systemic autoimmune disease (SAD), nonautoimmune diseases (NAD), and healthy blood donors (HBD).MethodsSerum from 275 SAD patients, 293 NAD patients, and 300 HBD were processed at 1:80 dilution using four HEp-2 kits according to the manufacturers’ instructions. Interkit reproducibility was determined for positive/negative results and patterns. The agreement of positive/negative results among kits for each sample was determined as the reactivity agreement score (RAS). The pattern reproducibility score (PRS) in each sample was calculated as a function of the number of kits showing equivalent patterns. Qualitative variables and ordinal variables were analyzed by the Chi-square and Mann-Whitney U tests, respectively.ResultsA total of 402 samples were nonreactive in all kits and were considered devoid of autoantibodies. Further analysis included the 466 reactive samples (238 SAD, 119 NAD, 109 HBD). Reactivity to the nucleus had the highest interkit reproducibility (RAS = 83.6), followed by the metaphase plate (RAS = 78.9), cytoplasm (RAS = 77.4), and nucleolus (RAS = 72.4). Interkit reproducibility was higher in SAD (RAS = 78.0) than in NAD (RAS = 70.6) and HBD (RAS = 71.3) groups. Samples with strong reactivity (++++/4 and +++/4) had higher interkit reproducibility than those with weak reactivity (+/4). In the SAD group, RAS for nuclear reactivity was 87.5% for strongly reactive samples as opposed to 4.4% for weakly reactive samples, and the same was observed for NAD and HBD samples. The most robust patterns were the centromere AC-3 (PRS = 78.4), multiple nuclear dots AC-6 (PRS = 73.6), nuclear coarse speckled AC-5 (PRS = 71.3), nuclear homogeneous AC-1 (PRS = 67.9), and the reticular cytoplasmic AC-21 (PRS = 68.6).ConclusionInterkit nonreproducibility in HEp-2/IFA is prevalent and occurs with the highest frequency with weakly reactive samples. International initiatives with the engagement of in vitro diagnostic industry are encouraged to promote the harmonization of the properties and performance of HEp-2/IFA commercial kits.

The diagnostic accuracy of biomarkers for diagnosis of primary biliary cholangitis (PBC) in anti-mitochondrial antibody (AMA)-negative PBC patients: a review of literature.

Primary biliary cholangitis (PBC), also known as primary biliary cirrhosis, is an autoimmune disease of the liver characterized by anti-mitochondrial antibodies (AMA) in 90%-95% of patients. The aim of this study was to evaluate the diagnostic value of several serum biomarkers in patients with PBC but negative for AMA. Some antinuclear antibodies (ANA) pattern, detected by indirect immunofluorescence (IIF), such as multiple nuclear dot (MND) and rim-like patterns are well-known to be specific for PBC. The corresponding nuclear antigens are the components of the nuclear pore complex (Gp210 for rim-like pattern) and Sp100, PML proteins (for MND pattern) detectable by immunoblotting and ELISA methods. More recently, new biomarkers have been evaluated in order to improve the diagnostic sensitivity, such as kelch-like 12 (KLHL12) and hexokinase-1. Considering these different serum biomarkers, studies evaluating their diagnostic role in AMA-negative PBC patients compared to AMA-positive ones and controls were included in this review. Pooled sensitivity and specificity were 37% and 85%, respectively. The overall PPV and NPV mean values were 45% and 83%. Even if all biomarkers are very specific for PBC, the overall sensitivity was poor and therefore is necessary to identify a marker with a greater sensitivity for PBC in AMA-negative patients.

Diagnostic autoantibodies for autoimmune liver diseases.

Autoimmune liver diseases are conditions of low prevalence that comprise the triad of autoimmune hepatitis, primary biliary cholangitis (cirrhosis) and primary sclerosing cholangitis and their poorly characterised overlapping syndromes. Diagnostic autoantibodies are associated with autoimmune hepatitis and primary biliary cholangitis but not with primary sclerosing cholangitis. Autoantibodies are useful disease markers that facilitate early diagnosis of autoimmune hepatitis and primary biliary cholangitis and allow for therapeutic intervention to prevent progression to liver cirrhosis and associated complications. Adult onset type 1 autoimmune hepatitis is associated with F-actin reactive smooth muscle autoantibody, antinuclear autoantibody in 60% of patients, and autoantibody to SLA/LP in 15–20%. Juvenile onset type 2 autoimmune hepatitis is associated with LKM-1 and LC-1 autoantibodies. Primary biliary cholangitis is associated with a mitochondria-associated autoantibody designated M2 in >90% of patients and with disease-specific antinuclear autoantibodies in 50% that bind to antigens in the nuclear core complex and in multiple nuclear dots. Autoantibodies to the nuclear core complex target gp210, nucleoporin p62 and nuclear lamin B receptor. Autoantibodies to multiple nuclear dots target Sp100 and PML antigens. Liver autoantibodies in asymptomatic patients with normal liver function may precede the subsequent development of overt autoimmune liver disease. For routine diagnostic immunology laboratories, initial screening for liver autoantibodies by immunofluorescence remains the method of choice with confirmation for reactivity with their target antigen by enzyme-linked immunosorbent assay (ELISA) or line blot when required.

Autoantibodies in breast cancer sera are not epiphenomena and may participate in carcinogenesis.

BackgroundThe objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases.MethodsWe performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens.ResultsAutoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative.ConclusionsThe distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women.Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1385-8) contains supplementary material, which is available to authorized users.

Incidental antinuclear autoantibody to sp100 and gp210 in subjects with normal liver function tests

Aim Primary biliary cirrhosis (PBC) specific antinuclear antibodies (ANA) are found in about 50% of patients with PBC. They are largely directed to sp100 and gp210 and are associated with ANA immunofluorescence (IIF) staining patterns of multiple nuclear dots (MND) and nuclear pore complex (NPC) respectively. They have not previously been reported in subjects with normal liver function tests (LFTs). We review the incidental incidence of these autoantibodies in our test population and their association with normal and cholestatic LFTs. Method Retrospective analysis of lineblots performed based on ANA IIF pattern of MND and NPC. LFTs were recorded from date of ANA testing and at one and two years of follow up. Results 187 lineblots were positive for autoantibody to sp100 and/or gp210 detected on the basis of ANA pattern to MND and NPC. Twenty-nine patients with anti-gp210 antibodies and 51 patients with anti-sp100 antibodies detected incidentally on the basis of ANA pattern remained biochemically and symptomatically stable during a follow up period of one year. Discussion The population identified here could serve as a basis for long term studies, contributing to our knowledge as the predictive value of these specificities detected incidentally is currently unknown.

Membrane strip affinity purification of autoantibodies.

A method for affinity purification of autoantibodies from a membrane strip using small volumes of human sera is described. The membrane strip is excised from a western blot containing a target antigen electrophoretically transferred from a sodium dodecyl sulfate (SDS) polyacrylamide gel. This method is a very useful alternative for affinity column chromatography, particularly when the antigen of interest is of low abundance in a HeLa cell extract. The protein mixture is resolved on a preparative SDS polyacrylamide gel and transferred to nitrocellulose membrane. A couple of strips are excised from either side of the blotted membrane and immunoblotted with specific antisera to identify the target band. Then the target band is excised horizontally and used for affinity purification. We have used this procedure to affinity purify antibodies to a 70,000 molecular weight protein derived from HeLa cell extract. A sham band, excised away from the target antigen, was used as a control for sham purification of autoantibodies. The autoantibodies purified in this manner reproduced the multiple nuclear dot anti-nuclear antibody pattern obtained using crude sera from 21 patients without primary biliary cirrhosis or anti-mitochondrial antibody.

Autoantibody profiling of patients with primary biliary cirrhosis using a multiplexed line-blot assay

ObjectiveTo evaluate the autoantibody profile in patients with primary biliary cirrhosis (PBC) using a new multiplexed line-blot assay specifically designed for the diagnosis of autoimmune liver diseases. MethodsSera of 58 consecutive PBC patients and 191 disease controls (144 with autoimmune liver diseases other than PBC, and 67 with non-autoimmune chronic liver diseases) were tested by both the multiplexed line-blot Autoimmune Liver Disease Profile 2 (ALD2) and by IIF on HEp-2 cells and on rat kidney/liver/stomach tissues. ALD2 contains the following PBC-associated antigens: AMA-M2, natively purified from bovine heart; M2-E3, a recombinant fusion protein including the E2 subunits of PDC, BCOADC and OGDC; sp100, PML and gp210 recombinant proteins. ResultsWith the ALD2 assay, a positive reaction to AMA-M2, M2-E3, sp100, PML and gp210 in PBC patients was observed in 77.6%, 84.5%, 34.5%, 15.1% and 18.9%, respectively, of the PBC sera. The overall sensitivity and specificity for PBC were 98.3% and 93.7%. Using IIF, positivity rates to AMA, and to antinuclear autoantibodies with membranous/rim-like and multiple nuclear dot patterns were 86.2%, 8.6% and 22.4%, respectively. The overall sensitivity and specificity for PBC of the IIF method were 86.2% and 97.9%, respectively. ConclusionsThe ALD2 line-blot showed a good diagnostic accuracy for PBC and a higher sensitivity than the IIF method to detect sp100 and gp210 autoantibodies.

Associations with Organ Involvement and Autoantibodies in Systemic Sclerosis: Results from the Canadian Scleroderma Research Group (CSRG)

Objective: Serum from SSc patients was analyzed centrally to determine ANA patterns and extractable nuclear antigens (ENAs) between lcSSc and dcSSc and associations with organ involvement. Methods: 1145 SSc patients had ANA and ENA analyzed by indirect immunofluorescence on HEp-2 substrate at a screening serum dilution of 1/160. Most ENA antibodies [Sm. U1-RNP, Ro52, SS-A/Ro60, topoisomeraseI (Topo1), SS-B/La, chromatin, ribosomal P and Jo1] were measured by laser bead immunoassay; and RNA polymerase III (RNAP) by ELISA. Results: ANA was positive in 95% (same in lcSSc, and dcSSc). Centromere pattern was present in 34%, speckled 22%, nucleolar 18%, homogeneous and speckled (H&S) 16%, multiple nuclear dots 6%. Anti-centromere Ab (ACA) occurred in 46% of lcSSc and 11% of dcSSc (P = 0.0001). ENAs that differed between lcSSc and dcSSc subsets were Topo1 (OR 2.4, P = 0.0001) and RNAP (OR 5.6, P 0.0001) more common in dcSSc. Overall, 15% had positive Topo1; usually with a H&S pattern (67%); Topo1 was associated with ILD on CXR (OR 2.3; 95% CI 1.5 - 3.5) and HRCT (OR 3.8; 95% CI 1.8 - 8.2). RNAP occurred in 18.5% (35.4% in dcSSc vs. 8.9% in lcSSc). Scleroderma renal crisis (SRC) was 13 times more likely if RNAP positive; P = 0.0001. ACA was only weakly associated with sPAP > 50 mmHg (OR 1.8; 95%CI 1.1 - 3.0). Conclusion: ANA homogeneous pattern alone is rare in SSc; ACA was significantly more common in lcSSc. Many ENAs are equal in lcSSc and dcSSc except RNAP and Topo1. RNAP has the highest OR of SRC. Topo1 is less strongly associated with ILD. Abstract word count: 249, Body word count 1246, Figures 2, Tables 2. Key Messages: 1) 95% of SSc has a positive ANA and ANA patterns in SSc include centromere, nucleolar, and homogeneous and speckled together; 2) Most ENAs are equal in both dcSSc and lcSSc except anti RNA polymerase III and topoisomerase I; 3) RNA polymerase III has the highest association (odds ratio) with scleroderma renal crisis, topoisomerase I is associated with interstitial lung disease; whereas anticentromere was not associated with elevated pulmonary arterial pressures on echocardiogram.

Diagnostic and clinical utility of antibodies against the nuclear body promyelocytic leukaemia and Sp100 antigens in patients with primary biliary cirrhosis

BackgroundThe lack of an immunoassay that detects antibodies to promyelocytic leukaemia (PML) protein, the primary biliary cirrhosis (PBC)-specific multiple nuclear dot (MND) antigen, has prompted us to develop a line immunoassay (LIA) for the simultaneous detection of PML and Sp100 MND-specific autoantibodies. MethodsPML and Sp100 were expressed in Escherichia coli, and analysed by SDS-PAGE and immunoblotting using a monoclonal antibody and MALDI-ToF fingerprinting. A quantitative PML and Sp100 LIA were developed and testing was performed in 150 anti-mitochondrial antibody (AMA) positive, 20 AMA-PBCs and 130 controls. ResultsThirty-five (23%) of 150 AMA+ PBCs (18 anti-MND+) were anti-PML+ (12%) or anti-Sp100+ (20%), 10 being anti-PML+/Sp100+, 5 single anti-PML+ and 20 single anti-Sp100+. Six (30%, 5 anti-MND+) AMA-PBCs were anti-PML+ or Sp100+. Only 2 (1.7%) pathological controls were anti-PML+ and/or anti-Sp100+. Levels of anti-PML correlated with those of anti-Sp100 (R=0.64, p<0.0001). The autoantibody profile largely remained unchanged over a 10year-follow up (52 patients, 352 samples). Anti-PML, Sp100 or MND-reactive PBCs were younger and had longer disease duration than the seronegative (p=0.06, for both). Anti-Sp100 levels correlated with the Mayo risk score (r=0.63, p=0.01). Anti-PML+/Sp100+ patients had more advanced disease compared to patients negative for anti-PML/Sp100 (p=0.04). ConclusionThe new line immunoassay offers a robust and accurate method for the detection of clinically-relevant PBC-specific anti-MND antibodies.

Patients With Autoimmune Hepatitis Who Have Antimitochondrial Antibodies Need Long-term Follow-up to Detect Late Development of Primary Biliary Cirrhosis

Patients with autoimmune hepatitis (AIH) who have antibodies against mitochondrial proteins (AMA positive) are believed to have an autoimmune syndrome that should be managed as AIH. Of patients with AMA-positive AIH, we report on 3 individuals to demonstrate how autoimmune liver disease can progress over time. Specific features of primary biliary cirrhosis (PBC) overlapped in time in these patients. Our observations indicate the importance of careful follow up of patients with AMA-positive AIH; health care professionals that treat such patients should therefore be aware of longitudinal clinical changes that might indicate development of PBC in this setting.

Antinuclear antibodies as ancillary markers in primary biliary cirrhosis

Antimitochondrial antibodies are the serological hallmark of primary biliary cirrhosis (PBC). Besides antimitochondrial antibodies, the autoantibody profile of PBC includes antinuclear antibodies (ANA) which are detectable by indirect immunofluorescence in up to 50% of PBC patients. Two immunofluorescence patterns are considered ‘PBC-specific’: the multiple nuclear dots and rim-like/membranous patterns. The target antigens of the multiple nuclear dots pattern have been identified as Sp100 and promyelocytic leukemia protein, whereas the rim-like/membranous pattern is given by autoantibodies recognizing multiple proteins such as gp210, nucleoporin p62 and the lamin B receptor. Other ANA, especially those already known in the rheumatological setting, such as anticentromere, anti-SSA/Ro and anti-dsDNA antibodies, can be frequently found in PBC, often coexisting in the same patient. In this article, we will report on recent progress in the antigenic characterization of ANA in PBC, their detection with both traditional assays and Western blot/ELISA with molecularly defined nuclear antigens, and we will discuss their clinical significance.

Clinical significance of conventional serum autoantibodies for various liver diseases in a Chinese population

The aim of this study was to investigate the prevalence of autoantibodies in various liver diseases and evaluate the performance parameters of AMA, ANA and their combinations for PBC. This study enrolled 115 PBC patients, 695 patients with other liver diseases and 105 healthy subjects. AMA were detected mainly in PBC patients, while ANA were detected in all categories. The high titer and multiple nuclear dot, rim-like and centromere (MRC) patterns of ANA were found in the majority of PBC patients, in contrast to moderate titers and other patterns. All the liver diseases but HAV and NAFLD had significantly independent association with ANA. The combination of MRC patterns and AMA showed superior performance parameters for PBC. Although ANA can occur in various liver diseases, high titer and MRC patterns of ANA are more specific for PBC. The combination of MRC patterns and AMA has superior performance for PBC.

Anti-mitochondrial antibodies: what’s new?

<h3>Introduction</h3> The method of choice for anti-mitochondrial antibody detection has been indirect immunofluorescence (AMA-IIF) on fresh frozen sections of rodent liver, kidney and stomach with an accepted sensitivity of between 90 and 95%. However, primary biliary cirrhosis (PBC) is a disease of variable prognosis and up to now there have been no assays that have been able to predict patients who are more likely to progress to liver failure. Recently, a line blot assay has become available (EUROIMMUN) that utilises both native PDC and the recombinant ‘triple' E2 subunit antigen M2-3E in addition to PBC-specific anti-nuclear antigens (ANAs), gp210 (nuclear pore), Sp100 (multiple nuclear dots) and PML (promyelocytic leukaemia nuclear bodies). <h3>Aim</h3> To investigate the performance of this assay in identifying patients with clinical PBC and to investigate the performance of this assay in identifying patients who are more likely to progress to more severe PBC based on profiling reactivities to M2, M2-E3 and the PBC-specific ANAs. <h3>Patients</h3> Patients included 48 consecutive AMA-IIF positive patients detected during routine autoantibody investigation, 37 patients being followed up in specialist gastroenterology clinics for PBC and 49 patients receiving a liver transplant for PBC. <h3>Results</h3> A review of the available clinical, biochemical and biopsy data on the 48 consecutive AMA-IIF positive patients found that patients who were anti-M2 and anti-M2-E3 positive were more likely to have clinical PBC at the time of testing, <i>p</i> ≤ 0.005 compared to those who were anti-M2/M2-E3 negative. These results were confirmed when all three groups were compared. The frequency of anti-M2 and anti-M2-E3 positivity was significantly higher in the PBC transplant group, <i>p</i>=0.0025 compared to the two groups. The frequency of anti-gp210 was also significantly higher in the PBC transplant group, <i>p</i>=0.026, supporting previous reports that anti-gp210 appears to be a significant risk factor for hepatic failure in PBC patients. We also confirmed that anti-Sp100 positivity was not clinically important in PBC patients. However, the frequency of anti-PML positivity was higher in the PBC transplant group. <h3>Conclusion</h3> We conclude that testing for anti-M2, anti-E3, anti-gp210 and anti-PML may be useful in identifying PBC patients who are at greater risk of disease progression.

PML Nuclear Body Component Sp140 Is a Novel Autoantigen in Primary Biliary Cirrhosis

Some patients with primary biliary cirrhosis (PBC) have antinuclear antibodies (ANAs). These ANAs include the "multiple nuclear dots" (MND) staining pattern, targeting promyelocytic leukemia protein (PML) nuclear body (NB) components, such as "speckled 100-kD" protein (Sp100) and PML. A new PML NB protein, designated as Sp140, was identified using serum from a PBC patient. The aim of this study was to analyze the immune response against Sp140 protein in PBC patients. We studied 135 PBC patients and 157 pathological controls with type 1 autoimmune hepatitis, primary sclerosing cholangitis, and systemic lupus erythematosus. We used indirect immunofluorescence and a neuroblastoma cell line expressing Sp140 for detecting anti-Sp140 antibodies, and a commercially available immunoblot for detecting anti-Sp100 and anti-PML antibodies. Anti-Sp140 antibodies were present in 20 (15%) PBC patients but not in control samples, with a higher frequency in antimitochondrial antibody (AMA)-negative cases (53 vs. 9%, P<0.0001). Anti-Sp140 antibodies were found together with anti-Sp100 antibodies in all but one case (19 of 20, 90%) and with anti-PML antibodies in 12 (60%) cases. Anti-Sp140 positivity was not associated with a specific clinical feature of PBC. Our study identifies Sp140 as a new, highly specific autoantigen in PBC for the first time. The very frequent coexistence of anti-Sp140, anti-Sp100 and anti-PML antibodies suggests that the NB is a multiantigenic complex in PBC and enhances the diagnostic significance of these reactivities, which are particularly useful in AMA-negative cases.

Multiple nuclear dots and rim-like/membranous IgG isotypes in primary biliary cirrhosis

Anti nuclear (ANA) immunomorphological patterns such as multiple nuclear dots (MND) and rim-like/membranous (RL/M) are considered highly specific but little sensitive for primary biliary cirrhosis (PBC) diagnosis. To evaluate frequency and clinical significance of MND and RL/M in PBC patients when investigated at the level of immunoglobulin G isotypes. MND and RL/M pattern have been tested in 141 PBC sera and 230 pathological controls using HEp-2 cells as substrate and anti- total IgG and individuaI IgG subclasses (IgG1, IgG2, IgG3, IgG4) as specific antisera. One hundred and fourteen of 141 (80%) PBC patients had RL/M or MND pattern when IgG subclasses were used as revealing reagents (vs. 34% when anti total IgG were used, p < 0.0001). The prevalent isotype was IgG1 for RL/M, and IgG2 for MND pattern. None of controls was positive. No clinical differences in terms of severity and outcome of disease have been observed in PBC patients positive for MND and RL/M when investigated with IgG isotypes. The research for RL/M and MND pattern at level of IgG isotype determines a wide gain in terms of sensitivity without a loss of specificity. In Italian PBC patients MND and RL/M pattern did not seem to characterize any subgroup of patients with a poorer prognosis.

Anti-nuclear antibodies of primary biliary cirrhosis recognize 78–92-kD and 96–100-kD proteins of nuclear bodies

The specificities of anti-nuclear antibodies (ANA) reacting with multiple nuclear dots (MND-ANA) present in about 15% primary biliary cirrhosis sera were studied by Western blot analysis with nuclear fractions from a human cell line. Reactivity with two broad bands of 78-92 kD and 96-100 kD of the insoluble fraction was present exclusively in MND-ANA-positive sera. Antibodies eluted from these proteins specifically retained the immunofluorescence reactivity of MND-ANA. Immunomorphological analysis by a pre-embedding technique revealed that the antibody specifically binds to nuclear regions resembling in size and number nuclear bodies. Since these structures are absent in immature rate endometrial cell and can be induced by diethylstilbestrol, we tested MND-ANA by immunofluorescence on cryostat sections of uteri from hormone-treated and untreated immature rats. A strong reaction of nuclear dots was observed predominantly in endometrial cells of hormone treated rats. We thus conclude that MND-ANA present in primary biliary cirrhosis sera are directed against 78-92-kD and 96-100-kD nuclear proteins located in nuclear bodies.

Patterns of anti-nuclear antibodies in patients with primary biliary cirrhosis and primary Sjögren syndrome

To investigate the anti-nuclear antibody (ANA) patterns in the diagnosis of primary biliary cirrhosis (PBC) and primary Sjögren syndrome (SS). Serum samples from 61 cases of PBC, 28 cases of primary SS, and 11 cases of overlap syndrome of PBC and SS were collected to detect the ANA with indirect immunofluorescence staining, and the difference in ANA patterns was analyzed between the PBC and SS patients. Antinuclear antibodies were detected in 85.2% of the PBC patients, with the following hierarchy of specificities: 37.7% being rim-like, 21.3% being discrete speckled, 18.0% being speckled, 8.2% being multiple nuclear dots, and 4.9% being anti-lamin; 89.3% of the primary SS patients was ANA positive with speckled antibody, only one of them presented discrete speckles (3.6%); and all of the PBC patients overlapped with SS (100%) were positive in ANA, and their contexture of ANAs was similar to those of the PBC patients: 45.5% being rim-like, 18.2% being discrete speckled, 18.2% being speckled, 18.2% being anti-lamin, and 9.1% being multiple nuclear dots. Rim-like pattern and multiple nuclear dots were statistically significant for the diagnosis of PBC, compared with primary SS (chi(2) = 14.236, 3.781, P < 0.01, < 0.05). The ANA patterns of PBC are different from those of primary SS. Among them, rim-like pattern and multiple nuclear dots are highly specific nuclear patterns of PBC and may be useful in diagnosing individuals without anti-mitochondrial antibodies.