How should a district general hospital immunology service screen for anti-nuclear antibodies? An ‘in-the-field’ audit

Anti‐nuclear antibody (ANA) testing assists in the diagnosis of several immune‐mediated disorders. The gold standard method for detection of these antibodies is by indirect immunofluorescence testing on human epidermoid laryngeal carcinoma (HEp‐2) cells. However, many laboratories test for these antibodies using solid‐phase assays such as enzyme‐linked immunosorbent assay (ELISA), which allows for higher throughput testing at reduced cost. In this study, we have audited the performance of a previously established ELISA assay to screen for ANA, making comparison with the gold standard HEp‐2 immunofluorescence test. A prospective and unselected sample of 89 consecutive ANA test requests by consultant rheumatologists were evaluated in parallel over a period of 10 months using both tests. ELISA and HEp‐2 screening assays yielded 40 (45%) and 72 (81%) positive test results, respectively, demonstrating lack of concordance between test methods. Using standard and clinical samples, it was demonstrated that the ELISA method did not detect several ANA with nucleolar, homogeneous and speckled immunofluorescence patterns. None of these ELISA^NEG HEp‐2^POS ANA were reactive with a panel of six extractable nuclear antigens or with double‐stranded DNA. Nonetheless, 13 of these samples (15%) originated from patients with recognized ANA‐associated disease (n = 7) or Raynaud's phenomenon (n = 6). We conclude that ELISA screening may fail to detect clinically relevant ANA that lack defined specificity for antigen.     

Autoantibodies profile in the sera of patients with Sjogren's syndrome: the ANA evaluation--a homogeneous, multiplexed system

BACKGROUND: Flow-based, multiplex bead arrays (MBA) have been developed for a variety of applications including the detection of antibodies to extractable nuclear antigens (ENA). It offers a rapid and sensitive method to assess multiple analyses in a single tube/well. PURPOSE: To evaluate the Athena Multi-Lyte ANA Test System utilizes Luminex Corporation's MBA technology for the detection of antinuclear antibodies (ANA) and ENA antibodies in the sera of patients with Sjogren's syndrome (SS). METHODS: MBA assay was used to detect ANA and ENA antibodies in the sera of 37 patients with SS and 96 sera from healthy subjects. RESULTS: All patients were women. Their mean age was 48.7 years and the mean disease duration was 7.27 years. ANA was found in 3 (3%) sera of healthy subjects by the AtheNA system and in 2 (2%) sera by the ELISA kit. A 99% concordance between the 2 assays was found. A 94.6% concordance between the 2 assays was found by testing the sera of patients with SS for ANA. By the AtheNA system, none of the sera of 37 patients with SS had autoantibodies reacting with Sm, Jo-1, dsDNA or histones. Anti-RNP antibody was found in 5.4% of the sera and 2.7% of the sera reacted with Scl-70 and histones. Anti-SS/A and anti-SS/B were identified in 84 and 76% of the sera, respectively. CONCLUSION: The AtheNa Multi-Lyte ANA Test System offers a sensitive and specific result for the detection of ANA and ENA antibodies in the sera of patients with SS.     

Prevalence of systemic autoimmune rheumatic diseases and clinical significance of ANA profile: data from a tertiary hospital in Shanghai,China

It is necessary and useful to explore prevalence of various systemic autoimmune rheumatic diseases (SARDs) in patients with suspicion of having SARDs and to characterize antinuclear antibodies (ANA) profile for identifying different populations (SARDs and non‐SARDs). A total of 5024 consecutive patients with available medical records were investigated, whose sera had been tested for ANA profile, including ANA, anti‐dsDNA and anti‐extractable nuclear antigen (ENA) antibodies, between 31 January 2012 and 26 March 2014. Only 594 (11.8%) patients were diagnosed with SARDs of those suspected with SARDs. The prevalence of systemic lupus erythematosus (SLE) was highest (3.2%), followed by rheumatoid arthritis (RA) (2.5%), primary Sjögren's syndrome (pSS) (1.7%), ankylosing spondylitis (AS) (1.5%), etc. Of females, SLE also showed the highest prevalence (6%), while of males, AS showed the highest prevalence (1.9%). The prevalence of most SARDs was closely associated with age, except mixed connective tissue disease (MCTD), and the variation characteristics among different age groups were different among various SARDs. The prevalence of ANA was significantly increased in most SARD patients [especially in SLE, systemic sclerosis (SSc) and MCTD]. For anti‐ENA antibodies, in contrast to some autoantibodies associated with multiple SARDs (e.g. anti‐SSA, SSB, nRNP), others were relatively specific for certain diseases, such as anti‐dsDNA, Sm, histone, nucleosome and Rib‐P for SLE, anti‐SCL‐70 for SSc and anti‐Jo‐1 for polymyositis/dermatomyositis (PM/DM). Of note, ANA profile appeared to be of little significance for AS, ANCA‐associated vasculitis (AAV), polymyalgia rheumatic (PMR), adult‐onset Still's disease (ASD) and Behcet's disease (BD). The younger were more likely to have the presence of anti‐dsDNA, Sm, histone or Rib‐P for SLE, and anti‐SSA for RA or MCTD. No significant differences for frequencies of ANA and anti‐ENA autoantibodies were found between sexes in most SARDs, with the exception of RA and AS. The present study suggests that, of patients with SARDs‐like clinical manifestations, the proportion of those with true SARDS is small, for most of whom tests for autoantibodies are necessary and useful to help make a prompt and precise diagnosis.     

Modelling autoimmune rheumatic disease: a likelihood rationale

Immunoglobulins (Igs) and autoantibodies are commonly tested in sera from patients with suspected rheumatic disease. To evaluate the clinical utility of the tests in combination, we investigated sera from 351 patients with autoimmune rheumatic disease (ARD) rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) and 96 patients with nonautoimmune rheumatic disease (NAD) (fibromyalgia, osteoarthritis, etc.). Antinuclear antibodies (ANA), rheumatoid factor (RF), antibodies against DNA and extractable nuclear antigens (anti‐ENA), IgG, IgA and IgM were measured for all patients. Logistic regression analysis of test results was used to calculate each patient's probability for belonging to the ARD or NAD group as well as likelihood ratios for disease. Test accuracy was investigated using receiver‐operating characteristic (ROC) plots and nonparametric ROC analysis. Neither concentrations of IgG, IgA, IgM, anti‐DNA nor anti‐ENA gave a significant effect on diagnostic outcome. Probabilities for disease and likelihood ratios calculated by combining RF and ANA performed significantly better at predicting ARD than utilization of the diagnostic tests in isolation (P < 0.001). At a cut‐off level of P = 0.73 and likelihood ratio = 1, the logistic model gave a specificity of 93% and a sensitivity of 75% for the differentiation between ARD and NAD. When compared at the same level of specificity, ANA gave a sensitivity of 37% and RF gave a sensitivity of 56.6%. Dichotomizing ANA and RF as positive or negative did not reduce the performance characteristics of the model. Combining results obtained from serological analysis of ANA and RF according to this model will increase the diagnostic utility of the tests in rheumatological practice.     

ANA as an entry criterion for the classification of SLE

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with highly variable clinical and immunological manifestations. In the classification of patients with this condition, the presence of an antinuclear antibody (ANA) is an important element, with new criteria from the American College of Rheumatology and European League against Rheumatism positioning ANA positivity by an immunofluorescence assay on HEp2-cells (HEp2-IFA) or by an equivalent solid phase assay as the entry criterion. This positioning is based on assumptions about the frequency of ANA positivity in SLE as well as the reliability of the assays. Studies indicate that these assumptions are still a matter of uncertainty since both types of assay show considerable variability and patients with SLE may display negative results in ANA testing. These findings suggest caution in positioning ANA positivity as an entry criterion for classification and point to the value of alternative serological approaches for ANA determinations.     

Routine immunofluorescence detection of Ro/SS-A autoantibody using HEp-2 cells transfected with human 60 kDa Ro/SS-A

BACKGROUND: Ro/SS-A autoantibodies associated with systemic lupus erythematosus (SLE) and Sjögren syndrome may be missed during routine screening for antinuclear autoantibodies (ANA) by immunofluorescence using HEp-2 cells. AIMS: To investigate the use of HEp-2 cells transfected with human 60 kDa Ro/SS-A for routine detection of these antibodies. METHODS: 10,500 sera were screened at a dilution of 1:200 for Ro/SS-A antibodies, identified by intense immunofluorescence staining in 10-15% of hyperexpressing cells of either the nucleus and nucleolus combined or the nucleus alone. RESULTS: Ro/SS-A antibodies were identified in 160/2100 ANA positive sera (8%), of which seven were ANA negative (titre < 200) and 33 had weak ANA titres (200) in 85-90% of non-hyperexpressing "background" cells. Enzyme linked immunosorbent assay (ELISA) confirmed the presence of Ro/SS-A antibodies in 110 newly diagnosed Ro/SS-A positive sera. Of these, 50 reacted with Ro/SS-A, 51 with Ro/SS-A and La/SS-B, and nine with Ro/SS-A and other extractable nuclear antigen (ENA) specificities. Fifteen sera which did not show Ro/SS-A antibodies by immunofluorescence tested positive for Ro/SS-A by immunodiffusion, counter-immunoelectrophesis, or ELISA; of these, 14 had ANA titres > 200. Clinical data from 95 Ro/SS-A positive patients showed that 52% had SLE, 24% Sjögren syndrome, 8% rheumatoid arthritis, and 16% other diseases. CONCLUSIONS: (1) HEp-2 cells transfected with human 60 kDa Ro/SSA are useful for routine immunofluorescence detection for Ro/SS-A antibodies with a positive predictive value of 100%; (2) sera positive for Ro/SS-A antibodies by immunofluorescence should be tested for ENA by other methods because > 50% of these sera will have another ENA reactivity in addition to Ro/SS-A; (3) detection of Ro/SS-A by immunofluorescence may be missed in the presence of high titre ANAs; (4) with a detection sensitivity of 91%, a negative immunofluorescence results for Ro/SS-A does not exclude the presence of this autoantibody.     

含IgG类抗Sm抗体对HEp2细胞增殖周期的影响

本文研究进入性含IgG类抗 Sm抗体对体外培养的HEp 2细胞的影响。结果表明 :经SLE患者IgG (含抗 Sm )作用后 ,HEp 2细胞G0 /G1期减少 ,G2 /M期升高 ,Tc明显延长 ,部分细胞发生凋亡。从时间关系上看 ,至 2 16h左右 ,HEp 2细胞周期复原 ,凋亡率的下降与HEp 2细胞偏离正常的程度呈正相关。提示进入性抗 Sm在SLE病损中的作用可能是诱发受侵细胞凋亡。     

Measurement of antinuclear antibodies: assessment of different test systems

The performance of rat liver and HEp-2 in the detection of antinuclear antibodies (ANA) was studied by two independent sites and compared against an ANA enzyme immunoassay (EIA) screen and EIA systems for the measurement of antibodies to double-stranded DNA (dsDNA) and ENA. Sixty-two sera from patients with connective tissue disease (CTD) and 398 from controls suffering from other disorders were included. The level of agreement was, for HEp-2 and rat liver (within one site), 82.0% (ANA positive/ANA negative) and 51.0% (ANA pattern); and for HEp2- and HEp-2 (between sites), 71.8 and 86.5%. On sera with the ANA homogeneous pattern, the measurement of anti-ENA EIA added little to the detection rate with anti-dsDNA EIA alone. On ANA speckled sera, the EIA reactivity depended on the reaction of the mitotic cells: while sera with positive mitoses reacted similarly to ANA homogeneous sera, in those with negative mitoses the measurement of anti-ENA added about 10% to the detection rate achieved with anti-dsDNA alone. The measurement of anti-Scl-70 and anti-Jo-1 did not markedly improve the positive rate with classical ENA (anti-SSA, -SSB, -Sm, and -RNP) alone, raising doubts about the cost efficiency of including these measurements in unselected sera. The ANA EIA identified patients with CTD at a rate similar to that for rat liver and HEp-2. However, up to 98% of the sera found to be negative by ANA EIA but positive by use of rat liver and HEp-2 were from controls. Thus, the ANA EIA may possible be used as an alternative screen, particularly in laboratories with a high frequency of sera from patients not suffering from CTD.     

Autoantibody to a novel neuronal antigen in systemic lupus erythematosus and in normal human sera.

A non-Fc receptor-bearing mouse neuroblastoma cell line, Neuro-2a, was used in indirect immunofluorescence tests to characterize the pattern of anti-neuronal activity of human sera in 41 cases of systemic lupus erythematosus (SLE), including seven cases of cerebral lupus, 30 "disease controls" (rheumatoid arthritis and chronic active hepatitis) and 30 healthy subjects. The immunofluorescence reaction with Neuro-2a gave a uniform ring fluorescence of the cell surface of cultured cells at 4 and at 37 degrees C, clusters were seen at 5 to 10 min and surface globules at 20 to 120 min. Titres of antibody to Neuro-2a in SLE ranged from less than 5 (seven cases), 5 to 20 (23 cases) and greater than or equal to 40 (11 cases). Titres of 80 to 160 were given by five of seven cases of cerebral lupus and two of 34 cases without cerebral lupus. Antibody to Neuro-2a was demonstrable in subjects without SLE, but to lower titres (less than 5-20). Of 11 SLE sera with antibody titres greater than or equal to 1:40, the antibody class was IgM in eight, IgG in two and both IgM and IgG in one. Absorption studies indicated that serum reactivity against Neuro-2a cells could be removed from some SLE sera with the cultured human neuroblastoma cell line, SK-N-SH, but not with mouse fibroblasts, mouse 3T3 cells, rat C6 glioma cells, rat transformed mesenchymal cells, nor by homogenates of mouse brain, heart, liver of kidney. Detection of antibody to Neuro-2a cell may be helpful in identifying patients with cerebral disease due to SLE.     

Antinuclear antibody detection using streptavidin-biotin-peroxidase complex on HEp-2 cell substrate.

The streptavidin-biotin-peroxidase complex (SABC) technique was compared to conventional indirect immunofluorescence (IIF) for the detection of anti-nuclear antibody (ANA) on HEp-2 cell substrate. SABC showed higher specificity and predictive value and gave more reproducible titres and clearer staining patterns than IIF in sera from a series of rheumatic disease patients. Sera from 80 patients with various types of rheumatic diseases and 20 without rheumatic disease were further tested using the SABC method. All systemic lupus erythematosus (SLE) sera were positive. The overall sensitivity was 95%, specificity 90% and predictive value 97% for rheumatic disease. The rim pattern was associated with SLE and mixed connective tissue disease. The nucleolar/homogeneous pattern was associated with scleroderma and SLE in remission. ANA titre and staining pattern have limited value in the clinical assessment of rheumatic disease; however, ANA has very high sensitivity for SLE and remains an excellent screening test.     

Multicenter evaluation study on a new HEp2 ANA screening enzyme immune assay.

The COBAS Core HEp2 ANA enzyme immune assay (EIA) was evaluated in a precision and a clinical sample study in comparison to indirect immunofluorescence assay (IFA) on HEp2-cells. In the precision study the COBAS Core EIA yielded intraassay coefficient variations (CVs) mostly below 9%, and interassay CVs between 4.7% and 10.4%. When comparing the COBAS Core EIA to IFA, the results corresponded well in healthy subjects, systemic lupus erythematosus, mixed connective tissue disease and rheumatoid arthritis. In the case of Sj?gren's syndrome and scleroderma patients the COBAS Core EIA yielded a lower rate of positive results compared to IFA. This discrepancy may be explained by the lack of detection of autoantibodies to nuclear antigens that can be identified only by IFA due to their compartmentalization and higher localized antigen density in HEp2 cells. The discrepancies in the group of dermato/polymyositis patients are due to the fact that the EIA contains mainly nuclear antigens and was able to detect only antibodies against the cytoplasmic Jo1 antigen that was added to the HEp2 nuclear extract. Routine sera were also evaluated; good agreement was found in sera from patients attending tertiary reference centres for autoimmune diseases but a higher number of discrepancies was reported in sera from unselected populations.     

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.     

Tissue specificity of antinuclear antibodies in scleroderma.

Studies of antinuclear antibodies (ANA) were carried out in 39 cases of systemic scleroderma and for comparison in 19 cases of systemic lupus erythematosus (SLE) using indirect immunofluorescence (IF) methods under standard conditions. The results on three different substrates--monkey esophagus, guinea pig lip and rat liver--are reported. In 48.7% of scleroderma cases ANA showed a substrate specificity. The highest percentage of positive results in scleroderma was obtained on monkey esophagus (97.4%) and the lowest on rat liver (61.5%). In SLE, in contrast, only about 13% of the sera displayed such specificity. If only sera with substrate specificity are considered, the positive results on monkey esophagus and rat liver are 94.7% and 21.1%, respectively. Titers of sera reacting positively on 2 or 3 substrates were mostly in agreement, although some sera both in systemic scleroderma and SLE showed higher titers on monkey esophagus. The IF pattern was usually the same regardless of the substrate. Tests for ANA in scleroderma should be performed on at least 2 substrates simultaneously.     

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.     

Autoantibodies to human nuclear antigen(s)-HNA-in connective tissue diseases and other disorders.

Autoantibodies reacting with nuclear antigen(s) on human cells (HNA) with weak or without reactivity on nuclei of other species have been found by the indirect immunofluorescence technique used in routine tests for the diagnosis of autoimmune diseases. Precipitin lines were obtained by counterimmunoelectrophoresis (CIE) only when human lymphocyte extracts were used and not with rabbit thymus acetone powder. By comparison with reference sera, the autoantibodies directed to HNA were found to be different from SSA/Ro antibodies and did not give the fluorescence pattern of anti nuclear mitotic apparatus (NuMA) antibodies on HEp-2 cells. The prevalence of sera with anti-HNA antibodies not associated with other antinuclear antibodies (ANA) is low (about 0.7% of ANA found in routine assay). In association with ANA of other specificities, the prevalence of anti-HNA antibodies, demonstrated after absorption of sera with rat liver acetone powder, was higher (about 1% of ANA positive sera). By treatment with physicochemical agents and enzymes, the HNA was found to be a DNA (glyco)-protein complex extractable with saline solution, resistant to 56 degrees C for 6 h and stable at pH values ranging from 3 to 10. Anti-HNA antibodies were found in patients with mild connective tissue diseases, but also in idiopathic interstitial pneumonia and in chronic hepatitis.     

Antinuclear Antibodies Detected by Indirect Immunofluorescence on HEp2 Cells and by Immunoblotting in Patients with Systemic Sclerosis

The HEp2 cell cultures appeared highly sensitive in detecting the antinuclear antibodies (ANAb) in systemic sclerosis, principally anticentromere antibodies of the CREST syndrome. The immunoblotting used with either complex cellular extracts from HeLa and rabbit thymus or purified nuclear components (high mobility group (HMG) proteins and histones) is able to identify precisely the ANAb targets and to contribute to diagnosis. With nuclear extracts of HeLa cells, the sera from 75.8% of CREST syndrome subjects stained 18 and 22 kD proteins. Corresponding antibodies were also detected in 72.7% of these patients, on HEp2 centromeres by indirect immunofluorescence. With the same extracts, 33.3% of sera from diffuse sclerosis/acrosclerosis patients contain antibodies staining 86, 73, 32 and 30kD. These sera also stain 77, 66 and 63kD from thymus extracts. Corresponding antibodies will be the anti-SCL-70 antibodies defined by double immunodiffusion. The anti-HMG antibodies were infrequent in systemic sclerosis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and consequently without interest for diagnosis. The anti-whole histones antibodies which are less frequent in diffuse sclerosis/acrosclerosis (35.7%) than in SLE (41.3%) recognize especially HI and H2A in the first diseases, HI and H2B in SLE and HI and H3 in RA.     

The nuclear pore complex protein Tpr is a common autoantigen in sera that demonstrate nuclear envelope staining by indirect immunofluorescence

We studied the autoantigen targets of 75 human sera that had antibodies to the nuclear envelope (NE) as identified by indirect immunofluorescence (IIF) on HEp‐2 cells. Several different IIF staining patterns could be identified when antibodies to different components of the nuclear membrane (NM) and nuclear pore complexes (NuPC) were identified: a smooth membrane pattern characteristic of antibodies to nuclear lamins, a punctate pattern typical of antibodies to the nuclear pore complex and more complex patterns that included antibodies to nuclear and cytoplasmic organelles. Western immunoblotting of isolated nuclear and NE proteins and immunoprecipitation of radiolabelled recombinant proteins prepared by using the full‐length cDNAs of the Translocated promoter region (Tpr), gp210 and p62 were used to identify specific autoantibody targets. Fifty‐two of the 75 (70%) sera bound to Tpr, 25 (33%) bound to lamins A, B or C, 15 (20%) reacted with gp210 and none reacted with p62. Sixteen (21%) did not react with any of the NE components tested in our assays. The clinical features of 37 patients with anti‐NE showed that there were 34 females and three males with an age range of 16–88 years (mean 59 years). The most frequent clinical diagnosis (9/37 = 24%) was autoimmune liver disease (ALD; two with primary biliary cirrhosis), followed by seven (19%) with systemic lupus erythematosus (SLE), four (11%) with a motor and/or sensory neuropathy, three (8%) with anti‐phospholipid syndrome (APS), two with systemic sclerosis (SSc), two with Sjögren's syndrome (SjS), and others with a variety of diagnoses. This report indicates that Tpr, a component of the NuPC, is a common target of human autoantibodies that react with the NE.     

Standardization of the immunofluorescence test for autoantibody to nuclear antigens (ANA): use of reference sera of defined antibody specificity

Standardization of the indirect immunofluorescence antinuclear antibody (IF-ANA) test can be improved for a given substrate with use of reference ANA sera, uniform assay conditions, and standardization of optical systems. To accomplish this, reference sera from the Arthritis Foundation with defined antibody specificities for nDNA, SS-B, RNP, Sm, nucleoli, and "speckled pattern" were reacted with commonly used IF-ANA substrates, mouse kidney sections, KB and HEp-2 tissue culture cells. Reagents and assay conditions used were those provided with the substrates in commercially available IF-ANA kits. A microscope slide with graded intensities of fluorescent beads was used to standardize microscope fluorescence intensity readings. The authors' fluorescence pattern and intensity results should be directly comparable to results obtained in other laboratories for the six antibodies for which reference sera are available. Although no defined sera are widely available for SS-A, Scl-70, PM-1, and centromere antibodies, the ability of each substrate to detect these antinuclear antibodies as well as mitochondrial, smooth muscle, ribosomal and microsomal antibodies also was tested. HEp-2 cells and KB cells were found to be superior to mouse kidney sections for detection of SS-A, Scl-70, PM-1 and centromere antinuclear antibodies. Mouse kidney sections were superior for screening of sera for the absence of ANA as well as for detection of smooth muscle and liver-kidney microsomal antibodies. Other antibodies were detected with equal sensitivity with all substrates and each of the three ANA kits used in the study performed satisfactorily. Use of reference sera as well as optical standardization is recommended for IF-ANA testing.     

Antinuclear antibodies detected by indirect immunofluorescence on HEp2 cells and by immunoblotting in patients with systemic sclerosis

The HEp2 cell cultures appeared highly sensitive in detecting the antinuclear antibodies (ANAb) in systemic sclerosis, principally anticentromere antibodies of the CREST syndrome. The immunoblotting used with either complex cellular extracts from HeLa and rabbit thymus or purified nuclear components (high mobility group (HMG) proteins and histones) is able to identify precisely the ANAb targets and to contribute to diagnosis. With nuclear extracts of HeLa cells, the sera from 75.8% of CREST syndrome subjects stained 18 and 22 kD proteins. Corresponding antibodies were also detected in 72.7% of these patients, on HEp2 centromers by indirect immunofluorescence. With the same extracts, 33.3% of sera from diffuse sclerosis/acrosclerosis patients contain antibodies staining 86, 73, 32 and 30kD. These sera also stain 77, 66 and 63kD from thymus extracts. Corresponding antibodies will be the anti-SCL-70 antibodies defined by double immunodiffusion. The anti-HMG antibodies were infrequent in systemic sclerosis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and consequently without interest for diagnosis. The anti-whole histones antibodies which are less frequent in diffuse sclerosis/acrosclerosis (35.7%) than in SLE (41.3%) recognize especially H1 and H2A in the first diseases, H1 and H2B in SLE and H1 and H3 in RA.     

肿瘤细胞组蛋白的免疫原性研究

为了寻找肿瘤患者血清中多种抗核抗体 (antinuclearantibodies,ANA )生成的新的可能免疫原 ;进一步论证细胞活化是核抗原性发生改变并诱导抗体产生的根本因素。提取SP2 / 0瘤细胞组蛋白免疫同系BALB/c小鼠 ,用ELISA方法检测IgG类抗组蛋白、抗dsDNA抗体 ,免疫荧光法检测ANA核型 ,免疫印迹法测定可溶性核抗原 (ENA )抗体。结果 :SP2 / 0瘤细胞组蛋白诱导同系BALB/c小鼠产生了IgG类抗组蛋白、抗dsDNA、抗Sm、抗SS A/SS B等多种自身ANA ,其核型有周边型、均质型、颗粒型等。推测肿瘤细胞组蛋白是诱导ANA生成的免疫原之一。