Recent advances in laboratory tests and the significance of autoantibodies to nuclear antigens in systemic rheumatic diseases

There are many types of ANA and they may react with different nuclear components varying from nucleic acids to both basic and acidic nuclear proteins. Recent investigations have demonstrated that it is important not only to detect the presence and quantity of the ANA, but also to identify the immunologic specificities of the ANA in a given patient. The specific identification of the immunologic specificity of the ANA helps in the diagnosis and management of therapy in the various rheumatic diseases. In recent years, much progress has been made in the improvements of sensitivity, specificity, and quality control of many of the clinical laboratory tests for detection and quantitation of ANAs. Distinct profiles of ANA characterize different rheumatic diseases. A number of ANAs are found in SLE, whereas other diseases are characterized by the presence or absence of a certain ANA or by differences in mean ANA titers. Specific ANAs have been used to isolate and characterize nuclear antigens at the molecular and functional levels.     

Relationship between snRNA species contained in nuclear antigens recognized by autoantibodies and the clinical profile in systemic rheumatic diseases

Antibodies to extractable nuclear antigens (ENA) are generally used in the diagnosis of connective tissue diseases. Using a rapid, very sensitive method we have shown that extractable nuclear antigens, which are now well-characterized at the molecular level, differ by their RNA content. The method was applied to the sera of 17 patients suffering from different connective tissue diseases. The results show that mixed connective tissue disease (MCTD) and other mild connective tissue diseases are characterized by the presence in the antigen of U1 small nuclear RNA (U1 snRNA) only. On the other hand, antibodies from 6 out of 8 patients tested with Systemic Lupus Erythematosus (SLE) recognize antigens exhibiting a more complex RNA pattern. Three of them precipitated all five snRNAs U2, U1, U4, U5, U6 whereas some snRNAs were lacking or quantitatively less important in precipitates obtained with the three others.     

Update on autoantibodies to intracellular antigens in systemic rheumatic diseases.

Autoantibodies to nuclear and intercellular antigens have been established as important diagnostic and specific markers for many of the systemic rheumatic diseases. Diseases that have demonstrated specific markers and profiles of autoantibodies include systemic lupus erythematosus, scleroderma, Sj?rgren's syndrome, mixed connective tissue disease, drug-induced lupus, and dermatomyositis/polymyositis. The study of molecular biology of the various autoantigens and autoantibodies has led to a better understanding of function and tissue pathogenesis. The clinical diagnostic significance of the various autoantigens and autoantibodies to nuclear and intracellular antigens is discussed in this article.     

Comparison and variation of different methodologies for the detection of autoantibodies to nuclear antigens (ANA)

Interest in the assessment of autoantibody specificity stems from the need for an autoantibody marker capable of predicting clinical events in autoimmune disorders. However, the multiplicity of epitopes present on autoantigenic particles, the quantitative and qualitative heterogeneity of autoantibodies, as well as the nature of the tests, mean that each of the assays used in their determination have different characteristics. The aim of this study was to compare the specificities of different ANAs using four commercial assays. The routine method used for the detection of ANA is indirect immunofluorescence on Hep-2 cells. The assays used were: counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA), and two immunoblotting assays. Kappa statistic was applied to evaluate the consistency between tests. Kappa index is a measure of agreement between categorical data. Kappa has a maximum of 1.00 when the agreement is perfect, a value of zero indicates no agreement better than chance, and negative values show worse than chance agreement. For SS-B antibodies, there was a good concordance between all four methods used (Kappa 0.66–0.74). For anti RNP antibodies, the results for CIE/ELISA (Kappa 0.60) were consistent as were the two immunoblot methods (Kappa 0.69). For anti Scl-70 (topoisomerase I) antibody, results from the ELISA and CIE methods were totally consistent (Kappa 1.00). In spite of the high prevalence of anti SS-A/Ro antibodies, the agreement between the methods was poor, without statistical significance. Finally, for Sm antibodies, more consistent results were obtained between CIE/ELISA (Kappa 0.51) and between one of the immunoblotting methods and ELISA (Kappa 0.54). In conclusion, CIE concurs mostly with ELISA for anti- RNP, Scl-70, Sm and SS-B antibodies, but with some disagreement for SS-A antibodies. J. Clin. Lab. Anal. 11:388–392, 1997. © 1997 Wiley-Liss, Inc.     

识别不同靶抗原的抗肾小球基底膜抗体与临床表型相关

目的研究识别不同靶抗原的抗肾小球基底膜(glomerular basement membrane,GBM)抗体与临床表型的关系。方法应用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA),以重组人a1、a2、a3、a4和a5(IV)NC1为固相抗原,测定97例抗GBM病患者血清中抗体的靶抗原,并分析其与临床病理表现的关系。69例患者血清进一步应用以正常人肾组织为底物的间接免疫荧光法(indirect immunofluorescence,IIF)检测抗体识别的抗原表位的位置。隐匿性抗原表位的暴露采用6M尿素对组织切片进行预处理,天然暴露抗原表位的检测则以未经处理的组织切片作为底物。结果所有患者血清均识别a3(IV)NC1,56.7%患者同时识别a1(IV)NC1,43.3%患者识别a2(IV)NC1,53.6%患者识别a4(IV)NC1,85.6%患者识别a5(IV)NC1。抗a3(IV)NC1抗体的水平是患者确诊时的血肌酐水平(r=0.308,P=0.003)以及肾脏病理肾小球中新月体的比例(r=0.492,P0.05)的独立决定因素。识别a5(IV)NC1的患者血清对天然暴露的抗原表位的识别率较高(67.3%比30.0%,P=0.026),且抗a5(IV)NC1抗体的水平与细胞性新月体的比例呈正相关(P=0.013)。合并抗中性粒细胞胞浆抗体(antineutrophil cy-toplasmic antibodies,ANCA)阳性的患者,对a1(IV)NC1(7.3%比26.2%,P=0.011)和a4(IV)NC1(7.7%比24.4%,P=0.023)的识别率较低。结论抗GBM抗体的主要靶抗原位于a3(IV)NC1,其抗体的水平是肾脏损害程度的决定因素。天然暴露的抗原表位可能与a5(IV)NC1相关,其抗体在疾病的发病机制中可能发挥重要作用。合并ANCA阳性的患者,其抗体识别的靶抗原较少。     

Association of HL-A 5 and immune responsiveness in vitro to streptococcal antigens

Lymphocytes, from randomly selected individuals having normal immune function, when incubated in vitro with varying concentrations of streptococcal antigens, responded in three ways: (a) response over the entire antigen concentration range, i.e., responders; (b) low response to only the highest antigen concentrations; and (c) no response at any antigen concentration. Frequency distribution analysis of these groups indicated that a significant association occurred between the ability to respond and HL-A 5.     

定量酶联免疫吸附试验检测各种结缔组织病患者血清中的抗可提取核抗原抗体

目的　定量检测各种结缔组织疾病 (CTD)患者血清中抗可提取核抗原 (ENA)自身抗体滴度 ,并探索其临床意义。方法　自制抗各种ENA单克隆抗体 ,以夹心酶联免疫吸附试验 (ELISA)定量检测 1 2 8例CTD患者、5 0例非CTD患者及 6 0名健康人血清的各种抗ENA自身抗体滴度。用健康人血清各ENA抗体滴度的均数加上 3倍标准差 ( x +3s)为cutoff值 ,界定各种ENA抗体检出率。结果　与健康对照组和非CTD患者组比较 ,抗Sm抗体在系统性红斑狼疮 (SLE)、抗ul RNP抗体在SLE和混合性结缔组织病 (MCTD)、抗SSA和SSB在干燥综合征 (SS)、抗Scl 70抗体在系统性硬化病 (PSS)、抗Jo 1抗体在多发性肌炎 /皮肤炎 (PM/DM)患者中的水平和阳性率都明显升高 (P <0 .0 1 )。结论　定量检测CTD患者血清中各种ENA抗体滴度 ,对CTD疾病的鉴别、诊断和分型具有重要价值     

定量酶联免疫吸附试验检测各种结缔组织病患者血清中的抗可提取核抗原抗体

目的定量检测各种结缔组织疾病(CTD)患者血清中抗可提取核抗原(ENA)自身抗体滴度,并探索其临床意义.方法自制抗各种ENA单克隆抗体,以夹心酶联免疫吸附试验(ELISA)定量检测128例CTD患者、50例非CTD患者及60名健康人血清的各种抗ENA自身抗体滴度.用健康人血清各ENA抗体滴度的均数加上3倍标准差(+3s)为cut off值,界定各种ENA抗体检出率.结果与健康对照组和非CTD患者组比较,抗Sm抗体在系统性红斑狼疮(SLE)、抗ul-RNP抗体在SLE和混合性结缔组织病(MCTD)、抗SSA和SSB在干燥综合征(SS)、抗Scl-70抗体在系统性硬化病(PSS)、抗Jo-1抗体在多发性肌炎/皮肤炎(PM/DM)患者中的水平和阳性率都明显升高(P<0.01).结论定量检测CTD患者血清中各种ENA抗体滴度,对CTD疾病的鉴别、诊断和分型具有重要价值.     

Association of class II human histocompatibility leukocyte antigens with rheumatic fever.

The association of class I and II HLA antigens with rheumatic fever and its manifestations was examined in 72 patients, including 48 blacks and 24 Caucasians. No significant association was found between class I antigens and rheumatic fever. In contrast, HLA-DR2 and HLA-DR4 phenotypes were encountered in a significantly higher frequency in black and Caucasian patients with rheumatic fever, respectively, compared with the control populations (P less than 0.005). The most significant association (P less than 0.005) of these DR antigens with a major manifestation of rheumatic fever was found for mitral insufficiency. In addition, a significant association was encountered between persistent elevation of antibody to the group A streptococcal carbohydrate and HLA-DR4 in Caucasian patients (P less than 0.04) or HLA-DR2 in the black patients (P less than 0.001). The frequency of HLA-DR2/4 heterozygotes among patients with rheumatic fever did not differ significantly from controls. These findings support the concept of a genetically determined susceptibility to rheumatic fever and, particularly, to rheumatic heart disease. The association of the clinical manifestations of rheumatic fever and the immune hyperresponsiveness to a streptococcal antigen could be ascribed to a disease-associated immune-response gene which is in linkage disequilibrium with the DR2 and DR4 alleles of HLA-DR locus on chromosome six.     

Evaluation of the LIA-ANA-Profile-17S for the detection of autoantibodies to nuclear antigens

ObjectivesThe diagnostic tests for autoimmune disease include screening for autoantibodies for nuclear antigens (ANA) and antibodies against extractable nuclear antigens (ENA). Using the line immunoassay (LIA) method, various kinds of ENA antibodies can be detected simultaneously. We evaluated the performance of the newly launched LIA-ANA-Profile-17S (Shenzhen YHLO Biotech, Shenzhen, China) as compared to a conventional LIA kit.MethodsResidual samples were collected from 200 patients who had been tested for ANA using indirect immunofluorescence. The LIA-ANA-Profile-17S was compared to the EuroLine ANA (Euroimmun, Oberlausitz, Germany) for the analysis of 17 different autoantibodies. The concordance rate and agreement between assays were determined. Samples showing discrepancies between the LIA-ANA-Profile-17S and EuroLine tests were further examined through additional analysis.ResultsThe overall agreement was moderate (kappa = 0.759, 95% CI = 0.712–0.805). Agreement between assays ranged from weak to almost perfect, except for those tests targeting nucleosomes, histones, and PM-Scl. Of the 57 disparate results between LIA-ANA-Profile-17S and EuroLine, 38 (66.7%) samples tested positive under an additional assay, showing variable patterns between types of autoantibodies. The positive rate of each autoantibody between LIA-ANA-Profile-17S and EuroLine did not differ significantly, except for anti-nucleosome and anti-histone assays in samples from patients diagnosed with systemic lupus erythematosus (P = 0.004 and 0.001, respectively).ConclusionsCompared to those from the conventional EuroLine assay, the LIA-ANA-Profile-17S results showed variable agreement in samples showing different prevalence of each autoantibody. The most frequently detected antibodies showed almost perfect agreement. The LIA-ANA-Profile-17S could play a role in the diagnosis of systemic autoimmune disease in ANA-positive samples.     

Absence of high-affinity calreticulin autoantibodies in patients with systemic rheumatic diseases and coeliac disease

Calreticulin has been reported to be an autoantigen in various autoimmune connective tissue diseases and in coeliac disease. Previous studies have used incubation buffers with low salt and low detergent concentrations (low stringency conditions) with serum albumin or other proteins as a blocking agent. Using these conditions we found a relatively high level of non-specific binding in many sera. Antibodies to proteins that are used as blocking reagents in ELISA (bovine serum albumin (BSA), ovalbumin, skimmed milk powder) are frequently present in sera, and these may cause false-positive results. Moreover, the low isoelectric point of calreticulin and its chaperone properties may give rise to false-positive results under low stringency conditions. We report that the use of a simple buffer without protein (50 mM Tris, pH 7.5, 1% Tween 20, 0.3 M NaCl) removes most of the problems with unwanted binding (high stringency conditions). Using the high stringency conditions, we screened sera from 107 patients with systemic lupus erythematosus, sera from patients with other systemic autoimmune diseases and from children with coeliac disease for the presence of high-affinity calreticulin autoantibodies by immunoblotting and ELISA. None of the sera contained high-affinity calreticulin antibodies. It is concluded that calreticulin is not a common autoantigen in patients with autoimmune connective tissue diseases or coeliac disease.     

The clinical significance of autoantibodies to soluble cellular antigens in systemic lupus erythematosus

The relationships between autoantibodies to soluble cellular antigens and clinical features in systemic lupus erythematosus (SLE) were investigated in a large clinical-serological study. The absence of these precipitins in serum was associated with a low prevalence of vasculitis and membranous nephropathy (MGN). Other significant findings were the associations between nRNP antibody and Raynaud's phenomenon and MGN, SSB antibody and sicca complex, PCNA antibody and a young age at onset, and Bu antibody and an old age at onset. However, the most impressive findings were in DA1-positive patients which showed a unique prevalence of photosensitive skin lesions, lymphoadenopathy and hepatosplenomegaly. The present study confirms the usefulness of antibodies to soluble cellular antigens in the classification of patients with SLE.     

Importance of detection of SS-A/Ro autoantibody in screening immunofluorescence tests for autoantibodies to nuclear antigens

SS-A/Ro autoantibodies are related to Sj?gren's syndrome and to several clinical subsets of lupus erythematosus. The most widely used laboratory procedure for ANA screening is the indirect immunofluorescence test (IF-ANA); the laboratory detection of anti-SS-A/Ro requires implementation and adherence to several technical and quality assurance recommendations. Using appropriate substrate cells containing the SS-A/Ro antigen, many so-called "ANA-negative" lupus erythematosus patients will demonstrate a positive IF-ANA.     

Current status of available standards for quality improvement of assays for detection of autoantibodies to nuclear and intracellular antigens

Considerable progress has been made in the improvement of clinical assays for the detection of autoantibodies to nuclear and intracellular antigens with the use of available World Health Organization (WHO) and Arthritis Foundation/Centers for Disease Control (AF/CDC) standards. The ultimate goal of standardization is for various clinical laboratory test results to be interchangeable and for an exchange of data to be done with confidence. This report discusses the available standards. In addition, significant technical problems and variations in methodologies for the detection of autoantibodies to intracellular antigens noted during a 4-year study by a European Consensus Study Group are detailed. Currently, there is a need for a future generation of reference preparations and standards that will show specific antibody reactivity on sensitive enzymes and immunoblotting assays. Standardization efforts should be done to characterize specific nuclear and cellular antigen preparations that may be of natural or of recombinant technology origin. © 1994 Wiley-Liss, Inc.     

Presentation, clinical features and outcome in different patterns of atherosclerotic renovascular disease

Atherosclerotic renovascular disease (ARD) is an increasinglyimportant cause of renal failure. However, important featuresof the clinical presentation are not fully described, and theoutcome after intervention by angioplasty remains controversial.Ninety-four patients with ARD diagnosed at angiography werereviewed. Twenty-four patients were diabetic. Thirty-nine patientshad unilateral renal artery stenosis or occlusion (group A),28 had bilateral stenosis (group B), and 27 had unilateral occlusionplus contralateral occlusion or stenosis (group C), Two yearsafter presentation, actuarial patient survival was 96%, 74.3%and 47.1% in groups A, B and C, respectively (p<0.001 forall differences); actuarial renal survival in surviving patientswas 97.3%, 82.4% and 44.7%, respectively (p<0.001 for alldifferences). Percutaneous transluminal balloon angioplasty(PCTA) was performed in 74 patients. Renal function improvedin only a minority of cases, but was stable in 73% of nondiabeticpatients 12 months after PCTA. Angioplasty was less effectivein diabetic subjects, with only 53.3% having stable renal functionat 12 months follow-up. Renal and patient survival were stronglyrelated to the initial angiographic findings. In nondiabeticsubjects, PCTA resulted in stabilization of renal function forat least one year in nearly threequarters of cases, which suggestsa benefit from intervention in this disease whose natural historyis otherwise of progression.     

2015年风湿免疫病主要进展

2015年风湿病的临床进展主要集中在针对类风湿关节炎、风湿性多肌痛、IgG4相关性疾病和银屑病关节炎管理指南和治疗策略的改变,同时更新了原发性干燥综合征和痛风的诊断标准。国内则针对系统性红斑狼疮相关肺动脉高压给出了相应的指南,并对妊娠围产期的系统性红斑狼疮患者给出了我国的围产期管理建议。2015年不仅关注风湿病本身,同时关注了风湿病的共患病, 尤其心血管疾病等并发症,并且更加强调患者教育。