数字荧光强度分析法在自身抗体滴度检测中的应用探讨

首次研究应用数码成像系统采集荧光图形并测定数字荧光强度，以数字荧光强度分析法代替血清稀释法测定ANA滴度。确定了最佳采图时间后，用数码成像系统Spot32采集4140例ANA阳性标本的荧光图像，用分析软件ipwin32(image-Pro plus）测定其数字荧光强度，同时将标本稀释检测滴度，确定不同滴度对应的数字荧光强度值，分析数字荧光强度与稀释滴度的对应关系，并比较不同ANA核型用两种方法确定的滴度的符合强度。结果表明：通过比较选择4s为最佳采图时间，经统计分析4s采集图像的数字荧光强度与稀释滴度的对应关系近似取值为：29－50对1：100，51－85对1；320，86－175对1：1000，176－215对1：3200，216－237对1；10000。对3140例标本进行数字荧光强度测定以及稀释滴度测定，两者总符合率为89．4％（2806／3140）。其中，以斑点型（1568／1586，98．9％）、均质斑点型（460／461，99．8％）和均质型（759／763，99．5％），符合率最高，总符合率为99．2％；抗仁核仁型（4／75，5．3％）、抗着丝点型（2／114，1．8％）、抗核糖体型（13／103，12．6％）以及其它特殊型（0／38，0％）符合率最低。因此，以数字荧光强度分析法代替血清稀释法确定ANA滴度的方法可应用于ANA的主要荧光图形斑点型、均质型及均质斑点型，应用数字荧光强度分析法较血清稀释法更客观、经济、简便。     

Reflex testing of speckled cytoplasmic patterns observed in routine ANA HEp-2 indirect immunofluorescence with a multiplex anti-synthetase dot-blot assay: a multicentric pilot study

Immunofluorescence on HEp2-cells is the standard diagnostic assay for the detection of anti-nuclear antibodies (ANA). Cytoplasmic speckled patterns are a common finding, and are associated with various antibodies, including anti-synthetase antibodies. However, classic ENA testing generally identifies only anti-Jo-1. Moreover, anti-synthetase syndrome is increasingly recognized as a pleomorphic entity, possibly presenting as isolated arthritis or interstitial lung disease. Sera referred for routine ANA testing were selected on the basis of the presence of a fine dense speckled cytoplasmic pattern (254 samples) and compared to control sera with negative cytoplasm (239 samples). All 493 samples were tested with a commercial synthetase profile dot-blot (D TEK - Alphadia-Alifax) including anti-Jo1, anti-PL7, anti-PL12, anti-EJ, anti-OJ, anti-KS, anti-ZO, anti-HA, anti-SRP, and anti-Ribosome P0. Retrospective clinical data was searched for positive patients. Dot-blot identified 18/254 (7.1%) positive sera in the samples with a cytoplasmic fluorescence pattern and 4/239 (1.7%) in the control group (χ2?=?8.4627; p?=?0.003625). Blot intensity was more intense in samples with concordant cytoplasmic staining (cytoplasmic negative 27?±?12.4; cytoplasmic positive 53.9 27?±?27.7; p?=?0.0027). In the positive samples, 8/18 had a highly compatible diagnosis (myositis, interstitial lung disease, arthritis), 7/18 an uncharacterized connective tissue disease, and 3 a diagnosis not associated with the presence of anti-synthetase antibodies. We evaluated the performance of a dot-blot assay for anti-cytoplasmic antibodies in a serologic cohort presenting a cytoplasmic speckled pattern found during routine ANA testing. This algorithm enabled the identification of a significant quota of patients with rare anti-synthetase antibodies and an incomplete or atypical clinical picture. Reflex testing strategies of speckled cytoplasmic patterns with multiplex assays containing cytoplasm-specific antigens, as opposed to standard ENA testing, may yield important data and for this reason should be implemented in routine ANA testing.     

A new diagnostic algorithm for pattern-oriented autoantibody testing according to the ICAP nomenclature: A pilot study

The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart.710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC-18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens.ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern.This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed.     

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).

Anti‐nuclear antibodies in patients with breast cancer

To study the prevalence of anti‐nuclear antibodies (ANA) in breast cancer patients and its association with tumour characteristics. Ninety‐one patients with breast mass detected by image studies and assigned to conduct diagnostic biopsy and eventual surgical treatment were studied for demographical, tumour data and presence of ANA. Serum of positive ANA patients was screened for the extractable nuclear antigen (ENA) profile. As comparison, 91 healthy individuals matched for age and from the same geographical area were included. In this sample 72 of 91 (79·1%) had malignant lesions (83% ductal infiltrative carcinoma). ANA was positive in 44·4% of patients with malignant tumour and in 15·7% of those with benign lesions (malignant versus benign with P = 0·03). Controls had ANA positivity in 5·4%, and when compared with tumour samples showed P < 0·0001. The most common immunofluorescence pattern was a fine dense speckled pattern. In the ANA‐positive patients with malignant lesions, seven had positivity for ENA profile (three for anti‐RNP and anti‐Sm, one for just anti‐RNP, two for anti‐Ro and anti‐La e two for just anti‐La). It was not possible to associate ANA positivity with tumour histological characteristics or staging or with patient's age. A negative association of ANA with hormonal (oestrogen or oestrogen plus progesterone) receptor status was found (P = 0·01). In this sample, there was a high prevalence of ANA positivity in breast cancer patients with a negative association with the presence of hormonal receptors. More studies are needed to understand the real value of this finding.     

Detection rate and antigenic specificities of antineutrophil cytoplasmic antibodies in chinese patients with clinically suspected vasculitis

The detection rate of antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with clinically suspected small vessel vasculitis was investigated, and their antigen specificity and demographic features were analyzed. A number of sera (n = 5,604) sent to our referral laboratory for ANCA screening were tested by indirect immunofluorescence (IIF), enzyme-linked immunosorbent assays (ELISAs) for myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Then the IIF-ANCA-positive sera that were negative for MPO- and PR3-ANCA were further tested by antigen-specific ELISA by using other five highly purified known ANCA antigens as solid-phase ligands. The known antigens included bactericidal/permeability-increasing protein (BPI), human leukocyte elastase (HLE), lactoferrin, cathepsin G, and azurocidins. Of the 5,604 sera, 267 (4.76%) sera were IIF-ANCA positive and 390 (7%) were antinuclear antibody (ANA) positive in the IIF assay. Of the IIF-positive samples, 213 were anti-MPO positive, 32 were anti-PR3 positive, and five cases were positive for both. Of the 48 sera positive for IIF-ANCA but negative for MPO- and PR3-ANCA, 13 sera (27%) recognized other target antigens, 7 sera recognized BPI, 5 recognized HLE, 1 recognize cathepsin G, and 1 recognized azurocidin. None of the sera recognized lactoferrin, and one serum sample recognized both BPI and HLE. The majority of ANCA-positive patients presented in summer or winter. There was no difference in gender (male/female ratio, 1:1.12) in ANCA-positive patients with a mean age of 53.1 years. The male/female ratio was 1.17:1 for patients over 60 years of age; however, it was 1:4 for patients under 20 years of age. We conclude that ANCA-related diseases are not rare in China, and the major antigens are MPO and PR3. When the IIF technique is used to detect ANCA, ANA should be carefully distinguished.     

Detection Rate and Antigenic Specificities of Antineutrophil Cytoplasmic Antibodies in Chinese Patients with Clinically Suspected Vasculitis

The detection rate of antineutrophil cytoplasmic antibodies (ANCA) in Chinese patients with clinically suspected small vessel vasculitis was investigated, and their antigen specificity and demographic features were analyzed. A number of sera (n = 5,604) sent to our referral laboratory for ANCA screening were tested by indirect immunofluorescence (IIF), enzyme-linked immunosorbent assays (ELISAs) for myeloperoxidase (MPO)- and proteinase 3 (PR3)-ANCA. Then the IIF-ANCA-positive sera that were negative for MPO- and PR3-ANCA were further tested by antigen-specific ELISA by using other five highly purified known ANCA antigens as solid-phase ligands. The known antigens included bactericidal/permeability-increasing protein (BPI), human leukocyte elastase (HLE), lactoferrin, cathepsin G, and azurocidins. Of the 5,604 sera, 267 (4.76%) sera were IIF-ANCA positive and 390 (7%) were antinuclear antibody (ANA) positive in the IIF assay. Of the IIF-positive samples, 213 were anti-MPO positive, 32 were anti-PR3 positive, and five cases were positive for both. Of the 48 sera positive for IIF-ANCA but negative for MPO- and PR3-ANCA, 13 sera (27%) recognized other target antigens, 7 sera recognized BPI, 5 recognized HLE, 1 recognize cathepsin G, and 1 recognized azurocidin. None of the sera recognized lactoferrin, and one serum sample recognized both BPI and HLE. The majority of ANCA-positive patients presented in summer or winter. There was no difference in gender (male/female ratio, 1:1.12) in ANCA-positive patients with a mean age of 53.1 years. The male/female ratio was 1.17:1 for patients over 60 years of age; however, it was 1:4 for patients under 20 years of age. We conclude that ANCA-related diseases are not rare in China, and the major antigens are MPO and PR3. When the IIF technique is used to detect ANCA, ANA should be carefully distinguished.     

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.     

Novel opportunities in automated classification of antinuclear antibodies on HEp-2 cells

The recommended method for antinuclear antibodies (ANA) detection is IIF but it is influenced by many different factors. In order to pursue a high image quality without artefacts and to reduce inter-observer variability, this study aims to evaluate the reliability of using automatically acquired digital images for diagnostic purposes. In this paper we present SLIM-system a comprehensive system that supports the two sides of IIF tests classification. It is based on two systems: the first labels the fluorescence intensity, whereas the second recognizes the staining pattern of positive wells. We populated a dataset of 600 images obtained from sera screened for ANA by IIF on Hep-2 cells. The error rate has been evaluated according to eight-fold cross validation method; the rates reported in the following are the mean of the tests. Performance of the system in positive/negative recognition ranges from 87% up to more than 94%. Staining pattern classification accuracy of main classes ranges from 71% to 74%. The system provides high and reliable identification of negative samples and a flexibility that permits to use this application for different purposes. The analysis of its perspective performance shows the system potential in lowering the method variability, in increasing the level of standardization and in reducing the specialist workload of more than 80%. Our data represent a first step to validate the use of Computer Aided Diagnosis (CAD), thus offering an opportunity for standardizing and automatizing the detection of ANA by IIF.     

Low positive rate of serum autoantibodies in colorectal cancer patients without systemic rheumatic diseases

Background: Antinuclear antibody (ANA) testing is useful for screening, diagnosis and follow-up of patients with systemic rheumatic diseases. Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard for ANA testing. However, ANA have also been detected in patients with different cancer types but without any autoimmune disease. To overcome these shortcomings, different automated solid-phase assays have been developed. Aim: To determine the positive rate of a new ANA detection method (EliA CTD Screen, Phadia, Germany), in CRC patients without systemic rheumatic diseases. Additionally, we compare this method with IIF. Materials and methods: Serum samples were obtained before a colonoscopy procedure in a patient cohort (n?=?186) with a high clinical suspicion of CRC. Samples for ANA detection in CRC patients were processed in parallel by IIF on HEp-2 and the solid-phase fluoroenzymeimmunoassay EliA CTD Screen (Phadia, Germany) on the Phadia 250 instrument (Phadia GmbH, Freiburg, Germany). Positive samples by IIF and/or CTD were tested with EliA single ANA assays (Phadia, Germany) on the Phadia 250 instrument (Phadia GmbH, Freiburg, Germany). Results: Forty-five patients diagnosed with CRC were included. Four cases were positive by CTD and 23 by IIF. Of the four positive patients by CTD, two were positive and one indeterminate for anti-dsDNA antibodies. Of the 23 positive by IIF, one patient was positive and another indeterminate for anti-dsDNA antibodies, and a third patient was positive for anti-U1RNP antibodies. Conclusions: The CTD assay shows a low false positive rate for detecting autoantibodies in a clinical context of CRC.     

Indirect immunofluorescence (IIF) of normal washed peripheral blood cells to demonstrate antineutrophil cytoplasmic antibodies (ANCA)

BACKGROUND: The "International consensus document on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA)" requires all sera to be examined by indirect immunofluorescence (IIF). However, commercial neutrophil slides are expensive, fluorescence patterns can be difficult to interpret, and coincidental antinuclear antibodies (ANA) cannot be demonstrated; in addition, in house cytospin neutrophil preparations are time consuming to prepare and deteriorate with time. AIMS: To compare the IIF demonstration of ANCA, using washed peripheral blood cell smears, with commercial neutrophil preparations and with ANCA positivity as demonstrated by enzyme linked immunosorbent assay (ELISA). METHODS: Serum fluorescence positivity, pattern, and intensity using washed peripheral blood cell smears were compared with the results obtained using commercial neutrophil slides (INOVA). Fluorescence positivity, pattern, and intensity of 500 sera from consecutive patients with suspected vasculitis tested with washed peripheral blood cells were compared with binding in ELISAs for proteinase 3 (PR3) and myeloperoxidase (MPO). RESULTS: IIF of washed peripheral blood cell smears detected seven of eight sera with cytoplasmic fluorescence (C-ANCA), and 11 of 12 sera with perinuclear fluorescence (P-ANCA) demonstrated using commercial slides. The two sera that were negative by IIF were also negative in the ELISAs for both PR3-ANCA and MPO-ANCA. Of the 500 sera examined, there were 35 (7%) with C-ANCA, 65 (13%) with P-ANCA, and eight (2%) IIF negative sera that were positive by either ELISA. There was a strong correlation between C-ANCA fluorescence and PR3-ANCA values (p < 0.0001), and a moderate to strong correlation between P-ANCA fluorescence and MPO-ANCA values (p < 0.001) when ANCA fluorescence was demonstrated with washed peripheral blood cell smears. CONCLUSIONS: Washed peripheral blood cells are a convenient and useful low cost alternative to commercial or cytospin neutrophil preparations for the IIF demonstration of ANCA.     

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.     

Analysis of human sera that are polyreactive in an addressable laser bead immunoassay

BACKGROUND: Following the introduction of addressable laser bead immunoassays (ALBIA) into our clinical laboratory, it was noted that certain sera would exhibit reactivity to numerous antigens in the array. To further understand the nature of this reactivity, we analyzed the reactivity of sequential sera that were identified over a 1 year period. METHODS: Sera that demonstrated reactivity to 6 or more of the 8 antigens in an ALBIA kit (QuantaPlex 8: chromatin, Sm, RNP, Scl-70, ribosomal P protein, SS-A/Ro, SS-B/La, Jo-1) were tested for autoantibodies by indirect immunofluorescence (IIF) on HEp-2 cell substrates, for IgG, IgM and IgA rheumatoid factor, chromatin and ribosomal P protein by ELISA and by LINE immunoassay (LIA) and immunoblotting (IB). RESULTS: In one calendar year, 40/4096 (0.8%) sera analyzed in a routine clinical laboratory setting demonstrated reactivity to 6 or more antigens in the QuantaPlex 8 kits. There was no common IIF pattern that could be attributed to the polyreactive sera. There was no apparent correlation of polyreactivity with IIF titers, indeed, 4/40 (10%) sera had a negative ANA at the screening dilution of 1/160. When subjected to IB, LIA and ELISA, polyreactivity to three or more antigens was confirmed for 12/40 (30%) of sera while 8/40 (20%) had reactivity to 1-2 antigens and 20 (50%) did not react with any antigens in these assays. Overall agreement of positive or negative tests between the ALBIA and IB, LIA and ELISA was 75% for chromatin, 50% for SS-A, 27.5% for Sm, 25% for Rib-P, 22.5% for RNP, 20% for Scl-70, 15% for Jo-1 and 7.5% for SS-B. 17/40 (42.5%) had a positive IgM, IgG or IgA rheumatoid factor, and 12/40 (30%) had all three isotype rheumatoid factors. CONCLUSIONS: On average, the agreement between ALBIA and other assays in this study of polyreactive sera was 30%. Approximately, one-half of sera that demonstrate reactivity to multiple autoantigens in a commercial ALBIA were confirmed to have reactivity to at least one autoantigen in another diagnostic assay and 30% could be regarded as polyreactive. Other sera, some of which had rheumatoid factor, appeared to have high background binding without demonstrating specific binding to any of the cognate antigens.     

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.     

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.     

肾脏疾病患者血清中ANCA的荧光模式及其意义

目的：进一步探讨肾脏疾病患者血清中抗中性粒细胞细胞质抗体(ANCA)的荧光模式及其意义。方法：应用间接免疫荧光(IIF)和ELISA方法检测了251例肾脏疾病患者血清中ANCA及其靶抗原。结果：在251例肾脏疾病患者中，ANCA阳性51例占20．3％，其中10例为cANCA阳性(10／251)；30例为cANCA阳性(30／251);11例为aANCA阳性(11／251)。结论：同时用IIF法和ELISA法检测ANCA，可以提高ANCA检出的阳性率；不同ANCA荧光模式与疾病的种类病情及预后密切相关；提高对不典型ANCA和pANCA伴ANA荧光模式的鉴别，有助于临床对血管炎和其它自身免疫性疾病的诊断。     

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.     

Anti-argonaute2 (Ago2/Su) and -Ro antibodies identified by immunoprecipitation in primary anti-phospholipid syndrome (PAPS)

Objectives. Primary anti-phospholipid syndrome (PAPS) is an autoimmune condition defined by anti-phospholipid antibodies (aPL) and thrombotic or obstetric events. Some PAPS can evolve into systemic lupus erythematosus (SLE) during follow-up. Few studies systematically examined lupus autoantibodies and their clinical significance in PAPS. The aim of our study is to analyze the clinical and laboratory correlations with lupus-related autoantibodies, detected by immunoprecipitation (IP), a technique not yet systematically applied to investigate autoantibodies in this condition.

Methods. Sera from 52 PAPS patients were screened by indirect immunofluorescence (IIF) antinuclear antibodies (ANA), IP of ³⁵S-labeled K562 cell extract, and ELISA [anti-Argonaute2 (Ago2, Su), 60kRo, 52kRo, La, dsDNA)]. Anti-Ago2/Su positive sera were also tested for anti-GW bodies (GWBs) by IIF double staining, using rabbit anti-Rck/p54 serum.

Results. First, 56% of PAPS patients (29/52) were ANA positive, mainly with speckled pattern. Anti-Ago2/Su antibodies were found in 13% (7/52), anti-Ro/SSA in 10% (5/52), anti-La in one case. The clinical profile of patients did not seem to be related to the presence of these antibody specificities. However, levels of IgG anti-β2 glycoprotein I antibodies were lower in anti-Ago2/Su positive patients (p = 0.02). None of anti-Ago2/Su or —Ro patients developed SLE during a 2-year follow-up. Ago2 is a key component of GWBs, however, only 1/7 anti-Ago2/Su serum showed a typical cytoplasmic GWBs staining.

Conclusions. Anti-Ago2/Su and -Ro antibodies are the two autoantibodies detected by IP in our PAPS cohort. Clarifying why Ago2/Su and Ro are specific targets of autoimmunity may help to understand the mechanisms of autoantibody production.     

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.     

International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA)

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples. Inclusion of the most recent positive sample in the IIF or ELISA may help demonstrate a change in antibody level. Reports should use recommended terms. Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of Wegener granulomatosis or microscopic polyangiitis and that decisions about treatment should not be based solely on the ANCA results.