International Consensus On ANA Patterns Research Articles

An automatic immunofluorescence pattern classification framework for HEp-2 image based on supervised learning.

Immunofluorescence patterns of anti-nuclear antibodies (ANAs) on human epithelial cell (HEp-2) substrates are important biomarkers for the diagnosis of autoimmune diseases. There are growing clinical requirements for an automatic readout and classification of ANA immunofluorescence patterns for HEp-2 images following the taxonomy recommended by the International Consensus on Antinuclear Antibody Patterns (ICAP). In this study, a comprehensive collection of HEp-2 specimen images covering a broad range of ANA patterns was established and manually annotated by experienced laboratory experts. By utilizing a supervised learning methodology, an automatic immunofluorescence pattern classification framework for HEp-2 specimen images was developed. The framework consists of a module for HEp-2 cell detection and cell-level feature extraction, followed by an image-level classifier that is capable of recognizing all 14 classes of ANA immunofluorescence patterns as recommended by ICAP. Performance analysis indicated an accuracy of 92.05% on the validation dataset and 87% on an independent test dataset, which has surpassed the performance of human examiners on the same test dataset. The proposed framework is expected to contribute to the automatic ANA pattern recognition in clinical laboratories to facilitate efficient and precise diagnosis of autoimmune diseases.

An Italian nationwide survey on the evolution of autoantibody diagnostics in autoimmune rheumatic diseases.

The advent of new technologies and the discovery of new antigenic targets of autoantibodies has led to significant changes in autoimmune diagnostics worldwide. To address the extent to which autoimmunology laboratories adhere to such innovation in testing and reporting practices, the Italian Society of Clinical Pathology and Laboratory Medicine launched a national survey to assess the current status of autoimmune diagnostics and to provide direction for further harmonisation. A questionnaire covering topics related to the diagnosis of autoimmune systemic rheumatic diseases was distributed to 152 Italian autoimmunology laboratories. The 59 closed-answer questions were subdivided into four main sections: 1. the setting (university, hospital or private laboratory) and the number of tests carried out; 2. the technologies used and their level of automation; 3. the analytical phase (antibody tests and methods), including awareness of the International Consensus on ANA Patterns (ICAP) initiative; 4. reporting of results and clinician relations. A total of 121 laboratories (79.6%) responded to the survey (15% universities, 70 hospitals, and 15% private laboratories). Indirect immunofluorescence is used by 94.8% of respondents, chemiluminescence by 78.4%, fluoro-immuno-enzymatic assays by 67.5%, immunodot by 52.6%, line-immunoassay by 47.4%, addressable laser bead immunoassay by 10.3% and radioimmunological methods by 10.2%. The great majority of respondents implemented complete automation of the listed methodologies. 65% of participants state that they add an interpretative comment in the report. 45% of participants enjoy a collaborative relationship with clinicians; counselling activities are provided by almost half of participants. Survey results indicated that almost all respondent laboratories have broadened their antibody panel and that high-throughput technologies have been widely introduced. Gaps identified by the survey include a still incomplete compliance with guidelines in antibody profiles (e.g. in antiphospholipid syndrome and rheumatoid arthritis and reporting of test results. Awareness of these differences provides insights that may further contribute to achieving harmonisation in autoimmune diagnostics.

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.

The Anti-Cytoplasmic and Anti-Mitotic Autoantibodies; Are These Antibodies Associated with Diseases?

Examination of antinuclear antibody (ANA) is used in diagnosis of systemic autoimmune diseases, and the indirect immunofluorescence (IIF) assay using HEp-2 cells is the gold standard method. HEp-2 allows the detection of multiple target antigen-directed autoantibodies. The guide “The International Consensus on ANA Patterns (ICAP)”, characterizes the patterns into three groups: nuclear, cytoplasmic, and mitotic. The majority of these are associated with autoimmune diseases, but some are rarely seen in autoimmune diseases or may be associated with conditions other than autoimmune disease. There is no consensus on how to report cytoplasmic and mitotic patterns-negative or positive. We aimed to examine the characteristics of patients that had cytoplasmic or mitotic staining in ANA evaluation by IIF. In our Medical Microbiology Laboratory, 18985 ANA tests of 16940 patients were studied between 01.01.2015-31.12.2019. Cytoplasmic or mitotic pattern was detected in 393 (2.07%) tests belonging to 385 patients. Cytoplasmic patterns suggestive of anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (ASMA), anti-Jo-1 and anti-ribosomal P-protein were not included. The most common patterns were anti-midbody, anti-spindle fibers, and anti-vimentin patterns. There were 66 rheumatology patients that were negative for ANA but had cytoplasmic or mitotic staining. There was no statistically significant difference between the diagnosis and patterns of these patients. We suggest that the ANA should be reported as “negative” in case of cytoplasmic or mitotic pattern unless the term anti-cell antibody is used. It should be noted in the description part of the report in order to distinguish significant cytoplasmic patterns and give an idea for some specific conditions.

The International Consensus on ANA Patterns (ICAP) in 2021-The 6th Workshop and Current Perspectives.

The establishment of the International Consensus on ANA Patterns (ICAP) in 2014-2015 was welcomed by members of the medical community as a significant improvement in guiding harmonization of ANA test interpretation and reporting. In the subsequent years, several itinerant meetings and continuous interaction with the community contributed to disseminate the ICAP harmonization on the immunofluorescence patterns observed in the indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) and to promote progressive improvement in the classification of HEp-2 IFA patterns. The 6th ICAP Workshop was held in person on September 6, 2021 as a satellite meeting of the 15th Dresden Symposium on Autoantibodies. This article summarizes the major discussions at the meeting as well as outlining the current plans for the ICAP committee.

How to report the antinuclear antibodies (anti-cell antibodies) test on HEp-2 cells: guidelines from the ICAP initiative.

Results of the anti-nuclear antibodies-indirect immunofluorescence assay (anti-cell antibodies test) on HEp-2 cell substrates should be communicated to clinicians in a standardized way, adding value to laboratory findings and helping with critical clinical decisions. This paper proposes a test report based on the practices informed by 118 laboratories in 68 countries, with recommendations from the International Consensus on ANA Patterns (ICAP) group. Major focus is placed on the report format containing endpoint titers, immunofluorescence patterns together with anti-cell (AC) nomenclature, remarks on follow-up or reflex testing, and possible other autoantibody associations. ISO 15,189 directives were integrated into the test report. Special situations addressed include serum screening dilutions and endpoint titers, relevance of immunofluorescence patterns with special attention to cytoplasmic patterns, mixed and compound patterns, and how to report different titers corresponding to multiple patterns or autoantibodies in the same sample. This paper suggests a subtitle for the HEp-2-IIFA, namely anti-cell antibodies test, which could gradually substitute the original outdated ANA nomenclature. This ICAP pro forma report represents a further step in harmonizing the way relevant clinical information could be provided by laboratories.

Strong Association of the Myriad Discrete Speckled Nuclear Pattern With Anti-SS-A/Ro60 Antibodies: Consensus Experience of Four International Expert Centers.

IntroductionThe morphological patterns in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA) reflect the autoantibodies in the sample. The International Consensus on ANA Patterns (ICAP) classifies 30 relevant patterns (AC-0 to AC-29). AC-4 (fine speckled nuclear pattern) is associated to anti-SS-A/Ro, anti-SS-B/La, and several autoantibodies. Anti-SS-A/Ro samples may contain antibodies to Ro60 and Ro52. A variation of AC-4 (herein designated AC-4a), characterized by myriad discrete nuclear speckles, was reported to be associated with anti-SS-A/Ro. The plain fine speckled pattern (herein designated AC-4b) seldom was associated with anti-SS-A/Ro. This study reports the experience of four expert laboratories on AC-4a and AC-4b.MethodsAnti-Ro60 monoclonal antibody A7 was used to investigate the HEp-2 IFA pattern. Records containing concomitant HEp-2 IFA and SS-A/Ro tests from Durand Laboratory, Argentina (n = 383) and Fleury Laboratory, Brazil (n = 144,471) were analyzed for associations between HEp-2 IFA patterns and disease-associated autoantibodies (DAA): double-stranded DNA, Scl-70, nucleosome, SS-B/La, Sm, and U1-RNP. A total of 381 samples from Dresden Technical University (TU-Dresden), Germany, were assayed for HEp-2 IFA and DAA.ResultsMonoclonal A7 recognized Ro60 in Western blot and immunoprecipitation, and yielded the AC-4a pattern on HEp-2 IFA. Analyses from Durand Laboratory and Fleury Laboratory yielded compatible results: AC-4a was less frequent (8.9% and 2.7%, respectively) than AC-4b (26.1% and 24.2%) in HEp-2 IFA-positive samples. Reactivity to SS-A/Ro occurred in 67.6% and 96.3% of AC-4a-pattern samples against 23% and 6.8% of AC-4b pattern samples. Reciprocally, AC-4a occurred in 24% and 47.1% of anti-SS-A/Ro-positive samples, and in 3.8% and 0.1% of anti-SS-A/Ro-negative samples. Data from TU-Dresden show that the AC-4a pattern occurred in 69% of 169 anti-SS-A/Ro-monospecific samples (62% of all anti-SS-A/Ro-positive samples) and in 4% of anti-SS-A/Ro-negative samples, whereas anti-SS-A/Ro occurred in 98.3% of AC-4a samples and in 47.9% of AC-4b samples. In all laboratories, coexistence of anti-SS-B/La, but not other DAA, in anti-SS-A/Ro-positive samples did not disturb the AC-4a pattern. AC-4a was predominantly associated with anti-Ro60 antibodies.ConclusionsThis study confirms the association of AC-4a pattern and anti-SS-A/Ro in opposition to the AC-4b pattern. The results of four international expert laboratories support the worldwide applicability of these AC-4 pattern variants and their incorporation into ICAP classification under codes AC-4a and AC-4b, respectively. The AC-4 pattern should be maintained as an umbrella pattern for cases in which one cannot discriminate AC-4a and AC-4b patterns. The acknowledgment of the AC-4a pattern should add value to HEp-2 IFA interpretation.

Application of Supervised Machine Learning to Recognize Competent Level and Mixed Antinuclear Antibody Patterns Based on ICAP International Consensus.

Background: Antinuclear antibody pattern recognition is vital for autoimmune disease diagnosis but labor-intensive for manual interpretation. To develop an automated pattern recognition system, we established machine learning models based on the International Consensus on Antinuclear Antibody Patterns (ICAP) at a competent level, mixed patterns recognition, and evaluated their consistency with human reading. Methods: 51,694 human epithelial cells (HEp-2) cell images with patterns assigned by experienced medical technologists collected in a medical center were used to train six machine learning algorithms and were compared by their performance. Next, we choose the best performing model to test the consistency with five experienced readers and two beginners. Results: The mean F1 score in each classification of the best performing model was 0.86 evaluated by Testing Data 1. For the inter-observer agreement test on Testing Data 2, the average agreement was 0.849 (κ) among five experienced readers, 0.844 between the best performing model and experienced readers, 0.528 between experienced readers and beginners. The results indicate that the proposed model outperformed beginners and achieved an excellent agreement with experienced readers. Conclusions: This study demonstrated that the developed model could reach an excellent agreement with experienced human readers using machine learning methods.

Russian-language adaptation of the international nomenclature of International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP)

Antinuclear antibodies (ANAs) represent a spectrum of autoantibodies targeted for various nuclear and cytoplasmic components of the cells. Indirect immunofluorescence assay (IIF) is the main detection method for “antinuclear factor”. A positive ANA test is usually reported as a titer and a pattern of fluorescence. The ANA patterns refers to the distribution of staining produced by antibodies that react with antigens located in nucleus and cytoplasm of HEp-2 cells. To standardize nomenclature and descriptions of the various fluorescence patterns of antinuclear factor (ANF), the Initiative of the International Consensus on ANA Patterns (ICAP) group was developed in 2014. The aim of ICAP is to promote consensus regarding nomenclature of ANA patterns, a microphotograph database, as well as classification depending on the employee skills. Information on the main characteristics, as well as specific clinical associations of the patterns is available at www.ANApatterns.org. In ANA classification trees, the patterns are indicated by the #AC (anticell pattern) alphanumeric code, being divided into nuclear, cytoplasmic and mitotic groups. Depending on the clinical significance and/or ease of recognition, this nomenclature focuses on the differences between the patterns described by specialists at competent and expert levels. Of the nuclear types, the most significant are homogeneous, speckled, dense fine-speckled, centromere, nucleolar, nuclear dots. The cytoplasmic types may be discerned into fibrillar, speckled, mitochondrial, Golgi, rods and rings. On leaders, behalf of the ICAP translation team is headed by the Full Member of Russian Academy of Sciences, Professor A.A. Totolian, under the auspices of the Russian Research Society of Immunologists. In this article, we present the Russianlanguage adaptation of the ICAP nomenclature, in order to ensure unification and standardization of ANA detection results in the patients with autoimmune diseases.

Current laboratory and clinical practices in reporting and interpreting anti-nuclear antibody indirect immunofluorescence (ANA IIF) patterns: results of an international survey

BackgroundThe International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns.MethodsTwo surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians.Results438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by > 85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by > 72% of laboratories.Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences.Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative.Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest.ConclusionThis survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive.

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.

Expression of ANA patterns by Indirect Fluorescent Assay in a local Hong Kong population from private sector

Abstract Objective Our aim was to investigate the association among age, sex and Antinuclear Antibody (ANA) patterns expressed in Hong Kong population for improving the autoimmune diagnosis by using ANA HEp-2 slides under fluorescent microscope. Methods A total of 65 blood samples from both male and female were collected and examined under fluorescent microscope. Haemolysed samples were excluded. In this research, a HEp-2 complete Kits with the methodology of indirect fluorescent antibody (IFA) was utilized for the detection and semi - quantification of antinuclear antibodies to distinguish positive ANA patterns classified in International Consensus on ANA Patterns (ICAP) for human body. Results In this experiment, we obtained that the number of positive samples was 6 (9%), the number of suspected (weak) positive samples was 4 (6%) and the number of negative samples was 55 (85%). In 6 positive samples, they were all females aged between 59 – 89. 5 in 6 positive samples in the result were over age 69 (83%). The positive ANA patterns included AC2 – Speckled, AC3 – Centromere, AC6 – Discrete Nuclear Dots, AC7 – Discrete Nuclear Dots, and AC8 – Nucleolar patterns separately. No AC-29 - DNA topoisomerase I was examined and resulted. We found that Hong Kong have a higher distribution of ANA positive frequency for female (9.23%) than male. Conclusion Our data indicated that ANA patterns were found in female aged above 69 is more likely to suffer from autoimmune disease related to ANA, providing an important foundation for considering and studying the trend in the elderly who are easier to have an ANA related autoimmune disease in Hong Kong.

Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists.

The indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting. We developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting. One hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. Based on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Rods and Rings Antinuclear Antibody Pattern in Non Hepatitis C Patients: A Series of Five Cases with Review of Literature

Antinuclear Antibody (ANA) is commonly found in various Autoimmune Diseases (AD) and their pattern help in narrowing down the differential diagnosis. International Consensus on ANA Pattern (ICAP) has recently introduced Rods and Rings (RR) pattern in their reporting guideline. Since its first identification, it has been considered to be exclusively associated with hepatitis C and its associated treatment {Ribavirin/Interferon-α (INF-α)}. However, later on, a few reports have described its association with other diseases and even in healthy population. In the present series, out of 25,242 clinical samples, five cases of RR-ANA pattern in non hepatitis patients were found and they were further investigated for their disease associations. We are documenting association of RR-ANA pattern with amoebiasis, hepatitis B, idiopathic thrombocytopenic purpura, Rheumatoid Arthritis (RA) and undifferentiated connective tissue disease. Documentation of such reports may help in better understanding of pathobiology and clinical relevance of this pattern.

V Brazilian consensus guidelines for detection of anti-cell autoantibodies on hep-2 cells

BackgroundThe V Brazilian Consensus for determination of autoantibodies against cellular constituents on HEp-2 cells, held in Brasilia (DF, Brazil) on August 27, 2016, discussed the harmonization between the Brazilian Consensus on ANA (BCA) guidelines and the International Consensus on ANA Patterns (ICAP) recommendations (www.anapatterns.org). Initial guidelines were formulated by the group of Brazilian experts with the purpose of guiding and enabling Brazilian clinical laboratories to adopt recommendations and to provide a common standard for national and international consensuses.MainbodyTwenty Brazilian researchers and experts from universities and clinical laboratories representing the various geographical regions of the country participated in the meeting. Three main topics were discussed, namely the harmonization between the BCA guidelines and latest recommendations of the ICAP initiative, the adjustment of the terminology and report on HEp-2 patterns, and a reassessment of quality assurance parameters. For the three topics, our aim was to establish specific guidelines. All recommendations were based on consensus among participants. There was concrete progress in the adjustment of the BCA guidelines to match the ICAP guidelines. To a certain extent, this derives from the fact that ICAP recommendations were largely based on the algorithm and recommendations of the IV Brazilian ANA Consensus, as consistently recognized in the ICAP publications and presentations. However, although there is great overlap between the two Consensuses, there are some point divergences. These specific items were individually and extensively discussed, and it was acknowledged that in several points ICAP improved recommendations previously issued by the Brazilian ANA Consensus and these changes were readily implemented. Regarding some specific topics, the BCA panel of experts felt that the previously issued recommendations remained relevant and possibly will require further discussion with ICAP. The term anti-cell antibodies was adopted as the recommended designation, recognizing that the assay addresses antibodies against antigens in the nucleus and in other cell compartments. However, the acronym ANA HEp-2 was maintained due to historical and regulatory reasons. It was also signalized that the latest trend in ICAP is to adopt the term Indirect Immunofluorescent Assay on HEp-2 cell substrate (HEp-2 IIFA). In addition, the quality assurance strategies previously presented were ratified and emphasized.ConclusionThe V BCA edition was successful in establishing an overall harmonization with the ICAP recommendations for interpretation of the HEp-2 IIFA test, pinpointing the perspectives in filling the remaining gaps between both initiatives.

Detection of Autoantibodies by Indirect Immunofluorescence Cytochemistry on Hep-2 Cells.

Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against cellular antigens for more than 50years. Originally using rodent tissue as substrate, the method was optimized by using the human immortal HEp-2 cell line derived from a larynx epidermal carcinoma. The HEp-2/IFA platform allows for optimal visualization of several cellular domains recognized by autoantibodies in the samples being tested. Serial dilution allows for the estimation of the concentration (titer) of the autoantibodies in the sample. Judicious analysis of the topographic distribution of the immunofluorescence (pattern) provides useful hints on the most plausible autoantigens being recognized, vis-à-vis the cognate autoantibodies. The importance of the HEp-2/IFA pattern has been recently emphasized by the International Consensus on ANA Patterns (ICAP), an initiative that established a comprehensive classification of the most relevant and prevalent HEp-2/IFA patterns (designated anti-cell (AC) patterns) and harmonized its nomenclature. The former designation "antinuclear antibody test" has been progressively replaced by the term "anti-cell antibody test," due to the recognition that the HEp-2/IFA method in fact allows the detection of autoantibodies to several cellular domains, such as the cytoplasm and mitotic apparatus.The performance of the HEp-2/IFA test is strongly influenced by several technical details, including cell culture conditions, cell fixation and permeabilization methods, choice and titration of fluorochrome-conjugated secondary antibody, use and choice of blocking solutions, washing buffers, and antifading mounting medium. The several steps of the procedure must be carefully performed in order to avoid the formation of false positive fluorescent artifacts. The quality control of the assay involves the use of serum standards for negative, low positive and strongly positive reaction in each run of the assay. In addition, every new lot or new brand of HEp-2 slides should be evaluated by using a panel of standard sera yielding the most relevant AC patterns. Special attention should be dedicated to the training of personnel for the analysis of the slides at the microscope. These should be able to identify possible artifacts, recognize all relevant AC patterns, and formulate possible reflex tests according to the observed AC patterns.

International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I.

AbstractThe indirect immunofluorescence assay (IFA) on HEp-2 cells is the reference method for autoantibody screening. The HEp-2 IFA pattern provides useful information on the possible autoantibodies in the sample. The International Consensus on Antinuclear Antibody Patterns (ICAP) initiative seeks to define and harmonize the nomenclature of HEp-2 IFA patterns. The most relevant and usual patterns have been assigned an alphanumeric code from anti-cell (AC)-1 to AC-28 and were organized into a classification algorithm (www.ANApatterns.org). The systemic sclerosis-associated autoantibodies to DNA topoisomerase I (Topo I) produce a peculiar composite 5-element HEp-2 IFA pattern (Topo I-like pattern) comprising the staining of the nucleus, metaphase chromatin plate, nucleolar organizing region, cytoplasm and nucleolus. In a recent assessment of the European Consensus Finding Study Group on autoantibodies, a well-defined anti-Topo I sample was blindly analyzed and classified according to ICAP AC patterns by 43 participant laboratories across Europe. There were wide variations among these laboratories in reporting nuclear, nucleolar and cytoplasmic patterns, indicating the inadequacy of the existing AC patterns to report the Topo I-like pattern. Several ICAP member laboratories independently demonstrated the overall consistency of the HEp-2 IFA Topo I-like pattern using HEp-2 slides from different manufacturers. The ICAP committee reviewed 24 candidate images and selected the four most representative images to be available on the ICAP website. The proper recognition of the AC-29 pattern should trigger suspicion of the presence of anti-Topo I antibodies, which may engender appropriate analyte-specific reflex tests to confirm the autoantibody specificity.

Report on the second International Consensus on ANA Pattern (ICAP) workshop in Dresden 2015.

The second meeting for the International Consensus on Antinuclear antibody (ANA) Pattern (ICAP) was held on 22 September 2015, one day prior to the opening of the 12th Dresden Symposium on Autoantibodies in Dresden, Germany. The ultimate goal of ICAP is to promote harmonization and understanding of autoantibody nomenclature, and thereby optimizing ANA usage in patient care. The newly developed ICAP website www.ANApatterns.org was introduced to the more than 50 participants. This was followed by several presentations and discussions focusing on key issues including the two-tier classification of ANA patterns into competent-level versus expert-level, the consideration of how to report composite versus mixed ANA patterns, and the necessity for developing a consensus on how ANA results should be reported. The need to establish on-line training modules to help users gain competency in identifying ANA patterns was discussed as a future addition to the website. To advance the ICAP goal of promoting wider international participation, it was agreed that there should be a consolidated plan to translate consensus documents into other languages by recruiting help from members of the respective communities.

From autoantibody research to standardized diagnostic assays in the management of human diseases - report of the 12th Dresden Symposium on Autoantibodies.

Testing for autoantibodies (AABs) is becoming more and more relevant, not only for diagnosing autoimmune diseases (AIDs) but also for the differentiation of defined AID subtypes with different clinical manifestations, course and prognosis as well as the very early diagnosis for adequate management in the context of personalized medicine. A major challenge to improve diagnostic accuracy is to harmonize or even standardize AAB analyses. This review presents the results of the 12th Dresden Symposium on Autoantibodies that focused on several aspects of improving autoimmune diagnostics. Topics that are addressed include the International Consensus on ANA Patterns (ICAP) and the International Autoantibody Standardization (IAS) initiatives, the optimization of diagnostic algorithms, the description and evaluation of novel disease-specific AABs as well as the development and introduction of novel assays into routine diagnostics. This review also highlights important developments of recent years, most notably the improvement in diagnosing and predicting the course of rheumatoid arthritis, systemic sclerosis, idiopathic inflammatory myopathies, and of autoimmune neurological, gastrointestinal and liver diseases; the potential diagnostic role of anti-DFS70 antibodies and tumor-associated AABs. Furthermore, some hot topics in autoimmunity regarding disease pathogenesis and management are described.

International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on reporting ANA results.

The International Consensus on ANA Patterns (ICAP) was initiated as a workshop aiming to thoroughly discuss and achieve consensus regarding the morphological patterns observed in the indirect immunofluorescence assay on HEp-2 cells. One of the topics discussed at the second ICAP workshop, and addressed in this paper, was the harmonization of reporting ANA test results. This discussion centered on the issue if cytoplasmic and mitotic patterns should be reported as positive or negative. This report outlines the issues that impact on two major different reporting methods. Although it was appreciated by all participants that cytoplasmic and mitotic patterns are clinically relevant, implications for existing diagnostic/classification criteria for ANA-associated diseases in particular hampered a final consensus on this topic. Evidently, a more concerted action of all relevant stakeholders is required. Future ICAP workshops may help to facilitate this action.