Antinuclear antibodies: A contemporary nomenclature using HEp-2 cells

Journal of Autoimmunity xxx (2010) 1e15

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Antinuclear antibodies: A contemporary nomenclature using HEp-2 cells Allan S. Wiik a, *, Mimi Høier-Madsen a, Jan Forslid b, Peter Charles c, Jan Meyrowitsch d a

Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, 171 76 Stockholm, Sweden c Translational Research, Kennedy Institute of Rheumatology, Imperial College London, 65 Aspenlea Road, London W6 8LH, United Kingdom d Percepton Ltd., Rialtovej 12, 2300 Copenhagen S, Denmark b

a b s t r a c t Keywords: Nomenclature Standardization HEp-2 cells Consensus formation Medical training ANA

The choice of terms used to describe indirect immunofluorescence (IIF) staining patterns of autoantibodies binding to HEp-2 cells is at present quite varied and disordered because no accurate consensus on names and descriptions exist. The aim of our study was to propose a logical and ordered IIF classification taxonomy based on 29 different selected IIF patterns. In a preliminary project carried out at Statens Serum Institut it was first shown by use of a software programme named DOORS developed by Percepton Ltd, that reading of digitized images of HEp-2 patterns on an LCD monitor could be used instead of traditional microscopy. Digitized images of HEp-2 patterns were then used in the EU supported project named CANTOR (June 1998eJuly 2000) aiming to reach consensus among three clinical immunology expert centres and collaborating to attain a classification version that could be used to qualitatively and quantitatively test and train image recognitions skills of laboratory technicians against expert consensus. The usability of this classification version was then tested in a course consisting of training and certification. The conclusion was that participants in the training programme clearly increased their perceptive skills using images, terms, descriptions and the graphic and statistic tools in the selfadministered DOORS programme and that software-assisted training could achieve a common and accurate level of visual pattern interpretation. All results from this project were reported to the European Commission but have not previously been published in scientific literature. This communication presents the final results of agreed image classifications. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Antinuclear autoantibodies (ANA) are clinically useful markers of certain chronic immuno-inflammatory diseases. The indirect immunofluorescence technique (IIF) used to detect ANA was described already in 1958 by Friou et al. [1] and IIF has become the standard method to screen for presence of ANA in patient sera. The use of tumor cell lines e.g. the laryngeal carcinoma cell line HEp-2 cells (ATCC-CCL 23) is now the preferred cell substrate for IIF

Abreviations: ANA, antinuclear antibodies; ANCA, anti-neutrophil cytoplasm antibodies; DM, dermatomyositis; DOORS, discrete object observation recognition system; FITC, fluoresceine isothiocyanate; IIF, indirect immunofluorescence; LEDGF, lens epithelium-derived growth factor; MCTD, mixed connective tissue disease; PBC, primary biliary cirrhosis; PCNA, proliferating cell nuclear antigen; PM, polymyositis; PML, promyelocytic leukemia; RNAP, RNA polymerase; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SSc, systemic sclerosis. * Corresponding author at: Digesmuttevej 10, 2970 Hørsholm, Denmark. Tel.: þ45 45867915. E-mail address: [email protected] (A.S. Wiik).

demonstration of ANA [2e4]. Though this paper deals with detection of ANA, it will also deal with other autoantibodies visualized by HEp-2 cell staining. In the present review the term “ANA” will thus cover all antibodies binding to HEp-2 cells, though only some of them are sensu strictu directed to nuclear antigens. Since the 1980-ies the clinical potential of detecting a positive ANA in a patient serum has progressed from merely being a marker in support for diagnosis of an autoimmune condition to becoming part of agreed diagnostic or prognostic criteria for certain diseases similar to clinically well-defined manifestations [reviewed in 4,5]. ANA detected by IIF using HEp-2 cells are found in frequencies between 100 and 50% of patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc), mixed connective tissue disease (MCTD), poly/dermato-myositis (PM/DM), Sjögren’s syndrome (SjS), Felty’s syndrome, drug-induced lupus-like disease, and juvenile chronic arthritis [5]. ANA directed to individual specific nuclear and cytoplasmic target molecules can be detected by a number of methods e.g. double immuno-diffusion, counter-immuno-electrophoresis, enzyme-linked immuno-sorbent assay (ELISA), antigen-specific

0896-8411/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2010.06.019

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bead fluorescence or luminescence techniques, and antigenspecific chip methods. These methods will not be mentioned further here, since the aim of this review is to look at the potential clinical and laboratory use and value of well-defined IIF results derived from expert interpretation of staining patterns seen on high quality HEp-2 cell slides. The usability of computer-assisted training to achieve visual pattern recognition consensus is also described. 2. Need for harmonization of IIF HEp-2 cell classification Use of unique names and unambiguous descriptions are imperative for interpreting and describing e.g. IIF HEp-2 cell patterns seen in routine serology. A high quality classification scheme can constitute a robust and valuable reference system. Attempts to attain accurate ANA classification can encourage experts to participate in further standardization, fuelled by recognition of new diagnostic sub-syndromes/phenotypes discovered to be associated with the presence of such antibodies. An agreed reference classification system can be continuously improved by experts who collaborate in consensus building studies that are on a proposed nomenclature like the one presented here. The clinical immunology laboratory has a very important role in supporting the chain of events that can lead to a correct diagnosis and phenotype of disease and thus to estimation of prognosis [4]. Both failing correct diagnosis in the clinical work-up and presenting a false positive result may lead to serious consequences for the patient, increased costs for the health care and social security systems give rise to loss of quality-of-life variables for the patients and their families. Therefore, optimizing diagnostic testing by establishing one standard nomenclature for positive IIF HEp-2 cell results, supported by unequivocal definitions, and reference images stored in one common database is compulsory for securing diagnostic quality. This will also alleviate meaningful consultation and interaction between clinical immunology laboratories. The autoimmune serology used for detecting non-organspecific autoantibodies by IIF HEp-2 cell technique is mainly related to diagnostics of chronic immuno-inflammatory rheumatic diseases and liver diseases, areas that are clinically quite complicated and diverse. Until now, very little textual information exists in the area of ANA and anti-cytoplasmic antibodies and most of the teaching has been undertaken in classrooms by experts using illustrative dia-positive slides or lecturing at a double-headed microscope. The nomenclature used has been confusing since one single term might cover antibodies with different staining characteristics and several terms could be used to describe one single pattern. This lack of consistency makes it difficult to compare fluorescence data derived from the literature. 3. European autoantibody consensus studies In the 8th European Workshop for Rheumatology Research (EWRR) in Corfu in 1988, chaired by Dr. Harolampos Moutsopoulos, the wide range of autoantibodies described in the literature was debated. Many participants had quite wide experience from their work as reviewers for medical journals and were concerned about the lack of efforts to advance the field. Also the very discrepant terms and results reported by different groups were discussed. Therefore, questions arose about the different factors that could lead to these inconsistencies, and the preliminary conclusion was that researchers might have been working with sera from rather heterogeneous patient populations, but also laboratory technicians could have used tests with low reproducibility. It was decided to invite various research laboratories in Europe to participate in workshops that would involve analyses of sera

from rheumatic disease patients for their content of autoantibodies to some common antigens e.g. dsDNA, nRNP, Sm, Ro(SSA), La(SSB), Scl-70, centromeres, ribosomal RNP and Jo-1. The aim was to examine the level of inter-laboratory concordance in detecting autoantibody specificities. Professors RN Maini (London) and WJ van Venrooij (Nijmegen) agreed to co-ordinate these studies with the help of head biomedical scientist Mr P Charles (London). Initially, 28 laboratories throughout Europe took part in analyzing patient sera for presence of autoantibodies using the techniques routinely used in their own laboratories. At that time laboratories mainly used IIF, counter-immuno-electrophoresis, immuno-diffusion, immunoblotting and ELISA. The first consensus study results were reported in the 9th EWRR chaired by Dr. Josef Smolen in Vienna. The early development of the European consensus studies were described in 1991 [6]. About a year later experts in the consensus study group were asked whether they would agree to submit their cookbook methods to the consensus study organizers, and these detailed operating procedures were subsequently sent around to all participating laboratories for orientation with a plea to return feedback suggestions for improvement. This led to the milestone publication of the book Manual of Biological Markers of Disease, edited by WJ van Venrooij and RN Maini, the first part of which was published in 1993 [7]. This first loose-leaflet part contained detailed descriptions of methods for IIF detection of ANA, methods for counter-immunoelectrophoresis and double immuno-diffusion, protein blotting, ELISA, immuno-precipitation of labelled proteins, immuno-precipitation of labelled RNA-containing autoantigens, methods to quantitate antibodies to dsDNA, to detect anti-neutrophil cytoplasm antibodies (ANCA) and antiperinuclear factor/antikeratin antibodies. The second loose-leaflet part of this book was published in 1994 [Supplement 1]. This part contained molecular descriptions of a large number of the antigens known to be recognized by autoantibodies at that time. The third loose-leaflet part of the book [Supplement 2] was published in 1996. This supplement focused on the various autoantibodies and autoantigens detected in rheumatoid arthritis (RA), SLE, SLE-overlap syndromes, SjS, SSc, PM/DM, systemic vasculitis, and primary biliary cirrhosis (PBC). Many authors contributing to the book and its supplements were expert participants from the European consensus studies. 4. Autoantibodies detected by use of IIF HEp-2 cell technique A great number of autoantibodies to cellular structures and organelles can be detected by IIF using HEp-2 cells as substrate. Up to the time of the publication of this method book it had been customary to look for ANA using frozen tissue sections of murine or rat liver or kidney as substrate. However, the use of cell lines was found to possess many advantages as pointed out by Humbel in 1993 [3]: cells cultured on glass slides lie evenly distributed and in a flat plane which allows detection of fluorescent conjugate molecules attached to any structure in the cell. With the advent of HEp-2 cells as the preferred cellular substrate, microscopists got the advantage of seeing cellular structures and organelles more distinctly, so that e.g. nuclear membrane, nucleoplasm, nucleoli, Golgi apparatus, ribosomes, mitochondria and cytoskeleton fibres were better recognized. This publication set up a logical hierarchy of staining patterns based on their location in the cell [3]. Characteristic details in IIF reactions involving the nucleus, the nucleoli, the mitotic spindle apparatus and the cytoplasm were described regarding antibodies binding to resting and dividing HEp-2 cells. Ever since its publication this glossary and the accompanying descriptions of IIF staining pattern has influenced ANA classification.

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However, this publication is not known to a wider audience and thus not often used as a reference for reporting ANA in scientific communications or in laboratory reports to clinics. Since different HEp-2 cell substrates may vary a lot in quality it is important to validate several cell preparations to select the best attainable and stick to the use of that. Thus, it was important for us to use just one big batch of HEp-2 cells (generously donated to us by ImmunoConcepts, Sacramento, California), selected on the basis of quality for the studies described below. 5. Attempts at refining ANA diagnostics using IIF It was now imperative to proceed towards harmonization of concepts and initiate standardization of ANA terminology. This involved agreement on a precise classification for global use, so that IIF ANA results could be read and interpreted identically around the world. Furthermore it became necessary to train the perceptive abilities of microscopists by challenging them with carefully preselected prototype test images, judged by experts as being classical representatives of reference IIF patterns and terms. For that reason a new exercise was added to the activities of the European Autoantibody Consensus Study Group during the last decade. In addition to the sera to be analyzed in the annual exercise, each participating laboratory centre also received a CD-ROM containing 20 IIF HEp-2 ANA images. These images had to be interpreted by the participating laboratory centres and reported back together with the serological results. Initially each image contained only one single staining pattern, but during the last few years even images with up to 4 different staining patterns in each slide have been used for the exercise. Participants receive written information on how to term and interpret the images by use of the taxonomy described below. Though this effort to reach consensus in ANA reading and interpretation has been well received by all centres, the actual gain in perceptive skills could not be quantitatively documented. 6. Computerized procedure to obtain harmonized HEp-2 classifications The software programme, DOORS (“Discrete Object Observation and Recognition System”) version 1.0, developed by Percepton Ltd, Copenhagen, was used in a preliminary project to clarify if reading of digitized images of HEp-2 patterns on an LCD screen could replace fluorescence microscopy readings of HEp-2 patterns. When this prerequisite had been confirmed (see below), DOORS was further developed and used in the EU-project CANTOR by participation of three European centres in London, Stockholm and Copenhagen in an attempt to harmonize a first HEp-2 cell classification version, that could be used and tested in a staged course for learning, training and certification/examination. The DOORS software programme is designed to help microscopists to combine three components into one entity: 1) the whole image with focus on image details, 2) an accurate verbal description and 3) a unique term for that image. Thus, the observer’s classification of an IIF HEp-2 image pattern had to be done using the following fixed procedure to ensure that the triad, image, detailed description and unique term, was used throughout: First, an encircled area of the image (either the whole image or a characteristic part of it) was studied in detail at 200 and 400 magnification, was interpreted aided by the descriptions of each reference image term, and finally the image was given a classification term from a taxonomy table (see below). The selected term automatically linked the selected term to the pattern on the screen, and the programme instantly showed whether the choice of term was correct or false.

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The visual and textual information helps the observer to memorize the associations and store them in their mind. When observers repeatedly use the same classification standard and judge images illustrating the same collection of image patterns, the intra-observer and inter-observer variability between observers can be calculated. The use of graphic and statistical tools embedded in the software allows participants to see their own results in between training sessions and before the certification session (examination). Thus, inexperienced observers can gradually reduce their faulty or self-biased classifications and become experts. The software programme reveals weaknesses in image recognition by allowing participants to see the misclassified image pattern along with the correct reference image and term. Thus, the learning efficiency of a classification course for one or more observers can be compared and documented. 7. Comparison between classifications by microscopy and by computer LCD screen evaluation A preparatory project in our laboratory at Statens Serum Institut in Copenhagen was carried out to assess whether reading of a particular microscopic field of an image could be interpreted with similar consistency on a computer LCD screen displaying the exact identical field examined in the microscope. Patient sera known to contain strong HEp-2 cell binding antibodies at a routine serum dilution of 1:160 were processed for ANA reading, using HEp-2 cell slides (ImmunoConcepts), and these were read in an incident light fluorescent Leica Aristoplan microscope from Leitz equipped with filters to display the typical yellow-green fluorescent colour of fluoresceine isothiocyanate (FITC)-conjugates. FITC-labelled rabbit antibodies specific for human Fcg-chains (DAKO, Glostrup, Denmark) were used at a dilution of 1:40. Four persons participated in the project which involved 86 sera. All slides were protected from fading by use of an anti-fading reagent. A servo-controlled two-axis scanner stage from Märzhäuser Wetzlar for 8 slides equipped with a computer-controlled motor at a stepping resolution of 0.1 my was connected to a computer by a serial RS-232 interface, programmed by Percepton Ltd. to ensure that observers were studying and classifying the precise identical pre-selected microscopic fields both in the microscope and on the computer screen. Digitized screen images were seen at a resolution of 480  220 TV lines recorded by a Kappa CF20 DX digital CCD colour video camera 1/200 -chip with microlenses 752 (H)  582 (V), a 1.4 lux (F 1.4) sensitivity and an analogue 24-bit video output for fluorescence microscopy. By use of a preliminary classification taxonomy of 9 different IIF ANA patterns and an intensity classification gradient of five different intensities, previously defined by persons chairing the project at Statens Serum Institut, four persons (A, B, C and D) took part in this comparative study. The 86 sera were classified five times by use of both microscopy and digitized images. To avoid fading the classifications had to be done during a period of one week. The digitized image fields were presented to the person in random order, tilted or shown upside-down to avoid memorydependent visual recognition caused by the rather short period set for carrying out the project. The Intra-Observer Variability for each of the four classification methods: ([microscopy pattern] [microscopy intensity], [computer image pattern], [computer image intensity]) was calculated for each observer in % by comparing for all five classification sets the average classification agreement between all classification sets and the calculated consensus set, in this example termed “intra-observer consistency”. This study showed that the average values found by evaluation of digitized images were higher both as regards pattern recognition (90 vs. 94), and intensity estimation (77 vs. 84).

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While some fading of the fluorescence might have taken place during the one week of study, this in our opinion could only partly explain the lower intra-observer consistencies of the microscopic readings. In conclusion, we now anticipated that IIF staining patterns judged from digitized images were at least as useful for further use in the study described below. 8. Harmonization efforts to create a HEp-2 cell classification version Supported by the results of the comparative study described above, a panel of laboratory experts from Stockholm, London and Copenhagen decided to use digitized images collected in each centre to agree on a set of unique reference classification types that could be incorporated into a HEp-2 classification format consisting of agreed names and definitions used in ANA serology. This led to the EU-funded project CANTOR which included technicians and experts working in the three laboratories as well as experts on study strategy selection and medical statistics. The project was coordinated by the Department of System Analysis, the Risø Science Institution, Roskilde, Denmark [8]. We anticipated that gradual harmonization of such a classification version could be done by use of repeated rounds of discussion about image patterns displayed on a computer screen aiming at converging our individual opinions towards a preliminary common set of standard names for use in a nomenclature for reference IIF staining patterns (prototypes) based on the article by Humbel [3]. The DOORS software was used to arrange these terms in the nomenclature in a tree-structured fashion into a classification format called the HEp-2 cell taxonomy (not shown). Here the taxonomy is shown in table form (Table 1).

Table 1 Taxonomy of HEp-2 cell staining patterns used in the CANTOR project. Membranous nuclear patterns Smooth membranous nuclear Punctate membranous nuclear Nucleoplasmic patterns Homogeneous nucleoplasmic pattern Large speckled nucleoplasmic Coarse speckled nucleoplasmic Fine speckled nucleoplasmic Fine grainy Scl-70-like nucleoplasmic Pleomorphic speckled (anti-PCNA) Centromere Multiple nuclear dots Coiled bodies (few nuclear dots) Nucleolar patterns Homogeneous nucleolar Clumpy nucleolar Punctate nucleolar Spindle apparatus patterns Centriole (centrosome) Spindle pole (NuMa) (MSA-1) Spindle fibre Midbody (MSA-2) CENP-F (MSA-3) Cytoplasmic patterns Diffuse cytoplasmic Fine speckled cytoplasmic Mitochondrial-like Lysosomal-like Golgi-like Contact proteins Vimentin-like Negative Undeterminable

8.1. Quantifying perceptive skills (Perceptometry) This taxonomy was now used for the visual classification system, characterized by a patented technology named ‘Perceptometry’, which made it possible for users to assign selected classification types to selected image areas. All executed classifications were saved locally on computers and were then merged into a common set of classifications in one database. Thus, it was possible by use of perceptometric tools to analyse intra-observer and inter-observer variability, using the statistical and graphic tools of DOORS to calculate and compare results by kappa statistics, by intra-observer and inter-observer variability for each individual observer, for each group, between individuals and between groups of observers.

8.2. Reference image selection The first step involved selection of digitized reference staining patterns representing each term in plenum and next to define these patterns down to minute details. Participants were furnished with a preliminary glossary based on the publication by Humbel [3], and they were asked to focus on the pattern seen in resting cells, in cells during DNA synthesis, in cells undergoing different phases of mitosis, patterns seen in the spindle apparatus and in the various large and small structures of the cytoplasm. Among the many electronic images stored locally in Copenhagen, London and Stockholm several candidates judged to be potential prototypic staining patterns and including cells representing all stages of the cell cycle were brought on CD-ROMs to a first meeting. Sitting down around a computer screen the 4 participating experts were asked to assign to each image pattern the IIF staining pattern term that fitted the best according to his/her experience. Each term and reference pattern which we agreed upon were included as samples for the final exercise. Twenty nine different patterns (27 positive, 1 negative and 1 borderline) were selected as being distinct and important for the exercise as reference patterns. Each reference pattern needed to be linked with an agreed term and an agreed description of the visual details. The next step was to attach to each pattern a term that not only described a pattern as being “granular, speckled, homogeneous” etc. but also took into account the accurate localization of the staining, e.g. nuclear membrane, nucleoplasm, nucleoles, cytoplasm etc. Where needed, the localization was included as a condition of the term. The characteristic distribution of the staining as e.g. “large speckled, coarse speckled, fine speckled, grainy, homogeneous” was thus followed by the localization term (nucleoplasmic, nucleolar, cytoplasmic). Particular care was taken to avoid any overlap of the terms which could lead to ambiguity. The hierarchy of terms was arranged in a localization-structured taxonomy as shown in Table 1. This taxonomy was used for the classification data input into the DOORS program. The descriptions covering characteristic features of all domains in interphase, metaphase and telophase cells were linked to each visual reference pattern seen during the introductory phase of the project. The descriptions included both positive staining features and negative characteristics that were important to define the term, e.g. “chromosomes should be negative in metaphase cells” etc. When around 40e60% of the cells showed some nuclear staining the positive cells were regarded as undergoing DNA synthesis (S-phase), a feature that is very important for recognition of some staining patterns, e.g. CENP-F, midbody (MSA-2), and the so-called anti-PCNA patterns (see below).

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8.3. Attempts to create a harmonized IIF image classification standard using software assistance Altogether more than 700 image sets shown at both 200 and 400 magnification have been included in the image library of the DOORS system through the years. Both experts, experienced technicians and beginner technicians took part in the study. Classifications agreed by the experts in Delphi rounds were used as reference. Table 2 presents the staged protocol for the experimental set up. The particular CANTOR project image databases contained all the classification categories mentioned. The database for baseline knowledge testing and for final examination consisted of 40 distinct single pattern image sets shown both at 200 and 400 magnifications. The database for the training phase consisted of 45 distinct images sets. These 85 (40 þ 45) image sets were different from the image sets used for construction of the 29 reference samples which were attached to the classification types of the taxonomy as reference image sets. The different phases of the study are illustrated in Table 2. One of the experts used DOORS to classify each image set three times, with the images presented in random order and flipped and rotated randomly in each session. Classification sets were exported by email by the system and then uploaded to a server at Percepton in Roskilde, Denmark, to make them available to all participants in the study. Images were available for download from this server, but were also distributed on CD-ROMs by ordinary mail. As can be seen from Table 2 the study protocol consisted of several phases. In each phase a participant had to classify the image set three times. Persons without prior microscopic image reading experience went through the same stages of our protocol as the experienced participants. In each training round the same 45 image sets were used but set up in a different random order and flipped over or upside-down. Care was taken to ensure that a suitable time interval occurred between each session to avoid short term memory bias of the results. The graphical and statistical tools of DOORS were used to evaluate the classification performance for each participant after each training cycle. Not only the kappa values which showed concordance rates with experts but also diagrams showing the discrepant results were accessible after each training session to help correct faulty and self-biased classification. If satisfactory results had been obtained during training the person could enter into the certification test (exam) after approval of a local expert. The results obtained at the start and at the end of the project are illustrated in Fig. 1. The rise of kappa values from 74% to 95% obtained by one

5

Kappa 1.00

100 95

0.90

Kappa value at start of course

0.80

Kappa value at en d o f c o u rs e

0.70

74

1 2 P a r ti c i p a n t s T

E

E

T

E

T

T

N

T

N

T

N

Fig. 1. Improvement in visual classification skills. E ¼ expert T ¼ trained N ¼ no prior experience.

totally inexperienced participant shows the learning efficiency by use of the software (shown on the far right of the figure). The promising results would indicate that an educational system like DOORS can be of great help for teaching, training and perhaps even standardization of image classification systems. 8.4. Proposed HEp-2 cell reference pattern encyclopedia The following photos constitute a proposed reference pattern encyclopedia, altogether covering staining patterns of the nuclear membrane, the nucleoplasm, the nucleoli, the mitotic spindle apparatus and the cytoplasm. Each of these carries a unique name and a description as accurate as deemed necessary. It is obvious that several IIF patterns can occur in one slide (one photo) because the serum studied contains more than one autoantibody. The definitions do not cover more than a first general picture of the most common ANAs surveyed at the time of the study. Since this review is intended for current as well as future use a number of additional details have been added recently by the lead author (written in smaller characters) e.g. in the section on fine speckled nucleoplasmic, grainy nucleoplasmic and certain cytoplasmic patterns. 8.4.1. Nuclear membrane (envelope) staining patterns 8.4.1.1. Smooth nuclear membranous pattern. A smooth membranous pattern (Fig. 2) can be caused by ANA binding to lamins A, B, C,

Table 2 Overview of the experimental strategy in the CANTOR project. Phases

Inexperienced

Experienced

Experts

Education

Reference images with tutor Test with reference images

Reference images with tutor Test with reference images

N.A.

Test with 40 images

Test with 40 images

Training test 1 Test with 45 images

Test with 45 images

Training test 2 Test with same 45 images Training test 3 Test with same 45 images

Test with same 45 images Test with same 45 images

Baseline test

Certification

N.A. Test with 40 images Test with 45 images Test with same 45 images Test with same 45 images

Test with 40 baseline Test with 40 baseline Test with 40 images images baseline images

Between baseline test, the 3 training tests and the certification test DOORS tools were used to judge results. N.A. ¼ not applicable.

Fig. 2. Smooth membranous nuclear staining.

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and/or some other integral membrane proteins [9]. This was described as - A smooth homogeneous ring-like fluorescence of the nuclear membrane in interphase cells. - Some samples with strong fluorescence may give an impression of whole nuclear staining - A similar pattern is seen in telophase cells. - In metaphase cells the fluorescence is diffusely localized in the cytoplasm, and chromosomal material is unstained.

8.4.1.2. Punctate nuclear membranous pattern. The punctate membranous pattern (Fig. 3) is produced by ANA directed to nuclear pore complex proteins [9]. The pattern was described as - A discontinuous punctate fluorescence along the nuclear membrane. When focusing through the nucleus the punctate staining can be seen on the surface of the entire nucleus. - A similar pattern is seen in telophase. - In metaphase the fluorescence is diffusely localized throughout the cytoplasm. - Some samples with strong antibodies may give an impression of whole nuclear staining. The text illustrates how these closely similar staining patterns were described to help microscopists focus on small characteristic details. The importance of differentiating between the two rather similar IIF patterns can be illustrated by their very different clinical associations, the smooth membranous pattern being associated with SLE, SjS, and sero-negative polyarthritis, while the punctate membranous pattern is associated with primary biliary cirrhosis PBC. 8.4.2. Nucleoplasmic staining patterns 8.4.2.1. Homogeneous nucleoplasmic staining pattern. Fig. 4a and b illustrates a homogeneous nucleoplasmic staining pattern with or without nucleolar staining characteristic of ANA directed to dsDNA, nucleosomes or other chromosome associated proteins e.g. histones [2,4]. Such ANA are mostly produced by patients with SLE, druginduced lupus, and juvenile chronic arthritis. This pattern was defined as follows: “A uniform diffuse fluorescence covering the entire nucleoplasm sometimes accentuated in the nuclear periphery can be associated with different antibodies, mainly directed to

Fig. 4. a. Homogeneous nucleoplasmic staining (positive nucleoli). b. Homogeneous nucleoplasmic staining (negative nucleoli).

chromosome components (DNA, histones a.o.). In some cases a more intense staining of the inner edge of the nucleus (nuclear rim) can be seen. Some samples may show an additional appearance of peripheral nucleolar staining. Nucleoli are often stained much like the surrounding nucleoplasm. In metaphase and telophase a homogeneous or peripheral chromatin staining is seen”. 8.4.2.2. Large granular nucleoplasmic pattern. Autoantibodies rendering a large granular nucleoplasmic pattern (Fig. 5) react with antigens in the nuclear matrix e.g. hnRNP and are found in patients with MCTD, sometimes together with anti-U1RNP [10]. The reaction pattern is characterized by “variably sized large speckles throughout the nucleoplasm and carries the popular name “nuclear matrix” staining. The distribution of IIF is due to reaction with interchromatin granules. Nucleoli and chromatin plates are negative. Metaphase and telophase cell cytoplasm contain diffusely localized speckles”.

Fig. 3. Punctate membranous nuclear staining.

8.4.2.3. Coarse granular nucleoplasmic pattern. Autoantibodies giving rise to a coarse granular nucleoplasmic staining pattern (Fig. 6) react with the nuclear RNP particles often called spliceosomes. They contain hnRNP and small nuclear RNA associated proteins, primarily the core proteins B/B0 , D, and E and the U1RNPassociated proteins A and C [10]. The antibodies to the D protein

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Fig. 5. Large speckled nucleoplasmic staining.

occur specifically in patients with SLE while the anti-U1RNP antibodies are apart of the clinical criteria for MCTD [11]. This characteristic IIF staining pattern was defined as follows: “Densely distributed, variously sized speckles, generally associated with larger speckles, throughout the nucleoplasm of interphase cells. Nucleoli are negative. Metaphase and telophase cell cytoplasm contain speckles with condensation around the chromatin plate which itself is negative. This pattern is most commonly associated with staining of nuclear spliceosomes”. Anti-U1RNP antibodies can be found in many different inflammatory rheumatic diseases, in which the patient manifests some symptoms overlapping with those seen in MCTD [4,12]. 8.4.2.4. Fine speckled nucleoplasmic staining. ANA giving rise to a fine speckled nucleoplasmic staining (in the selected image caused by antibodies to SSA(Ro)/SSB(La) (Fig. 7) were described as: “Fine speckled staining in a uniform distribution, sometimes very dense so that an almost homogeneous pattern is attained. Nucleoli may be positive (especially with anti-SSB/La antibodies) or negative. Cytoplasm of metaphase cells shows fine speckles and condensation around the chromatin plate which itself is negative. Nuclei of

Fig. 6. Coarse speckled nucleoplasmic staining.

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Fig. 7. Fine speckled nucleoplasmic staining.

telophase cells may be positive, sometimes being more strongly stained than nuclei of interphase cells”. This IIF pattern is commonly produced by ANA directed to the main autoantigens SSA (Ro) and/or SSB(La) in Sjögren’s syndrome/autoimmune epithelitis [13]. Anti-SSA(Ro)/SSB(La) antibodies in serum are now part of the revised international criteria for the disease [14]. As mentioned in the introduction several antibodies not included in our study can give rise to speckled nucleoplasmic staining. These are shortly mentioned for differential diagnostic purposes and for completeness sake. The first specificity is anti-Mi-2 antibodies which are directed to the central 75 kDa protein in the nucleosome remodelling and histone deacetylation complex. They give a fine speckled staining of interphase cells with negative nucleoli, and the dividing cells show negative chromosome plates [15]. A similar pattern is given by antibodies to the LEDGF (lens epithelium-derived growth factor), also called dense fine speckled. These antibodies decorate interphase nucleoplasm without the nucleoli but in contrast to the anti-SSA/SSB and anti-Mi-2 antibodies the anti-LEDGF antibodies stain the chromosomal plates of metaphase and telophase cells [16,17]. A fine speckled nucleoplasmic ANA decorating the interphase nuclei as well as the nucleoli can be seen in sera SSc/PM overlap patient sera containing strong antibodies to Ku antigen [18]. The chromosomes of dividing cells are negative. The Ku antigen is a complex antigen consisting of the enzymatically active DNAdependent protein kinase (DNA-PK) and its DNA-binding nonhistone p70/p80 heterodimers. 8.4.2.5. Fine grainy Scl-70-like pattern. An IIF staining pattern that we provisionally called the fine grainy Scl-70-like pattern is characteristic of antibodies in sera from patients with diffuse SSc (Fig. 8) harbouring antibodies directed to the enzyme DNA topoisomerase 1 (earlier called Scl-70 because of its immunoblotting binding pattern on extractable nuclear extracts) [19]. The nucleoplasmic staining is fine grainy to homogeneous with positive interphase cell nucleoli, and staining of the condensed chromatin seen in the mitotic cells. The following description was found to cover this IIF pattern: ”A uniform fine grainy staining of the nucleoplasm and the chromosomal areas of metaphase and telophase cells, often with accentuated positive nucleoli. The staining may, however, look homogeneous at lower magnifications. This staining is not only seen with anti-Scl-70 positive sera”.

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negative. This pattern is also known as “anti-PCNA” pattern (antiproliferating cell nuclear antigen), commonly seen in association with other patterns”. These antibodies are found in about 3% of all sera sent for ANA analysis, but is mainly found in SLE. The target antigen is a 34 kDa auxiliary protein of DNA polymerase d. It should be mentioned that several other ANA give rise to staining particularly of S-phase cells, but the IIF patterns are clearly distinguishable. The highly pleomorphic morphology of nucleoplasmic staining varying from fine to coarse speckled is so characteristic that it is rarely missed. One difficulty is that the anti-PCNA pattern is rarely seen alone but commonly occurs together with homogeneous nucleoplasmic or nucleolar patterns, probably because several sera found to be anti-PCNA positive derive from SLE patients.

Fig. 8. Fine grainy nucleoplasmic staining (Scl-70-like).

The main reason for adding this latter sentence was that a somewhat similar pattern can be seen with antibodies to the PM/ Scl antigen [20]. The nucleoplasmic staining of interphase cells is fine grainy but the nucleolar staining is clearly stronger and homogeneous [16]. The antigens involved are the PM/Scl-75 and PM/Scl-100 proteins of the PM/Scl complex found in the nucleoli but also partly in the nucleus and cytoplasm. The antibodies are found both in PM/SSc overlap patients and in patients with polymyositis or SSc only, possibly patients that will later develop overlap features. Anti-PM/Scl is the most common ANA encountered in juvenile myositis overlap syndrome.

8.4.2.7. Anti-centromere staining pattern. The next characteristic IIF ANA pattern to be mentioned here is the anti-centromere pattern (Fig. 10). These ANA were originally described in 1980 [22]. The description we reached at was: “Rather uniform discrete speckles located throughout the entire nucleus. Telophase and metaphase cells always show these speckles in the condensed chromosomal material. This pattern is produced by antibodies to the kinetochores of chromosomes, which may recognize several centromere proteins (mainly CENP-A, B, C, D, and E, but sometimes even CENP G and H)”. The antibodies are most commonly associated with limited SSc (earlier named the CREST syndrome), but are also frequently found in patients with Raynaud’s phenomena, teleangiectasias, lung involvement and a young age at disease onset [23]. Anti-centromere antibodies are seen in about 10% of patients with Sjögren’s syndrome and with biliary cirrhosis.

8.4.2.6. Pleomorphic nuclear pattern/PCNA pattern. A unique nuclear IIF pattern is given by antibodies to a proliferating cell nuclear antigen (PCNA) showing a pleomorphic nuclear staining of cells during the S-phase of DNA synthesis (Fig. 9) [21]. In our glossary this peculiar staining pattern was defined like this: “Nuclei of proliferating (S-phase) cells show a variety of speckles from fine to very coarse irregular speckles of the nucleoplasm (10e50% of the cells depending on the cell preparation). In some cells the nucleoli are stained as well. Resting (G-phase) and metaphase cells are

8.4.2.8. Multiple nuclear dot pattern. Antibodies giving rise to a multiple nuclear dot pattern (also named NSpI pattern) are directed to nuclear bodies, by some authors called PML bodies (Fig. 11) [4]. These can be described as: “5e10 dots per nucleus staining nuclear substructures, often named PML bodies (because of their pronounced expression in promyelocytic leukemic cells). Chromosomes of metaphase and telophase cells are negative. Cytoplasm of metaphase cells may be slightly stained”. Such antibodies can be directed to proteins in the PML bodies especially Sp-100, and PML protein. The antibodies are most frequently encountered in sera of patients with PBC but also in

Fig. 9. Pleomorphic nuclear staining (PCNA).

Fig. 10. Centromere staining.

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can be discerned [2,4]. The first to mention here is the homogeneous nucleolar IIF pattern (Fig. 13). “This pattern shows diffuse fluorescence of the entire nucleolus and no staining of chromosomes or the “nucleolar organizing regions” in the dense chromatin area of metaphase dividing cells. The cytoplasm is negative”. This pattern can be seen with antibodies to a 37 kDa phosphoprotein B23, a 110 kDa phosphoprotein nucleolin, PM/Scl protein complex, and Th/7-2 and To/8-2 RNA associated proteins of the RNase MRP/ RNase P complexes [16]. All of these antibodies are most commonly found in patients with some form of SSc or Raynaud’s syndrome, though some may occur in SLE and other systemic rheumatic diseases [4].

Fig. 11. Multiple nuclear dots staining.

some patients with immuno-inflammatory rheumatic diseases, sometimes co-occurring with anti-mitochondrial antibodies or antibodies to nuclear membrane pore protein gp210 [24]. Antibodies to the Sp-100 and the recently described Sp-140 proteins are highly specific for PBC [25]. Antibodies in patients with rheumatic disorders whose sera show multiple nuclear dot antibodies are not directed to the Sp-100 protein. Thus, for differential diagnosis specific assays for anti-Sp-100 antibodies need to be used in cases where multiple nuclear dots are found. 8.4.2.9. Coiled body pattern (few nuclear dots). A very discrete and often overlooked IIF staining pattern is the anti-coiled body pattern (Fig. 12). Antibodies to the p80 coilin protein in coiled bodies are generally rare. They can be found in patients with SjS, SLE, SSc, and Raynaud’s syndrome patients [26]. The characteristic IIF pattern can be described as: “2e6 dots per nucleus located in the nucleoplasm, often in close proximity to nucleoli. No staining of dense chromatin plates or cytoplasm of metaphase or telophase cells”. 8.4.3. Nucleolar staining patterns 8.4.3.1. Homogeneous nucleolar pattern. We now move to the nucleoli as structural targets of ANA where at least 3 IIF patterns

Fig. 12. Coiled body staining (few nuclear dots).

8.4.3.2. Clumpy nucleolar pattern. The second IIF pattern has been named clumpy nucleolar pattern due to the peculiar irregular staining of the nucleoli and coiled bodies (Fig. 14) [4]. We proposed the following description: “Brightly clustered larger granules corresponding to decoration of the fibrillar centres of the nucleoli as well as the coiled bodies. In mitotic cells the metaphase and telophase plate appears to have a fluorescent irregular “fan-like” edge. Metaphase cell cytoplasm may be slightly positive”. These antibodies target components of the U3-snRNP particles of the nucleoli, first and foremost fibrillarin localized in the dense fibrillar component of this organelle [19]. The anti-fibrillarin antibodies are selectively found in patients with SSc [27], and the antibodies occur in USA more commonly in black patients who manifest diffuse sclerosis combined with skeletal muscle and small bowel involvement. However, in white SSc patients the disease usually is limited [27]. In both races pulmonary hypertension is a threat indicating that SSc patients with anti-fibrillarin antibodies should be followed very closely to allow early treatment if any feature of incipient pulmonary hypertension occurs. 8.4.3.3. Punctate nucleolar pattern. The third nucleolar pattern has been named punctate due to the densely distributed but distinct grains seen in the nucleoli (Fig. 15). Another characteristic finding is staining of the nucleolar organizing region(s) located in the dense chromatin plate (arrow in Fig. 16). The description reached in our study was: “Small discrete grains mainly in the centre of the nucleoli. In metaphase cells 1e3 discrete speckles are seen within the chromatin body, corresponding to the nucleolar organizing regions. Metaphase cytoplasm is sometimes positive”.

Fig. 13. Homogeneous nucleolar staining.

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Fig. 14. Clumpy nucleolar staining.

Fig. 16. Centriole (centrosome) staining.

This nucleolar pattern is seen if anti-RNA polymerase (RNAP) I antibodies are found alone since this enzyme is uniquely found in nucleoli, but most commonly these antibodies occur together with anti-RNAP III and sometimes RNAP II antibodies that give rise to a speckled nuclear staining due to their presence in the nucleoplasm. The anti-RNAP I antibodies are specifically found in diffuse SSc patients, who develop the disease very rapidly from onset and frequently develop systemic arterial hypertension, heart, liver and kidney involvement [4,28]. Another punctate nucleolar staining pattern combined with nucleolar organizing region staining is found in rare cases of SSc especially in Spain, directed to the nucleolar antigen NOR 90 [29].

opposite sides of the dense chromatin plate in metaphase cells (Fig. 16). We found the following description covering the term: “The metaphase spindle displays a discrete fluorescent spot at each of the spindle poles lying perpendicular to one another. One or two bright spots closely adjacent to the nucleus are seen in the cytoplasm of interphase cells”. Anti-centriole antibodies are directed to multiple components of this microtubule-organizing centre e.g. pericentrin, ninein, Cep250, a- and g-enolases [30]. These ANA are rarely found in a routine laboratory setting since they occur only in few patients with partial signs of SSc or with Raynaud’s phenomena only, although they occur frequently in patients with mycoplasma infections [5].

8.4.3.4. Mitotic spindle apparatus staining patterns. Now we turn to antibodies that are not directed to sensu strictu nuclear antigens but are mentioned here because of their clear importance for cell biology and clinical sciences. 8.4.3.5. Anti-centriole (anti-centrosome) antibodies. The first one to mention is antibody to centrioles (centrosomes) visible as one to two bright organelles closely adjacent to the nuclear membrane in interphase cells and as two organelles characteristically located on

Fig. 15. Punctate nucleolar staining (arrows: nucleolar organizing regions).

8.4.3.6. Anti-spindle pole (anti-NuMa) antibodies. We found the following description for this pattern suitable: “Staining only of the triangular or “banana-shaped” pole area of the mitotic spindle in metaphase cells. Spindle fibres are negative. A more linear pattern of the poles are seen in telophase cells. A fine speckled nuclear pattern with negative nucleoli is mostly seen in resting and telophase cells, but these may also be negative” (Fig. 17).

Fig. 17. Spindle pole (NuMa) (MSA-1) staining.

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The main autoantigen is NuMa, a 240 kDa protein called centrophilin located in the close vicinity of spindle poles and in the nucleoplasm of interphase cells sparing the nucleoli [4]. A high frequency of anti-NuMa antibodies has been found in patients with mycoplasma pneumonia infections [31] but they are otherwise rare in rheumatic diseases. 8.4.3.7. Spindle fibre antibodies. Antibodies decorating only the spindle fibres themselves are easily distinguishable from antiNuMa because the resting cells are negative (Fig. 18). Description: “Staining of the whole spindle fibre apparatus from pole to the chromatin plate in metaphase cells. No staining of dense chromatin in resting or dividing cells”. These very rare antibodies target a protein called HsEg5 [32]. Anti-HsEg5 antibodies were first described in SLE, however, this association has not been clearly confirmed. 8.4.3.8. Midbody antibodies (MSA-2). These antibodies are named so because the most conspicuous IIF is concentrated in a peculiar way in the cleavage furrow area between two dividing cells during cytokinesis (Fig. 19) [3,4,16]. As can be seen there are several other characteristics in the staining pattern as described below: “In prophase and metaphase cells the fluorescence is localized in the chromosomal region as fine streaks perpendicular to the edge of the metaphase plate. In telophase cells the staining is restricted to the cleavage furrow and the narrow connecting midbody between cells that are completing cytokinesis. S and G2 phase cells are stained with discrete or patchy nuclear speckles. The pattern is due to antibodies to the MSA-2 antigen (Mitotic Spindle Antigen 2)”. The autoantigens targeted by such antibodies are not clearly defined. Anti-midbody is most often found in patients with SSc or Raynaud’s syndrome [4,5]. 8.4.3.9. CENP-F protein antibodies (MSA-3). This particular IIF staining pattern should be recognized with certainty since the main disease association is some form of cancer, most commonly lung and breast cancers [33]. The pattern was described like this (Fig. 20): “The most characteristic feature is seen in metaphase cells, where the pattern is constituted by 2 sets of dense large granules surrounding the chromosomal metaphase plate as a grip, often looking like a zipper. The surrounding cytoplasm is diffusely stained. In prophase cells a dense punctate decoration of the chromosomes is seen. A very fine dense nuclear speckled pattern

Fig. 18. Spindle fibre staining.

Fig. 19. Midbody (MSA-2) staining.

may be seen in some interphase cells. The pattern is due to antibodies to the MSA-3 antigen (“Mitotic Spindle Antigen-3”, which may be identical to the centromere protein CENP-F, an outer centromere protein)”. The CENP-F protein is not a native centromere component but a nuclear matrix protein which associates with kinetochores during the G2 phase. The frequency of malignancies in patients harbouring anti-CENP-F antibodies has been estimated very differently in various publications (50e80%). 8.4.4. Cytoplasmic staining patterns 8.4.4.1. Diffuse cytoplasmic pattern. A characteristic diffuse pattern is seen with antibodies to Jo-1 and other amino-acyl-tRNA synthetase antibodies e.g. PL-7, PL-12, OJ, EJ, Sc, KS [4,12] (Fig. 21). Description: “A very fine dense granular to homogeneous staining or cloudy pattern covering part or the whole cytoplasm”. Chromosomal material in metaphase cells is negative, while metaphase cytoplasm is positive”. At the time of study the following text was added to include anti-ribosomal P antibodies: “No staining of nuclei, but nucleoli may be homogeneously stained if antibodies are directed to ribosomal RNP proteins, precursors of which are found in nucleoli”.

Fig. 20. CENP-F (MSA-3) staining.

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Fig. 21. Diffuse cytoplasmic staining.

Fig. 22. Fine speckled cytoplasmic staining.

Thus, the diffuse cytoplasmic pattern can indicate presence of myositis-specific autoantibodies (Jo-1, PL-7, PL-12 etc.), but would indicate presence anti-ribosomal P antibodies specific for SLE if accompanied by nucleolar staining [4,12]. Antibodies to amino-acyl-tRNA synthetases like Jo-1 occur in particular PM patients, namely those that additionally exhibit other features of the so-called anti-synthetase syndrome: fibrosing alveolitis, arthritis, Raynaud’s phenomena and interstitial lung disease [34]. The lung manifestations may precede the onset of myositis. It is essential to notice that anti-tRNA synthetase antibodies only give rise to a positive cytoplasmic reaction in less than half of patients harbouring the antibodies. Thus, specific tests have to be used if the IIF HEp-2 cell result comes out negative. Antibodies to the ribosomal P0, P1, P2, proteins are highly specific for the diagnosis of SLE, and it has been proposed to include them as part of diagnostic criteria in the future. They occur in particular in patients manifesting neuro-psychiatric lupus, but are also prevalent in cases of active lupus nephritis [35]. The antibodies occur with different frequencies in different populations.

a reticular network. The major antigen cluster recognized is called M2, and is constituted by four major mitochondrial inner membrane proteins. Nuclei and nucleoli are negative, but strongly positive sera may give an impression of a speckled nuclear staining as well. Cytoplasm of dividing cells is strongly positive”. Presence of anti-mitochondrial antibodies at a dilution of 1:40 or higher is part of the diagnostic criteria for primary biliary cirrhosis The antibodies target E2 subunits of branched chain 2oxo-acid dehydrogenase complex as well as epitopes of the pyruvate dehydrogenase complex. Presence of anti-mitochondrial antibodies can predict later onset of PBC [36].

8.4.4.2. Fine speckled cytoplasmic pattern. This pattern was included to find out if diffuse and fine speckled patterns could be separated by participants in the study. We already knew that this pattern most likely was associated with presence of anti-tRNA synthetase antibodies just like the diffuse cytoplasmic pattern, in particular anti-Jo-1 (Fig. 22). The description reached at was: “Fine granules dispersed throughout the cytoplasm, becoming more evident towards the periphery of the cell, sometimes producing a “stardust”-like appearance. No staining of nuclei or nucleoli. This pattern is characteristic of antibodies to tRNA synthetases. Chromosomal material in metaphase cells is negative, but the cytoplasm contains fine granules like the resting cells”. Sera found to harbour such antibodies have been shown most frequently to target Jo-1 and derive from patients with the anti-synthetase syndrome. 8.4.4.3. Mitochondrial-like cytoplasmic pattern. The name mitochondrial-like was preferred because we could risk that autoantibodies to other finely dispersed organelles in the cytoplasm e.g. peroxisomes, signal recognition particles etc. could give a slightly similar pattern, and we did not have representatives of these antibodies. The pattern we included derived from a patient with primary biliary cirrhosis with known mitochondrial antibodies (Fig. 23) and this was described as follows: “Larger irregular granules extending from the nucleus throughout the cytoplasm in

8.4.4.4. Lysosomal-like cytoplasmic pattern. This pattern is very rare (Fig. 24) and the name does not imply that the organelles targeted in the figure are known. Again the pattern was included to test the perceptive discriminatory abilities of the participants. We described the patterns as: “Irregular intermediate to large organelles distributed throughout the cytoplasm, the pattern can be due to antibodies directed to organelles like lysosomes or peroxisomes. No staining of nuclei or nucleoli, but diffuse staining of the cytoplasm of dividing cells”. Obviously the possible clinical association of this pattern is impossible due to lack of knowledge about the

Fig. 23. Mitochondrial-like staining.

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them actin cables and tropomyosin. In our hands the HEp-2 cell slides from ImmunoConcepts rarely show reactivity with actin cables that consist of F-actin, possibly because of the fixation technique used. Also the tropomyosin-like structures are rarely seen. We described the image we used as reference pattern (Fig. 26) as follows: “Stress fibres lying in a simple focal plane just underneath the plasma membrane are stained. Nuclei, nucleoli and metaphase chromosomes are negative. Actin cables spanning the whole length of the cell close to the plasma membrane as well as shorter stress fibres with dendrite-like extensions and membrane ruffling are regularly stained”. This description thus does not try to separate the actin from the tropomyosin-like IIF reaction. There are several publications on the existence of autoantibodies to tropomyosin in the literature, but HEp-2 cells have not been used more than sporadically for their detection [38]. Antibodies to a number of microfilament proteins seem to be naturally occurring, polyreactive, and have low specificity for any particular autoimmune disease. Fig. 24. Lysosomal-like staining.

target organelle specificity. Antibodies to early endosomes and GW may give somewhat similar cytoplasmic staining [37]. 8.4.4.5. Golgi-like staining. Many autoantigens are known to be potential targets of antibodies to the Golgi complex [reviewed in 37]. The main autoantigen is assumed to be giantin. The staining pattern (Fig. 25) may be somewhat different but generally it is assumed that the IIF patterns involving the Golgi complex, lying closely adjacent to the nucleus of interphase cells, are easily recognized. All of the autoantigens are located on the cytoplasmic face of the Golgi cisternae. We described the pattern: “Staining of a polar organelle adjacent to and partly surrounding the nucleus, composed of irregular large granules. Nuclei and nucleoli are negative. Diffuse staining of the cytoplasm of dividing cells sometimes with accentuation around chromosomal material”. Anti-Golgi antibodies occur in a variety of rheumatic diseases e.g. SjS, SLE, RA and some overlap syndromes, but some anti-Golgi positive patients have non-autoimmune chronic disorders e.g. idiopathic cerebellar ataxia, lymphoma or pulmonary fibrosis. 8.4.4.6. Contact proteins. The surface of HEp-cells displays several proteins used for contact and interaction with other cell, among

Fig. 25. Golgi-like staining.

8.4.4.7. Vimentin-like staining pattern. Vimentin-like staining pattern (Fig. 27) [3] was described as: “Fibres dispersed in the cytoplasm, often concentrated around the nucleus. Nuclei, nucleoli and metaphase chromosomes are negative”. These antibodies are rare and they cannot at present be linked with any particular autoimmune condition, but the pattern was selected just for its morphological characteristics. As can be seen from the taxonomy tree there was also one box for undeterminable positive IIF. 8.4.4.8. Other antibodies. In addition to the antibodies described above there is interesting new literature on autoantibodies to endosomes, lysosomes, proteasomes, assemblyosomes, exosomes, and GW bodies that also can be visualized on HEp-2 cells [37]. Several of these antibodies are associated with autoimmune rheumatic or chronic neurological diseases. 8.5. Negative IIF results It is not necessary to show photos or describe what is seen in a negative HEp-2 cell preparation as seen with most healthy donor sera. However, it is very important to stress that a negative results does not rule out SLE or another autoimmune rheumatic disease. Likewise, even a strong positive ANA does not automatically indicate a diagnosis of an immuno-inflammatory rheumatic disorder

Fig. 26. Contact protein staining.

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Fig. 27. Vimentin-like staining.

(see dense fine speckled/anti-LEDGF above). However, it should be remembered that an autoantibody such as ANA, anti-mitochondrial antibody or anti-citrullin peptide antibody found in a serum of an apparently healthy person may be the first detectable immunological signature of latent rheumatic disease e.g. SLE, PBC and RA [36,39,40]. Also worth noting is that certain ANA such as anti-SSA(Ro) and anti-Jo-1 do not necessarily give a positive IIF result by use of HEp-2 cells as substrate, and thus supplementary methods have to be used to accurately determine whether they are present or not [41]. A special HEp-2 cell substrate has been developed to account for the weak expression of SSA/Ro antigen, engineered by transfecting some of the cells in HEp-2 cell slides with SSA/Ro-60, and the first studies from a large routine setting seem to indicate that this cell substrate is useful for detecting SSA/Ro antibodies [42]. Obviously, clinicians and laboratory experts need to agree whether antibodies binding to other structures that the nuclear membrane, nucleoplasm or nucleoli should be reported as “ANA negative” or if staining of some cytoplasmic structures expected to be of clinical importance should be reported as “positive ANA” or be mentioned in a supplementary text. 9. Discussion The results described above do not pretend to represent a real standardization of HEp-2 cell interpretation and nomenclature, but is meant to be a first harmonization step towards such standardization. Though preliminary, the nomenclature used here, can form a rational basis for further work on standardization of IIF pattern terminology, and for international discussion rounds to improve quality assurance in an area where few attempts have been made earlier to reach consensus on terms and definitions [43]. In addition it has not been attempted to quantitatively measure of the perceptive abilities of persons responsible for ANA interpretation and reporting. It is well known that false or missed diagnosis delays rational therapy and follow-up and adds substantially to long-term prognosis, as well as and health cost and individual patient suffering. Supported by scientific publications about high inter-observer variability reported within different image diagnostic fields there has been growing concern among health authorities and politicians how to detect and avoid various reasons for inaccuracy pertaining to diverse diagnostic methods, particularly in image diagnostic fields like rheumatology and immunology. This topic has been discussed at length in a recent supplement to American Journal of Medicine [44]

on how to implement self-calibrating feedback procedures to correct inaccurate image diagnostic classification. Today where cross-border diagnostics and treatments are starting to become available it is also imperative to attain global quality assurance of all aspects of diagnostics. This is a topic for serious discussions among politicians in the European Parliament at present. One main reason that the IIF ANA fields have not been moving forward lately may be the fast advent of new technologies that aim at high throughput testing and cost saving, without thoroughly investigating whether the change from IIF ANA screening to composite solid phase technologies (ELISA, dot blot, line blot, bead luminescence assay, microchips etc.) causes loss of important information for the clinician e.g. false positive or false negative results due to lack of a whole host of relevant autoantigens as antigen source [45e47]. Therefore, as quality assurance is an important goal for autoimmune sero-diagnostics, careful collaboration between patients, experienced clinicians and laboratory scientists is highly needed [46] and post-marketing studies must be done before general acceptance of an ANA screening method that is not based on IIF HEp-2 cell technique [47]. The caution to avoid premature acceptance is of particular importance in view of the long time frame of these chronic diseases where the autoantibody also signals prognostic outcome. In addition there are clear ethical aspects of all forms of diagnostics especially pertaining to chronic immuno-inflammatory diseases where early diagnosis and treatment is mandatory to avoid invalidity and loss of quality-of-life variables [48]. Finally, the diagnosis always will depend on the collaboration between clinicians, imaging specialists, laboratory diagnosticians, medical technologists, and the biotech industry. We also need international bodies to set new quality standards on how to keep all levels that interact in diagnostics informed about what is needed for advanced up-to-date diagnostics for the benefit of patients [49,50]. The self-administered system DOORS described for harmonizing image classification has now been developed further for internet use by Percepton to attain an interactive Web-based platform for teaching, harmonization and use in discussion fora, and people interested in further standardization of HEp-cell-based diagnostics are encouraged to contact the lead author of this article or consult the address www.percepton.com. The structured nomenclature described has now been used like an encyclopedia in the European Consensus studies during the last 6 years. A uniform recognition of antibodies giving rise to rare HEp-2 cell patterns will allow new discoveries regarding sub-syndromes/phenotypes of disease and their prognosis if clinical and laboratory scientists collaborate in multi-centre studies. Several other fields of medicine and technology that rely on subjective image interpretation, e.g. radiology, cytology, pathology etc. for may certainly benefit from using computer-assisted programmes like the DOORS program used here. Special address This manuscript has been written in honour of Professor Harolampos Moutsopoulos whose friendship and collaboration I have enjoyed for over 20 years. I still have great memories of the scientific meetings he has arranged through the years and will always remember the warm hospitality shown to me by him and his colleagues. Professor Moutsopoulos is indeed a great figure in autoimmunology as recognized by this special series of the journals [51e54]. Acknowledgements The participation of laboratory technicians in all three laboratories is gratefully acknowledged. Mr. Verner Andersen, Science

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Centre, Risø is warmly thanked for his skillful leadership during the CANTOR project. We want to thank ImmunoConcepts, Sacramento, California for the donation of one large batch of HEp-2 cells for this project. An EU grant (EU CANTOR project HC 4003 (HC)) supported the CANTOR project and thus, the EU Commission is gratefully acknowledged for making the study possible. References [1] Friou GJ, Finch SC, Detre KD. Interaction of nuclei and globulin from lupus erythematosus serum demonstrated with fluorescent antibody. J Immunol 1958;80:324e9. [2] Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol 1989;44:93e152. [3] Humbel RL. Detection of antinuclear antibodies by immunofluorescence, part 1. In: Maini RN, van Venrooij WJ, editors. Manual of biological markers of disease. Dordrecht: Kluwer; 1993. [4] Wiik A. 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Please cite this article in press as: Wiik AS, et al., Antinuclear antibodies: A contemporary nomenclature using HEp-2 cells, Journal of Autoimmunity (2010), doi:10.1016/j.jaut.2010.06.019