Clinical relevance of HEp-2 indirect immunofluorescent patterns

Introduction

Autoantibodies, as detected by the indirect immunofluorescence assay (IIFA) on HEp-2 cells (IIFA HEp-2), are recognised as important diagnostic markers in a plethora of autoimmune diseases, in particular the systemic autoimmune rheumatic diseases (SARD).1 Although somewhat dated by today’s standards, members of the American College of Rheumatology (ACR) prepared an evidence-based guideline for the usefulness of the HEp-2 IIFA results for diagnostic and prognostic purposes and also for meeting diagnostic criteria.2 That guideline was based on reactivity with nuclear antigens as detected by IIFA on rodent tissue or HEp-2 cells. More recently, the IIFA on HEp-2 cells was reinforced as the gold standard for autoantibody screening in SARD.3

Interestingly, the HEp-2 IIFA test reveals much more information than the mere absence or presence of autoantibodies, that is, the level of antibody as well as the HEp-2 IIFA pattern. Based on titration or appropriate evaluation of the fluorescence intensity, the antibody level can be determined and this information has general concordance with the clinical relevance of the test result. Indeed, higher antibody levels are better associated with SARD and have an increased likelihood to identify the autoantigen in follow-up testing.4–6 The importance of the level of autoantibodies is also recognised in the ACR guideline as well as by the recommendations issued by the European Autoimmunity Standardization Initiative (EASI) and the International Union of Immunologic Societies (IUIS) Autoantibody Standardization Subcommittee.2 7

The HEp-2 IIFA pattern may also reveal clinically relevant information. This information is not restricted to giving direction to follow-up testing for antigen-specificity, but, for instance, the centromere pattern is included in the classification criteria for systemic sclerosis,8 while the nuclear dense fine speckled pattern is reported to be more prevalent in apparently healthy individuals as compared with patients with SARD.9 To harmonise the names and descriptions of the distinct HEp-2 IIFA patterns, an ordered classification taxonomy was proposed.10 This proposal was subsequently elaborated on by the International Consensus on ANA Patterns (ICAP), initiated in parallel to the 12th International Workshop on Autoantibodies and Autoimmunity (2014) held in Sao Paulo, Brazil. During this workshop, a consensus was reached on the nomenclature and definitions of 28 HEp-2 IIFA patterns. Each HEp-2 IIFA pattern was ascribed an alphanumeric code from AC-1 to AC-28.11 The consensus nomenclature for each pattern and representative images were also made available online at the ICAP website (http://www.ANApatterns.org).

In addition to the nuclear patterns, important cytoplasmic and mitotic patterns may also be observed in HEp-2 IIFA analysis. Although reporting non-nuclear patterns is considered clinically relevant,7 for various jurisdictional reasons there is no clear-cut consensus viewpoint on reporting non-nuclear patterns as a negative or positive test.12 With the understanding that the term ‘Antinuclear antibody (ANA) test’ may be inappropriate to designate a test that also addresses autoantibodies to antigens in the cytoplasm and mitotic apparatus, an alternative name, anticellular antibodies, was suggested in the EASI/IUIS recommendations.7 Recent publications from ICAP have preferred the term HEp-2 IIFA as it covers the whole spectrum of patterns that can be observed when using the HEp-2 cells as substrate.13 14

Originally, the HEp-2 IIFA patterns were associated with diseases, but it was anticipated that many of these associations are only valid if the antigen-specificity was confirmed by follow-up testing. In subsequent ICAP workshops, it was agreed that the disease associations should be replaced by clinical relevance. In this current paper, we present the consensus on the clinical relevance of the distinct HEp-2 IIFA patterns as achieved by consecutive workshops and discussions among the executive ICAP members.

Materials and methods

For discussion about the structure of clinical relevance templates were prepared for AC-2 (LECA), AC-3 (JD) and AC-5 (MS). This formed the basis of a guideline for description of each AC pattern (EC). Of highest importance, it was agreed that the information should be objective and helpful for the clinician, the pattern–antigen associations should be put in the right clinical context and information should be evidence-based.

In preparation for the third ICAP workshop in Kyoto (2016), composition of the clinical relevance documents was started for the nuclear patterns (JD, LECA, MS), cytoplasmic patterns (CAvM, EKLC) and mitotic patterns (MH, TM). As far as already available, the documents were commented on by the ICAP executive board and, after appropriate adjustment, discussed with the workshop participants. The feedback from participants mainly focused on the structure of the information provided, on the required level of detail and the format of recommended follow-up testing.

In anticipation of the fourth ICAP workshop in Dresden (2017), the set of clinical relevance documents was completed for all patterns. Further comments from the ICAP executive board were included. The resulting documents were individually discussed with the workshop participants for nuclear (JD), cytoplasmic (CAvM) and mitotic (MH) patterns. Besides several substantive comments, there was general agreement that the information should be provided in tabular format at two distinct levels. The first level should contain information on relevant follow-up testing in the respective clinical context, the recommended follow-up tests should be commercially available and detailed test characteristics should not be given because of potential geographic and jurisdictional differences. Information based on case reports or small patient cohorts, as well as information on possible follow-up testing that is only available in specialised research laboratories, should only be provided in the second level information.

Tables for nuclear, cytoplasmic and mitotic patterns were prepared for first and second level information (JD). These tables were commented by the ICAP executive board and finalised by JD. Of note, since the starting point of the tables on clinical relevance is the HEp-2 IIFA pattern and not the clinically suspected disease, the tables do not list all autoantibodies related to the respective disease.

Results

Nuclear HEp-2 IIFA patterns

To date, a total of 15 nuclear HEp-2 IIFA patterns have been described, that is, AC-1–AC-14 and AC-29. Table 1 summarises the clinical relevance of these patterns.8 9 14–79 Since AC-29 was only recently described,14 the advice for follow-up testing for autoantibodies to topoisomerase I (Scl-70) in case of clinical suspicion of systemic sclerosis is also added as a note to the clinical relevance of AC-1. In particular, disease-specific immunoassays, like autoimmune liver disease profile, inflammatory myopathy profile, systemic sclerosis profile, are often only available in specialty clinical laboratories.

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For six nuclear HEp-2 IIFA patterns (AC-3, 5, 7, 8, 12 and 13), additional information about clinical relevance is summarised in online supplementary table S1. Although some assays for anti-CENP-A antibodies are commercially available, these antibodies are included in online supplementary table S1 because the majority of sera revealing the AC-3 pattern are also reactive with CENP-B. In contrast to CENP-A, CENP-B is included in many routine extractable nuclear antigens profiles.

Cytoplasmic HEp-2 IIFA patterns

Table 2 summarises the clinical relevance of the nine cytoplasmic HEp-2 IIFA patterns, that is, AC-15–AC-23.26 33 80–101 It is recognised that the distinction between AC-19 (dense fine speckled) and AC-20 (fine speckled) can be challenging. Moreover, within the spectrum of anti-tRNA synthetase antibodies, not all produce an HEp-2 IIFA pattern and only some anti-Jo-1 antibodies are considered to give the AC-20 pattern, while the other anti-tRNA synthetase antibodies (EJ, KS, OJ, PL-7 and PL-12) are more likely to reveal the AC-19 pattern. Solid information on the pattern of two additional anti-tRNA synthetase antibodies (Ha and Zo) is lacking. Overall, the relation between these two cytoplasmic HEp-2 IIFA patterns and the distinct anti-tRNA synthetase antibodies is subject to further discussion. In clinical practice, the complete spectrum of the anti-tRNA synthetase antibodies should be determined irrespective of the subtype of cytoplasmic speckled pattern, that is, AC-19 or AC-20.

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For seven cytoplasmic HEp-2 IIFA patterns (AC-15–AC-19, AC-22 and AC-23), more detailed information is provided in online supplementary table S2. In particular, for AC-16–AC-18, the clinical associations are quite diverse, depending on the antigen recognised. Overall, the clinical associations provided are primarily based on antigen-specific immunoassays and not on the HEp-2 IIFA pattern as such.

Mitotic HEp-2 IIFA patterns

The clinical relevance of the five mitotic patterns is summarised in table 3,102–122 with more detailed information in online supplementary table S3. As for the cytoplasmic patterns, clinical associations for the mitotic patterns are primarily based on antigen-specific immunoassays and not on the HEp-2 IIFA pattern as such.

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Discussion

In the current paper, we present the ICAP consensus on the clinical relevance of 29 HEp-2 IIFA patterns defined by ICAP.11 14 The consensus on clinical relevance is defined in the clinical context of the patient, that is, suspected disease, and includes recommended follow-up testing within the spectrum of antigen-specificities that are commercially available. Obviously, if follow-up testing identifies the antigen, the clinical relevance can be further refined.123

Defining the clinical relevance of HEp-2 IIFA patterns in the context of disease manifestations is meant to be an important tool for the clinician in the diagnostic work-up of patients suspected of SARD. Unfortunately, good data on the association between HEp-2 IIFA patterns and the distinct diseases are lacking, probably due to reasons summarised below. There are several reasons for not finding a perfect association between HEp-2 IIFA patterns and diseases. First, pattern assignment in clinical laboratories is rather inconsistent as shown by external quality assessments.14 124 125 This is exactly the reason why ICAP was initiated: the consensus on nomenclature and definitions of HEp-2 IIFA patterns allows to align pattern description across laboratories. Also, the integration of computer-aided immunofluorescence microscopy (CAIFM) may further improve the consistency in pattern assignments.126–131 As such, it is promising that several companies involved in CAIFM have declared their intention to accommodate to the ICAP classification. Second, even apparently healthy individuals may have autoantibodies as detected by the HEp-2 IIFA. Such autoantibodies, being either innocent bystander antibodies or predictive antibodies, may still be present on development of SARD and interfere with the SARD-related pattern. Interestingly, the pattern best associated with apparently healthy individuals is the nuclear dense fine speckled pattern (AC-2), but this association only holds if the specificity is confirmed as monospecific for DFS70.20 21 132 Third, the HEp-2 IIFA patterns may slightly differ depending on the cellular substrate used. For this reason, the ICAP website contains for each pattern multiple pictures taken from different brands of HEp-2 slides. Fourth, diseases like systemic lupus erythematosus and autoimmune inflammatory myopathies may be associated with distinct autoantibodies, each associated with a distinct HEp-2 IIFA pattern. If the autoantigens are ill defined, as is the case, for instance, in autoimmune hepatitis, only the most prevalent patterns are included. Altogether, it is evident that, with the exception of the centromere pattern (AC-3), all patterns are to be confirmed by antigen-specific immunoassay for a solid association with the respective autoimmune diseases.

While consensus statements have been generated for all 29 HEp-2 IIFA patterns, and it is highly recommended to report patterns,7 11 it is anticipated that laboratories may restrict their reports to the so-called ‘competent level’ patterns (http://www.ANApatterns.org).133 Although, for instance, the nucleolar patterns may not be reported as distinct entities (AC-8, AC-9 and AC-10), all three subtypes represent autoantibodies reactive with antigens associated with systemic sclerosis, either alone or in combination with autoimmune inflammatory myopathies. Follow-up testing, therefore, anyhow involves the systemic sclerosis multiparameter assay including all the relevant autoantibodies. Traditionally, only nuclear HEp-2 IIFA patterns have been considered as a true positive HEp-2 IIFA test, and this is most likely related to the time-honoured terminology ‘Antinuclear Antibody Test’,12 but it is evident from this report that even for nuclear HEp-2 IIFA patterns, the clinical associations are quite diverse. In particular, the nuclear dense fine speckled pattern (AC-2) seems to have an inverse association with SARD.9 134 On the other hand, the cytoplasmic HEp-2 IIFA patterns, and to a lesser extent the mitotic patterns, are also clinically relevant and may demand dedicated follow-up testing in daily clinical practice. Therefore, the ICAP executive board advocates that information on HEp-2 IIFA patterns should be reported to the clinician and should also be incorporated in diagnostic and classification criteria instead of the simple assignment ‘ANA-positive’.135

Although the HEp-2 IIFA has been considered the gold standard for autoantibody detection in SARD,3 the limitations of this assay are understood.136–138 Indeed, up to 35% of healthy controls may be positive if a screening dilution of 1/40 is used.139 Therefore, in the EASI/IUIS recommendations, it is advocated that each laboratory verifies that the screening dilution is defined by a cut-off set at the 95th percentile.7 However, by taking into account that the HEp-2 IIFA nowadays is ordered by a wide spectrum of clinical disciplines,1 the number of clinically unexpected positive results, that is, positive test results with no clinical evidence of an associated autoimmune disease, is ever increasing and may even equal the likelihood of a clinically true-positive result.140 141 A study performed in a community setting concluded that many patients with a positive ANA test are incorrectly given a diagnosis of systemic lupus erythermatosus and sometimes even treated with toxic medications.142 These arguments are used to introduce a gating strategy in order to restrict test-ordering to those cases that have a sufficiently high pretest probability for having a SARD. However, it can also be argued that patients with a low pretest probability should be tested using the HEp-2 IIFA in order to prevent true cases, especially those with very early disease manifestations, from being missed. This is a paradigm shift to disease prediction and prevention.143 In this strategy, the HEp-2 IIFA could be integrated in multianalyte ‘omic’ profiles for case finding and establishing an early diagnosis and preventing severe complications.143 144 Obviously, it is anticipated that the added value of the HEp-2 IIFA in this approach can be increased by incorporating information on both patterns as well as titres in combination with well-directed advices on follow-up testing.

Although the current consensus on the clinical relevance of HEp-2 IIFA patterns has come across after extensive discussion and debate within the ICAP executive board as well as with the workshop participants, the information provided is not based on a systematic review or meta-analysis of the existing literature. Because of the short history of ICAP, being founded in 2014, inclusion of older literature might have been hampered by potential differences in pattern nomenclature and definitions. For instance, the nuclear dense fine speckled (AC-2) and topo I-like (AC-29) patterns were previously often considered homogeneous, speckled or even mixed patterns. The centromere pattern (AC-3) or the cytoplasmic reticular/AMA (AC-21) patterns, on the other hand, are examples that probably have been less prone to change in pattern definition over time. The universal use of the ICAP nomenclature and pattern definitions, both in daily clinical practice as well as in the scientific literature, may enable systematic reviews in the future, and may well fine-tune current consensus based on expert opinions only.

In conclusion, the consensus statements on clinical relevance should be readily available to clinicians and this will enable further harmonisation of test-result interpretation with respect to HEp-2 IIFA patterns. Obviously, clinicians should be aware of the clinical suspicion for the respective patient, and therefore should order specific tests accordingly, also taking into account the anticipation of prevalence of HEp-2 IIFA negative (AC-0)13 results in SARD. The information on clinical relevance of HEp-2 IIFA patterns is intended to support the decision strategy of the clinician. Information presented in the online supplementary tables 1–3 is primarily intended to be used for complex cases in the consultation of the laboratory specialist by the clinician. Depending on various jurisdictional regulations, follow-up testing can be automated in predefined algorithms which eventually will shorten the diagnostic delay. Eventually, appropriate integration of HEp-2 IIFA pattern information may help to better define disease criteria and even enable a paradigm shift in the pretest probability paradox.