The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation

The presence of antinuclear antibodies (ANA) is a hallmark and classification criterion for a number of systemic autoimmune rheumatic diseases (SARD). ANA testing is usually performed as part of the initial diagnostic workup when suspicion of an underlying autoimmune disorder is high. The indirect immunofluorescence antibody (IFA) technique on HEp-2 substrate has been considered the traditional and preferred method for detecting ANA by some [1]. It allows detection of antibody binding to specific intracellular targets, resulting in diverse staining patterns that are usually categorized based on the cellular components recognized and the degree of binding, as reflected by the fluorescence intensity or titer [2, 3]. As a screening tool, the recognition of a well-defined HEp-2 IFA staining pattern may be helpful in determining the most likely specific autoantibodies present, as well as suggesting possible clinical associations for known specificities [3, 4]. In this regard, a positive HEp-2 IFA screening pattern can guide confirmatory testing and may also be useful for elucidating a specific clinical diagnosis or prognosis. Thus, the provision of HEp-2 IFA patterns and titers is considered to be clinically valuable with favorable utility in comparison with other methods for ANA detection [1‐11].

The nuclear IFA staining patterns most commonly recognized and reported by clinical laboratories include homogeneous, speckled, centromere, and nucleolar [1‐4, 12‐14]. Use of HEp-2 cell substrates, permits detection of additional nuclear staining patterns, as well as reactivity with cell constituents in compartments outside the nucleus (cytoplasmic) and cell components associated with mitosis (mitotic) [2, 4, 12‐14]. However, the reactivity and type of autoantigens associated with these patterns may vary among HEp-2 substrates from different manufacturers [15]. Furthermore, the expertise required to identify the different patterns and sub-classify their variants may not be universally available in clinical laboratories. Traditional legacy recommendations for reporting ANA patterns on HEp-2 cells continue to significantly influence clinical laboratory reporting [16, 17].

The first International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) was published in 2015 to systematize and update reporting of autoantibody patterns detected by IFA using HEp-2 cell substrates [12]. The goal of this initial and subsequent publications was to optimize usage of HEp-2 IFA patterns in patient care, by promoting standardization, harmonization and understanding of autoantibody test nomenclature and providing guidelines for test interpretation and reporting [4, 12‐15, 18]. To-date, 30 HEp-2 IFA nuclear, cytoplasmic and mitotic patterns have been elucidated by ICAP and presented in a classification tree (www.​anapatterns.​org). The ICAP guidelines indicate that ‘expert-level’ laboratories would report all the HEp-2 IFA patterns, whereas those designated as ‘competent-level’ laboratories would report 6 nuclear and 5 cytoplasmic HEp-2 IFA patterns [12].

In a previously reported survey administered in cooperation with the Association of Medical Laboratory Immunologists (AMLI), a significant number of respondents were unaware of the ICAP initiative, although a majority agreed on the need to standardize the nomenclature and reporting of HEp-2 IFA results [19]. Based on the responses from this survey, a consensus to improve ICAP awareness and further enhance HEp-2 IFA assessment through increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials. As others have also reported [20, 21], many laboratories around the world are inclined to adopt the ICAP nomenclature and embrace the recommendations provided in these consensus guidelines.

The objective of this study was to evaluate the performance of HEp-2 IFA interpretation based on nuclear, cytoplasmic and mitotic staining in an endeavor to characterize competency as outlined in the ICAP classification.

Using pre-tested and selected patient serum specimens, we report here the performance of 24 CL and IVD participants recruited from a professional organization focused on immunological laboratory testing and accustomed to the interpretation and reporting of HEp-2 IFA patterns. The specimens included examples from all 3 main categorical patterns (nuclear, cytoplasmic, or mitotic), were reported using ‘traditional’ and ICAP nomenclatures, and included patterns designated by ICAP as associated with both ‘competent’ and ‘expert’ laboratories. Our data demonstrates competence for participants in identifying and reporting common nuclear ANA patterns, but inconsistency in the decision to report and pattern reporting of cytoplasmic and mitotic patterns.

In recent years, efforts to standardize interpretation and reporting of HEp-2 patterns have led to a consensus nomenclature presented by ICAP, a group of experts [4, 12‐14] with the purpose of systematic reporting and optimizing the usage of HEp-2 IFA patterns in patient care [4]. In a previous study, we identified increasing awareness of this guidance; availability of reference materials for training and collaboration between professional organizations, IVD and CL amongst others as key elements necessary for improved harmonization of the HEp-2 IFA reporting [19].

In addition to accurately reporting binding of autoantibodies to defined cellular components, the survey also evaluated responses based on “traditional” categorization for nuclear patterns as well as the emerging ICAP nomenclature. As expected, all participants performed better with the more widely used or common traditional HEp-2 IFA nomenclature, which has more emphasis on limited nuclear staining features than required to correctly assign ICAP patterns. While the reason for this could be due to limited familiarity with ICAP, based on the data, other reasons for this can be inferred. First, the “traditional” categorization which can also be referred to as the ICAP “competent-level” is broad and minimizes the use of fine details and/or integrated pattern recognition in its interpretation. For example, most responders were capable of identifying ANA-003 and ANA-007 as nuclear speckled patterns but failed to accurately demonstrate the intended ICAP nomenclatures, coarse speckled/AC-5, and dense fine speckled/AC-2, respectively. In fact, a number of respondents classified the AC-2 DFS specimen as a mixed nuclear homogeneous and nuclear speckled pattern, as might be expected for traditional classification based on speckled staining of the nucleoplasm and intense chromatin staining. The combination requires integration to assign the nuclear DFS AC-2 ICAP pattern, rather than describing mixed homogeneous and speckled staining pattern with which it might be confused.

The specimen with antibodies to DNA topoisomerase I and the AC-29 staining pattern also demonstrated remarkable challenges of consistent ANA pattern reporting. Under ICAP, AC-29 is considered a sub-pattern of nuclear speckled staining [22], but only a minority of participants reported it as a nuclear speckled pattern using traditional nomenclature. Using “traditional” classifications, the pattern is a compound, mixed staining pattern in which the speckled component may not be perceived as dominant, even though it is consistently observed. In addition to the speckled nuclear staining, there is also staining of condensed chromatin in the mitotic cells, making it difficult to distinguish from homogeneous nuclear staining, as reported by majority of the CL, and nucleolar staining also is often present. Dellavance and colleagues [23] first reported on a composite of five unique HEp-2 staining attributes associated with positivity for anti-topoisomerase I which may not be consistently observed in all HEp-2 substrates and/or serum dilutions [22]. Among the value of the ICAP classification scheme is that interpretation of complex mixed staining may be better reported as a single unifying pattern. In support of this, laboratories accustomed to ICAP classification correctly reported the ICAP AC-29 topoisomerase pattern when asked to use the ICAP nomenclature, although they may not have reported it as a speckled ANA using traditional descriptions.

A recent multicenter analysis to evaluate the interpretation of HEp-2 IFA reported significant differences among laboratories in terms of qualitative results, patterns, and titers, particularly at low levels and in those with speckled patterns [24]. HEp-2 IFA titer determinations have been reported to have clinical significance in predicting risk for disease (healthy vs. disease) as well as association with specific autoantibodies [25‐29]. Our data confirm previous reports that ANA titers as reported by individual laboratories vary considerably, and point out another opportunity for harmonization of ANA reporting. With respect to the ICAP nomenclature, our data demonstrated clusters of participants based on the HEp-2 patterns reported by the participants. First, the majority of participants in this survey reliably read and interpreted the centromere, multiple nuclear dots, nuclear dense fine speckled, AMA and NuMA-like sub-patterns. The NuMA-like pattern is considered uncommon, and expected to be recognized by “Expert” level laboratories, but it has a characteristic appearance, and has clinically significant associations with a number of SARD [30]. Second, a significant group of participants could identify challenging ICAP-designated sub-patterns. These include the homogeneous nucleolar, cytoplasmic dense fine speckled, spindle fiber, nuclear coarse speckled, and anti-topoisomerase I patterns. Except for the AMA pattern, the overall performance of the CL participants for specimens with the cytoplasmic patterns was more variable, and lower than the IVD group. These observations have implications for defining competency for CL for cytoplasmic and mitotic patterns.

A minority of participants interpreted the nuclear fine speckled and cytoplasmic fine speckled sub-pattern specimens as intended. The data suggested that the HEp-2 patterns generated by those specimens had a sufficiently variable appearance, based on the kit manufacturer, and/or kit lot, to lead the specimens to appear as different ICAP categories in the hands of different participants. That hypothesis was confirmed by our direct review of the appearance from different laboratories (data not shown). The observations reinforce the need for harmonization of reagents, as well an enhanced training in pattern interpretation, in order to generate consistent results.

Analyses of the performance of the participants showed that the average accuracy with the expected patterns varied based on the hierarchical nomenclature categories and rater groups (CL vs IVD). Combined, both group of participants exceeded 80% average accuracy for two (nuclear and mitotic) of three group categorical patterns. The performance for both groups was more variable based on traditional and ICAP nomenclatures. However, the CL group had more varied average accuracy for both the traditional and ICAP nomenclatures with the ICAP nomenclature demonstrating significantly lower performnace. This may reflect how HEp-2 IFA patterns are reported in the CL and/or the experience of these participants. Notably, the participants in the IVD group had more years of experience than those on the CL group. Furthermore, only half of the CL participants routinely reported results for all three group categories, and the accuracy of the CL participants that reported all group category patterns routinely was comparable to the accuracy of the IVD group. Based on this observation, it is likely that a significant majority of participants that report all three group categories developed competencies for the more challenging (expert-level) patterns. However, although automation of ANA reading holds the promise of improved consistency and accuracy of ANA pattern recognition, automation-assisted reading in CL participants was not associated with a statistically significant improvement in accuracy.

The ICAP guidance is recognized as a potential roadmap towards the harmonization and standardization of HEp-2 IFA nomenclature [31, 32]. It is understood by its members and opinion leaders that this guidance will evolve, taking into consideration practical aspects for its adoption in clinical laboratories; diverse experience, ageing workforce, variability in reagents, microscopy and recent introduction of digital image readers [14, 19, 31]. Along these lines, this investigation is not without limitations. First, the intended responses (traditional nomenclature) for specimens with the cytoplasmic and mitotic patterns were not defined for specific sub-patterns (for example, cytoplasmic speckled or NuMa). This was intentional as it was largely unknown how CL report both patterns. The results obtained from this survey validates the approach, as the minority of laboratories reporting less than 3 main categorical patterns do report mitotic patterns considered expert-level on the ICAP classification tree [www.​anapatterns.​org, 12]. Second, the intended responses were monospecific and did not take mixed patterns into consideration. A number of participants reported mixed patterns for some of the specimens (data not shown), often with the intended dominant pattern reported together with minor additional pattern variants. Such reports were considered appropriate and in accordance for reporting patient results with more than one pattern [2]. Third, the survey included a limited number of participating CL including those with a significant interest and experience in ANA testing, which may not reflect the experience of a wider spectrum of international CL. Finally, some of the participants, particularly those in CL group, may have limited familiarity with the ICAP nomenclature, despite being associated with experienced laboratories.

The data presented confirm that standardization of reporting has not been achieved in performance of non-traditional HEp-2 patterns even by experienced and interested laboratories. This suggests the need and opportunities for further training and consensus-building. Using the ICAP nomenclature may have benefits for some sub-patterns and assigning competencies, notably for the mitotic and cytoplasmic main categorical groups and our data clearly demonstrate that recognition of the pattern associated with antibodies to topoisomerase is linked to familiarity with ICAP patterns. Furthermore, our data confirm previous observations that differences in the HEp-2 cell substrate can contribute to inconsistency in ANA sub-patterns interpretation and reporting [22]. Clearly, consistent ICAP sub-pattern reporting by laboratories is most meaningful if patterns are commutable using different sources of HEp-2 reagents. The relatively higher competencies of the IVD participants relative to the CL participants is of interest as some laboratories depend on IVD for training as gleaned from AMLI practice survey [19].

This study highlights significant competency for all participants in identifying the nuclear main categorical HEp-2 IFA patterns. This observation validates the ICAP competent-level classification for this group except for the anti-topoisomerase I antibody pattern. Our data also demonstrate opportunities for defining competencies and training for CL personnel in recognition of cytoplasmic and mitotic patterns.