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 experienced in the interpretation and reporting of HEp-2 IFA patterns. The specimens included examples from all 3 main categorical patterns (nuclear, cytoplasmic, or mitotic), 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 . 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 .
The term “antinuclear antibody’” is commonly used to refer to the HEp-2 IFA test for detection of antibodies to nuclear antigens. However, ICAP intentionally included standardized reporting of autoantibodies to HEp-2 cell compartments other than the nucleus, since some of the cytoplasmic and mitotic patterns are associated with systemic and organ-specific autoimmune diseases [2, 4, 12]. Thus, there has been a consensus suggestion to refer to ANA as anti-cellular antibodies  and, indeed, ICAP utilizes the anti-cellular (AC) nomenclature in their pattern classifications . Specimens with nuclear patterns made up the majority (58%) of the survey specimens, followed by the less commonly reported cytoplasmic (25%) and mitotic (17%) patterns. Of interest, relatively more CL in this survey indicated they reported mitotic than the cytoplasmic patterns, perhaps because some mitotic patterns such as the distinctive spindle pattern (AC-25, and AC-26) have been traditionally taught in conjunction with nuclear staining patterns. Amongst the responders, mitotic patterns were more accurately reported than the cytoplasmic patterns. While this survey may be limited by the number of participants, this observation is in contrast to the current expert-level classification by ICAP of all mitotic patterns, with implications for validation in a larger group of participants. On the other hand, except for the AMA (AC-21) pattern, the two other specimens with cytoplasmic patterns, which are intended to be of expert-level, did not achieve consensus. Cytoplasmic patterns are associated with diverse subtypes and different antigenic targets, many with defined clinical implications. For example, the AMA pattern is typically associated with primary biliary cholangitis (PBC), the dense fine speckled cytoplasmic pattern (AC-19) with anti-ribosomal P in SLE patients and the fine speckled pattern (AC-20) with some anti-tRNA synthetase antibodies (Jo-1) in patients with myositis [4, 24–27]. Other cytoplasmic sub-patterns include rods and rings associated with Hepatitis C infection treated with ribavarin and the cytoplasmic discrete dots pattern (AC-18) associated with GW bodies described in patients Sjögren's syndrome,neurological disease and other conditions [4, 28, 29].
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 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 speckled but failed to accurately demonstrate the intended ICAP nomenclatures, AC-5, and 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 , 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  first reported on a composite of five unique attributes associated with positivity for anti-topoisomerase I and HEp-2 IFA pattern which may not be consistently observed in all HEp-2 substrates and/or serum dilutions . 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 . 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 [32–36]. Our data confirm previous reports that ANA titers 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, as reported in a large cohort of Columbian patients . 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 relatively more variable, and lower than 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 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 the two different nomenclatures, traditional and ICAP. 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 reported results for all three group categories. When the results were stratified based on whether or not all three group categories are reported by the CL group, the performance for this subset was comparable to the IVD group. Based on this observation, it is likely that a significant majority of participants that report all three group categories may have developed competencies for the more challenging (expert-level) patterns. It is also likely that the IVD group may not be subjected to regulatory constraints like the CL participants and had increased flexibilities in reporting for this survey.
The ICAP classification tree indicates patterns that should be readily recognized (competent-level) versus patterns that would be more challenging and/or infrequent and distinguishable only when observers or technologists have attained the expert-level . The specimens chosen for this survey were chosen to cover a range of levels of expertise.
The ICAP guidance is recognized as a potential roadmap towards the harmonization and standardization of HEp-2 IFA nomenclature [38, 39]. 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, 20, 38]. 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 pattern 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 . 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 . 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 .