Implications for redefining the dense fine speckled and related indirect immunofluorescence patterns

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The long-awaited ‘pseudo-DFS pattern’

We thank Infantino et al. for the interest and comments on our manuscript [1]. In recent years there has been increasing appreciation of the ability of morphological patterns in the indirect immunofluorescence (IIF) assay (IFA) on HEp-2 cells (HEp-2 IFA) to direct further investigation of specific autoantibodies. The International Consensus on antinuclear antibody (ANA) patterns (ICAP) initiative has emphasized and refined this trend by orderly classifying and harmonizing the nomenclature of several relevant HEp-2 IIF patterns [2–4]. The ICAP initiative is intended to be dynamic in that the successive ICAP forums receive new information for consideration that often results in fine tuning the pattern repertoire (AC-0 to AC-29) [4,5]. Pseudo-DFS is on the list of these considerations. According to the ICAP modus operandi, any novel pattern must be thoroughly discussed by the steering committee and presented in an ICAP meeting that is open to all interested stakeholders. Once a consensus has been reached, the novel pattern qualifies for inclusion in the ICAP algorithm. In some instances, this may require more than one round of discussions, so that consensus is obtained among international experts. An additional aspect for building consensus on novel patterns is their expression on HEp-2 slides from different manufacturers. For the proposed ‘pseudo-DFS’ IFA pattern more data are needed to draw definite conclusions.

The difficulty in redefining the AC-2 (DFS) IIF pattern derives from the subtle features that characterize this pattern as a tightly packed, speckled staining of the whole interphase nucleus with heterogeneity in brightness, size and density of the several speckles and a similar staining pattern of the metaphase chromatin plate [6]. A variety of other nuclear speckled patterns staining the metaphase plate may be mistakenly classified as AC-2. The proposed pseudo-DFS pattern could provide a plausible addition to the ICAP nomenclature. However, it is feasible that this pattern is not caused by a single autoantibody specificity, but rather by different autoantibody specificities contained in different samples or even in some cases by samples with a mixture of autoantibodies. That being said, it should be emphasized that the correct recognition of the AC-2 pattern strongly speaks against the concomitant presence of other relevant ANAs, since most other ANAs tend to perturb the genuine AC-2 characteristics. In a recent study to establish an international anti-DFS70 reference material, it was shown that none of 741 consecutive bona fide AC-2 patterns had antibodies to common and relevant nuclear antigens [7]. This is clinically relevant, since the exclusive (i.e. monospecific) presence of anti-DFS70 antibodies is rarely observed in ANA associated rheumatic diseases (AARD) [8]. The authors’ comment that in the past the pseudo-DFS pattern was defined as homogeneous/speckled, speckled with positive metaphases, quasi-homogeneous. In our experience, those terms were often used for all samples that demonstrate a pattern that we now define as DFS (AC-2) or related patterns.

The next question refers to what possible antigens are targeted by autoantibodies associated with the pseudo-DFS pattern. There may be a number of candidate autoantigens and one possibility is that endogenous partners of DFS70 assembled in specific macromolecular transcription complexes, akin to the spliceosome [9], may be also be B-cell targets. Non-human antibodies against some of these interacting partners give HEp-2 IIF patterns similar to those produced by human anti-DFS70 antibodies, although not necessarily identical, and recognize proteins with molecular weights in the 60–70 kDa region. For instance, one such candidate, MeCP2, was considered as a novel autoantigen, but no human autoantibody reactivity to this protein was detected [10]

Finally, it should be emphasized that the DFS pattern (AC-2) is no different from other HEp-2 IIF pattern with respect to the definition of the underlying autoantibody specificity. For every single HEp-2 IIF pattern [centromere (AC-3) is a possible exception], we do need to confirm the autoantibody association using specific immunoassays, whenever available. The AC-2 pattern is also subjected to this rule and should be confirmed by anti-DFS70-specific immunoassays.

Declaration of interest

M Mahler and K Malyavantham are employed at Inova Diagnostics selling autoantibody assays. MJ Fritzler, C Casiano and L Andrade received consultant fees from Inova Diagnostics in the past. MJ Fritzler has received received speaking honoraria from Inova Diagnostics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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Funding

This paper was not funded.