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The P-ANCA pattern is defined as perinuclear indirect immunofluorescent (IIF) staining on ethanol fixed normal human neutrophils.1–3 This pattern is an artefact of ethanol fixation, dependent on the redistribution of certain cationic neutrophil granule proteins (such as myeloperoxidase (MPO), lactoferrin, and lysozyme) around the negatively charged nuclear membrane.1, 2 However, certain MPO-ANCA can produce cytoplasmic rather than perinuclear IIF staining,4 possibly related to a subpopulation of epitopes on MPO that do not redistribute with ethanol fixation.2 We now report that MPO-ANCA positive sera may produce a combination of P-ANCA and atypical cytoplasmic5 ANCA IIF patterns on certain ethanol fixed neutrophil substrates, potentially leading to interpretative and diagnostic difficulties.
Sera from six patients with biopsy confirmed microscopic polyangiitis (at different stages of disease activity) were selected because of initial difficulties in the interpretation of their IIF patterns on ethanol fixed neutrophil slides from Inova Diagnostics (San Diego, California, USA). All six sera were MPO-ANCA positive and proteinase 3-ANCA (PR3-ANCA) negative by the corresponding ORGenTec (Mainz, Germany) enzyme linked immunosorbent assay (ELISA). PR3-ANCA positive serum from a patient with biopsy confirmed Wegener's granulomatosis was also tested. To establish whether other ANCA antigen specificities were present, all sera were tested on the ORGenTec ANCA Combi-kit® ELISA containing proteinase-3, MPO, lactoferrin, elastase, cathepsin G, lysozyme, and bactericidal/permeability increasing protein (BPI). IIF was then repeated on all sera on two separate occasions using in house (kindly supplied by the Division of Immunology, Royal Brisbane Hospital) and two commercial (Inova Diagnostics (different batch) and Medical and Biological Laboratories (MBL, Nagoya, Japan)) ethanol fixed neutrophil slides. The IIF staining patterns and end point titres were determined by consensus.
Table 1 summarised the results. In four of the six sera, no reactivity other than MPO-ANCA was detected using the ANCA Combi-kit ELISA. Of the other two sera, one also contained lactoferrin-ANCA and the other lysozyme-ANCA. Nevertheless, in addition to P-ANCA staining, atypical cytoplasmic staining was consistently produced by all six MPO-ANCA sera on the Inova slides, but not on the MBL or in house slides. These findings were reproducible on two different batches of neutrophil slides from the former manufacturer.
Our small study demonstrates that sera containing MPO-ANCA may produce a combination of P-ANCA and atypical cytoplasmic IIF patterns on certain ethanol fixed neutrophil substrates. The recent International Consensus Statement recommends that such combined patterns be reported as “atypical ANCA”.3 Because atypical ANCA are not strongly associated with microscopic polyangiitis or Wegener's granulomatosis,3 an atypical ANCA IIF report on these sera could potentially erroneously lead the requesting clinician away from the correct diagnosis. However, in all six sera, the positive MPO-ANCA ELISA result would hopefully redirect attention towards a possible diagnosis of systemic necrotising vasculitis.
We have subsequently found that these combined IIF patterns do not occur with all MPO-ANCA positive sera on the Inova slides, and thus speculate that the phenomenon might be caused by factors in the ethanol fixation conditions of these slides resulting in the differential redistribution of different MPO epitopes. Therefore, we recommend that laboratories using this brand (and possibly other commercial brands) of ethanol fixed neutrophil slides be aware of this phenomenon, and consider repeating any sera producing such combined “atypical ANCA” IIF patterns on alternative ethanol fixed neutrophil substrates to clarify their “true” IIF pattern. Furthermore, antigen specific ELISA testing for MPO-ANCA and PR3-ANCA should also be performed on all such sera3 because combining IIF and ELISA in ANCA testing improves overall diagnostic specificity/predictive value compared with using either test alone.6