Different patterns of indirect immunofluorescence (IIF) in screening for two conditions are because of technical reasons and not necessarily interpretation errors, concludes a quality assessment study from Australia.

Eleven laboratories specialised in screening for antineutrophil cytoplasmic antibodies (ANCA) by IIF to confirm active Wegener’s granulomatosis or microscopic polyangiitis took part. They tested four sera chosen by the reference laboratory coordinating quality assurance testing of ANCA. Two showed staining of cytoplasmic granules (C-ANCA) typical of Wegener’s granulomatosis; one perinuclear staining and staining of myeloperoxidase (P-ANCA with MPO specificity); and one perinuclear staining (P-ANCA) typical of microscopic polyangiitis and irritable bowel syndrome.

The laboratories used different conjugates—their own or commercial conjugates—and different neutrophil substrates. Each laboratory used its usual protocol.

Most results conformed to the consensus pattern determined by the Australian ANCA Study Group. All neutrophil substrates affected cytoplasmic granularity and highlighting of the lobed nucleus—criteria used to separate C-ANCA from C-ANCA (atypical) which denotes other conditions. Eight assays from eight laboratories produced IIF deviating from the consensus pattern, with stained cytoplasmic granules for serum designated P-ANCA or P-ANCA with MPO specificity. These assays used different mixes of neutrophil substrate and included all six different conjugates, so the results were not limited to a particular substrate, conjugate, or combination of the two.

The international consensus on testing for ANCA stipulates using IIF. External quality control has indicated that among laboratories IIF patterns can vary with the same serum, and it was unclear whether this was wholly explained by observer error.