Are storage conditions of the substrate responsible for the variations of immunofluorescent perinuclear antineutrophil cytoplasmic antibodies (pANCA) pattern?

Abstract

Rationale Indirect immunofluorescence (IIF) on human ethanol-fixed neutrophils is the screening method to detect ANCA in vasculitis (AAV), inflammatory bowel disease (IBD), autoimmune liver disease (ALD). It has been shown that the IIF pattern may change when tests are performed in different laboratories depending on type of substrate and conjugate. AIM: To evaluate whether the variation in IIF pattern of pANCA may also depend on alterations of substrate related to the length of the storage period. Methods We evaluated sera from 2 AAV, 2 IBD and 6 ALD. Nine separate aliquots of sera were stored at −20°C. As substrate, ethanol-fixed human neutrophils purified from peripheral blood were used and slides preserved at −20°C. The IIF test was performed monthly, 9 times (from “time 0” to “time 8”). Results At “time 0” three sera (2 AAV, 1 ALD) showed a rim-like perinuclear staining with nuclear extension. Six sera (4 ALD, 2 IBD) showed a coarse perinuclear staining and 1 (ALD) an “intermediate” pattern, i.e. a coarse perinuclear with partial nuclear extension. The patterns of the 10 sera remained unmodified along time, except in 2 cases (1 ALD, 1 IBD), which, from “time 4” on, changed from a coarse into an “intermediate” pattern. Conclusions 1. Duration of substrate's storage does not seem to be the main factor responsible for variations in IIF titres and patterns of pANCA, when tested in the same laboratory. 2. The presence of distinct IIF patterns in different diseases confirms that pANCA target more than one still poorly defined antigen.