Abstract:

Objective To investigate the influence of cytoplasmic antinuclear antibody（ANA）on indirect immunofluorescence assay（IFA） based antineutrophil cytoplasmic antibody（ANCA）determination. Methods Totally，66 patients with non-ANCA-associated vasculitis with ANA patterns of cytoplasmic fibrillar linear（AC-15，2 cases），cytoplasmic fibrillar filamentous（AC-16，4 cases），cytoplasmic discrete dots/GW body-like（AC-18，7 cases），cytoplasmic dense fine speckled（AC-19，25 cases），cytoplasmic fine speckled（AC-20，13 cases）and cytoplasmic reticular/AMA（AC-21，15 cases）were enrolled from Renji Hospital of Shanghai Jiao Tong University School of Medicine from June to July 2023. IFA was carried out for ANCA，western blot was carried out for anti-extractable nuclear antigen（ENA）antibody spectrum and anti-autoimmune liver disease（AILD）antibody spectrum，and enzyme-linked immunosorbent assay（ELISA） was used for anti-double-stranded DNA（dsDNA） antibody，anti-nucleosome antibody and ANCA spectrum. Results Among 66 cases of cytoplasmic ANA，98.5%（65/66）were ANCA negative，and 1 case（1.5%） of atypical cytoplasmic ANA（cANCA）was ANCA negative. In ethanol-fixed human neutrophils，42.4%（28/66） were negative，30.3%（20/66）were atypical ANCA，22.7%（15/66）were perinuclear ANCA（pANCA），and 4.5%（3/66）were cANCA. The positive rate of formalin fixed neutrophils was 1.5%（1/66）. The 96%（24/25）of AC-19 samples were positive in ethanol-fixed substrate，48.0%（12/25）for pANCA，44.0%（11/25）for atypical ANCA，and 4.0%（1/25） for atypical cANCA. The positive rate of pANCA was higher when accompanied with anti-ribosomal P protein antibody than anti-ribosomal protein（Rib. P） antibody negative（64.7%，12.5%，P=0.02）. AC-21 mainly showed as negative and atypical ANCA in ethanol-fixed substrate，whose percentages were both 40.0%（6/15），13.3%（2/15） for cANCA，and 6.7%（1/15） for pANCA. AC-15，AC-16，AC-18 and AC-20 were mainly negative in ethanol-fixed substrate，and most of the positive samples in ethanol-fixed substrate were positive when accompanied with anti-ENA antibody spectrum. Conclusions Cytoplasmic ANA has little interference on formalin fixed neutrophils in IFA based ANCA determination，and the interference of cytoplasmic ANA in ethanol-fixed neutrophils mostly shows in AC-19 and AC-21. Interferences showing as pANCA or atypical ANCA may occur when these 2 ANA patterns combining with other nuclear antigens.

Key words: Antinuclear antibody, Cytoplasmic type, Antineutrophil cytoplasmic antibody, Interference