Improvement of indirect immunofluorescence technique to detect antineutrophil cytoplasmic antibodies and its impact on test positivity rate

• Published in: Brazilian journal of medical and biological research Vol. 56; no. 1; p. e12636
• Main Authors: Zarur, E B, Rodrigues, S H, Ambrogini, Jr, O, Ferraz, M L G, Mastroianni-Kirsztajn, G, Andrade, L E C, Souza, A W S de
• Format: Journal Article
• Language: English
• Published: Brazil Associacao Brasileira de Divulgacao Cientifica (ABDC) 01.01.2023; Revista Brasileira de Pesquisas Medicas; Associação Brasileira de Divulgação Científica
• Subjects: ANCA-associated vasculitis; Antibodies; Antibodies, Antineutrophil Cytoplasmic; Antineutrophil cytoplasmic antibodies; Autoantibodies; Autoimmune hepatitis; BIOLOGY; Chronic active hepatitis; Cross-Sectional Studies; Diagnosis; Enzyme-Linked Immunosorbent Assay; Fluorescent antibody technique; Fluorescent Antibody Technique, Indirect; Health aspects; Hepatitis; Humans; Immunofluorescence; Laboratories; Measurement; MEDICINE, RESEARCH & EXPERIMENTAL; Methods; MPO-ANCA; Myeloblastin; Peroxidase; PR3-ANCA; Proteinase 3; Ulcerative colitis; Vasculitis; Brazil; MPO-ANCA; PR3-ANCA; Autoimmune hepatitis; ANCA-associated vasculitis; Antineutrophil cytoplasmic antibodies; Ulcerative colitis
• ISSN: 0100-879X; 1414-431X; 1414-431X; 1678-4510
• DOI: 10.1590/1414-431X2023e12636

The indirect immunofluorescence (IIF) technique for antineutrophil cytoplasmic antibodies (ANCA) detection is subject to substantial differences across laboratories. This study aimed to assess the impact of improvements in the IIF-ANCA technique on the positivity rate of ANCA tests. A cross-sectional study was performed with serum samples from patients with ANCA-associated vasculitis (AAV), autoimmune hepatitis (AIH), and ulcerative colitis (UC). A paired analysis was performed for IIF-ANCA results using the traditional method and a modified protocol after a series of specific adjustments in the technique based on the protocol of IIF-ANCA test performed at a nation-wide private laboratory in Brazil. ANCA specificity was assessed by ELISA for anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) antibodies. Sixty-one patients were evaluated. The positivity rate of IIF-ANCA tests at disease presentation performed at the University reference laboratory was 32.3% in AAV, AIH, and UC patients, whereas the positivity rates of IIF-ANCA and ELISA tests in other laboratories were 75.0 and 72.7%, respectively. After modifications in the IIF-ANCA technique, there was a significant increase in the positivity rate (14.8 vs 34.3%; P=0.0002) and in median titers [1/40 (1/30-1/160) vs 1/80 (1/40-1/80); P=0.0003] in AAV, AIH, and UC patients. UC had the highest increment in positive results from 5.3 to 36.8%. There was poor agreement between MPO- or PR3-ANCA and both IIF-ANCA techniques. In conclusion, modifications in the IIF-ANCA protocol led to a significant improvement in its positivity rate and titers.