240. Subjectivity in the visual interpretation of ANCA screening by indirect Immunofluorescence testing
Creators: name: Polona Žigon1, 2
name: Saša Čučnik1, 3
Publisher: name: Zenodo
Year: 2022
Subjects: Vasculitis
ANCA
Abstracts
Giant Cell Arthritis
MPA
GPA
EGPA
Takayasu
IgA vasculitis

description: <strong>Background:</strong> The most commonly used screening test for ANCA is the indirect immunofluorescence (IIF) test with ethanol and formalin-fixed neutrophils. This is followed by separate specific measuring of antibodies to PR3 and MPO. The classic perinuclear ANCA staining pattern (P-ANCA) is characterized by perinuclear staining with nuclear extension and is usually accompanied by anti-MPO. The classic cytoplasmic ANCA pattern (C-ANCA) is characterized by granular, cytoplasmic staining with central or interlobular accentuation and is usually associated with anti-PR3. The P-ANCA pattern attributable to MPO-ANCA is actually an artifact as with formalin-fixed neutrophils, MPO-ANCA produces a C-ANCA pattern. Atypical P-ANCA pattern, which can be defined as perinuclear IIF that does not meet the criteria for classic P-ANCA, are usually associated with inflammatory conditions other than AAV. Although several automated processing and detection systems are available, many laboratories still perform conventional visual interpretation. Studies investigating the comparability of ANCA slide assessments are lacking. We aimed to evaluate interpretation between trained analysts for different ANCA patterns based on the subjectivity of the interpreter and to determine the proportion of samples in which formalin-fixed neutrophil slides are needed for final decision. <strong>Methods:</strong> All samples (n=560) referred to the Immunology Laboratory, Department of Rheumatology were simultaneously tested with ethanol- and formalin-fixed neutrophils and determined as either negative, P-ANCA, C-ANCA or atypical P-ANCA patterns by 6 trained analysts and correlated with MPO/PR3-ANCA ELISA results. All of the analysts had &gt;5 years of experience and were competent in reading and interpreting ANCA-IIF testing. <strong>Results:</strong> According to the consensus among analysts as well as anti-MPO/PR3 results, there were 455 (81.3%) negative, 14 (2.5%) C-ANCA, 49 (8.8%) P-ANCA and 42 (7.5%) atypical P-ANCA patterns among 560 samples. On average, analysts attributed a negative result to 78% of the samples, which is 3% less compared to the final results. On average, analysts attributed a C-ANCA result to 2.7% of samples, which is the same proportion as the final results. 5/14 samples with C-ANCA patterns were simultaneously positive for anti-PR3. For the latter, all analysts correctly chose the C-ANCA pattern. On average, analysts assigned 9.3% of samples to the P-ANCA result, which is comparable to the proportion of P-ANCA in the final results. 25/49 samples also had anti-MPO antibodies. Among the latter, analysts correctly decided on a P-ANCA pattern in 20/25 P-ANCA MPO + samples. In 5 samples, there was no consensus. The IIF on Ethanol-fixed neutrophils was mostly homogeneous with or without perinuclear rim staining, while formalin-fixed readings were either negative or negative with nuclear staining. On average, analysts assigned atypical P-ANCA result to 7.9%, which is comparable to the final results. On average 30% of samples had a negative reading on ethanol-fixed slides, concluding that in more than 70% of the samples additional testing on formalin-fixed neutrophils was needed. <strong>Conclusions:</strong> Analysts had the most comparable results for negative samples and for samples with C-ANCA pattern, with slightly more variability in P-ANCA and atypical ANCA patterns. Differences were found mainly in samples with low fluorescence intensity and unclear pattern. For routine performance of IIF ANCA it is suggested that samples are tested in both ethanol and formalin-fixed neutrophils and viewed in parallel by two analysts. However, the slides with an unclear pattern should be reviewed by a third one. The final report should further be agreed considering all available data, i.e. anti-MPO/PR3 antibodies and antinuclear antibodies, as detected on Hep-2 slides. <strong>Disclosures:</strong> None