A new cytological approach to improve the final diagnosis of Eosinophilic Granulomatosis with Polyangiitis (EGPA)

Introduction: Eosinophilic granulomatosis with polyangiitis (EGPA) is an uncommon systemic necrotizing vasculitis that affects small to medium sized vessels and is associated with severe asthma, allergic rhinitis, nasal polyposis and blood and tissue eosinophilic infiltration. Antineutrophil cytoplasmic autoantibodies (ANCA) are present in about 40% of cases. The presence of four or more of the above described findings yields a sensitivity of 85% and a specificity of 99.7% for the final diagnosis of EGPA. The aim of the study was to develop a new diagnostic tool to support the diagnostic and prognostic work-up of EGPA patients with the possibility to evaluate extracellular mediators in nasal secretion. Methods: Nasal secretions were gained from 40 patients of which 20 EGPA, 10 suspected EGPA and 10 controls after positioning the cotton pieces in the nasal middle meatus where they were left for 10 min. Subsequently the cotton pieces were put in 5 ml of saline solution and left in ice until processing. The liquid obtained from the squeezing of the cottons was centrifuged two times at 1600 RPM for 10 min. Supernatant was frozen after first centrifugation. Trypan Blue was added to 10 μl of sample to calculate the number of cells and their vitality. Finally, 100 μl of sample with containing 1.5–2.00 × 105 cells were centrifuged with Cytospin at 300 RPM for 15 min. The slides were stained with May Grunwald/Giemsa and cell population analysed at microscope with objective oil immersion 40× of magnification. The remaining cells were frozen or cultured. Results: The mean number of cells obtained after centrifugation was 2.00–4.08 × 106 cells/ml for EGPA patients and 6.42–8.62 × 105 cells/ ml for control group and suspected EGPA. Cytologic analysis allowed us to count an increase in the percentage of eosinophils in EGPA patients with active ENT disease and in some of the patients with a suspect of EGPA, where this percentage correlated with histologic evidence of disease. Conclusions: This method seems preliminarily a reliable cytologic examination allowing fresh cells isolation and analysis of extracellular mediators. Compared to the direct slither of the mucus on the surface of the glass, cytospin significantly reduced any dye accumulation. Further studies are ongoing to support the described method, in order to confirm the cytologic diagnostic reliability to identify prognostic biomarkers of EGPA.