Flow Cytometry Analysis of Eosinophils in Severe Asthma Patients.

Subject satisfying eligibility criteria will be asked to participate the study; all will receive a detailed explanation of the nature of the study, of the aims and objectives. After having signed informed consent, patients will be divided into two groups, based on their clinical status: controlled asthma group (group A) and severe asthma group (group B). Visits will be planned at recruitment, after a run-in period (3-months), meant to optimize first and second lines medical treatment and confirm diagnosis of severe asthma. After the run-in period, patients will be re-evaluated and appropriate medical therapy will be tailored (T0). To group B, therapy with Mepolizumab or Omalizumab will be given, according to standard guidelines. At T0 a blood sample will be collected (3ml) to evaluate eosinophils phenotype by flow cytometry (see below). After 6 months (T1/2) and12 months (T1) patients will be visited again and a blood sample will be taken for flow cytometry analysis.All samples will be properly stored and shipped for analysis, according to best of practice and state of art criteria, within 6 hours from sample's collection. Data will be collected and stored according to the GDPR (General Data Protection Regulation, EU 2016/679). Biological material collected will be used only for the indicated assay and excess material will be discharged. * Baseline evaluation (T'0) In case of acceptance, and after signing an informed consent, participants will be asked to collect a complete medical history, including allergies and Asthma related comorbidities; a comprehensive physical examination and respiratory function tests consisting of: flow-volume spirometry plus residual volume and DLCO determination. In case of a first diagnosis of asthma a bronchodilator response or a methacholine challenge tests will be obtained. In case of a preexistent diagnosis of asthma, previous examination confirming and proving the diagnosis will be collected. * Evaluation Tool The following data will be collected at T'0, T'1/2 (six months) and T'1 (twelve months): Clinical assessment: ACT, on demand or rescue therapy, acute exacerbations, medical visits, working absences, hospital admissions. Functional assessment: Flow volume curve, Residual volume, Ig E and eosinophils' count, FeNO, 6 minutes walk test. * Flow cytometry The investigators will evaluate, by multicolor flow cytometry, the expression of molecules on eosinophils'membrane, using a whole-blood staining protocol, so that blood samples will be minimally manipulated, thus reducing the possibility of artifacts. Briefly, peripheral blood samples will be processed within 6 hours from withdrawal. Samples will be stored at room temperature until processing. Blood samples will be stained, using fluorochrome conjugates antibodies directed towards eosinophils'surface molecules. In particular, the investigators will use antibodies against CD45, CD66, CD15, CD16 and Siglec-8, which will be used to identify eosinophils between other leukocytes; CD63, CD294 (CRTH2, prostaglandin D2 receptor), CD125 (IL-5 receptor), CD193 (receptor for several chemokines, as for example RANTES, Eotaxin, MCP-3, MIP1α), and HLA-DR: these molecules are upregulated with eosinophils activation, so they can have an altered expression in severe asthma patients. After 30 minutes incubation at 4°C, FACS lysing solution (BD Bioscience) will be used to remove erythrocytes, according to manufacturer's instruction. Samples will be resuspended in phosphate buffered saline (PBS) and acquired on a LSRFortessa X-20 (BD Bioscience) flow cytometer, equipped with four lasers.