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Introduction Studies on the pulmonary microbiome in patients with idiopathic pulmonary fibrosis have linked dysbiosis to risk of disease progression,exacerbations,and mortality.Data on the microbiome in connective tissue disease-associated interstitial lung disease (CTD-ILD)remain limited and inconsistent.The primary objective of this study is to standardize the sampling and analysis of bronchoalveolar lavage fluid(BALF).The secondary objective is to characterize the microbiome according to disease phenotype and exposure to immunosuppressants.Material and Methods We enrolled patients diagnosed with CTD-ILD, either treatment-naïve to immunosuppressive therapy or with progressive disease according to ATS/ERS criteria.Patients on corticosteroid therapy at doses higher than 10 mg/day of prednisone(or equivalent),with active infections or malignancies,were excluded.Clinical, radiological, and functional data were collected.On the same day as BAL, patients completed the Saint George’s Respiratory Questionnaire,Gastroesophageal Reflux Disease Impact Scale,and HAQ-DI.BAL was performed under general anesthesia via laryngeal mask to minimize contamination from the upper airways.Microbiome analysis was carried out on BALF and two internal controls:pure sterile saline solution and saline inoculated through the bronchoscope’s working channel. Peri-and post-procedural complications were recorded,along with patient-perceived discomfortassessed using a Numerical Rating Scale one week after the procedure.Results Clinical and demographic data are reported in table 1. All patients successfully completed the procedure, reporting a low level of perceived discomfort,with a median of 1 out of 10 on the NRS.For microbiome analysis, the number of bacterial genome copies in the control samples was near zero,confirming minimal contamination.Microbiological analysis also showed a specific bacterial profile in BALF samples compared with controls.All standard microbiological cultures were negative.Eleven BALF samples had a genetic content of fewer than 1000 reads and were not further analyzed.No statistically significant clinical-demographic differences emerged between these samples and those with more than 1000 reads. Among analyzed samples, the most represented phyla were Firmicutes, averaging 40.9% (±16.0), followed by Proteobacteria (26.4% ±28.0) and Bacteroidetes (22.0% ±12.0).The predominant bacterial genera were Prevotella and Streptococcus.An association was observed between a restrictive pattern in pulmonary function tests and greater alpha-diversity.Conversely,no statistically significant differences in beta-diversity were found,either by spirometric pattern or in relation to immunosuppressive _treatment.Conclusions BAL,performed using a standardized technique,minimized contamination and was well tolerated by patients,suggesting its potential integration into the diagnostic approach for CTD-ILD.Pulmonary microbiome analysis may represent a potential biomarker of disease.Abstract P.098 Table 1Baseline characteristics of patients