“Preliminary characterization of the macrophage population in bronchoalveolar lavage through Percoll separation and flow cytometry”.

Bronchoalveolar lavage (BAL) is a valuable biological matrix for studying the pulmonary microenvironment, as it provides access to cells and soluble mediators from the lower respiratory tract. However, BAL is technically challenging because of its variable cellularity, heterogeneous composition, and possible presence of mucus or debris, which may affect downstream analyses. The aim of this thesis was to establish and evaluate a methodological workflow for processing BAL samples and recovering macrophage-enriched cellular fractions. Whole BAL samples were first characterized by flow cytometry to define their cellular composition. Different macrophage-enrichment strategies were then tested, including plastic adherence, single-density Percoll separation, and discontinuous Percoll-gradient separation. Recovered fractions were assessed by cell counting, morphology, viability, flow cytometry, RNA extraction, and qPCR. Flow-cytometric analysis confirmed the heterogeneous nature of BAL samples, although macrophages represented the predominant cellular population in most cases. Plastic adherence resulted in marked cell loss, whereas discontinuous Percoll-gradient separation allowed the recovery of distinct macrophage-enriched fractions. The MIDDLE and BOTTOM layers showed different morphological and cellular features and were therefore analyzed separately. Selected Percoll fractions were used for preliminary molecular characterization. The expression of IL6, IL1B, and IL10 was evaluated as an exploratory readout of inflammatory and regulatory signals. qPCR analysis showed heterogeneous cytokine-expression profiles, with selected fractions, particularly from BAL4 and BAL7, displaying increased IL6 and IL1B expression, suggestive of a more pro-inflammatory molecular profile. Overall, this work supports the feasibility of integrating BAL characterization, Percoll fractionation, flow cytometry, and qPCR for the exploratory analysis of macrophage-enriched BAL fractions. The results remain preliminary and require validation in larger cohorts with broader molecular and phenotypic panels.