2026-05-17T00:20:33Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/234592024-11-28T20:23:32Zcom_20.500.12105_15322com_20.500.12105_2051col_20.500.12105_16967

00925njm 22002777a 4500 dc Santopolo, Giulia author Clemente, Antonio author Rojo Molinero, Estrella author Fernández, Sara author Álvarez, María Concepción author Oliver, Antonio author de la Rica, Roberto author 2022-11-17 Background: Phenotyping sputum-resident leukocytes and evaluating their functional status are essential analyses for exploring the cellular basis of pathological processes in the lungs, and flow cytometry is widely recognized as the gold-standard technique to address them. However, sputum-resident leukocytes are found in respiratory samples which need to be liquefied prior to cytometric analysis. Traditional liquefying procedures involve the use of a reducing agent such as dithiothreitol (DTT) in temperature-controlled conditions, which does not homogenize respiratory samples efficiently and impairs cell viability and functionality. Methods: Here we propose an enzymatic method that rapidly liquefies samples by means of generating O2 bubbles with endogenous catalase. Sputum specimens from patients with suspected pulmonary infection were treated with DTT, the enzymatic method or PBS. We used turbidimetry to compare the liquefaction degree and cell counts were determined using a hemocytometer. Finally, we conducted a comparative flow cytometry study for evaluating frequencies of sputum-resident neutrophils, eosinophils and lymphocytes and their activation status after liquefaction. Results: Enzymatically treated samples were better liquefied than those treated with DTT or PBS, which resulted in a more accurate cytometric analysis. Frequencies of all cell subsets analyzed within liquefied samples were comparable between liquefaction methods. However, the gentle cell handling rendered by the enzymatic method improves cell viability and retains in vivo functional characteristics of sputum-resident leukocytes (with regard to HLA-DR, CD63 and CD11b expression). Conclusion: In conclusion, the proposed enzymatic liquefaction method improves the cytometric analysis of respiratory samples and leaves the cells widely untouched for properly addressing functional analysis of lung leukocytes. Santopolo G, Clemente A, Rojo-Molinero E, Fernández S, Álvarez MC, Oliver A, et al. Improved cytometric analysis of untouched lung leukocytes by enzymatic liquefaction of sputum samples. Biol Proced Online. 2022 Nov 17;24(1):17. 10.1186/s12575-022-00181-z 1480-9222 Biological procedures online http://hdl.handle.net/20.500.13003/18591 36396988 L2020133668 2-s2.0-85142138997 https://hdl.handle.net/20.500.12105/23459 885724200001 Improved cytometric analysis of untouched lung leukocytes by enzymatic liquefaction of sputum samples