Automated EuroFlow approach for standardized in-depth dissection of human circulating B-cells and plasma cells.

Background: Multiparameter flow cytometry (FC) immunophenotyping is a key tool for detailed identification and characterization of human blood leucocytes, including B-lymphocytes and plasma cells (PC). However, currently used conventional data analysis strategies require extensive expertise, are time consuming, and show limited reproducibility. Objective: Here, we designed, constructed and validated an automated database-guided gating and identification (AGI) approach for fast and standardized in-depth dissection of B-lymphocyte and PC populations in human blood. Methods: For this purpose, 213 FC standard (FCS) datafiles corresponding to umbilical cord and peripheral blood samples from healthy and patient volunteers, stained with the 14-color 18-antibody EuroFlow BIgH-IMM panel, were used. Results: The BIgH-IMM antibody panel allowed identification of 117 different B-lymphocyte and PC subsets. Samples from 36 healthy donors were stained and 14 of the datafiles that fulfilled strict inclusion criteria were analysed by an expert flow cytometrist to build the EuroFlow BIgH-IMM database. Data contained in the datafiles was then merged into a reference database that was uploaded in the Infinicyt software (Cytognos, Salamanca, Spain). Subsequently, we compared the results of manual gating (MG) with the performance of two classification algorithms -hierarchical algorithm vs two-step algorithm- for AGI of the cell populations present in 5 randomly selected FCS datafiles. The hierarchical AGI algorithm showed higher correlation values vs conventional MG (r2 of 0.94 vs. 0.88 for the two-step AGI algorithm) and was further validated in a set of 177 FCS datafiles against conventional expert-based MG. For virtually all identifiable cell populations a highly significant correlation was observed between the two approaches (r2>0.81 for 79% of all B-cell populations identified), with a significantly lower median time of analysis per sample (6 vs. 40 min, p=0.001) for the AGI tool vs. MG, respectively and both intra-sample (median CV of 1.7% vs. 10.4% by MG, p<0.001) and inter-expert (median CV of 3.9% vs. 17.3% by MG by 2 experts, p<0.001) variability. Conclusion: Our results show that compared to conventional FC data analysis strategies, the here proposed AGI tool is a faster, more robust, reproducible, and standardized approach for in-depth analysis of B-lymphocyte and PC subsets circulating in human blood.

Cromoglicato de sodio tópico en la rinitis alérgica / Topical sodium cromoglycate in allergic rhinitis

En un estudio longitudinal, prospectivo, abierto y experimental, se utilizó solución de cromoglicato de sodio al 8%, aplicándose tópicamente en pacientes de edad pediátrica, en los cuales las edades oscilaron de recién nacidos a 17 años 11/12 meses. Se confirmó la etología alérgica de la rinitis mediante la sintomatología nasal, valoración objetiva, pruebas cutáneas, estudioo de eosinofilia nasal y sanguínea. Se incluyeron 33 pacientes en un estudioo, lo cuales recibieron el medicamento tópico nasal por espacio de 8 semanas, con valoración de parámetros subjetivos y objetivos cada 2 semanas. El objetivo principal del estudioo fue demostrar la utilidad del cromoglicato de sodio tópico en la rinitis alérgica, el grado de efectividad que presenta en nuestro medio con condiciones ambientales adversas y con severidad clínica marcada, así como el grado de seguridad que se logra con la droga en el tratamiento profiláctico de este padecimiento. El resultado final nos indicó una respuesta excelente en 23 pacientes (70%), regular en 8 (24% y malo en 2 (6%). Los efectos colaterales, son mínimos, lo que indica una buena tolerancia en el tratamiento preventivo de la rinitis alérgica. Se concluye la eficiencia del cromoglicato de sodio tópico en la profilaxis de la rinitis alérgica. Los resultados en la literatura reportados en otros estudioos son discretamente inferiores a los encontrados en el presente trabajo, lo que se atribuye al uso de solusión de cromoglicato de sodio al 8%, superior en concentración a la usada en otros trabajos reportados