title: Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface-only versus intracellular antigens with the Fix & Perm reagent.
abstract: Staining for intracellular markers with the Fix & Perm reagent is associated with variations in the scatter properties of leucocytes, limiting automated analysis of flow cytometry (FCM) data. Here, we investigated those variables significantly contributing to changes in the light scatter, autofluorescence, and bcl2 staining characteristics of peripheral blood (PB) leucocytes, after fixation with Fix & Perm. Our major aim was to evaluate a new mathematical approach for automated harmonization of FCM data from datafiles corresponding to aliquots of a sample treated with cell-surface-only versus Fix & Perm intracellular staining techniques. Overall, neither the anticoagulant used nor sample storage for <24 h showed significant impact on the light scatter and fluorescence properties of PB leucocytes; similarly, the duration of the fixation period (once >15 min were used) had a minimum impact on the FCM properties of PB leucocytes. Conversely, changes in cell/protein concentrations and the fixative/sample (vol/vol) ratio had a clear impact on the light scatter features of some populations of leucocytes. Accordingly, lower cell/protein concentrations were associated with lower scatter values, particularly for the neutrophils. Such changes could be partially corrected through the use of higher fixative to sample volume ratios. Despite the variable changes detected between aliquots of the same sample treated with cell surface-only versus intracellular staining procedures, the new mathematical approach here proposed and evaluated for automated harmonization of common parameters in both datafiles, could correct the FCM profiles of leucocytes derived from cells undergoing conventional fixation/permeabilization procedures, and made them indistinguishable from those corresponding to aliquots of the same sample treated with cell-surface-only staining techniques.
authors: Instituto de Pediatria e Puericultura Martagão Gesteira and Programa de Pós-Graduação em Clínica Médica, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil.
authors: Elaine Sobral
authors: ES
authors: da Costa
authors: Rodrigo Tosta
authors: RT
authors: Peres
authors: Julia
authors: J
authors: Almeida
authors: Quentin
authors: Q
authors: Lécrevisse
authors: María Elena
authors: ME
authors: Arroyo
authors: Cristina
authors: C
authors: Teodósio
authors: Carlos Eduardo
authors: CE
authors: Pedreira
authors: Jacques J M
authors: JJ
authors: van Dongen
authors: Alberto
authors: A
authors: Orfao
mesh_terms: D000328:Adult; D000941:Antigens / Q000032:analysis / Q000737:chemistry*; D000954:Antigens, Surface / Q000032:analysis / Q000737:chemistry*; D051017:Apoptosis Regulatory Proteins / Q000032:analysis / Q000737:chemistry; D005260:Female; D005404:Fixatives; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007962:Leukocytes / Q000737:chemistry*; D008027:Light; D008297:Male; D012542:Scattering, Radiation; D013194:Staining and Labeling
chemical_list: D000941:Antigens; D000954:Antigens, Surface; D051017:Apoptosis Regulatory Proteins; D005404:Fixatives; D005456:Fluorescent Dyes; C090468:TP53BP2 protein, human