Human peripheral B cells: a different cytometric point of view.
BACKGROUND
Human peripheral B lymphocytes, analyzed by current flow cytometers, frequently show complex patterns of morphological and fluorescence signals. However, fluorescence intensity values are commonly reported without any correlation to the cell surface area. We propose a different approach, based on the evaluation of the ratio of phenotype fluorescence intensity to forward scatter intensity, to determine the apparent fluorescence density of surface molecules.


METHODS
Starting from list mode acquired data, and after logical gating of live B cells, the analytical procedure suggests a serial scanning of the FSC versus SSC plot to obtain apparent fluorescence density of progressively larger cells.


RESULTS
This method, applied to normal human peripheral B lymphocytes, was able to detect the presence of steady and modulated (with respect to cell size) fluorescence densities for a variety of surface molecules. B cells from patients with B cell disorders displayed interesting alterations of the phenotype density values and distributions.


CONCLUSIONS
Our preliminary data show that, in human B cell cytometry, the apparent fluorescence density based method allows one to recognize variations in fluorescence intensities solely due to cell size differences and to disclose patterns of expression not detectable by the conventional intensity based approach.
Carbonari
Maurizio
M
Department of Clinical Medicine, University of Rome La Sapienza, Viale dell'Università 37, 00185 Rome, Italy. maurizio.carbonari@uniroma1.it
Tedesco
Tiziana
T
Fiorilli
Massimo
M
D000954:Antigens, Surface / Q000032:analysis* / Q000276:immunology*; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology*; D048429:Cell Size / Q000276:immunology; D003449:Cryoglobulinemia / Q000097:blood / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D014644:Genetic Variation; D006526:Hepatitis C / Q000097:blood / Q000276:immunology; D006801:Humans; D010641:Phenotype; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7 / Q000032:analysis / Q000276:immunology
D000954:Antigens, Surface; D005456:Fluorescent Dyes; D018127:Tumor Necrosis Factor Receptor Superfamily, Member 7