Naslov
Polychromatic flow cytometry in routine immunophenotyping : more colors may reveal hidden Immunophenotypes

Autori
Banić, Ivana ; Lokas Bulat, Sanrda ; Živković, Jelena ; Navratil, Marta ; Mrkić Kobal, Iva ; Polančec, Denis ; Turkalj, Mirjana

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
30th Congress of the International Society for Advancement of Cytometry : abstracts / - , 2015, Xx-xx

Skup
Congress of the International Society for Advancement of Cytometry (30 ; 2015)

Mjesto i datum
Glasgow, Ujedinjeno Kraljevstvo, 26.06.2015. - 30.06.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Flow cytometry ; PID ; Immunophenotypes

Sažetak
Monitoring changes in human immune cell populations such as lymphocytes, monocytes etc. in different clinical phenotypes is crucial. The distributed nature of the hematopoietic system makes it amenable to flow cytometric analysis. Today, both research and clinical laboratories addressing various immunological aspects heavily rely on flow cytometry for phenotypical and functional analyses of immune components in different disease settings, such as primary immunodeficiencies, as well as in the healthy immune system. Flow cytometry is currently the platform of choice in analyzing the complex components of the immune system- i.e. to separately characterize many phenotypically and functionally distinct subsets of leukocytes, any of which might be clinically relevant. Multi- colour flow cytometry assays, therefore, need to be developed with great care in order to ensure reliability of data generated therewith. The aim of this research was to analyze the phenotype of human peripheral blood monouclear cell subsets (lymphocyte and monocyte) in the diagnostics and monitoring of primary immunodeficiencies (PID) using polychromatic flow cytometry assays. Comprehensive immunophenotyping of whole blood was performed using polychromatic flow cytometry. Peripheral whole blood samples were stained with antibodies for cell surface markers: CD45, CD3, CD4, CD8, CD19, CD56 and CD16 as well as for two activation markers: HLA-DR and CD27. Samples were lysed and fixed with 1x FACSLysing solution (BD Biosciences, USA). Upon centrifugation, leukocyte pellets were resuspended in 1% paraformaldehyde/PBS. Acquisition of samples was performed using the Navios flow cytometer (Beckman Coulter, USA). Data was analyzed using FlowLogic™ software package (Inivai Technologies, Australia). Peripheral blood from more than 70 patients aging from 2-18 years was analysed in the same manner. Dual-panel platform that involved two six color antibody combinations and extensive analysis of FCS data files resulted with the finding of several interesting immunophenotypes: CD19dim+CD27high+ B cells, CD3+CD8+CD16high+T cells and CD3-CD4+ lymphocytes. In addition, three subpopulations of monocytes were resolved in each patient: CD14 high+ CD16-, CD14high+ CD16+ and CD14 -/dim+ CD16 high+. Polychromatic flow cytometry using expanded immune monitoring panel in routine immunophenotyping enabled the detection of mononuclear subsets that could not be resolved in procedures where typical four-color immunophenotyping assays are used. In studies of immune disorders, such as primary immunodeficiencies, a polychromatic approach using even only six colors may allow deeper insight and understanding of the complex interplay among immune cells and improve diagnostic procedures.

Izvorni jezik
Engleski

Znanstvena područja
Kliničke medicinske znanosti

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