Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.
BACKGROUND
Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8).


OBJECTIVE
We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM.


METHODS
Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM.


RESULTS
Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM.


CONCLUSIONS
Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.
Department of Pediatric Cardiology, Heart Center, University Leipzig, Leipzig, Germany. jobocsi@web.de
József
J
Bocsi
Dominik
D
Lenz
Anja
A
Mittag
Viktor Sebestyén
VS
Varga
Béla
B
Molnar
Zsolt
Z
Tulassay
Ulrich
U
Sack
Attila
A
Tárnok
D001562:Benzimidazoles / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D017252:CD3 Complex / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D016516:CD4-CD8 Ratio; D016827:CD8 Antigens / Q000032:analysis; D004247:DNA / Q000032:analysis; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods*; D047569:Laser Scanning Cytometry / Q000295:instrumentation / Q000379:methods*; D007962:Leukocytes / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D045663:Quantum Dots; D013194:Staining and Labeling / Q000379:methods; D019809:Streptavidin / Q000737:chemistry
D001562:Benzimidazoles; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D005456:Fluorescent Dyes; D001710:Biotin; D004247:DNA; D019809:Streptavidin; C017807:bisbenzimide ethoxide trihydrochloride