Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots

Jozsef Bocsi; Anja Mittag; Viktor Sebestyen Varga; Bela Molnar; Zsolt Tulassay; Ulrich Sack; Dominik Lenz; Attila Tarnok

doi:10.1117/12.644544

27 March 2006 Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots

Jozsef Bocsi, Anja Mittag, Viktor Sebestyen Varga, Bela Molnar, Zsolt Tulassay, Ulrich Sack, Dominik Lenz, Attila Tarnok

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Proceedings Volume 6096, Colloidal Quantum Dots for Biomedical Applications; 60960Z (2006) https://doi.org/10.1117/12.644544
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

Scanning Fluorescence Microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al. Cytometry 2004, 61A:1). The ratio of CD4+/CD8+ cells is an important in immune diagnostics in immunodeficiency and HIV. Therefor a four-color staining protocol (DNA, CD3, CD4 and CD8) for automated SFM analysis of lymphocytes was developed. EDTA uncoagulated blood was stained with organic and inorganic (Quantum dots) fluorochromes in different combinations. Aliquots of samples were measured by Flow Cytometry (FCM) and SFM. By SFM specimens were scanned and digitized using four fluorescence filter sets. Automated cell detection (based on Hoechst 33342 fluorescence), CD3, CD4 and CD8 detection were performed, CD4/CD8 ratio was calculated. Fluorescence signals were well separable on SFM and FCM. Passing and Bablok regression of all CD4/CD8 ratios obtained by FCM and SFM (F(X)=0.0577+0.9378x) are in the 95% confidence interval. Cusum test did not show significant deviation from linearity (P>0.10). This comparison indicates that there is no systemic bias between the two different methods. In SFM analyses the inorganic Quantum dot staining was very stable in PBS in contrast to the organic fluorescent dyes, but bleached shortly after mounting with antioxidant and free radical scavenger mounting media. This shows the difficulty of combinations of organic dyes and Quantum dots. Slide based multi-fluorescence labeling system and automated SFM are applicable tools for the CD4/CD8 ratio determination in peripheral blood samples. Quantum Dots are stable inorganic fluorescence labels that may be used as reliable high resolution dyes for cell labeling.

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Jozsef Bocsi, Anja Mittag, Viktor Sebestyen Varga, Bela Molnar, Zsolt Tulassay, Ulrich Sack, Dominik Lenz, and Attila Tarnok "Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots", Proc. SPIE 6096, Colloidal Quantum Dots for Biomedical Applications, 60960Z (27 March 2006); https://doi.org/10.1117/12.644544

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KEYWORDS

Luminescence

Atomic force microscopy

Quantum dots

Microscopes

Blood

Biological research

Microscopy

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