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Proceedings Paper

Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using Quantum dots
Author(s): Jozsef Bocsi; Anja Mittag; Viktor Sebestyen Varga; Bela Molnar; Zsolt Tulassay; Ulrich Sack; Dominik Lenz; Attila Tarnok
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Paper 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.

Paper Details

Date Published: 27 March 2006
PDF: 11 pages
Proc. SPIE 6096, Colloidal Quantum Dots for Biomedical Applications, 60960Z (27 March 2006); doi: 10.1117/12.644544
Show Author Affiliations
Jozsef Bocsi, Univ. Leipzig (Germany)
Anja Mittag, Univ. Leipzig (Germany)
Viktor Sebestyen Varga, Semmelweis Univ. Budapest (Hungary)
Bela Molnar, Semmelweis Univ. Budapest (Hungary)
Zsolt Tulassay, Clinical Gastroenterology Research Unit (Hungary)
Ulrich Sack, Univ. Leipzig (Germany)
Dominik Lenz, Purdue Univ. (United States)
Attila Tarnok, Univ. Leipzig (Germany)

Published in SPIE Proceedings Vol. 6096:
Colloidal Quantum Dots for Biomedical Applications
Marek Osinski; Kenji Yamamoto; Thomas M. Jovin, Editor(s)

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