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

Jozsef Bocsi, Anja Mittag, V. Varga, B. Molnár, Z. Tulassay, Ulrich Sack, Dominik Lenz, Attila Tarnok

Research output: Chapter in Book/Report/Conference proceedingConference contribution

2 Citations (Scopus)

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 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.

Original languageEnglish
Title of host publicationProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume6096
DOIs
Publication statusPublished - 2006
EventColloidal Quantum Dots for Biomedical Applications - San Jose, CA, United States
Duration: Jan 22 2006Jan 24 2006

Other

OtherColloidal Quantum Dots for Biomedical Applications
CountryUnited States
CitySan Jose, CA
Period1/22/061/24/06

Fingerprint

Fluorescence microscopy
Semiconductor quantum dots
Fluorescence
Color
Scanning
Microscopes
Flow cytometry
Dyes
Mountings
Labeling
Blood
Lymphocytes
Ethylenediaminetetraacetic acid
Antioxidants
Free radicals
Labels
DNA

Keywords

  • Bleaching
  • Fluorescence mounting medium
  • QD605
  • QD655
  • SBC
  • SFM

ASJC Scopus subject areas

  • Engineering(all)

Cite this

Bocsi, J., Mittag, A., Varga, V., Molnár, B., Tulassay, Z., Sack, U., ... Tarnok, A. (2006). Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using quantum dots. In Progress in Biomedical Optics and Imaging - Proceedings of SPIE (Vol. 6096). [60960Z] https://doi.org/10.1117/12.644544

Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using quantum dots. / Bocsi, Jozsef; Mittag, Anja; Varga, V.; Molnár, B.; Tulassay, Z.; Sack, Ulrich; Lenz, Dominik; Tarnok, Attila.

Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Vol. 6096 2006. 60960Z.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Bocsi, J, Mittag, A, Varga, V, Molnár, B, Tulassay, Z, Sack, U, Lenz, D & Tarnok, A 2006, Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using quantum dots. in Progress in Biomedical Optics and Imaging - Proceedings of SPIE. vol. 6096, 60960Z, Colloidal Quantum Dots for Biomedical Applications, San Jose, CA, United States, 1/22/06. https://doi.org/10.1117/12.644544
Bocsi J, Mittag A, Varga V, Molnár B, Tulassay Z, Sack U et al. Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using quantum dots. In Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Vol. 6096. 2006. 60960Z https://doi.org/10.1117/12.644544
Bocsi, Jozsef ; Mittag, Anja ; Varga, V. ; Molnár, B. ; Tulassay, Z. ; Sack, Ulrich ; Lenz, Dominik ; Tarnok, Attila. / Automated four color CD4/CD8 analysis of leukocytes by scanning fluorescence microscopy using quantum dots. Progress in Biomedical Optics and Imaging - Proceedings of SPIE. Vol. 6096 2006.
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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 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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