Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots

Cytometry A. 2006 Mar;69(3):131-4. doi: 10.1002/cyto.a.20217.

Abstract

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).

Aims: 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.

Conclusion: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzimidazoles / chemistry
  • Biotin / chemistry
  • CD3 Complex / analysis
  • CD4 Antigens / analysis
  • CD4-CD8 Ratio
  • CD8 Antigens / analysis
  • DNA / analysis
  • Flow Cytometry
  • Fluorescent Dyes / chemistry
  • Humans
  • Immunophenotyping / instrumentation
  • Immunophenotyping / methods*
  • Laser Scanning Cytometry / instrumentation
  • Laser Scanning Cytometry / methods*
  • Leukocytes / chemistry
  • Leukocytes / cytology
  • Leukocytes / immunology
  • Quantum Dots*
  • Staining and Labeling / methods
  • Streptavidin / chemistry

Substances

  • Benzimidazoles
  • CD3 Complex
  • CD4 Antigens
  • CD8 Antigens
  • Fluorescent Dyes
  • Biotin
  • DNA
  • Streptavidin
  • bisbenzimide ethoxide trihydrochloride