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

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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. © 2006 International Society for Analytical Cytology