Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations.
BACKGROUND
Flow cytometry (FCM) and laser scanning cytometry (LSC) are the routine techniques for fluorescent cell analysis. Recently, we developed a scanning fluorescent microscopy (SFM) technique. This study compares SFM to LSC (two slide-based cytometry, SBC, techniques) and FCM, in experimental and clinical settings.


METHODS
For the relative cell-frequency determinations, HT29 colorectal cancer cells and Ficoll separated blood mononuclear cells (FSBMCs) were serially diluted (from 1:1 to 1:1,000) and measured by each of the three techniques. For the absolute cell number determinations (only for SBC) FSBMCs were smeared on slides, then HT29 cells were placed on the slide with a micromanipulator (5-50 cells). Tumor cells circulating in the peripheral blood were isolated by magnetic separation from clinical blood samples of colorectal cancer patients. All samples were double-stained by CD45 ECD and CAM5.2 FITC antibodies. For slides, TOTO-3 and Hoechst 33258 DNA dyes were applied as nuclear counter staining.


RESULTS
In the relative cell frequency determinations, the correlations between the calculated value and measured values by SFM, LSC, and FCM were r(2) = 0.79, 0.62, and 0.84, respectively (for all P < 0.01). In the absolute cell frequency determinations, SFM and LSC correlated to a high degree (r(2) = 0.97; P < 0.01).


CONCLUSIONS
SFM proved to be a reliable alternative method, providing results comparable to LSC and FCM. SBC proved to be more suitable for rare-cell detection than FCM. SFM with digital slides may prove an acceptable adaptation of conventional fluorescent microscopes in order to perform rare-cell detection.
Bocsi
József
J
1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
Varga
Viktor Sebestyén
VS
Molnár
Béla
B
Sipos
Ferenc
F
Tulassay
Zsolt
Z
Tárnok
Attila
A
D017920:Antiporters; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D047569:Laser Scanning Cytometry / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008856:Microscopy, Fluorescence / Q000379:methods*; D009369:Neoplasms / Q000097:blood / Q000175:diagnosis / Q000473:pathology; D029701:Saccharomyces cerevisiae Proteins; D013223:Statistics as Topic
D017920:Antiporters; D029701:Saccharomyces cerevisiae Proteins; C099946:VCX1 protein, S cerevisiae