Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens.
BACKGROUND
Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population. Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application. We describe here a reliable and reproducible application of both three- and four-color multiparameter DNA analysis.


METHODS
After immunostaining of fresh samples of peripheral blood, bone marrow and single cell suspensions of lymph nodes from healthy and lymphoma patients, a methanol fixation for TO-PRO-3 and DRAQ5 staining was tested.


RESULTS
The red-excitable TO-PRO-3 on a FACSCalibur is limited to two-color antigen staining including fluorescein-isothiocyanate and phycoerythrin-labeled monoclonal antibodies due to its broad excitation spectrum. Although DRAQ5 is only applicable to flow cytometers equipped with a single argon laser emitting 488-nm light, its emission spectrum can be easily separated from the FITC, PE, and PE/Texas-Red emissions. DRAQ5 showed almost identical stoichiometric DNA binding characteristics as propidium iodide. Coefficient of variation produced by DRAQ5 staining is in the range of 3.5 and is adequate for detecting aneuploid amd near-diploid cells.


CONCLUSIONS
These advantageous features of DRAQ5 make it a reliable candidate for multiparameter clinical studies.
Plander
M
M
Department of Hematology, University Teaching Hospital of Vas County, Szombathely, Hungary.
Brockhoff
G
G
Barlage
S
S
Schwarz
S
S
Rothe
G
G
Knuechel
R
R
D000880:Anthraquinones; D014408:Biomarkers, Tumor / Q000032:analysis; D002232:Carbocyanines / Q000737:chemistry; D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D016130:Immunophenotyping; D008214:Lymphocytes / Q000737:chemistry / Q000166:cytology; D008223:Lymphoma / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D009589:Nitrogen Oxides / Q000737:chemistry; D015203:Reproducibility of Results; D013194:Staining and Labeling
D000880:Anthraquinones; D014408:Biomarkers, Tumor; D002232:Carbocyanines; D004273:DNA, Neoplasm; C406029:DRAQ5NO; D009589:Nitrogen Oxides; C098830:TO-PRO-3