DRAQ5-based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties.
BACKGROUND
Analysis of cell cycle kinetics offers important information regarding the behavior of normal and neoplastic cells. Most often, cell cycle determinations by flow cytometry (FCM) have been performed using whole-sample analysis with intercalating dyes like propidium iodide (PI). The cell cycle phase assessment in individual cell subsets in heterogeneous samples is best performed using combined antigen/scatter and DNA analysis. DRAQ5, a novel DNA binding dye that excites at 488 nm and emits in the far red spectra, rapidly penetrates intact live cells while preserving their light scatter properties and expression of surface antigens. We evaluated the ability of this dye to measure cell cycle phases in a variety of clinical hematolymphoid samples.


METHODS
We first compared whole sample DRAQ5 and PI cell cycle analyses in 26 clinical hematolymphoid samples. Next, we analyzed cell subpopulations in 39 samples of nonpathologic bone marrow by performing simultaneous CD45/CD34 and DRAQ5 staining. We assessed cell cycle characteristics specific to each population identified by CD45/CD34/side light scatter: lymphocytes, monocytes, immature and mature granulocytes, nucleated erythroid cells, and early precursors.


RESULTS
Whole sample DNA cell cycle analyses by DRAQ5 and PI showed no significant differences in S-phase. DRAQ5, however, produced slightly larger coefficients of variation. DRAQ5-based DNA content analysis was easily performed on the distinct marrow cell subpopulations, since light scatter and antigen expression were completely preserved. Significant differences in S-phase were noted between subpopulations of cells exhibiting different degrees of maturation.


CONCLUSIONS
Because of its simplicity of use, excitability with 488 nm lasers, and the ability to stain viable cells, DRAQ5 should prove most useful in the kinetic evaluation of normal and neoplastic hematolymphoid cell subsets identified by light scatter and antigenic expression.
Yuan
Constance M
CM
Department of Pathology, Shands Hospital, University of Florida College of Medicine, Gainesville, Florida 32610, USA. yuancm@pathology.ufl.edu
Douglas-Nikitin
Vonda K
VK
Ahrens
Kim P
KP
Luchetta
Gabriel R
GR
Braylan
Raul C
RC
Yang
Lijun
L
D000880:Anthraquinones; D018952:Antigens, CD34 / Q000378:metabolism; D000954:Antigens, Surface / Q000032:analysis* / Q000737:chemistry; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D004247:DNA / Q000032:analysis*; D006413:Hematopoietic System / Q000166:cytology / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008027:Light; D009589:Nitrogen Oxides / Q000032:analysis* / Q000737:chemistry; D011419:Propidium / Q000032:analysis / Q000737:chemistry; D012542:Scattering, Radiation; D013194:Staining and Labeling
D000880:Anthraquinones; D018952:Antigens, CD34; D000954:Antigens, Surface; C406029:DRAQ5NO; D009589:Nitrogen Oxides; D011419:Propidium; D004247:DNA; D017493:Leukocyte Common Antigens