David Bourget (Western Ontario)
David Chalmers (ANU, NYU)
Rafael De Clercq
Jack Alan Reynolds
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Analysis 66 (2):154–157 (2006)
BACKGROUND: The majority of flow cytometric DNA content analyses are performed on whole peripheral blood, bone marrow or tumor samples containing significant numbers of non-malignant cells which hamper specific DNA content analysis. Simultaneous analysis of DNA content and immunophenotype greatly improves the specificity of DNA-ploidy measurements. Therefore, a three-color flow cytometric assay using DRAQ5 was developed and validated. METHODS: The results of DNA content analysis using DRAQ5 and propidium iodide were compared using peripheral blood samples of 15 healthy volunteers. The reproducibility of the multiparameter DRAQ5 assay was assayed in 10 analytical runs and the sensitivity was evaluated with addition experiments. RESULT: Using DRAQ5, slightly wider CVs of the G0/G1 peak were obtained (average CV=3.29%). When DRAQ5 staining and immunophenotyping were combined, the within- and between-run imprecision was 1.98% and 1.67%, respectively and the DNA content of 25 DNA-hyperdiploid tumor cells (DNA-index=1.16) per 4.10(4) mononuclear cells (0.06%) could be determined. CONCLUSIONS: The multiparameter DRAQ5 assay has a superior sensitivity and specificity compared to the propidium iodide based method. Since at least 25 DNA-hyperdiploid cells per 10(4) DNA-diploid mononuclear cells could be detected, multiparameter DRAQ5 DNA content analysis could be used to study minimal residual disease
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