Abstract
Background: The NPE Analyzer® flow cytometer can simultaneously analyze the electronic nuclear volume (ENV) and DNA content of cells. This study describes the schematics, resolution, reproducibility, and sensitivity of biological standards analyzed on this unit.
Methods: Calibrated beads and biological standards (lymphocytes, trout erythrocytes [TRBC], calf thymocytes, and tumor cells) were analyzed for ENV versus DNA content. Parallel data (forward scatter versus DNA) from a conventional flow cytometer were obtained.
Results: ENV linearity studies yielded an R value of 0.999. TRBC had a coefficient of variation (CV) of 1.18 ± 0.13. DNA indexes as low as 1.02 were detectable. DNA content of lymphocytes from 42 females was 1.9% greater than that for 60 males, with a noninstrumental variability in total DNA content of 0.5%. The ENV/DNA ratio was constant in 15 normal human tissue samples, but differed in the four animal species tested. The ENV/DNA ratio for a hypodiploid breast carcinoma was 2.3 times greater than that for normal breast tissue.
Conclusions: The high-resolution ENV versus DNA analyses are highly reliable, sensitive, and can be used for the detection of near-diploid tumor cells that are difficult to identify with conventional cytometers. ENV/DNA ratio may be a useful parameter for detection of aneuploid populations.
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Krishan A, Wen JH, Thomas RA, Sridhar KS, Smith WF. NASA/American Cancer Society high-resolution flow cytometry project - II: Multiparametric analysis of DNA content and electronic nuclear volume in human solid tumors (manuscript submitted).
Thomas R, Eggleston W (1987). Flow cytometry. U.S. Patent No. 4,673,288.
Ploem JS (1967). The use of a vertical illuminator with interchangeable dichroic mirrors for fluorescence microscopy with incidental light. Z Wis Mikrosk 68: 129-142.
Thornthwaite JT, Sugarbaker EV, Temple WJ (1980). Preparation of tissues for DNA flow cytometric analysis. Cytometry 1: 229-237.
Fasman GD (1976). Selected Data for Molecular Biology In: The Chemical Rubber Co. (CRC) Handbook of Biochemistry and Molecular Biology Nucleic Acids Volume II, 3rd Edition, p 296.
Knobloch A, Vendrely C, Vendrly R (1957). The amount of deoxyribonucleic acid in a single trout sperm. Biochim Biophys Acta 24: 201.
Lee GM, Thornthwaite JT, Rasch EM (1984). Picogram per cell determination of DNA by flow cytofluorometry. Anal Biochem 137: 221-226.
Deloukas P, Schuler GD, Gyapay G, Beasley EM, Soderlund C, Rodriguez-Tome P, Hui L, Matise TC, McKusick KB, Beckmann JS, Bentolila S, Bihoreau M, Birren BB, Browne J, Butler A, Castle AB, Chiannilkulchai N, Clee C, Day PJ, Dehejia A, Dibling T, Drouot N, Duprat S, Fizames C, Bentley DR et al. (1998). A physical map of 30,000 human genes. Science 282: 744-746.
Harris P, Boyd E, Young BD, Ferguson-Smith MA (1986). Determination of the DNA content of human chromosomes by flow cytometry. Cytogenet Cell Genet 41: 14-21.
Morton NE (1991). Parameters of the human genome. Proc Natl Acad Sci USA 88: 7474-7476.
Mullaney PF, Crowell JM, Salzman GC, Martin JC, Hiebert RD, Goad CA (1976). Pulse-height lightscatter distributions using flow-systems instrumentation. J Histochem Cytochem 24: 298-304.
Leary JF, Todd P, Wood JC, Jett JH (1979). Laser flow cytometric light scatter and fluorescence pulse width and pulse rise-time sizing of mammalian cells, J Histochem Cytochem 27: 315-320.
Mullaney PF, Van Dilla MA, Coulter JR, Dean PN (1969). Cell sizing: a light scattering photometer for rapid volume determination, Rev Sci Instrum 40: 1029-1032.
Stal O, Hatschek T (1988). A rapid system for static cytofluorometry enabling the simultaneous determination of nuclear size and DNA content. Pathol Res Pract 183: 329-335.
Grover NB, Naaman J, Ben-Sasson S, Dolianski F (1969). Electrical sizing of particles I. Biophys J 9: 1398-1414.
Thomas RA, Cameron BF (1974). Electronic cell volume analysis utilizing the AMAC II transducer. J Histochem Cytochem 22: 626-641.
Petersen SE (1986). Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol. Cytometry 7: 301-306.
Cram LS, Lehman JM (1977). Flow microfluorometric DNA content measurements of tissue culture cells and peripheral lymphocytes. Human Genetics 37: 201-206.
Hoehn H, Johnston P, Callis J (1977). Flow-cytogenetics: sources of DNA content variation among euploid individuals. Cytogenet Cell Genet 19: 94-107.
Redkar A, Krishan A (1999). Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin fixed, paraffin embedded human breast tumors. Comm Clin Cytometry 38: 61-69.
Krishan A, Oppenheimer A, You W, Dubbins R, Sharma D, Lokeshwar B (2000). Flow cytometric analysis of androgen receptor expression in human prostate tumors and benign tissues. Clin Cancer Res 6: 1922-1930.
Thomas A, Krishan A, Robinson D, Sams C and Costa F (2001). NASA/American Cancer Society High-Resolution Flow Cytometry Project-I. Cytometry 43: 2-11.
Krishan A, Wen J, Thomas R, Sridhar K, Smith W (2001). NASA/American Cancer Society High-Resolution Flow Cytometry Project-III. Multiparametric Analysis of DNA Content and Electronic Nuclear Volume in Human Solid Tumors. Cytometry 43: 16-22.
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Thomas, R.A., Krishan, A. & Brochu, M. High resolution flow cytometric analysis of electronic nuclear volume and DNA content in normal and abnormal human tissue. Methods Cell Sci 24, 11–18 (2002). https://doi.org/10.1023/A:1024100705728
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DOI: https://doi.org/10.1023/A:1024100705728