Statistical Science

Statistical considerations in DNA flow cytometry

T. Lynn Eudey

Full-text: Open access

Abstract

Flow cytometry is frequently used for assessing individual cell characteristic(s) for a large number of cells. It has a variety of medical applications including assessing the quantity of intracellular DNA and detecting the presence of antigens such as CD4. Flow cytometric variables are evaluated for their clinical prognostic value, particularly in cancer, and are often used for clinical screening of diseased patients. The prognostic worth of these variables is questionable and is controversial in the medical community. This controversy is caused in part by the multiple methods of analysis and the lack of adherence to quality control standards. The analysis of flow cytometric data presents a number of interesting statistical problems, particularly in deconvolution of overlapping distributions and detection of abnormal subpopulation(s) of cells. The current methods incorporate subjective procedures, may use ill-founded assumptions and yield differing results. This article summarizes the flow cytometry process of measurement and reviews unsolved statistical and quality control issues pertaining to the analysis of flow cytometric data. DNA histogram analysis is used to exemplify these issues.

Article information

Source
Statist. Sci., Volume 11, Number 4 (1996), 320-334.

Dates
First available in Project Euclid: 17 September 2002

Permanent link to this document
https://projecteuclid.org/euclid.ss/1032280305

Digital Object Identifier
doi:10.1214/ss/1032280305

Keywords
Aneuploid DNA DNA histogram diploid deconvolution fluorochromes $S$-phase

Citation

Eudey, T. Lynn. Statistical considerations in DNA flow cytometry. Statist. Sci. 11 (1996), no. 4, 320--334. doi:10.1214/ss/1032280305. https://projecteuclid.org/euclid.ss/1032280305


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References

  • Alanen, K. A., Joensuu, H. and Klemi, P. J. (1989). Autoly sis is a potential source of false aneuploid peaks in flow cy tometric DNA histograms. Cy tometry 10 417-425. Alanen, K. A., Klemi, P. J., Joenssu, H., Kujari, H. and
  • Pekkala, E. (1989). Comparison of fresh, ethanol-preserved, and paraffin-embedded samples in DNA flow cy tometry. Cy tometry 10 81-85.
  • Bagwell, C. B. (1993). Theoretical aspects of flow cy tometry data analysis. In Clinical Flow Cy tometry: Principles and Application (K. D. Bauer, R. E. Duque and T. V. Shankey, eds.) Chapter 3. Williams & Wilkins, Baltimore.
  • Bagwell, C. B. (1996). A journey through flow cy tometric immunofluorescence analyses-finding accurate and robust algorithms that estimate positive fraction distributions. Clinical Immunology Newsletter 16 33-37. Baisch, H., Beck, H.-P., Christensen, I. J., Hartmann, N. R., Fried, J., Dean, P. N., Gray, J. W., Jett, J. H., Johnston, D. A., White, R. A., Nicolini, C., Zeitz, S. and Watson,
  • J. V. (1982). A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cy tometry. Cell Tissue Kinetics 15 235-249. Baldetorp, B., Bendahl, P.-O., Ferno, M., Alanen, K., Delle, U., Falkmer, U., Hansson-Aggesjo, B., Hockenstrom, T., Lindgren, A., Mossberg, L., Nordling, S.,
  • Sigurdsson, H., Stal, O. and Visakorpi, T. (1995). Reproducibility in DNA flow cy tometric analysis of breast cancer: comparison of 12 laboratories' results for 67 sample homogenates. Cy tometry (Communications in Clinical Cy tometry) 22 115-127.
  • Bauer, K. D., Duque, R. E. and Shankey, T. V., eds. (1993). Clinical Flow Cy tometry: Principles and Application. Williams & Wilkins, Baltimore.
  • Becker, R. L., Jr. and Mikel, U. V. (1990). Interrelation of formalin fixation, chromatin compactness and DNA values as measured by flow and image cy tometry. Analy tical and Quantitative Cy tology and Histology 12 333-341. Beerman, H., Kluin, P. M., Hermans, J., Van de Velde, C. J. H.
  • and Cornelisse, C. J. (1990). Prognostic significance of DNA ploidy in a series of 690 primary breast cancer patients. International Journal of Cancer 45 34-39.
  • Begg, A. C. (1995). The clinical status of Tpot as a predictor? Or why no tempest in the Tpot! International Journal of Radiation Oncology, Biology, physics 32 1539-1541. (Editorial.)
  • Benson, N. A. and Bray lan, R. C. (1994). Evaluation of sensitivity in DNA aneuploidy detection using a mathematical model. Cy tometry 15 53-58.
  • Carr, R. F. and Abaza, A. M. (1993). False aneuploidy in flow cy tometric DNA analysis of paraffin embedded tissue: effects of Carnoy's fixation. Cy tometry 14 668-672. Chance, J. T., Larsen, S. A., Pope, V., Measel, J. W. and Cox,
  • D. L. (1995). Instrument-dependent fluorochrome sensitivity in flow cy tometric analyses. Cy tometry Communications in Clinical Cy tometry 22 232-242. Chassevent, A., Daver, A., Bertrand, G., Coic, H., Geslin, J.,
  • Bidabe, M.-Cl., George, P. and Larra, F. (1984). Comparative flow DNA analysis of different cell suspensions in breast carcinoma. Cy tometry 5 263-267. Clark, G. M., Dressler, L. G., Owens, M. A., Pounds, G.,
  • Oldaker, T. and McGuire, W. L. (1989). Prediction of relapse of survival in patients with node-negative breast cancer by DNA flow cy tometry. New England Journal of Medicine 320 627-633.
  • Cusick, E. L., Milton, J. I. and Ewen, S. W. (1990). The resolution of aneuploid DNA stem lines by flow cy tometry: limitations imposed by the coefficient of variation and the percentage of aneuploid nuclei. Analy tical Cellular Pathology 2 139-148.
  • Darzy nkiewicz, Z. (1990). Acid-induced denaturation of DNA in situ as a probe of chromatin structure. Methods in Cell Biology 33 337-352.
  • Darzy nkiewicz, Z. (1993). The cell cy cle: application of flow cy tometry in studies of cell reproduction. In Clinical Flow Cy tometry: Principles and Application (K. D. Bauer, R. E. Duque and T. V. Shankey, eds.) Chapter 2. Williams & Wilkins, Baltimore.
  • Dean, P. N. (1985). Methods of data analysis in flow cy tometry. In Flow Cy tometry: Instrumentation and Data Analy sis (M. A. Van Dilla, P. N. Dean, O. D. Laerum and M. R. Melamed, eds.) Chapter 6. Academic Press, London.
  • Dean P. N. and Jett J. H. (1974). Mathematical analysis of DNA distributions derived from flow microfluorometry. Journal of Cell Biology 60 523-527. Dowle, C. S., Owainati, A., Robins, A., Burns, K., Ellis, I. O.,
  • Elston, C. W. and Blamey, R. W. (1987). Prognostic significance of the DNA content of human breast cancer. British Journal of Surgery 74 133-136.
  • Dressler, L. G. (1990). Controls, standards, and histogram interpretation in DNA flow cy tometry. Methods in Cell Biology 33, Chapter 17 157-171. Ferno, M., Baldetorp, B., Borg, A., Olsson, H., Sigurdsson, H.
  • and Killander, D. (1992). Flow cy tometric DNA index and S-phase fraction in breast cancer in relation to other prognostic variables and to clinical outcome. Acta Oncologica 31 157-165.
  • Fried, J. (1976). Method for the quantitative evaluation of data from flow microfluorometry. Computers and Biomedical Research 9 263-276.
  • Frierson, H. F. (1988). Flow cy tometric analysis of ploidy in solid neoplasms: comparison of fresh tissues with formalinfixed paraffin-embedded specimens. Human Pathology 19 290-294.
  • Frierson, H. F. (1991). The need for improvement in flow cy tometric analysis of ploidy and S-phase fraction. American Journal of Clinical Pathology 96 439-441.
  • Givan, A. L. (1992). Flow Cy tometry: First Principles. Wiley-Liss, New York. Gnant, M. F. X., Blijham, G., Reiner, A., Reiner, G., Rey nders, M., Schutte, B., van Asche, C., Steger, G. and Jakesz, R.
  • (1992). DNA ploidy and other results of DNA flow cy tometry as prognostic factors in operable breast cancer: 10 year results of a randomized study. European Journal of Cancer 28 711-716.
  • Gong, J., Traganos, F. and Darzy nkiewicz, Z. (1993). Simultaneous analysis of cell cy cle kinetics at two different DNA ploidy levels based on DNA content and cy clin B measurements. Cancer Research 53 5096-5099.
  • Grogan, W. M. and Collins, J. M. (1990). Guide to Flow Cy tometry Methods. Dekker, New York.
  • Hedley, D. W., Shankey, T. V. and Wheeless, L. L. (1993). DNA cy tometry consensus conference. Cy tometry 14 471- 500. (Opening statement on conference.) Hedley, D. W., Freidlander, M. L., Tay lor, I. W., Rugg, C. A.
  • and Musgrove, E. A. (1983). Methods for analysis of cellular DNA content of paraffin embedded pathologic material using flow cy tometry. Journal of Histochemistry and Cy tochemistry 31 1333-1335. Herbert, D. J., Nishiy ama, R. H., Bagwell, C. B., Munson,
  • M. E., Hitchcox, S. A. and Lovett, E. J., III (1989). Effects of several commonly used fixatives on DNA and total nuclear protein analysis by flow cy tometry. American Journal of Clinical Pathology 91 535-541.
  • Herman, C. J. (1992). Cy tometry DNA analysis in the management of cancer. Clinical and laboratory considerations. Cancer 69 1553-1556. Hiddemann, W., Schumann, J., Andreeff, M., Barlogie, B., Herman, C. J., Leif, R. C., May all, B. H., Murphy, R. F. and Sandberg, A. A. (1984a). Convention on nomenclature for DNA cy tometry. Cy tometry 5 445-446. Hiddemann, W., Schumann, J., Andreeff, M., Barlogie, B., Herman, C. J., Leif, R. C., May all, B. H., Murphy, R. F. and Sandberg, A. A. (1984b). Convention on nomenclature for DNA cy tometry. Cancer Genetics and Cy togenetics 13 181- 183.
  • Hitchcock, C. L. (1991). Variability in flow cy tometric results using identical archival samples. Cy tometry Supplement 5 46.
  • Hitchcock, C. L. and Ensley, J. F. (1993). Dissociating solid tumors. In Clinical Flow Cy tometry: Principles and Application (K. D. Bauer, R. E. Duque and T. V. Shankey, eds.) Chapter 6. Williams & Wilkins, Baltimore.
  • Hitchcock, C. L. and Scott, K. (1990). Optimization of techniques for flow cy tometric analysis of DNA from archival material (abstract). Cy tometry Supplement 4 102.
  • Hitchcock, C. L. and Scott, K. (1991). Flow cy tometric analysis of archival tissue: the use of a model tumor sy stem to examine the effects of technical parameters (abstract). Laboratory Investigation 64 122A. Isobe, H., Sridhar, K. S., Doria, R., Cohen, F., Raub, W. A.,
  • Saldana, M. and Krishan, A. (1995). Prognostic significance of DNA aneuploidy in diffuse malignant mesothelioma. Cy tometry 19 86-91.
  • Joensuu, H., Alanen, K. A., Klemi, P. J. and Aine, R. (1990). Evidence for false aneuploid peaks in flow cy tometric analysis of paraffin-embedded tissue. Cy tometry 11 431-437. Joensuu, H., Alanen, K., Falkmer, U. G., Klemi, P.,
  • Nordlin, S., Remvikos, Y. and Toikkanen, S. (1992). Effect of DNA ploidy classification on prognosis in breast cancer. International Journal of Cancer 52 701-706.
  • Joensuu, H. and Toikkanen, S. (1992). Identification of subgroups with favorable prognosis in breast cancer. Acta Oncologica 31 293-301. Kallioniemi, O.-P., Blanco, G., Alaveikko, M., Hietanen, T.,
  • Mattila, J., Lauslahti, K. and Koivula, T. (1987). Tumor DNA ploidy as an independent prognostic factor in breast cancer. British Journal of Cancer 56 637-642. Kallioniemi, O.-P., Joensuu, H., Klemi, P. and Koivula, T.
  • (1990). Inter-laboratory comparison of DNA flow cy tometric results from paraffin-embedded breast carcinomas. Breast Cancer Research and Treatment 17 59-61. Kallioniemi, O.-P., Vasakorpi, T., Holli, K., Isola, J. J. and
  • Rabinovitch, P. S. (1994). Automated peak detection and cell cy cle analysis of flow cy tometric DNA histograms. Cy tometry 16 250-255. Keny on, N. S., Schmittling, R. J., Siiman, O., Burshtey n, A.
  • and Bolton, W. E. (1994). Enhanced assessment of DNA/proliferative index by depletion of tumor infiltrating leukocy tes prior to monoclonal antibody gated analysis of tumor cell DNA. Cy tometry 16 175-183. Key hani-Rofagha, S., O'Toole, R. V., Farrar, W. B., Sickle
  • Santanell, B., De Cenzo, J. and Young, D. (1990). Is DNA ploidy an independent prognostic indicator in infiltrative node-negative breast adenocarcinoma. Cancer 65 1577-1582.
  • Kusuda, L. and Melamed, M. R. (1994). Display and correction of flow cy tometry time-dependent fluorescence changes. Cy tometry 17 340-342. Larsen, J. K., Munch-Peterson, B., Christiansen, J. and Jor
  • gensen, J. (1986). Flow cy tometric discrimination of mitotic cells: resolution of M, as well as G1, S and G2 phase nuclei with mithramy cin, propidium iodide and ethidium bromide after fixation with formaldehy de. Cy tometry 7 54-63.
  • Lewis, W. E. (1990). Prognostic significance of flow cy tometric DNA analysis in node-negative breast cancer patients. Cancer 65 2315-2320.
  • Marquardt, D. W. (1963). An algorithm for least-squares estimation of nonlinear parameters. Journal of Sociology and Industrial Applied Mathematics 11 431-441. McIntire, T. L., Goldey, S. H., Benson, N. A. and Bray lan,
  • R. C. (1987). Flow cy tometric analysis of DNA in cells obtained from deparaffinized formalin-fixed ly mphoid tissues. Cy tometry 8 474-478.
  • Mendelsohn, J. and Rice, J. (1982). Deconvolution of microfluorometric histograms with B splines. J. Amer. Statist. Assoc. 77 748-753.
  • Muirhead, K. A. (1993). Quality control for clinical flow cy tometry. In Clinical Flow Cy tometry: Principles and Application (K. D. Bauer, R. E. Duque and T. V. Shankey, eds.) Chapter 11. Williams & Wilkins, Baltimore.
  • Omerod, M. G. (1994). Analy sis of DNA-general methods. In Flow Cy tometry, A Practical Approach (M. G. Omerod, ed.), 2nd ed., Chapter 7. Oxford Univ. Press. O'Reilly, S. M., Camplejohn, R. S., Barnes, D. M., Millis, R. R.,
  • Rubens, R. D. and Richards, M. A. (1990). Node-negative breast cancer: prognostic subgroups defined by tumor size and flow cy tometry. Journal of Clinical Oncology 8 2040- 2046. Ottestad, L., Pettersen, E. O., Nesland, J. M., Hannisdal, E.,
  • Fossa, S. D. and Tveit, K. M. (1993). Flow cy tometric DNA analysis as prognostic factor in human breast carcinoma. Pathology, Research and Practice 189 405-410. Pellicciari, C., Mangiarotti, R., Bottone, M. G., Danova, M.
  • and Wang, E. (1995). Identification of resting cells by dualparameter flow cy tometry of statin expression and DNA content. Cy tometry 21 239-337.
  • Pelstring, R. J., Hurtubise, P. E. and Swerdlow, S. H. (1990). Flow-cy tometric DNA analysis of hematopoietic and ly mphoid proliferations: a comparison of fresh, formalin-fixed and B5-fixed tissues. Human Pathology 21 551-558.
  • Rabinovitch, P. S. (1993). Practical considerations for DNA content and cell cy cle analysis. In Clinical Flow Cy tometry: Principles and Application (K. D. Bauer, R. E. Duque and T. V. Shankey, eds.) Chapter 8. Williams & Wilkins, Baltimore.
  • Rabinovitch, P. and Jacobberger, J. (1995). DNA workshop. Presented at Third Annual Clinical Cy tometry Conference, Chesapeake Cy tometry Consortium. Shankey, T. V., Rabinovitch, P. S., Bagwell, B., Bauer, K. D., Duque, R. E., Hedley, D. W., May all, B. H. and Whee
  • less, L. (1993). Guidelines for the implementation of clinical DNA cy tometry. Cy tometry 14 472-477.
  • Shapiro, H. M. (1988). Practical Flow Cy tometry, 2nd ed. Alan R. Liss, New York.
  • Shapiro, H. M. (1995). Practical Flow Cy tometry, 3rd ed. Wiley Liss, New York. Sigurdsson, H., Baldetorp, B., Borg, A., Dalberg, M.,
  • Ferno, M., Killander, D. and Olsson, H. (1990). Indicators of prognosis in node-negative breast cancer. New England Journal of Medicine 322 1045-1053.
  • Silvestrini, R. (1994). Quality control for evaluation of the Sphase fraction by flow cy tometry: a multicentric study. Cy tometry Communications in Clinical Cy tometry 18 11-16. (Written by Silvestrini and the SICCAB Group for Quality Control of Cell Kinetic Determinations.)
  • Terry, N. H. A. and Peters, L. J. (1995). The predictive value of tumor-cell kinetic parameters in radiotherapy: considerations regarding data production and analysis. Journal of Clinical Oncology 13 1833-1836. (Editorial.) Theissig, F., Dimmer, V., Haroske, G., Kunze, K. D. and Meyer,
  • W. (1992). Use of nuclear image cy tometry, histopathological grading, and DNA cy tometry to make breast cancer prognosis more objective. Analy tical Cellular Pathology 3 351-360.
  • Toikkanen, S., Joensuu, H. and Klemi, P. (1989). The prognostic significance of nuclear DNA content in invasive breast cancer-a study with long-term follow-up. British Journal of Cancer 60 693-700. Uy terlinde, A. M., Baak, J. P. A., Schipper, N. W., Peterse, H.,
  • Matze, E. and Meijer, C. J. L. (1990). Further evaluation of the prognostic value of morphometric and flow cy tometric parameters in breast cancer patients with long follow-up. International Journal of Cancer 45 1-7.
  • Van Dilla, M. A. (1985). Overview of flow cy tometry: instrumentation and data analysis. In Flow Cy tometry: Instrumentation and Data Analy sis (M. A. Van Dilla, P. N. Dean, O. D. Laerum and M. R. Melamed, eds.) Chapter 1. Academic Press, London. Van Dilla, M. A., Dean, P. N., Laerum, O. D. and
  • Melamed, M. R., eds. (1985). Flow Cy tometry: Instrumentation and Data Analy sis, Analy tical Cy tology Series. Academic Press, London.
  • Verity Software House, Inc. (1996). Advertisement for ModFit LTTM. Cy tometry 22 front page.
  • Vindelov, L. L. and Christensen, I. J. (1990). A review of techniques and results obtained in one laboratory by an integrated sy stem of methods designed for routine clinical flow cy tometric DNA analysis. Cy tometry 11 753-770.
  • Watson, J. V. (1992). Flow Cy tometry Data Analy sis: Basic Concepts and Statistics. Cambridge Univ. Press. Weaver, D. L., Bagwell, C. B., Hitchcox, S. A., Whetstone, S. D., Baker, D. R., Herbert, D. J. and Jones, M. A.
  • (1990). Improved flow cy tometric determination of proliferative activity (S-phase fraction) from paraffin-embedded tissue. American Journal of Clinical Pathology 94 576-584.
  • Wheeless, L. L., Jr. (1991). Flow instrumentation and data analysis. In Diagnostic Flow Cy tometry (J. S. Coon and R. S. Weinstein, eds.) Chapter 2. Williams & Wilkins, Baltimore. Wheeless, L. L., Coon, J. S., Cox, C., Deitch, A. D., deVere White, R. W., Fradet, Y., Koss, L. G., Melamed, M. R., O'Connell, M. J., Reeder, J. E., Weinstein, R. S. and
  • Wersto, R. P. (1989). Measurement variability in DNA flow cy tometry of replicate samples. Cy tometry 10 731-738. Witzig, T. E., Gonchoroff, N. J., Therneau, T., Gibertson, D. T., Wold, L. E., Grant, C., Grand, J., Katzmann, J. A.,
  • Ahmann, D. L. and Ingle, J. N. (1991). DNA content flow cy tometry as a prognostic factor for node-positive breast cancer. Cancer 68 1781-1788. Zalupski, M. M., Maciorowski, Z., Ry an, J. R., Ensley, J. F.,
  • Hussein, M. E., Sundareson, A. S. and Baker, L. H. (1993). DNA content parameters of paraffin-embedded soft tissue sarcomas: optimization of retrieval technique and comparison to fresh tissue. Cy tometry 14 327-333.