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Reproducibility in DNA flow cytometric analysis of breast cancer: comparison of 12 laboratories' results for 67 sample homogenates

Baldetorp, Bo LU ; Bendahl, Pär-Ola LU ; Fernö, Mårten LU ; Alanen, Kalle; Delle, Ulla; Falkmer, Ursula; Hansson-Aggesjö, Britt; Hockenström, Thomas; Lindgren, Anders and Mossberg, Lotta, et al. (1995) In Cytometry 22(2). p.115-127
Abstract
Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S-phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra- and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non-diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples... (More)
Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S-phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra- and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non-diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid+tetraploid) vs. aneuploid (the remaining non-diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54-0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non-diploid; overall kappa = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall kappa = 0.47 (for low SPF vs. high SPF vs. "no SPF reported")]. Discrepancies were chiefly attributable to differences in the categorization of the S-phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near-diploid and small non-diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S-phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S-phase calculation and tetraploid definition. (Less)
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published
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keywords
Interlaboratory, reproducibility, flow cytometry, DNA ploidy, S-phase, proliferation, quality control, standardization, breast cancer
in
Cytometry
volume
22
issue
2
pages
115 - 127
publisher
John Wiley & Sons
external identifiers
  • pmid:7587742
  • scopus:0029040468
ISSN
0196-4763
DOI
10.1002/cyto.990220207
language
English
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yes
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cd36588f-3bf2-4202-9932-568dfc63e5a9 (old id 1108869)
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2008-07-24 17:32:22
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2017-07-09 04:24:36
@article{cd36588f-3bf2-4202-9932-568dfc63e5a9,
  abstract     = {Flow cytometric (FCM) DNA analysis yields information on ploidy status and the S-phase fraction (SPF), variables of prognostic importance in breast cancer. The clinical value of the SPF is currently being evaluated in prospective randomized trials. The widespread use of FCM DNA analysis emphasizes the importance of reproducibility (both intra- and interlaboratory). In this study, 67 nuclear suspensions of breast cancer samples were analyzed by 12 laboratories routinely performing FCM DNA analysis in breast cancer. No general guidelines were imposed; each laboratory used its own standard protocols. For DNA ploidy status (diploid vs. non-diploid), agreement was complete for 79% (53/67) of the samples, compared with 64% (43/67) of samples when tetraploidy was considered [i.e., euploid (diploid+tetraploid) vs. aneuploid (the remaining non-diploid)]. For the SPF, pairwise comparison of the results of all 12 laboratories yielded a mean Spearman's rank correlation of 0.78 (range: 0.54-0.93). For those 39 samples being categorized in low or high SPF by all laboratories, all agreed in 14 samples (36%). Similar patterns were obtained with kappa measures, agreement being good for ploidy status (diploid vs. non-diploid; overall kappa = 0.87 and 0.74 for euploid vs. aneuploid), but moderate for the SPF [overall kappa = 0.47 (for low SPF vs. high SPF vs. "no SPF reported")]. Discrepancies were chiefly attributable to differences in the categorization of the S-phase values, rather than in FCM procedures, other critical differences being in the detection and interpretation of near-diploid and small non-diploid cell populations, the definition of tetraploidy, and the choice and execution of the method used for S-phase estimation. Based on the observations of this study, detailed guidelines for FCM analysis and interpretation of data are proposed in the Appendix. Some issues remain, however, e.g., to standardize a method for S-phase calculation and tetraploid definition.},
  author       = {Baldetorp, Bo and Bendahl, Pär-Ola and Fernö, Mårten and Alanen, Kalle and Delle, Ulla and Falkmer, Ursula and Hansson-Aggesjö, Britt and Hockenström, Thomas and Lindgren, Anders and Mossberg, Lotta and Nordling, Stig and Sigurdsson, Helgi and Stål, Olle and Visakorpi, Tapio},
  issn         = {0196-4763},
  keyword      = {Interlaboratory,reproducibility,flow cytometry,DNA ploidy,S-phase,proliferation,quality control,standardization,breast cancer},
  language     = {eng},
  number       = {2},
  pages        = {115--127},
  publisher    = {John Wiley & Sons},
  series       = {Cytometry},
  title        = {Reproducibility in DNA flow cytometric analysis of breast cancer: comparison of 12 laboratories' results for 67 sample homogenates},
  url          = {http://dx.doi.org/10.1002/cyto.990220207},
  volume       = {22},
  year         = {1995},
}