B. Baldetorp et al., REPRODUCIBILITY IN DNA FLOW CYTOMETRIC ANALYSIS OF BREAST-CANCER - COMPARISON OF 12 LABORATORIES RESULTS FOR 67 SAMPLE HOMOGENATES, Cytometry, 22(2), 1995, pp. 115-127
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 evalu
ated in prospective randomized trials. The widespread use of FCM DNA a
nalysis emphasizes the importance of reproducibility (both intra- and
interlaboratory). In this study, 67 nuclear suspensions of breast canc
er samples were analyzed by 12 laboratories routinely performing FCM D
NA analysis in breast cancer. No general guidelines were imposed; each
laboratory used its own standard protocols. For DNA ploidy status (di
ploid vs. non-diploid), agreement was complete for 79% (53/67) of the
samples, compared with 64% (43/67) of samples when tetraploidy was con
sidered [i.e., euploid (diploid + tetraploid) vs. aneuploid (the remai
ning non-diploid)]. For the SPF, pairwise comparison of the results of
all 12 laboratories yielded a mean Spearman's rank correlation of 0.7
8 (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'')]. Discrepancie
s were chiefly attributable to differences in the categorization of th
e S-phase values, rather than in FCM procedures, other critical differ
ences being in the detection and interpretation of near-diploid and sm
all non-diploid cell populations, the definition of tetraploidy, and t
he choice and execution of the method used for S-phase estimation. Bas
ed on the observations of this study, detailed guidelines for FCM anal
ysis and interpretation of data are proposed in the Appendix. Some iss
ues remain, however, e.g., to standardize a method for S-phase calcula
tion and tetraploid definition. (C) 1995 Wiley-Liss, Inc.