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Sir There are a number of points which I believe should be raised with regard to the article by Mullen and Miller (1989) on DNA flow cytometry of breast cancer fine needle aspirates. First, I would point out that the authors have omitted to reference previous contributions on the same topic (Spyratos et al., 1987; Remvikos et al., 1988a, Briffod et al., 1989). Second, as a cytometrist, I believe that data file histograms should be presented (rather than redrawn ones) as these usually contain extensive information on the quality of the preparations (baseline, noise, skewed peaks, aggregates). Third, the method of calculation of S-phase values should always be stated. The most important weakness of the article is the definition of the variations on the histograms. For instance, a modification of more than 5% in DNA index is considered significant, where the standard deviation inherent in the method of calculation (internal standard) is of 4%. From a statistical point of view, a threshold of at least twice the standard deviation should be used, i.e. 8%. Incidentally, the committee on nomenclature of flow cytometry published its recommendations in 1984 (Hiddeman et al., 1984), stipulating that animal cells should definitely not be used as calibration standards. To my knowledge, these recommendations have been recognised by the Society of Analytical Cytology and are followed by most cytometrists. As for the modifications of cell populations, this is a very delicate point that should be treated with much caution. A variation in the proportion of the diploid cells could first of all be due to the presence of a variable contingent of normal cells (lymphoid infiltrate for example). Therefore, a precise score of the normal cells must be established on control smears before a threshold of 10% for a substantial difference between duplicate analyses can be validated. Breast cancers are heterogeneous. This has been shown by Auer et al. (1980), but also by our comparison of FCM and cytogenetics for the same tumour (Remvikos et al., 1988b), with sometimes a paradiploid and an aneuploid clone being simultaneously present. Variations in the proportions of the different cell populations in repeated samplings are therefore to be expected. A final point I wish to make concerns the usefulness of iterative FNA in the assessment of response to treatment. By analogy with in vitro experiments, modifications in the cell cycle distribution are primarily to be expected (for instance accumulation of cells in S or G2M). This was shown in the pioneering work of Vindelov et al. (1982) and has been confirmed for breast cancers (Spyratos et al., 1988; Remvikos et al., 1988c). DNA flow cytometry (FCM) is a promising method in clinical oncology, in particular because of the possibility of measuring DNA index and S-phase fractions (Kalionemi et al., 1988). It was recently shown that DNA-ploidy and Sphase fraction are significantly related to response to cytotoxic chemotherapy (Briffod et al., 1989; Remvikos et al., 1989). I believe that the article by Mullen and Miller can only bring unjustified confusion to the minds of clinicians who may be interested in FCM. Yours etc.,