Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas

Abstract

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H-ThdR) labeling index (LI).Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H-ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study.In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.Key terms: DNA histogram analysis, debris background subtractionThe fraction of cells in DNA synthesis phase is one of the important features derived from flow cytometric DNA distributions. Particularly in breast carcinoma biopsies, the S phase portions proved to be prognostically valuable indicators of the proliferative activity, and correct determinations are therefore required.Numerous data have been published on this subject; such data had been estimated either by the tritiated thymidine (3H-ThdR) labeling index GI) (6,14,17,27,30,32,37,38,40) or by DNA flow cytometry (3,5,7,20,29,31,33,34,39). Comparison of these data revealed that the cytometrically determined S phase portions were generally higher than those obtained by the alternative method 3H-ThdR-LI (15,16,(24)(25)(26)28,30), but satisfactory reasons for the differences were lacking.Although various methods and models for analyses of flow cytometric DNA distributions have been developed and studied exhaustively (survey in 2,10,42,44), little attention has been directed to other influences, such as the debris background level, which may cause errors which are often larger than those resulting from choice of an analytical model. Nevertheless, the few studies of Beck, Haag, and van der Linden (4,15,41) have addressed appropriate subtraction of the debris components on which the actual DNA distributions of intact cell nuclei are superimposed.The rationale of corrections is to define a mathematical function of the debris background distributions which can be subtracted from the originally recorded distribution. Two differ...

Comparison of S‐phase fractions measured by flow cytometry and autoradiography in human transplant tumors

“…SPF measurements by FCM are expected to furnish approximately similar results which can be achieved by elimination of the influences of background debris in the DNA histogram, as published recently from our laboratory (11). This study revealed that good agreement between S-phase fractions measured by DNA FCM and autoradiography could be obtained only if a procedure of background correction was applied to the histograms.…”

“…Nevertheless, because these results were not determined from identical samples, they cannot be compared directly. Moreover, the majority of the breast cancer samples in that study (11) contained low percentages of S-phase cells, with a mean of about 5%. The influence of the background correction on cases with high SPF (up to about 40%) were not examined.…”

“…The methods of preparation, staining, and measurement were performed according to Zante et al (31) as described recently (11).…”

“…Such reserve has been intensified by discrepancies between flow cytometrically (FCMI-measured SPF and the 3H-thymidine labeling index (TLI) in some tumors (6,11,14).…”

Effect of sonication on paraffin‐embedded tissue preparation for DNA flow cytometry

“…The use of a cup horn has the advantage that the specimens can be sonicated in small groups of samples and the samples are in closed test tubes and need not come into direct contact with the transducer. A preliminary study using different periods of time (2,4,5,6,7,8,10, and 15 min) indicated 4 min was optimal for most tissue types when using the equipment described above.…”