Cell DNA content measurements, including determination of tumor ploidy and S-phase fraction, have been performed on a wide variety of human tumors for > 20 years using flow cytometry. During this time many publications have discussed the clinical utility of cell DNA content measurements. A major impediment to the more widespread application of cytometric DNA content measurements has been the lack of agreement among these studies. Whereas some discrepancies can be attributed to poorly designed studies lacking sufficient follow-up or significant numbers of patients, in many cases the discrepancies are due to technical factors in flow or image cytometry.The terminology used to describe the results of flow cytometry studies is often confusing and not universally applied. Although a convention for nomenclature for all DNA cytometry was recommended in 1984 (l), the guidelines suggested are frequently not used in published studies. Cytometric studies should not use cytogenetic terminology (hypodiploid, peritetriploid, etc.), where no direct measurement of changes in the number or composition of individual chromosomes has been made. Rather, the terms DNA diploid and DNA aneuploid should be used, with identification of the degree of DNA content abnormality given by the use of the DNA index, or DI (ratio of mean or mode of sample G,/G, population divided by mean or mode of diploid reference cells).One critical aspect of the clinical applications of these measurements is the use of standardized procedures to prepare and analyze clinical samples and to analyze and interpret cytometry data. A number of studies have demonstrated significant intralaboratory, as well as interlaboratory variation in the results of DNA content analyses (2-5) and in the interpretation of flow cytometric data (6). The purpose of this work is to help increase the reliability and reproducibility of DNA content flow cytometry by pointing out important technical considerations for cytometry, to provide guidelines that logically follow from these considerations, and to provide a framework for the development of standards and standardization of DNA content flow cytometry. Although a number of reported studies have utilized image cytometry to provide DNA content analysis, the technical differences between image and flow cytometry suggest that guidelines for the clinical application of image cytometry be developed independently.In reviewing the published literature, it is clear that many studies fail to provide sufficient information to judge critically the quality of cytometric measurement. It is recommended that all publications using DNA content cytometry provide details of the technique used to isolate and prepare cells, data that indicate that the sample used for cytometry contains representative tumor material, details concerning the techniques used to stain cells or nuclei (dye concentration, enzyme concentrations in units, cell concentrations), and details of the techniques used to analyze DNA content histograms (debris and aggregation correction ...

Cell DNA content measurements, including determination of tumor ploidy and S-phase fraction, have been performed on a wide variety of human tumors for > 20 years using flow cytometry. During this time many publications have discussed the clinical utility of cell DNA content measurements. A major impediment to the more widespread application of cytometric DNA content measurements has been the lack of agreement among these studies. Whereas some discrepancies can be attributed to poorly designed studies lacking sufficient follow-up or significant numbers of patients, in many cases the discrepancies are due to technical factors in flow or image cytometry.The terminology used to describe the results of flow cytometry studies is often confusing and not universally applied. Although a convention for nomenclature for all DNA cytometry was recommended in 1984 (l), the guidelines suggested are frequently not used in published studies. Cytometric studies should not use cytogenetic terminology (hypodiploid, peritetriploid, etc.), where no direct measurement of changes in the number or composition of individual chromosomes has been made. Rather, the terms DNA diploid and DNA aneuploid should be used, with identification of the degree of DNA content abnormality given by the use of the DNA index, or DI (ratio of mean or mode of sample G,/G, population divided by mean or mode of diploid reference cells).One critical aspect of the clinical applications of these measurements is the use of standardized procedures to prepare and analyze clinical samples and to analyze and interpret cytometry data. A number of studies have demonstrated significant intralaboratory, as well as interlaboratory variation in the results of DNA content analyses (2-5) and in the interpretation of flow cytometric data (6). The purpose of this work is to help increase the reliability and reproducibility of DNA content flow cytometry by pointing out important technical considerations for cytometry, to provide guidelines that logically follow from these considerations, and to provide a framework for the development of standards and standardization of DNA content flow cytometry. Although a number of reported studies have utilized image cytometry to provide DNA content analysis, the technical differences between image and flow cytometry suggest that guidelines for the clinical application of image cytometry be developed independently.In reviewing the published literature, it is clear that many studies fail to provide sufficient information to judge critically the quality of cytometric measurement. It is recommended that all publications using DNA content cytometry provide details of the technique used to isolate and prepare cells, data that indicate that the sample used for cytometry contains representative tumor material, details concerning the techniques used to stain cells or nuclei (dye concentration, enzyme concentrations in units, cell concentrations), and details of the techniques used to analyze DNA content histograms (debris and aggregation correction t...

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