Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

Smeets, A. W. G. B.; Pauwels, R.P.E.; Beck, H. L. M.; Feitz, W. F. J.; Geraedts, J. P. M.; Debruyne, F.M.J.; Laarakkers, Lisette; Vooijs, G. P.; Ramaekers, Frans C. S.

doi:10.1002/cyto.990080103

Cited by 17 publications

(8 citation statements)

References 15 publications

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“…Unfortunately, the bulk of these data is not always comparable and, in some cases, is even conflict ing, especially as far as the recurrences and proliferative pattern are concerned. Conclusive results are not available and these discrep ancies may be explained by the differences in the tech niques used, the random (or not) selection of patients, the choice of the groups to be examined, the types and modalities of sample collection and the morphological criteria followed for classification [19].…”

Section: Discussionmentioning

confidence: 99%

Cellular DNA Content and Proliferative Activity Evaluated by Flow Cytometry versus Histopathological and Staging Classifications in Human Bladder Tumors

Vita¹,

Forte²,

Maggi³

et al. 1991

Eur Urol

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Section: Discussionmentioning

confidence: 99%

Cellular DNA Content and Proliferative Activity Evaluated by Flow Cytometry versus Histopathological and Staging Classifications in Human Bladder Tumors

Vita¹,

Forte²,

Maggi³

et al. 1991

Eur Urol

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“…Future studies should address techniques to determine the recovery of representative tumor cells or nuclei and to compare this recovery to the proportional number of tumor cells in the source tissue. (24)(25)(26)(27)(28)(29). Optimal techniques for the isolation of tumor cells from different tissues need to be established based on carefully designed studies.…”

Section: Samplingmentioning

confidence: 99%

Guidelines for implementation of clinical DNA cytometry

Shankey

Rabinovitch

Bagwell³

et al. 1993

Cytometry

373

173

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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 ...

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“…Collagenase is currently the most frequently used enzyme for preparing cell suspensions for cytogenetic analysis of solid tumors (12,19). Some reports on solid tumors, however, mention negative effects of collagenase treatment on FCM results, ranging from a poor yield (17) to a larger percentage of DNA diploid histograms (2,4,17). Here we describe our experiences with collagenase digestion and its effects on the ploidy of prostate tumor cells, investigated by FCM.…”

mentioning

confidence: 97%

Preferential loss of abnormal prostate carcinoma cells by collagenase treatment

König¹,

Dongen²,

Schröder³

1993

Cytometry

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Effects of two different methods of tumor disaggregation on flow cytometric ploidy distribution and intact cell yield were investigated. Either mechanical disaggregation or collagenase digestion was applied to 35 prostate tumor specimens. Seven collagenase-treated samples failed to yield any intact cells, whereas with mechanical disaggregation in all cases a sufficient number of intact cells were obtained. No differences in the FCM ploidy distribution of tumors with a DNA diploid stemline were observed comparing both techniques. In DNA aneuploid tumors, however, collagenase treatment had an adverse effect on the abnormal cell populations. In 14/17 of such tumors, the abnormal cell populations were significantly reduced; in eight of these the percentage of DNA aneuploid cells declined even below the minimum percentage (10%) that was defined for DNA aneuploidy. Since collagenase is a widely used enzyme for tissue disaggregation, especially in tumor cytogenetics, the presented data will have consequences for the interpretation of results obtained by methods involving the use of this enzyme.

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Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

Cited by 17 publications

References 15 publications

Cellular DNA Content and Proliferative Activity Evaluated by Flow Cytometry versus Histopathological and Staging Classifications in Human Bladder Tumors

Cellular DNA Content and Proliferative Activity Evaluated by Flow Cytometry versus Histopathological and Staging Classifications in Human Bladder Tumors

Guidelines for implementation of clinical DNA cytometry

Preferential loss of abnormal prostate carcinoma cells by collagenase treatment

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