Flow cytometric DNA content of fresh tumor specimens using keratin‐antibody as second stain for two‐parameter analysis

Abstract

Studies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium‐iodide for assessing DNA content and, on the other, a monoclonal keratinantibody for marking epithelial and tumor cells. Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid arid 146 (46%) as DNA “euploid.” By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (220) DNA euploid. The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%. In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA‐content assessment becomes more reliable.

Ploidy, expression of erbB1, erbB2, P53 and amplification of erbB1, erbB2 and erbB3 in non-small cell lung cancer

“…Stromal cells are seen as the optimal reference population [11]. By using anticytokeratin as a second stain for epithelial cells, the sensitivity of the interpretation of the histograms is markedly improved [11,12]. Most studies report an 80% prevalence of aneuploid tumours, which is in accordance with the present results [13,14].…”

Development of a Flow Cytometric Method to Determine DNA Ploidy of Oesophageal Cancer Cells Obtained by Forceps Biopsy Samples During Oesophago-Gastro-Duodenoscopy

“…As discussed above, selective destruction of aneuploid cells during preparation is unlikely. Undifferenti- ated tumours often loose specific cell antigens like cytokeratins [32]. Such undifferentiated tumours cells can not be discriminated from stromal cells, but there were no undifferentiated tumours in the study group.…”

Cell cycle analysis by flow cytometry: Principles and applications

“…Multiparametric analysis allows an increase in diagnostic sensitivity. Double labelling of DNA and cytokeratin (specific for epithelial cells) enables to lower the detection threshold of tumoural cells for epithelial malignant growth [66,157]. This is particularly useful to detect metastasis or for effusion analysis [91].…”