Flow cytometric monitoring of electronic nuclear volume in combination with DNA content can be used for refining the histopathological grading of human tumors.

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Nuclear size and chromatin texture are important parameters in histopathological grading of human breast tumors such as the ductal carcinoma in situ (DCIS). Most of the histopathological grading of tumors is based on examination of cells under a microscope and thus is qualitative rather than quantitative. In NPE Quanta flow cytometer (NPE Systems Inc, Pembroke Pines, FL), electronic nuclear volume can be measured rapidly (Cytometry 43: 2-22, 2001) and in combination with determination of DNA content could be used for grading of human tumors. Breast tumor biopsies were obtained from the Cooperative Human Tissue Network (CHTN), Birmingham, AL. A small piece of the tissue was minced in DAPI staining solution (3 μg/ml in phosphate buffered saline containing 0.1% nonidet-40). Trout nucleated red blood cells (TRBC) were used as internal controls. Samples were filtered thru a 40-micron nylon cloth before analysis in a NPE Quanta flow cytometer for nuclear volume and DNA content. In normal breast tissues, nuclei with diploid DNA content had electronic volume similar to that of the TRBC used as controls. In some of the DCIS samples, diploid and tetraploid cells had similar electronic nuclear volume while in some other DCIS samples, the tetraploid nuclei were larger with volume two to three time larger than that of the diploid cells. In contrast in some of the DCIS tumors with hexaploid sub-populations, the hexaploid nuclei were relatively smaller with mean volume twice that of the diploid nuclei. These observations show the large amount of variation in nuclear volume of cells isolated from DCIS samples. Our data suggests that use of electronic Coulter volume in combination with other cellular parameters such as DNA content or hormone receptor expression could be a useful tool for refining the grading of human tumors. Supported by Department of Army, Army Medical Research and Material Command, Contract grant number: DAMD-17-00-1-0342.