Instruments for rapid cytophotometric analysis of morphologically identified clinical cell material.

Two instruments for cytophotometric analysis are described. The first is a generally applicable object stage scanning microspectrophotometer for the wavelength region 240-700 nm. The microscopic image is shown on a monitor screen and delineation of the analyzed objects is made with the aid of a light pen. The second instrument is a high speed microscope photometer specially adapted for the measurement of DNA, nuclear protein, and projected nuclear area following combined Feulgen and Naphthol Yellow S staining. A two-dimensional CCD sensor measures the light intensity in the microscopic image with a resolution of 0.3 micron. The objects are selected by the operator pointing the light pen at the image screen. The integrated absorbance at two different wavelengths, together with the projected area, is automatically measured for each object and presented in histogram form. Cells in clusters or sections can be measured under operator control with the help of the light pen. The time required for measuring 100 cells is 5-10 min in an ordinary preparation. The application of these instruments to the grading of cellular atypia and in the prognostic evaluation of malignant tumors is demonstrated on material from developing squamous bronchial carcinoma and in invasive mammary and prostatic adenocarcinoma.

Cytochemical changes in the nucleus during tumour development.

The general background to tumour analytic work using quantitative optical cytochemical methods is first presented. An instrument complex, constructed especially for multiparameter work in cytopathological material has been developed. Nuclear changes have been followed in cell populations during their development through different grades of atypia to cancer and conspicuous cytochemical changes were observed. In a comprehensive series of clinically verified mammary carcinomas, a large percentage of cases was found in which the DNA values were within the normal range, while the others showed pronounced aneuploidy. A clear correlation was found between DNA profile-type and patient survival, the latter of which reflects the degree of malignancy in the individual case. The shift from resting state (G0) to growth activated G1-stage is initiated by a large increase of the nuclear proteins. Mammary tumours of a high malignancy grade, as judged by their DNA profile type, showed an especially great accumulation of nuclear protein and thus a high degree of activation. DNA-profile measurements, preferably combined with determinations of nuclear proteins can thus be used for judging malignancy grades in mammary tumours, which is also of considerable clinical interest. An as yet limited observational material also indicates similar situations in some other types of tumour.

Morphometric analysis of neoplastic transformation in rodent fibroblast cell lines.

Populations from seven pairs of neoplastic and nonneoplastic cell lines of common origin were compared for evidence of a change in morphotogical parameters accompanying neoplastic transformation. Projected cytoplasmic area, projected nuclear area, cell dry mass, nuclear dry mass, whole-cell absorption at 265 nm, and nuclear absorption at 265 nm were determined. Neoplastic transformation was consistently accompanied by three morphological features: (a) decrease in projected area of the lamellar cytoplasm; (b) decrease in the projected area of the nucleus; and (c) decrease in dry mass of the lamellar cytoplasm. Ultraviolet absorption was generally less in the neoplastic cells than in the nonneoplastic cells, while nuclear mass remained approximately the same. These results are interpreted as quantitative evidence that neoplastic transformation is accompanied by morphological change. The morphological events may be characterized as a loss in cell surface and nuclear membrane components.

Quantitative studies of direct and indirect immunofluorescent staining of salmonella bacteria.

Salmonella bacteria were stained with serial dilutions of anti-Salmonella conjugates of different F/P ratios and the staining intensity was measured quantitatively in an ultramicrofluorometer. In any given dilution of the conjugates, a stronger fluorescence was obtained with the more highly labelled conjugates. The dependence of fluorescence intensity of F/P ratio varied with the dilution of the conjugate. Similar results were obtained by the indirect immunofluorescence method. In four bacterial systems the direct and indirect immunofluorescent staining methods were compared quantitatively. The indirect method was 5 to 30 times more sensitive than the direct comparing the last dilutions giving a positive reaction by visual observations. The standard deviation of the intensity values of the stained bacterial cells was between 10 and 40 per cent of the mean. Different sources of variation in the quantitative measurement technique are discussed.