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Summary The method described herein for the quantitative evaluation of the amount of eu- and heterochromatin in a single interphase cell nucleus is based upon the measurement of optical density in Feulgen-stained nuclei with a scanning cytophotometer (UMSP I). The digitalized values for each grid point are entered via punched paper tape into a computer and processed therein. The position and optical density level of the grid points serve as criteria for the recognition of heterochromatin. The computer calculates the total DNA content of all such points, as well as the proportion of this heterochromatin DNA with respect to the entire DNA content of the cell nucleus. The heterochromatin contents so obtained agree with values obtained from a densitometric determination from photographs of cell nuclei. Agreement with cytogenetic experiments is also observed. The applicability of the method is demonstrated for the heterochromatin content of a variety of rodent species.

Introduction: Cervical cancer has a typical DNA distribution pattern, which is characterised by the appearence of atypical stemlines and DNA-values, that exceed the tetraploid range ( Atkin , Richards , and Ross , 1959 ; Sandritter und Fischer , 1962 ). Carcinoma in situ behaves itself like an invasive cancer with respect to its DNA-distribution-pattern ( Sandritter und Fischer , 1962 ; Steele , Manocha , and Stich , 1963 ; Caspersson , 1964 ). Dysplastic lesions of the cervix show polyploid DNA-values exceeding the tetraploid range ( Wied , Messina , and Rosenthal , 1966 ; Brandao , 1969 ; Hrushovetz , and Lauchlan , 1970 ). The appearence of aneuploid values in dysplastic cell populations is regarded as a special criterium of premalignancy ( Wagner , Sprenger und Blank , 1972 ; Sachs , Stegner und Bahnsen , 1972 ). The aim of this paper is to point out the possibilities of ascertaining cytological findings by DNA-cytophotometry. Material and Methods: 3 cases are described, which had suspicious Papanicolaou findings and which were then repeatedly controlled by cytophotometry. The conventionally fixed smears were stained according to Papanicolaou. The cytological diagnoses followed the principle of differential cytology ( Wagner , 1968). The histological diagnoses were done on cone biopsies. The Papanicolaou stained material was restained with acriflavine ( Bohm und Sprenger , 1968) for the DNA-determination. The Feulgen hydrolysis in hydrochloric acid eliminates the Papanicolaou dye. The automatic registering microphotometer based on the Leitz MPV I model ( Sprenger und Bohm , 1971 ) was used for single cell photometry. Prior to cytophotometry the acriflavine stained cells were identified cytomorphologically by phase contrast microscopy. The values of leucocytes and normal intermediate cells are used as a diploid standard. Results and Discussion: The first follow up case shows the progression of an intraepithelial precancerous lesion of the cervix with good agreement of cytological and cyto-photometric findings. The second observation shows the regression of an intraepithelial precancerous lesion agreeing with Papanicolaou and cytophotometric findings. The third observation documents the additional information obtained by cytophotometric DNA-determination in a case of repeatedly doubtful cytological findings. The 3 case reports denote the additional information of Feulgen-DNA-cytophotometry in cases with equivocal Papanicolaou cytology.

Bates and Cohen (1950 a, b) and jayle and Crepy (1952) have already reported on the microfluorometry of estrogens a few years ago. Fluorescence spectra were measured comparatively precisely and reported by Slaumwhite et al. (1953) and by Aitken and Preedy (1953). As for the limit of such measurements, Braunsberg et al. (1954) reported that a very minute amount could be detected.The present authors also reported on the fluorescence spectra of natural estrogens (Nakao and Aizawa, 1955) and described the method whereby the minimum amount could be determined.Numerous reports have appeared regarding the method of isolation of natural estrogens, such as the chromatography (Stimmel, 1944) and counter-current distribution (Engel et al., 1950) and more especially the microchromatography by Braunsberg et al. (1954).The present paper describes the separatory determination of estrogens in the blood and urine established by the writers.