Durchflußfluoreszenzzytophotometrisches Prescreening in der Zervixzytologie —Ein Methodenvergleich

Abstract

Introduction: Since the investigations of Sandritter et al. (1960) the DNA contents of the cell nucleus are regarded as a suitable, quantitative measuring unit for automatic prescreening in cervical cytology. The acriflavine Feulgen reaction ( Böhm and Sprenger, 1968 ; Sprenger et al., 1971 ) and the ethidium bromide dye ( Dittrich and Göhde, 1969 ) are both suitable as staining methods for the quantitative determination of nuclear DNA. Modifications of the ethidium bromide staining are obtanied by pepsin treatment prior to staining ( Berkhan, 1972 ; Sprenger et al., 1972 ) and by subsequent ultrasonic treatment ( Göhde et al., 1972 ; Sprenger et al., 1973 ). A self constructed prototype based on the Leitz MPV ( Sprenger et al., 1972 ) and the ICP 11, Fa. Phywe, Göttingen, are used as flow-through cytophotometers. The aim of this paper is to investigate, which one of the different preparations and photometers is best suited for the automatic prescreening in cervical cytology. Material and Methods: The smear material is taken by an Ayre spatula from the cervix and suspended in 0,9% saline. The suspended material is then washed twice in isotonic saline and within 5 hours fixed in absolute ethanol for at least 30 min. The smear material can be kept indefintely long in alcohol at 7°C. A control smears is Papanicolaou-stained. Acriflavine Feulgen staining: The cell scrapings are hydrolyzed in 2 N HCl at 28°C for 20 min. Acriflavine (Fa. Serva, Heidelberg) is used as a 0,01% Schiff solution according to Graumann (1953) without decolorisation with active carbon. The suspended cells are kept in the staining solution for 1 hour. Finally they are washed three times in acidified alcohol (3% HCl in 70% ethanol) and once in destilled water and then suspended in destilled water. Ethidium bromide staining: The cells are stained for 30 min. in a tris-buffer 0,001% ethidium bromide solution (Fa. Serva, Heidelberg). The pretreatment with pepsin is done in a solution of 0,2 g pepsin (1000 E/g) in 100 ml 0,2% HCl at 37°C for 15 min. The ultrasonic wave generator Sonifier B 12 (Fa. Bronson Sonic Power Co., Danbury, Connecticut) is operated with 60 W for 2,5 sec. The acriflavine or ethidium bromide stained suspensions is filtered two times through a meshtissue with 75 μ poresize in order to eliminate the larger aggregates. A self-constructed prototype or the ICP 11 from Fa. Phywe, Göttingen, are used as flow-through photometers. For fluorescence excitation of acriflavine and ethidium bromide the homogeneus glass filters BG 12 and BG 38 are used. Barrier filters are OG 590 for acriflavine and OG 550 for ethidium bromide. All filters Fa. Schott, Mainz, W.-Germany. In order to set instrument and determine the 2c position (diploid value) in the histograms, a suspension of bull sperms (1c) is used together with the acriflavine dye and a suspension of calf thymus lymphocytes (2c) with the ethidium bromide preparation. The evaluation of the flow-through cytophotometric histograms is calculated by a quotient which is given by the number of cells at the double distance of the 2c value divided by the number of cells at the 2c peak. Samples with a quotient ≥0,1 are regarded as flow-through cytophotometric positive, those with a quotient < 0,1 are classified as flow-through cytophotometric negative. A sample is not evaluable, when the total cell number in the 2c peak is less than 50% of the maximum cell number (1000 cells/canal). Results and Discussion: The self constructed prototype and the Phywe ICP 11 are equally suitable for flow-through cytophotometric measurements. Compared to the acriflavine Feulgen staining the ethidium bromide staining with pepsin pretreatment requires less time for preparation. A single cell suspension is realized by pepsin pretreatment. An insignificant loss of cells reduces the number of non evaluable samples. A diminished aggregate formation reduces the positive results. In contrast to acriflavine, the ethidium bromide staining with pepsin treatment yields a large number of false negative results, which are explained by the low relative frequency of atypical cells in a mixed population with normal cells. The false negative results of the ethidum bromide preparation can be probably diminished by a more detailed mathematical histogram analysis, which shall be capable to detect a small number of atypical cells. In acriflavine Feulgen stained preparations cell aggregates with numerous nuclei give high DNA readings, which imitate the augmented DNA contents of atypical cells. In such cases the histograms may be false positive. Cell sorting devices attached to flow-through cytophotometers may select cells with suspicious DNA contents. The well preserved cellular morphology of acriflavine preparation permits a recheck of the cells by the Papanicolaou method. A recheck of ethidium bromide preparations is impossible. The cellular morphology is heavely altered by the previous pepsin digestion.