E. Sprenger

Fluorescence cytophotometry: a valuable method for the quantitative determination of nuclear feulgen-DNA

SummaryA cytofluorometric apparatus with incident illumination for fluorescence excitation is described here.Cytophotometric fluorescence measurements (UV- and blue excitation) of acriflavine-acridine yellow-, coriphosphine- and pararosaniline-Schiff stained di-, tetra- and octoploid liver nuclei, leucocytes and sperms (Feulgen reaction) were found to agree with the absorbance data obtained from the same slide by means of the integrating microdensitometer.The stoichiometry of the fluorescence emission is discussed in detail. It is emphasized that not a linear, but an exponential relationship exists between the emitted fluorescence intensity and the concentration of the fluorescent substance.Cytofluorometry of Feulgen-stained nuclei has proved to be as reliable and fast as the absorbance scanning measurements at the intergrating microdensitometer.

Proportionalitätsfehler bei der Feulgen-Hydrolyse

SummaryComparing the Feulgen dye-content of different nuclei (liver cells, lymphocytes, granulocytes and sperms) after alcohol-fixation deviations were found between the measured Feulgen values and the DNA-content to be expected from the DNA-constancy law. The Feulgen values of lymphocytes and granulocytes proved to be lower (up to minus 20%) than those of diploid liver nuclei at all hydrolysis times, while in the postmaximal range of hydrolysis the values of the haploid sperms were too high (even higher than those of the diploid nuclei). Such differences did not appear when nuclei of the same cell type in different DNA- ploidy classes (liver nuclei) were compared.Those deviations of leucocytes and sperms were no longer found after fixation in methanol-formalin-glacial acetic acid and, in addition, could not be confirmed by UV-absorption measurements. For that reason we suppose them to be due to proportionality errors caused by variations in the hydrolytic behaviour of the different nucleoproteins.Thus fixation in methanol-formalin-glacial acetic acid seems to be more suitable for quantitative Feulgen measurements than alcohol-fixation, which may easily give rise to proportionality errors during Feulgen hydrolysis.Moreover, to avoid any false interpretation of Feulgen data we should suggest controll measurements using another independent method (f. e. UV-absorption), or — if this is impossible — to check the Feulgen values after different fixations and variant hydrolysis times (premaximal, maximal, postmaximal).ZusammenfassungBeim Vergleich des Feulgen-Farbgehaltes verschiedener Zellkerne (Leberzellen, Lymphozyten, Granulozyten und Spermien) traten nach Alkoholfixierung Abweichungen der gemessenen Feulgen-Werte von den nach dem Gesetz von der DNS- Konstanz zu erwartenden DNS-Gehalt dieser Kerne auf. Verglichen mit den Feulgen-Werten der diploiden Leberzellkerne ergaben Lympho- und Granulozyten bei allen Hydrolysezeiten zu niedrige (bis zu minus 20%), haploide Spermien im postmaximalen Hydrolysebereich zu hohe Feulgen-Werte (z. T. sogar höhere Werte als die diploiden Zellkerne). Innerhalb desselben Zelltypes wurden dagegen, auch beim Vergleich der verschiedenen Ploidiestufen der Leberkerne, keine Differenzen festgestellt.Da die an Leukozyten und Spermien beobachteten Abweichungen nach Methanol-Formalin-Eisessig-Fixierung nicht mehr auftraten und auch durch UV-absorptionscytophotometrische Messungen nicht bestätigt werden konnten, muß man annehmen, daß es sich um Proportionalitätsfehler handelt, die auf Hydrolyseunterschieden beruhen.Für die quantitative Feulgen-Cytophotometrie scheint daher die Methanol-Formalin-Eisessig-Fixierung besser geeignet zu sein als die Alkoholfixierung, bei deren Verwendung es leicht zu Proportionalitätsfehlern während der Feulgen-Hydrolyse kommen kann.

Durchflußfluorescenzcytophotometrie für ultraschnelle DNS-Messungen an großen Zellpopulationen

SummaryA flow-through cytophotometer based upon the Leitz-microscope photometer (MPV) is described. The light source is a high pressure Xenon arc HBO 100 in an arrangement for epiillumination.The flow system is designed in such a way that each cell, after previous Feulgenstaining in aqueous suspension, has to pass through the focal plane of the objective. This is achieved by a self-constructed flow channel with a vertical fluid stram carrying the stained cells along the optical axis of the microscope at a flow rate of 100 to 1000 cells per sec. After passage through the focal plane with simultanneous registration of the highest fluorescence reading the cells are flushed off by a horizontal water current.The fluorescence light impulses emitted by the cells are classified and stored by an impulse height discriminator, from which they may be recalled either analogically as a histogram (DNA distribution pattern) or digitally as a series of numbers.In addition, the fluorescence impulses can be visualized at the screen of an oscillograph. The flow rate of the fluorochromized cells is determined by means of a counter unit.The steps of preparation of the cell suspension as well as fixation, hydrolysis and Feulgen-staining with acriflavine-Schiff of the suspended tissue cells is described in detail.The possibilities of application of the instrument as a cyto-morphometric approach to cytology and cytochemistry automation are discussed.ZusammenfassungAusgehend vom Leitz-Mikroskopphotometer MPV mit Auflicht-Anregung wird ein Durchflußfluorescenzcytophotometer beschrieben, das es gestattet, die relativen Feulgen-DNS-Mengen von bis zu 1000 Zellen pro Sekunde zu bestimmen. Die Meßwerte werden in einem Impulshöhenanalysator klassifiziert und gespeichert. Auf Abruf wird der Speicherinhalt analog als Histogramm ausgeschrieben oder digital als Zahlenfolge ausgedruckt.Das Vorgehen bei der Zellsuspensionsherstellung, Fixierung, Hydrolyse und Färbung mit dem Feulgen-Farbstoff Akriflavin wird dargelegt.Anwendungsbeispiele werden besprochen.

Die Aussagekraft der Zellkern-DNS-Bestimmung bei der Diagnostik des Prostatakarzinoms

Abstract Introduction Punch biopsies and fine needle aspiration biopsies are of great interest in the differential diagnosis of benign and malignant changes of the prostate. The aim of this paper is to decide whether the determination of the nuclear DNA contents is suitable to differentiate between prostatic cancer and benign changes of the prostate. Material and Methods The material was obtained by punch biopsy of the prostate. Two imprint preparations were made from the tissue cylinder, were stained according to Papanicolaou and examined cytologically. The remaining material was embedded in paraffin, stained with hematoxylin-eosin and examined histologically. The Papanicolaou stained preparations were subjected to Feulgen hydrolysis and restained in acriflavine for the fluorescence cytophotometric nuclear DNA measurement ( Bohm and Sprenger , 1968 ). The Feulgen hydrolysis removes the Papanicolaou stain. The individual cell photometric measurements of the nuclear DNA were carried out on an automatically registering fluorescence cytophotometer ( Sprenger and Bohm , 1971a ). Punch biopsies from 32 patients were examined. Histologically there were 6 cases with benign hyperplasia, 13 cases with hyperplasia and chronic prostatitis, 10 cases with adenocarcinoma and 3 carcinomas of the anaplastic type. The nuclear DNA of a random population from the two imprint preparation was cytophotometrically determined and summarized in a histogram. Leukocytes and morphologically normal columnar cells were measured as a reference population to obtain the 2c value (DNA contents of a diploid chromosome set). The measured DNA distribution pattern in the random populations was characterized mathematically by: 1. The relative mean ploidy value (U) in the random population. FE = fluorescence units 2. The relative frequency of euploid and polyploid DNA values (Z) in the random population. N = number of measured cells The nuclear DNA content is defined as euploid or polyploid (x) when it fits the mean diploid value ± 25% or its multiples ± 25%. All cells out of this range x are defined as aneuploid and called y. An analysis of variance shows whether the U- and Z-values of the histological groups differ significantly from each other. Results and Discussion The histograms of the cases with benign hyperplasia of the prostate have a slender diploid peak as it is typical for benign cell populations. If a prostatitis complicates the hyperplasia the histogram shows a small number of cells in the tetraploid region. The DNA distribution pattern of carcinomas yields DNA values spread out to the 8c range. Benign lesions have diploid stemlines. The stemlines of the tumor cases are diploid, polypoid, aneuploid or not clearly recognizable. The analysis of variance of the U- and Z-values between hyperplasia with or without chronic prostatitis yields no significant differences. Between adenocarcinomas and anaplastic carcinomas of the prostate there are also no significant differences of the U- and Z-values. Therefore cases of hyperplasia with and without chronic prostatitis form one group and are opposed to a group comprising adenocarcinomas and anaplastic carcinomas. The analysis of variance of the U-values shows highly significant differences between the group of carcinomas and the hyperplasia group. The Z-values of the two groups differ also significantly. One case with an adenocarcinoma of the prostate was treated with estrogen. The DNA histogram revealed only a small number of cell nuclei with a DNA content higher than diploid. This type of a histogram may be interpreted as a beginning regression of the atypical cell population. Tumor cell populations and benign cells of the prostate can be distinguished by the cytophotometric determination of nuclear DNA.

The significance of DNA flow-through fluorescence cytophotometry for the diagnosis of prostate carcinoma.

Flow-through fluorescence cytophotometric determination of nuclear DNA content was employed for the diagnosis of prostate carcinoma. Fine needle aspiration biopsy material from the prostate of 220 patients was used for study. A false negative rate of 11.4% and a false positive rate of 29.7% were obtained when the results of flow-through photometry were compared with those of traditional cytodiagnosis. It was found that 4.5% of the specimens were unsuitable for cytologic diagnosis and 10.9% for flow-through cytophotometry. False negative DNA histograms may be due to two factors: either the number of tumor cells is small or there are tumor cells whose nuclear DNA content does not differ from that of a normal cell population. False positive findings result from proliferating cells in inflammatory activation. Errors in preparation of the material and mechanical mistakes, such as cellular clumping and coincidences, are less likely causes. The greater percentage of specimens which were inadequate for cytophotometry was due to the large number of cells needed for a utilizable flow-through photometric histogram. The high rate of false negative and false positive results (11.4% and 29.7%, respectively) argues against using flow-through photometric nuclear DNA determination for the diagnosis of prostate carcinoma.

Fluoreszenzzytophotometrische Bestimmung der Zellkern-DNS

SummaryAcriflavine-Feulgen (Böhm und Sprenger, 1968) and ethidiumbromide fluorescence after previous pepsin digestion (Göhde et al., 1971) yield corresponding DNA distribution patterns when applied to liver smears. With the time-consuming acriflavine staining (220 min) the cellular morphology is best preserved and the stained specimen may be stored for long periods.The rapidly obtainable ethidiumbromide staining (70 min) results in a selective fluorescence of DNA only after heavy alteration of the cellular morphology by pepsin digestion. Storage of the material is only possible for two days. The heavily altered cellular morphology, however, is rather unfavorable for prescreening in automated cytology. Specimens that were found to be suspicious are no longer suitable for conventional cytological diagnosis, because they have lost their cytoplasm and nuclear chromatin structure.ZusammenfassungDie Akriflavin-Feulgen-Färbung (Böhm und Sprenger, 1968) und die Ethidiumbromid-Fluorochromierung nach vorausgehender Pepsinbehandlung (Göhde et al., 1971) ergeben an Leberzellausstrich-Präparaten weitgehend gleichartige DNS-Häufigkeitsverteilungen. Bei der zeitlich aufwendigen (220 min) Akriflavin-Färbung ist eine optimale Darstellung der Zellmorphologie und eine jahrelange Archivierung der Ausstrichpräparate möglich.Die rasch durchführbare Ethidiumbromid-Färbung (70 min) bietet nur nach schwerer Alteration der Zellmorphologie durch die vorausgehende Pepsinbehandlung eine selektive Darstellung der DNS. Eine Archivierung von Ausstrichpräparaten ist nur für 2 Tage möglich. Die schwere Alteration der Zellmorphologie ist besonders bei durchflußphotometrischen Prescreeninguntersuchungen in der gynäkologischen Krebsvorsorge nachteilig. Zellpopulationen, die zu einer zytophotometrischen Verdachtsdiagnose geführt haben, sind durch Verlust des Zytoplasmas und der Kernstruktur einer konventionellen morphologischen Diagnostik nicht mehr zugängig.

The diagnosis of endometrial carcinoma by means of jet wash and DNA flow-through cytophotometry.

Flow-through cytophotometric determination of nuclear DNA content on jet wash material from the endometrium was employed in the diagnosis of endometrial carcinoma. One hundred and thirty cases were studied. In comparison to results from cytodiagnosis, flow-through photometry yielded a false negative rate of 31.6% and a false positive rate of 42.2%. The false negative findings resulted in part from the small relative frequency of atypical cells in a mixed population of normal and atypical cells in certain cases. Besides this, we often found carcinomas with a diploid DNA stem line, which could not be distinguished cytophotometrically from normal corpus endometrium (Sandritter, 1952; Atkin et al., 1959; Hustin, 1976). The flow-through photometrically false positive findings may have resulted either from cell aggregates or from a nuclear DNA content elevated over the diploid value in proliferating cells (D. Wagner et al., 1968; D. Wagner and Richard, 1968). The observed false negative and false positive rates demonstrate that flow-through photometric determination of nuclear DNA content is unsuitable for the diagnosis of endometrial carcinoma.

The mathematical evaluation of flow-through cytophotometric data in processing cervical cytology.

Summary The flow-through cytophotometric determination of cell nuclear DNA content should be employed as a cervical cytological prescreening procedure. The goal of a mathematical evaluation of measured results is the fewest possible number of false negative findings consistent with an acceptably small number of false positive findings which must be redone. The statistical method for deciding whether an individual belongs to one of two possible groups is discriminant analysis. The difference between each of eight, selected percentiles and the diploid peak were employed as parameters for the frequency distribution. Comparison of the numerical results of discriminant analysis to simultaneous determinations after Papanicolaou resulted in an empirically obtained critical value for the classification of the discriminant values. The application of this process results in rates of 33% false positive findings and 5% false negative findings.

Absorbance and Fluorescence Cytophotometry of Nuclear Feulgen DNA. A Comparative Study

Fluorescence cytophotometric measurements of intracellular substances, either those with native fluorescence or those treated with appropriate fluorescence staining are based upon physical laws that are different from those of absorbance cytophotometry.

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 ( Bohm and Sprenger , 1968 ; Sprenger et al., 1971 ) and the ethidium bromide dye ( Dittrich and Gohde , 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 ( Gohde 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, Gottingen, 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, Gottingen, 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 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.