W. Sandritter

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.

Autopsy and clinical diagnosis.

In 1096 cases of death (autopsy rate 63.8%) the accuracy of clinical diagnoses was investigated by comparing clinical diagnoses with recorded autopsy findings. -- In 81.3% of the cases the primary disease had been determined correctly. In more than half of these cases the immediate cause of death or an additional disease contributing to death had not been correctly identified. In 16% of the cases the diagnosis proved to be inadequate. -- In 2.6% of all cases the primary disease, cause of death and accompanying illnesses were misdiagnosed. Most of these patients had stayed in the hospital for a much shorter time than the rest of the patients. -- Among conditions clinically diagnosed as cirrhosis of the liver, pulmonary embolism, myocardial infarction, cerebral hemorrhage, and malignant tumors -- pulmonary embolism was by far the most frequent condition to go unrecognized, i.e. in 50% of th cases in which it was present. Primary liver cell carcinoma proved to be the malignant tumor most frequently not identified by clinical studies. -- Four clinical diagnoses (shock, septicemia, diabetes mellitus and uremia) were often unsupported by morphological findings. Yet there were 13 clinically undiagnosed cases of septicemia in which findings at post mortem examination revealed this condition. These cases also underline the importance of autopsies.

The pulmonary air-blood barrier of human shock lungs (a clinical, ultrastructural and morphometric study).

Interstitial edema in the alveolar septa is the first morphologically recognisable change to be observed in cases of shock. It is brought about by the altered function of the membranes of the damaged epithelium and endothelium in the alveolar wall. At the same time there is an impairment of gaseous exchange, which is rendered more difficult by the exudative process in the interstitium. Pari passu with these events there is injury to the cells of both the alveolar epithelium and the alveolar capillary endothelium. Both these processes are still reversible. The point of irreversibility appears to be reached--so far as time is concerned--at the end of the first week, after which the injurious effects on the cell are established, since the thin alveolar wall necessary for the exchange of gases becomes overgrown with bulky alveocytes (Tpye II), and the fibroblasts in thealveolar interstitium push the capillaries away from the surface of the alveolus. In most of the advanced cases of shock this process of thickening of the alveolar wall exceeds the critical value, and respiratory exchange is so impaired that satisfactory functioning of the lungs is no longer possible.

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.

DNA in Heterochromatin Cytophotometric Pattern Recognition Image Analysis among Cell Nuclei in Duct Epithelium and in Carcinoma of the Human Breast

Summary Feulgen-stained normal breast epithelium (4 cases) and breast carcinoma (8 cases) were analyzed by means of cytophotometric scanning measurements. Besides the DNA content, DNA concentration, and area of the cell nucleus, the number and absolute as well as relative amount of condensed chromatin was determined. In addition, the DNA concentration, area, and form of the condensed chromatin was evaluated. It was demonstrated that nuclear DNA is elevated (near tetraploid stemlines) in carcinoma cells, the DNA concentration is more highly variable, and that the elevated DNA amounts are largely attributable to elevated nuclear area values. The number of “heterochromatic” particles is more variable in tumor cell nuclei than in normal cell nuclei. Significantly more chromocenters were demonstrable in only one case. The absolute DNA content in condensed chromatin of carcinoma cells is also highly variable (significantly elevated only in a single case). The most important result of the foregoing investigation seems to be that the percentage DNA content in condensed chromatin is greatly decreased in all tumors (6%) as compared to normal cell nuclei (13%). Regarding the form of the condensed chromatin particles, the particles ares more spherical in tumor cells as contrasted to more pointed in normal cells. The results are discussed in relationship to biochemical determinations of heterochromatin.

DNA and the cell cycle.

The cell cycle and its significance for pathology is reviewed. The molecular basis and questions of regulation in the cell cycle are discussed, along with deviations from the normal cycle in the form of polyploidy formation and aneuploidy in tumor cells. Automated prescreening processes may play a role in the future in daily routine diagnosis. Methods of cytophotometry with the possibility for objective pattern recognition will become significant in pathology in many ways, both in research and in routine work.

CEA positivity in sera and breast tumor tissues obtained from the same patients

It is well known that the concentration of serum CEA correlates best with colorectal carcinomas but less well with other solid tumors. The aim of the present study was to elucidate further the poor relationship between confirmed primary mammary carcinomas and CEA serum levels, 108 of these tumors have hence been studied for their tissular CEA positivity and the results obtained were compared with the CEA serum values found preoperatively in the same patients. Techniques employed were the indirect immunoperoxydase method for the histologic studies and the enzymoimmuno-assay for the serum measurements. The discordance and concordance of the CEA results obtained with the two techniques were analyzed in respect to the histologic types of tumors classified following the ICD-O nomenclature. Our results seem to prove that: 1) although most tumors produce the antigen only a fraction of them releases it, and 2) the most common histologic type of tumor is the one secreting the antigen the less frequently. These observations are apparently independant of the tumor stages T1-T4, N- or N+, M-.

Initiation of DNA synthesis in a system of synchronized L-cells: effect of actinomycin D.

SummarySpecific biochemical events in the mitotic cycle of cells can be investigated in synchronous cell cultures. In the work presented here, synchronization was accomplished by mechanical selection of cells in mitosis. This method produces a cell population of 95% mitotic cells, representing an undisturbed physiological system, where factors can be investigated which initiate DNA synthesis. With the aid of Actinomycin D, a drug known to inhibit DNA-dependent RNA synthesis the problem was investigated whether specific transcription in the G1-phase or S-phase is necessary for the initiation and maintenance of DNA synthesis. Permanent contact of Actinomycin D with synchronous cell cultures leads to a total block of DNA synthesis, when administered in the G1-phase, and a decrease in the rate of DNA synthesis when applied during the S-phase. Pulses of 2 hours of Actinomycin at different times of the mitotic cycle lead to a diminished rate of DNA synthesis. These experiments are suggestive of a specific, temporally limited transcription during the G1-phase necessary for the initiation of DNA synthesis. Apparently the information for the synthesis of enzymes is produced during this period of the life cycle.The morphology of Actinomycin D-treated cells is characterized by two different features: the nucleoli assume a round, compact shape and appear to be split into a multitude of smaller granular subunits. The chromatin also undergoes a shift towards a fine granulation.

Morphologic development of human shock lung.

In the initial phase of shock, edema spreads throughout the alveolar interstitium even before injury occurs in the alveolar epithelium and endothelium. The endothelium and the epithelium are damaged only subsequently, causing reduction in the average barrier thickness of the epithelium and the endothelium. A point of irreversibility is reached by the end of the first week. While early cell regeneration may be observed within the alveolar endothelium and epithelium, proliferation of fibroblasts and fibrosis of the alveolar wall occur in addition to edema which spreads within the interstitium. This widening of the gas exchange barrier may be considered as the anatomic substrate of respiratory insufficiency induced by shock. This enlargement continues up to the moment when thickening of the alveoli impedes satisfactory functioning of the lung, and, as consequence, threatens the life of the patient.

Einfluß einer einstündigen Autolyse auf die quantitative Zytoarchitektur der Rattenleberzelle (Eine ultrastrukturell-morphometrische Studie)

Abstract Introduction Autolysis is very often a reason for cell damage and is also superimposed on many other cell damages. Already one hour of autolysis causes serious changes in cell metabolism, which can be demonstrated morphometrically. Material and Methods Experiments were made with 10 male adult Wistar rats. 5 animals were for control purposes and 5 animals had to undergo a 1-hours autolysis. The morphometric analysis of the liver parenchymal cells was based on the information of Weibel et al. (1968) with the help of a computer program. Results and discussion The consequence of the drop in energy caused by autolysis is an enlargement of the liver cell which is possibly due to a breakdown of the energy-dependent ionic pumps. One of the earliest observable cell changes is a so-called “Kern- wandhyperchromatosis” and chromatin condensation within the nuclei, whereby the Kernwandhyperchromatosis is seen to be a direct consequence of the increase in lactate and decrease of pH. ATP-sufficiency causes a disturbed function of mitochondrial membranes. The mitochondria are swollen, the number of mitochondrial grana is clearly reduced. An enlargement of the mitochondrial outer membrane takes place by folding while the surface of mitochondrial cristae remains unchanged. As a consequence of the altered membrane activities also the peroxisomes swell at reduced numerial density. At unchanged total volume of RER the surface of the granulated membranes of the RER decrease by 50%. This decrease caused by ribosome detachment of the granulated membranes corresponds to the enlargement of the degranulated membrane parts of the endoplasmic reticulum. The vesiculation is caused by an unspecific damage of cytoplasm. While the density of its volume and the membrane surface remain unaltered, the SER also shows a tendency to small vesiculation caused by an unspecific damage of cytoplasm. The increase in number and volume of the lysosomes and vacuoles of unknown origin speaks for a lysosomal activity. The cell compartment responsible for protein synthesis shows the most impressive morphometric and morphologic changes, which eventually can be explained by a decrease of protein synthesis which is needed to obtain enough energy for a well operating physiological equilibrium.