Wittekind's hematological stain with pure azure B-eosin and its possible applications: a reply to Dr. Bins.
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Biomedical subjects
Publications and source records attributed to O Leder.
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In a case of spherocytosis combined with ulcus cruris, Doppler sonography and phlebography revealed a chronic venous insufficiency, together with fibrinous precipitates in capillary and venous vessel walls. Some haemolysis and fragmentation of red cells in the biopsy was also observed. It is suggested that the haemodynamic alteration due to chronic venous insufficiency may have played the main role in the development of the ulcers, although the rheological disturbance caused by the spherocytosis may have had an aggravating effect. Circulatory disorders should be excluded, if skin ulcers are to be classified as haematological disorders.
Hormone cytology smears have been classified using a standard method of graph theory. The Euclidean distance was chosen as a suitable measure of the dissimilarity between smears. Maximum cliques were defined as groups to which no further element can be added without invalidating the group-defining distance. Both the type and number of cells found in the smear influence the classification. The absolute number of cells appears to be especially appropriate for the discrimination of the different proliferation stages of vaginal epithelium. However, the elaboration of an adequate sampling procedure is necessary for obtaining smears from the lateral wall of the vagina, as well as for the improved preparation of the smears themselves.
A short-term safety study on the liver- and spleen-extract Factor AF2 has been carried out. Daily injections of 4 or 33.3 mg/kg body weight for six weeks were not followed by drug-induced changes.
The results of light and electron microscopy of 18 punch biopsies of sunburst varicosities are presented. Sunburst varicosities are widened cutaneous veins located within the superficial vessel net as well as within descending branches, often with an asymmetrically thickened wall. The thickened vessel walls contain collagenous and muscle fibers. Only a few elastic fibers have been detected, as opposed to numerous oxytalan fibers. Electron microscopy reveals an interfibrillar collagenous dysplasia, lattice collagen, and some matrix vesicles. The center of sunburst vessels is found at depths ranging from 175 to 382 microns below the stratum granulosum.
The standardized stain composed of pure azure B and eosin, as published by Wittekind and colleagues in 1986, demonstrated granules in neutrophilic leucocytes that were much coarser than those seen after staining with conventional Romanowsky-Giemsa methods. These granules belong to at least two classes. Their identification cannot be achieved by means of the morphologic characteristics of single granules; a multivariate analysis of the granulation as a whole, and a comparison with specifically stained primary granules is required. In particular, this study on unbiased cell samples showed that with Wittekind's method, the primary granules in peripheral neutrophils are stained. Further study of clinical smears revealed an enhanced dye uptake by the secondary granules. The staining behavior of the granules is related to the leukocyte count.
Adequate methods of sampling are by far the most neglected procedures in stereological work. This is true of both theory and practice. Indeed, it is often forgotten that Delesse not only introduced the stereological method, but also specified the necessary conditions for its use. To help make good this deficiency methods of stereological sampling are reviewed. In particular, the ratio estimate is discussed and examples are given. Estimation of the percentages of each cell class in populations of blood cells and cells from vaginal smears have problems in common. In most cases the bias of the ratio estimate is trivial. The formula for approximate calculation of the standard error of the ratio estimate requires more sampling units than most people realize. In addition, in artifical smears, a more even distribution of the cells is recommended.
The estimation of the composition of the vaginal smear of the cytological hormone diagnosis is given by a sampling method. The results show that the cells should be distributed in a more uniform way on the slide than it is the case within conventional smears.
Modern theory and experience question the reliability and suitability of sampling per judgment being the method for the evaluation of the percentage of cells in clinical cytology till now. For the reduction of this gap empirical studies were undertaken in order to receive proper estimates of blood smears. A simple method for the differential blood count is to take 30 or more strips of the width of the field of vision of the microscope perpendicular to the smearing direction as sampling units at random or by systemtaic selection with a random starting point. For the class of cells under consideration as well as for whole the cell population on the slide, the coefficient of variation of the mean of the cells within different strips has to be less than 0,1. Smears of a width of 10 mm instead of the usual 22 mm seems to be preferable. Under such conditions more than 1.000 cells are to be counted for a reliable estimate of the more frequent classes of cells. It is not supposed that this tedious method will be carried out by technicians but modern apparatus are presumed to give consistent estimates in such a way.
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Ca ions are highly cardiotoxic if their influx into the myocardial fibres becomes abundant. The intracellular Ca overload initiates a deleterious high-energy phosphate deficiency by excessive activation of Ca-dependent intracellular ATPases and by impairing the phosphorylating capacity of mitochondria. This Ca-induced high-energy phosphate exhaustion is a crucial point in the etiology of the myocardial fibre necroses produced in rats by large doses of beta-adrenergic catecholamines, particularly isoproterenol, or by a number of other cardiotoxic agents. Accordinly, the myocardium is sensitized to necrotization by factors which favour Ca overload (dihydrotachysterol, 9alpha-flourocortisol acetate, NaH2PO4). Conversely, the structural integrity of the hearts can be protected by any substance or procedure which prevents an excessive intracellular Ca accumulation, particularly by inhibitors of the transmembrane Ca influx, such as verapamil, D 600 or prenylamine.
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Skeletal and heart muscle fibers undergo severe functional and structural alterations, resulting in necrotization as soon as extracellular Ca ions penetrate excessively into the sarcoplasm, so that the capacities of the Ca binding or extrusion processes become insufficient. In mechanically injured skeletal muscle fibers, this necrotization process begins in the neighborhood of the membrane lesion where a large Ca inward transport takes place. Accordingly, elimination of Ca from the Ringer solution or an outward electric current which blocks the influx of extracellular Ca prevents the onset of necrotization, whereas additional Ca or an inward electric current which augments the influx of Ca potentiates the course of degradation. The crucial reaction in the production of necroses in skeletal and heart muscle fibers is a high energy phosphate deficiency which results (a) from excessive activation of Ca-dependent intracellular ATPases, and (b) from Ca-induced mitochondrial destruction. This applies especially to myocardial fiber damage caused by large doses of beta-adrenergic catecholamines such as isoproterenol. The number and size of the isoproterenol-induced cardiac lesions are obviously determined by the extent and, particularly, by the duration of the Ca-mediated high energy phosphate penury. Substances which sensitize the myocardium to catecholamine-induced necrotization (9-alpha-fluorocortisol, dihydrotachysterol, NaH2PO4) act by potentiating intracellular Ca overload and high energy phosphate breakdown. Conversely, verapamil D 600, and other Ca-antagonistic compounds protect the structural integrity of the heart muscle fibers by restricting transmembrane Ca influx and, consequently, ATP and creatine phosphate exhaustion.
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The crucial point in the pathogenesis of isoproterenol-induced myocardial necrotization is an abundant intracellular Ca accumulation leading to high energy phosphate exhaustion. Accordingly, in the early stage of the isoproterenol-induced necrotization process, the onset of ATP and creatine phosphate breakdown strictly parallels the acute Ca gain. In this type of necrosis, the Mg losses from the myocardium appear as a concomitant phenomenon. The hearts can be protected against the deleterious Ca overload and necrotization by increasing the plasma concentration of Mg, K, or H ions in order to counterbalance Ca according to the ration (see article). On the other hand, if Mg, K, or H ion concentrations are too low, isoproterenol-induced Ca uptake and myocardial lesions are potentiated.