bluuebunny/crossref_metadata_2025 huggingface dataset:
ULTRASTRUCTURAL AUTORADIOGRAPHIC DETECTION OF INTRACELLULAR IMMUNOGLOBULINS WITH IODINATED Fab FRAGMENTS OF ANTIBODY THE COMBINED USE OF ULTRASTRUCTURAL AUTORADIOGRAPHY AND PEROXIDASE CYTOCHEMISTRY FOR THE DETECTION OF TWO ANTIGENS (DOUBLE LABELING)
Authors: GONATAS NICHOLAS K. , STIEBER ANNA , GONATAS JACQUELINE , GAMBETTI PIERLUIGI , ANTOINE JEAN CLAUDE , AVRAMEAS STRATIS
Published: April 2011

Intracellular immunoglobulins of paraformaldehyde-fixed rat lymph node cells were identified with ultrastructural autoradiography of 125I Fab of sheep antirat Ig antibody. We observed excellent intracellular penetration and specific localization of the iodinated Fab fragments of the antirat Ig antibody. In contrast, 125I sheep antirat IgG or conjugates of peroxidase and Fab-antibody fragments did not show satisfactory intracellular penetration. Simultaneous detection of plasma membrane and intracellular immunoglobulins has been achieved with the use of iodinated Fab of sheep antirat Ig and peroxidase-labeled Fab of sheep antirat Ig and subsequent processing of the cells for cytochemical (peroxidase) and autoradiographic study. The simultaneous demonstration of specific (antiperoxidase) antibody and Ig molecules is also feasible.
ULTRASTRUCTURAL STUDY OF MEMBRANE-BOUND ENZYMES IN THYMOCYTES
Authors: MONNERON ARIANE , BENICHOU JEAN-CLAUDE , PASTEUR INSTITUT , FLORENTIN YVETTE , GUERRY ELIANE
Published: April 2011

Calf thymocytes in suspension, as well as isolated calf thymocyte nuclei, were incubated in the presence of several phosphorylated substrates. 5'-Nucleotidase was easily detected on the plasma membrane of thymocytes (external side), but could be demonstrated on isolated nuclei only to a small extent. No other substrates were detectably hydrolyzed by isolated nuclei except adenosine triphosphate and 3'-thymidine monophosphate. The surface of whole cells was found to be much more reactive. A 3'-nucleotidase activity was shown to occur on the plasma membrane of a number of thymocytes and produced large lead phosphate deposits, some of them protruding into the cytoplasm. Enzymic activities splitting β-nicotinamide adenine dinucleotide phosphate and uridine diphosphate glucose were also readily detectable on the surface of cells. Since the pattern of the lead phosphate deposits and the number of reactive cells varied with the added substrate, and since cells were compared with their isolated nuclei, the positive reactions were considered to indicate the presence (on the exposed membranes) of the corresponding enzymes on the exposed membranes.
A MORE SENSITIVE AND SPECIFIC HISTOCHEMICAL PEROXIDASE STAIN FOR THE LOCALIZATION OF CELLULAR ANTIGEN BY THE ENZYME-ANTIBODY CONJUGATE METHOD
Authors: WEIR E. EARL , PRETLOW THOMAS G. , PITTS ANNETTE , WILLIAMS EDWIN E.
Published: April 2011

In an attempt to increase the sensitivity and specificity of immunohistochemical procedures using peroxidase-labeled antibody, we have modified the technique. This modification takes advantage of the pH optimum of horseradish peroxidase. The best buffer salts, hydrogen peroxide concentration, time of staining, fixation after incubation with labeled antibody and counterstains were determined empirically. Compounds similar to the substrate are known to undergo photooxidation, and specificity was enhanced by keeping the solutions in the dark.
PROBLEMS OF SPECIFICITY IN THE USE OF A STRONTIUM CAPTURE TECHNIQUE FOR THE CYTOCHEMICAL LOCALIZATION OF OUABAIN-SENSITIVE, POTASSIUM-DEPENDENT PHOSPHATASE IN MAMMALIAN RENAL TUBULES
Authors: FIRTH J. A.
Published: April 2011

The p-nitrophenyl phosphate-strontium procedure for the localization of the phosphatase component of Na-K-activated adenosine triphosphatase was evaluated using rat renal cortex as a test tissue. The results obtained by light microscopy were unexpected in that reaction product was found only on the brush borders of proximal tubule cells; this reaction was ouabain-resistant, K-independent and partially Mg-dependent, but could be completely inhibited by l-tetramisole. Electron microscopy showed that a reaction was also present on the cytoplasmic surfaces of the lateral and basal plasma membranes of the proximal and distal tubule cells. That seen in the distal tubule was sensitive to ouabain but not to l-tetramisole, whereas that in the proximal tubule showed a mixture of ouabain-sensitive and l-tetramisole-sensitive components. It is concluded that the procedure as originally described is not specific, demonstrating alkaline phosphatase as well as Na-K-adenosine triphosphatase, but that this problem may be overcome by the use of an alkaline phosphatase inhibitor.
A NEW METHOD FOR THE INVESTIGATION OF THE CAPTURE REACTION IN PHOSPHATASE CYTOCHEMISTRY III. EFFECTS OF THE COMPOSITION OF THE INCUBATION MEDIUM ON THE TRAPPING OF PHOSPHATE IONS IN A MODEL SYSTEM
Authors: CORNELISSE C. J. , VAN DUIJN P.
Published: April 2011

A model system developed for the study of diffusion problems in lead salt enzyme cytochemistry served as a basis for the experiments reported in the present paper. Phosphate leakage, which was investigated in polyacrylamide films during incubation in media containing lead, could be expressed by a graphical parameter related to the average displacement of the phosphate ions prior to the onset of lead phosphate precipitation in the films. This parameter can be used as a measure for the efficiency of the capture reaction in cytochemical reactions in tissue sections. The concentration of lead ions, and to a lesser extent that of the phosphate ions, was found to be critical for the degree of phosphate diffusion in this system. Addition of 0.3% of β-glycerophosphate to the lead medium gave increased phosphate diffusion, and trapping was markedly improved by added chloride ions. Raising of the molarity of the acetate buffer resulted in impaired trapping efficiency. Similar findings were made in a parallel study on the kinetics of lead phosphate precipitation in supersaturated solutions with a nephelometric method. The results of the present investigation indicate that the film model system is highly suitable for the study of factors affecting the rapid immobilization of diffusible products of cytochemical reactions.
LOCALIZATION OF LYSOZYME IN NORMAL RAT TISSUES BY AN IMMUNOPEROXIDASE METHOD
Authors: KLOCKARS MATTI , OSSERMAN ELLIOTT F.
Published: April 2011

The immoperoxidase-immunoglobulin bridge technique has been utilized for the localization of lysozyme (LZM) in the cells and tissue of the normal rat. Specific LZM staining was found in the proximal tubules of the kidney, Paneth cells of the small intestine and alveolar macrophages. LZM staining was also demonstrated in macrophages in the perifollicular sinusoids of an activated lymph node. The immunoperoxidase method appears to have significant advantages over previously described methods for LZM localization, and it should also be adaptable to electron microscopic studies.
RESOLUTION IN AUTORADIOGRAPHY USING SEMITHIN SECTIONS
Authors: SALPETER MIRIAM M. , BUDD G. C. , MATTIMOE SHARON
Published: April 2011

Resolution studies were performed on light microscope autoradiographic specimens of 0.5-1 µ thicknesses coated with Ilford L4 and Kodak NTB2 and AR10 emulsions. A radioactive line source, previously employed for electron microscope autoradiography (Salpeter, Bachmann and Salpeter, 1969; Salpeter and Salpeter, 1971), was used. It was found that with tritium labeling the half-distance (HD) was approximately 3500 Å. This value was not significantly altered with increasing section beyond 0.5 µ or emulsion thickness beyond ~2,000 Å. A 0.5-µ section labeled with 14C had an HD value of 7000 Å when coated with a monolayer of Ilford L4 or Kodak NTB2 emulsion. Increasing section or emulsion thickness did increase this value. It was found that the density distributions, normalized in units of their own HD, gave a good fit to the set of universal curves derived from electron microscope autoradiographic specimens. These universal curves are thus applicable for analyzing light microscope autoradiographic specimens. Some tritium HD values applicable to high voltage microscopy are also given.
EFFECTS OF ALDEHYDE FIXATION AND OF PREGANGLIONIC DENERVATION ON ACETYLCHOLINESTERASE AND BUTYROCHOLINESTERASE OF CAT AUTONOMIC GANGLIA
Authors: KOELLE GEORGE B. , DAVIS RICHARD , KOELLE WINIFRED A.
Published: April 2011

Fixation of cat stellate ganglia by perfusion in situ with cold 4% formaldehyde in Krebs-Ringer solution, followed by immersion in the same solution for 3 or 6 hr, produced inactivation of approximately one-half the acetylcholinesterase (AChE) and butyrocholinesterase (BuChE); after 21 hr, further inactivation occurred. Similar fixation with glutaraldehyde (1, 2 and 4%) inactivated over three-quarters of the AChE and BuChE at 3 hr, but there was little further loss after 21 hr. Following sectioning of the preganglionic trunk, the AChE activity of the cat superior cervical ganglion fell to approximately 20%, and BuChE fell to approximately 50%; this confirms an early report by Sawyer and Hollinshead, which has been questioned in part because of subsequent histochemical findings. The basis for the discrepancy is discussed, along with its bearing on the interpretation of histochemical observations in general.
IMMUNOCYTOCHEMICAL OBSERVATIONS ON HYPERSENSITIVITY SKIN REACTIONS TO HORSERADISH PEROXIDASE AND TO ANTIHORSERADISH PEROXIDASE γ-GLOBULIN
Authors: STRAUS WERNER
Published: April 2011

Delayed skin reactions were produced in rabbits with relatively low serum antibody titers by a single injection of a small amount of horseradish peroxidase (HRP) or of rat anti-HRP γ-globulin in complete Freund's adjuvant into the footpads. One to 2 days after skin testing, the reaction for the specific antibody was observed in the delayed skin lesions at the following locations: on the surface of lymphocytes and monocytes (macrophages); in the perinuclear region of small and medium sized lymphocytes; in blast cells; and in cytoplasmic granules of basophil leukocytes. The possible relationship of these antibody locations to delayed type or basophil cutaneous hypersensitivity was discussed. In mixed Arthus-type-delayed type hypersensitivity skin reactions to HRP in animals with high serum antibody titers, a different location of the antibody was observed. In confirmation of earlier data, the reaction occurred in cytoplasmic granules of macrophages and polymorphonuclear leukocytes and in association with collagen fibers, and antigen-antibody complexes accumulated especially in the walls and areas surrounding blood vessels. These localizations of the antibody were considered to be characteristic for Arthus-type hypersensitivity. When rabbits were double immunized against HRP and egg albumin, the reaction for (cytophilic) antibody to HRP on the surface of monocytes, lymphocytes and granulocytes in the upper dermis was as strong in the skin lesion elicited by egg albumin as in that elicited in the same animal by HRP. Lymphocytes with perinuclear antibody appeared in mixed Arthus-type-delayed type hypersensitivity skin reactions to HRP 1-2 days after skin testing but many plasma cells were seen only after 4-5 days in most cases. Observations are also reported on: the persistence of the antigens (HRP and rat anti-HRP γ-globulin) in the skin lesions; the acid phosphatase reaction in cells infiltrating the skin lesions; and cell contact, especially between lymphocytes and macrophages.
HISTOCHEMICAL DEMONSTRATION OF (Na+-K+)-ACTIVATED ADENOSINE TRIPHOSPHATASE
Authors: GUTH LLOYD , ALBERS R. WAYNE
Published: April 2011

Rats were perfused with a l2% solution of polyvinyl pyrrolidone (pH 7.25). Frozen sections were prepared from spinal cord and kidney and incubated under conditions that are known to be optimal for demonstrating the p-nitrophenylphosphatase activity of (Na+-K+)-adenosine triphosphatase (ATPase). After incubation, the sections were treated sequentially with cobalt chloride and ammonium sulfide to visualize the reaction product. The reaction product was localized perferentially to the neuropil of the spinal cord and to the distal tubules of the kidney. In addition to the histologic localization, the following observations indicate that the histochemical reaction is specific for demonstrating (Na+-K+)-ATPase activity: (a) the conditions that are optimal for the p-nitrophenylphosphatase activity of purified (Na+-K+)-ATPase were also optimal for our histochemical method; (b) the histochemical reaction was inhibited by ouabain; and (c) the p-nitrophenylphosphatase reaction occurred under conditions that inhibit alkaline phosphatase activity. An additional advantage of this procedure is that it employs a perfusion solution which, although it contains no fixative, preserves the integrity of the tissue after freezing to a degree suitable for light microscopic examination.
ELECTROPHORESIS OF ELASTASE-LIKE ESTERASES FROM HUMAN NEUTROPHILS
Authors: SWEETMAN F. , ORNSTEIN L.
Published: April 2011

Multiple esterases from individual leukocyte types of normal adult human beings have been demonstrated after separation by cationic disc gel electrophoresis. Two substrates, l-naphthyl N-acetyl-dl-alanine (NAcAla) and l-naphthyl butyrate, were used routinely in a simultaneous coupling azo dye method to demonstrate the esterases. Cytochemically, the neutrophil granules stain very poorly with α-unsubstituted carboxylic acid esters such as 1-naphthyl butyrate or 1-naphthyl acetate but stain selectively and intensely at pH 7.0 with two classes of naphthol esters; α-halo-substituted esters such as 1-naphthyl 2-bromobutyrate or naphthol AS-D chloroacetate or α-amino-substituted carboxylic acid esters such as l-naphthyl N-acetyl-dl-alanine or l-naphthyl N-acetyl-l-alanyl-l-alanyl-l-alanine. Cationic zymograms showed three major neutrophil esterases which hydrolyze NAcAla and l-naphthyl N-acetyl-l-alanyl-l-alanyl-l-alanine vigorously and l-naphthyl butyrate very poorly, which provided us with the first evidence that these enzymes might be proteolytic. Cationic zymograms of a mixture of purified enzymes (reference enzymes), pancreatic elastase, chymotrypsin and trypsin, were compared to zymograms of neutrophil extracts after assaying with NAcAla. The neutrophil esterases and elastase hydrolyzed NAcA1a vigorously; chymotrypsin and trypsin hydrolyzed it moderately. More precise characterization of the neutrophil esterases was obtained with chloromethyl ketone inhibitors which have the advantage of being irreversible and also can be synthesized with a substrate-like moiety which confers specificity. Neutrophil extracts and the mixture of reference enzymes were preincubated before electrophoresis with either N-acetyl-l-alanyl-l-alanyl-l-alanine chloromethyl ketone (NAcAla3CK), N-acetyl-l-alanyl-l-phenylalanine chloromethyl ketone (NAcAlaPheCK) or N-tosyl-l-lysine chloromethyl ketone (TLysCK). Elastase was selectively inhibited by NAcAla3CK, chymotrypsin by NAcAlaPheCK and trypsin by TLysCK. Neutrophil "lysosomal" and neutrophil-enriched cell extracts which were treated before electrophoresis with the chloromethyl ketone inhibitors at the same concentrations as used for the reference enzymes were almost completely inhibited by the NAcAla3CK except for one enzyme fraction. This fraction was inhibited by NAcAla3CK when neutrophil preparations were electrophoresed and then exposed to the inhibitor before assaying for activity. The neutrophil enzymes were much less sensitive to the TLysCK and NAcAlaPheCK. The evidence from these studies strongly supports the conclusion that the major esterases in human neutrophil granules are elastase-like esterases.
ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF PROLACTIN IN RAT ANTERIOR PITUITARY BY USE OF THE UNLABELED ANTIBODY ENZYME METHOD
Authors: PARSONS JONATHAN A. , ERLANDSEN STANLEY L.
Published: April 2011

The National Institute of Arthritis, Metabolism and Digestive Diseases antirat prolactin antiserum, used widely for radioimmunoassay of rat prolactin, was used in the unlabeled antibody-peroxidase-antiperoxidase complex method for the ultrastructural immunocytochemical staining of rat prolactin cells. Sections were exposed to antiprolactin antiserum (1:400-1:100,000) or to antiovine luteinizing hormone antiserum (1:1,000) for 3 min or 4 or 48 hr. Specificity of prolactin staining was demonstrated by the absence of immunologic staining of other pituitary cells and by negative results with prolactin cells when normal serum or antiovine luteinizing hormone antiserum was used. Intensity of prolactin staining was directly related to the concentration and duration of exposure to antiprolactin antiserum. Prolactin secretion granules, located peripherally or within Golgi regions, were uniformly stained. Immunologic staining for prolactin was not observed within cisternae of the endoplasmic reticulum. These results show that the National Institute of Arthritis, Metabolism and Digestive Diseases antirat prolactin antiserum is specific for prolactin by ultrastructural immunocytochemical criteria and that antiserum dilutions appropriate for radioimmunoassay can be used in immunologic localization studies.
THE INFLUENCE OF CHANGING CELL KINETICS ON FUNCTIONAL DIFFERENTIATION IN THE SMALL INTESTINE OF THE RAT A STUDY OF ENZYMES INVOLVED IN CARBOHYDRATE METABOLISM
Authors: DE BOTH N. J. , PLAISIER H.
Published: April 2011

The activity of a number of enzymes involved in carbohydrate metabolism was measured in different cellular compartments of the intestinal epithelium by microchemical techniques. The enzyme activities were related to different cell positions along crypt and villus and to cell age. Enzyme activities in proliferating, differentiating and functional cell compartments of the intestine of normal rats were compared with those in which the cell kinetics had been modified. The effect of increased proliferative activity within the crypt of normal animals was studied in the intestine during recovery after low radiation doses. The effect of increased life-span was investigated in germ-free animals. The specific activity of α-glucosidase, present in microvilli, was found to increase considerably during cell differentiation and subsequent cell migration along the villus. Its specific activity remained unchanged in isolated intestinal loops deprived of dissaccharide substrate for 6 weeks. Lactate dehydrogenase and, to a lesser extent, glucose 6-phosphate dehydrogenase show a similar pattern of activity to α-glucosidase. In contrast hexokinase and isocitrate dehydrogenase were equally distributed among crypt and villus cells and no clear differences were observed with increasing cell age. Increased proliferation in the crypts during recovery after low radiation doses resulted in a marked decrease in both crypt and villus cells of activity of α-glucosidase. The germ-free state of the intestine also significantly influences the pattern of α-glucosidase but has little influence on the other enzymes tested.
ULTRASTRUCTURAL IMMUNOCYTOCHEMICAL LOCALIZATION OF LYSOZYME IN THE PANETH CELLS OF MAN
Authors: ERLANDSEN STANLEY L. , PARSONS JONATHAN A. , TAYLOR THOMAS D.
Published: April 2011

Human lysozyme was localized immunocytochemically at the ultrastructural level within Paneth cells of man by use of the unlabeled antibody enzyme method. Specific staining for lysozyme was observed over secretion granules in the apical cytoplasm, within the region of the Golgi apparatus and within some, but not all, lysosomes. No staining was observed within the cisternae of the rough endoplasmic reticulum or other cellular organelles. In control experiments comparing semiadjacent sections of the same Paneth cell, substitution of either normal rabbit serum for rabbit antihuman lysozyme antiserum (specificity control) or normal sheep serum for sheep antirabbit immunoglobulin G antiserum (method control) completely eliminated specific staining for lysozyme. The intensity of staining for lysozyme was related to both the titer and length of exposure to antilysozyme antiserum. Specific staining was obtained in tissue embedded in Araldite or Epon and was facilitated by etching with hydrogen peroxide. No staining was observed after prolonged fixation in glutaraldehyde or treatment with uranyl acetate in block.
HISTOCHEMICAL PROPERTIES OF THE ANTRAL GASTRIN CELL
Authors: LARSSON L.-I. , SUNDLER F. , HÅKANSON R. , GRIMELIUS L. , REHFELD J. F. , STADIL F.
Published: April 2011

The antropyloric mucosa of rat, rabbit, cat, pig and man was investigated using certain modifications of the original Falck-Hillarp procedure. In rabbit, cat, pig and man, combined formaldehyde-ozone treatment induced intense fluorescence in a large number of epithelial cells. By subsequent treatment of the sections for immunohistochemical detection of gastrin (immunofluorescence or immunoperoxidase labeling), it could be shown that these cells were identical with the gastrin cells. The gastrin cells of cat and man could be visualized also by treatment with formaldehyde-HCl, a feature not shared with the gastrin cells of rabbit and pig. The gastrin cells in the rat antrum gave no fluorescence with either formaldehyde-ozone or formaldehyde-HCl. From histochemical model experiments and cytospectrofluorometric analyses it appears probable that the formaldehyde-ozone-induced fluorescence reflects the presence of a peptide with NH2-terminal tryptophan. The chemical background of the fluorescence induced by formaldehyde-HCl treatment is as yet obscure. The gastrin cells of all animals studied stained with silver in the Grimelius method. Argentaffin and diazo staining as well as silver staining according to Hellerström and Hellman were carried out on sections from human antrum only. The gastrin cells were negative with these stains, but they stained with lead-hematoxylin. These properties support the view that the gastrin cells are identical with the so-called G cells.
ACRIDINE ORANGE STAINING OF EPITHELIAL CELLS IN STRONG SALT SOLUTION
Authors: ADAMS LAWRENCE R.
Published: April 2011

Epithelial cells may be stained with acridine orange in strong solutions of salt to emit red-orange cytoplasmic fluorescence and green nuclear fluorescence under excitation with blue illumination.
FLUORESCENCE SPECTROSCOPIC AND FADING BEHAVIOR OF EHRLICH'S HYPERDIPLOID MOUSE ASCITES TUMOR CELLS SUPRAVITALLY STAINED WITH ACRIDINE ORANGE
Authors: GOLDEN JAMES F. , WEST SEYMOUR S.
Published: April 2011

Corrected fluorescence emission spectra and measurements of fluorescence intensity and fluorescence fading were recorded from Ehrlich's hyperdiploid mouse ascites (EHD) tumor cells supravitally stained with acridine orange (AO). After equilibration of the staining reaction, some EHD tumor cells with dye content greater than 5 x 10–15 mole AO/cell spectroscopically exhibit both red and green fluorescence while the rest fluoresce only green. This behavior contrasts with that previously observed for normal mouse leukocytes. Below 5 x 10–15 mole AO/cell, all EHD tumor cells fluoresce green, but the intensity varies considerably from cell to cell. The fading rate of the green fluorescence appears to be a linear function of fluorescence-exciting light intensity. Absolute light intensity measurements, a necessity for interexperiment comparisons and automation, were based upon a phosphor particle standard. The results presented are indicative of the possibilities for automated cytopathology that can be uncovered by rapid recording, corrected spectrum microspectrofluorophotometry and related biophysical cytochemical techniques.
ERYTHROCYTE MORPHOLOGY AND CENTRIFUGAL "SPINNER" BLOOD FILM PREPARATIONS
Authors: BACUS JAMES W.
Published: April 2011

The centrifugal "spinner" blood film preparation technique was investigated with respect to the preparation of blood films with the retention of conventional erythrocyte morphology. It was determined that the initial spinning produced a monolayer blood film while the duration of spinning affected the separation of the red cells in the monolayer and altered their individual morphologic characteristics. The spinner preparation resulted in a slide with the total surface similar in morphologic characteristics to one thin slice, or cross-sectional area, across a typical wedge preparation. The spinning time in effect determined the position of the cross-section. In many series of slides from the same blood, spun at increasing time intervals, at least one slide in each series contained erythrocytes whose morphology matched the "best" area of the corresponding wedge slide. The effects of red cell concentration and plasma viscosity were varied experimentally, to assess independently the effect of each on monolayer formation and morphology. The results indicated that it was necessary to spin for a longer time to overcome the viscosity effects due to the red cell concentration and also those due to plasma. Two evaluation criteria, "separation" and "central pallor development," were shown to produce different spinning time estimates. These differences were more pronounced at higher blood viscosities than at lower blood viscosities.
COLOR INFORMATION IN BLOOD CELLS
Authors: CHENG GEORGE C.
Published: April 2011

Two hundred color spectra of blood cells treated with Wright-Giemsa stain were measured by using an Olympus microspectrophotometer. The wavelength of the light source was varied from 400 to 700 nm. Several basic pictorial regions in the blood smear were investigated. These regions included the background, basophil, eosinophil, neutrophil, lymphocyte, monocyte and the red blood cell. A subset of the color spectrum curves was used as references. The remaining spectrum curves were used as testing samples and were shown to individuals who had no prior knowledge of leukocyte color information. All of the persons tested could correctly identify the testing samples with the reference samples. This experiment showed that color information in blood cells is not only useful but also sufficient for leukocyte differentiation for the samples that have been investigated.
PREPARATION OF CLINICAL GYNECOLOGIC SPECIMENS FOR AUTOMATED ANALYSIS AN OVERVIEW
Authors: WHEELESS LEON L. , ONDERDONK M. A.
Published: April 2011

This paper surveys the problems and attempted solutions for preparation of human gynecologic cytology specimens for automated analysis. Topics discussed include: (a) specimen collection, (b) mucus elimination, (c) cell dispersal, (d) specimen filtration and (e) artifact.
BLOOD GRANULOCYTE STAINING WITH ACRIDINE ORANGE CHANGES WITH INFECTION
Authors: MELAMED M. R. , TRAGANOS F. , ADAMS L. R. , KAMENTSKY L. A.
Published: April 2011

Peripheral blood granulocytes from patients with infections take up acridine orange supravitally to produce a greater range of elicitable red cytoplasmic fluorescence per cell than is obtained when infection is absent. It is suggested that this may be another indication of leukocyte mobilization in infection, although the mechanism involved is not yet understood.
THE DESIGN, OPERATION AND EVALUATION OF A HIGH SPEED AUTOMATIC METAPHASE FINDER
Authors: GREEN DARYLL K. , NEURATH PETER W.
Published: April 2011

An automatic television microscope coupled to a PDP 9 computer is described which detects metaphase cells on slides stained by one of several generally used staining procedures. It determines the presence of a candidate cell by the number of chromosomes on a first pass. On a second pass it selects suitable cells by the size, eccentricity and radius of gyration of the distribution of the events. The finding speed is about 1 cm2/min for the first pass and about 6 sec/cell selected. The quality of the cells selected was checked by cytogeneticists in two laboratories. Depending on the criteria used, 10-50% of the cells selected were found to be of good quality, useful for manual or machine analysis. It was concluded that a finder of this performance could substantially advance the practice of manual chromosome analysis and would be essential for computerized systems of rapid karyotyping.
METAPHASE SPREAD DETECTION AND FOCUS USING CLOSED CIRCUIT TELEVISION
Authors: JOHNSON ELBERT T. , GOFORTH LAWRENCE J.
Published: April 2011

Automatic focus and detection of metaphase chromosome spreads have been implemented in an automated light microscope system and performance has been evaluated. The video signal from a standard closed circuit television camera is used to generate focus quality and spread presence parameters that are input to a computer. The computer, through its operating software, controls lateral and vertical motion of the microscope stage to search the slide for metaphase chromosome spreads and to focus them. The video signal processing required to generate the parameters, its implementation and the operating software are described. The evaluation of spread detection performance emphasizes the need for well prepared slides with clean backgrounds. The performance of two methods of obtaining a focus parameter is compared. The first method involves measuring the high spatial frequency content of the image; the second method uses the distribution of optical density within the image.
COMPUTER-BASED ELECTRONIC CELL VOLUME ANALYSIS WITH THE AMAC II TRANSDUCER
Authors: THOMAS R. A. , CAMERON B. F. , LEIF R. C.
Published: April 2011

The separation of the sensing electrodes from the power electrodes, which is the central concept of the electronic cell volume parameter of the AMAC transducers, has been demonstrated to prevent artifacts due to electronic rise time, bubble formation and changes in the suspending medium. The use of a laminar flow transducer (designed as a full bridge circuit) and a low noise amplifier produced pulses with high signal to noise ratios and spectra with low coefficients of variation. Data acquisition and analysis with a digital computer along with graphics output are discussed. The superiority of the constant voltage bridge and transformation equations for this circuit are presented. A method of characterizing the shape factor for nonspherical particles is derived and applied to human erythrocytes. Preliminary capacitance measurements of erythrocytes and leukocytes are discussed. The resolution of the AMAC II system is demonstrated, with homogeneous polystyrene latex spheres and normal human erythrocytes. Data demonstrating an increased size heterogeneity in a human hemoglobinopathy (sickle cell anemia) is presented. The usefulness of electronic cell volume analysis by the AMAC II transducer in monitoring the separation of cells has been demonstrated with a bovine serum albumin neutral density separation of irreversibly sickled cells from nonirreversibly sickled cells.
CELL CYCLE ANALYSIS FROM COMPUTER SYNTHESIS OF DEOXYRIBONUCLEIC ACID HISTOGRAMS
Authors: GRAY JOE W.
Published: April 2011

A method is presented for the computer synthesis of deoxyribonucleic acid (DNA) histograms, based on estimates of cell distribution around a life cycle, DNA synthesis rate and instrumentally introduced dispersion. Synthetic histograms are compared with experimental DNA histograms for two cell systems; in one, the cell population is in asynchronous exponential growth (steady state), in the other, the cell population is recovering from a block at the G1/S phase interface (nonsteady state). The average phase transit times and the associated dispersions are determined, using a DNA histogram synthesis technique, for the nonsteady state population.
A MICROSPECTROFLUOROMETER WITH EPI-ILLUMINATION OPERATED UNDER COMPUTER CONTROL
Authors: PLOEM J. S. , DE STERKE J. A. , BONNET J. , WASMUND H.
Published: April 2011

Until recently the recording of fully corrected excitation and fluorescence spectra from fluorophores in microscopic preparations has been a time-consuming procedure. A new type of microspectrofluorometer with epi-illumination has been developed. This instrument is operated on line with a digital computer permitting automatic operation of monochromator motors, electrical shutters and correction of spectra. The electrical shutters are used for a rapid change between low intensity dia- and strong epi-illumination. Exposure to epi-illumination is limited to the short period of recording the spectra. A new optical system permits centering of structures to be measured, using the binocular head for viewing the illuminated image of the monochromator slit and the fluorescent image excited with dia-illumination simultaneously. New objectives and eyepieces with a high light collecting power permit the visualization of weak fluorescence obtained with low intensity dia-illumination.
AUTOMATED CLASSIFICATION OF NORMAL AND ABNORMAL LEUKOCYTES
Authors: BRENNER J. F. , NEURATH P. W. , SELLES W. D. , NECHELES T. F. , GELSEMA E. S. , VASTOLA E.
Published: April 2011

The development of an automated system for counting and classifying normal and abnormal leukocytes in peripheral blood smears is described. General requirements are discussed and the results of a simulation experiment are presented. A sample of 1572 leukocytes, divided equally among 17 types, was photographed and analyzed using computerized pattern recognition techniques. Various geometrical, color and texture parameters were extracted from the cell images and an optimal set of 20 were used in several computerized classification runs. Training on one-half of the sample and classifying the other half resulted in an over-all correct classification of between 67 and 77% depending on the definition of classification error. When only normal cells are considered, correct classification is obtained for 9l.5% of the cells.
HEMATOLOGIC AUTOMATION BY USE OF CONTINUOUS FLOW SYSTEMS
Authors: SAUNDERS A. M. , SCOTT F.
Published: April 2011

The principle of continuous flow analysis, originally employed for chemistry tests, has been adapted for hematologic procedures. Instruments are available for cell counting and hematocrit determination and, more recently, for white blood cell differential counts. By decreasing chance for human error, these instruments increase the accuracy of routine tests. Automation also helps ease the burden of an ever-increasing workload.
THE HEMALOG D WHITE CELL DIFFERENTIAL SYSTEM
Authors: MANSBERG H. P. , SAUNDERS ALEX M. , GRONER W.
Published: April 2011

The Hemalog D system performs automated differential white cell counts utilizing principles of cytochemistry, electro-optical measurement and signal logic processing. Using 0.4 ml of anticoagulated whole blood and an automated continuous flow staining technique, the system is designed to process a new sample each minute. The decision logic classifies individual cells by size and intensity of staining as they flow through detectors designed to measure light loss and light scattering simultaneously. With this system, 10,000 cells/ sample are classified in less than 1 min. Alcian Blue identifies basophils when used in the presence of quaternary ammonium salts and other counterions. Monocytes are the only cells stained in an esterase method using α-naphthol butyrate at pH 6.1 with hexazonium pararosanilin as coupler. This unstable diazonium is produced on stream continuously from stable components. Size and peroxidase staining serve to classify the remaining cells. Lymphocytes and large mononuclear cells are unstained, neutrophils have moderate peroxidase activity, while eosinophils stain very strongly. Threshold detection logic for light scattering and absorption differentiate the neutrophils, eosinophils and lymphocytes. The distribution of peroxidase activity in neutrophils serves as an index of maturity of these cells. The clinical evaluation of this index is reported elsewhere. Results are reported as percentage and as cells per cubic millimeter. A total white blood cell count is also reported and the pattern of cell measurements for 10,000 cells is provided as a picture with diagnostic information content. Advantages of this system in rapid screening result from improved precision of automation, the processing of large numbers of cells and the saving of labor. Precision also makes the acquisition of sequential data on single patients more meaningful and diagnostically valuable.
HIGH RESOLUTION SHADING CORRECTION
Authors: SCHULTZ MORTON L. , LIPKIN LEWIS E. , WADE MARTA J. , LEMKIN PETER F. , CARMAN GEORGE M.
Published: April 2011

Quantitative cytology requires accurate representation of a specimen's optical densities. As the requirements for measurement precision increase, instrument-induced errors become increasingly more difficult to reduce to the point at which their effect on experimental data is insignificant compared to the measured parameters. Shading induces a significant amount of amplitude ambiguity to data obtained from a scanning system. A method of shading correction on single pixels is introduced as a new way to reduce some errors that currently plague scanning systems.
AUTOMATED AUTORADIOGRAPHIC GRAIN COUNTING IN HUMAN DETERMINED CONTEXT
Authors: LIPKIN LEWIS E. , LEMKIN PETER , CARMAN GEORGE
Published: April 2011

An automated autoradiographic grain counter which dichotomizes results into nuclear and background categories for multiple human determined classes of Romanowsky-stained cells has been constructed. Additional instrument capabilities and functions are indicated as applicable to appropriately prepared material. The operation of the instrument is summarized. Its performance characteristics and its potential role in general quantitative cytology, cytokinetics research and cancer chemotherapy will be evaluated by projected field trial instruments.
SIMULTANEOUS DIFFERENTIAL STAINING BY A CATIONIC CARBOCYANINE DYE OF NUCLEIC ACIDS, PROTEINS AND CONJUGATED PROTEINS II. CARBOHYDRATE AND SULFATED CARBOHYDRATE-CONTAINING PROTEINS
Authors: GREEN MARIE R. , PASTEWKA JULLIA V.
Published: April 2011

The cationic carbocyanine dye l-ethyl-2-[3-(l-ethylnaphtho[l,2d]thiazolin-2-ylidene)-2-methylprolpenyl]-naphtho[1, 2d]thiazolium bromide stains several classes of macromolecules differentially in histologic sections. Most proteins are red at pH 4.3 and pink or unstained at pH 2.8. The caseins are blue at pH 4.3 and unstained at pH 2.8. Nuclei stain purple, mast cells stain red-purple, cartilage stains purple and mucoproteins stain blue-green at both hydrogen ion concentrations. The nature of some of the macromolecules involved in these color reactions has been determined by the use of chemical and enzymatic procedures and by staining films of glycosaminoglycuronoglycans, proteins and conjugated proteins. The method requires less than 1 hr staining time for most tissues; the stain is stable when kept in the dark and has the advantage of distinguishing several macromolecules in tissues simultaneously. The simplicity of the method and the ability to discriminate classes of macromolecules in tissues should make this stain valuable in pathologic diagnosis.
THE UNLABELED ANTIBODY ENZYME METHOD OF IMMUNOCYTOCHEMISTRY QUANTITATIVE COMPARISON OF SENSITIVITIES WITH AND WITHOUT PEROXIDASE-ANTIPEROXIDASE COMPLEX
Authors: PETRALI JOHN P. , HINTON DENNIS M. , MORIARTY GWEN C. , STERNBERGER LUDWIG A.
Published: April 2011

Araldite sections of rat pituitary intermediate lobe were used with anti-17-39 adrenocorticotropin in the unlabeled antibody enzyme method to compare electron microscopic immunocytochemical staining by peroxidase-antiperoxidase complex (PAP) with antiserum or purified antibody to peroxidase followed by peroxidase (PO). Quantitation of normalized optical densities of secretory granules offered high significance comparison (P < 0.0001) of experimental with control values and of experimental values with each other. Use of purified antibody (prepared by a new density gradient method) yielded four times higher sensitivity than antiserum to PO, while a 6.5-fold increase would have been expected from the degree of contamination of anti-PO in the serum by nonanti-PO immunoglobulin. Use of PAP was four to five times more sensitive than purified anti-PO and 20 times more sensitive than antiserum to PO. The increased sensitivity of PAP is explained by the high over-all binding affinity of PO for anti-PO in the cyclic PAP molecule, thus preventing the losses of PO that occur during washing when anti-PO and PO have been applied in sequence. Identification of the characteristic, cyclic PAP molecules affords confirmation of specific staining at high resolution. In the sequential application of anti-PO and PO, no PAP molecules are formed, thus making distinction of specific from nonspecific deposition of enzyme reaction products ambiguous. With the use of anti-17-39 ACTH and the intermediate lobe, the unlabeled antibody enzyme method was 16,000-100,000 times more sensitive than radioimmunoassay.
5'-NUCLEOTIDASE AND ACID PHOSPHATASE OF SPINAL CORD QUANTITATIVE HISTOCHEMISTRY IN CAT AND MOUSE SPINAL CORDS AND IN MOUSE BRAIN
Authors: SURAN ANITA A.
Published: April 2011

Measurements of 5'-nucleotidase in mouse spinal cord showed that enzymic activity was unequally distributed, being twice as high in dorsal gray as in ventral gray matter, and that white matter had still lower activity. Similar determinations with cat spinal cord revealed no corresponding regional difference in 5'-nucleotidase activity in the gray matter and, again, a lower activity in white matter. In cat spinal cord the enzyme activities were lower than in corresponding areas of mouse cord. Essentially all of the 5'-nucleotidase activity was associated with particulate fractions of spinal cords of both species as well as with sedimentable proteins from mouse brain. Phosphatase activity in fresh mouse brain homogenates was much higher in the supernatant than in the particulate fraction; however, no appreciable phosphatase activity could be detected in either freshly homogenized or lyophilized spinal cord sections obtained from cat or mouse. The effects of some histochemical reagents and inhibitors on both enzymic activities were also measured.
LOCALIZATION OF CARBONIC ANHYDRASE IN AVIAN GASTRIC MUCOSA, SHELL GLAND AND BONE BY IMMUNOHISTOCHEMISTRY
Authors: GAY CAROL V. , SCHRAER HARALD , FALESKI EDWARD J. , SCHRAER ROSEMARY
Published: April 2011

Antisera to purified avian erythrocyte carbonic anhydrase (CA) were produced in rabbits and the γ-globulin fractions were isolated and purified by ammonium sulfate precipitation and diethylaminoethyl-Sephadex chromatography. The anti-CA immuno-γ-globulins were tested by immunoelectrophoresis and judged to be highly specific for CA. Fluorescein isothiocyanate-conjugated goat antirabbit γ-globulins were used in the indirect fluorescent antibody localization of CA in cryostat sections. Specific fluorescence of CA was observed in gastric mucosal lining cells, shell gland columnar and tubular gland cells, red blood cells, erythroblasts and osteoclasts. Specific fluorescence was absent in the several immunologic controls, including the blocking test. Specific fluorescence was also lacking in tissue constituents which contain no detectable amounts of CA, i.e., muscle, connective tissue, blood vessels and nuclei. A significant finding in this study is the occurrence of cross-reactions between antibodies to red blood cell CA and CA from other tissues, indicating the immunologic similarity of CA from different tissue sources.
VISUALIZATION OF DEOXYRIBONUCLEIC ACID MOLECULES BY PROTEIN FILM ADSORPTION AND TANTALUM-TUNGSTEN SHADOWING
Authors: ABERMANN REINHARD , SALPETER MIRIAM M.
Published: April 2011

Preparatory procedures and high resolution shadowing of deoxyribonucleic acid (DNA) molecules for electron microscopy are described. They involve a two-stage mounting of the DNA on cytochrome c monolayers and shadowing with tantalum-tungsten. Conditions are determined that produce a smooth substrate and reproducible binding of the DNA strands in a randomly coiled manner without obvious stretching artifacts. When limiting factors such as excess buffer salts are eliminated, the DNA strands have the over-all dimensions expected from the naked molecule.
PROPANOIC ACID-FERRIC OXIDE HYDROSOLS DIFFERENTIAL CELL SURFACE BINDING AND ITS RELATION TO MEMBRANE LIPID
Authors: COOPER GEORGE W. , MILLER LOUIS H.
Published: April 2011

Acetic acid-ferric oxide hydrosol was bound as a monolayer of particles on the outer surface of plasma membranes of various cells. Substitution of propanoic for acetic acid produced novel binding patterns, the aggregation of colloid at focal sites being independent of in situ negative surface charge. This pattern was observed on some cells (leukocytes, platelets, malaria-infected erythrocytes and malaria parasites) but not on others (normal erythrocytes) and on regional areas of certain cells (i.e., midpiece of sperm). Alteration of the aliphatic portion of lipids abolished aggregation. Hydrolysis of zwitterions of phospholipids or neuraminic acid and partial enzymatic degradation of membrane proteins had no effect on the phenomenon. We propose that aggregation of positively charged colloid particles depends on the association of the aliphatic tail of propanoic acid with the lipid region of the membrane. Positioning of carboxyl groups of propanoic acid in the hydrophilic region of the membrane would increase negative surface charge. Focal aggregation of ferric oxide in butanoic but not in lactic acid was consistent with this hypothesis.
ULTRASTRUCTURAL STAINING OF THIN SECTIONS WITH IRON HEMATOXYLIN
Authors: BRISSIE R. M. , SPICER S. S. , HALL B. J. , THOMPSON N. T.
Published: April 2011

Verhoeff's iron hematoxylin (VIH) applied to epoxy thin sections provides ultrastructural staining of selected sites including elastic fibers, ribosomes, heterochromatin in nuclei of somatic cells, chromatin clumps in germ cells and the nucleolonema and ovoid body of the nucleolus of primary spermatocytes. Granules in rat parotid acinar cells show several staining patterns not otherwise easily recognized. Mucous droplets or granules of goblet or Paneth cells of the ileum stain. Staining of defined polymers in epoxy thin sections indicates that VIH reacts with polycations and polyanions. Ethanolic hematoxylin alone at pH 1.3 imparts electron opacity to heterochromatin, ribosomes, parotid granules and elastica but yields no staining for light microscopy. A hematoxylin-FeCl3, solution stains mucus of goblet and Paneth cells. The latter staining, which requires FeCl3 in the solution, can be eliminated by saturating the solution with NaCl and, accordingly, depends on ionic binding. Staining of elastic fibers and parotid acinar granules depends on hydrogen bonding, since saturation of the VIH or hematoxylin solution with urea blocks the ultrastructural staining. Urea does not, however, alter the staining of nuclear structures or ribosomes.
Differential permeation of Artemia cysts and cucumber seeds by alcohols.
Authors: Smith C W , Siegel S M
Published: April 2011

The rate of penetration of the simpler alcohols into brine shrimp cysts and cucumber seeds was studied. In solutions below 70% the rate of penetration is related to lipid solvent capacity of the alcohol. In concentrations above 70%, particularly in absolute alcohols, methanol penetrates brine shrimp rapidly and ethanol penetrates slowly. All the other alcohols tested did not pepetrate the dormant structures. Ethionine and deuteroxy-methanol did not affect the rate of penetration of methanol. It is suggested that in dehydrated membranes the lipid moiety is protected by a continuous sheet of protein. Methanol, which is fairly similar to water, is probably able to penetrate the membrane by initiating a conformation change in the protein, exposing the lipid which subsequently dissolves in the methanol thus destroying the membrane.