Image analysis combined with quantitative cytochemistry. Results and instrumental developments for cancer diagnosis

This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermediate electron-acceptors in quantitative cytochemistry

Summary The efficacy of Meldola Blue (MB), a new intermediate electronacceptor, has been compared with that of phenazine methosulphate (PMS) in the assay of oxidoreductase activity in cryostat sections; various tetrazolium salts have been used as the final electron-acceptors. Three enzymes: succinate dehydrogenase, glucose 6-phosphate dehydrogenase and lactate dehydrogenase were investigated, the activity in sections being quantitated by scanning and integrating microdensitometry. Phenazine methosulphate was superior to Meldola Blue in transferring reducing equivalents from reduced coenzyme to all the tetrazolium salts examined.     

Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry

Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.     

On quantitative histo- and cytochemistry and general reflections

Reproduced, with modification, from Carlsberg Res. Commun. 49, 255–258 by kind permission of the Editor at Springer-Verlag.     

Detection of metabolic changes in hepatocytes by quantitative cytochemistry

Summary Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by (1) a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and (2) well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.In honour of Prof. P. van Duijn     

Detection of metabolic changes in hepatocytes by quantitative cytochemistry

Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.     

Qualitative and quantitative analysis of erythrocyte surface membrane sialyl residues using affinity cytochemistry with special reference to diabetic patients

The adenocarcinoma produced by transplantation into nude mice of a neoplastic human salivary intercalated duct cell line was treated with 0.1 ml of minimal essential medium (MEM) containing dibutyryl cyclic AMP (dB-cAMP) at a final concentration of 1 mM daily for 28 days and examined morphologically and immunohistochemically. The dB-cAMP treatment resulted in a marked suppression of tumor growth. In addition, tumor nests with a myoepithelial cell phenotype characterized by the presence of microfilament systems reactive with antimyosin and anti-S-100 protein sera were often observed in the treated tumors, but not in untreated controls. These findings lead us to suggest that neoplastic intercalated duct cells can be induced to differentiate into myoepithelial cells and that levels of cAMP within the cells may regulate this cytodifferentiation.     

mTRAQ-based quantitative analysis combined with peptide fractionation based on cysteinyl peptide enrichment

In the present study, the fractionation scheme for cysteinyl peptide enrichment (CPE) was combined with the mass differential tags for relative and absolute quantification (mTRAQ) method to reduce sample complexity and increase proteome coverage. Cysteine residues of the proteins were first alkylated using iodoacetyl PEG₂–biotin instead of other conventional alkylating agents such as iodoacetamide. After trypsin digestion, amine groups were labeled with mTRAQ, and these labeled peptides were fractionated according to the presence or absence of cysteine residues using avidin–biotin affinity chromatography. With these approaches, we were able to divide the peptides into the two fractions with more than 90% fractionation efficiency for standard protein and MCF7 cell lysate. When the fractionation strategy was applied to colorectal cancer tissue samples, we were able to obtain quantitative information that was consistent with the previous study based on mTRAQ quantification, implying that the cysteine-based fractionation method does not affect mTRAQ quantification. We expect that the mTRAQ-based quantitative analysis combined with peptide fractionation through the CPE strategy would allow for deep-down analysis of proteome samples and ultimately for increasing proteome coverage with simultaneous quantification for biomarker discovery.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

The use of an inexpensive,general purpose microcomputer in quantitative cytochemistry

Summary The histochemical localization of alkaline phosphatase in sheep parathyroid gland was investigated using light and electron microscopy. The reaction products of enzyme activity were observed by light microscopy in pericytes. By electron microscopy they were limited to the intercellular spaces between the gland cells, being exclusively confined to the external surface of plasma membrane.     

Freeze-fracture cytochemistry with polymyxin B

Summary The peptide antibiotic polymyxin B, has been used as a label in a freeze-fracture cytochemical study of anionic phospholipids in the plasma membrane of uterine epithelial cells. The reagent produces mainly circular lesions visible on both P and E faces of fractured membranes. IMPs are found to be associated with lesions and we consider the possible association of integral membrane proteins with anionic phospholipids as well as the mechanisms of lesion formation.     

The application of quantitative cytochemistry to the study of diseases of the connective tissues

The connective tissues are a complex organisation of tissues, cells and intercellular materials spread throughout the body and are subject to a large number of diseases. Such complexity makes the study of the metabolism of the connective tissues in health and more particularly in disease states difficult if one uses conventional biochemical methodology. Fortunately the techniques of quantitative cytochemistry, as developed in recent years, have made it possible to study the metabolism of even such complex and refractory connective tissues as bone. Using properly validated assays of enzyme activity in unfixed sections from various tissues a number of the diseases of the connective tissues have been studied. For example the synovia from patients with rheumatoid arthritis and related conditions have been studied using these techniques and marked alterations in the metabolism of the synovial lining cell population of this tissue have been demonstrated. These alterations in metabolism are believed to be related to the destruction of cartilage and bone found in such diseases. Investigations of the metabolism of the chondrocytes of articular cartilage in a strain of mice which spontaneously develops osteoarthritis has revealed a lack of certain key enzymes of carbohydrate metabolism in precisely those areas where degradation of the matrix of articular cartilage begins suggesting a causal relationship between these events. These same techniques have been used to study the cellular kinetics and metabolism of the dermis and epidermis in the disfiguring disease, psoriasis. The metabolism of healing bone fractures, the diagnosis and treatment of the mucopolysaccharidoses and the metabolic effects of currently used anti-inflammatory and anti-rheumatic drugs have also been examined. Perhaps the most exciting aspect of these studies has been the development and use of the technique of the cytochemical bioassay (CBA) to study hormonally mediated diseases of the connective tissues. Such studies have recently shed new light on the molecular lesion in pseudohypoparathyroidism. Though still in their relative infancy the studies described in this review show the potential inherent in the use of quantitative cytochemistry for the study of diseases of the connective tissues.     

A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry

Many peroxidase inhibitors have been used in horseradish peroxidase (HRP) mediated immunostaining and in situ hybridization to quench background peroxidase activity. However, the efficacy of these inhibitors has been controversial, partially due to the lack of a quantitative study. Tyramide signal amplification (TSA) is much more sensitive than other HRP-mediated methods but its super-sensitivity also demands effective inhibition of background peroxidase activity. In searching for an effective peroxidase inhibitor, we have systematically evaluated the efficacy of several peroxidase inhibitors by quantifying the fluorescence intensity in cultured fibroblasts and tissue sections treated with the inhibitors. For cultured cells, 0.05 mM of phenylhydrazine and 1 unit/ml of glucose oxidase gave only moderate inhibition of HRP activity while 1 mM of sodium azide (NaN₃), 3% of hydrogen peroxide (H₂O₂), NaN₃/H₂O₂ combined and 0.02 N hydrochloric acid (HCl) provided more complete inhibition. However, the inhibitory effect of NaN₃/H₂O₂ is reversible upon removal of the inhibitors and followed by incubation and wash to mimic antibody interactions. Similar results were obtained from rat skin wound tissues that have strong endogenous peroxidase activity. Our results recommend the use of HCl and caution the use of phenylhydrazine, glucose oxidase, NaN₃ and H₂O₂ as potent peroxidase inhibitors.