The measuring of cell features

The usefulness of various feature sets for discriminating between different cell populations cannot be assessed without considering the entire automated cytology system, from specimen preparation through scanning, cell search, cell segmentation, artifact rejection and feature extraction to object classification and multivariate data analysis methods. These different stages in the analysis procedure are discussed in this paper and illustrated by examples from the author's experience of cell analysis studies, ranging from single-cell classification experiments to a test of a complete automated system under realistic conditions on a set of 397 cell specimens.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

Acriflavine-Feulgen stilbene staining: a procedure for automated cervical cytology with a television based system (LEYTAS).

A sample preparation and staining procedure for automated cytology with a TV based system (LEYTAS) is described. It consists of a centrifugation technique and automated acriflavine-Feulgen stilbene staining of cervical specimens. The advantages of using both the fluorescence and the absorption image of acriflavine-Feulgen stilbene stained cervical cells for a television based system are discussed.     

Cytopress: automated slide preparation of cytologic material from suspension

This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation. Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.     

Determination of liquid-based cervical cytology specimen adequacy using cellular light scatter and flow cytometry.

BACKGROUND: The majority of cervical cytology specimens are being collected in liquid-based preservatives (LBP). However, the assessment of specimen adequacy, as mandated by The Bethesda System (TBS), is still being performed at the time of slide review. We present a rapid, flow cytometric method for assessing specimen adequacy. METHODS: Three LBPs were compared for cell-surface antigen preservation. A three-color antibody panel was used to confirm the light scatter profile of specific cells in a liquid-based cervical cytology specimen. Using forward and orthogonal light scatter alone, we were able to assess the adequacy of liquid-based cytology specimens in all LBPs tested. RESULTS: The number of polymorphonuclear neutrophils (PMNs), endocervical (columnar) cells, ectocervical (squamous) cells, and debris in 120 liquid-based cervical cytology samples was quantified in less than 10 min. Using cutoffs of > 20% PMNs, < 1.0% endocervical cells, < 1.0% ectocervical cells, and < 500 total cells per milliliter, light scatter correlated with microscopic determination of adequacy with a correlation coefficient of 0.99. CONCLUSIONS: This rapid method allows the quantitative determination of cervical cytology adequacy in liquid-based cytology preparations prior to the preparation of slides for morphologic assessment.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

Cytologic evaluation of conjunctival epithelium using Cytobrush-S: value of slide preparation by ThinPrep technique

Cytologic evaluation of conjunctival epithelium using Cytobrush-S: value of slide preparation by ThinPrep technique
Recent clinical trials have indicated that an automated smear apparatus (ThinPrep process) of sample preparation has great diagnostic sensitivity. In this study, conjunctival brush cytology prepared using the ThinPrep method was applied in ocular surface disorders especially for dry eye status. To assess its diagnostic value in cellular samples, 17 patients with keratoconjunctivitis sicca (KCS) and 10 normal volunteer patients were examined using this technique. Conjunctival cells from normal controls revealed fine chromatin and polyhedral cytoplasm without having keratinized cytoplasm. On the other hand, the cellular samples from KCS revealed increased keratinized cells with pyknotic nuclei. They also contained extremely elongated cells. In KCS patients, the mean number of keratinized cells was significantly higher (34.1 cells/300 cells) than that of the normal control group (0.2 cells/300 cells). In patients with KCS, inflammatory cell counts were also higher than those of normal controls. Conjunctival cytology by means of the ThinPrep method obviously deserves additional trials as an adjunct in the cytology of dry eye states, especially in quantitative ocular evaluation for various ocular lesions.     

A brief history of the lysigenous gland hypothesis

This paper summarizes the early history of the lysigenous gland concept, traces its evolution to recent times, and explores some possible causes of conflicting reports of lysigeny and schizogeny. Secretory cavities and ducts are generally thought to form either through the schizogenous separation of cells or through lysigeny (cell lysis). Gland lysigeny was first proposed in 1857 by Karsten, who believed that plant tissues represent solidifications of humoral fluids and that plant secretions are formed by the resorption of previously solidified cells. The lysigenous gland concept has modernized as our understanding of cytology has improved, but it was established early, from Karsten’s hypothesis, long before the influence of artifacts of specimen preparation was appreciated. Different methods of specimen preparation, including variations in the mounting media used to mount freehand sections, may have caused some of the discrepancies between the findings of lysigenists and schizogenists. Tschirch and Haberlandt promoted the schizolysigeny concept, and believed that the conflicting reports resulted from incomplete observations of a developmental process that included both schizogenous separation of cells and cell lysis to form secretory cavities and ducts. Both lysigeny and schizogeny have been reported in the recent literature, although most reports of lysigeny have been opposed by conflicting observations of schizogeny, and lysigeny may represent a false category of gland development caused by the misinterpretation of artifacts.     

Sedimentation velocity separation: a preparation method for cervical samples.

A preparation procedure, aiming at monolayer deposition of cervical exfoliative material on glass slides for high resolution prescreening has been developed. The main features of this procedure are centrifugal deposition after suspension and sedimentation of samples over isopycnic medium of 1.026 density. Fractioning of the separation column after centrifugation at 50 X g yields two preparations with leukocytes, bacteria and cellular debris predominantly located on the first slide and epithelial cells on the second one. The degree of spatial cellular isolation as well as the amount of diagnostically relevant cells per slide seem to fit the requirements of automated high resolution analysis.     

Distinguishing cell types or populations based on the computational analysis of their infrared spectra

Infrared (IR) spectroscopy of intact cells results in a fingerprint of their biochemistry in the form of an IR spectrum; this has given rise to the new field of biospectroscopy. This protocol describes sample preparation (a tissue section or cytology specimen), the application of IR spectroscopy tools, and computational analysis. Experimental considerations include optimization of specimen preparation, objective acquisition of a sufficient number of spectra, linking of the derived spectra with tissue architecture or cell type, and computational analysis. The preparation of multiple specimens (up to 50) takes 8 h; the interrogation of a tissue section can take up to 6 h (～100 spectra); and cytology analysis (n = 50, 10 spectra per specimen) takes 14 h. IR spectroscopy generates complex data sets and analyses are best when initially based on a multivariate approach (principal component analysis with or without linear discriminant analysis). This results in the identification of class clustering as well as class-specific chemical entities.     

A comparison of fluorescent stains for the assessment of viability and metabolic activity of lactic acid bacteria

Lactic acid bacteria (LAB) are used as starter or probiotic cultures in the food and pharmaceutical industries and, therefore, rapid and accurate methods for the detection of their viability are of practical relevance. In this study 10 LAB strains, belonging to the genera Enterococcus, Lactococcus, Leuconostoc, Lactobacillus, Streptococcus and Weissella, were subjected to heat and oxidative stresses and cell injury or death was assessed comparing different fluorescent probes (Syto 9; Propidium Iodide, PI; 4,6-diamidino-2-phenylindole, DAPI; 5,(6)-carboxyfluorescein diacetate, cFDA) to identify the stain combination which most reliably allowed the detection of live/metabolically active and dead cells. Protocols for specimen preparation and staining were optimized and a simple procedure for automated cell counts was developed using NIH ImageJ macros. Cysteine and semi-solid agar solution were efficiently used as anti-fading agent and mounting medium, respectively. The double staining cFDA-PI apparently offered the best and most versatile indication of both cell metabolic activity and membrane integrity. An excellent correlation between manual and automated cell counts for the majority of strain/stain combinations was found. This work provides a simple protocol for specimen preparation and staining based on the use of safe, easy to prepare and inexpensive reagents as compared to other methods. Additionally, the automated cell count procedure developed can be applied to several bacterial species and allows an increase in the number of experimental trials and the reproducibility and sensitivity of the analysis.     

Intraocular lymphoma. Cytologic diagnosis and the role of immunologic markers

We retrospectively reviewed the cytology and immunohistochemistry of vitreous cells from 14 patients with benign or malignant intraocular diseases. Nine patients had intraocular lymphoma and five had benign forms of diffuse uveitis. The most useful cytologic criteria for lymphoma included an irregular nuclear outline, a coarse chromatin pattern and prominent nucleoli. In several cases, it was necessary to obtain more than one specimen to establish the diagnosis of lymphoma. When vitreous fluid was mixed with a balanced salt solution prior to processing, a resulting artifact interfered with interpretation of the specimen. A diagnosis of lymphoma was established by cytology in all nine patients; immunohistochemical techniques were less sensitive than was cytology.     

Polysaccharides and associated components of mesophyll cell-walls prepared from grasses

Mesophyll cells have been prepared from rye-grass leaves by a procedure similar to that used by Edwards and Black¹ for crab-grass. Successive filtrations through nylon cloth of 300-, 80-, 45-, and 15-μm mesh yielded suspension consisting chiefly of single cells, some broken. Passage through a French pressure cell brought the total breakage to near 100%. Microscopic examination of the walls showed that very little of the cell contents remained. The walls were thin (0.1–0.2 μm) and had numerous pit fields; torn surfaces revealed a randomly oriented, fibrillar structure. Determinations of the polysaccharide content, protein, phenolic esters, lignin, and ash accounted for ～85% of the dry matter. The proportion of glucose in the polysaccharides of these walls was similar to that in the leaf cell-wall fraction as a whole, but the proportions of arabinose, galactose, and uronic acid were higher, and that of xylose lower. The acetyl content was 1.0–1.2%. The walls were readily digested by fungal culture filtrates, by snail digestive juice, and by immersion in the sheep rumen in nylon bags. These findings are discussed in relation to the digestion of forage cell-walls in ruminants.     

Multidimensional slit-scan prescreening system: preliminary results of a single blind clinical study

A multidimensional slit-scan flow system was developed to serve as an automated prescreening instrument for gynecological cytology. A 2-year single blind clinical study was carried out to evaluate system performance. Cellular material was collected by scraping the uterine cervix and stained in suspension with acridine orange. Seven hundred and forty specimens (701 patients) including 156 abnormal specimens representing a broad spectrum of abnormality were analyzed. Approximately 50,000 cells were analyzed for each specimen. The system false-positive rate was 17.6% while the false-negative rate was 2.8%. All misclassified abnormals were specimens with cellular changes consistent with a slight dysplasia of nonkeratinizing type. The instrument in its present configuration appeared sensitive to the entire spectrum of abnormality existing in the female genital tract and it classified as abnormal any specimen containing on the order of 0.1% (or greater) abnormal cells.     

The preparation of cervical scrape material for automated cytology using gallocyanin chrome-alum stain.

A method is described for preparing cervical scrape specimens for automated analysis on the Cerviscan prescreening system. In order to reduce the cellular clumping found in cervical scrape material, cells are collected in suspension, syringed to disaggregate the cell clumps, and then pipetted onto a glass to give a monolayer of cells. The cells are then stained with gallocyanin chrome-alum to give the required quantitation of nucleic acid content, using a rapid staining procedure. Experimental results are given which show that specimens prepared by this method are more suitable for automated analysis than the conventional Papanicolaou stained preparation.     

Standardization of fine needle aspiration cytology of the breast -- comparison of Auto Cyto Fix and conventional smears.

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

Standardization of fine needle aspiration cytology of the breast – comparison of Auto Cyto Fix and conventional smears

In Japan, there are some problems with fine needle aspiration (FNA) cytology of the breast, such as insufficient smeared cells, air-drying artefact and excessive erythrocytes. Liquid-based cytology has been found to solve these problems. Equipment for such preparations has been developed, but can be expensive to purchase and operate. We developed Auto Cyto Fix 1000 (ACF), which is inexpensive and automatically smears and fixes cells. The purpose of this study was to compare the various cytological features of conventional and ACF specimens. We evaluated whether the ACF method would be able to replace the conventional method. Forty-eight FNA specimens of breast were studied. All specimens were prepared by the direct smeared (DS) and ACF methods and evaluated for unsatisfactory cell collection, air-drying artefacts, background findings and epithelial cell findings. Although ACF specimens were prepared using the cells remaining in the needle and syringe after preparing DS specimens, the cellularity of two of the ACF specimens was better than that of the corresponding DS specimens. ACF specimens never showed air-drying artefact. Unlike DS specimens, which have many erythrocytes in the background, erythrocytes were filtered out and the background of ACF specimens was clean. We believe that many problems attributable to conventional FNA specimen preparation have been solved in this study. Preparation using the ACF apparatus can reduce running costs and can be used to prepare FNA specimens of the breast for cytological examination as an alternative to the conventional method.     

Binding of Griffonia simplicifolia I lectin to rat pulmonary alveolar macrophages and its use in purifying type II alveolar epithelial cells

We report that the isolectin Griffonia simplicifolia I-B4 isolated from G. simplicifolia seeds binds to rat alveolar macrophages present in frozen sections of lung tissue or bronchoalveolar lavage fluid. G. simplicifolia I-B4 does not bind to alveolar epithelial cells. We established that G. simplicifolia I-B4 binds to the macrophages via interaction with terminal alpha-D-galactopyranosyl residues present on these cells. This was substantiated by demonstrating that binding is inhibited either by the haptenic sugar alpha-D-galactopyranoside or by treating the cells with coffee bean alpha-galactosidase. Because murine laminin is known to contain terminal alpha-D-galactopyranosyl end-groups, and because we found that an anti-laminin antiserum binds to rat alveolar macrophages, we suspect that G. simplicifolia I-B4 may be binding to laminin present on the macrophages. To isolate alveolar type II epithelial cells from rat lungs, we developed a method that utilizes the lectin G. simplicifolia I. When proteinase-derived suspensions of pulmonary cells are incubated with G. simplicifolia I, the macrophages agglutinate and can be removed by filtration through nylon mesh. After incubating the resulting cellular suspension in tissue culture, the adherent cells are 94 +/- 2% (S.D.) type II cells. When compared to cells isolated by repeated differential adherence, the lectin-prepared type II cells have similar morphology and staining characteristics, form domes in monolayers and incorporate similar amounts of palmitate into disaturated phosphatidylcholine. We believe that the procedure outlined in this report provides a simple and effective method to isolate type II alveolar epithelial cells from rat lungs.     

Sample preparation with an automated robotic workstation for organic acid analysis by gas chromatography-mass spectrometry

We attempted to automate sample preparation for analysis of organic acids by gas chromatography-mass spectrometry using a computer-controlled, automated robotic workstation that is integrated and connected to the gas chromatography-mass spectrometry (HP-5890/5971) system. Of the two methods developed, one employed solvent extraction, while the other utilized a silica, solid-phase extraction cartridge. Both automated methods were compared to a manual, solvent extraction procedure used routinely in our laboratory. Normal, spiked urine, and urine from patients with a variety of metabolic abnormalities were analyzed. The robotic workstation did not meet all our requirements for a rapid, reliable, laboratory device. Recoveries with the automated procedure were less than with the manual method, and some organic acids important in the diagnosis of inborn errors of metabolism were not detected. Additionally, the robotic device had mechanical and design problems that made it slower and less reliable than the manual procedure.     

Diagnosis of adenoid cystic carcinoma of the lung by bronchial brushing: a case report

BACKGROUND: A diagnosis of pulmonary adenoid cystic carcinoma on exfoliative cytology specimen is very uncommon. The diagnostic cytologic material typically is obtained following a tissue biopsy. No previous report of the diagnosis has been made on bronchial brushing cytologic material when the procedure preceded a tissue biopsy. CASE: A 44-year-old man who used to smoke cigarettes and was otherwise well complained of persistent cough for the past 6 months. A chest radiograph revealed a mass lesion in the left hilum. Computed tomography of the chest disclosed an irregular and spiculated soft tissue mass in the left apical anterior segment. Bronchial brushing via bronchoscope was performed, revealing carcinoma cells consistent with an adenoid cystic carcinoma on cytology. A bronchial biopsy and subsequent left upper lobectomy were performed, confirming the diagnosis of adenoid cystic carcinoma of the lung associated with tumor extension to the epithelial surface. CONCLUSION: A diagnosis of bronchial adenoid cystic carcinoma is possible on bronchial brushing. However, as a method in exfoliative cytology, the usefulness of bronchial brushing in diagnosing this tumor is limited by the neoplasm's proximity to the mucosal surface and whether the mucosa has been breached.