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Optical configuration of the laser scanning cytometer. The laser beam is directed through the objective of a BX-50 Olympus microscope to excite fluorescently labelled cells on a microscope slide. Emitted fluorescent light is passed back through the objective lens and reflected through appropriate filters to photomultiplier tubes. Images of epifluorescent or bright field microscopy can be captured after relocation with a CCD camera linked to an image analysis system.
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Sputum induction is an important non-invasive technique for measuring airway inflammation in asthma. Cell numbers are often too low for flow cytometric analysis. Laser scanning cytometry (LSC) is a novel technique that allows objective multicolour fluorescence analysis of cells on a microscope slide.
LSC was used to determine sputum eosinophil and...
Context in source publication
Context 1
... current instrument set up incorporates two photomultiplier tubes for the detection of green or red fluorescent signals and a scatter sensor (fig 1). In order to calculate the proportion of a particular cell subset as a percentage of the total we labelled the blood and sputum samples with propidium iodide and used its bright red nuclear staining pattern for software contouring and hence capture of all nucleated cells or nucleated cell remnants. ...
Citations
... Further post-mortem studies supported this theory [43,44], and with the advent of bronchoscopy, several studies showed increased numbers of epithelial cells in bronchoalveolar lavage (BAL) [45][46][47] and desquamation visible in endobronchial biopsies on light microscopy [48,49] and using electron microscopy [50]. However there has been debate as to whether these findings represent artefactual damage from tissue sampling/processing [51], and in keeping with this, there are studies showing no difference in epithelial cells in BAL [52][53][54][55] or sputum [56,57], studies showing no difference in desquamation on bronchial biopsies [58][59][60], and postmortem studies which have not seen increased desquamation [61]. It is worth noting though that several of these studies did show a difference e.g. higher numbers of epithelial cells in asthmatic sputum, higher degree of epithelial desquamation in asthmatic biopsies, although this was not statistically significant, often due to small numbers. ...
Our knowledge and understanding of asthma have evolved over time, leading to new and improved treatments for this disease. Despite existing treatments however, there remains to date a significant proportion of asthmatics who remain poorly controlled, with unmet needs. Most existing treatments are based on the Th2-driven inflammation model of asthma, however there is increasing recognition of the importance of the epithelium in asthma pathogenesis. It has been proposed that the asthmatic epithelium is chronically damaged and unable to repair, with increased permeability as a result. Existing treatments do not address the epithelial damage directly, however there are now available recombinant growth factors that have been shown to have beneficial effects on epithelial healing. Our hypothesis was that modification of the epithelium, in effect boosting its repair using recombinant human keratinocyte growth factor (rhKGF), would lead to improvement in clinical parameters.
This was explored in several fashions. Firstly a randomised, double-blind, placebo-controlled clinical trial was performed using 20 poorly controlled, moderate asthmatics, with the active treatment group receiving parenteral rhKGF. Assessments before and after drug administration included objective, clinically relevant, measures of asthma such as airway hyperresponsiveness (AHR) measurements, spirometric measures, exhaled nitric oxide measurements and peak flow recording. Subjective, patient-centred assessments were also made using questionnaires to assess asthma control and quality of life, and bronchoscopy was performed to obtain samples to measure biological effects of the drug. KGF treatment resulted in a significantly greater improvement in the primary outcome of mannitol AHR, together with greater improvements in quality of life in the active treatment group compared to placebo. Other features (such as methacholine AHR, asthma control questionnaire scores, spirometric values, exhaled nitric oxide and peak flow variability) did not differ significantly between the groups, although this may be due to a greater than expected placebo response. Biological outcomes also did not differ significantly between the groups, although this may have been due to the sampling time-point used.
Concurrently to the clinical trial above, in vitro experiments were performed on cell cultures of epithelial cells from asthmatic and healthy donors, to verify and further explore the effects of KGF on an asthmatic epithelium. Specifically mechanical wounds were inflicted on the cultures, with assessment of the repair process using wound imaging, measurement of trans-epithelial electrical resistance (TER) and permeability to FITC-labelled dextran, in the presence and absence of KGF. As a subset of these experiments, some cultures were exposed to mechanical compression using air pressure, as a mimic for bronchoconstriction, to see if KGF was effective in these circumstances. Results confirm a biological effect for KGF on wound repair in the asthmatic epithelium, which can also partially overcome the deleterious effect of compression on wound healing. An intrinsic difference in wound healing between asthmatic and healthy cohorts, as previously reported, was not apparent.
Lastly the potential of nuclear medicine imaging, to assess epithelial permeability, was explored, for its potential use in future studies of asthma treatments addressing the epithelium directly. Unfortunately this was halted after a pilot study suggested potential methodological flaws – the results and conclusions from this pilot study are presented here, with suggestions for future studies in this area.
... Improved information of MC infiltration would have been obtained by methods including specific antibodies to mast cell subtypes and basophils, as used in immunocytochemistry or laser scanning cytometry. 27,28 However, in a study on cellular infiltrates in allergic children based on findings of specific antibodies to mast cells and other cells in mucosal biopsies, the results were in agreement with our study, as unexpected absence of mast cells in the epithelium was found in a proportion of allergic children sensitized to aeroallergens. 29 In the future, microarray-based studies of genes and transcripts in cells will probably result in superior information on predictive biomarkers, as this technique has shown genes exclusively expressed in mast cells and other cells in subjects with current allergy. ...
The ability to predict the development of allergic diseases in infants is important. Predictive biomarkers are wanted to improve the risk evaluation in addition to known heredity of allergy. Biomarkers taken during infancy need to be evaluated through longitudinal studies into adulthood. The objective of this study was to analyse the occurrence of metachromatic cells in the nasal mucosa during infancy (MC(infancy)) and evaluate the cells as predictive biomarkers of allergy development.
Previously, MC(infancy) occurrences were analysed in 64 infants with and without allergy heredity, and related to allergy development at 18 months and 6 years of age. In this third follow-up at 18 years of age, current allergy symptoms were analysed. MC(infancy) findings were related to the cumulative number of allergic subjects. The predictive values of MC(infancy) and known heredity were compared.
The cumulative number of subjects with allergy was 46, probable allergy 5, and no allergy 13. Detected MC(infancy) predicted allergy with high accuracy (31/33), but negative MC(infancy) findings did not exclude the risk (15/31). In the group of allergic subjects positive MC(infancy) were found in 31/46 (67%), positive heredity in 37/46 (80%) and one/both factors positive in 43/46 (93%). Detection of MC(infancy) could precede the debut of allergy symptoms by many years.
Detected MC(infancy) predicted allergy development, but absence of MC(infancy) did not exclude the risk, and therefore this biomarker was not found to be adequate. There is a further need to find biomarkers with high ability to both predict and exclude the risk.
... However, the use of sputum for molecular genetic analyses is limited by its cellular heterogeneity, which includes about 1% bronchial epithelial cells. [8][9][10] The large excess of macrophages and neutrophils that account for [95% sputum cell population could obscure detection and quantitation of neoplastic changes occurring in the bronchial epithelial cells. 7 Therefore, enrichment of bronchial epithelial cells before the actual detection procedure is needed to improve the efficiency and accuracy of genetic and cytologic diagnosis of lung cancer in sputum samples. ...
... A sample was considered adequate if it had more than 2 3 10 7 cells, less than 4% oral squamous cells, and [50% alveolar macrophages, as described previously. 8,9 Magnetic Cell Sorting (MACS) ...
Sputum is an easily accessible diagnostic material for lung cancer early detection by cytologic and molecular genetic analysis of exfoliated airway epithelial cells. However, the use of sputum is limited by its cellular heterogeneity, which includes >95% macrophages and neutrophils and only about 1% bronchial epithelial cells. We propose to obtain concentrated and purified bronchial epithelial cells to improve early detection of lung cancer in sputum samples.
Sputum was collected from patients with stage I nonsmall-cell lung cancer, cancer-free smokers, and healthy nonsmokers. Magnetic-assisted cell sorting (MACS) with anti-CD14 and anti-CD16 antibody beads were used to enrich bronchial epithelial cells by depleting macrophages and neutrophils from sputum. Fluorescence in situ hybridization (FISH) analysis for detection of FHIT deletion and cytology were evaluated in the enriched specimens.
The bronchial epithelial cells were concentrated to 40% purity from 1.1% of the starting population, yielding an average of 36-fold enrichment and at least 2.3 × 105 cells per sample. Detecting FHIT deletions for lung cancer diagnosis produced 58% sensitivity in the enriched sputum, whereas there was 42% sensitivity in the unenriched samples (P = .02). Cytologic examination of the enriched sputum resulted in 53% sensitivity, as compared with 39% sensitivity in unenriched sputum (P = .03). Furthermore, only 2 cytocentrifuge slides of the unenriched sputum were needed for the analyses, as compared with up to 10 cytocentrifuge slides required from the unprocessed specimens.
The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer. Cancer (Cancer Cytopathol) 2008.
... Samples of human sputum suitable for analysis are obtained in specialist respiratory practice by sequential saline aerosol inhalation at concentrations of 3%, 4% and 5% for 5 min each. Subjects expectorated for approximately 2 min and sputum samples were collected in a sterile 30 ml universal container on ice (Woltmann et al 1999). ...
... More detailed results and a method comparison using this method and traditional manual differential cell counting are presented elsewhere (Woltmann, Ward et al 1999). ...
Biological samples from human tissues are characterized by complexity and heterogeneity. The ability to make rapid, reliable, quantitative fluorochromatic measurements on clinical samples allows the development of new and practical assays that could influence diagnosis and treatment in a variety of clinical applications. Laser scanning cytometry (LSC) is a very versatile and adaptable technology that allows for the quantitative analysis of cell samples that are unsuitable for flow cytometry by virtue of their presentation and context. Crucially, it allows the direct visualization of cells and rare events and the correlation of imagery with fluorochromatic measurements. In this chapter, we describe early experiments in the study of cytotoxic drug uptake and resistance in human tumor cells and in the study of sputum cells from asthmatic patients, which harness the specific capabilities of LSC to practical clinical problems.
... Epithelial shedding is characteristic of bronchial asthma. Bronchial washings reveal that epithelial cells are shed in clusters composed entirely of columnar cells, and asthma patients have an increased number of epithelial cell clumps (creola bodies) in their sputum (Woltmann et al., 1999). According to electron microscopy the basal cells remain attached to the basal lamina (BL). ...
Shedding of airway epithelial cells is a common finding in asthma. In this study, the attachment of the airway epithelial cells to the basal lamina (BL) was investigated by transmission electron microscopy (TEM) of biopsies from patients with atopic asthma and healthy controls. The following parameters were quantitatively determined: the height of the epithelium and of the columnar cells, the number of basal cells per 100 microm of basal lamina, the contact surfaces of basal cells or columnar cells with the basal lamina, and between basal cells and columnar cells. In order to compare the quantitative method with previous literature data, measurements were also carried out on rat airway epithelium. Compared to the rat, the columnar cell height in the human is increased, basal cells are smaller, and there is a larger contact area between basal cells and basal lamina, as well as between basal and columnar cells. The contact area between columnar cells and basal lamina is hence less in the human airway. The contact area between columnar cells and basal lamina in asthmatics is significantly less than in healthy controls, due to larger intercellular spaces. It is concluded that attachment of columnar cells to the basal lamina occurs mainly indirectly, via desmosomal attachment to basal cells, and that direct attachment of columnar cells to the basal lamina is weakened in asthmatics.
... Epithelial damage is a key feature of asthma. As a result of inflammation, a large portion of columnar epithelial cells shed and form Creola bodies, detected in sputum and during bronchoscopy in asthmatic patients [3]. This cycle of damage and repair has been proposed as a key mechanism leading to thickening of the airway wall, and other pathologic alterations collectively characterized as airway remodeling [4], which in turn has been associated with incompletely reversible airway narrowing, bronchial hyper-responsiveness and asthma symptoms [5]. ...
Human rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro.
Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry.
RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation.
RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.
... The laser scanning cytometer is a novel microscope-linked and computer-operated instrument that measures fluorescence and optically scans cells labeled with fluorescent probes on a microscope slide. 39 ...
Since the 1980s, sputum induction by inhalation of hypertonic saline has been successfully used for diagnosing Pneumocystis carinii pneumonia in patients infected with HIV. In recent years, sputum induction and its subsequent processing has been refined as a noninvasive research tool providing important information about inflammatory events in the lower airways, and it has been used for studying various illnesses. In asthma, one application is to use sputum inflammatory indices to increase our understanding of complex relationships between inflammatory cells, mediators, and cytokine mechanisms. In chronic obstructive pulmonary disease, sputum assessment could be used as a screening test before deciding on long-term corticosteroid treatment. In tuberculosis, sputum induction is a valuable diagnostic tool for HIV-seropositive patients who do not produce sputum. Sputum induction appears to be a relatively safe, noninvasive means of obtaining airway secretions from subjects with cystic fibrosis, especially from those who do not normally produce sputum. Moreover, sputum induction can also be used in chronic cough and lung cancer. Generally, induction is performed through ultrasonic nebulizers, using hypertonic saline. It is recommended that sputum be processed as soon as possible, with complete homogenization by the use of dithiothreitol. We have also shown in this article an example of a protocol for inducing and processing sputum employing a nebulizer produced in Brazil.
... Aggregated cells were gated out using an algorithm in the LSC software that finds and marks multiple cells. Individual proTNF-a-, TACE-, IL-6-or IL-10-positive cells were identified and quantified using Compucyte software on the basis of integrated green (proTNF-a, TACE, and IL-6) or orange (IL-10) fluorescence [35,36]. ...
... It remains to be shown whether TACE represents a marker of inflammation for CAP; however, this is unlikely, given that TACE is ubiquitously expressed and attempts to up-regulate its expression have proved unsuccessful [11]. Moreover, because TACE becomes down-regulated after cleavage of its substrates [36], its usefulness as a prognostic determinant in CAP seems limited. What is evident from our studies on ELF cells from both infected and uninvolved lungs of patients with CAP is that down-regulation of TACE does occur in vivo during acute pulmonary inflammation. ...
Bronchoalveolar lavage fluid recovered from infected and uninvolved lungs of patients with community-acquired pneumonia (CAP;
n=16) on day 6±0.8 was analyzed for cytokine, soluble receptor, and antagonist levels. The role of tumor necrosis factor (TNF)–α–converting
enzyme (TACE) in the resolution of the local inflammatory response was investigated. TNF-α, interleukin (IL)–1β, and IL-6
were elevated in the infected versus uninvolved lobe, whereas IL-10 was not. Epithelial lining fluid (ELF) cytokine levels
correlated with intracellular cytokine expression. Levels of proTNF-α were reciprocally related to TNF-α ELF levels. Levels
of soluble receptors, generated by TACE cleavage of membrane-bound precursors, were compartmentalized to infected ELF. TACE
was down-regulated by internalization in cells from the site of infection. These data demonstrate that, in vivo during CAP,
TACE has a role in regulating resolution of the local inflammatory response by modulating levels of pro- and counterinflammatory
mediators
... The process whereby the software identifies pixelated clusters in individual cells is called "cell capture". To date, the most common method of capturing cells is for the software to identify nuclei stained with a fluorescent dye [11][12][13]. To obtain immune receptor data from the cell surface and cytoplasm after nuclear capture, a "contour" is generated at a fixed distance from the nuclear perimeter. ...
... LSC has successfully been used to examine the phenotype of many cell types including blood leukocytes [15] and eosinophils and bronchial epithelial cells in sputum [11], but has not been used to study the AM immune phenotype. LSC may be ideally suited to the study of paediatric AM. ...
... The two previously reported methods for detecting cells using the LSC involve either staining the nucleus with a nucleic acid dye [11][12][13] or contouring according to scatter properties [14]. Nuclear contouring has been successfully employed for cells with single nuclear profiles and can discriminate different cell populations in peripheral blood according to the nuclear size and density of chromatin condensation [12]. ...
Laser scanning cytometry (LSC) generates quantitative information on immune receptor expression from cells cytocentrifuged onto a microscope slide. In children, the description of developmental changes in immune receptor expression on alveolar macrophages (AM) has been limited by the small number of cells recovered by bronchoalveolar lavage (BAL). The applicability of LSC to the study of AM from normal children was therefore assessed. AM were obtained by BAL of normal children following intubation prior to elective surgery. The ability of LSC to identify the cytoplasm of AM was assessed using either: 1) autofluorescence; 2) forward scatter; 3) nuclear staining with propidium iodide; or 4) a fluorescent-labelled monoclonal antibody to CD68, a pan-macrophage antigen. LSC could only reliably identify individual AM when stained with CD68. The sensitivity for detecting single whole AM using CD68 was 0.97 and the positive predictive value was 0.88, respectively, with excellent repeatability. In addition, a range of immunofluorescence parameters were generated for CD68. It is concluded that laser scanning cytometry is suited to the study of immune receptor expression from small numbers of paediatric alveolar macrophages, when CD68 is used for cell identification.
... This material can also be used for immunophenotyping as described for hematological malignancies (54) and melanoma metastases (55); the latter study compared results obtained by LSC with those obtained by immunohistochemistry and quantitative realtime polymerase chain reaction (PCR). Other samples with low cell numbers can be analyzed by LSC, such as sputum (56). In the future, samples may include ascites and pleural effusion as described for image analysis (57) or liquor, saliva, and nasal secretion, among others. ...
This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings.
