Flow cytometric enrichment for respiratory epithelial cells in sputum

Kraemer, Petra S.; Sanchez, Carissa A.; Goodman, Gary E.; Jett, James R.; Rabinovitch, Peter S.; Reid, Brian J.

doi:10.1002/cyto.a.20041

Cited by 11 publications

(15 citation statements)

References 53 publications

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“…Statistically, there was only a tendency of higher intracellular GSH levels in lymphocytes in DTT samples compared to D-PBS samples (p = 0.06). Cam5.2 is a pan-epithelial marker [46], CD15s is expressed on granulocytes, while CD3 and CD4 are well-known markers for T cells. These markers were used for classification of the cell populations. …”

Section: Resultsmentioning

confidence: 99%

Novel Method to Process Cystic Fibrosis Sputum for Determination of Oxidative State

et al. 2009

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Background: Induced sputum is the most commonly used method to analyze airway inflammation in cystic fibrosis (CF) patients ex vivo. Due to the complex matrix of the sample material, precise and reliable analysis of sputum constituents depends critically on preanalytical issues. Objectives: Here we compared the commonly used method for sputum processing by dithiothreitol (DTT) with a novel mechanical method in regard to basal cellular parameters, neutrophil markers and glutathione (GSH) levels. Methods: Sputum samples from CF patients were processed in parallel with or without the use of DTT. The key improvement of the mechanical method was the processing in many very small aliquots. Cellular and humoral markers were assessed and compared according to Bland-Altman. Results: Total cell count, cell viability, differential cell count, neutrophil elastase levels and flow cytometrically analyzed neutrophil markers (CD63, CD11b, DHR) did not differ between the two methods. Intracellular and extracellular GSH levels were significantly higher in DTT-treated samples (p = 0.002). Conclusion: The mechanical sputum-processing method presented had a similar yield of cells and fluids as the conventional DTT method and the advantage of omitting the introduction of reducing agents. This method allows a more reliable analysis of redox-dependent airway inflammation in sputum cells and fluid from CF patients than methods utilizing DTT.

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Section: Resultsmentioning

confidence: 99%

Novel Method to Process Cystic Fibrosis Sputum for Determination of Oxidative State

et al. 2009

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“…Biopsies of NSE were minced to release nuclei (22), stained with the DNA binding dye 4V ,6-diamidino-2-phenylindole, and nuclei from NSE cells were identified on the basis of side scatter and 4V ,6-diamidino-2-phenylindole fluorescence as described previously (23). Briefly, this sorting strategy is based on the retention of dense cytoplasm around the nuclei of squamous cells that nonspecifically binds 4V ,6-diamidino-2-phenylindole.…”

Section: Methodsmentioning

confidence: 99%

“…Thus, these nuclei have both a high degree of side scatter due to the complexity of the cytoplasm around the nucleus, and fluoresce brightly with 4V ,6-diamidino-2-phenylindole, in contrast to the Barrett's intestinal epithelial nuclei, which take up less 4V ,6-diamidino-2-phenylindole and have significantly lower side scatter. In experiments with lung bronchial sputum samples, such a sorting procedure was able to generate populations with <1% columnar epithelium (23). DNA from the flow-sorted cells was isolated as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

Neosquamous Epithelium Does Not Typically Arise from Barrett's Epithelium

Paulson

Sanchez

et al. 2006

Clinical Cancer Research

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Purpose: Neosquamous epithelium (NSE) can arise within Barrett's esophagus as a consequence of medical or surgical acid reduction therapy, as well as after endoscopic ablation. Morphologic studies have suggested that NSE can develop from adjacent squamous epithelium, submucosal gland ducts, or multipotent progenitor cell(s) that can give rise to either squamous or Barrett's epithelium, depending on the luminal environment. The cells responsible for Barrett's epithelium self-renewal are frequently mutated during neoplastic progression. If NSE arises from the same cells that self-renew the Barrett's epithelium, the two tissues should be clonally related and share genetic alterations; if NSE does not originate in the self-renewing Barrett's, NSE and Barrett's esophagus should be genetically independent. Experimental Design: We isolated islands of NSE and the surrounding Barrett's epithelium from 20 patients by microdissection and evaluated each tissue for genetic alterations in exon 2 of CDKN2A or exons 5 to 9 of the TP53 gene. Nine patients had p16 mutations and 11 had TP53 mutations within the Barrett's epithelium. Results: In 1of 20 patients, a focus of NSE had a 146 bp deletion in p16 identical to that found in surrounding Barrett's epithelium. The NSE in the remaining 19 patients was wild-type for p16 or TP53. Conclusion: Our mutational data support the hypothesis that, in most circumstances, NSE originates in cells different from those responsible for self-renewal of Barrett's epithelium. However, in one case, NSE and Barrett's epithelium seem to have arisen from a progenitor cell that was capable of differentiating into either intestinal metaplasia or NSE.The stratified squamous epithelium that normally lines the esophagus is established in the 4th month of embryonic development (1) and usually persists until death. In some individuals, the squamous epithelium is replaced by a specialized intestinal metaplasia in response to chronic gastroesophageal reflux disease, a condition termed Barrett's esophagus (2). Patients with Barrett's esophagus are at a 30-to 40-fold increased risk for the development of esophageal adenocarcinoma, a cancer that has increased rapidly in incidence over the past 30 years (3). The mechanisms of conversion from squamous to intestinal epithelium are not known, but it has been hypothesized that the mucus-producing Barrett's specialized intestinal epithelium provides greater protection against the erosive effects of reflux than squamous epithelium (4).Treatment for Barrett's esophagus consists primarily of surgical and medical interventions to control gastroesophageal reflux. Proton pump inhibitors, used in the United States since the late 1980s, bind irreversibly to the H + /K + ATPase and prevent the production of acid by gastric parietal cells (5, 6). Proton pump inhibitors have become the treatment of choice for reflux and are remarkably effective, capable of reducing acid output by >95% (7). The development of effective acid suppression therapies has led to the observatio...

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“…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. 10 …”

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confidence: 99%

Magnetic enrichment of bronchial epithelial cells from sputum for lung cancer diagnosis

Qiu

Todd

et al. 2008

Cancer

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BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS The enrichment of bronchial epithelial cells could improve the diagnostic value of sputum and the efficiency of genetic and cytologic analysis of lung cancer.

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Flow cytometric enrichment for respiratory epithelial cells in sputum

Cited by 11 publications

References 53 publications

Novel Method to Process Cystic Fibrosis Sputum for Determination of Oxidative State

Novel Method to Process Cystic Fibrosis Sputum for Determination of Oxidative State

Neosquamous Epithelium Does Not Typically Arise from Barrett's Epithelium

Magnetic enrichment of bronchial epithelial cells from sputum for lung cancer diagnosis

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