Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens

Ioana M. Sonea From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Kristi Harkins From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Michael J. Wannemuehler From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Albert E. Jergens From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Ellen A. Merten From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Randy E. Sacco From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Joan E. Cunnick From the Department of Biomedical Sciences, (Sonea, Merten), the Cell and Hybridoma Facility (Harkins), the Veterinary Medicine Research Institute (Wannenmuehler, Sacco), and the Departments of Veterinary Clinical Sciences (Jergens) and Microbiology, Immunology, and Preventive Medicine (Wannenmuehler, Cunnick), College of Veterinary Medicine, Iowa State University, Ames, IA 50011.

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Abstract

Objective

To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis.

Sample Population

Mucosal biopsy specimens from 10 adult dogs.

Procedure

Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis.

Results

A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 ± 21.5 × 106) and endoscopic (7.2 ± 3.4 × 106) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7 ± 20.4 × 106), but collagenase digestion of endoscopic biopsy specimens was less rewarding.

Conclusion and Clinical Relevance

A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals. (Am J Vet Res 1999;60:346-353).

Abstract

Objective

To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis.

Sample Population

Mucosal biopsy specimens from 10 adult dogs.

Procedure

Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis.

Results

A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 ± 21.5 × 106) and endoscopic (7.2 ± 3.4 × 106) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7 ± 20.4 × 106), but collagenase digestion of endoscopic biopsy specimens was less rewarding.

Conclusion and Clinical Relevance

A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals. (Am J Vet Res 1999;60:346-353).

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