FLOW CYTOMETRIC ANALYSIS OF CANINE COLONIC MUCOSAL LYMPHOCYTES OBTAINED FROM ENDOSCOPIC BIOPSIES

Authors: SONEA, IOANA - IA STATE UNIV., AMES, IA; HARKINS, KRISTI - IA STATE UNIV., AMES, IA; WANNEMUEHLER, MICHAEL - IA STATE UNIV., AMES, IA; JERGENS, ALBERT - IA STATE UNIV., AMES, IA; MERTEN, ELLEN - IA STATE UNIV., AMES, IA; Sacco, Randy; CUNNICK, JOAN - IA STATE UNIV., AMES, IA

Submitted to: American Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: Intestinal diseases are commonly seen in dogs. Traditionally, diagnosis is based on the evaluation of biopsies of affected gut. This can provide valuable information about the architecture of the intestinal tissue, but little information concerning changes in white blood cell populations. Changes in white blood cell populations would suggest involvement of the immune system in intestinal disease. There are techniques available for the study of white blood cells in the affected intestine. Previously, the study of these intestinal cells required the removal of a segment of bowel. We have developed a method to isolate a representative sample of cells from biopsies. This minimally invasive technique can be used to study the white blood cells in the gut of client animals, and may prove an invaluable method for studying or evaluating canine intestinal disease.

Technical Abstract: We have validated the use of colonic biopsies obtained via endoscopy as a source of mucosal lymphocytes for flow cytometric analysis in the dog. A total of 10 adult dogs were used. Mucosal lymphocyte subsets obtained from excised colon were compared with mucosal lymphocyte subsets obtained from biopsies taken with endoscopic forceps in 6 adult dogs. Endoscopic colonic cbiopsies from 4 other adult dogs were used to further define whether obtained mucosal lymphocytes were predominantly of intra-epithelial 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 and B cells) were determined using flow cytometric analysis. Large numbers of viable mucosal lymphocytes were obtained after dissociation of the colonic epithelium from excised gut (45.5 +/ 21.5 x 10**6) and endoscopic biopsies (7.2 +/ 3.4 x 10**6). Lymphocyte subsets obtained with either method were identical for each animal and consisted predominantly of intra-epithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded large numbers of viable lymphocytes from the lamina propria (56.7 +/- 20.4 x 10**6), but collagenase digestion of endoscopic biopsies was less rewarding. Thus, a method to extract a representative sample of viable intra-epithelial mucosal lymphocytes from endoscopic biopsies for flow cytometric analysis has been developed. This minimally invasive technique can be used to study the mucosal lymphocyte populations of client animals and may prove an invaluable method for studying or evaluating canine intestinal disease.