Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa
Por:
Carrasco, A, Mane, J, Santaolalla, R, Pedrosa, E, Mallolas, J, Loren, V, Fernandez, M, Fernandez-Banares, F, Rosinach, M, Loras, C, Forne, M, Andujar, X, Vidal, J, Viver, JM and Esteve, M
Publicada:
1 mar 2013
Resumen:
An ideal method of immune cell isolation should provide maximum cell yield without disturbing functional properties. Intestinal endoscopic biopsies, in contrast to surgical samples, allow the study of all disease stages but have the drawback of a minimum amount of tissue available, making protocol optimization mandatory. We compared for the first time two methods of separation of colonic epithelium and five methods of lamina propria cell isolation for colonic biopsy specimens (mechanical, enzymatic and organ culture protocols). Lymphocyte number, viability and phenotype (CD45 +, CD103 +, CD3 +, CD4 +, CD8 +, CD19 +, CD16-56 +) were analyzed by flow cytometiy. Neither of the two epithelial detachment protocols achieved proper epithelial separation, though the high intensity ion chelation method was more accurate. Maximum cell yield of lamina propria lymphocytes without phenotypic modification was obtained with overnight smooth enzymatic digestion. High dose collagenase incubation caused a marked decrease in CD4 + lymphocytes of the lamina propria as compared to low enzymatic method (p = 0.004). Mechanical and biopsy culture are not advisable methods because of the low cell yield, and phenotypic alterations and high contamination rate, respectively. (C) 2013 Elsevier B.V. All rights reserved.
Filiaciones:
Carrasco, A:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Mane, J:
Autonomous Univ Barcelona, Hosp Univ Germans Trias & Pujol, Germans Trias & Pujol Fdn, Gastroenterol & Nutr Res Unit,Hlth Sci Res Inst, Barcelona, Catalonia, Spain
Santaolalla, R:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
:
Autonomous Univ Barcelona, Hosp Univ Germans Trias & Pujol, Germans Trias & Pujol Fdn, Gastroenterol & Nutr Res Unit,Hlth Sci Res Inst, Barcelona, Catalonia, Spain
Mallolas, J:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
:
Autonomous Univ Barcelona, Hosp Univ Germans Trias & Pujol, Germans Trias & Pujol Fdn, Gastroenterol & Nutr Res Unit,Hlth Sci Res Inst, Barcelona, Catalonia, Spain
:
Germans Trias & Pujol Fdn, Hlth Sci Res Inst, Flow Cytometry Unit, Barcelona, Catalonia, Spain
Fernandez-Banares, F:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Rosinach, M:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Loras, C:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Forne, M:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Andujar, X:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Vidal, J:
CATIAB, Flow Cytomeby Unit, Barcelona, Catalonia, Spain
Viver, JM:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
Esteve, M:
Univ Barcelona, Dept Gastroenterol, Hosp Univ Mutua de Terrassa, Res Fdn Mutua de Terrassa, Barcelona 08221, Catalonia, Spain
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