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Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials

McGowan, I and Anton, PA and Elliott, J and Cranston, RD and Duffill, K and Althouse, AD and Hawkins, KL and De Rosa, SC (2015) Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials. PLoS ONE, 10 (5).

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Abstract

© 2015 McGowan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
McGowan, Iimcgowan@pitt.eduIMCGOWAN
Anton, PA
Elliott, J
Cranston, RDrdc27@pitt.eduRDC27
Duffill, K
Althouse, AD
Hawkins, KL
De Rosa, SC
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorGray, Clive M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, or Units > Magee-Women's Research Institute
Date: 1 May 2015
Date Type: Publication
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Journal or Publication Title: PLoS ONE
Volume: 10
Number: 5
DOI or Unique Handle: 10.1371/journal.pone.0126454
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Medicine
Refereed: Yes
Date Deposited: 23 Aug 2016 14:10
Last Modified: 13 Oct 2017 22:57
URI: http://d-scholarship.pitt.edu/id/eprint/28473

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