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      Sampling Site Matters When Counting Lymphocyte Subpopulations

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          Abstract

          Clinical and scientific work routinely relies on antecubital venipunctures for hematological, immunological or other analyses on blood. This study tested the hypothesis that antecubital veins can be considered to be a good proxy for other sampling sites. Using a hematocytometer and a flow cytometer, we analyzed the cell counts from samples coming from the radial artery, the dorsal hand veins and the antecubital veins from 18 volunteers. Most surprisingly, we identified the greatest difference not to exist between arterial and venous circulation, but between the distal (radial artery & dorsal hand veins) and proximal (antecubital veins) sampling sites. Naïve T cells had a higher cell count distally compared to proximally and the reverse was true for effector memory T cells. Despite these differences there were high correlations between the different sampling sites, which partially supports our initial hypothesis. Our findings are crucial for the future design and interpretation of immunological research, and for clinical practice. Furthermore, our results suggest a role for interval lymph nodes in the trafficking of lymphocytes.

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          The vast majority of CLA+ T cells are resident in normal skin.

          K Yamanaka, Nina Mirchandani, Rebecca Dowgiert … (2006)
          There are T cells within normal, noninflamed skin that most likely conduct immunosurveillance and are implicated in the development of psoriasis. We isolated T cells from normal human skin using both established and novel methods. Skin resident T cells expressed high levels of CLA, CCR4, and CCR6, and a subset expressed CCR8 and CXCR6. Skin T cells had a remarkably diverse TCR repertoire and were mostly Th1 memory effector cells with smaller subsets of central memory, Th2, and functional T regulatory cells. We isolated a surprising number of nonexpanded T cells from normal skin. To validate this finding, we counted T cells in sections of normal skin and determined that there are approximately 1 x 10(6) T cells/cm(2) normal skin and an estimated 2 x 10(10) T cells in the entire skin surface, nearly twice the number of T cells in the circulation. Moreover, we estimate that 98% of CLA(+) effector memory T cells are resident in normal skin under resting conditions. These findings demonstrate that there is a large pool of memory T cells in normal skin that can initiate and perpetuate immune reactions in the absence of T cell recruitment from the blood.
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            Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

            Holden T. Maecker, Holli Dunn, Maria Suni … (2001)
            Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.
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              A model for harmonizing flow cytometry in clinical trials.

              Elaine Reed, Sally Amos, Hua Yeh … (2010)
              Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.
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                Author and article information

                Contributors
                : Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2012
                Publication date (Electronic): 25 July 2012
                Volume: 7
                Issue: 7
                Electronic Location Identifier: e41405
                Affiliations
                [1 ]Centre for Health Economics Research and Modeling Infectious Diseases, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium
                [2 ]Translational Cancer Research Unit, Oncology Center, GZA Hospitals Sint-Augustinus, Antwerp, Belgium
                [3 ]Department of Medical Oncology, University of Antwerp and Antwerp University Hospital, Antwerp, Belgium
                [4 ]Interuniversity Institute for Biostatistics and Statistical Bioinformatics, Hasselt University, Hasselt, Belgium
                [5 ]Department of Laboratory Medicine, Antwerp University Hospital, Antwerp, Belgium
                [6 ]Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium
                [7 ]Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Antwerp, Belgium
                [8 ]Centre for the Evaluation of Vaccination, Vaccine and Infectious Disease Institute, University of Antwerp, Antwerp, Belgium
                [9 ]School of Public Health and Community Medicine, University of New South Wales, Sydney, Australia
                University of Freiburg, Germany
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Performed the experiments: BO DP RM ES. Analyzed the data: BO. Wrote the paper: BO DP NH VVT PVD PB ES. Conceived the study: BO DP. Designed the study: BO DP RM VVT PVD PB ES. Supervised the study: NH PVD PB ES.

                Article
                Publisher ID: PONE-D-11-08927
                DOI: 10.1371/journal.pone.0041405
                PMC ID: 3405139
                PubMed ID: 22848485
                SO-VID: b6541f15-a048-419a-8ab6-0aa5db506c7c
                Copyright statement: Copyright @ 2012
                License:

                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.

                History
                Date received : 5 March 2012
                Date accepted : 21 June 2012
                Page count
                Pages: 7
                Funding
                The authors thank the University of Antwerp for the financial support received. B.O., D.P. and E.S. thank the Research Foundation Flanders (FWO) for the financial support they have received. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Immunology
                Subject: Immune Cells
                Subject: B Cells
                Subject: NK cells
                Subject: Monocytes
                Subject: T Cells
                Subject: Immune System
                Subject: Lymphoid Organs
                Subject: Immunologic Techniques
                Subject: Mathematics
                Subject: Statistics
                Subject: Biostatistics
                Subject: Medicine
                Subject: Clinical Immunology
                Subject: Immune Cells
                Subject: B Cells
                Subject: NK cells
                Subject: Monocytes
                Subject: T Cells
                Subject: Immune System
                Subject: Lymphoid Organs
                Subject: Immunologic Techniques
                Subject: Clinical Research Design
                Subject: Dermatology
                Subject: Inflammatory Diseases
                Subject: Skin Infections
                Subject: Hematology
                Subject: Platelets
                Subject: Red Cells
                Subject: White Cells

                ScienceOpen disciplines: Uncategorized
                Data availability:
                ScienceOpen disciplines: Uncategorized

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