Article

Optimizing a Multicolor Immunophenotyping Assay

ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, 40 Convent Drive, Room 5509, Bethesda, MD 20892, USA.
Clinics in Laboratory Medicine (Impact Factor: 1.35). 10/2007; 27(3):469-85, v. DOI: 10.1016/j.cll.2007.05.002
Source: PubMed

ABSTRACT Flow cytometry-based immunophenotyping assays have become increasingly multiparametric, concomitantly analyzing multiple cellular parameters. To maximize the quality of the information obtained, antibody conjugate panels need to be developed with care, including requisite controls at every step. Such an optimization procedure for multicolor immunophenotyping assays is time consuming, but the value of having a reliable antibody conjugate panel that provides for sensitive detection of all molecules of interest justifies this time investment. This article outlines important considerations and procedures to undertake for the successful design and development of multicolor flow cytometry panels.

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Available from: Yolanda D Mahnke, Dec 01, 2014
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    • "This has been described in detail previously (Wood, 2006; Mahnke & Roederer, 2007). The following checks should be performed: ● Steric hindrance test: Label cells with (i) the complete set of antibodies and (ii) with each individual antibody as single stains. "
    British Journal of Haematology 03/2014; 165(4). DOI:10.1111/bjh.12789 · 4.96 Impact Factor
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    • "were added to ascertain the previously identified circulating progenitor cell populations in human PB (Estes et al., 2010a,b). All antibodies were titered based on the mean fluorescence intensity with fluorochrome conjugate coupling to specific antigens optimized for the four-antibody plus viability marker panel (Baumgarth and Roederer, 2000; Mahnke and Roederer, 2007; Tung et al., 2007; Estes et al., 2010a). "
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    ABSTRACT: A subset of peripheral blood hematopoietic stem and progenitor cells of bone marrow origin is elevated in humans with solid cancers before treatment and declines with therapy. This biomarker of angiogenesis is not specific to tumor type and has great potential in the objective assessment of treatment response in clinical trials. This pilot study was designed to develop a biomarker of neoangiogenesis in dogs for the diagnosis of cancer, the measurement of treatment response, and the provision of objective data in clinical trials. Polychromatic flow cytometry was used to quantify two subsets of circulating hematopoietic stem and progenitor cells in dogs with spontaneous solid tumors before (n=8) and after (n=3) treatment, and normal controls (n=6). Pro-angiogenic peripheral blood cells of bone marrow origin were detected in all eight cases and the six normal controls; however, there was no statistically significant difference between the two groups. Interestingly, an apparent decline in pro-angiogenic cells was observed after treatment. Bone marrow derived hematopoietic cells appear to contribute to tumor angiogenesis in dogs, as has been previously reported in humans. While the methodology for pro-angiogenic cell quantification in a small number of dogs in the current study did not result in a significant difference from normal controls, an optimized canine polychromatic flow cytometry protocol holds great promise in the development of a canine cancer model and for the objective measurements of treatment response in clinical trials.
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    • "Markers of interest for use in multicolor flow cytometry are assigned to three categories: primary, secondary, and tertiary (Chattopadhyay et al, 2006, 2010; Perfetto et al, 2004; Mahnke YD & Roederer M, 2007). Primary markers are those that are highly expressed on cells, without intermediate fluorescence (i.e., they exhibit on/off expression). "
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