Comparison of CyTOF assays across sites: Results of a six-center pilot study

Michael D. Leipold*, Gerlinde Obermoser, Craig Fenwick, Katja Kleinstuber, Narges Rashidi, John P. McNevin, Allison N. Nau, Lisa E. Wagar, Virginie Rozot, Mark M. Davis, Stephen DeRosa, Giuseppe Pantaleo, Thomas J. Scriba, Bruce D. Walker, Lars R. Olsen, Holden T. Maecker

*Corresponding author for this work

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    Abstract

    For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments.We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods.All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values. <. 30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.

    Original languageEnglish
    JournalJournal of Immunological Methods
    Volume453
    Pages (from-to)37-43
    ISSN0022-1759
    DOIs
    Publication statusPublished - 2018

    Keywords

    • Clustering
    • CyTOF
    • Mass cytometry
    • Multicenter
    • Normalization
    • Standardization

    Cite this

    Leipold, M. D., Obermoser, G., Fenwick, C., Kleinstuber, K., Rashidi, N., McNevin, J. P., ... Maecker, H. T. (2018). Comparison of CyTOF assays across sites: Results of a six-center pilot study. Journal of Immunological Methods, 453, 37-43. https://doi.org/10.1016/j.jim.2017.11.008
    Leipold, Michael D. ; Obermoser, Gerlinde ; Fenwick, Craig ; Kleinstuber, Katja ; Rashidi, Narges ; McNevin, John P. ; Nau, Allison N. ; Wagar, Lisa E. ; Rozot, Virginie ; Davis, Mark M. ; DeRosa, Stephen ; Pantaleo, Giuseppe ; Scriba, Thomas J. ; Walker, Bruce D. ; Olsen, Lars R. ; Maecker, Holden T. / Comparison of CyTOF assays across sites: Results of a six-center pilot study. In: Journal of Immunological Methods. 2018 ; Vol. 453. pp. 37-43.
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    abstract = "For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments.We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods.All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values. <. 30{\%}. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.",
    keywords = "Clustering, CyTOF, Mass cytometry, Multicenter, Normalization, Standardization",
    author = "Leipold, {Michael D.} and Gerlinde Obermoser and Craig Fenwick and Katja Kleinstuber and Narges Rashidi and McNevin, {John P.} and Nau, {Allison N.} and Wagar, {Lisa E.} and Virginie Rozot and Davis, {Mark M.} and Stephen DeRosa and Giuseppe Pantaleo and Scriba, {Thomas J.} and Walker, {Bruce D.} and Olsen, {Lars R.} and Maecker, {Holden T.}",
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    language = "English",
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    pages = "37--43",
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    Leipold, MD, Obermoser, G, Fenwick, C, Kleinstuber, K, Rashidi, N, McNevin, JP, Nau, AN, Wagar, LE, Rozot, V, Davis, MM, DeRosa, S, Pantaleo, G, Scriba, TJ, Walker, BD, Olsen, LR & Maecker, HT 2018, 'Comparison of CyTOF assays across sites: Results of a six-center pilot study', Journal of Immunological Methods, vol. 453, pp. 37-43. https://doi.org/10.1016/j.jim.2017.11.008

    Comparison of CyTOF assays across sites: Results of a six-center pilot study. / Leipold, Michael D.; Obermoser, Gerlinde; Fenwick, Craig; Kleinstuber, Katja; Rashidi, Narges; McNevin, John P.; Nau, Allison N.; Wagar, Lisa E.; Rozot, Virginie; Davis, Mark M.; DeRosa, Stephen; Pantaleo, Giuseppe; Scriba, Thomas J.; Walker, Bruce D.; Olsen, Lars R.; Maecker, Holden T.

    In: Journal of Immunological Methods, Vol. 453, 2018, p. 37-43.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Comparison of CyTOF assays across sites: Results of a six-center pilot study

    AU - Leipold, Michael D.

    AU - Obermoser, Gerlinde

    AU - Fenwick, Craig

    AU - Kleinstuber, Katja

    AU - Rashidi, Narges

    AU - McNevin, John P.

    AU - Nau, Allison N.

    AU - Wagar, Lisa E.

    AU - Rozot, Virginie

    AU - Davis, Mark M.

    AU - DeRosa, Stephen

    AU - Pantaleo, Giuseppe

    AU - Scriba, Thomas J.

    AU - Walker, Bruce D.

    AU - Olsen, Lars R.

    AU - Maecker, Holden T.

    PY - 2018

    Y1 - 2018

    N2 - For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments.We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods.All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values. <. 30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.

    AB - For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments.We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods.All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values. <. 30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.

    KW - Clustering

    KW - CyTOF

    KW - Mass cytometry

    KW - Multicenter

    KW - Normalization

    KW - Standardization

    U2 - 10.1016/j.jim.2017.11.008

    DO - 10.1016/j.jim.2017.11.008

    M3 - Journal article

    VL - 453

    SP - 37

    EP - 43

    JO - Journal of Immunological Methods

    JF - Journal of Immunological Methods

    SN - 0022-1759

    ER -

    Leipold MD, Obermoser G, Fenwick C, Kleinstuber K, Rashidi N, McNevin JP et al. Comparison of CyTOF assays across sites: Results of a six-center pilot study. Journal of Immunological Methods. 2018;453:37-43. https://doi.org/10.1016/j.jim.2017.11.008