Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study

Liset Westera, Tanja Van Viegen, Jenny Jeyarajah, Azar Azad, Janine Bilsborough, Gijs R. Van Den Brink, Jonathan Cremer, Silvio Danese, Geert D'Haens, Lars Eckmann, William Alvis Faubion, Melissa Filice, Hannelie Korf, Dermot McGovern, Julian Panes, Azucena Salas, William J. Sandborn, Mark S. Silverberg, Michelle I. Smith, Severine VermeireStefania Vetrano, Lisa M. Shackelton, Larry Stitt, Vipul Jairath, Barrett G. Levesque, David M. Spencer, Brian G. Feagan, Niels Vande Casteele

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Objectives:Flow cytometry (FC) AIDS in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor.Results:Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG.Conclusions:Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

Original languageEnglish (US)
Article numbere126
JournalClinical and Translational Gastroenterology
Volume8
Issue number11
DOIs
StatePublished - Nov 2 2017

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Multicenter Studies
Flow Cytometry
Prospective Studies
Ionomycin
Inflammatory Bowel Diseases
Blood Cells
Analysis of Variance
Acquired Immunodeficiency Syndrome
Acetates
Biomarkers
Staining and Labeling
T-Lymphocytes
Pharmaceutical Preparations
Population

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Westera, L., Van Viegen, T., Jeyarajah, J., Azad, A., Bilsborough, J., Van Den Brink, G. R., ... Casteele, N. V. (2017). Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study. Clinical and Translational Gastroenterology, 8(11), [e126]. https://doi.org/10.1038/ctg.2017.52

Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study. / Westera, Liset; Van Viegen, Tanja; Jeyarajah, Jenny; Azad, Azar; Bilsborough, Janine; Van Den Brink, Gijs R.; Cremer, Jonathan; Danese, Silvio; D'Haens, Geert; Eckmann, Lars; Faubion, William Alvis; Filice, Melissa; Korf, Hannelie; McGovern, Dermot; Panes, Julian; Salas, Azucena; Sandborn, William J.; Silverberg, Mark S.; Smith, Michelle I.; Vermeire, Severine; Vetrano, Stefania; Shackelton, Lisa M.; Stitt, Larry; Jairath, Vipul; Levesque, Barrett G.; Spencer, David M.; Feagan, Brian G.; Casteele, Niels Vande.

In: Clinical and Translational Gastroenterology, Vol. 8, No. 11, e126, 02.11.2017.

Research output: Contribution to journalArticle

Westera, L, Van Viegen, T, Jeyarajah, J, Azad, A, Bilsborough, J, Van Den Brink, GR, Cremer, J, Danese, S, D'Haens, G, Eckmann, L, Faubion, WA, Filice, M, Korf, H, McGovern, D, Panes, J, Salas, A, Sandborn, WJ, Silverberg, MS, Smith, MI, Vermeire, S, Vetrano, S, Shackelton, LM, Stitt, L, Jairath, V, Levesque, BG, Spencer, DM, Feagan, BG & Casteele, NV 2017, 'Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study', Clinical and Translational Gastroenterology, vol. 8, no. 11, e126. https://doi.org/10.1038/ctg.2017.52
Westera, Liset ; Van Viegen, Tanja ; Jeyarajah, Jenny ; Azad, Azar ; Bilsborough, Janine ; Van Den Brink, Gijs R. ; Cremer, Jonathan ; Danese, Silvio ; D'Haens, Geert ; Eckmann, Lars ; Faubion, William Alvis ; Filice, Melissa ; Korf, Hannelie ; McGovern, Dermot ; Panes, Julian ; Salas, Azucena ; Sandborn, William J. ; Silverberg, Mark S. ; Smith, Michelle I. ; Vermeire, Severine ; Vetrano, Stefania ; Shackelton, Lisa M. ; Stitt, Larry ; Jairath, Vipul ; Levesque, Barrett G. ; Spencer, David M. ; Feagan, Brian G. ; Casteele, Niels Vande. / Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study. In: Clinical and Translational Gastroenterology. 2017 ; Vol. 8, No. 11.
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abstract = "Objectives:Flow cytometry (FC) AIDS in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor.Results:Mean interlaboratory CV ranged from 1.8 to 102.1{\%} depending on cell population and gating strategy (LGLS, 4.4-102.1{\%}; LGCS, 10.9-65.6{\%}; CG, 1.8-20.9{\%}). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG.Conclusions:Central gating was the only strategy with mean CVs consistently lower than 25{\%}, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.",
author = "Liset Westera and {Van Viegen}, Tanja and Jenny Jeyarajah and Azar Azad and Janine Bilsborough and {Van Den Brink}, {Gijs R.} and Jonathan Cremer and Silvio Danese and Geert D'Haens and Lars Eckmann and Faubion, {William Alvis} and Melissa Filice and Hannelie Korf and Dermot McGovern and Julian Panes and Azucena Salas and Sandborn, {William J.} and Silverberg, {Mark S.} and Smith, {Michelle I.} and Severine Vermeire and Stefania Vetrano and Shackelton, {Lisa M.} and Larry Stitt and Vipul Jairath and Levesque, {Barrett G.} and Spencer, {David M.} and Feagan, {Brian G.} and Casteele, {Niels Vande}",
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T1 - Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study

AU - Westera, Liset

AU - Van Viegen, Tanja

AU - Jeyarajah, Jenny

AU - Azad, Azar

AU - Bilsborough, Janine

AU - Van Den Brink, Gijs R.

AU - Cremer, Jonathan

AU - Danese, Silvio

AU - D'Haens, Geert

AU - Eckmann, Lars

AU - Faubion, William Alvis

AU - Filice, Melissa

AU - Korf, Hannelie

AU - McGovern, Dermot

AU - Panes, Julian

AU - Salas, Azucena

AU - Sandborn, William J.

AU - Silverberg, Mark S.

AU - Smith, Michelle I.

AU - Vermeire, Severine

AU - Vetrano, Stefania

AU - Shackelton, Lisa M.

AU - Stitt, Larry

AU - Jairath, Vipul

AU - Levesque, Barrett G.

AU - Spencer, David M.

AU - Feagan, Brian G.

AU - Casteele, Niels Vande

PY - 2017/11/2

Y1 - 2017/11/2

N2 - Objectives:Flow cytometry (FC) AIDS in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor.Results:Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG.Conclusions:Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

AB - Objectives:Flow cytometry (FC) AIDS in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor.Results:Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG.Conclusions:Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

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