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. 2014 Jul:409:44-53.
doi: 10.1016/j.jim.2014.05.021. Epub 2014 Jun 23.

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays

Janet S Staats  1 , Jennifer H Enzor  2 , Ana M Sanchez  3 , Wes Rountree  3 , Cliburn Chan  4 , Maria Jaimes  5 , Ray Chun-Fai Chan  6 , Amitabh Gaur  6 , Thomas N Denny  3 , Kent J Weinhold  2
Affiliations

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays

Janet S Staats et al. J Immunol Methods. 2014 Jul.

Abstract

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.

Keywords: Antigen specific; Flow cytometry; Grading; ICS; Proficiency testing; Site performance.

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Figures

Fig. 1
Fig. 1
Scatter plot variance shown with and without target channels.
Fig. 2
Fig. 2
Backgating improves magnitude of functional response by including CD3 dim+ antigen-specific cells in the CD3 gate. Mock site analysis (2A) versus centralized analysis (2B).
Fig. 3
Fig. 3
Backgating to include dim CD8+ cells improves magnitude of measured response. Mock site analysis (3A) versus centralized analysis (3B).
Fig. 4
Fig. 4
Minor compensation error leads to a false positive response (FP). Mock site analysis (4A) versus centralized analysis (4B).
See this image and copyright information in PMC

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