QUAliFiER: An automated pipeline for quality assessment of gated flow cytometry data
Effective quality assessment is an important part of any high-throughput flow cytometry data analysis pipeline, especially when considering the complex designs of the typical flow experiments applied in clinical trials. Technical issues like instrument variation, problematic antibody staining, or reagent lot changes can lead to biases in the extracted cell subpopulation statistics. These biases can manifest themselves in non–obvious ways that can be difficult to detect without leveraging information about the study design or other experimental metadata. Consequently, a systematic and integrated approach to quality assessment of flow cytometry data is necessary to effectively identify technical errors that impact multiple samples over time. Gated cell populations and their statistics must be monitored within the context of the experimental run, assay, and the overall study.
We have developed two new packages, flowWorkspace and QUAliFiER to construct a pipeline for quality assessment of gated flow cytometry data. flowWorkspace makes manually gated data accessible to BioConductor’s computational flow tools by importing pre–processed and gated data from the widely used manual gating tool, FlowJo (Tree Star Inc, Ashland OR). The QUAliFiER package takes advantage of the manual gates to perform an extensive series of statistical quality assessment checks on the gated cell sub–populations while taking into account the structure of the data and the study design to monitor the consistency of population statistics across staining panels, subject, aliquots, channels, or other experimental variables. QUAliFiER implements SVG–based interactive visualization methods, allowing investigators to examine quality assessment results across different views of the data, and it has a flexible interface allowing users to tailor quality checks and outlier detection routines to suit their data analysis needs.
We present a pipeline constructed from two new R packages for importing manually gated flow cytometry data and performing flexible and robust quality assessment checks. The pipeline addresses the increasing demand for tools capable of performing quality checks on large flow data sets generated in typical clinical trials. The QUAliFiER tool objectively, efficiently, and reproducibly identifies outlier samples in an automated manner by monitoring cell population statistics from gated or ungated flow data conditioned on experiment–level metadata.
- Braylan RC: Impact of flow cytometry on the diagnosis and characterization of lymphomas, chronic lymphoproliferative disorders and plasma cell neoplasias. Cytometry A 2004, 58A:57–61. CrossRef
- Hengel RL, Nicholson JK: An update on the use of flow cytometry in HIV infection and AIDS. Clin Lab Med 2001,21(4):841–856.
- Illoh OC: Current applications of flow cytometry in the diagnosis of primary immunodeficiency diseases. Arch Pathol Lab Med 2004, 128:23–31.
- Kiechle FL, Holland-Staley CA: Genomics, transcriptomics, proteomics, and numbers. Arch Pathol Lab Med 2003,127(9):1089–1097.
- Mandy FF: Twenty-five years of clinical flow cytometry: AIDS, accelerated global instrument distribution. Cytometry A 2004, 58A:55–56. CrossRef
- Orfao A, Ortuno F, de Santiago M, Lopez A, San Miguel J: Immunophenotyping of acute leukemias and myelodysplastic syndromes. Cytometry A 2004, 58A:62–71. CrossRef
- Bagwell CB: DNA histogram analysis for node-negative breast cancer. Cytometry A 2004, 58A:76–78. CrossRef
- Keeney M, Gratama JW, Sutherland DR: Critical role of flow cytometry in evaluating peripheral blood hematopoietic stem cell grafts. Cytometry A 2004, 58A:72–75. CrossRef
- Bashashati A, Brinkman RR: A survey of flow cytometry data analysis methods. Adv Bioinf 2009, 584603:2009.
- Krutzik PO, Irish JM, Nolan GP, Perez OD: Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol 2004,110(3):206–221. CrossRef
- Maecker H, McCoy P, Nussenblatt R: Standardizing Immunophenotyping for the Human Immunology Project. Nat Rev Immunol 2012,12(3):191–200.
- Pozarowski P, Darzynkiewicz Z: Analysis of cell cycle by flow cytometry. Methods Mol Biol 2004, 281:301–312.
- Pala P, Hussell T, Openshaw PJ: Flow cytometric measurement of intracellular cytokines. J Immunol Methods 2000,243(1–2):107–124. CrossRef
- Vermes I, Haanen C, Reutelingsperger C: Flow cytometry of apoptotic cell death. J Immunol Methods 2000,243(1–2):167–190. CrossRef
- Lehmann AK, Sornes S, Halstensen A: Phagocytosis: measurement by flow cytometry. J Immunol Methods 2000,243(1–2):229–242. CrossRef
- Shulman N, Bellew M, Snelling G, Carter D, Huang Y, Li H, Self SG, McElrath MJ, De Rosa SC: Development of an automated analysis system for data from flow cytometric intracellular cytokine staining assays from clinical vaccine trials. Cytometry Part A : j Int Soc Anal Cytology 2008,73(9):847–856. CrossRef
- Nelson EK, Piehler B, Eckels J, Rauch A, Bellew M, Hussey P, Ramsay S, Nathe C, Lum K, Krouse K, Stearns D, Connolly B, Skillman T, Igra M: LabKey Server: an open source platform for scientific data integration, analysis and collaboration. BMC Bioinf 2011, 12:71. CrossRef
- Hahne F, Le Meur N, Brinkman R, Ellis B, Haaland P, Sarkar D, Spidlen J, Strain E, Gentleman R: flowCore: A Bioconductor software package for high throughput flow cytometry data analysis. BMC Bioinf 2009, 10:106. CrossRef
- Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JYH, Zhang J: Bioconductor: Open software development for computational biology and bioinformatics. Genome Biol 2004,5(10):R80. CrossRef
- Hahne F, Khodabakhshi A, Bashashati A, Wong CJ, Gascoyne RD, Weng A, Seyfert-Margolis V, Bourcier K, Asare A, Lumley T, Gentleman R, Brinkman R: Per-channel basis normalization methods for flow cytometry data. Cytometry Part A 2010, 77A:121–131.
- Sarkar D, Le Meur N, Gentleman R: Using flowViz to visualize flow cytometry data. Bioinformatics 2008,24(6):878–879. CrossRef
- Gosink JJ, Means GD, Rees WA, Su C, Rand HA: Bridging the Divide between Manual Gating and Bioinformatics with the Bioconductor Package flowFlowJo. Advances in Bioinformatics 2009, 809469:2009.
- Rew R, Davis G: NetCDF: an interface for scientific data access. Computer Graphics and Applications, IEEE 1990,10(4):76–82. CrossRef
- Mike Jiang NG Greg Finak: ncdfFlow: ncdfFlow: A package that provides ncdf based storage for flow cytometry data. [http://bioconductor.org/packages/2.10/bioc/html/ncdfFlow.html]
- Chambers JM: Programming with Data: A Guide to the S Language. 175 Fifth Avenue, NY 10010, USA: Springer–Verlag, New York, Inc.; 2004.
- Lang DT: XML: Tools for parsing and generating XML within R and S-Plus. 2012,. [ http://CRAN.R-project.org/package=XML]. [R package version 3.9–4]
- Chan C, Felber P, Garofalakis M: Efficient filtering of XML documents with XPath expressions. VLDB J—Int J Very Large Data Bases 2002,11(4):354–379. CrossRef
- Yu H: Rmpi: Interface (Wrapper) to MPI (Message-Passing Interface). 2010,. [ http://CRAN.R-project.org/package=Rmpi]. [R package version 0.5–9]
- Knaus J: snowfall: Easier cluster computing (based on snow). 2010,. [ http://CRAN.R-project.org/package=snowfall]. [R package version 1.84]
- Urbanek S: multicore: Parallel processing of R code on machines with multiple cores or CPUs. 2011,. [ http://CRAN.R-project.org/package=multicore]. [R package version 0.1–7]
- Feng S, Ekong U, Lobritto S: Clinical and histological predictors of operational tolerance in pediatric liver transplant recipients. The 2011 Joint International Congress of ILTS , ELITA , & LICAGE Valencia, June 22–25, 2011; Spain Edited by Lake, J R and Roberts, JP 17(suppl 1):S107.
- QUAliFiER: An automated pipeline for quality assessment of gated flow cytometry data
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
- Online Date
- September 2012
- Online ISSN
- BioMed Central
- Additional Links
- Flow cytometry
- Quality assessment
- BioConductor package
- Industry Sectors
- Author Affiliations
- 1. Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA, 98109, USA
- 2. Tolerance Assays and Data Analysis, the Immune Tolerance Network, University of California, San Francisco, 3 Bethesda Metro Center, Bethesda, 20814, MD, USA
- 3. Department of Statistics, University of Washington, Box 354322, Seattle, 98195, WA, USA