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Open AccessArticle

Assessment of Protocol Impact on Subjectivity Uncertainty When Analyzing Peripheral Blood Mononuclear Cell Flow Cytometry Data Files

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Wolfson School of Mechanical, Electrical and Manufacturing Engineering, Loughborough University, Loughborough, Leicestershire LE11 3TU, UK
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Department of Aeronautical, Automotive, Chemical and Materials Engineering, Loughborough University, Loughborough, Leicestershire LE11 3TU, UK
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GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK
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LGC Group, Queen’s Road, Teddington, Middlesex TW11 0LY, UK
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Author to whom correspondence should be addressed.
Academic Editors: Georgios S. Markopoulos, George Vartholomatos and George Alexiou
Methods Protoc. 2021, 4(2), 24; https://doi.org/10.3390/mps4020024
Received: 25 February 2021 / Revised: 26 March 2021 / Accepted: 28 March 2021 / Published: 30 March 2021
Measured variability of product within Cell and Gene Therapy (CGT) manufacturing arises from numerous sources across pre-analytical to post-analytical phases of testing. Operators are a function of the manufacturing process and are an important source of variability as a result of personal differences impacted by numerous factors. This research uses measurement uncertainty in comparison to Coefficient of Variation to quantify variation of participants when they complete Flow Cytometry data analysis through a 5-step gating sequence. Two study stages captured participants applying gates using their own judgement, and then following a diagrammatical protocol, respectively. Measurement uncertainty was quantified for each participant (and analysis phase) by following Guide to the Expression of Uncertainty in Measurement protocols, combining their standard deviations in quadrature from each gating step in the respective protocols. When participants followed a diagrammatical protocol, variation between participants reduced by 57%, increasing confidence in a more uniform reported cell count percentage. Measurement uncertainty provided greater resolution to the analysis processes, identifying that most variability contributed in the Flow Cytometry gating process is from the very first gate, where isolating target cells from dead or dying cells is required. This work has demonstrated the potential for greater usage of measurement uncertainty within CGT manufacturing scenarios, due to the resolution it provides for root cause analysis and continuous improvement. View Full-Text
Keywords: measurement uncertainty; Flow Cytometry; subjectivity; variation; data analysis measurement uncertainty; Flow Cytometry; subjectivity; variation; data analysis
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MDPI and ACS Style

Grant, R.; Coopman, K.; Silva-Gomes, S.; Campbell, J.J.; Kara, B.; Braybrook, J.; Petzing, J. Assessment of Protocol Impact on Subjectivity Uncertainty When Analyzing Peripheral Blood Mononuclear Cell Flow Cytometry Data Files. Methods Protoc. 2021, 4, 24. https://doi.org/10.3390/mps4020024

AMA Style

Grant R, Coopman K, Silva-Gomes S, Campbell JJ, Kara B, Braybrook J, Petzing J. Assessment of Protocol Impact on Subjectivity Uncertainty When Analyzing Peripheral Blood Mononuclear Cell Flow Cytometry Data Files. Methods and Protocols. 2021; 4(2):24. https://doi.org/10.3390/mps4020024

Chicago/Turabian Style

Grant, Rebecca; Coopman, Karen; Silva-Gomes, Sandro; Campbell, Jonathan J.; Kara, Bo; Braybrook, Julian; Petzing, Jon. 2021. "Assessment of Protocol Impact on Subjectivity Uncertainty When Analyzing Peripheral Blood Mononuclear Cell Flow Cytometry Data Files" Methods Protoc. 4, no. 2: 24. https://doi.org/10.3390/mps4020024

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