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

ABSTRACT

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.