Operator measurement uncertainty contributions within post-analytical flow cytometry data [poster]

Operator measurement uncertainty contributions within post-analytical flow cytometry data [poster

<i>Mycobacterium avium</i> Infection Induces H-Ferritin Expression in Mouse Primary Macrophages by Activating Toll-Like Receptor 2

<div><p>Important for both host and pathogen survivals, iron is a key factor in determining the outcome of an infectious process. Iron with-holding, including sequestration inside tissue macrophages, is considered an important strategy to fight infection. However, for intra-macrophagic pathogens, such as <i>Mycobacterium avium</i>, host defence may depend on intracellular iron sequestration mechanisms. Ferritin, the major intracellular iron storage protein, plays a critical role in this process. In the current study, we studied ferritin expression in mouse bone marrow-derived macrophages upon infection with <i>M. avium</i>. We found that H-ferritin is selectively increased in infected macrophages, through an up-regulation of gene transcription. This increase was mediated by the engagement of Toll like receptor-2, and was independent of TNF-alpha or nitric oxide production. The formation of H-rich ferritin proteins and the consequent iron sequestration may be an important part of the panoply of antimicrobial mechanisms of macrophages.</p> </div

Effect of <i>Mycobacterium avium</i> infection on intramacrophagic ferritin.

<div><p>Bone marrow-derived macrophages were obtained from C57Bl/6 mice and infected with <i>M. avium</i>, as described in Material and Methods, or left uninfected. A - At different time points, macrophages were lysed and the amount of ferritin was quantified by ELISA. Data are presented as ng of ferritin per mg of total protein. The results are shown as average ± SD from one experiment performed in triplicate out of four independent experiments. Superscripts indicate statistical significance between M. avium-infected and uninfected, within the correspondent time-point, as follows: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. B – BMM uninfected or infected with <i>M. avium</i> for 24h were incubated for 6h with (⁵⁵Fe) ferric ammonium citrate. Total protein (18 µg) was loaded (in duplicates) in native PAGE and exposed to autoradiography to analyze protein-bound iron. A single band was detected corresponding to cytosolic H/L ferritin. The values indicate the average relative band intensity for each condition. C – BMM infected with <i>M. avium</i> for 4h, 1 and 3 days and respective uninfected controls were tested for IRP-IRE binding activity, by gel retardation assay. 2% of 2-mercaptoethanol (2-ME) fully activates IRP binding activity and shows equal loading. BMM treated with iron or deferoxamine (DFO) were tested in a separated gel to confirm the reliability of the assay. D – BMM were treated with the transcriptional inhibitor actinomycin D or with vehicle. After an 8h-infection with <i>M. avium</i>, the BMM were lysed and H- and L-ferritin were quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. ***<i>p</i><0.001, NS not significant. </p> <p>E – At different time points, total RNA was collected from macrophages and the expression levels of ferritin genes was quantified by qRT-PCR, and normalized to <i>Hprt1</i>. Results are shown as fold increase in <i>M. avium</i>-infected macrophages in comparison with uninfected ones. Data are presented as average ± SE from one experiment performed in triplicate from a total of two independent experiments. </p></div

Effect of <i>M. avium</i> infection on ferritin content in the absence of TNF-alpha, iNOS and TLR-2.

<p>Bone marrow-derived macrophages were obtained from C57Bl/6 (WT), TLR-2^-/-, TNF-alpha^-/- and NOS2^-/- mice. BMM were infected and the ferritin content was quantified as described in Figure 1. The results are shown as average ± SD from one experiment performed in triplicate out of two independent experiments.</p

TLR-2 activation leads to increased expression of H-ferritin.

<p>A, B – BMM from C57Bl/6 (WT) and TLR-2^-/- mice were left uninfected or infected for 24h with <i>M. avium</i>. The H-ferritin fold increase in infected BMM in comparison with uninfected ones is shown at the protein level (A) and mRNA (B). C – BMM were treated with the TLR-2 agonist FSL-1 for 24h, and the levels of H- and L-ferritin was quantified by ELISA. Results show the average + SD from one experiment performed in triplicate out of three independent experiments. Statistical differences as described in Figure 1.</p

Assessment of protocol impact on subjectivity uncertainty when analyzing peripheral blood mononuclear cell flow cytometry data files

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 re-search 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 re-spectively. 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 re-spective protocols. When participants followed a diagrammatical protocol, variation between participants reduced by 57 %, increasing confidence in a more uniform re-ported cell count percentage. Measurement uncertainty provided greater resolution to the analysis processes, identifying that most variability contributed in the Flow Cytom-etry 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

Understanding the contribution of operator measurement variability within flow cytometry data analysis for quality control of cell and gene therapy manufacturing

Flow Cytometry is a measurement technique used in Quality Control and in-process measurements of biomanufactured Cell and Gene Therapy products. However, it contains a number of sources of measurement variation at; sample preparation, instrument setup, analysis, and post-analytical data analysis stages. The latter sees variation introduced from operator subjectivity, which is investigated here to understand what effects the interpretation of diagrammatical protocols have on inter-operator analysis. 36 operators from different sites were given a series of histograms to analyse, gating a shifting peak. This was repeated with diagrammatical protocols to apply gates which reduced inter-operator variation by up to 92%. Various control limits include and exclude different results and when adjusted with a log transform differences in outlier discrimination have been found. This research supports the use of Flow Cytometry diagrammatical protocols to reduce the contribution of inter-operator variation and measurement uncertainty associated within Cell and Gene Therapy manufacturing scenarios

Quantifying operator subjectivity within flow cytometry data analysis as a source of measurement uncertainty and the impact of experience on results

Flow cytometry is a complex measurement characterization technique, utilized within the manufacture, measurement, and release of cell and gene therapy products for rapid, high-content, and multiplexed discriminatory cell analysis. A number of factors influence the variability in the measurement reported including, but not limited to, biological variation, reagent variation, laser and optical configurations, and data analysis methods. This research focused on understanding the contribution of manual operator variability within the data analysis phase. Thirty-eight participants completed a questionnaire, providing information about experience and motivational factors, before completing a simple gating study. The results were analyzed using gauge repeatability and reproducibility techniques to quantify participant uncertainty. The various stages of the gating sequence were combined through summation in quadrature and expanded to give each participant a representative uncertainty value. Of the participants surveyed, 85% preferred manual gating to automated data analysis, with the primary reasons being legacy (“it’s always been done that way”) and accuracy, not in the metrological sense but in the clear definition of the correct target population. The median expanded uncertainty was calculated as 3.6% for the population studied, with no significant difference among more or less experienced users. Operator subjectivity can be quantified to include within measurement uncertainty budgets, required for various standards and qualifications. An emphasis on biomanufacturing measurement terminology is needed to help understand future and potential solutions, possibly looking at translational clinical models to engage and enhance better training and protocols within industrial and research settings