Assessment of Automated Flow Cytometry Data Analysis Tools within Cell and Gene Therapy Manufacturing.

Flow cytometry is widely used within the manufacturing of cell and gene therapies to measure and characterise cells. Conventional manual data analysis relies heavily on operator judgement, presenting a major source of variation that can adversely impact the quality and predictive potential of therapies given to patients. Computational tools have the capacity to minimise operator variation and bias in flow cytometry data analysis; however, in many cases, confidence in these technologies has yet to be fully established mirrored by aspects of regulatory concern.

Systematic Design, Generation, and Application of Synthetic Datasets for Flow Cytometry.

Application of synthetic datasets in training and validation of analysis tools has led to improvements in many decision-making tasks in a range of domains from computer vision to digital pathology. Synthetic datasets overcome the constraints of real-world datasets, namely difficulties in collection and labeling, expense, time, and privacy concerns. In flow cytometry, real cell-based datasets are limited by properties such as size, number of parameters, distance between cell populations, and distributions and are often focused on a narrow range of disease or cell types.

Pragmatic Micrometre to Millimetre Calibration Using Multiple Methods for Low-Coherence Interferometer in Embedded Metrology Applications.

In-situ metrology utilised for surface topography, texture and form analysis along with quality control processes requires a high-level of reliability. Hence, a traceable method for calibrating the measurement system's transfer function is required at regular intervals. This paper compares three methods of dimensional calibration for a spectral domain low coherence interferometer using a reference laser interferometer versus two types of single material measure.

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 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.

Current trends in flow cytometry automated data analysis software.

Automated flow cytometry (FC) data analysis tools for cell population identification and characterization are increasingly being used in academic, biotechnology, pharmaceutical, and clinical laboratories. The development of these computational methods is designed to overcome reproducibility and process bottleneck issues in manual gating, however, the take-up of these tools remains (anecdotally) low. Here, we performed a comprehensive literature survey of state-of-the-art computational tools typically published by research, clinical, and biomanufacturing laboratories for automated FC data analysis and identified popular tools based on literature citation counts.

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.

Establishing the variation of blood metrics within UK Biobank to inform cell therapy manufacturing processes.

Understanding blood component variation as a function of healthy population metrics is necessary to inform biomanufacturing process design. UK Biobank metrics were examined for variation in white blood cell count as an analog to potential manufacturing starting material input. White blood cell count variation of four orders of magnitude (6.

Focus variation microscope: linear theory and surface tilt sensitivity.

In a recent publication [3rd International Conference on Surface Metrology, Annecy, France, 2012, p. 1] it was shown that surface roughness measurements made using a focus variation microscope (FVM) are influenced by surface tilt. The effect appears to be most significant when the surface has microscale roughness (Ra≈50  nm) that is sufficient to provide a diffusely scattered signal that is comparable in magnitude to the specular component.

A meta-analysis of biological variation in blood-based therapy as a precursor to bio-manufacturing.

Currently cellular therapies, such as hematopoietic stem cell transplantation (HSCT), are produced at a small scale on a case-by-case basis, usually in a clinical or near-clinical setting. Meeting the demand for future cellular therapies will require a robust and scalable manufacturing process that is either designed around or controls the variation associated with biological starting materials. Understanding variation requires both a measure of the allowable variation (that does not negatively affect patient outcome) and the achievable variation (with current technology).

Computer-aided lens assembly.

We propose a computer-aided method of lens manufacture that allows assembly, adjustment, and test phases to be run concurrently until an acceptable level of optical performance is reached. Misalignment of elements within a compound lens is determined by a comparison of the results of physical ray tracing by use of an array of Gaussian laser beams with numerically obtained geometric ray traces. An estimate of misalignment errors is made, and individual elements are adjusted in an iterative manner until performance criteria are achieved.

Nonperturbing measurements of spatially distributed underwater acoustic fields using a scanning laser Doppler vibrometer.

Localized changes in the density of water induced by the presence of an acoustic field cause perturbations in the localized refractive index. This relationship has given rise to a number of nonperturbing optical metrology techniques for recording measurement parameters from underwater acoustic fields. A method that has been recently developed involves the use of a Laser Doppler Vibrometer (LDV) targeted at a fixed, nonvibrating, plate through an underwater acoustic field.

Sensitivity errors in interferometric deformation metrology.

Interferometric measurement techniques such as holographic interferometry and electronic speckle-pattern interferometry are valuable for measuring the deformation of objects. Conventional theoretical models of deformation measurement assume collimated illumination and telecentric imaging, which are usually only practical for small objects. Large objects often require divergent illumination, for which the models are valid only when the object is planar, and then only in the paraxial region.

Synthetic aperture interferometry: in-process measurement of aspheric optics.

A scanning probe consisting of a source and receive fiber pair is used to measure the phase difference between wave fronts scattered from the front and rear surfaces of an aspheric optic. This system can be thought of as a classical interferometer with an aperture synthesized from the data collected along the path of the probe. If the form of either surface is known, the other can be deduced.