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Fluorescence and Light Scatter Calibration Allow Comparisons of Small Particle Data in Standard Units across Different Flow Cytometry Platforms and Detector Settings.

ABSTRACT: Flow cytometers have been utilized for the analysis of submicron-sized particles since the late 1970s. Initially, virus analyses preceded extracellular vesicle (EV), which began in the 1990s. Despite decades of documented use, the lack of standardization in data reporting has resulted in a growing body of literature that cannot be easily interpreted, validated, or reproduced. This has made it difficult for objective assessments of both assays and instruments, in-turn leading to significant hindrances in scientific progress, specifically in the study of EVs, where the phenotypic analysis of these submicron-sized vesicles is becoming common-place in every biomedical field. Methods for fluorescence and light scatter standardization are well established and the reagents to perform these analyses are commercially available. However, fluorescence and light scatter calibration are not widely adopted by the small particle community as methods to standardize flow cytometry (FCM) data. In this proof-of-concept study carried out as a resource for use at the CYTO2019 workshop, we demonstrate for the first-time simultaneous fluorescence and light scatter calibration of small particle data to show the ease and feasibility of this method for standardized FCM data reporting. This data was acquired using standard configuration commercial flow cytometers, with commercially available materials, published methods, and freely available software tools. We show that application of light scatter, fluorescence, and concentration calibration can result in highly concordant data between FCM platforms independent of instrument collection angle, gain/voltage settings, and flow rate; thus, providing a means of cross comparison in standard units. © 2020 International Society for Advancement of Cytometry.

SUBMITTER: Welsh JA

PROVIDER: S-EPMC8482305 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:An iterative method is proposed to calibrate radiation sensitivities of an arbitrary two-dimensional (2D) array of detectors. The array is irradiated with a wide open- field beam at the central position, as well as at laterally and longitudinal shifted positions; the 2D beam profile of the wide field is reconstructed iteratively from the ratios of shifted images to the central image. The propagation errors due to output variation and inaccurate array positioning are estimated and removed from the reconstructed beam profile by an error-locking scheme with narrow open-field irradiations. The beam profile is interpolated when necessary and then compared to raw detector responses to determine sensitivities. Two additional methods were implemented for comparison: 1) the commercial iterative calibration method for MapCHECK2 with translation and rotation operations; 2) a labor-intensive noniterative method without the issue of error propagation. A MapCHECK2 2D detector array was used to validate the proposed method with the 6 MV photon beam from a Varian iX linear accelerator. All calibration methods were repeated three times. A total of 5, 9, and 29 irradiations were required to implement the commercial method, the proposed method and the noniterative method respec- tively. Moreover, a 5 mm positioning error was intentionally introduced into the calibration procedures of the commercial and the proposed method to test their robustness. Under the normal operation condition of the linear accelerator and with careful alignment of the MapCHECK2, the deviations of the calibrated sensitivities of the proposed method and commercial method with respect to the noniterative method were 0.30% ± 0.29% and 0.92% ± 0.63% respectively; when the 5 mm positioning error was presented, these two methods resulted in deviations of 0.40% ± 0.36% and 3.58% ± 1.94%, respectively. A patient study suggested that, due to this 5 mm positioning error, the mean DTA (dose to agreement) passing rate by the commercial method was 2.7% lower than that by the noniterative method, whereas the proposed method led to a comparable passing rate. It is evident from this study that the proposed iterative method leads to within 1% mean calibration results to established methods. It requires much fewer number of measurements than noniterative method and is more robust against the positioning error than the commercial iterative method. The method also eliminates the need of rotation operations and, therefore, is applicable to inline detector arrays without rotation function, such as electronic portal imager device (EPID).

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Fluorescence and Light Scatter Calibration Allow Comparisons of Small Particle Data in Standard Units across Different Flow Cytometry Platforms and Detector Settings.

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