Dataset Information

Evaluating flow cytometer performance with weighted quadratic least squares analysis of LED and multi-level bead data.

ABSTRACT: We developed a fully automated procedure for analyzing data from LED pulses and multilevel bead sets to evaluate backgrounds and photoelectron scales of cytometer fluorescence channels. The method improves on previous formulations by fitting a full quadratic model with appropriate weighting and by providing standard errors and peak residuals as well as the fitted parameters themselves. Here we describe the details of the methods and procedures involved and present a set of illustrations and test cases that demonstrate the consistency and reliability of the results. The automated analysis and fitting procedure is generally quite successful in providing good estimates of the Spe (statistical photoelectron) scales and backgrounds for all the fluorescence channels on instruments with good linearity. The precision of the results obtained from LED data is almost always better than that from multilevel bead data, but the bead procedure is easy to carry out and provides results good enough for most purposes. Including standard errors on the fitted parameters is important for understanding the uncertainty in the values of interest. The weighted residuals give information about how well the data fits the model, and particularly high residuals indicate bad data points. Known photoelectron scales and measurement channel backgrounds make it possible to estimate the precision of measurements at different signal levels and the effects of compensated spectral overlap on measurement quality. Combining this information with measurements of standard samples carrying dyes of biological interest, we can make accurate comparisons of dye sensitivity among different instruments. Our method is freely available through the R/Bioconductor package flowQB. © 2017 International Society for Advancement of Cytometry.

SUBMITTER: Parks DR

PROVIDER: S-EPMC5483398 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Evaluating flow cytometer performance with weighted quadratic least squares analysis of LED and multi-level bead data.

Parks David R DR El Khettabi Faysal F Chase Eric E Hoffman Robert A RA Perfetto Stephen P SP Spidlen Josef J Wood James C S JC Moore Wayne A WA Brinkman Ryan R RR

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20170203 3

We developed a fully automated procedure for analyzing data from LED pulses and multilevel bead sets to evaluate backgrounds and photoelectron scales of cytometer fluorescence channels. The method improves on previous formulations by fitting a full quadratic model with appropriate weighting and by providing standard errors and peak residuals as well as the fitted parameters themselves. Here we describe the details of the methods and procedures involved and present a set of illustrations and test ...[more]

PMID: 28160404

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Project description:A CT scanner measures the energy that is deposited in each channel of a detector array by x rays that have been partially absorbed on their way through the object. The measurement process is complex and quantitative measurements are always and inevitably associated with errors, so CT data must be preprocessed prior to reconstruction. In recent years, the authors have formulated CT sinogram preprocessing as a statistical restoration problem in which the goal is to obtain the best estimate of the line integrals needed for reconstruction from the set of noisy, degraded measurements. The authors have explored both penalized Poisson likelihood (PL) and penalized weighted least-squares (PWLS) objective functions. At low doses, the authors found that the PL approach outperforms PWLS in terms of resolution-noise tradeoffs, but at standard doses they perform similarly. The PWLS objective function, being quadratic, is more amenable to computational acceleration than the PL objective. In this work, the authors develop and compare two different methods for implementing PWLS sinogram restoration with the hope of improving computational performance relative to PL in the standard-dose regime. Sinogram restoration is still significant in the standard-dose regime since it can still outperform standard approaches and it allows for correction of effects that are not usually modeled in standard CT preprocessing.The authors have explored and compared two implementation strategies for PWLS sinogram restoration: (1) A direct matrix-inversion strategy based on the closed-form solution to the PWLS optimization problem and (2) an iterative approach based on the conjugate-gradient algorithm. Obtaining optimal performance from each strategy required modifying the naive off-the-shelf implementations of the algorithms to exploit the particular symmetry and sparseness of the sinogram-restoration problem. For the closed-form approach, the authors subdivided the large matrix inversion into smaller coupled problems and exploited sparseness to minimize matrix operations. For the conjugate-gradient approach, the authors exploited sparseness and preconditioned the problem to speed up convergence.All methods produced qualitatively and quantitatively similar images as measured by resolution-variance tradeoffs and difference images. Despite the acceleration strategies, the direct matrix-inversion approach was found to be uncompetitive with iterative approaches, with a computational burden higher by an order of magnitude or more. The iterative conjugate-gradient approach, however, does appear promising, with computation times half that of the authors' previous penalized-likelihood implementation.Iterative conjugate-gradient based PWLS sinogram restoration with careful matrix optimizations has computational advantages over direct matrix PWLS inversion and over penalized-likelihood sinogram restoration and can be considered a good alternative in standard-dose regimes.

Dataset Information

Evaluating flow cytometer performance with weighted quadratic least squares analysis of LED and multi-level bead data.

Publications

Evaluating flow cytometer performance with weighted quadratic least squares analysis of LED and multi-level bead data.

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