Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

Gaucher, J.C.; Grünwald, David; Frelat, G.

doi:10.1002/cyto.990090608

Cited by 9 publications

(14 citation statements)

References 8 publications

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“…Two previous reports by Gaucher et al (1) and Steen (7) carefully characterized flow cytometers in terms of Q and B and discussed measures of sensitivity. Both investigations used dim light flashes from an LED to measure the optical noise contribution from photoelectron statistics.…”

Section: Comparison With Previous Workmentioning

confidence: 99%

“…If the average number of photoelectrons is n, then the standard deviation SD e at the photocathode is SD e ϭ ͱn (1) The signal processing converts photoelectrons to a linear channel in a histogram with a gain factor G. Note that the gain defined here includes all aspects of the signal conversion process not simply PMT and amplifier gain. In the process of amplification by the amount G, the noise is smoothed to a normal distribution with standard deviation still determined by the original Poisson statistics.…”

Section: Theoretical Considerationsmentioning

confidence: 99%

“…Previous studies by Pinkel and Steen (4) and Steen (7) and by Gaucher et al (1) provide a rigorous theoretical and empirical basis for thoroughly analyzing the fluorescence sensitivity of a flow cytometer. This report builds on their work, analyzes the factors that most affect practical measurement of Q and B, and describes simple methods for determining Q and B.…”

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confidence: 98%

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Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity

Chase¹,

Hoffman

1998

Cytometry

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Section: Comparison With Previous Workmentioning

confidence: 99%

Section: Theoretical Considerationsmentioning

confidence: 99%

mentioning

confidence: 98%

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Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity

Chase¹,

Hoffman

1998

Cytometry

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“…To address the quantitative characterization of fluorescence sensitivity, the underlying physics of optical detectors needs to be taken into account (Wood and Hoffman, 1998). A theoretical model for the CV of dimly fluorescent particles (Gaucher et al, 1988;Steen, 1992) can be developed based on optical detection efficiency, Q (number of photoelectrons per fluorochrome molecule analyzed), and background light, B. B can be expressed in units of the equivalent number of fluorochrome molecules that would produce that background.…”

Section: The Q and B Methods To Characterize Instrument Sensitivitymentioning

confidence: 99%

Standardization, Calibration, and Control in Flow Cytometry

Hoffman

2005

CP Cytometry

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Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions are provided for common terms to alert the reader to critical distinctions in meaning. The unit presents extensive background on particle types and cautions and goes on to describe practical aspects of methods to standardize and calibrate instruments, in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration.

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“…Inter-instrument differences can be substantial, complicating staining panel design, and efforts to standardize multi-cytometer or multicenter studies (6,7). Fortunately, the factors that drive these differences can be quantified to predict staining panel success across multiple instruments by measuring the sensitivity and resolution of each detector on each instrument.…”

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confidence: 99%

Q and B values are critical measurements required for inter‐instrument standardization and development of multicolor flow cytometry staining panels

Perfetto¹,

Chattopadhyay²,

Wood

et al. 2014

Cytometry Pt A

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Much of the complexity of multicolor flow cytometry experiments lies within the development of antibody staining panels and the standardization of instruments. In this article, we propose a theoretical metric and describe how measurements of sensitivity and resolution can be used to predict the success of panels, and ensure that performance across instruments is standardized (i.e., inter-instrument standardization). Sensitivity can be determined by summing two major contributors of background, background originating from the instrument (optical noise and electronic noise) and background due to the experimental conditions (i.e., Raman scatter, and spillover spreading arising from other fluorochromes in the panel). The former we define as B cal and the latter we define as B sos . The combination of instrument and experiment background is defined as B tot . Importantly, the B tot will affect the degree of panel separation, therefore the greater the degree of B tot the lower the separation potential. In contrast, resolution is a measure of separation between populations. Resolution is directly proportional to the number of photoelectrons generated per molecule of excited fluorochrome and is known as the "Q" value. Q and B tot values can be used to define the performance of each detector on an instrument and together they can be used to calculate a separation index. Hence, detectors with known Q and B tot values can be used to evaluate panel success based on the detector specific separation index. However, the current technologies do not enable measurements of Q and B tot values for all parameters, but new technology to allow these measurements will likely be introduced in the near future. Nonetheless, Q and B tot measurements can aid in panel development, and reveal sources of instrument-to-instrument variation in panel performance. In addition, Q and B values can form the basis for a comprehensive and versatile quality assurance program. Published 2014 Wiley Periodicals Inc. Key termsinter-instrument quality control and standardization; Q and B values; panel development; panel success; panel design POLYCHROMATIC flow cytometry provides a powerful means to dissect cellular characteristics and function (1). The technology is limited, however, by the complexity of experimental design. Cell surface and intracellular proteins must be detected using carefully titrated antibodies conjugated to fluorescent dyes. The optimal combination of these antibody-fluorochrome conjugates must be determined empirically through iterative experiments (2). This process typically requires months of experimentation, while only limited guidance is available. Some approaches focus on the spectral overlap of fluorochromes but these approaches are flawed. Cell populations are often easily discriminated even when spectral overlap (compensation) is high

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Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

Cited by 9 publications

References 8 publications

Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity

Resolution of dimly fluorescent particles: A practical measure of fluorescence sensitivity

Standardization, Calibration, and Control in Flow Cytometry

Q and B values are critical measurements required for inter‐instrument standardization and development of multicolor flow cytometry staining panels

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