Characterization of Flow Cytometer Instrument Sensitivity

Hoffman, R. A.; Wood, James C. S.

doi:10.1002/0471142956.cy0120s40

Cited by 44 publications

(71 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Fixed nuclei are used as standards for DNA measurements or tools for linearity of fluorescence verification (2,3). Stabilized cells can potentially be used as controls and calibrators (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). One approach being developed using CD4 T cells for immunofluorescence (see below), which assumes the antigen expression is truly stable across individuals and stability of the product is well validated (18,19).…”

Section: Fluorescencementioning

confidence: 99%

“…In the first cell calibrator approach, a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g. CD4 binding sites) (4,5) can be used to translate the linear fluorescence intensity scale to an ABC scale. It is highly recommended that a single clone of the antibody amenable to labeling with different types of fluorophores associated with various fluorescence channels be used for the scale conversion.…”

Section: Linear Scale To Biological Scale Transformation Using Biologmentioning

confidence: 99%

See 1 more Smart Citation

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part III – analytical issues

Davis¹,

Dasgupta

Kussick

et al. 2013

Cytometry Part B Clinical

112

View full text Add to dashboard Cite

Section: Fluorescencementioning

confidence: 99%

Section: Linear Scale To Biological Scale Transformation Using Biologmentioning

confidence: 99%

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part III – analytical issues

Davis¹,

Dasgupta

Kussick

et al. 2013

Cytometry Part B Clinical

112

View full text Add to dashboard Cite

“…In contrast to light scatter, flow cytometer fluorescence intensity measurements and performance are readily calibrated in absolute units to facilitate inter-lab standardization (21)(22)(23). Fluorescence-based approaches can involve the use of a specific fluorescent antibody to trigger the detection of a particular sub-set of EVs, but a more general approach is to use a fluorescence membrane probe to trigger detection of any membrane bound nanoparticle (24,25).…”

mentioning

confidence: 99%

High sensitivity flow cytometry of membrane vesicles

et al. 2015

View full text Add to dashboard Cite

Extracellular vesicles (EVs) are attracting attention as vehicles for inter-cellular signaling that may have value as diagnostic or therapeutic targets. EVs are released by many cell types and by different mechanisms, resulting in phenotypic heterogeneity that makes them a challenge to study. Flow cytometry is a popular tool for characterizing heterogeneous mixtures of particles such as cell types within blood, but the small size of EVs makes them difficult to measure using conventional flow cytometry. To address this limitation, a high sensitivity flow cytometer was constructed and EV measurement approaches that allowed them to enumerate and estimate the size of individual EVs, as well as measure the presence of surface markers to identify phenotypic subsets of EVs. Several fluorescent membrane probes were evaluated and it was found that the voltage sensing dye di-8-ANEPPS could produce vesicle fluorescence in proportion to vesicle surface area, allowing for accurate measurements of EV number and size. Fluorescence-labeled annexin V and anti-CD61 antibody was used to measure the abundance of these surface markers on EVs in rat plasma. It was shown that treatment of platelet rich plasma with calcium ionophore resulted in an increase in the fraction of annexin V and CD61-positive EVs. Vesicle flow cytometry using fluorescence-based detection of EVs has the potential to realize the potential of cell-derived membrane vesicles as functional biomarkers for a variety of applications. V C 2015 International Society for Advancement of Cytometry

show abstract

“…Instrument optimization is crucial because Q and B tot values for each detector reflect the quality of the fluorescence optical pathway, which consists of the laser used to generate photons, the filters used to separate specific wavelengths, and the detectors that convert photon measurements into photoelectron pulses. Reduced Q values and/or increased B tot values affect the ability to distinguish bright and dim staining from background (11,12). Q and B tot values can be used to monitor an instrument's performance; however, in this study we also demonstrate the value of these measurements in predicting performance of putative staining panels.…”

mentioning

confidence: 67%

“…The formula, as described here, was modified from the separation parameter described by Hoffman and Wood in 2007 (11). Specifically, SI ps can be defined as the "distance" needed to separate a labeled cell population from the minimum level of detection in a sample stained with multiple mAb-conjugates.…”

Section: Discussionmentioning

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

View full text Add to dashboard Cite

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

show abstract

Characterization of Flow Cytometer Instrument Sensitivity

Cited by 44 publications

References 8 publications

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part III – analytical issues

Validation of cell‐based fluorescence assays: Practice guidelines from the ICSH and ICCS – part III – analytical issues

High sensitivity flow cytometry of membrane vesicles

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

Contact Info

Product

Resources

About