Determination of background, signal‐to‐noise, and dynamic range of a flow cytometer: A novel practical method for instrument characterization and standardization

Abstract

A well-defined scale calibration in flow cytometry can improve many aspects of data acquisition such as cytometer setup, instrument comparison and sample comparison. The theory for scale calibration was proposed by Steen over two decades ago, but it has never been put into regular use due to the lack of a widely available precision light source. The introduction of such a light source, the quantiFlash , gave this possibility. Here, we describe how this light source can be used to characterize a cytometer's PMT performance. We, therefore, characterized the instrument's response over the entire PMT voltage range. As a consequence, we propose a practical method to characterize a cytometer's signal-to-noise ratio (SNR) and dynamic range (DNR). This allows the selection of a voltage/gain corresponding to a PMT's maximum efficiency and hence the lowest electronic noise, which can help with experiment design. We further introduced a decibel (dB) scale for the presentation of SNR and DNR values. SNR and DNR are stand-alone values that allow the direct comparison of different instruments. Finally, with this method, it becomes clear that increased SNR comes at the expense of DNR and thus the limiting factor of modern cytometers is the DNR. © 2017 International Society for Advancement of Cytometry.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…After installation through a service engineer or exchange of components (e.g., lasers, filters, or PMTs), the status of the instrument is documented in a so‐called “baseline.” A lot of information (not all of it is listed here) about the linear range of each PMT (important for proper measurement and compensation (see Chapter II Section 1.3 Measuring SOVs/compensation controls), electronic noise and background (B r , SD EN ), detector efficiency (Q r ), as well as sensitivity (Peak ratio between negative and positive population) and quality of laser alignment (%rCV) is stored in this file. All the introduced values are summarized in Table with a very brief explanation and are described in much greater detail elsewhere .…”

Comments on “Determination of Background, Signal‐to‐Noise, and Dynamic Range of a Flow Cytometer: A Novel Practical Method for Instrument Characterization and Standardization”

“…The corresponding dot plots for the pulse area data are shown in Figure 2B(i), (iii), and (v) (similar to fig. 3C(iii)); . All of the linear scaled and biexponential scaled pulse peak and pulse area dot plots show nearly “spherical” uncorrelated 2D Gaussian distributions.…”

“…Published in November 2017, the publication “Determination of Background, Signal‐to‐Noise, and Dynamic Range of a Flow Cytometer: A Novel Practical Method for Instrument Characterization and Standardization” by Giesecke et al appeared in Cytometry Part A. Over the last 2 years, the publication has generated many questions by users who have been unsuccessful in repeating the procedures described. They have repeatedly turned to me and others to help clarify what they might be doing wrong.…”

Phosphoflow cytometry and barcoding in blood platelets: Technical and analytical considerations

“…CytoFLEX Daily QC Fluorospheres were used to check the cytometer's optical alignment and fluidic stability. Gain settings were optimized with an LED pulser (quantiFlash) . Automatic compensation was performed with BD CompBeads, and FCS files were analyzed on Cytobank.…”