Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design
Flow Cytometry Core, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland.Cytometry Part A (Impact Factor: 2.93). 03/2013; 83A(3). DOI: 10.1002/cyto.a.22251
After compensation, the measurement errors arising from multiple fluorescences spilling into each detector become evident by the spreading of nominally negative distributions. Depending on the instrument configuration and performance, and reagents used, this "spillover spreading" (SS) affects sensitivity in any given parameter. The degree of SS had been predicted theoretically to increase with measurement error, i.e., by the square root of fluorescence intensity, as well as directly related to the spectral overlap matrix coefficients. We devised a metric to quantify SS between any pair of detectors. This metric is intrinsic, as it is independent of fluorescence intensity. The combination of all such values for one instrument can be represented as a spillover spreading matrix (SSM). Single-stained controls were used to determine the SSM on multiple instruments over time, and under various conditions of signal quality. SSM values reveal fluorescence spectrum interactions that can limit the sensitivity of a reagent in the presence of brightly-stained cells on a different color. The SSM was found to be highly reproducible; its non-trivial values show a CV of less than 30% across a 2-month time frame. In addition, the SSM is comparable between similarly-configured instruments; instrument-specific differences in the SSM reveal underperforming detectors. Quantifying and monitoring the SSM can be a useful tool in instrument quality control to ensure consistent sensitivity and performance. In addition, the SSM is a key element for predicting the performance of multicolor immunofluorescence panels, which will aid in the optimization and development of new panels. We propose that the SSM is a critical component of QA/QC in evaluation of flow cytometer performance. Published 2013 Wiley- Periodicals, Inc.
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ABSTRACT: 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 Bcal and the latter we define as Bsos. The combination of instrument and experiment background is defined as Btot. Importantly, the Btot will affect the degree of panel separation, therefore the greater the degree of Btot 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 Btot 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 Btot 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 Btot values for all parameters, but new technology to allow these measurements will likely be introduced in the near future. Nonetheless, Q and Btot 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.
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ABSTRACT: Fluorescence-labeled peptide-MHC class I multimers serve as ideal tools for the detection of antigen-specific T cells by flow cytometry, enabling functional and phenotypical characterization of specific T cells at the single cell level. While this technique offers a number of unique advantages, MHC multimer reagents can be difficult to handle in terms of stability and quality assurance. The stability of a given fluorescence-labeled MHC multimer complex depends on both the stability of the peptide-MHC complex itself and the stability of the fluorochrome. Consequently, stability is difficult to predict and long-term storage is generally not recommended. We investigated here the possibility of cryopreserving MHC multimers, both in-house produced and commercially available, using a wide range of peptide-MHC class I multimers comprising virus and cancer-associated epitopes of different affinities presented by various HLA-class I molecules. Cryopreservation of MHC multimers was feasible for at least 6 months, when they were dissolved in buffer containing 5–16% glycerol (v/v) and 0.5% serum albumin (w/v). The addition of cryoprotectants was tolerated across three different T-cell staining protocols for all fluorescence labels tested (PE, APC, PE-Cy7 and Quantum dots). We propose cryopreservation as an easily implementable method for stable storage of MHC multimers and recommend the use of cryopreservation in long-term immunomonitoring projects, thereby eliminating the variability introduced by different batches and inconsistent stability. © 2014 International Society for Advancement of Cytometry
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