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- MATERIALS AND METHODS
- LITERATURE CITED
Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures for routine, clinical flow cytometry. We investigated the effect of light exposure, handling, and storage conditions on the fluorescence characteristics of some common phycoerythrin tandem fluorochromes. In general, PE-Cy5 showed the lowest degradation rates, whereas PE-Cy7 showed the highest. During storage, long-term degradation rates were lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate decreased to 0.3%/month. As degradation rates were minimized, we studied the effect of slow degradation of a set of tandem dye conjugates on compensation matrix values over several months. Finally, we explored the effect of slow degradation on flow cytometric analysis using the same compensation settings for extended periods for an analysis template with preset regions and gating strategies. © 2009 International Society for Advancement of Cytometry
THE emission spectra of most fluorochromes are fairly constant. The conjugation of fluorochromes to proteins (e.g., antibodies) may affect their quantum efficiency (i.e., absolute fluorescence intensity) but shifts their entire spectrum by only a few nanometers (1). In contrast, tandem dyes are known to display a considerable degree of variability in fluorescence intensities within different regions of each emission spectrum. A tandem dye is a combination of two fluorochromes (donor and acceptor dye) such that the difference between the excitation and emission maxima (Stokes shift) is significantly increased relative to that of the donor dye. The higher Stokes shift is accomplished by means of fluorescence resonance energy transfer (FRET) between the donor molecule (e.g., phycoerythrin) and acceptor molecule (e.g., cyanine 7) (2–5). The optimum energy transfer level depends on the acceptor-to-donor molar ratio, distance, and orientation and results in significant reduction of donor-derived emission, while self-quenching of the acceptor is avoided (3, 6, 7). The delicate balance between donor and acceptor is the reason that emission spectra of tandem dyes are affected by manufacturing and protein conjugation processes as well as by an apparent instability of fluorescence characteristics. The instability (i.e., degradation) of tandem dyes is caused by photon-induced oxidation and results in relative short reagent shelf-lives. Despite the widely accepted notion that tandem dyes demand protection from light exposure to minimize changes in fluorescence characteristics (4, 6, 8–12), essentially no literature is available that describes these changes quantitatively.
A better understanding of how specific conditions affect the stability of tandem dye conjugates can lead to considerable improvements in the quality of flow cytometric measurements. Moreover, as the number of clinical applications increases, flow cytometry can be expected to focus on reducing operator-dependent processes. Automation of equipment setup procedures is one of the many important steps required for flow cytometry to become a standard technology that can be reliably and reproducibly used in a clinical setting.
While new fluorescent reagents are being developed, tandem dye conjugates are widely used in multispectral flow cytometry. Information about the expected rates of tandem dye degradation may be used to adopt more efficient spectral compensation setup procedures and could possibly reduce the frequency at which compensation settings require adjustment (13).
In this study, we investigated fluorescence characteristics of some common protein-conjugated phycoerythrin (PE) tandem dyes under various conditions of light exposure during typical cell-labeling procedures. Changes in FRET efficiency during postpurchase storage conditions were determined on particle-bound tandem dye conjugates to assess the challenges that might be encountered during spectral compensation when using compensation particles (14). A specific packaging strategy, designed to avoid any exposure to light, was tested and compared with a typical packaging method. We used our findings to follow changes in compensation matrix values and then determined the impact of these changes on flow cytometric data analysis. This study provides tools to extend the shelf-life of tandem dye conjugates and possibly minimize sample to sample variability.
MATERIALS AND METHODS
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- MATERIALS AND METHODS
- LITERATURE CITED
To assess changes in PE leakage (i.e., FRET efficiency) among a variety of tandem dye conjugates that are typically used in a flow cytometry laboratory, a total of 18 PE-tandem dye conjugated products were obtained from BD Bioscience (San Jose, CA), Beckman Coulter, Inc. (Miami, FL), BioLegend (San Diego, CA), eBioscience (San Diego, CA), Invitrogen (Carlsbad, CA), and Serotec (Raleigh, NC). Each tandem dye conjugate was bound to protein G-coated polystyrene particles (Spherotech, Inc., Lake Forest, IL) prepared in polystyrene tubes at ambient temperature in PuraFlow sheath fluid (Beckman Coulter, Inc).
Custom Lot numbers of biotin-coated polystyrene particles (Spherotech Inc.) irreversibly bound to streptavidin-conjugated PE-Texas Red (PE-TxRd, BD Bioscience), -PE, -PE-Cy5, -PE-Cy7 (eBioscience), or -PE-Alexa Fluor® 750 (Invitrogen) were individually dispensed in storage buffer. The storage buffer consisted of 0.016 molar phosphate buffered saline (pH 7.4) with 0.02% sodium azide and 0.2% bovine serum albumin. One set of reagents was dispensed into solid white vials and stored in a refrigerator for general laboratory use. A second set was dispensed in many subsets of single-use aliquots into solid brown vials, packaged in light-tight bags, and stored in a dedicated refrigerator that was only opened to obtain samples at times of measurements.
Measurements of relative fluorescence intensities in the short-term stability tests were performed on a refurbished FACScan (488 nm excitation, 585/42 BP and 650 LP detection filters; Cytek, Fremont, CA). Light exposure was measured at the location of the test samples with a Luna-Star F light meter (Gossen, Nürnberg, Germany). Exposure values (EV) were converted to Lux according to Lux = 2.5 × 2EV.
Measurements of relative fluorescence intensity in the long-term stability tests were performed monthly (white vial packaged reagents for up to 155 days, stricter light-protected reagents for up to 196 days) on a customized LSR-II equipped with 532 nm laser excitation (BD Bioscience). PE, PE-TxRd, PE-Cy5, and PE-Cy7 (as well as PE-Alexa Fluor® 750) emissions were detected through a 575/25, 625/30, 660/40, and 780/60 bandpass filters, respectively. Quality control assays to assess the reproducibility and stability of the flow cytometer were performed with eight-peak Rainbow particles (RCP-30-5A, Spherotech Inc.).
PE leakage was determined as the amount of donor (PE) dye emission measured relative to that of the acceptor dye (e.g., Cy7). Tandem dye degradation was expressed as the increase in the PE leakage over a period of time. Degradation rates were calculated based on the slope of the line that fitted the data points plotted in a PE leakage versus time graph.
Matrix values for spectral compensation matrices were determined using FlowJo (Tree Star, Inc., Ashland, OR).
To simulate sample analysis using the same compensation settings over an extended period of time, a single cell sample labeled with four colors (PE, PE-TxRd, PE-Cy5, and PE-Cy7) was used to generate an analysis template with preset, fixed regions. Hence, biological variability and variability due to sample preparation were avoided. The four-color-labeled cell sample was prepared using the mononuclear fraction of normal human peripheral blood. After two wash steps, cells were labeled with PE-conjugated mouse anti-human CD127 (Biolegend, San Diego, CA), PE-TxRd-conjugated mouse anti-human CD4 (Invitrogen), PE-Cy5-conjugated mouse anti-human CD25 (Biolegend), and PE-Cy7-conjugated mouse anti-human CD8 (BD Biosciences) monoclonal antibodies for 15 min at ambient temperature, protected from light. The same cells were used to prepare one unstained, four singly positive, and four Full-Minus-One control samples.
Listmode files acquired at various time points of tandem dye degradation were used to establish individual time point derived spectral compensation matrices. Listmode files of the singly-positive controls were used to establish values for a spectral compensation matrix that represents the appropriate matrix for analysis of the four-color-labeled cell sample. Data from the Rainbow particles controls were used to correct all measurements for day-to-day variability in instrument performance. These corrected values in the compensation matrices were adjusted for differences in reagents used in the particle samples and those used in the cell sample. An adjustment factor was determined by calculating the ratios between the cell sample-derived compensation matrix values and those of the first time point (Day 1) derived matrix. This set of adjustment factors was then applied to each time point derived matrix to correctly reflect the changes as they would occur in the cell sample-derived matrix.
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- MATERIALS AND METHODS
- LITERATURE CITED
Instability and variability of tandem dye conjugated products are well recognized problems in flow cytometry. Consequently, clinical laboratories and core facilities are required to perform specific Quality Assurance/Quality Control (QA/QC) procedures to ensure accurate sample analysis (15, 16). In addition, the dynamic fluorescence characteristics of these products contribute to the necessity of establishing spectral compensation values at frequent time intervals and limiting the use of analysis templates with preset regions and gating strategies. Limiting variability and increasing stability are important steps toward reproducibility and, by extension, toward simplifying QA/QC and equipment setup procedures.
Our results indicate that PE-Cy5 conjugates are least sensitive to light exposure, PE-Cy7 conjugates are the most sensitive, and PE-TxRd falls in between. Given that degradation of these tandem dyes can occur within minutes of ambient light exposure, a substantial degree of variability can be introduced to a set of samples when cell-labeling procedures involve large numbers of tubes or under other circumstances where light protective measures are difficult to maintain.
We show how handling PE-Cy7 conjugates in a dimly lit environment considerably increases its stability (up to fivefold). The results suggest that variability as introduced during sample preparation can be greatly reduced when sample preparation takes place in a dark laminar flow hood while ambient light in the laboratory is kept on.
Our tests also illustrate the degree of variability in light sensitivity typically present within a small group of commercially available PE-tandem dye conjugates. Variability is at least in part due to differences in manufacturing processes that are applied to create the optimal ratio between donor and acceptor dye. As mentioned elsewhere (17), it therefore appears virtually impossible to manufacture, so called, perfectly matched isotype controls for tandem dye conjugated antibodies. Furthermore, differences in subsequent protein conjugation processes contribute to variability through differences in the ratio between the number of fluorochrome and protein molecules (F/P ratio). We were unable to obtain the F/P ratio of most reagents used in this study. In the few cases where F/P ratios were obtained, the ranges given were so large (e.g., between 0.5 and 1.5) that they did not add clarity to our findings. The introduction of stricter product release criteria might significantly reduce the current level of variability among tandem dye products albeit at possibly high costs.
Although we have taken multiple measures to protect the PE-tandem dye conjugates from light, we, like others, show the importance of light-protective packaging to minimize degradation of tandem dyes during shipping and storage (12). In addition, we show that degradation of conjugated PE-tandem dyes may still occur at a low rate (generally <1%/month) when stored in a refrigerated, light-free environment. However, more batches and different conjugated products will need to be tested to determine whether tandem dye degradation under these light-protective storage conditions is a general phenomenon.
Currently, it is uncertain to what extent either single-use aliquots or a dedicated storage place minimizes degradation of PE-tandem dyes. Even though it is likely that single-use aliquots have an advantage over multiuse aliquots because of less exposure to light and oxygen, we expect that the light-protective color of the vial contributes the most to stability (12). Additional studies with larger samples will have to be performed to address these aspects definitively, and our results demonstrate the importance of minimizing light exposure.
As reported by Agrawal et al. (13), compensation settings may not always need adjustment when detector voltage and cell type remain unchanged. Certainly, this would also require a stable flow cytometer in which the overall performance remains unchanged. Furthermore, it is essential that the emission spectra of fluorochromes are stable. Therefore, we assessed stability of the tandem dye emission spectra and methods to maximize it. We have been able to reduce tandem dye degradation to a level where spectral compensation is barely affected. This opens up opportunities to explore the use of predetermined compensation matrices and analysis templates with preset regions and gating strategies. The ability to use such simplified approach for clinical samples might allow the more widespread use of flow cytometry in a clinical setting, at least for well separated cell populations. Our examples of analysis templates indicate that the use of a predetermined compensation matrix can be successful for periods of up to 196 days when the populations of interest are widely separated (CD4+ or CD8+ cells) on the condition that the flow cytometer is stable. However, we suspect that variability in day-to-day performance of a typical flow cytometer does not allow applying this method. Analysis of nondiscrete populations (such as CD4+/CD25bright cells) through preset regions without adjusting previously set compensation values may quickly result in inaccuracies. Accurate analysis of nondiscrete populations is determined by the ability to distinguish “positive” from “negative” populations (17). Although analysis of nondiscrete populations using tandem dyes and predetermined compensation matrix values results in erroneous analysis, differences in operator-dependent gating can overshadow this.
Variability among tandem dye products requires that compensation settings are determined for every lot number. Unless further development of manufacturing processes of tandem dye conjugates results in products with predetermined FRET efficiencies, successful use of prelabeled compensation particles to set up spectral compensation as proposed in Ref.14 would clearly not be an easy task when PE-tandem dyes are involved.
New developments in flow cytometry equipment (18), software (19), and reagents (20) in addition to consensus on sample preparation and analysis methods can ultimately result in a significant simplification of and cost reduction in clinical sample analysis.