TEMPERATURE EFFECTS ON FLUOROPHORE CYTO 2014 Booth #801 CONJUGATED MICROSPHERE STANDARDS

Kathryn Kilbride, Mary Meza Bangs Laboratories, Inc., 9025 Technology Drive, Fishers, IN 46038

ABSTRACT analyses as they offer both true spectral matching Conclusions: and true environmental responsiveness. When used Background: Both internally-dyed and surface-labeled micro- as an instrument QC tool, fluctuations in quality assurance programs must spheres have their place in a comprehensive flow intensity reflect instrument-related performance, encompass both the complexity of the instruments, cytometry QC program. It is important to recognize, as well as factors in the microenvironment (buffer / themselves, and the analyses that are run. Micropar- however, that standards with surface-immobilized suspending solution). ticles that are surface conjugated with fluorophore fluorophore are more susceptible to sub-optimal play an important role in such QC programs, and their handling, including prolonged and/or repeated expo- Fluorochromes each have unique properties, and may use is central to quantitative analyses in particular. sure to room temperature. It has been demonstrated be more or less sensitive to different environmental that proper handling is essential for maintaining the factors. For example, is known to be Like stained biologic samples, surface-labeled mi- integrity of quantitative fluorescence standards. These pH-responsive, and both temperature dependency crospheres are sensitive to their environments (buffer findings argue for the careful handling and storage and sensitivity to certain buffer additives has been pH, osmolarity, temperature). This responsive nature of fluorochromes and fluorophore-labeled reagents, observed (unpublished data). Though we have found makes them ideal surrogates for stained cell samples; as well, and underscore the value of surface-labeled R- (R-PE) microspheres to offer extreme- consequently, they can be similarly susceptible to microsphere standards as cell surrogates in quality ly stable performance over time, and have observed suboptimal handling. Though formal guidelines are control programs. them to be less sensitive than fluorescein-conjugated provided for handling and storage of Quantum™ microspheres to certain buffer conditions, the effects MESF microspheres, we undertook studies to better INTRODUCTION of temperature (i.e. deviations from refrigerated stor- understand the effects of temperature on FITC and age) had not previously been fully characterized. R-PE versions of these surface-labeled standards. In flow cytometry, a comprehensive program of quality assurance and standardization is essential for achiev- MATERIALS AND METHODS Methods: ing accurate and consistent results within a study, and Three types of trials were undertaken to investigate the generating comparable data between instruments and Quantum™ MESF FITC (Cat. 555) and PE (Cat. 827) effects of different handling conditions on Quantum FITC laboratories. Microsphere standards aid in defining kits were used as the surface labeled populations and and Quantum R-PE MESF microspheres. These included: the instrument’s capabilities and limitations in terms feature one blank bead population, and four or five of sensitivity, precision and accuracy, and provide a populations surface-labeled with increasing amounts 1.) A single day study of MESF bead populations to means for ensuring that the instrument is stable and of fluorochrome. Internally-labeled Full Spectrum assess fluorescence response upon removal from suitable for use. They are also helpful in microspheres (Cat. 885) were run as controls. refrigerated (4-8°C) storage and holding at room understanding the effects of extraneous factors such temperature (RT) thereafter. as temperature, humidity, and electronic noise. Quantum™ FITC MESF microspheres 2.) A longitudinal study to assess fluorescence stability A collection of microparticles is typically enlisted to when utilized in accordance with specified guidelines. QC the full breadth of components, systems and Quantum™ PE MESF configurations of an instrument. Microspheres may microspheres 3.) A longitudinal study to assess fluorescence stability be of different sizes to approximate various cell types; of microspheres when repeatedly acclimated to RT. they may be labeled with TM different fluorophores to match specific lasers/detec- Full Spectrum microspheres Internally-labeled (hard-dyed) Full Spectrum™ tors; and they may feature beads were run as controls, to discern normal instru- different labeling strategies (internally or surface ment fluctuation. labeled with fluorochrome). Three trials were undertaken to investigate the tem- perature-responsiveness of Quantum™ PE MESF and Results: Microsphere standards that are internally-labeled Quantum™ FITC MESF microspheres. These included: When samples were prepped and run immediately with fluorochrome offer static performance. They are upon removal from refrigerated storage, only normal not environmentally-responsive as the fluorophore 1.) A single day study of FITC and PE bead populations fluctuation, and no temperature-dependent effect molecules are entrapped within the bead, and do to assess fluorescence response upon removal from on fluorescence was observed for each population. not come into contact with the suspending solution, refrigerated (4-8°C) storage and acclimation to / However, when surface-labeled microspheres were where they might be subject to a range of conditions. holding at room temperature (RT) thereafter. repeatedly acclimated to RT prior to runs / return to When these types of standards are run on a routine refrigerated storage, PE-labeled microspheres experi- (daily) basis, fluctuations in fluorescence intensity 2.) A longitudinal study of Quantum™ PE and enced a gradual, yet progressive loss of fluorescence reflect normal daily variation of the instrument and its Quantum™ FITC MESF microspheres to assess intensity. In contrast, FITC-labeled microspheres were macro environment (laboratory temperature / humidity, fluorescence stability when utilized (removed from found to be sensitive to the first prolonged (30 min.) vibrational ‘noise,’ etc.). and immediately returned to refrigerated storage, < exposure to elevated temperature, though they proved 5min) multiple times in a week. to be relatively stable through subsequent exposures. Surface-labeled microspheres feature the same FITC and PE standards exhibited excellent stability fluorochromes that are used to label cells. They are 3.) A longitudinal study assessing fluorescence stability when returned to refrigerated storage immediately (< dynamic standards, as the surface-immobilized fluo- of PE and FITC MESF kits when utilized after removal 5 min.) Full Spectrum microspheres exhibited stable rochrome is exposed to the same conditions as are from refrigerated storage, acclimated to RT (30min), fluorescence across conditions. labeled cells, e.g. buffer pH / osmolarity / tempera- sampled / run, and returned to refrigerated storage. ture. Their use is central to quantitative fluorescence Beads were run ~every day through the work week. Microspheres were run on a FACSCalibur (488nm laser, CONCLUSIONS FL1 / FITC and FL2 / PE detectors) using CellQuest Pro software and a 1024 scale. Samples include one drop Microsphere standards aid in defining a flow cytom- (~50μL) of each bead population and 400μL diluent. eter’s capabilities and limitations in terms of sensitiv- Internally-dyed Full Spectrum™ microspheres were ity, precision and accuracy, and provide a means for run each day and charted alongside the surface-la- ensuring that the instrument is stable and suitable for beled FITC and PE populations to monitor other aspects use. They are also helpful in understanding the effects of instrument fluctuation / stability. of extraneous factors such as ambient temperature, humidity and electronic noise. Both internally-dyed RESULTS and surface-labeled microspheres have their place in a comprehensive QC program, and are well-suited to their respective roles, as instrument and (particularly Single Day Study, Response of FITC and PE Figure 2: Quantum™ MESF and Full Spectrum™ microsphere microspheres to RT (1024 scale used) samples were prepped and run immediately upon removal from quantitative fluorescence) study standards. refrigerated storage. The kits remained stable under these conditions. Microspheres were run immediately upon removal It is important to recognize, however, that standards from the refrigerator (8:00 a.m.), and every 30 with surface-immobilized fluorophore are at greater risk than internally-dyed microspheres if subjected minutes thereafter until 4:00 p.m., with the bottles Longitudinal Study, Response of FITC and PE to sub-optimal handling. We have demonstrated that remaining at RT after their initial removal from the re- MESF Kits with Acclimation to RT before Each PE, typically considered to be one of the most robust frigerator. FITC - labeledPeak microspheres 1 were foundPeak to 2 Run (~30min out of refrigerator) 370.0 590.0 365.0 585.0 and stable fluorochromes, exhibits marked sensitivity be sensitive to 360.0the first prolonged (30min)580.0 exposure to 355.0 575.0 350.0 570.0 to the cumulative effects of temperature cycling. This room temperature,345.0 but exhibited stable 565.0fluorescence Microspheres were removed from the refrigerator, 340.0 560.0 335.0 555.0 was demonstrated by an immediate and gradually thereafter. A temperature330.0 effect on the fluorescence550.0 allowed to acclimate to room temperature, samples 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 10:00 10:30 11:00 11:30 12:00 12:30 intensity of PE was not observed. Both internally-dyed10:00 10:30 11:00 11:30 12:00 12:30 prepped, and stock bottles returned to the refrigerator progressive loss of fluorescence intensity when beads Peak 3 Peak 4 were acclimated to RT and then returned to refriger- Full Spectrum 765.0and surface-labeled PE MESF910.0 beads (~30min). The kits were run at a rate of ~4-5 times 760.0 905.0 755.0 900.0 ated storage each day. In contrast, FITC microspheres were stable under750.0 these conditions and895.0 timeframe. / week over an 8-month period. Both kits exhibited a 745.0 890.0 740.0 885.0 were found to be sensitive to the first prolonged (30 Peak 1 Peak 2 735.0 880.0 progressive loss of fluorescence intensity. 370.0 590.0 730.0 875.0 365.0 585.0 725.0 870.0 min) exposure to room temperature, though they 360.0 580.0 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00

355.0 575.0 10:00 10:30 11:00 11:30 12:00 12:30 10:00 10:30 11:00 11:30 12:00 12:30 350.0 570.0 proved to be relatively stable through subsequent 345.0 565.0 340.0 560.0 335.0 555.0 Full Spectrum™ exposures. These findings highlight that negligent 330.0 550.0 585 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 10:00 10:30 11:00 11:30 12:00 12:30 10:00 10:30 11:00 11:30 12:00 12:30 583 or sub-optimal handling of microsphere standards, 581 PE population 579 Peak 3 Peak 4 577 765.0 910.0 particularly those with surface-immobilized fluoro- 575 760.0 905.0 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00

755.0 900.0 10:00 10:30 11:00 11:30 12:00 12:30 750.0 895.0 phore, may impact long-term stability and introduce 745.0 890.0 740.0 885.0 735.0 880.0 a source of variation. Standards must be handled in 730.0 875.0 725.0 870.0 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 8:00 8:15 8:30 9:00 9:30 1:00 1:30 2:00 2:30 3:00 3:30 4:00 10:00 10:30 11:00 11:30 12:00 12:30 10:00 10:30 11:00 11:30 12:00 12:30 accordance with their specific recommendations for

Full Spectrum™ highest confidence and stability. Both types of surface 585 583 Figure 1: FITC, PE and Full Spectrum™ microspheres run every 30 labeled standards exhibited excellent stability when 581 579 minutes following removal from the refrigerator. An initial tempera- 577 handled as recommended. 575

8:00 8:15 8:30 9:00 9:30 ture1:00 effect1:30 2:00 2:30 3:00 was3:30 4:00 observed for FITC, but only normal fluctuation, and no 10:00 10:30 11:00 11:30 12:00 12:30 temperature-dependent effect on fluorescence, was observed for PE and Full Spectrum populations.

Longitudinal Study, Response of FITC and PE MESF Kits Using Standard Handling Recommen- dations (≤ 5min out of refrigerator)

Microspheres were removed from the refrigerator, samples prepped, and stock bottles returned to the refrigerator (≤ 5min). Microspheres were run at a rate of ~2-3 times / week over a 12-month + period. The kits remained stable under these storage Figure 3: Quantum™ MESF microsphere samples were prepped and use conditions. and run after removal from refrigerated storage and acclimation to RT. The same bottles were returned to refrigerated storage after the runs. Both FITC and PE-labeled microspheres experienced a gradual, yet progressive, loss of fluorescence intensity, though the loss was much more extreme for PE. This effect is highlighted by the fluorescence profile of the internally-labeled Full Spectrum™ population.