1. ACQUISITION RATES EFFECT ON MULTIPARAMETRIC RARE EVENT ANALYSIS : AN INSTRUMENT COMPARISON John Tigges, Vasilis Toxavidis, Heidi Mariani Beth Israel Deaconess Medical Center/Harvard Stem Cell Institute 3 Blackfan Circle Boston, MA 02215 The current paradigm in Flow Cytometry is the increasing number of laser lines, multiparameter instrument capabilities, and increasing number of antibodies; flow cytometric applications have become more complex and with greater variability. One of the major challenges pertaining to instrumentation is dealing with reliability of the optics and the laser lines. It is of extreme importance to be able to shape the laser beams elliptically, focus them to the cellular interrogation point, and not lose laser power during this process. In conjunction with lasers and optics, the fluidics system plays an integral part in obtaining the best data possible from the cellular preparation. Event rate, determined by amount of pressure applied to the sample, rate of fluid through the system, and cellular concentration, is the driving force behind obtaining reproducible and reliable data. However, with advances in optical alignment and fluidic (hydrodynamic) focusing, even rare event analysis can be obtained at varying speeds without compromising data efficiency. Separation, and resolution show noticeable differences. Major discrepancy on Thy1.1 Tregs. Separation, and resolution show noticeable differences. Major discrepancy on Thy1.1 Tregs.  DsRed TRegs- Gallios avg. % gated=6.44LSR II avg. % gated=6.45Difference=0%  Thy1.1 TRegs-Gallios avg. % gated=10.48LSR II avg. % gated=6.72Difference=36%  LT HSC-Gallios avg. % gated=30.21LSR II avg. % gated=28.68Difference=5% MFI is significantly better on the Gallios. This is true across the board of variable speeds. MFI is significantly better on the Gallios. This is true across the board of variable speeds.  DsRed TRegs- Gallios avg. MFI=41.38 LSR II avg. MFI=37.30Difference=10%  Thy1.1 TRegs- Gallios avg. MFI=26.96 LSR II avg. MFI=24.77 Difference=8%  LT HSC- Gallios avg. MFI=27.76 LSR II avg. MFI=18.64 Difference=33% Average difference for % gated is 13.67% and average difference for MFI is 17%. Average difference for % gated is 13.67% and average difference for MFI is 17%. Median % Total difference is approximately 50% (Both Thy1.1 TRegs and LT HSC). Median % Total difference is approximately 50% (Both Thy1.1 TRegs and LT HSC). The bar graphs below demonstrate differences amongst % Total, % Gated, and MFI at the varying speeds for all sample types. The bar graphs below demonstrate differences amongst % Total, % Gated, and MFI at the varying speeds for all sample types. Hematopoietic Stem Cells (HSC) from Whole Bone Marrow and Regulatory T Cells were analyzed at varying speeds with differing numbers of parameters used and on two commercially available FACS analyzers. HSCs were labeled with FITC, PE-Cy7, APC, APC-Cy7, and Pacific Blue; TRegs were labeled with GFP, DSRed, and PE-Cy7; and Thy 1.1 TRegs were labeled with FITC, PE, APC, APC-Cy7, and DAPI. Cells were then acquired on both a Becton Dickinson SORP LSRII equipped with a 561nm laser and a Beckman Coulter Gallios at speeds of 5K, 10K, 15K, 20K, and 25K for Tregs. The SORP LSR II is equipped as follows: 488nm- FITC 530/30; 561nm- PE 585/15, DsRed 585/15, PE Cy7 780/60; 640nm- APC 670/14, APC Cy7 780/60; 405nm- Pacific Blue 450/50; UV- DAPI 450/50. The Gallios is equipped as follows: 488nm- FITC 525BP, PE 575BP, PE Cy7 755LP, DsRed 575BP (acquired using the PE channel; Gallios is not equipped with a 561nm); 633nm- APC 660/20, APC Cy7 755LP; 405nm- Pacific Blue 450/40, DAPI 450/40 (acquired using Pacific Blue channel with violet excitation). Both instruments underwent initial quality check according to manufacturer’s guidelines (Becton Dickinson- Cytometer Set-up and Tracking Beads; Beckman Coulter- Flow Check Pro Beads). Instrument performance was then verified using Spherotech 8 peak rainbow beads. Samples were acquired on High sample rate, manufacturer’s preset threshold off FSC (Gallios=100; LSRII=5000), and 10,000 Total events. Samples were analyzed with Beckman Coulter’s Kaluza 1.0 Software. All plots and gates remained consistent for each sample type. In both cases, no major intra-instrument variation due to speed was apparent in cellular number, % total, % gated or Mean Fluorescent Intensity. However, most notably, differences were viewed inter-instrument in % Total, % Gated, and MFI. This was particularly noticeable at the higher speeds analyzing the rare event populations (% Total ≤ 2). Overall separation and resolution were significantly different independent of speed. While an instrument can show consistency and reliability throughout variable event rate, it is not an obvious indicator of an instrument’s overall performance. The ability of an instrument to deliver a sample to the interrogation point effectively without any loss of laser intensity at that point or to the PMT is crucial to data acquisition and analysis. This is abundantly clear in rare event analysis, where cells are minimal and any loss of signal is detrimental. We have shown this to be true of the Gallios system with its patented Boulevard technology and enhanced fluidics system. Program Number 164 INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS DsRed TRegs Thy 1.1 TRegs Long Term HSCs DsRed TRegs Thy1.1 TRegs LT HSC For additional information please contact: John Tigges Flow Cytometry Core Beth Israel Deaconess Medical Center jtigges@bidmc.harvard.edu