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How to Get the Best Performance out of your CyTOF. Carina Torres CyTOF User Group Meeting CYTO 2013 23 May 2013. Outline. History CyTOF Workflow Instrument Startup Optimization Running Samples Instrument Shutdown Troubleshooting Progress Report…1 ½ years later. CyTOF at Lilly.
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How to Get the Best Performance out of your CyTOF Carina Torres CyTOF User Group Meeting CYTO 2013 23 May 2013
Outline • History • CyTOF Workflow • Instrument Startup • Optimization • Running Samples • Instrument Shutdown • Troubleshooting • Progress Report…1 ½ years later
CyTOF at Lilly A group of Lilly Senior Research Scientists went to a Garry Nolan talk CyTOF installed, placed in Flow Core Experiments began 2010 2011 2012 2013 September Purchased CyTOF (#9) Panel Validation Phenotyping Data analysis Bendall, S.C., et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332 (6030): 687-696, 2011.
Daily Workflow: Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma
Instrument Startup CLOSE VG2 VG1 TP2 TP1 Argon BP Ex Vent
Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma
Instrument Startup 20 min
Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma
Instrument Startup DI H20
Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma
Instrument Startup • Chiller turns on • Start-up sequence • Plasma ignites • Chiller turns on • Start-up sequence • Plasma ignites
Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Prepare Nebulizer and Syringe Pump Start Plasma
Optimization: Performance Check and Tuning ►To get the best resolution between detection channels ►Makes sure TOF values for CS and IR are <5% of peak height, calibration doesn’t typically need to be changed Mass Calibration ►Optimize to ensure efficient detection of ions ►The dual count coefficient is calculated to generate the dual signal scale that is used to quantify ion signals ►Measure sensitivity by doing a dual counts tests to ensure 159Tb dual counts are >100,000 ►Check Oxide ratio as an indication of plasma robustness. The ratio of 155Gd dual count /159Tb dual count should be less than 3%
Tuning Solution Optimization: Performance Check and Tuning
Tuning Solution MassResolution Optimization: Performance Check and Tuning Tb >400 Example: 10425/6(2)= 868 Peak height: x= 10425 (TOF) y= 525,000 (intensity) ½ max signal intensity =6
Tuning Solution MassResolution Mass Calibration Optimization: Performance Check and Tuning Ir Cs 9537 11457 <5% of peak height <5% of peak height 11457 9537
Tuning Solution MassResolution Mass Calibration Detector gain Optimization: Performance Check and Tuning All Tb 629242 136495 Max signal intensity Tb pulse counts = 4.61 (4-5)
Tuning Solution MassResolution Mass Calibration Detector gain Optimization: Performance Check and Tuning Dual Count Calibration Dual Count test Oxidation All 155Gd dual count 159Tb dual count =<3% 159Tb dual count >100,000 2754 218626 =1.26% CYTOF is Ready!
Automated QC at Lilly QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error
Automated QC at Lilly QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error
Automated QC at Lilly Dual count calibration Dual counts plot
When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows
When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows
When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows
Verify Mass Calibration Cs Ir 9537 11457 <5% of peak height <5% of peak height 9533 11453
When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows
Optimize Detector Voltage . . . . . . . . . . . . . . .
When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >5% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows
When Specs Are Not Met Observation Possible Solution Parameter <400 Call for service ToF values for Cs and Ir at beginning of peak are >3% of peak height Verify ToF values and enter new values in the mass calibration tab Mass Calibration 159Tb ratio is<4 or >5 optimize detector voltage Check X-Y alignment Optimize gas flows 159Tb dual count <100,000 Optimize current Swap nebulizer 155Gd dual count 159Tb dual count >3% Optimize gas flows
Optimize Current All
Re-Check QC Mass Calibration Dual Counts Plot
Running Samples: User Guidelines • Make sure sample loop is clean: run H20, check Mass Cal screen to verify
Running Samples: User Guidelines 600 30 10
Running Samples: User Guidelines • Filter samples and count cells. Resuspend cells to achieve collection rate • of ideally 300-500 events/sec • Clean with H20 between samples to minimize carryover • Clean instrument when done running samples (Wash solution and H20)
Instrument Shutdown • Turn off HPLC • Remove nebulizer and store in 3% Citranox
Troubleshooting: Common Issues Cause Solution Problem 1. Plasma won’t ignite Turn it on Argon off Use Ultra High Purity Argon Argon not pure enough Double check gas Incorrect Gas? Localize source Air Leak Plasma ignites Look for air leak in the area of sample introduction Plasma does not ignite Check the torch, injector, coil for condensation
Troubleshooting: Common Issues Cause Solution Problem 2. No/low signal Sample line capillary pinched Inspect capillary line Sample line capillary inserted improperly Reinsert capillary Check spray/swap nebulizer Clogged nebulizer Check gas flows Insufficient gas flows
Troubleshooting: Unusual Circumstances Cause Solution Problem 1. • Pankaj came out • and found the • leak. Replaced • torch and replaced • RFG coil • Leak in one • of the argon • lines • Arcing because • the coil not • aligned • Oxidation on • the coils • Clean/Replace • coil
Troubleshooting: Unusual Circumstances Cause Solution Problem 2. Poor contact of skimmer/reducer with the contact ring on the interface. This may be due to oxidation on the contact ring or on the reducer itself. Vigorously cleaned the contact ring on the interface with methanol. Eventually, Daniel came out to replace the interface
Troubleshooting: Unusual Circumstances Cause Solution Problem 3. Originally thought it was a faulty syringe pump. Turned out to be a bad valve. Replaced the entire valve assembly
Troubleshooting: Unusual Circumstances Cause Solution Problem 4. Clean/swap nebulizer Partially clogged/dirty nebulizer Sample capillary not positioned properly Reinsert capillary Bad syringe Replace syringe
Troubleshooting: Unusual Circumstances Cause Solution Problem 5. Cleaned ignitor pin with sandpaper Tungsten contamination; but from where? 180-190 180-190