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How to Get the Best Performance out of your CyTOF
3/31/2017 How to Get the Best Performance out of your CyTOF Carina Torres CyTOF User Group Meeting CYTO 2013 23 May 2013
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Outline History CyTOF Workflow Progress Report…1 ½ years later
Instrument Startup Optimization Running Samples Instrument Shutdown Troubleshooting Progress Report…1 ½ years later
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CyTOF at Lilly 2010 2011 2012 2013 CyTOF installed, Experiments began
3/31/2017 A group of Lilly Senior Research Scientists went to a Garry Nolan talk CyTOF installed, placed in Flow Core Experiments began 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): , 2011.
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3/31/2017 Preparing your Lab
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Daily Workflow: Instrument Startup
3/31/2017 Daily Workflow: Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Start Plasma Prepare Nebulizer and Syringe Pump
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Instrument Startup TP1 TP2 BP VG1 VG2 CLOSE Argon Ex Vent 3/31/2017
TP1,TP2,BP :for pumps VG1,VG2: vacuum levels INTRF: temperature and cooling Ex Vent: exhaust
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Instrument Startup Start Plasma Check lights on instrument
3/31/2017 Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Start Plasma Prepare Nebulizer and Syringe Pump
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3/31/2017 Instrument Startup 20 min
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Instrument Startup Start Plasma Check lights on instrument
3/31/2017 Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Start Plasma Prepare Nebulizer and Syringe Pump
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3/31/2017 Instrument Startup DI H20
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Instrument Startup Start Plasma Check lights on instrument
3/31/2017 Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Start Plasma Prepare Nebulizer and Syringe Pump
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Instrument Startup Chiller turns on Start-up sequence Plasma ignites
3/31/2017 Instrument Startup Chiller turns on Start-up sequence Plasma ignites Chiller turns on Start-up sequence Plasma ignites
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3/31/2017 Instrument Startup
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Instrument Startup Start Plasma Check lights on instrument
3/31/2017 Instrument Startup Check lights on instrument Turn Heater on Instrument setup Card Cage Tab Click Heater on oC Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument Start Syringe pump (HPLC) Start Plasma Prepare Nebulizer and Syringe Pump
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3/31/2017 Instrument Startup I usually wait about 30 minutes for the plasma to stabilize and then I start tuning according to the optimization protocol that DVS recommends.
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Optimization: Performance Check and Tuning
3/31/2017 Optimization: Performance Check and Tuning ►To get the best resolution between detection channels Mass Calibration ►Makes sure TOF values for CS and IR are <5% of peak height, calibration doesn’t typically need to be changed ►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 I usually wait about 30 minutes for the plasma to stabilize and then I start tuning according to the optimization protocol that DVS recommends. Order is critical because dual count calibration requires optimal detector gain and sensitivity and oxidation are dependent on dual count calibration Before I start this whole process I fill a 3 ml syringe…. ►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%
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Optimization: Performance Check and Tuning
3/31/2017 Optimization: Performance Check and Tuning Tuning Solution
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Optimization: Performance Check and Tuning
3/31/2017 Optimization: Performance Check and Tuning Tuning Solution Mass Resolution Tb Example: 10425/6(2)= 868 >400 Peak height: x= (TOF) y= 525,000 (intensity) ½ max signal intensity =6
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Optimization: Performance Check and Tuning
3/31/2017 Optimization: Performance Check and Tuning Tuning Solution Mass Resolution Mass Calibration Cs Ir 9537 11457 <5% of peak height <5% of peak height 9537 11457
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Optimization: Performance Check and Tuning
3/31/2017 Optimization: Performance Check and Tuning Tuning Solution Mass Resolution Mass Calibration Detector gain All Tb Max signal intensity Tb pulse counts 629242 136495 = 4.61 (4-5)
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CYTOF is Ready! Optimization: Performance Check and Tuning
3/31/2017 Optimization: Performance Check and Tuning Tuning Solution Mass Resolution Mass Calibration Detector gain Dual Count Calibration Dual Count test Oxidation All 155Gd dual count 159Tb dual count 159Tb dual count >100,000 =<3% 2754 218626 =1.26% CYTOF is Ready!
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Automated QC at Lilly 3/31/2017 QC Table Pulse Dual Mass Mass PPM
Counts Counts Intensity Error Error My bioinformatics guy recognized how meticulous the tuning process was and he took the initiative to write some software for me that has reduced my qc process to maybe 5 minutes if the instrument doesn’t need troubleshooting.
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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
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Automated QC at Lilly Dual count calibration Dual counts plot
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When Specs Are Not Met Observation Parameter Possible Solution <400
3/31/2017 When Specs Are Not Met Parameter Observation Possible Solution <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
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When Specs Are Not Met Observation Parameter Possible Solution <400
3/31/2017 When Specs Are Not Met Parameter Observation Possible Solution <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
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When Specs Are Not Met Observation Parameter Possible Solution <400
3/31/2017 When Specs Are Not Met Parameter Observation Possible Solution <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
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Verify Mass Calibration
3/31/2017 Verify Mass Calibration Cs Ir 9537 11457 <5% of peak height <5% of peak height 9533 11453
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When Specs Are Not Met Observation Parameter Possible Solution <400
3/31/2017 When Specs Are Not Met Parameter Observation Possible Solution <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
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Optimize Detector Voltage
3/31/2017 Optimize Detector Voltage . . . . . . . . . . . . . . .
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When Specs Are Not Met Observation Parameter Possible Solution <400
3/31/2017 When Specs Are Not Met Parameter Observation Possible Solution <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
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When Specs Are Not Met Observation Parameter Possible Solution <400
3/31/2017 When Specs Are Not Met Parameter Observation Possible Solution <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
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X-Y Alignment of the Interface
3/31/2017 X-Y Alignment of the Interface All
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3/31/2017 Optimize Gas Flows All
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3/31/2017 Optimize Current All
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3/31/2017 Re-Check QC Mass Calibration Dual Counts Plot
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Running Samples: User Guidelines
3/31/2017 Running Samples: User Guidelines Make sure sample loop is clean: run H20, check Mass Cal screen to verify
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Running Samples: User Guidelines
600 30 10
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Running Samples: User Guidelines
Filter samples and count cells. Resuspend cells to achieve collection rate of ideally events/sec Clean with H20 between samples to minimize carryover Clean instrument when done running samples (Wash solution and H20)
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Instrument Shutdown
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3/31/2017 Instrument Shutdown
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Instrument Shutdown Turn off HPLC
Remove nebulizer and store in 3% Citranox
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Troubleshooting: Common Issues
3/31/2017 Troubleshooting: Common Issues Problem Cause Solution 1. Plasma won’t ignite Argon off Turn it on Argon not pure enough Use Ultra High Purity Argon Incorrect Gas? Double check gas Air Leak Localize source Plasma ignites Look for air leak in the area of sample introduction Plasma does not ignite Check the torch, injector, coil for condensation
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Troubleshooting: Common Issues
3/31/2017 Troubleshooting: Common Issues Problem Cause Solution 2. No/low signal Sample line capillary pinched Inspect capillary line Sample line capillary inserted improperly Reinsert capillary Clogged nebulizer Check spray/swap nebulizer Insufficient gas flows Check gas flows
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Troubleshooting: Unusual Circumstances
3/31/2017 Troubleshooting: Unusual Circumstances Problem Cause Solution 1. Leak in one of the argon lines Pankaj came out and found the leak. Replaced torch and replaced RFG coil Arcing because the coil not aligned Oxidation on the coils Clean/Replace coil Collateral damage to the gas line As the plasma flows through the torch….Cushion of argon to cool the glass, inadequate flow (pre-set)
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Troubleshooting: Unusual Circumstances
3/31/2017 Troubleshooting: Unusual Circumstances Problem Cause Solution 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 Current wire applies the current to the reducer cone which concentrates the beam, became damaged and wasn’t conducting current. Insert pic of good contact
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Troubleshooting: Unusual Circumstances
3/31/2017 Troubleshooting: Unusual Circumstances Problem Cause Solution 3. Originally thought it was a faulty syringe pump. Turned out to be a bad valve. Replaced the entire valve assembly Syringe pump was moving but the syringe wasn’t
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Troubleshooting: Unusual Circumstances
3/31/2017 Troubleshooting: Unusual Circumstances Problem Cause Solution 4. Partially clogged/dirty nebulizer Clean/swap nebulizer Sample capillary not positioned properly Reinsert capillary Bad syringe Replace syringe Uneven pattern….
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Troubleshooting: Unusual Circumstances
3/31/2017 Troubleshooting: Unusual Circumstances Problem Cause Solution 5. Tungsten contamination; but from where? Cleaned ignitor pin with sandpaper
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Progress Report: 1 ½ years later
3/31/2017 Lilly CyTOF Projects: Multi-parametric, high content immunophenotyping LRAP with the Sanford Burnham Institute to investigate signaling pathways >100 expts: 60% validations & optimizations 40% phenotyping Validated core panel: 16 parameters What have we done so far? *Replaced Rhodium with Cisplatin for viability Joined Cytobank; Transitioning from FlowJo to Spade for data analysis ICP-MS characterization *Reference: Feinberg, H.G., et al. A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 81 (6): , 2012
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Acknowledgements DVS Sciences Andrew Glasebrook Mark Daniels
3/31/2017 Acknowledgements Andrew Glasebrook Mark Daniels Elaine Conner Marian Mastrangelo Han He DVS Sciences Pankaj Chaudhari Leslie Fung Ted Young Tad George Ron Cohn Daniel Tong John Fitchett
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