how to get the best performance out of your cytof...2015/10/02 · how to get the best performance...
TRANSCRIPT
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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
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Outline
• History • CyTOF Workflow
• 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
A group of Lilly Senior Research Scientists went to a Garry Nolan talk
CyTOF installed, placed in Flow Core
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.
Experiments began
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Preparing your Lab
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Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument
Check lights on instrument
Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC
Start Syringe pump (HPLC)
Start Plasma
Daily Workflow: Instrument Startup
Prepare Nebulizer and Syringe Pump
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Instrument Startup
TP1 TP2
VG1 VG2
BP Argon
Ex Vent
CLOSE
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Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument
Check lights on instrument
Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC
Start Syringe pump (HPLC)
Start Plasma
Instrument Startup
Prepare Nebulizer and Syringe Pump
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Instrument Startup
20 min
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Instrument Startup
Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument
Check lights on instrument
Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC
Prepare Nebulizer and Syringe Pump
Start Plasma
Start Syringe pump (HPLC)
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Instrument Startup
DI H20
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Instrument Startup
Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument
Check lights on instrument
Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC
Start Plasma
Start Syringe pump (HPLC)
Prepare Nebulizer and Syringe Pump
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Instrument Startup
• Chiller turns on • Start-up sequence • Plasma ignites
• Chiller turns on • Start-up sequence • Plasma ignites
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Instrument Startup
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Instrument Startup
Make sure Argon is on ▪ 100 psi at wall ▪ 50 psi on instrument
Check lights on instrument
Turn Heater on Instrument setup Card Cage Tab Click Heater on 200oC
Start Plasma
Start Syringe pump (HPLC)
Prepare Nebulizer and Syringe Pump
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Instrument Startup
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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
►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
Tuning Solution
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=6
Peak height: x= 10425 (TOF) y= 525,000 (intensity)
½ max signal intensity
Example: 10425/6(2)= 868
Optimization: Performance Check and Tuning
Mass Resolution
Tuning Solution
>400
Tb
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<5% of peak height <5% of peak height
Optimization: Performance Check and Tuning
Mass Resolution
Mass Calibration
Tuning Solution
9537 11457
Cs Ir 11457 9537
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Optimization: Performance Check and Tuning
Mass Resolution
Mass Calibration
Detector gain
Tuning Solution
Max signal intensity Tb pulse counts
629242 136495
= 4.61 (4-5)
Tb All
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All
Mass Resolution
Mass Calibration
Detector gain
Tuning Solution
Optimization: Performance Check and Tuning
Dual Count Calibration
Dual Count test Oxidation
=<3%
=1.26%
155Gd dual count 159Tb dual count
2754
218626
159Tb dual count >100,000
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Automated QC at Lilly
QC Table Pulse Dual Mass Mass PPM Counts Counts Intensity Error Error
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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
Mass Calibration
Parameter Observation Possible Solution
<400 Call for service
159Tb ratio is<4 or >5 optimize detector voltage
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
Check X-Y alignment Optimize gas flows Optimize current
Swap nebulizer
Optimize gas flows
159Tb dual count <100,000
155Gd dual count 159Tb dual count
>3%
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Mass Calibration
Parameter Observation Possible Solution
<400 Call for service
159Tb ratio is<4 or >5 optimize detector voltage
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
Check X-Y alignment Optimize gas flows Optimize current
Swap nebulizer
Optimize gas flows
159Tb dual count <100,000
155Gd dual count 159Tb dual count
>3%
When Specs Are Not Met
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Mass Calibration
Parameter Observation Possible Solution
<400 Call for service
159Tb ratio is<4 or >5 optimize detector voltage
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
159Tb dual count <100,000
Check X-Y alignment Optimize gas flows Optimize current
Swap nebulizer
Optimize gas flows 155Gd dual count 159Tb dual count
>3%
When Specs Are Not Met
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Ir Cs
9533 11453
<5% of peak height <5% of peak height
Verify Mass Calibration
9537 11457
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Mass Calibration
Parameter Observation Possible Solution
<400 Call for service
159Tb ratio is<4 or >5 optimize detector voltage
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
Check X-Y alignment Optimize gas flows Optimize current
Swap nebulizer
Optimize gas flows
159Tb dual count <100,000
155Gd dual count 159Tb dual count
>3%
When Specs Are Not Met
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.
. . . .
. . . . .
. . . . .
Optimize Detector Voltage
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Mass Calibration
Parameter Observation Possible Solution
<400 Call for service
159Tb ratio is<4 or >5 optimize detector voltage
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
Check X-Y alignment Optimize gas flows Optimize current
Swap nebulizer
Optimize gas flows
159Tb dual count <100,000
155Gd dual count 159Tb dual count
>3%
When Specs Are Not Met
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Mass Calibration
Parameter Observation Possible Solution
<400 Call for service
159Tb ratio is<4 or >5 optimize detector voltage
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
Check X-Y alignment Optimize gas flows Optimize current
Swap nebulizer
Optimize gas flows
159Tb dual count <100,000
155Gd dual count 159Tb dual count
>3%
When Specs Are Not Met
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X-Y Alignment of the Interface
All
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Optimize Gas Flows
All
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Optimize Current
All
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Re-Check QC
Mass Calibration
Dual Counts Plot
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• Make sure sample loop is clean: run H20, check Mass Cal screen to verify
Running Samples: User Guidelines
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Running Samples: User Guidelines
600 30 10
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• Filter samples and count cells. Resuspend cells to achieve collection rate of ideally 300-500 events/sec
Running Samples: User Guidelines
• 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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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
1. Cause Solution Problem
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
Plasma does not ignite
Look for air leak in the area of sample introduction
Check the torch, injector, coil for condensation
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Troubleshooting: Common Issues
2. Problem
No/low signal
Cause
Sample line capillary pinched
Sample line capillary inserted improperly
Inspect capillary line
Reinsert capillary
Clogged nebulizer Check spray/swap nebulizer
Insufficient gas flows
Check gas flows
Solution
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• Pankaj came out and found the leak. Replaced torch and replaced RFG coil
Troubleshooting: Unusual Circumstances
• Leak in one of the argon lines
1. Cause Solution Problem
• Clean/Replace coil
• Arcing because the coil not aligned • Oxidation on the coils
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Troubleshooting: Unusual Circumstances
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
2. Cause Solution Problem
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Troubleshooting: Unusual Circumstances
Originally thought it was a faulty syringe pump. Turned out to be a bad valve.
3. Replaced the entire valve assembly
Cause Solution Problem
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Clean/swap nebulizer
Troubleshooting: Unusual Circumstances
Cause Solution Problem 4. Partially clogged/
dirty nebulizer
Sample capillary not positioned properly
Reinsert capillary
Bad syringe
Replace syringe
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180-190
Tungsten contamination; but from where?
Troubleshooting: Unusual Circumstances
Cleaned ignitor pin with sandpaper
Cause Solution Problem 5.
180-190
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Progress Report: 1 ½ years later
What have we done so far?
>100 expts: 60% validations & optimizations 40% phenotyping
Validated core panel: 16 parameters
*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): 467-475, 2012
Lilly CyTOF Projects: • Multi-parametric, high content
immunophenotyping
• LRAP with the Sanford Burnham Institute to investigate signaling pathways
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Acknowledgements
Elaine Conner Marian Mastrangelo Han He
Andrew Glasebrook DVS Sciences Pankaj Chaudhari Leslie Fung Ted Young Tad George Ron Cohn Daniel Tong
Mark Daniels John Fitchett