choosing the right liquid handler and verifying its ......calculated volume (µl) 49.81 47.04 51.25...
TRANSCRIPT
© 2020 Artel1
Choosing the Right Liquid Handler and Verifying its
Performance
Jason GravesArtel
SLAS 2020
© 2020 Artel2
Agenda
▪ Review Requirements and Considerations for Liquid Handling Needs
▪ Verifying Pipetting Performance
▪ Questions and Comments
© 2020 Artel3
Why Do You Need an ALH (Automated Liquid Handler)?
▪ Scaling up a method or assay
▪ Increase capacity
▪ Free up operator time
▪ Quality issues
▪ Increase accuracy
▪ Decrease variability
▪ Improve reproducibility
© 2020 Artel4
Considerations for Choosing an ALH
▪ Budget
▪ $$$
▪ Facilities
▪ Space
▪ Utilities
▪ Environment
© 2020 Artel5
Application Specific Requirements
▪ Single Purpose System
▪ Focus on specific needs
▪ Multiple Methods
▪ Flexibility
© 2020 Artel6
Liquid Handling Options
▪ Deck Positions and Configurations
▪ Large versus small systems
▪ Labware height capabilities
▪ Pipetting Formats
▪ Single channel, 8-channel, 96 well heads, 384 well heads
▪ Volume range capabilities
▪ Tip type availability
▪ Devices
▪ Grippers, shakers, incubation, heating/cooling
© 2020 Artel7
Liquid Handler Configuration Requirements
▪ Labware
▪ Types
▪ Plate Type and Density
▪ Tubes
▪ Reservoirs
▪ Amount
▪ Movement
▪ Devices
© 2020 Artel8
Liquid Handler Configuration Requirements
▪ Transfer Requirements
▪ Format
▪ Full plate (96 well, 384 well, 1536 well)
▪ Columns or rows
▪ Single wells (cherry picking, pooling)
▪ Pipetting transfers
▪ Volumes
▪ Liquid types
▪ Critical vs non-critical
© 2020 Artel9
ALH Pipetting Verification
▪ ALH performance
▪ IQ/OQ on installation
▪ Regular calibration checks
▪ Post crash or relocation
▪ New tips or labware
▪ Development work
Liquid Class Development !!!
© 2020 Artel10
Liquid Class Development
▪ Specific to liquid type, labware, volume range and temperature
▪ Accuracy and Reproducibility
▪ Small volumes
▪ Viscous or sticky solutions
▪ Cold or warm solutions
© 2020 Artel11
How to Develop a Liquid Class
▪ Start with basic transfer parameters and watch the pipetting a few times
▪ If it looks good – move on to dial in the volume
▪ If it doesn’t look good, then adjust
▪ Pipetting speeds
▪ Air gaps
▪ Blowout volumes and speeds
▪ Prewetting
▪ Pauses
▪ Aspirate and dispense heights
© 2020 Artel12
How to Develop a Liquid Class
▪ Select at least three volumes
around your target range
▪ Dispense target volumes
▪ Measure dispensed volumes
▪ Plot Target volume against
Measured volume
▪ Calculate slope and y-intercept
▪ Input slope value into the ALH
scaling factor
▪ Input Y-intercept value into ALH
offset value
▪ Save new ALH method
Targ
et
Volu
me (
µL)
Measured Volume (µL)
Slope (y/x) = ALH Scaling Factor
Y-Int = ALH Offset Volume
Example (n = 96) Pre-adjust Post-Adjust
Target volume (µL) 2.5 2.5
Measured volume (µL) 2.83 2.55
Rel. inaccuracy (%) 13.34 2.04
Precision (%CV) 2.91 4.14
© 2020 Artel13
Multichannel Verification System
© 2020 Artel14
MVS Key System Capabilities
▪ Verifies the accuracy and precision of all ALH (1-384 tips)
▪ Measures performance over a wide volume range of 0.0001 to 350µL
▪ Measures aqueous and non-aqueous volume transfers including DMSO
▪ Obtains volume statistics, tip-by-tip and well-by-well
▪ Provides highly accurate, standardized results, traceable to national and
international standards
▪ Eliminates the need for rigorous environmental controls
▪ Simplifies verification and calibration- easy –to-use for any skill level
© 2020 Artel15
How the MVS (and PCS) works
▪ Employs a dual-dye, dual-wavelength,
ratiometric absorbance-based
measurement method for calculating the
dispense volume.
▪ Dyes of known concentration are
dispensed into a well-characterized
microtiter plate. The plate is mixed on a
plate shaker to ensure solution
homogeneity. Absorbance readings are
taken at 520 nm and 730 nm.
=
730
520
r
bTS
A
A
a
aVV
© 2020 Artel16
Starting Liquid Class Report
© 2020 Artel17
In Process Optimization of Liquid Class
© 2020 Artel18
MVS Assisted Optimization Liquid Class
© 2020 Artel19
Liquid Class Data Before and After
Starting Liquid Class Optimized Liquid Class
© 2020 Artel20
Sources of Variability for Volume Measurements
▪ Liquid handler
▪ Reagents
▪ Tips
▪ Temperature
▪ Operator
contribute directly to the
liquid dispensing
variability
affect the
measurement process
▪ Mixing
▪ Detector
▪ Plate type
© 2020 Artel21
Sources of Variability for Volume Measurements
Artel MVS removes all contributors to variability in liquid handling calibration…
except the liquid handler and the tips.
Leaving you with exactly the two things you want to measure!
© 2020 Artel22
Accuracy is Absolutely Essential
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8
Calculated Volume (µL) 49.81 47.04 51.25 52.72 50.16 51.56 50.12 51.09
Inaccuracy -0.37% -5.92% 2.50% 5.44% 0.32% 3.12% 0.23% 2.19%
Artel MVS volume (µL) 50.01 49.98 50.03 50.00 49.96 50.02 49.97 50.01
Inaccuracy 0.02% -0.04% 0.06% 0.00% -0.08% 0.04% -0.06% 0.02%
1 volume
measurement
applied to 8
calibration curves
Artel MVS ensures consistent volume accuracy from day to day, person to person!
© 2020 Artel23
How I Used the MVS to Optimize qPCR Transfers
▪ Method steps:
▪ Transfer 3-7µl from 96 well plate into all four quadrants of a 384 well plate
▪ Transfer 7-3µl from 96 well plate into all four quadrants of the same 384 well
plate for a 10µl reaction volume
▪ Mix well
▪ The MVS made it possible to accurately measure the transfers for both the
master mix and the template from the 96 well plate into the 384 well plate.
▪ I was also able to verify that the mixing step was working properly and be
confident that we were mixing at the right volumes for the right number of times.
© 2020 Artel24
How I Used the MVS to Optimize DNA Bead Cleanup Transfers
▪ Method requirements:
▪ Span-8 transfers from trough containing bead reagent to 96 well plate to
keep costs down
▪ Multichannel pipetting with 96 well head for remaining transfers for speed
▪ Critical steps are bead transfer, proper mixing for DNA capture and washing,
and final elution steps
▪ MVS alternate solutions made it possible to mock bead transfer out of the
reagent trough with Span-8
▪ Aqueous MVS solutions were used to dial in the mixing steps and final elution
with the 96 well head
© 2020 Artel26
Thank you