understanding error contribution of liquid handling and ... · liquid handlers, plate readers, etc....

© 2012 ARTEL

Understanding Error Contribution of Liquid Handling and Analytical Equipment to Overall Assay Performance

ELRIGManchester, EnglandSeptember 6, 2012

John Thomas Bradshaw, Ph.D.Artel, Inc.

© 2012 ARTEL

The world would be a simpler place if the “Black Box” existed!

The “Black Box”

input output

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Automation and Analytical Lab Equipment (e.g., liquid handlers, plate mixers, plate readers) can be powerful tools when their performance is Measured and Controlled.

Automation is My Friend

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What Do You WANT and EXPECT From Your“Robot Friend”?

Simple goals for your liquid handler (or plate reader, etc). It should… Reproducibly deliver the target volume with high

accuracy & precision

“Behave” – it should have consistent, predictable behavior according to the programmed methods and scripts (volume transfer, washing, mixing, alignment with labware, etc.)

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To know, the performance must be Measured and Controlled.

How to know if your Robot Friend is “Behaving”he

ight

= x

head diameter = y

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Assessing Liquid Handler Performance

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How is performance of an automated liquid handler evaluated, monitored and/or maintained? MVS Gravimetric methods Fluorescence/Absorbance methods Combination Other home-brew methods

Assessing Liquid Handler Performance

Accuracy and precision should be assessed on a tip-by-tip basis – some of the above methods do not capture this important information without a lot of time, skill and effort

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OverviewOverview of MVS Dual-Dye Photometric Method

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Output Report:Performance Well-by-well and Tip-by-tip

Well-by-well data

Failed wells

Failed tip

Tip-by-tip data

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Artel MVS Multichannel Verification System:Becoming a Standard

Beckman - World-wide use of Artel MVS by service team for OQ of automated liquid handlers

Tecan - Integration of Artel technology into QC Kit for verification of automated liquid handlers

Forensics labs, bio-pharma labs, government test labs, etc.

For more info on the MVS, please visit the Technopath/Artel booth.

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Liquid Handler Precision and Accuracy:The Impact of Not Knowing

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Monitoring Liquid Handler Performance at 10 L: Precision Only

All experiments A – G seem relatively similar

Precision only measurements – the amount of volume, may or may not be ‘correct’, but all the volume transfers are repeatable; all tips are

performing the same.

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By measuring accuracy, reagent concentration can be determined tip-by-tip and well-by-well and the assay’s characteristics are more defined, enabling a

more quantitative methodology.

Monitoring Liquid Handler Performance at 10 L: Precision and Accuracy

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Monitoring Liquid Handler Performance at 10 L:Knowing Accuracy, and Therefore, Concentration

6.80 L

9.97 L

10 L of critical compound (2 mM), 40 L total working volume

Method D – (6.80 L)(2 mM)/(40 L) = 0.34 mMMethod G – (9.97 L)(2 mM)/(40 L) = 0.499 mM

% difference in compound concentration = 35.2%(if experimental total working volume equals theoretical)

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Liquid Handler Optimization

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Optimizing Liquid Handler Performance After Sequential Parameter Adjustment

Sequential Parameter Adjustment

Albert & Bradshaw, J. Assoc. Lab. Autom., 2007, 12, 172 – 180.

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Optimizing Liquid Handler Performance After Sequential Parameter Adjustment

By tweaking and measuring (iteratively), liquid handler methods can easily be optimized when there is a standardized “yard stick” to compare results

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Facilitating a Bench-to-Automation Transfer of a PCR Assay

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Facilitating Method Transfer: Assay Transfer and Liquid Handling Steps

Target volumes delivered to the working assay

Who or what is used for the volume transfer steps? How are the volumes

measured? Quantified?

Target volumes delivered to the

‘transferred’ assay

Are the volumes equal or comparable?

=

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Troubleshooting After Transferring a Manual PCR Assay to Automation: ACCURACY MATTERS (1 of 2)

≠

Manual Method Automated Method• 10 µL Sample – In Excess; not a critical volume transfer step• 15 µL Master Mix – CRITICAL STEP; accuracy required• Thermocycler for 1.5 hours

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Accuracy Matters: Transferring & Troubleshooting a Manual PCR Assay to Automation (2 of 2)

Manual PCR assay “works” Automated PCR assay does not work

MVS used for liquid handler optimization, automation accurately dispenses critical target volume (15 L)

Automated PCR assay still does not work

MVS used to assess technician pipetting proficiency The technician was over-pipetting master mix (15.67 L)

The automation was adjusted to deliver the same volume as the manual method

Automated PCR assay worked – SUCCESS! “The automation was not to blame”

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Microtiter Plate Readers:How well do they agree?

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Microtiter Plate Readers = Vertical Beam Spectrophotometers

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Absorbance Comparison - Experiment

96-well plate filled with different concentrations of red dye solution

Plate measured on test readers and on a reference reader

6 different reader types were tested against a reference reader

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Plate Reader Absorbance of Several Readers vs. a Reference System

Ref Plate Reader = Single-channel, interference filterPlate Reader A = Single-channel, monochromatorPlate Reader B = Single-channel, interference filterPlate Reader C = Single-channel, interference filterPlate Reader D = Single-channel, monochromatorPlate Reader E = 8-channel, monochromatorPlate Reader F = 8-channel, interference filter

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Photometric Noise as a function of Optical Density

Solution-filled plates were measured ten times each reader.

The CV for each well was calculated and a pooled CV was determined at each absorbance level.

Reader A Reader B Reader CNominal

Absorbance %CV %CV %CV0.15 0.160% 0.460% 0.162%0.25 0.110% 0.340% 0.112%0.5 0.080% 0.210% 0.082%1.2 0.060% 0.170% 0.057%2.4 0.090% 0.260% 0.088%

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Plate Reader Absorbance of Several Readers vs. a Reference System

Ref Plate Reader = Single-channel, interference filterPlate Reader A = Single-channel, monochromatorPlate Reader B = Single-channel, interference filterPlate Reader C = Single-channel, interference filterPlate Reader D = Single-channel, monochromatorPlate Reader E = 8-channel, monochromatorPlate Reader F = 8-channel, interference filter

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Plate Reader E (8-Channel, monochromator) vs. Reference Plate Reader

Channels behave independently from one another. This one unit essentially acts like 8 separate

spectrophotometers.

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Plate Reader E (8-Channel, monochromator) vs. Reference Plate Reader

Channel differences become more apparent when compared to the median of the channels.

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Measuring CV After Mixing:Aspirate/Dispense Cycles vs. MVS Shaker

1-tip, 16 reps, three different target

volumes; 96-well plate

After each dispense, 3 “common” aspirate/dispense

cycles used to mix

Read plate with MVS

Same plateShaken on MVS Plate Shaker

(1) Re-read plate with MVS reader

(2) Shake Again(3) Read plate a

third time

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The original mixing method (aspirate/dispense steps) was not efficient. Customer had to notify other sister sites using same method. Note – diffusion time helped the mixing process for the 2-uL data in the original mixing method.

Measuring CV After Mixing:Aspirate/Dispense Cycles vs. MVS Shaker

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2 L (P20 tool) 10 L (P200L tool)

Directly Comparing Identical Robots & Methods at Multiple Locations

- Three different forensics laboratories (in three different cities)- Same robot models (Biomek 3000)- Same head and tip types- Exact methods and techniques employed in each lab

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Tech 1 Tech 2

J. Assoc. Lab. Autom., 2007, 12, 172-180.

Directly Comparing Operator Pipetting Proficiency *

* Two different technicians dispensing 20 L of same reagents in the samelaboratory with the same 8-tip, electronic pipette within minutes of each other

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Conclusions: Importance of Knowing Equipment Behavior

Liquid handlers, plate readers, etc. are powerful tools, but need to be Measured and Controlled.

Measuring volume accuracy and precision for the assay of interest will help build confidence in the capabilities of your liquid handler.

Not Measuring = Not Knowing

© 2012 ARTEL

Thank youArtel Corporate Headquarters25 Bradley DriveWestbrook, Maine 04092, USA

Tel: (207) 854-0860 Fax: (207) 854-0867 Email: [email protected]: www.artel-usa.com

understanding error contribution of liquid handling and ... · liquid handlers, plate readers, etc....

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