The Importance of Liquid Handling Details and Their Impact on your Assays

Transcription

1 The Importance of Liquid Handling Details and Their Impact on your Assays European Lab Automation Hamburg, Germany May 30, 2012 John Thomas Bradshaw, Ph.D. Artel, Inc.

2 Automation is My Friend Automated liquid handlers (Mr. Robot) can be powerful tools when their performance is Measured and Controlled.

3 What Do You WANT and EXPECT From Your Liquid Handler? Simple goals for your liquid handler. 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.)

4 Assessing Liquid Handler Performance How is performance of an automated liquid handler evaluated, monitored and/or maintained? MVS Gravimetric methods Fluorescence/Absorbance methods Combination Other home-brew methods Accuracy and precision should be assessed on a tipby-tip basis some of the above methods do not capture this important information without a lot of time, skill and effort

5 Overview Overview of MVS Dual-Dye Photometric Method

6 Output Report: Performance Well-by-well and Tip-by-tip Tip-by-tip data Well-by-well data Failed wells Failed tip

7 Precision and Accuracy: The Impact of Not Knowing

8 Monitoring Liquid Handler Performance at 10 ml: 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.

9 Monitoring Liquid Handler Performance at 10 ml: Precision and Accuracy 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.

10 Monitoring Liquid Handler Performance at 10 ml: Knowing Accuracy, and Therefore, Concentration 9.97 ml 6.80 ml 10 ml of critical compound (2 mm), 40 ml total working volume Method D (6.80 ml)(2 mm)/(40 ml) = 0.34 mm Method G (9.97 ml)(2 mm)/(40 ml) = mm % difference in compound concentration = 35.2% (if experimental total working volume equals theoretical)

11 Liquid Handler Optimization

12 Optimizing Liquid Handler Performance After Sequential Parameter Adjustment Sequential Parameter Adjustment Albert & Bradshaw, J. Assoc. Lab. Autom., 2007, 12,

13 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

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

15 Facilitating Method Transfer: Assay Transfer and Liquid Handling Steps Target volumes delivered to the working assay = Target volumes delivered to the transferred assay Who or what is used for the volume transfer steps? How are the volumes measured? Quantified? Are the volumes equal or comparable?

16 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

17 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 ml) Automated PCR assay still does not work MVS used to assess technician pipetting proficiency The technician was over-pipetting master mix (15.67 ml) The automation was adjusted to deliver the same volume as the manual method Automated PCR assay worked SUCCESS! The automation was not to blame

18 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 (1) Re-read plate with MVS reader (2) Shake Again (3) Read plate a third time Shaken on MVS Plate Shaker Same plate

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

20 Identifying a Bad-tip-in-the-box

21 Identifying A Bad Tip In the Box-of-Tips Situation: 8-tip tool on a Biomek Disposable generic tips 1-use tips wet dispense 1 ml or 15 ml 12 reps across 96-w plate Generic Tip Box 96-well destination plate 1. Pick up tips 2. Aspirate target 3. Wet-dispense target into buffer 4. Dispose of tips

22 Repeatedly Identifying A Bad Tip (8-tip robot, wet dispense, 12 reps across 96-w plate, HEAT MAP) Plate 1, Box 1 well 9D off Plate 2, Box 2 tip 9D did not deliver properly, again Plate 3, Box 3 (place rotated) 180 deg rotation of plate, showing Plate 3, well Box3 4E = 9D well location well 4E off Plate 4, Box 4 tip 9D swapped with tip 1D before run.

23 Monitoring Sequential Dispenses Uncovering Systematic Error

24 Target Volume Accuracy Decreases After 5 th Replicate Across Plate After 5 th rep, the mean volume decreases for the next 5 reps 70 ul 110 ul

25 Target Volume Accuracy Decreases After 5 th Replicate Across Plate Drop in accuracy after 5 th replicate

26 Standardized Platform for Volume Verification

27 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 2 ml (P20 tool) 10 ml (P200L tool)

28 Directly Comparing Operator Pipetting Proficiency * Tech 1 Tech 2 J. Assoc. Lab. Autom., 2007, 12, * Two different technicians dispensing 20 ml of same reagents in the same laboratory with the same 8-tip, electronic pipette within minutes of each other

29 Conclusions: Importance of Knowing Liquid Handler Performance & Behavior Liquid handlers 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

30 Thank you 25 Bradley Drive Westbrook, Maine USA Tel: (207) Fax: (207)