Assay Transfer Troubleshooting

Nov 10, 2014

What’s important when transferring an assay? General troubleshooting tips introduce a real assay transfer mystery (solved!)

Assay transferAutomating an assay—or transferring an assay from the benchtop to a liquid handling robot—is not always as straightforward as one would hope. Many assays are complex and involve numerous individual components and variables. You have to pay attention to the small details when approaching an assay transfer, details such as fluctuations in the environment (temperature/humidity); reagents and their associated lot characteristics and impurities; workflow; labware differences; growth conditions; technique and method development for the automation; types of controls to employ; contamination; light levels; if the instruments are calibrated (plate readers, pipettes, pH meters, etc.) and many more.

Liquid handling details in particular play a key role in a successful assay transfer, but these details and variables are often underappreciated. For instance, most lab personnel think they can operate pipettes with accuracy, precision, and speed. But do they know? Without actually knowing how much volume is transferred within the benchtop assay, transferring the assay to automation might lead to unnecessary troubleshooting tasks, wasting time and resources. In one example, a laboratory incorrectly spent days troubleshooting their liquid handler when in fact the error in their assay transfer was a completely different liquid handling issue (download the poster to see this lab’s approach to diagnosing and troubleshooting the method transfer).

While that troubled assay transfer story had a successful resolution, what’s the best way to solve assay transfer problems? When your manual and automated results don’t match up, where do you begin your troubleshooting?

A quick search of the internet reveals troubleshooting guides for specific assays and methods, but surprisingly, no general guide or framework on how to approach troubleshooting. Below we’ve put together a few suggestions on how one might approach troubleshooting.

General Troubleshooting Guidelines

Troubleshooting is an art that is perhaps under-taught in many labs. There are many ways to approach it—the important things to bear in mind are that you should question every assumption, and that little details can make a big difference. Writing everything out and making lists can also help.

1. Understand the assay, materials, equipment, etc. Make sure to implement calibration and training programs. Develop good documentation.
2. Write out expected outcome(s). Be as specific and detailed as possible.
3. Write out specific problem(s). Again be as specific and detailed as possible.
4. Write out your workflow, and based on steps 2 and 3, identify the most likely trouble spots. These are good places to begin, but you may need to expand.
5. At each trouble spot, identify all the potential variables. Question your solutions, reagents, and equipment and notice when these are different. For example, if you’re just repeating an experiment, identify what’s different between the two trials—are you using the same solutions, pipettes, reagents, pipette tips (different pipette tip characteristics can affect how much liquid is transferred)?

You can create a troubleshooting checklist to make sure that nothing gets missed (here are a few suggestions):

  • » Instruments and equipment
  • » Reagents and samples
  • » Labware and materials
  • » Liquid handling variables
  • » Storage conditions
  • » Experiment conditions
6. Don’t be afraid to ask for help. Forums and guides abound. Plus there are often nice technical service people on the other end of the phone line.
7. Once a difference has been found, create appropriate studies and controls to test your theory and rejoice in finding the reason behind the problem, or loop back to the top and try again!

Every detail can be important, and accuracy matters. Just to name a few detailed approaches: When using different equipment, make sure that they are similarly calibrated or that differences can be corrected for. Verify that you applied any corrections in the calculations. Take time to verify the reagent bottles, lot numbers, and that storage conditions are optimal. Evaluate differences between the two assays with respect to liquid handling, labware, reagent concentrations, operator-to-operator differences, and compare accuracy of all critical volume transfers.

Conclusions

So what’s important when transferring an assay? If your assay transfer went smoothly then the important factors and variables were successfully covered. But if you’re having problems with an assay transfer, then what’s important are the details—assay conditions, reagents, equipment, liquid handling, and so on. And sometimes it’s the details that you least expect, the ones where you assume that your liquid handling is accurate, but a second check shows that it’s not.


Additional Resources

Learn more about assay transfer


Download Poster: “Accuracy Matters When Quantitative, Manually-pipetted Assays Graduate to Automation – A Story in Diagnosing and Troubleshooting”


Keith AlbertAbout the Author

Keith Albert

Keith is an expert in liquid handling performance management, providing customized services across a wide range of platforms, applications, liquid volumes, and liquid classes. As Senior Applications Specialist, Keith combines his extensive knowledge and experience with rigorous science in his work in liquid handling system performance research, customer education, and assay/method validation.