Assay failures are common in the clinical lab, here are 5 of my favorites.
Troubleshooting assay failures is a second full-time job in R&D. Here are my top 5 'favorite' root causes!
If you've worked in R&D, deployed a process into production, and had it running for any amount of time, you've probably been called in to troubleshoot a failure at some point.
Sometimes failures can be attributed to 'blameless' problems like instrument malfunctions or problems with a reagent lot.
Those are boring though, so I'm not going to talk about them and instead I am going to point fingers.
Some of my favorite root causes are as follows:
1) Changes to Temperatures or Timing - Processes are validated at specific temperatures, incubation durations and timing of steps. Even minor changes to any of these things can cause a process to fail. Too short and the process doesn't complete, too long and everything in your sample disappears, not the right temperature and nothing happens, incubated in a water-bath instead of a thermal cycler and your sample evaporates, try to setup 15 plates at a time and the first plates incubate way longer than the last plates. Timing and temperature are everything in molecular testing and shouldn't be changed without input and validation from the development team.
2) Reagent/Tip Waste Overflows - Empty the waste when it's full. Don't push it down because you think you have enough room to squeeze in another run, don't ignore the fill lines on the waste carboys, and definitely don't let any of these wastes overflow if they're flammable. This is especially important on robots where many deck components are electrified and can easily 'spark' a disaster if something like an ethanol waste container overflows!
3) Not Following Deck Setup Prompts - "The robot crashed and I don't know why." Did you follow the deck setup prompts to make sure you put everything in the right place? **Blinks** "No, I just click through all of those screens and set it up like I was trained." Le sigh. This is why we can't have nice things, people, and also why totally unnecessary scans and deck checks are added to automated processes just to be sure that when an important thing is listed in a prompt, that important thing actually gets done.
4) Repeat Pipettors - "It said pipette XX volume so I used a repeat pipette to do everything all at once." Please don't ever do this. Repeat pipettes have their place, for like bulk dispensing of wash buffers. They should not ever be used for activities where precision is required. For example, when setting up enzymatic reactions, pipetting concentrated buffers before dilution, or any other activity that requires very accurate volume dispensing.
5) Everything R&D Creates is Perfect, Except for When It's Not - 'Continuous improvement' is the mantra of the lab industry. We all try our hardest to do our best, but we can't know everything, and while it's fun to point fingers (even at ourselves!), these root cause analyses always lead to better processes!
Brian Krueger