troubleshooting – Lablogatory

Blue Chips – Troubleshooting Ion Torrent Data

Ah, the blue chip – not much fun to see after spending a day preparing the libraries and running clonal amp overnight. There are a couple possible explanations for a blue chip, and you can figure them out by looking at the metrics of the run.

Test Fragments

The test fragments serve as a control for the sequencing run. They are spiked into the mixture of library ISPs before they are loaded on the chip. These will allow you to figure out where the problem occurred if you encounter a blue chip. If the Test Fragments are detected and are of sufficient quality, then this means the sequencing run worked and the problem most likely occurred before sequencing, during library prep or clonal amplification. If the Test Fragments are not detected, then it could mean one of two things – one – the clonal amplification did not work for either the library or the Test Fragment ISPs, or – two – the sequencing run was somehow at fault. Let’s take a look at both examples.

Troubleshooting a Blue Chip

In the event you see a blue chip, first, check to see what kinds of ISPs showed up after the analysis. For the chip pictured above, there were ISPs that had product on them, as you can see in the Live category (6,475,553 ISPs or 95.3% of the ISPs, shown in the screenshot below). This means clonal amp was successful for a small number of the library ISPs. Next, there were also Test Fragments detected, at 433,392 ISPs or 6.7% of the total ISPs. Scroll down to the bottom of the page, and you will see how the Test Fragments sequenced. We like to see the Percent 50AQ17 and Percent 100AQ17 at least in the 80’s, but even still, you can see that these were detected and were sequenced. Because of this, the sequencing run looks to be fine, so most likely the problem occurred before sequencing. In this case, we believe the library prep did not yield the expected 100pM concentration, so the library pool was over-diluted prior to clonal amplification. The library prep was repeated, and clonal amplification was run on the new pool of libraries, and the sequencing was successful.

In this next example, we have the other possibility. This chip was blue as well (this is a 520 chip, instead of a 530, to explain the different sized pictures).

First, there are only 2.7% Live ISPs, so even lower than the chip above. But the even stranger thing was that there were 0.0% Test Fragments, and at the end of the analysis, there were absolutely no ISPs left to be analyzed, library or Test Fragment. This was the only time we had ever seen a chip like this; generally, if we had blue chips, they were like the previous example. We looked at our library pool quant and it was in the expected range, so we did not believe it was a library prep issue. The sequencing initialization was successful and did not have any errors, so we did not believe it was a sequencing problem. We repeated clonal amplification with the same library pool and had successful sequencing. In speaking with our Field Application Scientist, it was decided it must have been a failure of one of the reagents of the clonal amplification – either a Taq was not present or something, so the clonal amplification never occurred, or something similar.

Hopefully you will not experience too many of these blue chips, but if you do, I hope you are a little more prepared to troubleshoot! Happy sequencing!

-Sharleen Rapp, BS, MB (ASCP)^CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine.

Troubleshooting Complex Instrumentation

When we talk about technological advances in laboratory medicine, the discussion usually focuses on analyzers, methodology, or ancillary equipment that makes testing more accurate or efficient (hello conveyer belts and molecular testing; goodbye bleeding time). While all of those are valid conversations, one component that gets left out of the mix is troubleshooting. After all, an instrument that runs 600 tests an hour is nothing more than a place to put sticky notes if it’s not working properly.

When I first started in the laboratory, “troubleshooting” more often than not meant “put a ‘do not use’ note on the analyzer and call in the service rep.” Unless the fix was something simple (like removing a jammed cartridge), we left it to the professionals. That attitude gradually changed, however. When I left the bench, most of my coworkers thought nothing of “lifting the lid” to change tubing, observing the inner workings of an analyzer as it operated, and repairing an instrument with the assistance of a service rep over the phone. In fact, manufacturers trained us fairly extensively in troubleshooting each time we bought a new analyzer. Thanks to the ubiquity of the internet, it is commonplace for a service rep to remotely take control of an analyzer in order to diagnose and repair software (and even some hardware) issues while offsite.

So what’s next? Videoconferencing software so a service rep can see a malfunction in action, maybe, or virtual reality software that can assist a bench technologist with complex repairs. Maybe even analyzers that diagnose or repair themselves!

What do you think? What is the next innovation in clinical laboratory instrumentation in terms of troubleshooting?

–Kelly Swails, MT(ASCP), is a laboratory professional, recovering microbiologist, and web editor for Lab Medicine.

Under the Hood

I like to keep some humor in the lab so when I see a technologist with a panel off a machine trying to troubleshoot an issue I will say “Uh-Oh, why do you have the hood up?” It’s a little tension breaker, especially if they are stressing about having their instrument down. It also acts as a little reset button so I can go through the troubleshooting steps with them. As technologists, we are modern day mechanics. We use instruments much more than we perform manual testing, and we are expected to be able to troubleshoot instruments that are more complex than the current day automobile.

Acquiring new instrumentation can be a lab changing experience. Each instrument has its quirks and special requirements. The vendors usually offer on site or even off site training for staff once the instrument is purchased. Who you send to these training sessions is just as important as the quality of training they receive. These sessions are where your staff will learn maintenance, operation, and most importantly troubleshooting. When your shiny new analyzer goes down, and it will, the time it takes to get it back up and running affects productivity, turnaround time, and staff morale. Nothing is more detrimental to a staff’s morale then coming into work and the first thing they hear is that the instrument they are on that day is already down. Having experienced that exact thing I can tell you it takes the wind right out of you.If it happens consistently you will see a decreased engagement by staff.

Whom should you send for analyzer training? You should have a good mix of talent and maybe some of the lower performing staff. This assures that you are keeping your talented staff engaged and shows weaker performers that you are invested in building them into a top performer. The question becomes, how do I make sure that the people I send get the most out of their experience? Let them know they will be responsible for presenting the material they learned to the rest of the staff once they get back from training. If any of your staff have an issue with that they are not the ones you should send. These small presentations will help with team building as well as solidifying the information for the key operator.

As leaders we must pick our key operators very carefully. When these choices become important is most likely when we won’t be in the office. Observe the staff that likes to troubleshoot instruments or that keep a level head once instruments are down. You want to make sure that once the hood goes up you have the best mechanic for the job.

-Matthew Herasuta