Randy True  0:00
‍So the safety part is about protecting y'all. I do need to give the disclaimers that this is not formal lab or biosafety training and that the site managers and personnel and volunteers, you are all ultimately responsible for maintaining appropriate training and certifications and and also with compliance with local, state and federal regulations. We're providing this information on a best effort basis during this public health emergency. And we're trying to highlight the key things, you know, that we pay attention to on a safety basis. So it's not it's not by any means complete. I included some links here. Y'all are being being an EMS here, you're probably pretty familiar with safety protocols medical protocols. So, basics, basic PPE is involved, mask gloves, lab coats. I usually prefer a face shield to goggles because mine fog up. We do have these listed in our protocol as a checkbox. The key is to have a face shield to stuff in the bunker. Pardon?

Speaker 2  1:09
‍Sorry, Mandy. We've got goggles in the bunker. As a checkbox. The key... Pardon? Sorry, I didn't...

Speaker 0  1:12
‍We've got goggles moved up here.

Speaker 2  1:22
‍And then Randy for masks, is this, I'm assuming N95 to work with this stuff?

Randy True  1:33
‍We do sometimes use medical masks as well. I prefer N95s especially when I'm at our lab because there are a bunch of other people. From a, Yeah, from an infection control point, the N95s are preferred if you have them. From a contamination, when we get the contamination side, which is about protecting the test, a medical mask is fine. So in terms of sample handling, it's better if the incoming sample tubes are actually in biohazard bags, or if you are doing collection, if you're having all 20 people come to the same place, you could put them in a rack and just put that in a closed bin and save a lot of plastic. Again, some of that is your call in terms of your infection control procedure. The sample tubes, we do call for them to be wiped with alcohol after debagging. So the step with the highest infection control risk is when you open the tubes with the dry swabs for adding the 1X inactivation saline solution. So we prefer to do this in a well ventilated place. The current deployments in Florida are in medical office rooms, which just have windows. And so they aren't doing it like this, where you have an open garage. You know, labs typically have biosafety cabinets or chem hoods. Those are good places to do it. So there's a sort of a few options here, but I think it is important to highlight that this is the step to pay the most attention to is this very first step of opening the tube to add the inactivation solution. After that, it gets vortexed and heated, and then that it's a chemical plus heat inactivation, which denatures all the proteins and lysis the virus. And so it's not even a positive sample would not be infectious after that step. So in terms of chemical safety, I provided a link here to the SDSs involved with our test. And the two main chemicals are TCEP and EDTA, which are the two components of the main components of the inactivation solution. This inactivation solution is at two, You work with it at two concentrations, the 100x, which is what you use to make the inactivation saline solution, which is what you add to the samples. So give extra caution when you handle the 100x inactivation solution. So we provided an eye wash station. And so you should know where that is and and have a sink, but and know where the sink is as well. All this is probably old hat to y'all. Okay, so let's jump into something probably that's not as much old hat to y'all, but it is to Katrina, which is contamination from a molecular biology perspective. So we're going to focus on four types of contamination And contamination control really is the name of the game here with this, this RNA assay. Actually, a high school teacher that we collaborated with very early on, she had never got an RNA assay to work in her lab until we provided her kits last fall. And she tried with her students and she said, didn't work. And I asked her, well, did you use everything that we gave you? Did you use anything from your lab? And she said, oh, we use the water. I said, just only use what we gave you. She did, and then it worked first time and it worked for her students. So what I'm gonna try to do here is kind of convey the kind of the mindset to get into in the key highlights. And it's worth just pausing for a second. And I'd like to click on this link if it's gonna work here. This summarizes the important aspects of RNAs contamination quicker than I can. And then I think helps, will help us kind of make a point.

NEB Narrator  5:59
‍Preventing RNA degradation is trickier than preventing DNA degradation. RNAses are present in all cell types from prokaryotes to eukaryotes and can sometimes survive prolonged boiling or auto-claving. So what are the major sources of RNAs contamination in the lab? Aqueous solutions and reagents, environmental exposure as RNAs are in the air, on both surfaces and in dust, and from human skin and hair. How can you prevent this contamination? Always wear gloves in the lab and change them often, especially after contact with skin, hair, doorknobs, keyboards, or animals. Use RNAs-free solutions and RNAs-free certified disposable plasticware and filter tips. Maintain a separate area for RNA work and carefully clean the surfaces. Decontaminate glassware by baking at 180 degrees Celsius or higher for several hours or by soaking in freshly prepared 0.1% DEPC water or ethanol for one hour, followed by draining and autocleaning. Decontaminate polycarbonate or polystyrene materials such as electrophoresis tanks by soaking in 3% hydrogen peroxide for 10 minutes. DEPC treatment of solutions is accomplished by adding one ml of any remaining amino acid residue.

Randy True  7:16
‍I'm intentionally skipping through this because they start talking about a bunch of stuff that's common to normal molecular biology labs. But this gets to a real key point, which is this lab that we have set up next door to y'all is really different from a normal lab because we're only doing one thing there, this COVID test. And this COVID test is quite simple compared to most tests that are done in a clinical lab or certainly most protocols that are done in a molecular biology research lab. So you might be wondering, oh, like, why are we setting up a COVID lab in a molecular RNA assay testing lab in a firehouse. Doesn't that take a real lab? And in some sense, yes, what you're going to have is actually a real lab. But in another sense, it's so pared down and the paring down is what enables us to be successful. I set up a lab in my garage and I did a did a FaceTime call with this Stanford professor that former Stanford professor who is collaborating with another nonprofit and he said, He said, you know, I said, what do you think of the lab I set up? And he's like, he's like, looks great. I was like, do you think I'm going to have problems, contamination, cleanliness? He's like, that's better than almost any lab at a, at a university because it's yours. You have it controlled. It's only for this and you'll be in great shape. And we have been, you know. So the key is to understand kind of how to avoid these several types of contamination. So let's just go through it real fast again. They did it on the video, but RNAs is this enzyme that degrades RNA, which is way more fragile than DNA, and it's everywhere. Getting an RNA assay to work is way harder than getting a DNA assay to work because again this stuff is everywhere and if you contaminate your tube your bag of tubes or any solution or something it can give you unreliable even sometimes intermittently bad results and it can be very frustrating to track down. The place we care about this RNAs contamination is primarily in this amplification reaction. And the reason is because the inactivation reaction, you're putting a nose swab with tons of RNAs in it into that reaction. And that's part of the purpose of it is to degrade RNAs. So we really focus on the second step of this test. How do you know, you know, how do we know if you have this problem? Well, the positive controls won't work. And so another key thing that makes us able to do this test in this kind of environment with newly trained firefighters is the controls, when you use them properly and when you design the test to use the controls, give you a great indication of when the test is working and when it's not. So what are we going to do to avoid the RNAs is, number one, is access control. And I sort of already saw some of this going on. I saw Peter in the lab like leaning over the amp table and I was like, oh no, don't touch anything with your bare hands. So that's the first thing.

Speaker 3  10:39
‍I'll come back later, Deacon.

Randy True  10:42
‍That's the first thing is that, you know, I've seen this problem too when we were giving tours, three, four fire chiefs and everybody were coming in and then it's like, oh no, like you don't want to, they can just, if they don't know, they can just touch something and cause a real annoying problem. So I would recommend putting a lock on the door, putting the sign up and just controlling access to it. I mean, do definitely feel free to give tours and stuff, but just make sure people don't touch anything. There is a station, a desk station where you can touch your computer or other stuff without gloves, but it's a certain area. All the rest of the area, you just think you've got to have gloves on. And even in addition to gloves, you really want a lab coat so that you don't even get your forearm touching anything. And it's just better to be extra careful about this and just to get in the mindset where skin doesn't touch anything that's clean in the stations. So I encourage a process to do a good cleaning up front and then keep things clean. Danny Chavez said he cleans his lab every day, and I think that's probably recommended from good lab practice. I actually don't for our lab here because it takes time and I prefer to be extra careful and keep the key things in bins that I keep on a shelf. So you can also use foil to put over things to keep them clean, keep dust off. You want to be extra careful with the reaction plastics and Katrina will help kind of point out what those are. You want to be wary of cross contamination. By cross contamination, I mean you touch a pen with your bare hands, and now you get finger grease on the pen. And then you're wearing gloves, you think you're okay, but then you go and touch something that's greasy, then you can pick up some and transfer. Now, you could drive yourself crazy thinking, well, how many degrees of cross contamination do I got to worry about? Worry about the first degree and just try to get some basic procedures and kind of mindset in place. And then you still got to do the work. You can't drive yourself crazy. Another key thing that we do to deal with RNAs contamination is we just we stage things so that we can just replace them and you can just get in you can you can get a new bottle of saline you can get new bag of tubes and you want to avoid the what I call the matlock trap which is like trying to sleuth and figure out where where the problems are coming from. It's better just to replace things, I could tell you some, I learned this one the hard way. Okay, so That's RNAs contamination. Do you guys have any questions about that?

Speaker 2  13:51
‍Nope. No, but I'm sure Randy's work, firefighters would probably look a little intimidated by all this stuff. This is not normally our area. Yeah. But I think the takeaway is, yeah, we're touching stuff and make sure we're clean.

Speaker 0  14:09
‍Yep. Would, would gowns work as well as lab coats?

Randy True  14:15
‍Yeah, gowns will work fine. Just anything that's kind of covering your skin. I actually jotted down on my follow-up that we should have included a couple of disposable lab coats just to account for multiple people in there. We did provide one kind of regular lab coat. But long sleeve shirt is also better than nothing, particularly if it's clean. Okay, I'll try to hurry quickly through the other kinds of contamination and some of these are best kind of learned along with the protocol. Particularly problematic one is positive control contamination. This is what screwed the CDC on the rollout of their kit and really put our whole country behind the eight ball at the beginning of this pandemic. This manifests as your negative controls aren't negative, they're showing up positive results or in the middle in conclusive results. So it's pretty obvious you have this. It can be hard to rectify because, especially if it's in the manufactured materials. So part of this is on our side. But the part that you worry about on your side is to follow the protocol and there's some special handling steps and procedures when you touch and deal with the positive control tube. You have to use the positive control tube with every heat cycle, every run, because it confirms that the reagents are still good. It's a critical part of the overall quality and assurance. So you do have to touch it and use it for every run. So I have some sort of recommendations in terms of just using one hand. We have a key glove change. And then we also just keep the positive controls in bags in the bottom of the freezer. And these procedures have worked really well for us for a long time now and haven't had any problems. So I think we'll be good to follow those. Okay, so sample cross contamination. By this we mean transferring material from one sample tube to another. So that's like, for example, if you open a tube and there's a drop on the tube and then you get that on your glove and then you open another tube and get that on the other tube on the threads or something in a way that can get inside. You know, if you have this problem, well, actually you don't know that you have this problem if all your tubes are negative, you could be cross contaminating all over the place. And you would never know if they're all negative, But if you do have a positive, you'll see other neighbor tubes get contaminated or come up positive. So a telltale sign of that would be neighbor positives and more than one positive in a batch. Again, following the protocol, being careful when you handle the sample tubes and the lids. If you do get a drop, wipe it up and clean it. And I suggest noting if that happens on a run sheet so that if you do get some positives and you can figure it out, you can look at it more carefully. Okay, so the last contamination I'm going to talk about is amplicon. And this one is really bad. So amplicons are the product of the DNA of the amplification. And they are DNA. They're not actually RNA. Sometimes they're called products. So I showed here the diagram for PCR and I showed the diagram for LAMP. LAMP is an alternative amplification to PCR and it is like biochemistry wizardry. It uses six primers instead of two and it forms these loop structures. And this happens all at a single temperature, whereas for the PCR, you ramp it up and down. And so LAMP produces 10 times as much DNA per volume as PCR in one third the amount of time and at a single temperature. So it's like a nuclear explosion of DNA production, whereas PCR is like a very controlled sort of firecrackers kind of going off. So the key here is that with LAMP, we wanna even be extra careful with amplicon contamination. And the reason I say if you get this, it's death, is because it can contaminate the entire lab and really require you to move the lab and start from scratch. I've even heard horror stories about me to do that in a new building. A lot of times this is because you open the tubes in order to do some other analysis. So I hadn't, you know, I'd never heard of LAMP before last summer. We've run PCR in the lab that I was in charge of at my former company, so totally familiar with PCR, never heard a lamp. And when I reached out to the team that developed the tests that we use, the first thing that the grad student who ran the test told me was never open the tubes. So that's the key thing. I don't see any reason why y'all would. We never have. And we've never had this amplicon contamination, but since it's so bad, I feel like I have to highlight it. And the way we deal with this is that we treat the tubes, the amplified tubes, the heater, the light box, everything in that area as if it's radioactive. And this area is like a black hole, anything that goes into it doesn't come out. So the pens that go in there, the post-it notes, you just leave them over there. And then whenever you touch them, the tubes or the heater, whatever, you again treat it like your gloves are radioactive and you take them off and you throw them away. And so it may feel wasteful, but don't worry about wasting gloves. It's, you will waste way more if you ever get any kind of contamination. It's just a part of the process. Semiconductor industry wastes even more gloves. Glove change is critical. Again, if you follow these rules, I think we won't have a problem. There is actual extra protection from this with a thermo-lay-bio-nucleotide, big words, it's some chemistry protection that helps where these amplicons, if they carry over, they get degraded in the first part of the temperature cycle. So don't worry about that if it doesn't make sense. It's just be aware. The main thing is to be aware of this AMP area and the fact that we just treat it like it's super radioactive. Not radioactive, not because we're worried about it hurting us, but just for this contamination perspective. OK, you all have any questions on the contamination?