Drawing the Line: The Rationale Behind Blood Tube Collection Order

- This is Lab Medicine Rounds, a curated podcast I'm your host, Justin Kreuter, a transfusion medicine pathologist and assistant professor of Laboratory Medicine and pathology at Mayo Clinic. Today we're routing with Dr. Brooke Katzman, assistant professor of laboratory medicine pathology and consultant in the Clinical Core Laboratory service division at Mayo Clinic. Thanks for joining us today, Dr. Katzman.

- Thanks for having me. - You know, so we wanted to have you here to talk about kind of the rationale behind blood tube collection order.

And I know before we started recording, I was confessing to you that, that I oftentimes am struggling with what color top do I need for what tests. And so I'm really jazzed to kind of get this a little bit straightened out with you maybe for our audience, since we're really a diverse audience here. Why is it important that kind of different sample collection tubes are collected in a particular order?

- Sure. Yeah. So the, the majority of our collection tubes have some sort of additive added to that particular tube. And so really that's what it comes down to is being able to make, make sure that those don't cross into other tubes. So for example, when a needle's inserted into the tube there, there's a slight chance that that ne needle will pick up some of that additive. So for example, blood splashing against the needle.

And then the issue is then when that next tube is collected, there's a risk that it'll carry over into that tube leading to a contamination by that additive. And this, I would say, is more of an indirect contamination where it's, it's happening again because of a, a splash or, you know, transfer from the needle. One example of maybe more of a direct contamination would be if a pH phlebotomist realizes that, you know, they collected the tube out of the wrong order, let's say for example, EDTA.

So potassium EDTA is one of the additives in our light purple tubes. And so rather than restarting, re poking that patient, they then pour that tube over into the SST tube, for example. And so now you have a serum a tube that now contains EDTA. And so you go to test that and, and now you have some really discrepant results. So both of these scenarios, test results for some of those analytes, they're gonna either be falsely elevated or falsely decreased.

And, you know, we know that has a, has a direct impact on patient care. So you risk misdiagnosis mistreatment often re results in a recollection of a specimen due to a spurious result, whether it's detected by the lab or detected by providers where it's just not consistent with clinical picture. So really to avoid this, the Clinical Laboratory Standard Institute, which is CLSI, they've set forth a a standard recommended order of collection within their guidelines.

And this is really what all laboratories and phlebotomy units are supposed to be following. - You know, I I I'm glad you kind of brought up this idea

of kind of this, I guess you were talking about as direct or indirect contamination, right? So, you know, I'm sure there's some folks in the audience where yeah, they could understand, oh, I collected the wrong tube and, and sort of innocently feel like, oh, let me put that into the right tube.

Not appreciating that there is, there are particular additives in these different tubes and how that could actually cause an interference in, in the lab testing, like you're saying, which, you know, there's gonna be additional costs, right? If the test needs to get right, rerun, potentially inappropriate care for the patient, if somebody's acting on false results as it may not get picked up. If it is picked up, there's even a delay then to get that additional testing.

So I guess what you're saying is like, when we make a mistake, we just need to call foul it ourselves and Yeah, - And you know what, often it's in the best intentions, right? A phlebotomist doesn't wanna restick a patient, they wanna say, you know, I already have this blood, why can't I use this, put it in this tube. So that's, that's the challenge. It comes from a good place, but we just have to, I think it's important just to realize why exactly we're doing what we're doing.

- Yeah. And to understand how, you know, gratefully it's nice that when we have phlebotomist, people that are trained in to do this, to take this role, but I know nurses also train in and do phlebotomy. I, I remember when I was in medical school, I, I did a, a number of collections from my own patients. So, so we have a lot of people on the team that may be involved and, and this is important knowledge to understand. So I think we're gonna dive in.

What, what is the particular order? Can you kind of like walk us through that, that rationale? Because I think that might get at that point about that concern about potential carryover. Like why is the order of the rainbow - The order the way it, that it's, yeah, yeah, yeah. So this is kind of a, I'll just say is a very simplified version of really what the intent is is or what, what the differences are. So the first collection would be a, a blood culture tube or a blood culture bottle.

And, and those are, you know, those are gonna be for specimens to, to measure bacteria, identify bacteria. We want basically the cleanest specimen that we can for that. The next tube is what we call our light blue top or our sodium citrate tube. These are tubes that are often used for coagulation testing.

And just another other important pre-analytic component is that they have to be completely filled to ensure that that proper ratio of blood to anticoagulant sodium citrate in this case is the anticoagulant. The next group would be the serum tubes. And those could be those with or without a clot activator or a gel. And so we think of, excuse me, sssts serum separator tubes. They may have a gel in it that once you spin it, it separates the blood from the serum.

They can be gold in color, but these tubes are typically used for general chemistry testing. The next are the heparin tubes. So these tubes can again, also be with or without gel. They can be either dark green, light green tops. Typically, again, chemistry tests are performed using these specimens. This is gonna result in plasma. And we specifically use these in STAT testing so that we can rapidly test the samples.

The, the difference with the serum is that we need it to, we need about 30 minutes for that specimen to clot before we can test that sample. So with plasma we can test immediately. The next tube type would be the EDTA potassium EDTA tube. This would be with or without a gel separator. Again, these are often lavender or light pink, purple in color. And these are gonna be used primarily for hematology and blood bank testing, which I'm sure you're very familiar with.

And then the next would be sodium fluoride or potassium oxalate. These tubes contain those particular molecules because they serve as glycolytic inhibitors. These basically preventing the breakdown of of glu glucose. And so these are gray top tubes and we use them again as I mentioned, typically for glucose testing where we wanna prevent any sort of glycolysis that can occur.

And then finally the yellow top tubes, these would be, these are tubes that contain an A, C, D, D solution, either A or B, really being for genetic testing. That's what these tubes are used for. And then there are some others that come, you know, after that. And it really is dependent on institution if there's research samples or things like that. - Wow. Okay. I really appreciate this. You kind of laid it out there. And one of the things I just want to go back to,

'cause I know it's often a point of, of conversation, is the difference between serum and plasma. Right? And you kind of nicely highlight kind of a stat version of this, and this is what I love for our clinicians to understand what's the difference between serum plasma and then for pathologists who are in practice to think about what are the timings and what are the practicalities for, for running.

So can you elaborate a little bit on what is the difference between serum plasma and kind of why that serum is gonna have that kind of 30 minute additional time tacked onto it? - Sure. Yeah. So serum, as I mentioned, that's going to require clotting. So that sample has to sit for 30 minutes, clot has to be generated, you need to separate that. And that is what's used for, for a lot of our testing. As I mentioned, that plasma sample, that's what we use in our stat labs.

So again, doesn't need any sort of clotting to occur. I will say that there are some differences in the quality of the specimens. Serum is typically a much cleaner specimen compared to plasma. So we try to use that when we can. But understanding turnaround team time needs, we will all of the testing performed in the hospital, clinical laboratory uses plasma and we typ, we use the plasma separator tubes or the PSTs.

- Wow, that's awesome to understand that, you know, there, there's pluses and minuses, right? I think as you're kinda laying that out, you know, a cleaner sample with serum, but it's gonna take longer to kind of produce that serum. In other words, for the coagulation to go down and, and kind of become devoid of, of clotting factors and fibrin in your sample as opposed to, Hey, I need it right now. There's not time for that.

- Right. And ultimately, I mean, it really comes down to the specimen that's chosen is what's acceptable by the manufacturer for the tests that are performed. Some tests can only be performed using plasma, some are only validated for serum. So we really have to, that's kind of what's guiding what tubes are collected.

- That idea, that concept. I, I know there's probably a decent percentage of our audience that's familiar with like, you know, what is, is an approved sample type for a particular test, but maybe for our clinical audience and, and our student audience that's listening, you know, what do we mean by that is, you know, I think sometimes people struggle with, you know, here's the patient's blood. Like why can't we test for this analyte?

- Yeah. So it really comes down to the two different flavors of tests. I'll, I'll say, so we have FDA approved tests. These have gone through the rigors of being reviewed by the FDA, the manufacturers submitted it, and there are specific claims in what we call the package insert that states it needs to be tested using this specimen type. It can be tested only on this patient population. These are the values that you would expect. These are the values that can be reported.

I know this often comes up, why can't I have a lower result? While that's not the, the, the manufacturer has not validated that, and therefore, if it's not included in their package insert, we would be basically going off-label, similar to how you would be prescribing a drug off-label for, for, you know, when it's used for its non intended purpose. Hmm. So then the next would be the lab developed tests.

And those are ones that, you know, we've often for esoteric measure, for esoteric analytes, but we, we measure it using, let's say a mass spec or, or some other instrument. But that test is developed with parameters that are within the lab. So developed by the laboratory, validated by the laboratory. And I know there's a lot of regulatory discussion around this right now, and there are changes coming, but for all intents and purposes from a stat lab, we like to use FDA approved tests.

So these are kits, you load reagents on an instrument and that testing is performed according to the specs by the, by the manufacturer. - Hmm. Right. So you, those are the ones where I guess because it's been validated that way, you, you, you're, you're I guess, confident that there's not gonna be any interference. - Yeah. So that, but, and those interferences are often listed in the manufacturer's package insert.

So one of the interference that we deal with a lot is hemolysis. So the presence of free hemoglobin, this can cause an interference with the wavelengths that are used for some of the tests, or it just, you're dumping contents out of the cell and therefore it's not really a true measurement of, you know, that particular analyte. So those things are spelled out and, and we still, I i I just wanna clarify this. We still have to validate that manufacturer's claim.

So the minimum requirement we do is called par precision accuracy, reportable range, and then reference interval. Those are the normal values. So that is kind of the bare minimum that we have to do. We often do far more than that as, as you can imagine. But those are the requirements to still show can we get the same values that the manufacturer claims? And, and we check this for things like precision manufacturer claims, we can go down to this value with this amount of precision.

And so we wanna know that, that, that is the same situation on our instruments in our hands. - Well, you got me kind of going down this pathway. I'm really nerdy and curious. I'm gonna have to invite you back again to talk about, you know, analytes and interferences. But - That's my, that's my, that's where my heart lies. Pre-analytic interferences are my favorite. If you can, if there's a favorite part of the testing phase,

pre-analytic is definitely mine. I've spent a lot of - My, I feel like academic - Career investigating interferences. - Well, I feel that we'll definitely bring you back to come back to kind of the, the order of collection. And you brought up, you know, when errors and mistakes are made, are there ways that we can kind of catch these errors? Are, I mean, I guess once it's done, I mean, if something puts somebody puts it in a different tube, it's nearly impossible to detect.

Is that, is that right? Or do we have ways of we - Yeah, we do have some tricks up our sleeves, fortunately. So I'll just say in general, you know,

teaching toward the process of correct order of draw is, is just critical. You know, we spend a lot of time on this. We have our trainers and trainees in a one-to-one fashion. So they're training one-to-one, and really they can't perform any patient testing until they've proven that they can, you know, operate using our standard operating procedures without any sort of assistance. So that type of coaching and mentoring really does establish the muscle memory.

So the, you know, repeating it over and over, it kind of becomes ingrained in your mind, this is not something that I do every day. And so therefore, you know, I am probably more rusty than, than our phlebotomist. But I think that is, is kind of critical to setting the stage or, or trying to prevent this from happening in the first place.

And we do, you know, we also do direct observations and things like that to be able to catch issues where, where people may be making these types of mistakes, but from a lab perspective, we can co, we can identify these incorrect collections using some creative ways to do this. So we kind of leverage the inaccurate results that are produced.

So if a specimen is collected inaccurately, we use those inaccurate results to help tip us off to say, Hey, this probably wasn't collected in the correct order. So an example I'll give for you is potassium, EDTA. So EDTA is a molecule, it's sequesters, divalent and trivalent metal ions. And so it's possible that EDTA contamination could be identified by observing a decrease in the concentration of some of our commonly measured cation.

So that would be magnesium and calcium, but we can also see large elevations in potassium. So I mentioned the tube additive is potassium, EDTA, and so we'll see very, very high potassium concentrations. And, and all of these components are not compatible with life. We, we, we can recognize that. And what we use is our, our middleware on the instruments to flag those samples and say, Hey, you might wanna take a look at this.

And, and it's based off of those patterns, a low magnesium, a low calcium in the presence of a high potassium up, we're thinking EDTA contamination. And so in that case, we can reject that specimen, prevent any results from being reported, and we can recollect that specimen. - That's awesome. And that's, and you're running a STAT lab and so even in the high pressure environment, this is functions well, - Correct.

Yes. We, we rely pretty heavily on some of those middleware rules to help help us and guide us and detect some issues with specimens. - And I guess last question I wanted to ask, you know,

are there some best practices for kind of reinforcing this idea of order of draw in the training? Or is it just one of these things of nothing to it, but to do it and repeat, repeat, repeat? - Yeah, I mean, that helps. Repetition, as I mentioned, helps, but I think the first thing I'll say, and this is true of not just laboratory collections, but laboratory testing in general, really helping to un have our techs understand the why behind it, behind it, why is it so important?

And I think sharing, you know, what would happen if you didn't do this correctly? And then translating that into the patient impact all of our staff, they wanna do the right thing. They wanna make sure that they're giving, they're providing samples that provide the best results for our patients. And so I think it's really helpful to, to help them understand that their work is not only valuable, but the way they do it is critical for accurate testing.

So that in general, I think is probably the best, the best way to address order of draw and, and maintain, you know, appropriate procedures for that. The next, I I sort of mentioned this, but regular observations, you know, more frequently assessing what people are doing. It helps identify that problem, but it also helps us identify other opportunities for improvement. So not just the order of draw process, but all facets of collections.

It helps give us an idea of what may be problem spots, what may be issues in our SOPs that we need to address. So I think doing that along with positive reinforcement is really, you know, a useful tool to help encourage the desired behavior.

And, and this is happening, you know, we often, we, we do our annual competency, but doing those direct observations more frequently, monthly, for example, allows you to catch any issues, you know, a bit earlier than you would with just an annual competency review. - I can imagine that that also kind of normalizes the experience of being observed, right? Correct. If it's happening more frequently, yes. Rather than like, oh, here's my observation, so now I'm really gonna do things correctly.

If it gets more exactly into the general habit, you know, maybe that makes it a little bit more of a welcoming environment and also maybe puts me just doing my normal routine. - Right. Right. And I'll say the, I mean the, the last thing I'll offer is from the laboratory perspective. So, you know, if we detect these errors or we suspect them, it's really important that we share that back with the phlebotomy team and particularly in a timely fashion.

This kind of goes back to the direct observations. If we can do this more quickly, we can, you know, nip it in the bud so to speak, and try to adjust these issues. Maybe it's something that a, that a phlebotomist is just, they just aren't unsure about the process and they keep doing.

And so if we can kind of catch that and prevent it, and we accomplish this through using our lab event management system, so lems, that allows the phlebotomy team to perform, you know, targeted education and address any issues or behaviors as they occur so that we can try to prevent these from happening. And, and we do a great job at this.

Every once in a while we have, we kind of have these flags and say up looks like EDTA contamination, but in general we do a great job of managing that and, and making sure that we are doing it correctly.

- Wow. We've been rounding with Dr. Brooke Katzman, understanding the why behind the Rainbow of Tube collection. Thanks for joining us Dr. Katzman. - Thank you so much. Thanks for having me. - If you've been interested in registering for Mayo Clinic Laboratories upcoming phlebotomy conference, please check out the the show notes. And thank you for taking the time to talk with us. To all of our listeners, thank you for joining us today.

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