We're going to talk about the lab and the lab considerations in a forensic drug test. Obviously, Donna referred to them in general earlier, and we're going to talk our ways through that. I was here briefly this morning when the hands went up around professional MROs and people who are green, and obviously I recognize a number of you in the audience. But before I get started, particularly for the new MROs, I want to define three terms, because these cause confusion, and I'm going to try to use them correctly as I go through my presentation. But these can cause confusion. Initially, there is a screen. When we talk about a lab test, there is a screen, and we're going to go through what that is. But essentially, it's a test that rules out the negative specimens. And then there's a second test in the lab called a confirmation, and that confirmation confirms the presumptive screening test result, which can be of two types. A screening result can show the presence of a drug or drug metabolite, and therefore the confirmation is a test that will confirm the presence of that drug or drug metabolite. And then there is a presumptive adulterant or presumptive invalid specimen. And then obviously the confirmation test will prove the presence of an adulterant or an invalid specimen, and we'll go through that in some detail. And then there's a third step, which is the step that you guys perform, and that's the verification or validation of that lab result. And I won't discuss that today. Kent will discuss that tomorrow. And some of what I say today will become clearer when Kent goes through his presentation on the MRO duties tomorrow. So today I'm going to discuss lab testing for drugs and drug metabolites, and also for specimen validity purposes. The goal of a forensic drug testing lab is to do those tests in a manner which is legally defensible. And although there are very few challenges today to the lab testing results, the primary challenges and arbitrations, et cetera, are to the collection process, there still may obviously be challenges to a lab test. So the lab performs these tests in a manner that is legally defensible. And here is a lab flowchart. And how many of you have ever been to a SAMHSA certified lab? Actually, more than normal. If you're expecting to see CSI, don't. They are essentially P factories. And by that I mean they receive urine specimens in the back door, either on third shift or first shift, they accession those in the receiving department on this diagram, they test them, and then they report electronically in the majority of cases those results to you. And when they do this testing, they do it in batches. They don't do it individual specimen by individual specimen. They test batches of specimens. And those batches can be 50, 100. For screening purposes, for confirmation purposes, they can be 20 or 30 or 50. So it's a batch of specimens. What ensures the accuracy and reliability of what they do is the quality control that they use within the lab and the data review steps that they have within the lab. And you can be assured after so many decades that those are finely tuned processes in the vast majority, if not all, of these labs. So what you get at the end of the process is a result on an individual specimen that's been tested within batches and then certainly quality controlled and data reviewed far more significantly than any clinical chemistry result or anything that comes out of a clinical lab. Now these labs also process not just DOT specimens. They process non-DOT specimens. And the majority of them use similar processes, if not identical processes, for non-DOT and DOT specimens. And they process, the big ones process up to 10,000 or more specimens a night. So when I talk about a pea factory, I talk about a pea factory. Because we're talking about thousands of specimens a night. And obviously you need to have sophisticated quality control. So we have a receiving department. We have the screening department, which we'll go into in a few minutes. And then there's data review of the screening results. And the negatives will then be reported to you. And obviously the vast majority of the specimens received are negative. So those negatives will be reported to you. The receiving department has to be separately secured from the rest of the lab. That is, individuals who work in that department cannot have access to other departments, screening or confirmation, and vice versa, with some exceptions. The vast majority of people who work in the screening and confirmation department cannot have access to the receiving department. So there are separately secured portions of the lab for storage of the specimens and for people who work in there. The non-negatives go on for confirmation. There's data review. And then the non-negatives are reported to you. Confirmed non-negatives, either for drugs or drug metabolites or substitution or adulteration, primarily invalids, have to be stored for at least 12 months. The vast majority of these labs will keep any specimen that has been legally challenged almost indefinitely. But they have to be stored for at least 12 months. Negatives that go through the confirmation process or through the general process, generally labs throw those away within five to seven days of testing. Obviously storage of all these urine samples could become a considerable problem. And everything that happens in this process is done under chain of custody. So every time somebody touches the bottle, they will put an entry into a chain of custody. Every time somebody removes an aliquot, an aliquot is a sample of the original specimen. Anytime anybody removes an aliquot from that specimen, then they will document that on a chain of custody. These are probably the most sophisticated requirements for chains of custody in the nation. They're far superior to anything that's done in the vast majority of crime labs. So the chain of custody documentation in here is certainly very sophisticated. So let's talk about drug testing. Let's talk about what a screening test is. It's an immunoassay test. And generally that has to meet the requirements of the FDA. It's used to eliminate negative specimens from further consideration. And what's a negative specimen? It's one whose drug or drug metabolite concentration is less than a pre-established cutoff. So let me give you a couple of examples. The cutoff for marijuana metabolite is 50, 5-0. Obviously if that number comes back at 10, it's a negative. But if it comes back at 49 or even 49.9, it's also a negative. Even though if you went ahead and confirmed that specimen, in all likelihood it would contain marijuana metabolite. But if it comes back at 49.9, it's a negative specimen. There's no statistical analysis around the cutoff. There's no plus or minus two standard deviations. There's none of that. It is a black and white line in the sand that says 49.9 is negative. So that is a negative specimen. On the converse, if that specimen came back at 50 or 50.1, it would be considered to be presumptive positive and go on for confirmation. So that cutoff that you see is a fast and hard line around which a specimen is defined as negative or presumptively positive. Now what is a cutoff? It's in reality something that's on a continuum. When these were set up in the first blush back in the 80s, there was a limit to the technology. If we had to go and do that again today, we would use much lower cutoffs because the technology is much improved. And we've seen, as we'll go through in a minute, a lowering of cutoffs, particularly in the last round of modifications to the SAMHSA rules that have driven the cutoffs lower in most cases. One exception was opiates. Opiates used to have a 300 nanogram per mil cutoff. They now have a 2,000 nanogram per mil cutoff. And I'll go through the reasons for that tomorrow when I talk about the toxicology of these drugs. So a cutoff is used to divide specimens into negative and presumptive positives. In the DOT world, they're mandated. Outside the DOT world, certain companies have lower, higher cutoffs that are in use in the DOT world, and the companies obviously test for more drugs than the DOT world tests for. So you'll see some variety outside of the DOT world in certain companies, but again, remember that there are state regulations that also drive what testing can and cannot be done in that state. Okay, here are the screening cutoffs that are in use today. And these changed back in fall of last year. Marijuana metabolite is 50. That remains the same. The cocaine metabolite cutoff for screening went from 300 to 150. The opiate metabolites or opiates stayed at 2,000, but you'll see the footnote. The footnote is that the immunoassay that's used for that screening be targeted to morphine. So that means that it's preferentially detecting morphine, but will also detect other opiates. There was an addition for 6-acetylmorphine that had to be added to the screening panels at a 10 nanogram per mil cutoff. PCP remained at 25. And amphetamine and methamphetamine under amphetamines lowered from 1,000 to 500, and then MDA was added at a 500 nanogram per mil cutoff. So there was lowering. There was obviously sophisticated performance testing of the labs before these were put in place. And at this point, everybody can do these analyses well. Okay, initial drug test. We talked about it being an immunoassay. We talked about it being FDA approved. There was an additional requirement that the lab must validate this test before using it, and certainly the inspectors, as I'll talk about in a few minutes, will test that and inspect for that. And then a lab may conduct a second initial test if there's some specificity issues and do that to rule out a cross-reactivity substance. And Kent will go through that tomorrow, but it really was in there for some issues that arose with an older enzyme immunoassay with drugs such as Flagyl and others that may have cross-reacted to that. Okay, I mean, talking about an immunoassay and what is the input to the result, this is just like any other clinical chemistry test. It is, it's a test that you will see widely used in all clinical labs. The differences are that there's a specimen aliquot which is handled under chain of custody. That chain of custody obviously applies in this case to the batch, not to each individual aliquot in that batch. There's a data review step before results are released. Those of you who are familiar with clinical chemistry labs, that data review step is often automated and there's no actual physical review of the results. But in a toxicology lab of this type, there's a need for physical review of the results. And then there's some internal quality control which I'll go through. Otherwise, this is just like a clinical chemistry test. Let's talk about the internal quality control. And this is where the meat of the reliability of the program is. Internal, open, and blind. What's an open quality control? It's a quality control specimen whose identity is known to the technician running the test. And there's a requirement that there's at least 10% of quality control specimens in any batch. There's additional requirements that those quality control specimens challenge the cutoff. So they can't be positive controls that are two to three times the cutoff. They have to be positive controls that challenge the cutoff. In addition to that open, there's an internal requirement for blind quality control. A blind quality control specimen in this program is one that is inserted in the receiving department by the people who receive the specimens into the batch. And there's a requirement that be at least 1% of the batch. The analyst who tests the specimens doesn't know which of the specimens is that blind. The data reviewer that reviews the specimens before reporting will break that code. And if there's an inconsistent result, for example, if that specimen should have been positive for cocaine metabolites but was tested negative for cocaine metabolites, then the whole batch of aliquots will be discarded and the whole process will begin again with the original specimen containers. So there are strict requirements around open and blind quality control within the lab. Let's go down to external blind. They're mandated under CFR Part 40. They're submitted by MROs, DERs, whomever. The lab gets the result and reports the lab result to the MRO. Obviously we have no idea which of the specimens we receive are these external blinds. If you receive a result that's inconsistent, for example it should have contained cocaine metabolites and came back negative, what are you required to do? Notify the feds, as the gentleman said. You should notify DOT and DOT will immediately begin an investigation of the lab. Way back when, that was an issue because the stability of some of these external blind specimens was not the best. But that is a very, very rare occurrence today. So I would not expect most of you in this room to ever come across that. Certainly going forward, I would not expect that to happen. There's also an external open performance testing program that is run by the National Lab Certification Program. Every quarter, the lab will get a number of specimens that challenge not just the drug testing portion of what they do, but also challenge the specimen validity portion of what they do. And if there is an inconsistent result or the lab performs badly on those specimens, it will trigger an immediate inspection of that lab. And these specimens are not necessarily the easiest in the world to do. So they really do test the proficiency of the drug testing lab. And then there's the final requirement, which is the National Lab Certification Program inspection, which happens every six months and continues for two to three days every six months. And the inspectors will inspect every non-negative positive specimen to make sure that chain of custody, quality control, et cetera, was followed. So this is a very detailed inspection. And in the larger labs that I talked about at the beginning, it takes teams of six to eight inspectors to complete those. So there's a lot of inspection, performance testing, and quality control that goes into this program. And that's obviously the key to its reliability. Okay, here are some of the immunoassays that are used. The third bullet, the FPIA, is actually going out of production. The vast majority of labs use an enzyme immunoassay, the first bullet, and the varieties within that. These are tried, true, tested technologies that have been in use for numbers of decades. Okay, confirmatory test. It's a second procedure performed on a different aliquot, obviously not the same, to identify an agent or the presence of a specific drug or metabolite. And it uses a different chemical principle from the immunoassays. And it used to be that you could only use gas chromatography, mass spectrometry, GC-MS, but they've added a number of other mass spectrometry technologies to the list. These are all widely used, not just in drug testing, but in endocrinology, pharmaceutical testing, sports testing, et cetera. So they're, again, well-tried, well-tested technologies, and you'll see a number of labs, I think, begin to use the more modern ones of these as we go forward. And, again, that test must be validated through a very sophisticated program before it's used to test the specimens. Now, here are the cutoffs for the confirmations. And you will notice, obviously, an immediate difference between these and the ones that we use for screening. Let's talk about the most obvious one, which is marijuana. The marijuana metabolite cutoff for screening was 50, 5-0. For confirmation, it's 15. The reason for that is that the immunoassay for marijuana will detect a number of THC metabolites, and the confirmation test will only detect one. So, obviously, you would expect the confirmation cutoff to be lower than the screening cutoff. And that's essentially the same philosophy with the other drugs, where you'll see differences between the screening cutoffs and the confirmation cutoff. Cocaine metabolite, the cocaine metabolite that is monitored is only one of them. And then when you look down to the amphetamines, methamphetamine, amphetamine, and the MDMA analogs, you will see again a lowering of the confirmation cutoff compared to the screening cutoff. Let me tell you what the chemical names are for these MDMA analogs. Obviously, MDMA is ecstasy. That's not its chemical name. It's methylandioxymethamphetamine. And MDA is methylandioxyamphetamine. And methylandioxyamphetamine is a metabolite of MDMA, as is amphetamine, a metabolite of methamphetamine. And then MDEA is EVE, or methylandioxyethylamphetamine. These drugs, obviously, as Kent mentioned this morning, are widely used, generally in the younger population. But obviously, younger populations age and may continue to use the drugs. So these are the confirmation cutoffs that are in use today. There's a couple of, well, we'll come to that in a minute. This is the confirmation assay. This is identical, in a sense, to the immunoassays we talked about. Again, sophisticated quality control, chain of custody of the aliquots, data review. Otherwise, it's the same technology that's widely used in a variety of clinical chemistry-type labs. But again, the need for data review and chain of custody. Okay, there's a couple of little wrinkles, as I call them. For a methamphetamine specimen to be positive by confirmation, let me back up one step. For a methamphetamine specimen to be positive, it A, must have tested positive under the screen. That is, was greater than, equal to or greater than 500 nanograms per mil for the amphetamine's immunoassay test. And B, it must contain at least 250 nanograms per mil of methamphetamine by confirmation. And in addition, at least 100 nanograms per mil of amphetamine. Now, the reason for that is one only. Obviously, throughout the last couple of decades, everybody's read about how you can convert ephedrine and pseudoephedrine to methamphetamine, right? I mean, it's been all over the press. Way back when, way back when being almost 30 years, 25 years ago now, it was discovered that during the confirmation process, if there were large amounts of ephedrine or pseudoephedrine in the urine specimen, that confirmation process could convert that ephedrine or pseudoephedrine to very small amounts of methamphetamine. You wouldn't convert it to amphetamine because amphetamine is the metabolite and there was nothing chemically going on in the confirmation process that would have done that conversion to amphetamine. So in those situations, what was seen was methamphetamine only. No amphetamine. So back then, HHS and DOT put in a temporary reporting rule that's now become, over many decades, a permanent reporting rule that requires the lab to confirm the presence of amphetamine, the metabolite, and to do that with a concentration of at least 100 nanograms per mil. If I was taking a test tomorrow, I might remember the next slide. Methamphetamine. This is four bullets of what the lab would report to you. This is what the lab reports to you. Methamphetamine is 2,000. That's clearly above 250. Amphetamine is 600. It's clearly above 250. The lab would report to you positive methamphetamine and positive amphetamine. If the methamphetamine was 2,000 and the amphetamine was 200, methamphetamine clearly above 250, amphetamine between 100 and 250, the lab would report to you positive methamphetamine. Be assured that amphetamine is above 100 because that's part of the inspection program. Be assured that's the case, but the lab would report to you positive methamphetamine. If the methamphetamine was 2,000, again clearly above 250, and the amphetamine was 75, that would be reported to you as negative. In all likelihood, that is a urine specimen from a methamphetamine user, but because the amphetamine is less than 100, it's reported to you as a negative. Again, as I said, if I was taking a test tomorrow. And then the final bullet is pretty clear. Amphetamine at 600, it's above 250, be reported to you as positive amphetamine. There are no similar rules in place for MDMA and MDA. And the reason for that obviously is that the ephedrine and pseudoephedrine phenomenon of being converted to methamphetamine does not happen. There's no conversion to MDMA anywhere in the GC mass spec or confirmation process. Okay, now there's another series of actually little wrinkles that came up with the newer rules. As I mentioned, 6M now has to be screened for. If that screen is positive or presumptive positive, the lab will go ahead and confirm with a cutoff of 10 nanograms per mil. And then obviously during that confirmation process, they will also confirm for the presence of morphine. 6M is an indicator of heroin use. What's another name for heroin? Diacetylmorphine. It is also called horse, but I was looking for the more chemically correct name. Diacetylmorphine is converted to 6-acetylmorphine. There's no other reason for acetylmorphine to be in a urine specimen unless the individual has used heroin. The 6-acetylmorphine is then converted to morphine, which is by far the primary and largest metabolite in concentration of heroin. So if you have a 6M screen positive, you would expect obviously an opiate screen positive also, and you would expect morphine to be present in the urine. If that morphine is greater than 2,000 nanograms per mil, and the 6M is greater than 10, the lab would report to you positive morphine, positive 6M. If that morphine is less than 2,000 nanograms per mil, and the 6M is greater than 10 nanograms per mil, the lab would report to you positive 6M. You wouldn't see a positive report for morphine because the concentration is not above 2,000. In that situation, you as the medical review officer have to call the lab and ask them if there's any morphine present in that urine specimen. That is, is morphine present above the limit of detection but below 2,000? And in all likelihood, it's going to be present at a pretty significant concentration, maybe 1,500, maybe 1,800, but certainly nothing that you should worry about, and the lab will confirm that presence to you. Now, if there's no morphine present in that urine specimen but the 6M is present, I'm not sure how that can happen, by the way, but if that did happen, then you have to report that finding to the DOT because it would be such a rare finding, it would deserve investigation. And again, I can't imagine how that would happen, but certainly if it did happen, then it would need to be investigated. So that's a little wrinkle that came up with the changes that went into effect last fall around 6M and opiate testing. Okay, let's talk a little bit about reconfirmations. How do you reconfirm a specimen? Two situations. One, obviously, at the donor's request, and Kent will go through the procedure for that tomorrow, but you confirm at the donor's request. If you confirm at the donor's request, which bottle do you test? The lab. The lab will test bottle B. Bottle B is the donor specimen. We as a lab could not touch it. Only upon his or her authority can we touch it. If you as a medical review officer have questions about the validity of the lab test result, you can certainly request that lab to go back and reconfirm presence of drug or drug metabolite on bottle A. You can't ask them to touch bottle B, but you can go and ask them to touch bottle A. And to be honest with you, when I talked about the methamphetamine issue that arose a few hundred years ago, it seems now, that was because some alert medical review officers just couldn't get their head around that donor being a methamphetamine user and requested that the lab go back and reconfirm. They couldn't reconfirm in a couple of instances. So that was driven by that process. You do have that authority as an MRO to go back and request the lab to look at bottle A. Very rarely done, but you do have that authority. So when we talk about a reconfirmation, obviously everything is done under chain of custody. Bottle B has to be forwarded to another SAMHSA certified lab. I, as Quest, cannot retest a specimen that I've done in my Quest location lab. It has to go to another lab. And the donor will tell you that, or the MRO will have some arrangement with the lab to do his or her retests. That process can be driven many different ways. Now when we talk about a reconfirmation, we talk about retesting it by the confirmation technology, GC-MS in this slide, but without regard to any cutoff. So we don't use cutoffs in a reconfirmation. Now why is that? There's two reasons. Let's talk about THC metabolite. Confirmation cutoff is 15. Maybe that specimen test at 15.2, 15.3 nanograms per mil. Statistically, you would expect possibly to be less than 15 if you reconfirmed it. So obviously from a statistical variation around a mean point of view, there's a reason not to use a cutoff on reconfirmations. The second reason is that THC metabolite has been shown to decrease in concentration over time. Now in today's world where there's some strict timelines established, you probably are not going to see it go to zero, but there are recorded instances of where after several months, the THC concentration has effectively become zero. What you might see is that the THC concentration out of lab A is 25, and then the THC concentration on the specimen out of lab B may be 15, 16. Now the first look at that, you might say to yourself, they're different. But no, THC concentrations can decrease. So that would not be necessarily a surprise. It might be rare, but it certainly wouldn't be unique. So that's why we certainly look at just the presence or absence of the drug or drug metabolite for reconfirmation. Now what does presence mean? It means it's present in the specimen above what's called the limit of detection or LOD used by that lab. In the early days, some labs had fairly high LODs, and you can be assured that the unions knew which labs they were, because their chances of getting a failed to reconfirm result was better in those labs. There's now a requirement under the SAMHSA programs that the lab must be able to demonstrate proficiency for reconfirmations at 40% of the cutoff. So in THC's case, it must be able to demonstrate proficiency at 6 nanograms per mil, and it's challenged with that in the proficiency testing program. If it's a reconfirmation for methamphetamine, the lab must confirm the presence of amphetamine as well. Not necessarily at the concentration of 100 that I mentioned earlier, the lab must confirm the presence of amphetamine as well as confirm the presence of methamphetamine in a reconfirmation for methamphetamine. Okay. This is, I'm not sure why this slide is in here. These are initial testing facilities that were proposed under the changes to DOTs. These are not in place today. We do not expect them to be in place tomorrow or next year or the year after. But if you are involved with the Nuclear Regulatory Commission, any testing that they might do, then some of the nuclear power plants have these facilities on site. So if you're involved in Nuclear Regulatory Commission, then you may very well come up against these types of facilities. Okay. Let's talk about specimen validity testing. And this is where things can get a little confusing if you're new to the business. For many years, even if you were a seasoned MRO, it was confusing. But there's some clarity being brought to it in the last five or so years, and I'm going to go through what that clarity is in the next series of slides. What we're looking at here, as Donna said, there's many different ways to beat the drug test. How many of you have ever read a small book by Abbie Hoffman, which is actually titled, Beat the P-Test? It just shows you're too young. That's all that shows. It's a classic. If you find one, you need to keep a copy, because it's a classic. He wrote, I think it was like 90 pages. He published it. Actually, Penguin Books published it in the mid-1980s. I don't know if you can get it through Amazon on the Kindle or anything like that yet. But he basically went through how to beat a drug test, and he came to the conclusion that the best and most simplest way to do it was to just drink lots of fluid. And that conclusion, quite frankly, is still the right one. But some people go to extremes. I've certainly been involved in situations where people have catheterized themselves, washed their bladders out, and then put negative urine, maybe from a friend, back into themselves. All I advise is that when you do that, you make sure your friend is negative. Because that's got to be extremely embarrassing. I can't take questions. I'll get back to you later. So the specimen validity test looks at whether that specimen is substituted, adulterated. And we're going to describe a new term as we go through here. Not necessarily a new term, but a different term which is invalid. And there are certain things that you will do as an MRO for each of those, dependent upon the finding, if the specimen is proven to be substituted, adulterated, or invalid. And that's where some clarity will come in tomorrow. I'm going to go through what the lab does, what the criteria are for those tests, and then Kent will bring some clarity to the MRO role in that tomorrow. If you go on the web and type in drug testing, you're not going to find Quest or LabCorp or any of your medical review officer companies. You're going to find things like no drug test, steal the drug test, etc. And a lot of companies, small entrepreneurial companies that market products to do this. And let me talk a little bit about what they market as an adulterant and generally what's been talked about in the last few years as adulterants. Generally they market adulterants that are oxidizing agents. They have the property of being a very strong oxidizing agent. And as an oxidizing agent, they oxidize THC metabolite. So they will destroy the THC metabolite that's present in a urine specimen. Some of them work quickly, some of them work slowly, but they all have that property of being able to oxidize THC metabolite. They don't oxidize the amphetamines, because they cannot be oxidized. They don't oxidize the cocaine metabolite, because that cannot be oxidized. They don't oxidize PCP, again, because that cannot be oxidized. In the opiates, they can oxidize morphine. Morphine is subject to oxidation, but not very efficiently. It's not going to take a morphine concentration and produce a zero out of that over time. So they're marketed to destroy the presence of the THC metabolite. And they're very effective at that. But if the donor smokes dope and then takes cocaine, they're going to have a positive cocaine metabolite in their urine. They may not have a positive THC metabolite, but they are going to have a positive cocaine metabolite. Let's talk about a few other adulterants. Obviously, people use bleach. They use soap. They use salt. They use Lysol, lye. A lot of household chemicals have been added to urine over time. Some of those are effective. Some of those are less than effective. Let's talk about bleach. Bleach, obviously, will have a very strong odor to it. So where is the best place to catch that odor? Collection site. If you think that somebody who's got to receive hundreds of urine specimens a night is actually going to smell each one, you've got to be kidding. That doesn't happen. So the collection site is the best place to try to detect a lot of these because a lot of them have characteristic odors. What about soap? You shake a urine bottle, it will froth naturally. You shake a urine bottle that contains urine and soap, it will froth unnaturally. There'll be a lot of froth in that urine container. Getting those sorts of remarks help the labs determine which specimens are adulterated and which are not. OK, so let's talk about substitution. It's based on creatinine and pacific gravity, as you would expect. I want to talk a little bit about the history of this, because the history of this shows how well you can adapt, I guess, over time to a changing situation. The first rattle out of the box on substitution was actually 10 years or so ago now. And the National Lab Certification Program went back and looked at four different data sets. They looked at data from a number of different studies, as outlined on this slide. And then they said, in the 47 studies, if you chose a reporting criteria of creatinine of less than 5 and pacific gravity of less than 001 or greater than 020, then if you reported that, you would find no exception in those 47 studies for that criteria. That definition or those findings were immediately challenged for two reasons. They were challenged because there were very few paired specimens in it. That is, a specimen that had creatinine and pacific gravity done on it at the same time. Some studies had creatinine, some pacific gravity, others had hosmolality. A lot of variation around the type of specimens and what testing was done on those specimens. Also at that particular time, a number of trans-oceanic flight attendants, primarily lightweight flight attendants, were reported as substituted and terminated from their positions. How many of you fly the Pacific or the Atlantic? How many of you have ever watched how much water a flight attendant drinks? In fact, they're advised to drink it a lot, a ton, in fact. So they immediately challenged these criteria as being unrealistic. And they challenged it and said, our members, because obviously it was a union, can produce urine specimens that have a creatinine of less than 5 and a pacific gravity of less than 0.03. So what DOT did, it went ahead and did a water loading study. Loaded up some individuals, predominantly female, with water and tested them over a period of hours. And of the 500, 113 satisfied the criteria for dilute, but none were reported as substituted. And around that time, I did an N of 1. I drink about nine shots of espresso a day. If I don't get my nine shots, I'm a bear. But nevertheless, I drink about nine shots. So I said to myself, I'm going to drink nine shots first thing in the morning, and then I'm going to drink water throughout the morning and see what I can get my creatinine and specific gravity to be. I can tell you I felt awful by the time I finished. And I was in steady state in the sense of what was going in was coming out. But I could not get my creatinine down below 7 and my pacific gravity below about 0.045 or something. I couldn't get to a substituted urine. And then Cheryl Barbanel reported a study in which she looked at a large number of specimens, and neither could she find in that large number of specimens any that were not seriously ill that produced a substituted urine. And then in 2003, which seems not too long ago, there was a symposium held in Tampa where two of the flight attendants who had been dismissed had actually gone through water loading experiments at a SAMHSA certified lab, had their creatinine and specific gravity tested at that lab, and were shown to be able to produce urines with a creatinine of less than 5 and a specific gravity of 0.03 or less. Now, their creatinines weren't 1 or 2. Their creatinines were 4.5, 4.6, but they were less than 5. That caused changes in the regulations. And we'll get to those changes in a minute. But it caused changes in the regulations. And at the same time that that changes occurred, there were criteria that were passed on the labs. The labs had to do certain things around specimen substitution and adulteration. So let's talk through these slides. I'm not going to read every one here. But a certified lab, creatinine, specific gravity, pH, oxidizing adulterants. Remember, I talked about oxidizing agents. Additional validity test if required based on observations, et cetera. So a lab now has to do, and has had to do this for at least in the last four to five years, a number of tests around specimen validity. And then when we talk about adulteration, we're talking about this being confirmed adulteration. That is, we've done the same type of tests that we did for a drug. We did a screen. And then we confirmed that drug with another chemical principle, primarily mass spectrometry. A confirmed adulterant is a screen. And we have confirmed the presence of that adulterant. This becomes important in the definition I'm going to move to in the next slide. So the presence of the adulterant has been confirmed, or as SAMHSA called it, verified. So when we talk about pH, pH of less than 3 or greater than 3, a specific acid is used or needed to get to a pH of less than 3. Would hurt like hell to pee a specimen with less than 3 pH. A pH of greater than or equal to 11, that's a very strong alkaline. Bleach isn't going to get you there. That's a very strong alkaline. So this is clearly an adulterated specimen. Nitrite is one of those oxidizing agents. Nitrite's also presence in urine from what? UTI. So there is a cutoff for nitrite, 500 micrograms per ml. Literature data shows that you can get to about 125 from a UTI. You can't get any higher than that. So obviously, this is fourfold what you might expect in extreme UTIs. Chromium, halogen, pyridine, glutaraldehyde, they're all oxidizing type agents. They have the impact on THC metabolite. Obviously, surfactant is soap. And then the presence of any other adulterant is verified. People add an alcohol or something of that nature to it, and you verify it. That would be an adulterant. You would not expect to see, obviously, isopropanol in a urine specimen, and not most urine specimens anyway. So those are adulterants. The urine specimen is reported as substituted. Now, this is the rules that were issued by SAMHSA, Substance Abuse and Mental Health Service Administration, HHS. Urine is reported as substituted when the creatinine is less than 2. Obviously, in the situation with the flight attendants, that was not the creatinines that they could produce. The Pacific gravity is less than or equal to 0010. We've now gone to four significant figures from three. And I will advise all of you that when you do the test tomorrow, if you're taking the test tomorrow, to be very careful about where those zeros are. They sort of blend into each other as you read exam questions. Or the Pacific gravity is greater than or equal to 0200. When might you see, very rarely, a creatinine of less than 2 and a Pacific gravity of greater than 0200? Generally, that's not urine. Or it is urine that somebody's added some of those Gatorades that are yellow in color, those types of high potassium, high sugar drinks, in an attempt to fool the test. So these are generally not urine specimens, although they're called substituted. People have obviously substituted urine post-collection for that material, or substituted that material for urine post-collection. So that's a substituted specimen. The prior slides were adulterated. And this is just a reminder of when a specimen is reported as dilute. Creatinine is greater than or equal to 2, but less than 20. Pacific gravity between 0010 and 0030. This is a dilute specimen. OK, invalid. Talk about invalid. This was a new definition. An invalid specimen, at least in the substituted arena, is fairly clear. It's when there's an inconsistent creatinine and Pacific gravity. And this slide contains some examples of that. Creatinine value that you wouldn't expect compared to that Pacific gravity, or vice versa. That is defined as invalid. And Kent will go through your MRO role in that specimen tomorrow. Another criteria is pH is greater than or equal to 3, but less than 4.5. And then there's some defining marks on the upside. That's because normal urine pH is generally between 6 to 7 and 1.5. 4.5 is pretty low for a normal pH, and 9 is pretty high. So again, this is because you wouldn't expect those values of pH in a normal population, and everybody decided that was an invalid specimen. OK, here is the slide that sometimes causes confusion. This is the same series of compounds that I showed you, and the end of the sentence said is verified. In this case, it is determined. The difference, a drug or drug metabolite is confirmed by a mass spectrometric technique. You can certainly confirm a number of these adulterants with techniques using different chemical principles, and mass spec being one of them, ion chromatography being another. But they're so rarely performed, and most SAMHSA certified labs do not do that type of testing, and it's expensive to do, several hundred dollars in most cases, that most labs actually don't do that. What they do is they take another aliquot of the original urine specimen, and they retest it, if I want to call it that, using the same procedure that they used to do the screen. So they use the same colorimetric procedure, or whatever they did the screen with, they use it again just to determine that there's still an adulterant in that specimen, that they didn't get the wrong aliquot or something. And then they will say, it's determined, we got the same test twice, we're now going to call that invalid. Because we haven't verified the presence of the adulterant, we've just redone our screening test, therefore we suspect strongly it contains an adulterant, we're going to call it invalid, and then you guys are going to take over as MROs. And you have a fairly easy choice when it comes to this type of thing. There's maybe one exception that Kent will go through, but generally it's a fairly easy choice tomorrow. OK, then we go to the bottom, interference with the immunoassay, interference with the mass specs, and then my favorite, the physical appearance of the specimen is such that it may damage the lab's instruments. Not sure what that quite means, whether it's thick and gooey or flaming or something of that type. I've never seen a report of that, but that is on the list. So we've got adulterated specimens, we've got substituted specimens, and we have invalid specimens. OK, now we have a difference, the only difference that occurs in the regulations anywhere between SAMHSA and DOT. The rules that cover substitution testing for federal employees, not federally covered employees, but federal employees, and the rules that are in place for federally covered employees such as DOT, substitution testing is the only place, from a lab point of view, that they differ. And they differ because DOT said, we consider the finding of less than 5 micrograms per mil of creatinine, sorry, 5 milligrams per decaliter of creatinine. We consider that finding, if it's less than 5 milligrams per decaliter, to be so rare and so unusual that we require you, MROs, to do some special things. And essentially what it requires, because we, the lab, will report these to you. For example, we'll report you a 4.6 milligram per decaliter creatinine at a specific gravity of 0009. There's four significant figures again. That's less than 0010. We'll report those to you. And that requires you to do what? Contact the DER and do an immediate recollection under direct observation, because it's such a rare occurrence. And SAMHSA said, and for federal employees, what SAMHSA has said, for a federal employee to be deemed as substituted, or his or her urine to be substituted, the creatinine has to be 2 or less. And specific gravity has to be 0010 or less. They don't have that gray range between 2 and 5. So if you're working in that area, you need to remember that difference. It's the one place where DOT and SAMHSA differ. OK, let's talk about validity testing retests. This is different from reconfirmation for drugs. A retest of a specimen for specimen validity purposes uses the same criteria as the original. So if you're retesting for substitution, you use the same criteria. If you're retesting for the presence of the adulterant, you use the same criteria. So there's a difference between drug testing and validity testing in the retests. We talked about the types of adulterants. There's all sorts. We talked about the books. This is a perfect example of one that's pretty sophisticated chemistry. Obviously, it can be hidden pretty quick. It's a very small tube. Donna said you've got to empty your pockets out. You can put this in a sock. I mean, nobody's going to find this on you. It's actually a mix of peroxidase and hydrogen peroxide. And the hydrogen peroxide is the oxidizing agent. Oxidize away the THC metabolite. And then the peroxidase comes behind it and destroys the hydrogen peroxide. Pretty sophisticated. And they've gotten very clever over time in developing these adulterants. That's why it becomes very expensive to confirm them. Because it's very expensive technology needed to go ahead and confirm stuff like that. This is still my favorite. Fine purifier and cleanser, suitable for precision instruments, jewelry, and bodily fluids. It's simply detergent soap. Clear detergent soap that was diluted down 50 to 100-fold. And that $3 bottle that you bought from the supermarket, right, put in these little tubes that I showed you, and you sell on the internet for $50 for each tube. Sort of margins that I really would like to see in my lab, but can't do that. OK, the urinator, Donna mentioned the Wizzator is more graphically correct. We report results this way for the primary testing, and Kent will go through this tomorrow for MROs. And then obviously, we have a series of reports that we also have for split testing. Splits always have failed to reconfirm or reconfirmed in front of the report. I mean, obviously, we split. We're not doing the range of tests that we do. And initially, we're just focusing in on one aspect, drug positive, substitution, or adulterant. Litigation packages contain copies of data. I certainly said up front, lab results are not challenged, or very rarely challenged. But nevertheless, the labs produce a number of litigation packages. And certainly, you as MRO may have seen a number of those. I strongly advise that if you are part of an arbitration hearing, and any question about the lab result comes up to call the lab and have them have a representative present. And then sometimes the dragon wins. This is not a program that's going to detect every drug user in the workplace. It's a program that's designed to deter drug use. Remember, I talked about right up front, 49.9 THC metabolite on a screen. That undoubtedly contains THC metabolite, right? So people adulterate, substitute. This is a deterrence program. I once heard a talk from a statistician who went through the specificity and sensitivity data around drug testing. And his conclusion was that if you needed to detect every drug user in a workplace, then you needed to have a random drug testing program that was at a 600% or more level. And there's no way that that's going to happen in today's regulated world. And it will be far too expensive. The return on investment may well be very, very low. So sometimes the dragon wins. OK, I'll take questions during the break.

lab considerations

forensic drug test

screen

confirmation

presumptive adulterant

verification

urine specimens

quality control measures

mass spectrometry

specimen validity testing