Hello, this is Michael Peat, and I'm going to talk to you about forensic drug testing laboratory considerations in this presentation. I have a second presentation that is forensic testing laboratory considerations for specimen validity testing, and they both include the same definitions in terms of process, so I'd like to describe them before I get into the details of the drug testing program. Initial test in both cases is considered to be a screening test, and that is the terminology used by the forensic drug testing labs. A screening test simply rules out negative specimens from further consideration, and it identifies presumptive non-negative specimens, and as we go through this presentation and the second one, I will use those terms in various places. The result of a negative screen is a negative report to the medical review officer. The result of a confirmation test, or as it's called in specimen validity testing, a validation test, is one of five options. It can be considered a positive non-negative. It can be considered a negative non-negative, that is a non-negative presumptive specimen that has confirmed negative. It can be considered an adulterated, substituted, or an invalid result. Those three terms, or the last three terms, apply to specimen validity testing mostly, and I will define them in the second part of my talk, but just for the completeness of definitions, I've included them here. There are other definitions or terms that a lab might use. First is a term called aliquot, which is A-L-I-Q-U-O-T, and aliquots are actually samples of the original specimen, small samples, can be a milliliter or two, or more maybe for a confirmation test, but they're samples of the original specimen, bottle A, that are tested in the areas of the lab, which I will define in a few slides. A batch, or a batch of aliquots, is what the lab uses for testing purposes. It can include 50, 100 specimens for screening. It can include 10, 15 specimens for confirmation, as long as that batch certifies particular requirements in terms of quality control, et cetera, then that's the batch that will be tested by the lab. Aliquots are put into their batches in what's called the receiving area of the lab, and I will discuss that in a few minutes. When you think of a forensic drug testing lab, you may think that it's a forensic lab of the nature you see on programs such as CSI. That's not the case. They are actually P-factories. Specimens come into the back of the lab, or the rear door, delivered by various couriers. They're transferred to the receiving area, they're received into the lab system, computer system, they're aliquoted, the batches are built, they're tested, and then the results are reported electronically to the medical review officer. Several of the FDT labs that are certified by the NLCP, the National Laboratory Certification Program, do thousands of specimens a night. Not all of them are obviously DOT-regulated specimens, but they tend to follow DOT regulations and HHS protocols for testing all specimens. That's obviously important for the production flow of the lab, it's important for the quality of what all drug testing results are, and it certainly streamslines the process. So why, given that they process these large numbers of specimens per night, is the reliability of the lab the most important, or the most impressive, of the flow of a specimen from the donor to the medical review officer? It is because over the year, these labs have built impressive quality assurance programs. There's been a concerted effort by HHS and others in the government to build a very strict inspection program, which they certainly have, and I will discuss those in more detail in a further slide, but those are the major reasons why the reliability of a forensic drug testing lab today is so firm that it can be used for this type of testing. As I said, lots of this, what I will talk about in the first part, will also apply to the second part, but I'm going to refrain from discussing specimen validity testing until that part of the talk. So let's move on to the drug testing portion and talk about what the goals of a drug testing lab are. So the four bullets on these slides define the goals of a drug testing lab. The first is to reliably discriminate between those specimens which are positive or negative in terms of drug or drug metabolite, to determine if a specimen is valid, and to identify those specimens that are substituted or adulterated. The most important of these bullets is that they perform these tests in a legally defensible manner, which is part of what we talked about earlier in terms of their reliability. In today's world, there's few challenges to the lab results. Primarily they're in arbitration hearings and primarily it's part of the total hearing and there's very few, if any, lab results overturned in that manner today. It could happen, but I don't know of a case in the last several years or maybe even a decade where it has. So the goals are to do this type of testing and to do it in a legally defensible manner. I'm going to talk in the next slide about what a drug testing lab might look like from a flow perspective, as I think that will give you an overview of what we're going to talk about in the following slides. This is a laboratory flowchart. Specimen collection has been covered by Dr. Smith and I'm not going to repeat any of the details from that presentation. Specimens are received in the receiving area from couriers, either laboratory couriers or FedEx or some type of courier. During the receiving process, their identity is confirmed, the chain of custody is reviewed and completed. Specimens may be given their lab accession number or not, and if not, the lab will use the chain of custody number. Bottles A and B never leave that area. There are exceptional circumstances in which they might, but routinely they do not. Aliquots are removed from Bottle A, aliquots are batched, and it's that batch of aliquots that are sent to screening. Security of the receiving area is high. Nobody whose job function warrants them to be in that area is allowed in that area. If they need to be, then they have to be escorted and sign in and sign out. After screening of the batch, we'll cover that in a few slides, results are reviewed and the negatives from that batch would then be reported to the medical review officer. Presumptive non-negatives are batched for confirmation, and a confirmation can result in two different results, a negative, the non-negative specimen may confirm as a presumptive non-negative specimen may confirm as a negative, and obviously more frequently as a non-negative result. Both those, the negatives and non-negatives, are then reported to the MRO. Confirmed non-negative specimens are stored for at least 12 months under frozen conditions. Although there is no guideline in the regulations for negatives, they are generally kept for between five and seven days. All the transfers in this process of the bottles and of the aliquots and handling thereof are under chain of custody, and that is very different from some drug testing labs and certainly very different from some forensic science labs. So all those processes, all the transfers, are under chain of custody. So what is an initial test, a.k.a. a screening test? It is an immunoassay screen to eliminate negative urine specimens from further consideration. The initial test shall use an immunoassay, meets the requirements of the Food and Drug Administration for commercial distribution. The current guidelines do provide for the use of alternative screening tests, for example, Ultra HPLC MS-MS, but I do not know of any lab using those today. So what is a negative specimen? A negative specimen is one whose apparent concentration of a drug or metabolite is less than the pre-established cutoff concentration for that drug or drug metabolite. This cutoff is a line in the sand as far as this program is concerned. If a drug is present just below the cutoff, then it is a negative. So, for example, THC, the cutoff for a screening test is 50, 5-0. If that specimen tests at 49.9, it is a negative. The lab cannot go back and test it again. They can only go back and test it again if the quality control failed in that batch, but they cannot routinely establish a protocol where they statistically test a specimen around the cutoff to see how many times it tests positive or twice negative. That is not allowed and never has been allowed in this program. So it's a line in the sand. Now, does that mean that that specimen contains no drug or metabolite of a drug? No. In all likelihood, it would contain a drug or drug metabolite. For example, in the situation I just described, if a specimen was 49.9, it would undoubtedly confirm for the present of the THC metabolite. But the lab cannot go on to do any further testing on that specimen provided the quality control is passed in the batch if the cutoff, if it is below the cutoff. And that, as I said, is a line in the sand. So the next slide will define a positive specimen, which obviously would be the difference of this. So this is a definition of a positive specimen. It's one whose apparent concentration of drug or metabolite is greater than or equal to the pre-established cutoff concentration for that drug or metabolite. So in the situation that I just mentioned, for the marijuana metabolite or THC metabolite, if it tested positive at 50 or 50.1 or 50.6, it would be considered to be a presumptive non-negative specimen and be sent on for confirmation. Again, of course, the quality control has to satisfy the requirements from the SOP for that screening test before it would be sent on for confirmation. So let's talk a little bit about what a cutoff is. So what is a cutoff? Well, it's an arbitrary point on a continuum of possible drug or metabolite concentrations. And it's used to devise specimens into negatives and positives. When the program was first established in the 80s and 90s, the continuum was quite broad and the testing technologies, I'm not going to say crude, but certainly not what they are today. If this program was established now, then those testing technologies would be much more advanced and the concentration of these cutoffs would be much lower because the technology has improved. But the concept is the same. It's still an arbitrary point and it's still used to devise specimens into negatives and positives. Administration of the cutoffs is normally by regulation. We're talking primarily DOT in this program, but also the federal government in other programs, or by a company itself in the company's program. So it's by regulation or by company. There are two sets of cutoffs used. One is used for the screening tests, and the second one is used for the confirmation tests. And the next slide is going to consider the screening tests. These are the mandated screening cutoffs by HHS, and those are incorporated into the DOT regulations. And these are ones that have been in use for many years now. The list has grown over the years. Initially it was five drug classes and seven drugs. It's now seven drug classes and 14 drugs. Some cutoffs have also changed. For example, cocaine metabolites has decreased, codeine and morphine has increased over the years, and the THC metabolite has stayed the same for a number of years now. Fencyclidine also has stayed the same. 6-acetylmorphine was added almost a decade ago, and the amphetamines have changed from 1,000 to 500, and MDMA and MDA were added as drugs several years ago. MDMA is methylene deoxymethamphetamine, or ecstasy, and MDA is methylene deoxyamphetamine, which is a metabolite of ecstasy, just as much as amphetamine is a metabolite of methamphetamine. And we'll have a lot more to say about amphetamine and methamphetamine in a few slides. Our next slide reminds us of the requirements for a screening assay. These are the requirements for the screening tests, or the initial tests. It must be an FDA-approved immunoassay. The lab must validate this test before testing specimens, and there are strict protocols defined by the National Lab Certification Program for this validation. There are a sort of catch-all phrase covered under the third bullet here, but I'm not going to go through that in any detail because it still involves recognition by the FDA. The lab may conduct a second initial test using a method of different specificity to rule out cross-reacting compounds. That clause is contained in the HHS guidelines. I don't know of a lab today that does that, and if it does that, it has to validate it, quality control it, et cetera, and that's an expensive process in today's world. So the requirements, Bullets 1 and 2, are the most important of those. Now the next slide includes a core of the testing protocol for immunoassays, and by core, I mean the factors that go into an immunoassay and reporting a result from one. On the left, under reagents slash calibrator, method, instrumentation, those are factors that are included in a clinical assay as well as a forensic drug testing assay. Reagents and calibrators are bought from manufacturers. Methods are provided by manufacturers. Instrumentation is obviously bought by the lab from manufacturers. So all those are common to both clinical and forensic drug testing assays. It's the column on the right in blue that's different. I'm going to go from the bottom up here. Internal quality control for a forensic drug testing assay has to be per batch. There are requirements for the number of quality control specimens per batch. There are requirements as to what their concentration should be, that is they should challenge the cutoff, and there's requirements for their performance per batch. On the other hand, quality control in a clinical lab can be done once a day, can be done once a week in certain cases, and there are no sophisticated requirements for what the concentration of the quality control specimen should be. So that's very different. The two uses of quality control, clinical versus forensic drug testing, are very different. And as I mentioned, chain of custody, aliquot chain of custody, is very important in a forensic drug testing lab. It will be the batch of aliquots the chain of custody follows, but in a clinical lab there's obviously no requirements for a chain of custody. And finally, data review. In a forensic drug testing lab, a certifying scientist will review all the results of the screen. So they'll review the quality controls, the positives, the negatives, the chain of custody documentation, and only after all those reviews have been performed that certifying scientist will release results to identify presumptive non-negatives or for those results to be transmitted to the medical review officer. Again, there's no requirements within a clinical lab for data review of that nature, and data review is often performed automatically by the instrument, and sometimes that automated results are reported directly to the client without any human intervention. So there's big differences on the right side of this diagram between a forensic drug testing lab and a non-drug testing lab. The next slide I'm going to talk to is about the quality assurance programs and the inspections that are carried out on forensic drug testing labs certified by SAMHSA or HHS, and this is the key and the meat, so to put it, of the reliability of these labs today. So let's talk about the levels of quality control and quality assurance in a forensic drug testing lab. Number one indicates that a 10% of the batch has to be internal open slash blind quality control specimens. What is the difference of an open and blind quality control? An open quality control in this case is inserted into the batch by the technician who is performing the screening test. Generally, they have to be 75 or 125% of the cutoff. They do not or cannot be negative completely, and they cannot be five or six times of the cutoff. They have to challenge the cutoff, and that is built into the HHS guidelines. So those are the open quality controls. They're used, obviously to determine the stability of the calibration and very rarely today does the calibration become unstable but if it does these open quality control specimens are designed to pick that up. Blind quality control on the other hand is inserted into the batch by the receiving technician who builds that batch so the receiver may have a number of blind specimens given a lab accession number they insert it into the batch randomly and that batch is then sent off to be tested. Obviously under that scenario the screening tech does not know which is the blind quality control. Blind quality control has to be at least one percent of the quality controls of the batch size so in that case if it's a hundred specimens at least one has to be a blind quality control the total has to be ten percent of that hundred or ten most labs do ten open and one blind in that scenario but there has to be a combination of open and blind. The identity of the blind quality control specimen is broken by the reviewer that I talked about on the last slide and that reviewer will approve the batch based on that quality control specimen the blind being identified as positive or negative whatever the result should be and if that result is not met for example if a positive cocaine specimen or benzolecanine specimen comes back negative then the results of all that batch are thrown out the specimens are reallocated and the process starts over. It is very very rare that that happens but it does happen occasionally and this process is designed primarily to show the specimens have not been misallocated somebody misallocated a specimen again very very different from a clinical testing lab. Secondly there are what's called open performance testing specimens which are tested quarterly it's supplied by the National Lab Certification Program to the lab for performance testing it tests the lab in its detection of drugs drug metabolites specimen validity the ability to reconfirm specimens which I'll come to in a few minutes it tests the labs ability to do all of that and it tests it stringently in the sense that it provides specimens around the cutoff specimens close to the reconfirmation cutoff so the challenge that I mentioned so all of this is designed to make sure a lab can perform which is different in large degree to the tests are done in a clinical lab just to show their proficiency it's not necessarily the latter isn't anyway not necessarily seeing how a lab can perform it's seeing how efficient it is. The second major part of the Quality Assurance Program is this National Lab Certification Program this is a requirement that labs go through every six months it's two to eight trained inspectors for two to three days depending on the size of the lab so this is an important inspection and in today's world nearly all non-negatives are reviewed by this inspection team every six months it's an expensive process it can cost some of the bigger drug testing labs a hundred thousand dollars a year or so but it's important to maintain the quality assurance and it's important to ensure the users of these drug testing labs of the accuracy of the results I'm going to switch gears somewhat and start to talk about confirmation in the next slide. A confirmation test is a second analytical procedure performed on a different aliquot of the original specimen to identify an agent or the presence of a specific drug or metabolite that is taken from the HHS guidelines the important things to note different aliquot second analytical procedure to identify an agent or the presence of a specific drug or metabolite what it doesn't mention is this test has to be quantitative the screening test has to be in a sense semi quantitative has to be accurate precise around a cutoff for the screening assay this test requires the lab to perform a confirmation test and a quantitative test or and a quantitative test so it has to determine the concentration of the drug or drug metabolite in that particular specimen. Now we're going to move to a confirmation test of what are these confirmation tests. Confirmation test or the analytical method for that confirmation must combine a chromatographic separation and a mass spectrometric identification and we've listed some combined procedures here in the first bullet GC-MS was the first one used gas chromatography mass spectrometry and a lot of labs still retain that technology for confirmations there are others including what's called hybrid technologies MS-MS UPLC high-resolution mass spec that are in use these are more expensive than GC-MS but coming down in price from an instrument perspective the labs in any way they use these have to validate them before they can test specimens and again that validation and the review of it is part of the inspection program that I just talked about so these tests will result not only in the identification of a drug or drug metabolite but also in its determination of the concentration of that drug or drug metabolite. So I'm going to talk about what drugs are confirmed for. These are the confirmation cutoffs mandated by HHS and DOT. Just a few comments THCA is the metabolite of THC and the concentration for confirmation is lower than that in the screening assay which was 50 benzalekonine is a metabolite of cocaine and its concentration is lower than that of the screening assay for cocaine metabolites 6-monoacetylmorphine 6-acetylmorphine or MAM is the initial metabolite of heroin which is diacetylmorphine and its detection in the urine specimen above this cutoff of 10 is conclusive proof of heroin use and there's no other explanation for that and we'll talk more about that in the toxicology part of my presentation. The other drugs are the same as the drug classes screened for in the initial assay or the screening assay. The next two slides document the screening cutoffs and the confirmation cutoffs. This is the first of those two slides and on the left you can see the screening cutoff in nanograms per milliliter and on the right the confirmation cutoff. Let's talk about marijuana metabolites. The screening cutoff is 50 and the confirmation for this THC acid metabolite is 15. That's because THC acid is about a third of the total metabolite picture and as you can see 15 to 50 is about 1 to 3 and we'll talk a little bit about that in the toxicology part of my presentation. The other issues that I want to point to make are codeine and morphine opiates are 2000. Part of that is the issues caused by poppy seeds and again that would become part of my toxicology program module. Hydromorphone, hydrocodone, oxycodone, oxymorphone are the opioids tested for and 6-acetylmorphine is the initial metabolite of heroin and conclusive proof of heroin use if detected above these cutoffs. So we're going to move on to the second slide that includes the other drugs. This second slide includes PCP, amphetamines, MDMA and MDA as screens and confirmation of PCP and methamphetamine, amphetamine, MDMA and MDA. These amphetamines to methamphetamine and amphetamine, these are important things and drugs to consider which I will do in a couple of slides because they're obviously fairly routinely detected and one of the major reasons for testing in this program. Ecstasy and its metabolite MDA is very rarely detected. Occasionally it is but it's very rarely detected. So we're going to move on to the core diagram as I called it for screening and talk about it from a confirmation perspective. So this is the core diagram as I refer to it for a screening assay that deals with confirmations. The left part of it is very similar to the screening, reagents, calibrator, method, instrumentation. They're all identical between a forensic drug test and a clinical test. Nothing is changed, documentation requirements, etc. are almost the same between a clinical accreditation inspection and a forensic drug testing inspection. On the other side, internal quality control and adequate chain of custody, obviously differences between a forensic drug testing lab and a clinical lab. The clinical lab may not require either but the forensic drug testing lab requires both. The key to a confirmed result is this data review step. This is a positive certifying scientist who generally has been in the lab a long time, has technical expertise, very experienced in the lab's protocol and the review of data and they will review all the data for the test including the screening result, including the quality controls and they will review all the chain of custody including the external chain of custody form and if all that data and chain of custody are good, they will release the result to the MRO. They obviously also serve as the touch point for the MROs if they have technical questions and a number of them have built close relationships with MROs over the year. It is your trusted resource within the lab because they're very, very familiar with the lab procedures and protocols and chain of custody documentation. So I'm going to move on now to the methamphetamine reporting requirements of this program. There is a history to this slide and the history is worthy of note. In the 1990s, an alert medical review officer with a methamphetamine result in front of him as positive interviewed the driver and determined that the result and the driver profile, the personality etc. of the driver didn't match. He requested a retest of Bottle A and as an aside to the story, you as a medical review officer will have access to Bottle A and if you have any concerns about the result from the lab, I certainly would recommend that you get Bottle A retested. I wouldn't recommend that you do this under normal circumstances. I would only recommend that you do that if you are really troubled by the result you got from the lab. In the circumstance that I started the story with, the retest of Bottle A came back with a negative methamphetamine. After many backwards and forwards between the lab, the MRO and the other labs and the program, the National Lab Certification Program, it was decided to send Bottle A to a second certified lab who confirmed the absence of methamphetamine. Turns out the driver had been using mega quantities of ephedrine. Obviously in those days there was no GPS, so trucks were not covered across the globe by GPS and the driver was probably using this ephedrine to drive longer hours. However, in the confirmation process, not in the body of the driver, but in the confirmation process, the ephedrine, which had large quantities in the urine specimen, converted to methamphetamine in some situations, particularly when there was heat and acid involved. So we trace quantities of methamphetamine, but methamphetamine nevertheless, and it did not convert to amphetamine. Obviously if methamphetamine had been used, you would have expected the driver in this case to have metabolized it to amphetamine. Now that's no big surprise today. We all know that methamphetamine can be made from ephedrine or pseudoephedrine pretty quickly and easily. You have to be careful not to blow yourself up in the garage, but it can be made pretty clean and easily today. That's why you cannot buy ephedrine and pseudoephedrine off the counter, so to put it in the drug store you have to get it from the pharmacist. So because of this, HHS and DOT mandated that not only specimens that would be reported as positive methamphetamine must also contain certain amounts of amphetamine, it's metabolite to be reported positive for methamphetamine. In today's world, that is, positive specimens for methamphetamine must contain 250, that's the confirmation cutoff, nanograms per ml of methamphetamine, and at least 100 nanograms per ml of amphetamine to be reported as positive for methamphetamine. And this rule has been in place with varying concentrations ever since those first instances in the 1990s. So what does that really mean? And that's shown by examples in the next slide. So these are some examples of how a lab might report results to you as the MRO. The first bullet, methamphetamine of 2000, amphetamine of 600, clearly both those by confirmation are above the cutoff, the cutoff of confirmation being 250 for each. So that clearly would be reported to you as a positive methamphetamine and amphetamine and you would then interview the donor with those results in hand. The second bullet, methamphetamine is still 2000, the amphetamine however is 200. So that's above the 100 mentioned in the prior slide for amphetamine and it's above 250, which is the concentration cutoff for methamphetamine, that would be reported to you as positive methamphetamine. The amphetamine is below 250, it doesn't meet the required cutoff but it is above 100 which validates that methamphetamine at 2000 and that would be reported to you as positive methamphetamine. And again you as a medical review officer would proceed with that information. The third bullet is methamphetamine at 2000 with an amphetamine at 75, 2000 is above 250, amphetamine is below 100, that would be reported to you as negative even though the methamphetamine is above 250. You would not know that if methamphetamine was present in that specimen, you would only know that it took a little longer possibly to get a result but you wouldn't know why and that specimen would come back to you as negative. Now this reporting protocol is examined on every NLCP inspection just to ensure that it's being followed. So when you get a positive methamphetamine only result, you can be assured that lab has determined the concentration to be somewhere between 100 and 249. So an important slide to bear in mind if you're going to take the MRO certification exam and the final bullet is just showing you an amphetamine positive at 600 above 250 and a report from the lab as positive methamphetamine. The next two slides I'm going to discuss only briefly some drugs not included in the program because they're commonly found today in the toxicology world but I just want to cover them pretty quickly. On this slide I've included some information on the cannabimomimetics and the methcathinone derivatives or bath salts. Let's talk about cannabimomimetics for a second or two. There are a large number of these today. 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forensic drug testing

laboratory considerations

drug testing process

screening tests

confirmation test

medical review officer

reliability in drug testing labs

quality assurance programs