Dr. Sample will now continue drug testing with alternative matrices. Okay, why don't we switch to hair testing now? We've had a good session on oral fluid testing. As we look at hair testing, it's been used historically for quite some time, for over a century, in forensic toxicology since the 1890s, environmental toxicology for more than 50 years, epidemiological studies have been used to assess gestational drug exposure, and it's been used for drugs of use testing since the 1980s. But as I mentioned earlier, hair is a solid matrix, 65 to 95 percent of its protein, one to nine percent lipids. There are small quantities of trace elements, polysaccharides, water. And as we look at the growth stages of hair, and I'm really talking head hair right now in terms of these stages, as well as the hair growth rates in a bit. So there's an antigen phase that persists for a number of years. There is an active growing stage at any one time, 80 to 90 percent of your hair is in that active growing stage. There's a catagen phase, which lasts a couple weeks, and you can think of it as a transition phase, and then the telogen phase, which is a resting phase in which 10 to 20 percent of your hair is in at any given time. But what's important to remember is that if you have, you know, two side-by-side strands of hair, they may not be in the same stage at the same time. So each stage, you know, each hair shaft is growing at its own rate, and it's not uniform across the scalp, which means you can have different concentrations across the hair that is harvested for drug testing. So hair is not homogeneous in nature, and that's why it's important why laboratories generally don't test a single strand of hair. They test several strands of hair in order to create that homogeneity that we're accustomed to in both urine and oral fluid drug testing. So the growth rate for hair is varied depending upon the specific location of the hair, and even on the head, it grows at different rates. We generally think of hair growing at half an inch per month, or 1.3 centimeters, but that's for the vertex. That's how you get to that 90-day detection window for a collection of the commonly utilized 3.9 centimeters of hair. So, you know, while an individual's hair may be longer, laboratories really only test that closest 3.9 centimeters. And by, you know, using that segment of head hair, we think of hair as having this up-to-90-day look-back window for repetitive use. But because it's a solid matrix, laboratories to prepare the sample for analysis may cut it, prepare the sample for analysis, may cut it, they may grind it, they may pulverize it, some combination of several of those, but you want to make the hair smaller, finer, than testing that whole length or strand of hair. It's washed, and washed can include some combination of water, organic solvents, you know, methanol, phosphate buffers, etc. There could be an extraction or digestion process to remove drug from the hair matrix, and then purification, just like we do in urine or oral fluid testing, and that typically would involve solid phase extraction or liquid-liquid extraction. And while the testing oftentimes is done utilizing immunoassay, typically ELISA or the enzyme-linked immunosorbent assay technique, some laboratories may also use alternate technology, such as gas chromatography, mass spectrometry, or chromatography, mass spectrometry, or some sort of MS-MS version of GC or LC being the inlet. And more recently, at least one laboratory has been utilizing fast mass spectrometry that doesn't utilize any chromatography, and this is a screen, so you don't need to worry about that separation in time necessarily, but they're using laser diode thermal desorption or LDTD mass spectrometry, mass spectrometry. So how do drugs get into the hair? So it's possible for drugs to diffuse from the blood to the actively growing hair follicle. It may enter the hair through secretions from the sweat, from sebaceous gland secretion, and the other, and you know, what would be a particular concern would be environmental contamination. And you know, that presents, environmental contamination presents one of the main interpretive issues in hair testing. Somewhat related to that, there have been allegations of hair color, and by extension, ethnic bias, but I'll talk a little bit about that, and you know, we'll see what the science says. So this is a summary of a study that looked at the effect of pigmentation on coding deposition in hair using rats. So what was found was that pigmented, rats with pigmented hair incorporate about three times more coding than non-pigmented rats. And Long Evans rats, which have both black and white hair, the concentration of coding in the pigmented hair was 44 times higher than the concentration in the non-pigmented hair from the same animals. So the first study involved different rats with different colored hair. Second study involved, you know, comparing an individual rat with dark, and both dark and white colored hair, and there are certainly differences seen in the concentrations. There are also large differences seen, not just for coding, but also morphine and nor-coding. So, you know, the conclusion I think one can take from that is that pigmented hair possesses a greater capacity to incorporate coding and its metabolites than non-pigmented hair. So these are basic drugs, and by basic, I mean like acid base, not necessarily a basic, like, baseline. In another study, they looked at pigmented, effective pigmentation on coding and phenobarbital deposition in hair using rats. Phenobarbital is an acidic drug. Again, replicating the coding was incorporated at a greater rate than phenobarbital. Phenobarb was found to be present in the same concentration of both pigmented and non-pigmented hair, and one of the, you know, bottom line take-home messages here is that the data suggests that hair pigmentation greatly affects weak base incorporation, but not weak acid incorporation into hair. And consequently, phenobarb may also be an ideal drug to separate out factors other than pigmentation, which involved an incorporation of drugs into hair. So I think the bottom line message from this is that, at least in my mind, clearly darker hair might be more likely to incorporate certain drugs, particularly basic drugs, at a higher rate than lighter hair or acidic drugs. But the question is, at the end of the day, does that make a difference in drug testing results? So it's one thing to have a statistically significant difference, but is there, as we say in the clinical world, is there a clinically significant difference? So this study looked at hair color and race differentials and positivity rates. So is that, were those hair color and race differences driving differences in positivity rates, or is it simply a matter of differences in drug preference by different ethnic groups? So there are 2,000 randomly selected hair samples, 500 negative, 500 positive each for amphetamines, cocaine, and marijuana. Looked at the relationship between hair color and hair test results. And 4,000 specimens were equally divided between Caucasians and African Americans. And evaluated the relationship between hair color and urine test results. So one study, 2,000 samples looked at hair. Another study, 4,000 samples looking at urine specimens, comparing Caucasian and African American results. In the interest of time, really have these questions up here more as reference for you, but you really want to know, does the outcome of hair analysis have to do, is there a consistent relationship with the hair color? Is there a consistent relationship with the ethnic classification? And similarly for urine, do those positive outcomes have a consistent relationship with hair color? Or perhaps do the urine and hair testing outcomes, if you're consistent with, across all drugs, are they generally grouped or documented between, general group based on documented ethnic or racial differential prevalence for drugs? And what's in red here, I think, really the key message is that these differences they saw were driven primarily differences in use rates for particular drugs by the two ethnic groups and not by differential use across drugs between, sorry, I'm going to restate that. Driven primarily by differences in use rates for the particular drugs and the differential use of those drugs across racial and ethnic subcultures. So it's not a matter of hair color, it's not a matter of ethnicity, it really may be a matter as you're looking at these observed differences in positive prevalence rates, may be simply related more to racial or ethnic more to drug use patterns, not ethnicity or color. So as I mentioned earlier, you're talking about the take home messages, certain basic drugs have greater affinity for hair than other, especially acidic drugs, the epidemiological data suggests that there's neither a hair color nor an ethnic bias in the drug test results. Also important to remember that even if you suspect that ethnicity or color plays a role, the ethnicity or color doesn't manufacture drug, the drug metabolite is present due to use, therefore is not a false positive. And if we look across all specimen types, differences in drug metabolism, clearance rates, fluid intake, if you're talking about oral fluid testing, it doesn't, fluid intake doesn't impact oral fluid testing, all can result in differences in the likelihood of detecting drugs in a particular specimen. So I think the scientific data at this point clearly shows that there really isn't a hair color or ethnic bias, the remaining, you know, one of the other remaining interpretive issues is environmental contamination and whether that could cause a positive result. So there may be certain occupations, so there may be certain occupations, DEA agents, policemen, crime lab technicians, that might be more likely to be exposed to environmental cocaine, or, you know, perhaps via smoking, for example, cocaine or methamphetamine or PCP or opiates. And does a combination of unique metabolites, for example, carboxy-THC, laboratories measure carboxy-THC, they don't measure all they could, THC parent, in order to rule out environmental contamination. So by looking at unique metabolites, metabolite ratios, performing some sort of wash correction, some laboratories may analyze the wash from a confirmatory analysis and subtract the concentration in the wash from the measured concentration in the hair sample, or some multiple of that wash concentration is subtracted from the drug concentration in the sample. Other laboratories may analyze that wash by immunoassay, and if there's an indication that drug is present in the wash, take additional steps. Again, all of these are designed to preclude the reporting of a positive due to environmental contamination. Nonetheless, even with all these steps that laboratories take, cocaine does remain the most controversial. So most of the studies that have looked at environmental cocaine have involved the application of dry cocaine or soaking the hair in a solution using cut hair. So most of the studies, well there have been a few that have been in vivo environmental contamination, most of them are in vitro, so you run the risk of incorporation to those cut ends. Some of the published data has demonstrated that standard wash procedures, metabolites, criterion wash correction are effective. Other studies have suggested that they're not fully effective. In the case of cocaine, which probably remains the most talked about in terms of having a possible impact from environmental contamination, hydroxycocaine metabolites have been proposed as unique metabolites to increase the specificity of reporting, but it comes at a cost. There's decreased sensitivity, thereby resulting in the detection of fewer cocaines, fewer cocaine positives, I should say. And certain of those hydroxycocaine metabolites may be formed by hair treatments, or maybe presidents treat cocaine, so it remains a complicated issue. Complicated issue. So, as I mentioned earlier, in 2020, HHS published the proposed hair mandatory guidelines. Same drug panel as we've been talking about for urine and oral fluid, amphetamine, methamphetamine, MDMA, MDA, cocaine and benzalekamine, opioids, including, you know, codeine morphine, as well as the hydros and oxy compounds, 6-acetylmorphine, testing for marijuana metabolite, not the parent compound, and PCP. Most of these proposed cutoffs were consistent with what was then current industry standards, although the THC confirmatory cutoff in those proposals was 50% of what was then still as the current industry standard. There were no metabolite rules, for example, an amphetamine rule. Historically, in earlier proposed HHS regulations and what is industry standard today for non-regulated testing for cocaine, no requirement for ratios of cocaine to benzalekamine, or the presence of other cocaine metabolites, such as norcocaine or cocaethylate, which is a non-regulated norcocaine or cocaethylate. And there's also no specified concentration for codeine morphine without a medical explanation for the reporting of a verified positive. But again, emphasizing here, these are proposed rules, HHS only, and there are no final rules for either HHS or DOT testing. So, for those proposed rules in 2020, HHS received 211 comments, and they specifically requested information on color and ethnic bias, the criteria to rule out environmental contamination, the impact of hair treatments, and they also proposed the collection of—I'm sorry, I've got to stop. I lost it. Rhonda? Starting over, when HHS published their proposed hair mandatory guidelines in 2020, they specifically requested comments on a number of items, and in total received 211 responses. They requested information on color, hair color, and potential for ethnic bias in hair testing, criteria that may be used to rule out environmental contamination, as well as the impact of hair treatments on the accuracy and reliability of the hair test results. They were also proposing at that time something rather unique, in that they were asking about whether an alternate specimen should be collected as well, and that should be tested prior to reporting a positive. So an alternate specimen could either be collected simultaneously or after the lab positive, and because an alternate specimen was being collected, they were proposing that no split testing of hair for a positive. Obviously, they requested comments on the specific analytes and cut-offs that were included in the panel, whether or not specimen validity testing should be required or authorized, information about the collection material, the guides, whether they should be transparent or foil, the specimen containers, which historically have been enveloped, and, again, should those have some sort of window or be transparent, and the testing reasons. So, for example, should hair testing only be used for pre-employment and random? So they received 211 comments. They have taken those under advisement and are in the process of working through the generation of either new proposed guidelines or, on the basis of that feedback, or actual final guidelines for publication. My crystal ball isn't too good on saying when we're going to see that, but, you know, it's been, at this point, nearly, well, it hasn't been more than three years since those proposed guidelines were published, so it will be, you know, hopefully sometime soon we'll be hearing whether it will be new proposed guidelines or final guidelines. But hair testing is not coming to federal testing anytime soon. So here's the slide again comparing positivity by different specimen types. We'll focus more on the hair in this slide. Not surprisingly, hair has consistently had a higher positivity rate than urine testing always has and continues to. It really isn't surprising since hair detects a pattern of repetitive use, whereas urine is more of a, you know, single point in time or very recent use. But we see some striking differences as we look at some particular analytes, like cocaine, and you'll see in a second methamphetamine. So here you can see much, much higher positivity rates for cocaine as compared with either urine or oral fluid. Methamphetamine has the same type of picture, much, much higher positivity rates on hair than the other two specimen types. Personally, I attribute that to the fact that both cocaine and methamphetamine are basic drugs and as a reflection of how well those two drugs are incorporated into hair. It's not a difference in usage patterns between people subject to hair testing versus urine or oral fluid testing. It's all chemistry and physiology here. And, you know, looking at the, not that data, but another data set, a slightly different take here, is Quest Diagnostics performed a study of paired and urine hair specimen. So look at the positive prevalence rates. There are 30,000 non-negative specimens that were evaluated between January 2004 and June of 2005. And the data was categorized into three separate buckets. Specimens that were positive in urine but negative in hair, specimens that were positive in hair, and negative in urine, and specimens that were positive in both, individuals, I should say, that were positive in both hair and urine specimens. And, you know, building on those previous two slides, if we look at cocaine and methamphetamine, in the case of cocaine, specimens that were positive in urine and negative hair, only 2%. So what that means is if you, if one was to rely on hair testing, you'd find 98% of those cocaine positive users and you'd only miss 2%. In the case of methamphetamine, it's not much different. 5.9% were positive in urine, negative in hair. 71% positive in urine, negative in hair. And, you know, only 23% were positive in both. Moving down to marijuana, it's almost like a coin toss. It doesn't matter which specimen type you're going to choose, you're going to miss a third of the, roughly a third of the positives. So about a third of the specimens were positive in urine only, a third in hair only, and a little bit higher, but, you know, in round numbers, roughly a third positive in hair and urine. So, you know, certainly as you look at this paired collection data, or simultaneously collected data, there are marked differences in the positive prevalence rates based on the specimen type that's being used. And I attribute a large part of that to the specific drug and how acidic or how basic it is. So my last slide on hair, you know, summarizes the advantages and disadvantages. So it's long window detection, really detects that repetitive use. It's a lifestyle test that we're looking at hair testing. Hair is easy to collect and handle and store, far easier to do a second collection capability if need be. No, it's still going to have different results. I never recommend a retest or a, probably wouldn't be a split, but a retest using a subsequently collected specimen. But at least hair might have a greater possibility for that. And, you know, two subsequent positives certainly rules in. A second positive, a second specimen that's negative doesn't rule out or doesn't refute in my mind the results of that first test. Hair testing is non-invasive, although if you spent a lot of money for your do, you may argue that hair testing actually is more invasive than some other specimen types. And again, because an observed collection, you know, beating, subverting that test is likely to be more difficult than it is with urine. But a disadvantage is that it doesn't detect recent use. It means it's not good for post-accident, reasonable suspicion testing. Great for pre-employment and random, not so good for some of the other testing reasons. Even with the studies that I summarized earlier regarding environmental contamination and hair bias, those remain controversial issues, remain controversial. When HHS published their proposed guidelines just three years ago, still may not fully understand the mechanism of drug deposition into hair. And, you know, the science is beginning to increase, particularly with respect to some of the unique metabolites for cocaine. But they're still a newer science and there are relatively few controlled administration studies. So now totally switching to away from alternate specimens and moving to alternate testing technologies. So let's talk about near donor testing first. So point of collection testing devices, POCT, it's not point of care. There's no care here when we're talking about forced drug testing. But it's a point of collection device. So most of those devices are visually read. It utilizes an immunoassay. It's an immunochromatographic method that's visually read, although there may be some that have an instrumented read using some sort of automated reader. And there may be some non-immunoassay devices. But the bulk of these are immunochromatographic devices that are used both for oral and oral-to-oral testing. That are used both for oral fluid as well as urine testing. There's no instant testing for hair. And as you look at the device types, you know, physically, they can be very different. There could be some that you pipette a small amount of specimen into a well on the device, and it then migrates, and the administrator can visually interpret those results. It may be a submersion type, like a DIP card. Some may have an integrated cup. There may be some combinations of multi-component kits. So how do those immunoassay tests work? The lateral flow chromatographic immunoassay. So there is an antibody that is with the dye. At the base fluid, the liquid is added. The two interact and migrate up. And that antibody dye complex, if it forms at the test line, that means that that antibody was free. It wasn't displaced. The dye wasn't displaced from the antibody by drug. And you'd have a negative result. So it may be counterintuitive from what you're accustomed to with the tests such as pregnancy tests, looking at HCG. So for these types of tests, a negative line is represented by a color line. A positive test is represented by no line. And then they also have what I like to call a process control line. And really, that's not a QC, not a control in the classical sense. All that does is verify that fluid and reagents went from the bottom of the device or beginning of the device up to the end or top of the device to make sure that it flowed correctly. So what are some of the issues with this technology? One is the efficacy. While those tests can be very good for certain analytes, they may not be very sensitive for other analytes. The biggest example for this situation is in testing oral fluid for THC. Those devices are woefully insensitive as compared with traditional laboratory cutoffs or HHS cutoffs for THC and oral fluid. Based on those standard cutoffs, if you're using an instant oral fluid device, you may miss 70% of the positives that a laboratory test may pick up. For the other drugs, it does a great job as compared with laboratory testing. There may not be SVT incorporated. Laboratories routinely, almost always, do some sort of specimen validity testing when they're doing urine drug testing. Not necessarily with oral fluid, but certainly with urine testing. I think almost all non-regulated workforce drug testing for drugs of abuse in urine includes some component of specimen validity testing. Many of the POCT devices don't have any sort of SVT test incorporated. The FDA has regulatory oversight over these devices and would require FDA clearance. They view them as IVDs. Some states, either by statute or regulation, requires the use of FDA-cleared devices or may have other requirements with respect to those POCT devices. You have specimen collector training and device training. That training needs to be done for that specific device that's used. You need to train people on interpretation. The colored line is a red line. Are those individuals colorblind? Where colorblindness occurs, red is one of the most common types of colorblindness. What type of quality control or quality assurance is going to be performed? Unlike laboratory testing where every batch of specimens includes a control, there is no real control outside of utilizing a device, which has both time and cost implications, to run a control. And if you're going to run a control, at what frequency? These are only presumptive results. Just like initial testing in the laboratory, it's only presumptive and action should not be taken without sending the specimen to a laboratory for confirmation. When it gets sent to the laboratory, does the laboratory only perform confirmation or do they re-screen it? Only if it is presumptively positive on that lab screen do they perform confirmatory testing. What's the role for an MRO for negative POCT results? What you need to remember is that this type of testing technology cannot be used for federally regulated testing. If an employer wishes to use this type of testing, they can do so, but that's under a company policy. It can't be done as part of any DOT test. It certainly couldn't be done on the same specimen prior to doing a DOT test. The last slide I'll leave you with is this concept of an Instrumented Initial Test Facility, or IITF. It seemed like a great idea when a number of us were sitting around a table at the Drug Testing Advisory Board talking about how to get better quality near-donor testing experience. This concept of an IITF was developed. It is essentially an HHS or National Laboratory Certification Certified Screening Facility. It has all of the same requirements that a certified lab does from receipt of the specimen through screening. In the event something is presumptively positive, it would then be sent to a fully certified HHS lab for re-screening and then confirmation. Unfortunately, while this approach could be used for HHS testing, it could also be used for NRC testing. It was partly thought of as a way to assist with NRC testing. In the past, a number of sites had on-site screening labs. It was thought to be a way to meet the need there. However, because of the enabling law, not a regulation, not a rule, but the law for DOT testing that requires both screening and confirmation to occur at the same location, the IITF cannot be used as a part of the DOT program. I want to thank you for your attention and look forward to your questions.

hair testing

drug detection

racial bias

environmental contamination

point-of-collection testing

Instrumented Initial Test Facility

urine and oral fluid comparison

cocaine contamination