Hello, this is Michael Peet and I'm going to talk to you about hair testing and some of the lab procedures and pharmacological aspects of that. Hair specimens have gained a lot of attraction and attraction over the past two decades and it's now one of the most commonly tested specimens outside of the DOT HHS regulated arenas. Dr. Smith reviewed the status of DOT approval HHS and DOT approval of hair and where we are in that process. We have yet haven't seen any proposed mandatory guidelines. However, hair is in common use outside of the regulated business primarily for pre-employment reasons and certainly that is because you can take three centimeters of hair and you have approximately three months of drug history within that hair and we'll talk a little bit about that in a later slide. I'm going to review what we know today. We know more than we did five years ago and some of that is good and some of it is not so good. But in general, if you have sophisticated technologies available, sophisticated immunoassays and sophisticated mass spec procedures, you can detect drug in almost any hair. It's a question of how much drug is present in that hair. So I'm going to move first to comparing hair and urine as sort of general specimens for drug testing. So this slide includes some pros and cons comparing hair to urine. Obviously one of the pros is it's a less invasive collection and it is directly observed. Multiple sampling for hair can give a drug history under certain circumstances that has been challenged of late by some data that I will talk about in a future slide. It's obviously more resistant to adulteration and substitution, although there are products that claim to be able to beat the drug test in hair. And there's easier shipment and storage of a hair specimen to the lab than there is for urine. Cons are variation in detection windows. Generally you can't expect to do a post-accident test on a hair specimen because the drug hasn't incorporated into the hair in that short period of time. And there also is some debate about how much drug you have to use before it appears in hair. That's the second bullet, less is known about the science. We have more sensitive procedures are needed and those mass spectrometry technologies are more than capable today of testing hair and they're more than becoming cheaper to buy and the labs can obtain them. Proficiency testing programs, however, are very difficult to establish for hair. And the one that I know of best is the European one. There's none as far as I know that are available within the US. So that is certainly a con when you look at establishing a hair testing program for federal and regulated employees. So I'm now going to go to the next slide which includes the screening cut-offs for hair and the one after that will include the confirmation cut-offs. These are the screening cut-offs used by two labs who perform a lot of hair testing today. Lab A screening cut-offs and Lab B's are both in picograms per milligram of hair. So you can see the sensitivities that are needed to be able to detect these drugs in hair specimens. Marijuana TAC is one in Lab A and two in Lab B. The other drugs here are comparable although Lab B tends to be a little higher than Lab A. We have seen no proposed cut-offs from the federal government but my feeling would be it would be a combination of these and certainly I expect marijuana to be either one or two because that is needed to detect the concentrations of marijuana metabolite and other cannabinoids in hair. So now I'm going to move to the confirmation cut-offs. Let's talk about the confirmation cut-offs. Again, we're looking at picograms per milligram. You can see in both labs marijuana metabolite THCA confirmation cut-off is 0.1 picograms per milligram of hair. That obviously is a very low concentration and requires sophisticated mass spec technology and care and experience to be able to perform that assay. Cocaine, opiates, opioids, PCP are similar between the two labs and in the 100s of picograms per milligram of hair. It's important to realize that in some of these cases, particularly cocaine, there is a need to include the metabolites of cocaine and not just benzolecanine. So you can see in lab A, benzolecanine, cocaine, cocaethylene, which is a product that's formed between cocaine and ethanol and then norcocaine, which is another metabolite of cocaine. It's requirements that those all be detected at a 300 picogram per milligram concentration where in lab B is the same except it's lowered this concentration for cocaine to 50. This is important as we will talk about external contamination potential on hair and the determination of metabolites in the hair specimen to avoid accusations that the hair was contaminated with cocaine. So again, you can see very low concentrations and we're going to talk a little bit about why that is in a few minutes, but secondly, the need to detect metabolites. The next slide is going to start the discussion around pharmacology. These are four bullets that relate to the pharmacology of hair or more correctly, to the structure of hair. The last bullet relates to some data that I've already mentioned, but we'll mention again. Obviously, hair consists of a follicle or a bulb and a shaft and as Donna has said, the aim is to cut the shaft close to the head and certainly not pull the shaft from the head. Three stages of growth, antigen, catagen and telogen. Antigen accounts for about 85% of scalp hair follicles are in this phase and a lower percentage for other body hair. The HHS DOT program will not, I'm sure, incorporate other body hair, but some of the programs in use today will do so if head hair cannot be obtained. And then you can see there's a vertex region of the scalp where it grows at 0.4 millimeters per day or one centimeter a month. So in general, hair grows at one centimeter a month and many procols call specified collection of about 1.5 inches, which is approximately 90 days worth of hair. And again, that is something that is routinely done now. The hair is collected, packaged, sent to the lab, weighed and then treated as I will mention in a few minutes. I'm going to keep talking about pharmacology on the next slide. Drug is incorporated into hair by several different pathways. Passive diffusion into the follicle base from blood is embedded into hair as bands grow and that amount of drug deposited is proportional to the blood concentration and the distance of the band from the surface can estimate time of use. There is no intent in any of the workplace drug programs in existence today using hair to do those sorts of determinations and I'm sure they won't be within the HHS DOT regulations either. The other way is through sweat glands and the sebaceous glands. It's absorbed into the shaft and after the formation of the shaft and then obviously grows with the shaft during that process of growth. So these are the ways a drug gets into hair. We really know very little about which of these are the most prevalent and we know very little whether some drugs prefer to passively diffuse or whether some drugs are more present in hair because of the sweat glands, etc. We know very little about those exact processes and I'm not sure that matters per se in these programs but obviously it would be nice from a scientific point of view to establish that. So the next slide is some talking points around hair and I'm going to move to that slide now. These are some talking points that relate to hair testing in the lab. It's the same approach as urine testing as an initial immuno test or screening test followed by confirmation use max spectrometric procedures. The latter will include some of the more sophisticated particularly LC-MS, MS or Ultra HPLC MS, MS or even time of flight MS. So it's going to be a different ball game for inspectors who inspect these labs because you would expect the inspection team to have some experience and knowledge of these sophisticated mass spec procedures when they do their inspections. Chain of custody and QC protocols are similar to urine testing and that's again you know not to be a big surprise all these programs include those sort of protocols. However as I mentioned PT programs are difficult to develop. There is a lab certification program available through the College of American Pathologists but that program does not have a PT proficiency testing component that deals with real hair specimens but there is one available. I've done some inspections both within the U.S. and outside of the U.S. under that program and it's interesting to see the approaches used in hair testing by both the American labs and some of the international labs. So I'm going to move on to more talking points. This slide deals with external contamination. There is worry and concern that labs have to have external contamination wash procedures and there's no consensus on the procedure to be used. The lab surely has to demonstrate that no drugs are washed out of the hair with that wash procedure and it has to demonstrate that that wash procedure removes any drugs that are sitting on the hair and not in the hair and there are some reports that some of those surface contaminants move into the hair during the wash cycle. That's why it's important that drug metabolites are detected. It's extremely important that the lab can show that that drug has been in the human body through its metabolism and certainly that's the case for cocaine but all drugs it's the same that there has to be a demonstration that metabolites can be detected. I talked a little bit about that earlier and the labs that are doing non-workplace hair testing today certainly satisfy those requirements. These are some summary bullets. Adsorption from deposition by sweat and absorption from blood and serum is the most common theory that that drugs get into hair during those mechanisms. which has been used outside of the workplace for some criminal investigations etc. There's no proposal to use it inside the workplace but that has been called into question because of diffusion among the shaft as the hair grows and not distinct bands remaining as distinct bands to document use. Detailed dose to concentration studies are not available although those have begun to be available there are no detailed studies for all of the drugs that we're talking about. Again I'm not sure that's important because we are just looking at detection and not trying to determine them in the time of use and it's not suitable as I mentioned for reasonable cause or post-accident testing because of the time lag between drug use and the incorporation into hair shafts. That time lag could be seven days maybe quicker than that for some drugs but it may be up to seven days. So that's this slide and now I'm going to talk about some hair versus urine data. I have three slides that compare hair and urine testing. The first is the study design and the next two slides are the comparison of the urine positives and hair positives. So we're looking at positive prevalence rates for hair and urine which are paired whether there's concordance between hair and urine test results, same donor, same collection date. Positive rates are higher in hair than urine for most drugs as I'll show and both specimens detect some positives that the other does not and when you look back on what I've told you in the last few slides that shouldn't surprise you but as we'll see that some of the drugs there's quite a variation. So the next two slides are going to cover that prevalence comparison. So these are the drugs and the hair specimens and urine specimen data. This is approximately 193,000 paired specimens that were analyzed by Quest between 2004 and 2009. 73% of these were pre-employment, 12% were random and 15% were other. If you look at the data and go across the percent difference, the percent difference is the comparison from hair to urine. So for example overall positive rate was 12.6 for hair and 7.6 for urine. And amphetamines, methamphetamine and cocaine in particular, the positive rate was much higher in hair than it was in urine. In marijuana it was essentially the same, in opiates urine was more effective at detecting the opiates than hair specimens and PCP was essentially the same. So you can see that from a pre-employment perspective you would get an increased positive rate from amphetamine, methamphetamine and cocaine use if you use the hair specimen compared to a urine specimen collected at the same time. So this is interesting data and it certainly supports some of the data that's been used by the hair testing lab to advocate for hair testing. The next slide is going to look at the similar comparison but in a different manner. This is approximately 30,000 non-negative hair specimens between 2004 and 2009 analyzed at Crest Laboratories. Now if you can see in the three columns, urine positive and hair negative is in the left column, urine negative and hair positive is in the middle column and urine and hair both positive is in the right column. So the overall 18.4% were urine positive and hair negative, 52% were urine negative and hair positive and 31% approximately both specimens were positive. And as you go down you can again see the differences between a urine positive and a hair negative and a urine negative and a hair positive. The significant differences for the amphetamines and cocaine, marijuana, opiates and marijuana and PCP are approximately the same and opiates have more urine positives, hair negatives than urine negatives, hair positives. Opiates and opioids may be one of the most difficult drug groups to do in hair from an analytical point of view and that may account for some of the differences in that particular drug group. But again you can see dramatic increases in the number of positives for amphetamine, methamphetamine and cocaine. The next slide I'm going to talk a little bit about some Brazilian data. Brazil has a drug testing program for its drivers that is a little unique. When the drivers in Brazil need to have the Brazilian equivalent of a CDL renewed, they're required at that time to provide a hair specimen, if they can't provide hair they're required to provide fingernails and those specimens are sent to a hair testing lab and results of those specimens go back to the donor and the donor is required to send those results to the Department of Transportation or the Brazilian equivalent of that. There are thousands of specimens a day, hair specimens a day analyzed in Brazil. Probably not at this point, July 2020 because of COVID-19 but yes there are thousands of specimens collected a day under that CDL equivalent program. This shows the overall data in terms of positive rates for the drugs which are listed on the right side and I want to talk a little bit about cocaine which is in this slide because I have more data on cocaine on the next slide. These slides 1 and 2 are courtesy of Dr. Barry Sample from Quest Diagnostics. Analysis by Quest Diagnostics showed cocaine ranks as the most commonly detected drug in toxicology tests for Brazilian drivers renewing their licenses from March 2016 to March 2019. You can see if you look at the pie chart 73% of the positives in Brazil in hair are cocaine far less for amphetamine 3%, methamphetamine 1%, opiates 10% or approximately 10% and marijuana 13%. So in Brazil this is surprising, the number of cocaine positives that you detect in driver's hair but it's certainly present both in Quest and in other hair testing labs in Brazil. So that's my last slide on this topic and I believe I've set the stage for some more questions if you wish around hair and I've also set the groundwork which I believe will be used by HHS when they establish cut-offs and testing protocols and collection protocols for hair analysis. So thank you for your attention and I look forward to seeing you on another presentation.

hair testing

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pharmacological aspects

pre-employment tests

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