Decisions regarding the portion of youth participants tested for recent SU is informed by national estimates of prevalence of use . Youth must be naïve to alcohol and recreational drugs at in-person study enrollment, while prescription drug use is permitted in youth participants at the initial baseline visit . Bio-specimens addressing recent, past 3-month and lifetime SU exposures are assessed. Tests of recent drug use will provide the opportunity to evaluate the reliability of self-reports of current drug use. Given potential alcohol or drug effects on test results , youth participants testing positive for recent drug exposure at any follow-up onsite visit, will be asked to reschedule, and to return for testing on another date, drug and alcohol free. Youth reporting a history of non-prescription drug use, or participants suspected of substance intoxication, once they arrive to the lab for study assessments, will be tested for recent drug use, even if they have not been selected randomly for testing before their arrival. Because the effects of drug exposure and misuse of prescribed drugs on brain and cognitive development in youth is a primary focus of the ABCD Study, a reported history of use is not exclusionary at follow-up annual visits, and the assessment of past 3-month and lifetime SU exposures are of high value. The ABCD research team is in the process of developing protocols for future bio-specimen collection, but, have not yet formalized additional bio-specimens. Given the protocol for future onsite visits beyond 1-year follow-up remains undetermined, it is not included in this report.Oral fluid will be collected for toxicology testing of 7 drugs. In follow-up years, urine will be collected and tested at the beginning of each onsite visit, supplemented by breathalyzer testing for a random selection of subjects, to rule out recent non-prescription drug, nicotine, and alcohol use prior to neurocognitive assessment. Onsite drug testing will be completed for alcohol use and a broad range of commonly used substances at 1-year follow-up. At baseline, a positive drug test is exclusionary for initial enrollment into ABCD, as are self or parent/guardian report of youth ingestion of more than 1 whole drink,cannabis grow racks more than one whole cigarette or the equivalent amount of another tobacco product, any marijuana, or misuse of any drug .

Hair samples will be collected from youth subjects during each annual onsite visit for future confirmation of the presence or absence of SU. Hair provides an extended window of drug metabolite detection , and therefore can help confirm drug use despite irregular and/or infrequent drug ingestion. While not without some disadvantages , hair is relatively easy to handle and store, less susceptible to adulterants, and provides longer detection times compared to other biological matrices that have shorter detection windows . By banking hair samples for future testing, results can be used in combination with oral fluid and self-report to ensure that a ‘clean’ baseline is confirmed. This is especially important for the sub-sample of individuals who escalate to substantial levels of drug use by an early age , as these individuals will provide the most crucial test for neurocognitive and brain structure/function differences predating their escalation, or emerging after that escalation.Oral fluid will be collected for toxicology testing using a Draeger 5000 Drug Test Unit, which provides a qualitative test for 7 drugs. Oral fluid drug screening has advantages over conventional methods including reduced biohazard generation, ease of collection, and less susceptibility to adulteration . Oral fluid concentrations are also more tightly correlated to blood than urine concentrations allowing inferences of impairment and flexible detection windows. The Dräger Drug Test 5000 screening device is used to test oral fluid at baseline and each follow-up year to identify recent use of amphetamine, benzodiazepines, cannabis , methamphetamine, cocaine, methadone, and 3,4-methylenedioxymethamphetamine. The Draeger unit was selected over other on-site testing devices because of the high sensitivity for THC, one of the most common substances of abuse. The Draeger provides a lower THC cut-off concentration compared to other testing devices and therefore higher sensitivity and detection accuracy. The Dräger Drug Test 5000 is increasingly utilized for roadside testing . The Dräger system consists of an analyzer, test cassette oral fluid collector, and buffer cartridge. To perform a screening test, the participant refrains from eating or drinking for 10 min.

The test cassette contains a cellulose pad that is moved from one side of the mouth to the other for approximately one minute until the volume adequacy indicator turns blue to confirm a sufficient volume. The sample is then inserted into the Dräger analyzer with 3 mL buffer for drug stabilization. All drug results are displayed on the analyzer device within 5–8 min as “positive” or “negative.” The test is repeated for cases in which the results are unexpected based on self-report and/or clinical observation; repeat test results, and whether results are in-line with self-report and clinical observation are coded. Oral fluid drug testing can be influenced by several factors, including frequency of SU, body fat, and method of ingestion, however work by Huestis and colleagues suggest that 5 ng/mL concentration cut-off provides high diagnostic sensitivity, specificity, and efficiency for oral fluid cannabinoid detection .In addition to the screening measures for SU at the time of lab visits, the ABCD consortium is also collecting hair samples from all participants at each on-site lab visit for a wider detection window to provide longer-term information on history of drug consumption. Approximately 10 percent of participants will have hair analysis conducted at baseline, as the cost would be prohibitive to analyze every sample at every time-point. However, all hair samples are archived for each participant at each site, and future analyses can be done on hair samples for participants who endorse drug use as it becomes more prevalent as the ABCD cohort progresses through adolescence. Participants are instructed that we will cut a sample that is ½ inch wide by two strands deep from the back of the head below the crown. After collection, the sample is placed in foil tightly and sent to Psychemedics. Gas chromatographymass spectrometry and liquid chromatography-mass spectrometry procedures are used to test for the following parent drugs and metabolites: alcohol ethyl glucurolide , cannabis (11-Nor-9-carboxy-THC and cannabidiol , methamphetamine and methylenedioxy-methamphetamine , amphetamine, opiates , and cocaine/benzoylecgonine . Commonly used hair procedures do not impact quantitative results, and samples are relatively easy to collect and store compared to other biological matrices.

Limitations include hair that is too short . This is particularly a challenge in testing pre-pubertal children, where hair on other parts of the body are less available than in older youth and young adults.A hallmark of adolescence is reproductive maturation, known as puberty. Puberty heralds the onset of adolescence, and the hormonal surges that occur during this period of time impact the ‘environment’ of the developing brain. Pubertal maturation influences trajectories of, and sex differences in, brain development and behavior , including SU during adolescence . While much has been discovered in the last decade about the impact of pubertal hormones on adolescent brain and cognitive development (for a review, see , much is yet to be learned, particularly in connection with resilience or risk for SU during adolescence, and related mental health problems. To the best of our knowledge, the ABCD study is the largest most comprehensive study collecting pubertal hormone data longitudinally across adolescence,cannabis grow system and the ability to connect them to brain development, cognition, behavior, genetics and SU. Differences in onset of pubertal timing and maturation, and associations with SU and mental health vary as a function of sex, race and region . Thus, investigating these important hormone associations with neuro development across the United States in a representative sample, both racially and regionally, is essential. Important to the ABCD project, behavioral risk factors begin to emerge during pubertal onset and do so in a sex-specific fashion, with an increased prevalence of SU and externalizing disorders in boys compared to girls . Adolescence is a period of prolonged sensitivity to environmental factors, when the maturing central nervous system is particularly sensitive to insult . How SU impacts the relationships between pubertal onset, mental health, and neuro development remain unclear, and is therefore an objective of the ABCD Study. DHEA, testosterone and estradiol is being assessed each year spanning across both pre- and well past post- pubertal stages of development. It is important to note that pubertal maturation is assessed independently of gonadal hormone measures , and is a key interacting factor for understanding relationships between hormone levels, brain development and SU.Pubertal hormones are assessed in participating adolescents through the collection of a single salivary bio-specimen each year throughout the 10-year duration of ABCD.

This method provides a quick, accurate and reliable method for measuring numerous key gonadal hormones from a single sample. The source of pubertal hormones found in saliva come from several glands in or near the mouth . Compared to collecting blood, the non-invasive nature of the salivary method requires less training for the administer, and eliminates the need for coordination with a phlebotomist. Saliva contains lower levels of pubertal hormones compared to levels found in blood, yet saliva levels are highly correlated with the free blood serum levels that typically exert biophysiological effects .Like serum hormone levels, salivary hormone levels exhibit circadian patterns, making participant waking time , and time of day of saliva collection important variables for statistical analyses and interpretation of results. Oral hygiene, injury resulting in bleeding or inflammation, and food particles can alter levels of hormones in the sample, and/or interfere with accuracy of the assays to assess hormone levels. To adjust for these confounds, notes on the color of the sample should be taken into consideration when running statistical analyses with salivary hormone data . The amount of time it takes a participant to produce a saliva sample can influence the concentrations of hormone levels. The duration of collection time can be impacted by the flow rate of saliva production, which may vary as a function of certain medications, making duration of sample collection an important factor to consider for analyses and interpretation of results. The average sample collection time for the ABCD cohort of 9–10 year olds is approximately 5.25 min . For girls, additional fluctuations in gonadal hormones occurring across the menstrual cycle are captured by collecting key factors, such as: age of onset of menstruation, type of contraceptives used, regular or irregular cycles, length of cycles, and date of last menstrual cycle. Given the cyclic nature of estradiol, factors relating to menstruation are key for understanding possible decreases in estradiol levels across years.Researchers examining ABCD hormone data should be aware that approximately 4% of the currently existing hormone data is affected by some type of experimental error. Many of these errors can be found within the ABCD data set under the research associates’ notes, or become obvious upon close inspection of the data entered relating to factors described in above sections. Bacterial growth in the saliva sample is blocked upon freezing of the sample. However, logistical challenges can sometimes prevent immediate freezing . Errors in data entry occur. As can be seen in Fig. 3, some participants are listed as waking up after 15:00 ; however participants typically arrive much earlier in the day for testing/scanning, making this a data entry error. All saliva samples are stored at −20 to −80 °C before shipping on dry ice for analyses. The deep freezers are subject to malfunctioning ; thus notes about samples thawing, which can significantly impact hormone levels, need to be considered. Upon receipt of frozen samples shipped from each ABCD site, Salimetrics completes all notes on sample quality and thawing, conducts assays, and the initial data entry of hormone levels. Samples are run in replicates, and key details of each hormone assay can be found on their website . Given the sheer quantity, saliva samples shipped from each site every 2 months, and subsequently analyzed in batches at Salimetrics; thus initial assessment for possible batch effects should be conducted before moving on with further analyses within a selected ABCD sub-sample.Genetics plays a crucial role in personality traits , psychiatric illness, including substance abuse disorders.