Hair Analysis in Clinical and Forensic Toxicology

Brazil

Chapter 1

Anatomy and Physiology of Hair, and Principles for its Collection

Gail Audrey Ann Cooper,    Cooper Gold Forensic Consultancy Ltd, Fife, Scotland, UK

The incorporation of drugs and metabolites into the growing hair follicle provides the basis for a historical record of past use and exposure to drugs. Hair growth rates vary depending on the type of hair and also vary from person to person but using an average head hair growth rate of 1 cm/month allows an estimate of the timescale of drug use/exposure in the months prior to sample collection. The key structures of the anatomy involved in the formation of the hair follicle and incorporation of drugs are described and special attention given to the three main routes of incorporation: from the bloodstream, sweat and sebum bathing the hair, and from numerous sources of external contamination. The mechanisms of incorporation are also addressed focusing on the role of melanin and the question of a dose–response relationship.

Finally, sample collection protocols are discussed in addition to highlighting case-specific differences for workplace drug testing, postmortem investigations, and drug-facilitated crimes.

Keywords

Hair physiology; hair growth; incorporation mechanisms; sample collection; external contamination

1.1 Introduction

The main advantage of hair as a testing matrix is the ability to provide historical detail of an individual’s exposure to drugs following chronic use, but it is also possible to detect drugs in hair following a single exposure [1]. Unlike many traditional biological samples collected for toxicological investigations such as blood, urine, or oral fluid, drugs incorporated into the hair matrix remain relatively stable for many months or even years.

As a consequence of these advantages, hair samples are routinely collected and analyzed as part of criminal investigations (drug-related deaths, drug-facilitated crimes (DFCs), child protection) and for monitoring drug misuse (drug rehabilitation programs, workplace drug testing). The number of laboratories offering hair testing continues to increase and although there are currently no recognized standardized methodologies, the importance of adherence to industry best practice and recognition of the implementation of international quality standards (e.g., ISO/IEC 17025:2005) for testing laboratories who provide this service has been reported [2].

With continual improvement in instrument sensitivity and recognition of the importance of method validity with acceptable detection limits, the main challenges to expert evidence relating to hair testing are not as a result of unsuitable testing methodologies but are most commonly a consequence of over interpretation of the analytical findings. This is invariably due to a lack of understanding of the many factors affecting the presence of drugs in hair and no consideration given to the role of alternative routes of incorporation or indeed the degradation of drugs in hair over time.

Although researchers have reported finding arsenic in hair collected from the Emperor Napoleon Bonaparte [3] and cocaine in the hair of Peruvian mummies [4] centuries after their deaths, drug concentrations in hair decrease over time due to natural wash out [5]. The stability of drugs in hair is dependent on both the morphology and physicochemical properties of hair [6]. On a daily basis, hair is subjected to exposure to sunlight and weathering that has been shown to reduce concentrations of cannabinoids [7], methadone, cocaine and their metabolites, and heroin metabolites in hair [8]. External contamination and loss of drugs from hair is facilitated by diffusion into and out of the hair in the presence of water [9]. Although daily shampooing does not significantly affect drug concentrations [10,11], drying your hair, curling or straightening your hair with heat can damage or destroy the cuticle providing routes for contamination and loss of incorporated drugs.

Further damage is evident when hair is subjected to harsher cosmetic treatments [10,12–16]. Studies reported in the literature are varied with respect to the drugs and cosmetic treatments investigated but are all in agreement that dyeing, perming, relaxing, and bleaching have a deleterious effect on the drug concentration detected in hair. Decreases in drug concentrations were more prominent for bleached hair compared with dyed hair and damaged hair showed significantly lower concentrations.

In order to fully understand and interpret the results of hair tests, it is essential that the forensic toxicologist has a clear understanding of what factors influence the incorporation of drugs into the growing hair shaft including contamination but must also consider the factors affecting loss of the incorporated drug. The focus of this chapter will be on the routes of incorporation and the factors influencing incorporation. This chapter will also address the importance of the sample collection process and will highlight case-type-specific collection protocols to ensure the collection of a representative sample for all toxicological investigations.

1.2 Hair Anatomy and Physiology

Hair covers almost the entire surface of the human body with the exception of the outer surface of the lips, the palms of the hands, soles of the feet, and some parts of the external genitalia [17]. The main purpose of body hair is to protect the surface of the skin from injury and to help regulate body temperature. The appearance of body hair varies from fine almost colorless hair found on most parts of the body surface to thicker, longer hair on the scalp.

The hair shaft, visible above the surface of the skin, consists of tightly packed fully keratinized cells with the cuticle forming an outer protective layer [18]. The cuticle is susceptible to damage from a number of sources including, exposure to light or heat, chemical treatments including bleaching and perming, and also physical damage. As a consequence, over time the surface of the hair may be compromised exposing the inner layers and is particularly evident in the distal ends of the hair.

The interior structure of the hair shaft contains cortical cells forming the cortex which encompasses the bulk of the hair shaft and also contains cuticle cells where melanin, the principle pigment in hair, is located. The innermost region of hair within the cortex is the medulla and may be continuous along the length of the shaft, discontinuous, or completely absent.

The hair follicle is embedded 3–4 mm below the surface of the skin and is the key structure responsible for hair growth. The structure of the hair follicle is illustrated in Figure 1.1.

Figure 1.1 Structure of the hair follicle.

The main structures of the hair follicle include the outer root sheath (ORS), the inner root sheath (IRS), and the root bulb. The ORS forms a bulge area at the base of the erector pili muscle, close to the sebaceous gland and is believed to be the source of stem cells critical for hair follicle development and pigmentation [19]. The IRS provides support for the growing hair producing intracellular binding material and directing the growing hair upward [20].

The cells located within the lower region of the root bulb, the matrix, are mitotically active while the upper region of the root bulb contains the keratogenous region. At the base of the root bulb is the dermal papilla which contains the blood supply. The IRS degrades as the growing hair dehydrates and keratinization takes place [21].

Close to the hair follicle are the sebaceous and apocrine glands both of which secrete directly into the follicle. The apocrine glands, unlike the sebaceous glands, are not present over the entire surface of the body but are localized in the axilla and pubic regions. The eccrine sweat glands are located close to the follicle but secrete near the exit of the hair follicle not directly into the follicle [22].

1.3 Classification of Hair Types

The three basic hair types on the human body are classified as vellus, intermediate, and terminal. Vellus hair covers the majority of the body surface of both children and adults, is fine, short, and non-pigmented, and is also found on the eyelids and forehead. Where vellus hair is produced by non-sexual hair follicles and unaffected by hormones, intermediate and terminal hairs are influenced by hormones and changes during puberty. Terminal hair is found on the scalp, beard, eyebrow, eyelash, armpit, and pubic areas and in contrast to vellus hair is coarse, long, and pigmented with a large cross-sectional area. Intermediate hair has characteristics of both vellus and terminal hairs and is found on the arms and legs of adults.

Human hair is also commonly classified using the ethnic subgroups of African, Asian, or European. An alternative classification system proposes eight different subgroups based on whether the hair is straight or curly and provides a more objective approach than a subjective ethnicity-based approach [23]. The classification system involves the measurement of three parameters: the curve diameter, the curl index, and the number of waves.

1.4 Hair Growth Rates

A number of factors affect hair growth including age, stage of development, sex, pregnancy, metabolic and genetic disorders, nutrition, and seasonal changes [24,25]. Hair growth is a cyclical process driven by changes in the activity of cytokines (hormones) resulting in individual hairs on the body at various stages of the growth cycle [26]. The three recognized stages in hair growth are the anagen, catagen, and telogen phases. The anagen or growth phase can last for several years and is characterized by the formation of the hair shaft protruding from the surface of the skin. It is estimated that 85% of the hairs on the human scalp are in the anagen phase. The catagen or transitional phase follows this period of active growth. During this stage, cell division stops, the hair shaft becomes fully keratinized, and the bulb begins to degenerate, the length of which varies depending on the type of hair. The telogen or resting phase follows the catagen phase and is a period when there is no hair growth but the dermal papilla remains in the resting phase. It is estimated that 10–15% of all hairs are in the telogen phase at any given time with the length of time increasing with age and is also dependent on the hair type. The hair is easily removed during this stage and is often referred to as shedding. A short time later the growth phase recommences by stimulating stem cells from the bulge area of the ORS [27].

The Society of Hair Testing (SoHT) recommends utilizing an average growth rate of 1 cm/month for head hair [28]. The variation in the reported growth rates for scalp hair is considerable and is further compounded by the variability of growth rates for different hair types as summarized in Table 1.1 [18,29–40].

Table 1.1

Published Growth Rates for Different Human Hair Types (Centimeters/Month)

Scalp hair is preferred because it has the fastest growth rate with the highest percentage of follicles in the anagen phase. In comparison, pubic hair has a slower growth rate with a longer resting phase while beard hair is thicker with the slowest growth rate. When analyzing non-head hair samples, it is important to consider not only the variation in growth rates but also the variation in the proportion of hair actively growing or in the resting phase [18]. Where head hair is not available, pubic hair is considered a good alternative but additional consideration should be given to the potential for contamination from urine.

The majority of the published growth rates for scalp hair range from 0.6 to 1.5 cm each month supporting the average growth rate of 1 cm/month. However, this is an oversimplification as a growth rate of as high as 3.36 cm/month has been reported and these rates are for adults hair only [18]. There is a significant lack of published information relating to the variation in growth rates for younger children or teenagers. Hair growth rates in children were reported in a small study published in 1964 [41] with 13 females and 7 males whose ages ranged from 3 to 9 years. Hair growth rates were faster for males across all regions of the scalp ranging from 0.300 to 0.355 mm/day for males and 0.273 to 0.331 mm/day for females.

It is recognized that in early life many infants are born with a full head of hair while others have little or no hair for many months and those born with hair may lose some or all of their hair in the first year of life. Barth [42] described the emergence of hair on the body of the fetus from 9 weeks to actively growing hair follicles with roots covering the surface of the scalp at 20 weeks’ gestation. The hair follicles change from the anagen phase to the catogen phase during the 26th to 28th week gestation period and then on to the telogen phase in what is described as a progressive wave from the frontal to parietal regions of the scalp. Many of the telogen hairs are shed in utero while other hairs continue to grow until near birth before also entering the telogen phase.

Consideration should always be given when interpreting timescales represented by the length of a hair sample not only as a consequence of the variation in recognized growth rates but also to the age of the subject. In addition, as it has been estimated to take approximately 7–10 days for the growing hair to reach the surface of the scalp, hair cut from the scalp does not represent the most recent period of hair growth.

1.5 Hair Color

Pigmentation within the hair shaft accounts for as little as 0.1–5% of the hair mass with proteins and lipids accounting for 65–95% and 1–9%, respectively [18]. Differences in hair color are a consequence of the variation in the type and quantity of melanin present. Follicular melanogenesis or pigment formation takes place within organelles named melanosomes which are present within specialized cells named melanocytes [43]. This process takes place exclusively within the hair follicle and is regulated by enzymes, receptors, and proteins during the anagen phase of active hair growth [44].

Four types of melanin are thought to determine the color of hair, namely eumelanin, pheomelanin and their oxidative products, oxyeumelanin, and oxypheomelanin [45]. In general darker colors of black and brown hair are associated with predominantly eumelanin, with lighter shades associated with increasing amounts of oxyeumelanin. Pheomelanin results in red shades of hair color with lighter shades being associated with increasing amounts of oxypheomelanin.

1.6 Mechanisms of Drug Incorporation

The exact mechanisms by which drugs or analytes of interest are incorporated into hair and the factors affecting their stability are not fully understood. It is believed that drugs and trace elements circulating in the body are incorporated into hair during periods of increased metabolic activity and cell division synonymous with the anagen growing phase [46]. There are however three recognized routes of incorporation of drugs into hair including directly from the blood supply, from sebum and sweat bathing the hair, and from external contamination. Figure 1.2 illustrates the three recognized routes of incorporation of drugs into hair [18,47]. The extent to which each of these routes contributes to drug incorporation is unclear or indeed to what extent they each exert their influence, but what is clear is that it does vary from drug to drug [21].

Figure 1.2 Incorporation routes into the hair follicle.

There are different models of incorporation proposed that attempt to explain the drug profiles observed in hair. The most simplistic model involves passive diffusion of drugs directly from the blood supplying the hair follicle. With passive diffusion it would be expected that drug concentrations in hair would correlate with the drug concentration in the blood at the time of hair synthesis [48,49]. This model does not however explain the different metabolic profiles seen in hair and blood. In blood, parent drugs are less commonly detected in comparison to the corresponding primary metabolites, while in hair the presence of the parent drug is more common, for example, cocaine and 6-monoacetylmorphine (6-MAM), the primary metabolite of heroin, are generally found in higher concentration in hair than their metabolites, benzoylecgonine and morphine, respectively.

The biochemical concept was proposed to explain the endogenous incorporation of drug molecules into growing hair [50]. This concept tentatively explains the high parent drug to metabolite ratio in hair, the dependence of incorporation on the physicochemical properties of the drug, the incorporation of drugs into non-pigmented hair, and the dependence of drug content on hair pigmentation.

It is clear that hair has different affinities and binding capacities for different drugs with unique binding mechanisms for each drug [51,52]. Drug pKa, structure, size, lipophilicity, protein binding capacity, and melanin affinity are the factors known to affect the incorporation and binding of drugs [29,53,54]. Basic drugs, such as amphetamines and cocaine, incorporate into hair to a greater extent than neutral or acidic drugs and are consequently present in higher concentrations in hair compared with benzodiazepines and cannabinoids.

1.7 Incorporation from the Bloodstream, Sebum and Sweat

Lipid solubility of a drug is a critical factor in determining the rate of transport from the blood stream across the cell membrane into the growing root bulb. The pH gradient from plasma, pH 7.3, to more acidic conditions within the melanocytes/keratinocytes (pH 3–6) provides advantageous conditions for basic drugs to incorporate preferentially to more acidic drugs. This was clearly demonstrated using structurally related compounds, rhodamine (cation) and fluorescein (anion), administered intraperitoneally resulting in distinct fluorescent bands of rhodamine corresponding to each daily dose [55]. Although fluorescein was detected in the matrix cells during formation, it was not detected in the keratinized hair.

Other routes must also be considered to help explain the drug profile observed in hair. Drugs have been detected in both sweat and sebum and as both of these secretions bathe the follicle and hair shaft as they grow, there will be contribution to the drugs incorporated into hair [56,57].

Evidence to support the role of sweat incorporation of ethyl glucuronide (EtG) into hair was reported following detection of EtG in beard hair 9 h after a single dose of alcohol (ethanol), however, the predominant route was incorporation from the blood stream directly into the root bulb [58]. EtG concentrations increased to a maximum of 72–242 pg/mg within 2–4 days post dose and gradually decreased to below the limit of quantitation by day 8–10 as the root bulb grew and emerged from the surface of the skin.

1.8 Incorporation from External Contamination

Drug contamination from the environment must also be considered including passive exposure from being close to individuals smoking drugs (e.g., heroin, crack cocaine, cannabis), through directly handling the drugs, touching surfaces contaminated with drugs and then touching your own or someone else’s hair. In particular, children, and infants to a greater extent, are at high risk of exposure if living within a household where drug misuse is prevalent [59–61]. Consideration should also be given to the unborn fetus who will be exposed to drugs if their mother continues to misuse during her pregnancy [62–65].

External contamination and the role it plays in drug incorporation has been extensively investigated by researchers, with the greatest challenge in attempting to recreate realistic contamination scenarios. Consideration should also be given to the condition of the hair sample which may be damaged through natural wear and tear or chemical treatment and also to the porosity of the hair, all of which will affect the incorporation of drugs from external contamination.

Studies have demonstrated significant contamination of drug-free hair exposed to smoke containing cannabis [66], blood containing cocaine [67] and heroin metabolites [68], and from the direct application of cocaine to the hair [69].

Site-specific contamination should also be taken into account, including the potential for urine and gland secretions contaminating pubic hair and pieces of epidermis contaminating beard hair when collected by shaving. Concentrations of EtG measured in different hair sample types varied significantly and raise concerns about the use of pubic or axillary hair samples as alternative specimens to head hair in the assessment of chronic use of alcohol due to positive and negative biases, respectively [70].

The SoHT provides guidelines for washing to remove gross contamination from the surface of the hair, however, the selective removal of drugs on the surface while preserving drugs incorporated into the hair matrix is an unlikely outcome when using both organic solvents and aqueous solutions. In one study, complete decontamination was not achieved by participating laboratories who were sent drug-free hair samples contaminated with cocaine. The laboratories all used different wash protocols but none of them were able to decontaminate the hair [71].

Proposals for discriminating between contamination and incorporated drugs have been proposed [72,73] and may provide sufficient confidence in the future to be accepted as a standardized approach but until such time, the potential role of external contamination must always be considered when interpreting positive hair tests.

External contamination is discussed in greater detail in Chapter 3.

1.9 Dose–Response Relationship

Although controlled dosage studies with animals and humans have supported a linear relationship between dose and drug concentrations found in hair for certain drugs [48,74–77], the vast majority of studies have demonstrated that a linear relationship does not exist [78–82]. The most likely explanation is due to inter-subject variation with a number of additional variables including hair color and cosmetic treatments being of particular significance.

Following controlled low and high dosage regimes of methamphetamine, good intra-subject correlation was observed with methamphetamine and amphetamine concentrations measured in hair [83]. A large inter-subject variation was also reported in this study, however, when corrected for melanin content demonstrated a good correlation, supporting a dose-related relationship for the incorporation of methamphetamine and its metabolite amphetamine and resulting concentrations measured in hair.

Evidence of a dose-response relationship for methamphetamine was reported under controlled conditions and correcting for the melanin content. However, controlled conditions are not representative of real-life exposure and the role of contamination must also be considered. Children exposed to methamphetamine were reported to have methamphetamine/amphetamine concentrations in hair of similar magnitude to those measured in adults misusing methamphetamine [84,85].

1.10 Melanin Binding

A number of studies have investigated the incorporation of different drugs into hair involving animal models which have provided useful information on the role of melanin [86–91]. Darker hair contains more pigmentation (melanin) than light colored hair and so animals with both pigmented and non-pigmented hair were administered drugs of different basicity. The findings supported the greater binding affinity of basic drugs to melanin as they were present in higher concentrations in pigmented hair. Neutral and acidic drugs demonstrated no greater affinity to pigmented or non-pigmented hair.

It is important to remember when relating these findings to human hair that animal and human hair differs with respect to growth cycles and structure which may result in different drug distribution.

Differing incorporation profiles of fatty acid ethyl esters in rat hair compared with human hair were reported [92] and was also observed for erectile dysfunction drugs and their metabolites [93]. Sildenafil and its primary metabolite, desmethyl sildenafil, were measured in pigmented and non-pigmented rat hair and in two human hair samples collected from individuals suspected of using illegal supplies of sildenafil. The concentrations in pigmented hair were higher than those measured in non-pigmented hair supporting the role of melanin in drug incorporation. However, the metabolite concentration was significantly higher than the parent drug in rat hair with a mean ratio of drug to metabolite of 0.1 (no range reported) and was in direct contrast to the ratio observed in the two human hair samples of 0.83 and 3.6. The authors acknowledged previously published research that also observed higher metabolite concentrations in rat hair for sildenafil but they also reported a similar profile for human hair samples analyzed, not consistent with their findings [94]. In both studies the number of human hair samples tested was small and therefore a much larger study is required to investigate the drug to metabolite profiles in human hair. The effect of the hair sample preparation conditions used to extract the drugs from the matrix was also reported as influential on the apparent ratios measured. In addition, both the concentrations measured and the parent drug to metabolite ratios varied for the other erectile dysfunction drugs investigated (mirodenafil and vardenafil). Although structurally related compounds, other factors affect their incorporation in additional to melanin.

Kim et al. [95] investigated the role of melanin with respect to the incorporation of synthetic cannabinoids in hair and found melanin was not a significant factor in their incorporation into hair.

Additional factors affect the incorporation of drugs into hair including the amount and type of melanin present and binding to keratin [96].

1.11 Sample Collection Protocols

Guidance is available on recommended best practice for the collection of hair samples, including specific recommendations for postmortem hair sampling, workplace drug testing, and DFCs [97–100]. The majority of the steps involved with the collection process are the same irrespective of the case type, but there are case-type-specific protocols that must also be considered.

The SoHT recommends cutting hair as close as possible to the scalp from the vertex region of the head as illustrated in Figure 1.3 [101]. Collection of a sample from the vertex region is preferred as this is the site where there is least variation in growth rates compared with other regions of the scalp or when compared to other body hair types. In addition, hair growing on the scalp has the highest percentage of follicles actively growing and not in the resting phase.

Figure 1.3 Vertex region of the scalp.

The volume of hair required for analysis is a lock of hair proportionate to the thickness of a pencil. The collection of a sufficient volume of hair is critical to ensure the completion of all necessary tests. Not surprisingly, the most common concern from both collectors and the donor is that the amount of hair sampled should not leave a visible bald patch and is of particular concern for parents of small children or individuals suffering from baldness or thinning hair. To avoid distress to individuals, the collection of smaller hair samples from multiple sites within the vertex region is also an acceptable protocol. The smaller individual locks should then be combined into one larger lock of hair and aligned with the root end identified.

In addition to collecting sufficient volume to ensure all tests required can be carried out, it is particularly important when carrying out segmental analysis of hair. Cutting hair into smaller segments of most commonly 0.5–3 cm in length provides a more detailed historical profile of an individual’s exposure and may also provide a means of determining external contamination in postmortem cases [102]. However, smaller segments will further compromise the volume available for testing.

Although specialist facilities are not required for the collection of scalp hair, when head hair is not available, alternative collection sites should be considered including intimate samples (e.g., pubic hair). In these circumstances, the privacy of the donor must be prioritized while ensuring the integrity of the collection process.

Collection of hair samples should only be carried out by a competent collector who must recognize and adhere to good practice guidelines for sample collection to eliminate the potential for contamination of the sample(s) and will ensure chain of custody is maintained throughout the process. Le Beau et al. [103] also highlighted the importance of ensuring the sample is collected as close to the scalp as possible following an evaluation of the hair samples taken by collectors with varying levels of experience. The length of hair remaining on the scalp after cutting was 0.8 ± 0.1 cm leading to significant impact on the timescale of the corresponding segments from the collected samples and the subsequent interpretation of the findings.

Hair samples are routinely collected by medical personnel (general practitioner, forensic medical examiner, nurse) but the collector does not require medical qualifications. The collector may however be required to give evidence in a court of law and should therefore be prepared to demonstrate their competence through experience and appropriate training records.

1.12 Collection Procedure

Clear instructions must be provided with the hair collection kit to provide guidance and to allow the collector to check the contents are all present and correct. The collection kit should be sealed so that the collector can check that the seals have not been tampered with prior to opening the kit to use for the first time and to check the contents. Scissors used to cut the hair sample should be cleaned with an alcohol-free wipe or sterilized prior to use. The standard contents of a hair collection kit should include the following:

• Chain of custody form

• Foil and collection envelope

• Security seals

• Evidence bag (optional)

• Transportation envelope (optional)

• Instructions for collection of a hair sample

The main purpose of the chain of custody form is to document details relating to the collection and handling of the sample and to facilitate identification of the sample. Duplicate or carbon copies of the form may be required if there are multiple agencies involved. In the case of workplace drug testing, the anonymity of the donor must be maintained therefore anonymized copies of the form are required for dispatch with the sample to the laboratory for analysis.

A secure mail service is recommended to ensure chain-of-custody is maintained, and specialist courier services are available with tracking systems and secure transportation to allow complete traceability of the samples from the collector to the laboratory.

The following step-by-step guide summarizes the collection process:

Step One: Cut a lock of hair close to the scalp from the vertex region and align the hair identifying the root end (Figure 1.4).

Step Two: Fold the foil lengthways and avoid folding in the middle as this will kink the hair making it difficult to handle (Figure 1.5).

Step Three: Place the foil-wrapped hair sample within the collection envelope, seal, initial, and date.

Step Four: Place the completed chain of custody form and sealed collection envelope into the evidence bag and transportation envelope and send to the laboratory for analysis.

Figure 1.4 Cut hair aligned with the root end identified.

Figure 1.5 Fold the foil lengthways to secure the aligned hair.

For long-term storage, hair samples should be stored at room temperature, dry, and away from direct sunlight.

1.12.1 Workplace Drug Testing

The European Workplace Drug Testing Society published guidelines for European workplace drug and alcohol testing in hair in 2010 [98]. Within the guidelines the collection of hair was covered in detail as a critical part of the process and in particular the role of the collector. An immediate supervisor of the donor or coworker, relative or close friend may not perform the collection. Additionally, an employee of the drug testing laboratory may not collect a sample from the donor unless it can be demonstrated that the sample could not be linked to the donor.

Access to the collection facility should be restricted and entry to the room prohibited during the collection process. The chain of custody form should have a minimum of three parts or copies: one for the testing laboratory, one for the donor, and one retained by the collector. A list of information to be recorded on the form was also detailed within the guidelines including a declaration from the donor on their use of prescribed medication, sample authenticity, correctness of sample labeling and packaging, and permission for the sample to be analyzed at the testing