Forensic & Investigative Sciences Program Theses and Dissertations

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Automated derivatization and identification of controlled substances via total vaporization solid phase microextraction (Tv-Spme) and gas chromatography-mass spectrometry (Gc-Ms)
(2018) Hickey, Logan D.; Goodpaster, John
Gas chromatography-mass spectrometry (GC-MS) is one of the most widely used instrumental techniques for chemical analyses in forensic science laboratories around the world due to its versatility and robustness. The most common type of chemical evidence submitted to forensic science laboratories is seized drug evidence, the analysis of which is largely dominated by GC-MS. Despite this, some drugs are difficult or impossible to analyze by GC-MS under normal circumstances. For these drugs, derivatization can be employed to make them more suitable for GC-MS. In Chapter 1, the derivatization of primary amino and zwitterionic drugs with three different derivatization agents, trifluoroacetic anhydride (TFAA); N,O-bis(trimethylsilyl)trifluoroacetamide + 1% trimethylchlorosilane (BSTFA + 1% TMCS); and dimethylformamide dimethylacetal (DMF-DMA), is discussed. The chromatographic performance was quantified for comparison between the derivatives and their parent drugs. Peak symmetry was compared using the asymmetry factor (As), separation efficiency was measured by the number of theoretical plates (N), and sensitivity was compared by measuring the peak areas. In Chapter 2, derivatization techniques were adapted for an automated on-fiber derivatization procedure using a technique called total vaporization solid phase microextraction (TV-SPME). TV-SPME is a variation of SPME in which a small volume of sample solution is used which can be totally vaporized, removing the need to consider the equilibrium between analytes in the solution and analytes in the headspace. By allowing derivatization agent to adsorb to the SPME fiber prior to introduction to the sample vial, the entire derivatization process can take place on the fiber or in the headspace surrounding it. The use of a robotic sampler made the derivatization procedure completely automated. In Chapter 3, this on-fiber derivatization technique was tested on standards of 14 controlled substances as well as on realistic samples including simulated “street meth”, gamma-hydroxybutyric acid (GHB) in mixed drinks, and hallucinogenic mushrooms, and was also tested on several controlled substances as solid powders. Future work in this area is discussed in Chapter 4, including adapting the method to toxicological analyses both in biological fluids and in hair. Some of the expected difficulties in doing so are discussed, including the endogenous nature of GHB in the human body. The presence of natural GHB in beverages is also discussed, which highlights the need for a quantitative addition to the method. Additional method improvements are also discussed, including proposed solutions for complete derivatization of more of the analytes, and for decreasing analysis time.
Characterization of Simple Sequence Repeats in Phormia Regina Miegen (Diptera: Callphoridae)
(2024-08) Waletzko, Cassandra; Picard, Christine; Walsh, Susan; Owings, Charity
Phormia regina Meigen is a forensically relevant species of blow fly, common in North America and used to estimate the minimum postmortem interval in forensic casework. It is also possible to use blow flies to survey the environment for biotic and abiotic information drawn from both larval and adult stages. There are both forensic and environmental uses for genetic analysis of blow flies. Blow fly kinship is especially useful for detecting postmortem movement of a corpse or to assess abundance of carrion in a given location. To test genetic relationships between individuals, discriminatory loci such as microsatellites, or polymorphic tandemly repeated sequences of DNA are necessary. Here, we characterize novel microsatellites generated from the genome of P. regina. Thirty-four candidate polymorphic loci with conserved flanking regions, have been isolated. To date, seven are heterozygous and polymorphic testing in two lab populations and one wild population. The simple sequence repeats characterized here complement existing loci (N = 6) for greater discrimination for testing relationships between individual flies.
Critical Comparison of Total Vaporization- Solid Phase Microextraction vs Headspace- Solid Phase Microextraction
(2021-05) Train, Alexandra; Goodpaster, John; Manicke, Nicholas; Picard, Christine
Solid Phase Microextraction (SPME) is a popular sampling technique that can be paired with Gas Chromatography/Mass Spectrometry (GC-MS). SPME-GC-MS is used in forensic chemistry due to its simplification of the sample preparation process. Headspace-Solid Phase Microextraction (HS-SPME) is a technique where the sample is heated to generate volatiles in the headspace of the vial. A SPME fiber is then inserted into the vial and the compounds in the headspace will bind to the fiber. Total Vaporization- Solid Phase Microextraction (TV-SPME) is a technique that is derived from the HS-SPME technique. In Chapter 1, the critical comparison of HS-SPME and TV-SPME is discussed. Samples including marijuana, essential oils, and CBD oil were utilized to compare the two techniques. The compounds of interest in marijuana are the three main cannabinoids: cannabinol (CBN), cannabidiol (CBD), and tetrahydrocannabinol (THC). The sample preparation and GC-MS parameters were kept the same for all samples to determine which SPME technique works best for these sample types and yielded the greatest sensitivity. It was found that HS-SPME shows greater sensitivity with CBN and equivalent sensitivity with essential oils, THC and CBD. In Chapter 2, the detection of synthetic cannabinoids utilizing liquid-liquid injection as well as HS-SPME and TV-SPME is discussed. The detection of these compounds is important because this type of drug has become more prevalent in the United States because they can be chemically altered slightly so they still have the effects of a drug but can evade drug legislation. The detection of synthetic cannabinoids using liquid injection was found to be successful but detection using HS-SPME and TV-SPME was found to be unsuccessful. In Chapter 3, the analyses of real and artificial saliva utilizing HS-SPME and TV-SPME is discussed. Determining the compounds present in real saliva and artificial saliva will be of importance for future research into determining if the presence of drugs in saliva can be analyzed with these techniques. The analyses of real and artificial saliva were found to be successful using HS-SPME, without derivatization, and TV-SPME, with and without derivatization. Many of the compounds present in the real saliva were detected and were confirmed to be compounds regularly found in saliva by other scientific literature.
Design and implementation of gas chromatography-mass spectrometry (GC-MS) methodologies for the analysis of thermally labile drugs and explosives
(2016-11-18) Ash, Jordan R.; Goodpaster, John V.
Gas Chromatography/Mass Spectrometry (GC/MS) is an analytical technique that sees frequent use in labs across the world. It is also one of the most common instruments found in forensic science laboratories. This technique can efficiently and accurately separate and identify a broad range of compounds that may be present in evidence submitted for analysis. In this work, the versatility of this instrument was applied to new methodologies for the detection of explosives and illicit drugs. The analysis of explosives by GC/MS is common but can be problematic. The thermally sensitive nature of some explosives can cause them to degrade when introduced to the high temperatures of a GC/MS inlet. This project looked at the design and implementation of a way to separate and detect a variety of nitrate ester explosives in a short amount of time. In addition to this, a new technique known as Total Vaporization-Solid Phase Microextraction (TV-SPME) was utilized as a pre concentration technique. The parameters for TV-SPME were statistically optimized for a low level of detection. The combination of these areas allowed for the separation of ethylene glycol dinitrate, nitroglycerin, erythritol tetranitrate, and pentaerythritol tetranitrate with a detection limit as low as 50 parts per trillion (ppt). Degradation products such as 1-mononitroglycerin, 1-3-dinitroglycerin, and 2-mononitroglycerin were also successfully identified. The problem of thermally labile compounds extends to the world of illicit drugs. In the second project, several derivatization schemes were developed for common controlled substances. N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was used for silylation, trifluoroacetic anhydride (TFAA) was sued for acylation, and (N,N-Dimethylformamide dimethyl acetal (DMF-DMA) for alkylation. Three different compound classes totaling 15 different drugs were investigated. N,N-Dimethylformamide dimethyl acetal (DMF-DMA) is presented as a novel way of derivatizing several drugs of interest. Primary amines and zwitterions were derivatized with this reagent to much success, specifically: amphetamine, 2-(4-Iodo-2,5-dimethoxyphenyl)ethan-1-amine (2C-I), pregabalin, and gabapentin.
The Detection and Identification of Explosives by Canines and Chemical Instrumentation
(2022-05) Reavis, Madison Dylan; Goodpaster, John V.; Manicke, Nicholas; Bors, Dana
With bombings in the United States on the rise for the first time since 2016, the detection and identification of explosives remains of pertinent interest to law enforcement agencies. This work presents two soon-to-be published research articles that focus on the detection and identification of explosives by both chemical instrumentation and canines. The first article, Quantitative Analysis of Smokeless Powder Particles in Post-Blast Debris via Gas Chromatography/Vacuum Ultraviolet Spectroscopy (GC/VUV), utilizes gas chromatography/vacuum ultraviolet spectroscopy (GC/VUV) to determine the difference in chemical composition of two smokeless powders in both pre- and post-blast conditions. The compounds of interest in this study were nitroglycerin, 2,4- dinitrotoluene, diphenylamine, ethyl centralite, and di-n-butyl phthalate. Concentration changes between pre- and post-blast smokeless powder particles were determined as well as microscopic differences between pre- and post-blast debris for both smokeless powders in all devices. To our knowledge, this is the first use of GC/VUV for the quantification of explosives. The second article, An Odor-Permeable Membrane Device for the Storage of Canine Training Aids, proposes the use of an odor-permeable membrane device (OPMD) as a standardized storage method for canine training aids. It is hypothesized that the OPMD would minimize cross-contamination between training aids, and that the OPMD could be used for canine training as well as storage. The goal of this research is to use flux and evaporation rate to quantify the explosive odor that escapes from the OPMD compared to unconfined explosives. Preliminary data suggests that there is an exponential relationship between relative boiling point and evaporation rate. It has been determined that compounds with higher boiling points have lower evaporation rates than compounds that have lower boiling points. The materials studied thus far are known odor compounds produced by explosive formulations. These include nitromethane, nitroethane, 1- nitropropane, r-limonene, and toluene.
Electrochemical Characterization of Common Cutting Agents Found in Illicit Drugs
(2023-08) Bloom, George; Deiss, Frederique; Manicke, Nicholas; Rainey-Beymer, Christina
Nationwide use of illicit drugs has continued to rise over the last few decades, with more than a two-fold increase in global seizures from 2016 and 2020. Most seized drug samples are complex mixtures of drugs and cutting agents, which can complicate the detection and quantification of the illicit drugs in the sample. The presence of these cutting agents can however be beneficial for source tracing purposes, as the majority of cutting agents are selected based on availability in the area where the bulk drug was prepared. The goal of this work was to conduct a systematic study of the electrochemical characteristics of the most common cutting agents found in illicit drugs using unmodified, commercially available glassy carbon electrodes. The long-term goal is to establish an extensive database of electrochemical characterizations of cutting agents and illicit drugs encountered by law enforcement using unmodified, commercially available electrodes to help expand the developing field of forensic electrochemical analyses. This database could then be referenced for the identification of unknown samples to determine the presence of possible illicit drugs and cutting agents that are present to help guide the analyst in further testing. The standard methods for drug detection include a combination of laboratory testing and field-deployable assays ranging from colorimetric tests to gas chromatography-mass spectrometry instrumentation. These detection methods, as well as relevant literature were investigated in Chapter 1. The most used screening methods for illicit drugs are colorimetric tests; however, these assays are prone to false positives. Chapter 1 introduces the existing applications and current research efforts in forensic electrochemistry by describing relevant electrochemical sensors and methods and examining in particular their performance regarding accuracy, sensitivity, and low-cost claims. This overview highlights the broad possibilities of electrochemical analysis in forensics as well as the opportunities when applied to detection and quantification of illicit drugs, demonstrating the current needs for more systematic and consistent characterizations of cutting agents found in seized-drug samples. Chapter 2 details the material, reagents, and experimental conditions, showing their simplicity, and the standard electrochemical and preparative equipment used geared towards an easy implementation in any analytical laboratory. Chapter 3 describes the systematic voltametric characterizations performed on thirteen common cutting agents: phenacetin hydrochloride, levamisole hydrochloride, diphenhydramine hydrochloride, quinine, acetaminophen, ascorbic acid, caffeine, lactose, inositol, mannitol, glucose, sodium bicarbonate and calcium carbonate. In addition to the common, information-rich cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV) were used as these pulsed electroanalytical methods are typically considered more sensitive than CV and often employed for quantitative analyses of species present at low concentrations (Chapter 3). Overall, DPV resulted in voltammograms with peaks shaped closer to the ideal redox peaks, also referred to as ‘better defined’, thus enhancing the analytical performance of the assay. For example, In the analysis of diphenhydramine hydrochloride, DPV permitted the measurement of an oxidation with a peak displayed at 1.0 V vs Ag/AgCl, which was not observable when performing CV or SWV. On the other hand, SWV provided noticeably greater intensities of peak current, which allowed for a better detection of the difficult-to-observe redox reactions of quinine occurring at -0.4, 0.0 and 0.4 V vs Ag/AgCl. Some chemical species when present in seized drugs can alter the pH of the tested samples, such as ascorbic acid. Changes in pH will impact the redox activity of the pH-dependent electroactive species present in a sample, thus we investigated how pH of the solvent affected the observation of the redox peaks of those susceptible cutting agents, namely ascorbic acid, quinine, diphenhydramine hydrochloride, and levamisole hydrochloride (Chapter 4). Of particular interest was a significant change in the electrochemical characterization of these species when the pH was varied around their pKa values. Additionally, the composition of the solvent, or supporting electrolyte (SE) solution, can in some cases result in interactions with the analytes in the sample; the study of caffeine with different SE in Chapter 4 illustrates this situation. Specifically, sulfuric acid was the most suited SE of those tested for caffeine analysis. The impact of successive voltametric scans, on the analysis of chemical species were also investigated, using acetaminophen and quinine, demonstrating the development of additional redox peaks in some situations that could provide additional elements towards a more individualized electrochemical profile for cutting agents (Chapter 4). The influence of the material of the working electrode on the electrochemical characterization of cutting agents was explored. Solutions of ascorbic acid, acetaminophen, quinine, and diphenhydramine hydrochloride were electrochemically characterized using a glassy carbon and a platinum working electrode, while ascorbic acid was also characterized on gold and silver electrodes. These examples demonstrate the adaptability of this electroanalytical method with various commonly used electrodes. (Chapter 4). In Chapter 5, we applied similar electrochemical method to the identification of cutting agents and illicit drugs in two-component mixtures. Specifically, these trials included mixtures of fentanyl with a cutting agent at a relative ratio of 1 : 100, using as cutting agents ascorbic acid, diphenhydramine hydrochloride, or glucose, demonstrating the ability of this simple electrochemical method using common commercial electrodes to simultaneously detect illicit drugs and cutting agents.
Electrochemical Characterization of Fentanyl for Forensic Analysis
(2021-08) Sellnau, Natalie; Deiss, Frederique; Goodpaster, John; Manicke, Nicholas
The use and abuse of fentanyl has risen drastically over the last several decades. The abuse of this substance has created a hazardous situation for law enforcement and first responders because they could arrive at locations and not necessarily know that they will encounter fentanyl or a fentanyl analog. Fentanyl analogs are substances that have a similar structure to fentanyl, and while the analogs may have additional or altered groups on the molecule, the backbone structure remains similar. This work focus on the electrochemical characterization of fentanyl as a stepping stone for the detection of both fentanyl and later fentanyl analogs by electrochemistry. The metabolic reaction of fentanyl is an N-dealkylation to norfentanyl, occurring in the liver, and can be mimicked by electrochemistry through the irreversible oxidation of fentanyl. This electrochemical reaction is hypothesized to generate electroactive metabolites in solution. The combination of the visualization of both the irreversible oxidation with the development of the additional metabolic electrochemical peaks would constitute a unique electrochemical signature for fentanyl and fentanyl analogs towards a universal rapid screening assay. The electrochemical behavior of fentanyl was characterized in depth using multiple electrochemical techniques such as cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). The optimization of the supporting electrolyte, the potential range, and methods to decrease the background current were explored with CV. To work towards a more portable system, screen printed electrodes were used. The observation of the metabolic peaks remained challenging, and different methods were attempted to achieve it. The quantification of fentanyl was successfully demonstrated using the different electrochemical systems proposed in this work. The electrochemical characterization of fentanyl and the optimization of multiple experimental parameters were the first step in developing a universal, rapid, electrochemical sensing method for the detection of fentanyl and fentanyl analogs.
Evaluation of Odor Compounds Sensed by Explosives-Detecting Canines
(2013-08-14) Kitts, Kelley M.; Goodpaster, John V. (John Vincent); Lee, Lei; Siegel, Jay A.; Picard, Christine
Canines are regularly utilized by law enforcement agencies to detect explosives. However, the mechanism by which canines respond to explosive vapors is not well understood, leading to difficulties in canine training and testing. It is known that the amount of vapor generated from explosive compounds is dependent upon several factors including sample amount, vapor pressure, and the degree of confinement. Underlying these factors is the basic process of evaporation of an unconfined explosive, which is rucial to understanding how explosive vapors behave in other, more confined, systems. In Stage One of this study, evaporation rates were determined for several explosive liquids using an analytical balance. These rates were compared to one another as well as to theoretical models for the evaporation of liquids. In general and as expected, mass decreased linearly with time and evaporation rates decreased logarithmically as boiling point increased. Several examples of solvent “pinning” on a metal surface were also observed. While an empirical model for the evaporation of unconfined explosive liquids was developed, a comprehensive model for the escape of explosive vapors from sealed containers (i.e., a suitcase, knapsack, or IED container itself) is needed. The second part of Stage One of this study was to determine that the flow rate of explosive vapors escaping from relatively large orifices does not conform to Fick’s Law of Diffusion. Fick’s model states that the flow rate is linearly dependent upon the cross sectional area of the orifice and the material’s diffusion coefficient. Instead, the flow rate was found to be linearly dependent upon the diameter of the orifice due to the tendency of the flow to diffuse outwards from its circular edge. A clear relationship between flow rate and diffusion coefficient was seen, however. Additional uncertainty arises concerning the complexity of the odor generated from explosive compounds. Because explosive vapors are often complex (they consist of multiple chemical compounds), confusion exists regarding the cause of canine alert; that is the “odor compound” that allows for canine detection of various explosives. Although 2, 4- dinitrotoluene (DNT) has been explored as a potential odor compound, the possibility of a nitrated explosive inherently producing nitrated gas upon decomposition has not. Stage Two of this study focused on evaluating nitrate as a potential cause of canine alerts. An LC/MS method for the detection of nitrate ions in Composition C-4 and flake trinitrotoluene (TNT) was developed and tested. Instrumental analysis was not successful in detecting nitrate ions in any of the explosives tested. The lack of nitrate was confirmed using a diphenylamine color test for nitrates, thus eliminating nitrate as an odor compound and cause of canine alert to nitroaromatic compounds. 2, 4-DNT has been introduced as a potential odor compound of TNT, however, the mechanisms behind its vapor emission have not been thoroughly explored. More specifically, due to the “sticky” nature of the 2, 4-DNT isomer, the effects of surface adhesion to container walls are of concern. In particular, whether the amount of material lost to surface adhesion is significant enough to effect canine detection of TNT. A second focus of Stage Two explored this concern. A GC/MS method for the detection and separation of TNT and DNT isomers in liquid extracts was developed and the amount of 2, 4-DNT residues adhering to container walls was quantified. These values, compared to the amount 2,4-DNT expected to saturate each container (determined by the Ideal Gas Law), showed a significant preference of 2,4-DNT in the solid phase as opposed to in the gas phase. The amount of residue adhering to the walls of a gallon can differed from expected values by nearly 70%. The amount of material extracted from a quart can exceeded expected values by 137%. The apparent sticky nature of 2, 4-DNT resulted in a significant loss of material needed to fully saturate a container and thus canine detection success may be affected. In the final stage of this study, theories regarding odor compounds and odor availability of nitromethane, TNT, and Composition C-4 were tested using certified explosives-detecting canines. These trials included thirty-three canine-handler teams from eight government agencies. The odor availability of nitromethane was tested by placing varying volumes of nitromethane in containers with differing degrees of confinement and studying the effects on canine detection success. The odor availability trial showed no significant effect of sample amount or degree of confinement on canine detection so long as the sample volume was sufficient to saturate its container. In this study that volume was determined to be < 1 mL. Detection of 2, 4-DNT, TNT-NESST (Non-Hazardous Explosives for Security Training and Testing), and flake TNT were also studied using certified canines. The purpose of this was to identify the odorant responsible for canine alert to the explosive TNT. These trials showed a significant response to 2, 4-DNT compared to TNT and its training aid; this suggests that 2, 4-DNT is the primary cause of canine alerts to TNT. Additionally, Composition C-4 and RDX-NESTT were tested along with potential odor compounds that included the manufacturing solvent, cyclohexanone, the energetic “taggant” 2, 3-dimethyl-2.3-dinitrobutane (DMNB), the plasticizer dioctyladipate (DOA) and its degradation product 2-ethyl-1-hexanol. While some response to DMNB and cyclohexanone was seen, the most significant response was to the actual Composition C-4. This suggests that the cause of canine alert to Composition C-4 is the explosive mixture as a whole and not a single chemical component of the mixture
Evaluation of storage conditions on DNA used for forensic STR analysis
(2014) Beach, Lisa Renae; Picard, Christine; Goodpaster, John V. (John Vincent); Randall, Stephen Karl, 1953-
Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.
Forensic applications of associating human scalp hair morphology and pigmentation analysis at the microscopic and molecular level
(2017-08) Stubbs, Wesli Kay; Walsh, Susan; Picard, Christine; Berbari, Nicholas
Criminal investigation and the science behind evidence analysis is an ever- growing niche, and forensic DNA phenotyping (FDP) is no exception. For years the only information given to authorities regarding DNA found at a crime scene was STR analysis and matching to a comparative DNA sample from a known source. However, what happens when there is no suspect to compare DNA profiles, or the case involves a missing person where the only available piece of evidence is a biological sample found at the scene? Before FDP, not much could be done with the DNA sample and the investigation would be stalled. Now it is becoming possible to statistically predict an individual’s visual characteristics using FDP. Currently, with the use of Irisplex, HIrisplex, and HIrisplex-S, statistical analyses and predictions can be done for categorical eye, hair, and skin color by looking at specific genes and their associative SNPs, such as HERC2 and OCA2. The more that is understood about trait-determining genes and their functional significance with regards to our physical traits, the more phenotypes can be added to these prediction tools. In an effort to discover additional genes associated with human phenotypes, this study looked at thirty-two pigmentation-associated candidate genes, and eleven hair structure and morphology associated genes in owl monkey pelage samples. Although the samples were not of human origin, it is important to point out the high conservation between humans and their non-human primate relatives. The owl monkeys used in this study were helpful for tracking expression levels of genes controlling differentpigmentation and hair structure types, because each monkey had intra-individual variation in thickness and in coat color which allowed the generation of potential candidate genes for human investigation. Of the 43 total candidate genes analyzed, 36 had successful amplification, and 28 showed a significant difference in expression when comparing the different samples. The second part of this study was to compare quantitative characteristics of human hair in physical samples and two-dimensional (2D) photos. A test set of 45 individuals had 3-5 hairs from the vertex of their head plucked and analyzed, and a 2D photograph was taken of their scalp hair. The idea was to be able to make quantitative phenotypes in hair (such as hair width, amount of curl) from 2D imagery, when physical samples are not available for analysis. This is due to the fact that the majority of genotype-phenotype databases consist solely of photographic imagery, and seldom have hairs that can be microscopically prepared for analysis. Defining measurable phenotypes from 2D photos that strongly correlate with their physical counterparts allow for the generation of a more accurate phenotype for future genome wide association studies (GWAS) within and outside this laboratory that study hair thickness and hair curl. Three different quantitative phenotypes were compared between the microscopic and 2D photo- analysis.

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