How can blood tell us when a crime happened?

Researchers at the University at Albany have determined how to track changes in blood stains to determine their age using Raman spectroscopy.

Featured image credit: Geralt from Needpix

In a criminal investigation, blood evidence is a huge bonus for investigators. Blood can give important insight into the nature of the crime itself. More importantly, it is a source of DNA, which scientists use for profiling to assign blood to a specific person. More recently, researchers wanted to see if blood could also help determine when a crime was committed.

Time since deposition (TSD) tells how long it has been since blood was deposited. If an investigator determines that blood found at a crime scene has a TSD of one week, they can estimate that the crime was committed a week ago. TSD can also help investigators determine if blood found is actually relevant to the crime. For example, weeks-old blood found at the scene of a crime that happened the night before would not be relevant to the investigation.

There are several methods for determining the TSD of blood, such as UV/Vis spectrophotometry and high-performance liquid chromatography. Most of these tests will destroy the sample, which can make it hard to do further testing, such as DNA profiling. Additionally, these methods are still in the experimental phase and are not accurate or reliable. To resolve these issues, researchers at the University at Albany led by Dr. Igor Lednev have looked to a spectroscopic technique called Raman spectroscopy. The Lednev group has done extensive work with Raman spectroscopy, and have demonstrated its ability to analyze several types of evidence such as GSR and drugs.

Spectroscopy measures the interactions of a material with light. In Raman spectroscopy, an instrument measures the amount of light a material scatters when it has been hit with a laser. The amount and the energy of the scattered light depends on the chemical structure of the materials tested, so every material will produce its own unique Raman spectrum (Fig. 1).

Figure 1. General process of Raman spectroscopic testing. Credit: Jalissa Thomas

In previous research, the Lednev group demonstrated that Raman spectroscopy can distinguish different body fluids using their unique spectra caused by their differing biological components. These components will change, however, as the body fluid dries and ages. Exposure to oxygen in the air causes certain compounds in blood to oxidize, particularly the iron-containing compound hemoglobin. This causes a change in the charge of the iron, which changes the structure of the compound and how it interacts with light. Because of this, the researchers hypothesized that they should be able to track the changes in blood composition over time to estimate the TSD.

The researchers collected Raman spectra for blood from a male and female donor at various time points ranging from one hour to one week. They then performed two statistical tests on the spectra: correlation analysis and partial least squares regression (PLSR). Correlation analysis described the order in which the changes in the spectra happened. PLSR allowed the researchers to determine whether the changes in the spectra were random or related to blood age, and ultimately to make TSD predictions.

The Lednev group determined that some Raman signals that were caused by hemoglobin changed with age (Fig. 2). One specific signal was the 377 cm-1 peak, which was identified as a marker for metHb, the oxidized form of hemoglobin (Fig. 3). This peak increased as the blood aged. The 1637 cm-1 peak (identified as the oxygen marker) decreased with time. These changes showed that as the blood aged, the amount of oxygen in the blood decreased while amount of oxidized hemoglobin increased.

Figure 2. Raw blood spectrum showing the Raman signals referenced in the paper. Credit: Jalissa Thomas

The correlation analysis found that two other specific signals in the Raman spectrum changed together over time. These peaks (1224 cm-1 and 1252 cm-1)represent different shapes of metHb. The 1224 cm-1 peak represent the protein in a sheet form, whereas the 1252 cm-1 represents its random coil form. The analysis found that the 1224 cm-1 peak decreased while the 1252 cm-1 peak increased, and that the decrease in the sheet-metHb peak occurs first. This indicated that the metHb loses its sheeted structure, and then begins to aggregate, or cluster, into randomly coiled structures. Lednev and co. used this data to confirm the changes of hemoglobin in aging blood and how they could be monitored to determine TSD (Fig. 3).


Figure 3. Process of hemoglobin aging in blood. Credit: Hemoglobin structure from Yikrazuul, sheet structure from Thomas Shafee, and random coil structure from Daniele Pugliesi, (Wikipedia, available via CCL)

The researchers built a prediction model using PLSR to determine the ability of this method to predict TSD of non-tested samples. They used the male donor’s blood to make the model and then assessed the accuracy of the model’s predictions using the female donor’s blood. The changes in blood spectra allowed for the PLSR program to predict the age of blood stain samples. The program estimated the TSD of these blood samples correctly and consistently. 

The Lednev group demonstrated that Raman spectroscopy could be used to accurately determine the TSD of blood by tracking the changes in clean aged blood spectra. In the real world, however, blood samples will be subject to various climates and/or environmental contamination that can hinder this analysis. In order to make this technique implementable in the field, the effects of various conditions, such as different temperatures, levels of sunlight exposure, and humidity, as well as blood deposited on various surfaces (e.g. wood, tile) and materials (e.g. cotton clothing, upholstery), must be researched. This information will be important for forensic investigators to take into account when using this method to analyze blood evidence in real cases.

Title A Raman “Spectroscopic clock” for bloodstain age determination: the first week after deposition
AuthorsKyle C. Doty, Gregory McLaughlin, Igor K. Lednev
OrganizationUniversity at Albany, Albany, NY
Year2016
JournalAnalytical and Bioanalytical Chemistry
Linkhttps://link.springer.com/article/10.1007/s00216-016-9486-z

An alternative method for detecting THC in body fluids

Researchers at Oregon State University examine a more efficient method for the detection of THC in body fluids.

Credit for featured photo: Michal Jarmoluk from pixabay

Marijuana is one of the most commonly used psychoactive drugs in the country. Marijuana contains the well-known compound THC (D9-tetrahydrocannabinol), which is responsible for many of its effects, such as increased heart rate and reddening of the eyes. When THC enters the body, the compound can end up in various body fluids such as saliva, urine, and blood. This fact allows toxicologist to detect THC for a variety of forensic applications. Among these applications are preventing drug abuse and determining if someone is driving under the influence.

Figure 1: Instruments, such as the one above, are typically used to analyze drugs. Above is a gas chromatography machine, which can be used to confirm the identity of drugs. Credit: creative commons

Current methods for the detection of THC and other illicit drugs require extensive sample preparation and long data processing times. This can waste precious time in a forensics investigation. Surface Enhanced Raman Spectroscopy (SERS) has potential for being a quicker but accurate alternative. In Raman spectroscopy, light is used to irradiate a sample, however, the energy that passes through the sample (or rather, is scattered by it) is different than the energy used to irradiate it. A spectrum can be created from this shift in energy, or “Raman” shift, and the unique chemical information can then be used to identify a compound. SERS uses a substrate (often a precious metal like gold or silver) to make the signal for the Raman spectrum stronger, making it possible to analyze miniscule amounts of sample.

Figure 2: In SERS, a sample is deposited on silver or gold nanoparticles, which enhances the Raman signal.

While SERS has been used to detect THC in a methanol solution (since it serves as a standard that can be used to calibrate the system and prepare it for more complex analysis), work has yet to be done on using SERS to detect THC in body fluids. For this reason, the Micro- & Nano-photonics Research Group at Oregon State University sought to determine if SERS could be used to detect THC in blood plasma and saliva.

THC was added at various concentrations to methanol (the positive control), blood plasma, and purified saliva. The solutions were loaded onto the SERS substrate, and the Raman spectra of the samples were collected using a Raman microscope. Using advanced statistical methods like principal component analysis, Sivashanmugan et al. showed that their method could be used to detect THC at various concentrations, as low as 10­-12 M, in the selected body fluids. That is 1 mol of THC for every 1 trillion liters of solution. Such a low detection limit is especially important in forensic science, since small traces of body fluids are often retrieved from crime scenes for analysis.

In addition to looking at THC in various body fluids, Sivashanmugan et al. monitored the degradation of THC in body fluids over time. When THC is present in body fluids, it undergoes a process known as metabolization, where it turns into less active compounds that can be eliminated from the body with greater ease. One of the metabolites of THC is THC-COOH, which has a carboxylic functional group attached to the main ring. Since metabolites like THC-COOH are present in body fluids after marijuana use, rather than THC itself, toxicologist test for the presence of these metabolites in body fluids. In order to look at the degradation, or metabolization, of THC in body fluids over time, Sivashanmugan et al. used SERS to collect the Raman spectra of THC in raw saliva over the course of 9 hours. Using advanced statistical methods, they were able to characterize the metabolization of THC into metabolites such as THC-COOH in raw saliva.

The method developed by Sivashanmugan et al. detects THC in various body fluids as well as characterizes the metabolization of THC into THC-COOH. This technique has potential to be applied as a sensing technique for the detection of marijuana abuse. Due to the shorter sample preparation and data analysis time associated with SERS, implementing this method could save forensic toxicologist valuable time.

Title Trace Detection of Tetrahydrocannabinol in Body Fluid via Surface-Enhanced Raman Scattering and Principal Component Analysis
Authors Kundan Sivashanmugan, Kenneth Squire, Ailing Tan, Yong Zhao, Joseph Abraham Kraai, Gregory L. Rorrer, Alan X. Wang
Journal ACS Sensors
Year March 2019
Link https://pubs.acs.org/doi/10.1021/acssensors.9b00476
Citation Sivashanmugan, K.; Squire, S.; Tan, A.; Zhao, Y.; Kraai, J. A.; Rorrer, G. L., Wang, A. X. Trace Detection of Tetrahydrocannabinol in Body Fluid via Surface-Enhanced Raman Scattering and Principal Component Analysis. ACS Sens. 2019, 4, 1109-1117.

Detecting drugs and explosives in fingerprints

Researchers at SUNY Albany show that fingerprints are more than just identifiers – they can link a suspect directly to a chemical with minimal sample preparation

Credit for feature photo: Pixabay

In the forensics world, finding fingerprints is a huge boon; their presence and subsequent match to a known standard can quickly link a suspect to a crime scene. But what if we could get more than just identity from fingerprints – say whether the donor touched drugs, psychoactive plants or explosive compounds beforehand?

Figure 1: What if after someone touches an explosive device, we could analyze the compounds on the fingerprint they leave elsewhere? Credit: Michael Gaida from Pixabay.

While this is not a new idea, previous collection methods were only able to obtain either the chemical information (the things they touched) or the fingerprint itself, but not both. Technicians would swab the fingerprint and smudge the distinctive whorls, loops and arches beyond usability in an effort to identify the drugs or explosive compounds present. If the CSI techs wanted the identification, they abandoned analyzing for any illicit substances to preserve the fingerprint pattern.

Dr. Rabi Musah and her student Kristen Fowble at SUNY Albany took a different approach. Musah used a technique called mass spectrometry imaging (MSI) – specifically Laser Ablation-Direct analysis in real time- Imaging Mass Spectrometry (LADI-MS) – to simultaneously obtain the chemical information and the fingermark pattern without destroying the sample. The technique is relatively new (less than three years old) and uses the location, or spatial distribution, of chemicals in the fingerprint to obtain a picture, much like how in a digital photo, pixels make up each point of the photo image.

Musah’s method used a laser to ablate or remove pieces of the fingerprint. These portions contain a bunch of molecules that are either endogenous or exogenous. Endogenous, or normally present compounds originate from or are excreted onto the skin, including proteins, lipids (like cholesterol), salts and organic molecules. Exogenous molecules are those not normally present, such as the heroin or acetylsalicylic acid from an aspirin pill that the fingerprint donor touched.

As the laser moves across the fingerprint and the microscopic portions are ablated from the collection device, such as a piece of tape, the mass spec collects information on how much and where all these molecules are located (Fig 2). The quantity of the molecule at each location is plotted to the picture using a heat map, where a higher concentration corresponds to an yellow color, and lower concentrations correspond to a deep red color. Places with no compound present are black. The stark difference between the presence and absence of chemicals create a nice contrast between the fingerprint and the background.

Figure 2: General flowthrough of mass spec imaging (MSI) using LADI-MS in this study. Figures for steps 1 and 2 provided by wikiHow How to Dust for Fingerprints (available via Creative Commons CC BY-NC-SA 3.0).

Using an endogenous molecule like cholesterol, researchers recreated a picture of the fingermark, with the ridges and patterns intact. At the same time, they collected information on an exogenous molecule like cocaine. Overlaying the two pictures of chemical spatial distribution showed that the fingermark is the origin of the exogenous molecule of interest. In addition, Fowble & Musah were able to identify cocaine in a cocaine-laden fingerprint four days after the initial deposition.

Though the technique sounds highly technical, LADI-MS has several advantages that decrease sample manipulation compared with other MSI methods. Because the technique is performed in an ambient environment (i.e. room temperature and pressure), there is no special step necessary for sample introduction, such as HPLC-MS which has a separation step before introduction to the mass spec. Furthermore, unlike other techniques that require the sample to be dissolved first, LADI-MS analyzes the collected fingerprint directly. Less manipulation means lower chances of contamination from outside sources or losing the miniscule sample.

Additionally, though the current study focused on only three exogenous compounds (cocaine, active ingredients in psychoactive plants, and pseudophedrine, used to make methamphetamine in illicit meth labs), this technique has great potential to identify compounds in complex mixtures. Further investigation could determine if the technique can identify drugs or chemical metabolites in the blood from a bloody fingerprint, thereby demonstrating if the donor had drugs in their system at the time of deposition.

Fowble and Musah’s novel technique maximizes the amount of information obtained from a simple fingerprint while decreasing the technical manipulation needed for analysis. 

Title Simultaneous imaging of latent fingermarks and detection of analytes of forensic relevance by laser ablation direct analysis in real time imaging-mass spectrometry (LADI-MS)
AuthorsKristin L. Fowble, Rabi A. Musah
JournalForensic Chemistry
PublisherElsevier
Year2019
Link https://www.sciencedirect.com/science/article/pii/S2468170919300700