Bloody Clocks Do Tell Time

Credit for featured photo: NelC and DPP Law

Someone has just discovered a body! Forensic analyst rush to the scene and begin their examination of the body. What is one of the first things these analysts determine? Post-mortem interval, or PMI.

PMI is an estimation of the time interval during which an individual died. Determining the PMI firstly allows investigators to create a timeline of events for the crime, and secondly helps rule out suspects who may have an alibi. However, the methods used to determine PMI, such as examining the body temperature, body stiffness, and degree of decomposition, do not provide information about when the actual crime occurred. Say a bloodstain was deposited on concreate due to an assault, but the victim did not die until hours later. Methods used to determine PMI would indicate when the victim died, not when the assault occurred.

EUPOL Afghanistan: Crime Scene Management
Figure 1: Forensic analyst examining a “dead body” found at a “crime scene”. Credit: EUPOL Afghanistan Media (CC).

Another piece of evidence commonly found at crime scenes may be able to estimate the time since a crime occurred; blood. Blood is the most commonly found body fluid at a crime scene. There are a variety of forensic uses for the analysis of blood deposited at a crime scene. Bloodstain pattern analysis, genetic analysis, and the analysis of foreign materials (such as drugs or poison) in blood can provide important information to a case. Forensic scientist can also determine the age of a bloodstain, defined as the time since blood was leaked from the body and formed the bloodstain (Fig 2). This information is an important factor in determining the time since a crime occurred, rather than just the PMI. 

Figure 2: This figure shows the metabolic changes blood undergoes as it ages, along with the corresponding changes in color. This color change is often used to approximate the age of a blood stain. Credit: Niara Nichols.

While current methods for the determination of bloodstain age look at the color of the bloodstain, Kang et al. from Eulji University in Korea looked into using common metabolites in blood to determine the body fluid’s age. Kang et al. focused on hemoglobin, the protein responsible for blood’s red color that captures and transports oxygen throughout the body. When hemoglobin is exposed to the external environment, the protein undergoes various chemical transformations, producing compounds similar in structure to the chemical called metabolites. Kang and his colleagues used High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) to analyze bloodstains after various timepoints to determine if different metabolites of blood, such as oxidized hemoglobin (HbO2), methemoglobin (Met-Hb), and hemichrome (Fig 2), could indicate a bloodstain’s age.

Figure 3: HPLC-MS machines, like the one above, are often used in forensic labs in order to analyze samples. Credit: fiddledydee (CC).

Scientist collected blood from healthy donors and let them absorb onto filter paper to generate bloodstains that mimicked blood absorbed onto clothing. They let the bloodstains sit in the dark for up to three weeks. After the appropriate amount of aging, the scientists analyzed the bloodstains using HPLC-MS/MS. HPLC-MS/MS is a method commonly used in forensic science to separate (the HPLC part) and identify (the MS part) compounds in a mixture. The MS analysis was conducted in tandem (MS/MS), meaning the MS was ran using two adjacent mass spectrometers, in order to increase sensitivity and get more detailed results.

Kang et al. used statistical analysis to select 62 different molecular features and discriminate between the different bloodstain ages. By using the METLIN database, a large database containing tandem MS data, they were able to associate those molecular features with five different metabolites. The blood concentration levels of the five metabolites changed significantly over the three-week period. For example, L-tryptophan and ergothioneine, showed a significant decrease in concentration between the bloodstain ages (Fig 4). Continuing this analysis with the other metabolites, Kang et al. used advanced statistical analysis to classify the bloodstains by age by analyzing the amounts of the metabolites present in the bloodstain with 75% accuracy.

Figure 4: Reprinted (adapted) with permission from (Seok, Ae Eun; Lee, Jiyeong; Lee, You-Rim; Lee, Yoo-Jin; Kim, Hyo-Jin; Ihm, Chunhwa Ihm; Sung, Ho Joong; Hyun, Sung Hee; Kang, Hee-Gyoo. Estimation of Age of Bloodstains by Mass-Spectrometry: A Metabolomic Approach. Anal. Chem. 2018, 90, 21, 12431-12441.). Copyright (2019) American Chemical Society.

Kang and his colleagues concluded their study by stating that more work needs to be conducted on this method, most likely because there is room for improved instrumentation and the variety of conditions that can exist at a crime scene need to be taken into consideration. They hypothesized that Triple-Quadrupole MS Quantification (TQMSQ) may provide more selective results than MS/MS since the multiple mass filters associated with TQMSQ allows for greater selectivity and the analysis of smaller amounts of sample. Kang et al. plans to use this instrument in place of MS/MS in future studies. Finally, Kang et al. plans to analyze blood under various temperatures and humidity as well as age stains past three weeks in order to demonstrate that metabolites are robust indicators of bloodstain age.

 Title Estimation of Age of Bloodstains by Mass-Spectrometry: A Metabolomic Approach
AuthorsAe Eun Seok, Jiyeong Lee, You-Rim Lee, Yoo-Jin Lee, Hyo-Jin Kim, Chunhwa Ihm, Ho Joong Sung, Sung Hee Hyun, Hee-Gyoo Kang
JournalAnalytical Chemistry
Year2018
Linkhttps://pubs.acs.org/doi/10.1021/acs.analchem.8b01367
CitationSeok, Ae Eun; Lee, Jiyeong; Lee, You-Rim; Lee, Yoo-Jin; Kim, Hyo-Jin; Ihm, Chunhwa Ihm; Sung, Ho Joong; Hyun, Sung Hee; Kang, Hee-Gyoo. Estimation of Age of Bloodstains by Mass-Spectrometry: A Metabolomic Approach. Anal. Chem. 2018, 90, 21, 12431-12441.

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