Forensic Age Estimation defines an expertise in forensic medicine, which aims to define in the most accurate way the chronological age the person of an unknown age involved in judicial or legal proceedings [1]. Age estimation of unknown human bodies is important in crime investigation or mass disaster investigation because the age at the time of death can guide investigators to get the most probable and narrowed matches from a large number of matches. Aging is a complicated process characterized by a progressive decline in physical, mental, and reproductive capacities, leading to a loss of function, increased susceptibility to disease, and ultimately the end of life [2]. These progressive process leads to have various changes in the human body, which plays a key role in the determination of the age of the person. Age Estimation is necessary in the identification and creation of biological profiles, which can then compared with suspects, victims, or missing persons. There are many conventional morphological methods for estimation of age such as examining bones, skull, and teeth morphologically [3] or analyzing the amino acid racemization of teeth [4]. However, what if there is nobody/teeth available for morphological examination? These methods have limited use in the investigation because they depend on the availability of sample sources such as teeth, bones, or other identifiable body parts having physical features. Here the Age Estimation through biological fluid samples comes into focus. Because there is a high probability of the presence of various biological fluids such as blood, urine, saliva, semen, vaginal fluid on the scene of the crime.

Age Estimation through Biological Samples

Biological samples recovered from crime scenes can play an essential role in criminal investigations, providing vital information as to the involvement of particular individuals. A substantial number of studies have conducted with the aim of characterizing the entire metabolome of human bodily fluids including saliva, urine, serum, blood using mass spectroscopy, and other analytical methods. Also, various methods that would allow to estimate the age of crime suspects or missing persons from biological fluid samples collected at the scene of crime have devised as follows-

Blood                                                                                                                                        

Blood is one of the most commonly encountered biofluids found at the crime scenes of homicides and assaults.

DNA Rearrangements – Recently developed DNA based method for human age estimation takes advantage of the fundamental characteristics of immune cells known as T cells. These T cells produce circular DNA (known as SJ TRECs) as a by-product of the process of T-cell specific DNA rearrangements. The quantification of SJ TRECs helps in the estimation of age as their number declines at a constant rate with age [5].

DNA Methylation – This involves the addition of a methyl group to the cytosine (nucleotide of DNA). Age-associated DNA methylation changes have been studied and these studies indicate that, average DNA methylation levels increase in the genome throughout the first year of life and after that, the levels are relatively stable [6]. After reaching adulthood, DNA methylation decreases across the genome [7].

Telomere Shortening – Telomere is a repetitive sequence of nucleotides present at the end of chromosomes, for its protection. The quantitative relation between age and changes in telomere length also considered as a parameter for the prediction of human age [8]. The estimation of human age, according to telomere shortening is a method used in consideration of gender [8].            

Saliva

Saliva is usually deposited on floors, skin, cloth, paper, in bite marks found in homicides, sexual and other assaults, drugs, and alcohol abuse.

Salivary Protein Levels – The quantification salivary protein changes are associated with age. The more proteins are present in the adolescent group followed by children and lastly in the adult’s group [9].

DNA Methylation – The two marker proteins in saliva are specially analyzed for age prediction. The methylation profiles of this age markers show high correlations with age [10].    

Urine

A urine sample can also present on scenes of crime as a biological sample.    

Testosterone Levels – Testosterone is a sex hormone produced in small amounts in females as compared with males. The levels of the testosterone in urine vary with age in both males and females. The levels are at a peak in the adolescent group and decrease with age [11].           

Semen                                                                                                            

Semen stain is one of the most important biological evidence in sexual crime scenes.

DNA Methylation – For estimation of age through semen, DNA methylation is considered as the most effective age-predicting marker because the level of DNA methylation in the genome found in the semen sample changes according to the age of individuals [12]. DNA methylation has been regarded as the most promising age-predictive biomarker during the examination of blood, saliva, semen samples. Some environmental and genetic factors such as sex, diseases, diet variations, genetic disorders of individual, environmental effects like rain, wind, heat on samples present at scene effects on the changes in these markers. Thus, during the age estimation, these factors have been considered for the best result. 

References

1.     Forensic Medicine – From Old Problems to New Challenges. Edited volume by Duarte Nuno Vieira. DOI: 10.5772/19261.

2.     Fontana L, Partridge L, Longo VD. Extending healthy life span – from yeast to humans. Science. 2010; 328(5976): 321–326.

3.     Ritz-Timme S, et al. Age estimation: The state of the art in relation to the specific demands of forensic practise. Int. J. Legal Med. 2000; 113: 129–136. doi: 10.1007/s004140050283.

4.     Ohtani S, Yamamoto T. Age estimation by amino acid racemization in human teeth. J. Forensic Sci. 2010; 55: 1630–1633. doi: 10.1111/j.1556-4029.2010.01472.x.

5.     D. Zubakov, F. Liu, M.C. van Zelm, J. Vermeulen, B.A. Oostra, C.M. van Duijn, G.J. Driessen, J.J.M. van Dongen, M. Kayser, A.W. Langerak. Estimating human age from T-cell DNA rearrangements. Current Biology, 2010; DOI: 10.1016/j.cub.2010.10.022

6.     Martino DJ, Tulic MK, Gordon L, et al. Evidence for age-related and individual-specific changes in DNA methylation profile of mononuclear cells during early immune development in humans. Epigenetics. (2011); 6:1085–1094. doi: 10.4161/epi.6.9.16401.

7.     Jones MJ, Goodman SJ, Kobor MS. DNA methylation and healthy human aging. Aging Cell. (2015); 14:924–932. doi: 10.1111/acel.12349.

8.     Estimation of Human Age According to Telomere Shortening in Peripheral Blood Leukocytes of Tibetan Fu Ren et al. Am J Forensic Med Pathol.2009 Sep.

9.     Drashti Bhuptani, Satish Kumar, Maarisha Vats, and Rahul Sagav. Age and gender related changes of salivary total protein levels for forensic application Forensic Odontostomatol. 2018 May; 36(1): 26–33.

10.  Yuya Hamano, Sho Manabe, Chie Morimoto, Shuntaro Fujimoto, and Keiji Tamaki Forensic age prediction for saliva samples using methylation-sensitive high resolution melting: exploratory application for cigarette butts Sci Rep. 2017; 7: 10444.

11.  WalterFutterweit, Neal L.McNiven, Roger Guerra-Garcia, NormanGibree, MichaelDrosdowsky, George L.Siegel, Louis J.Soffer, Ira M.Rosenthal, Ralph I.Dorfman. Testosterone in human urine.

12.  A validation study of DNA methylation-based age prediction using semen in forensic casework samples.Lee JW, et al. Leg Med (Tokyo). 2018.PMID: 29413993.

Author: Ekta B. Jadhav.       

M.Sc. Forensic Science.

Intern at Dept. of Forensic Science & Criminal Investigation,

Legal Desire Media & Insights.