Age estimation of bloodstains: a preliminary report based on aspartic acid racemization rate

This study describes an innovative application of a well-established method of age determination. The conventional method of aspartic acid racemization (AAR) is based on estimation of the d-l-aspartic acid ratio in slow turnover tissues, such as tooth tissue, to reflect the age of an individual. This method has been recently applied to age estimation in forensic investigations, and is also widely used for archeological dating of fossils. We suggest that the aspartic acid racemization method could be applied to a significant, although unresolved, forensic issue: that of bloodstain dating. Standard kinetic experiments were used to describe the characteristics of the racemization reaction in bloodstains, which were then employed to estimate the age of various samples. The soluble protein fraction of a bloodstain produced a stronger correlation between elapsed time and d-aspartic acid content than total amino acid fractions. According to our preliminary results, the time lapse after the creation of a bloodstain can be determined ex vivo by measuring the extent of aspartic acid racemization. Our analysis highlights the need for further study into the preservation and composition of bloodstains to assist in further development of this pioneering application.     

The application of amino acid racemization in the acid soluble fraction of enamel to the estimation of the age of human teeth

Estimation of age-at-death for skeletonised forensic remains is one of the most significant problems in forensic anthropology. The majority of existing morphological and histological techniques are highly inaccurate, and show a bias towards underestimating the age of older individuals. One technique which has been successful in forensic age estimation is amino acid racemization in dentine. However, this method cannot be used on remains where the post-mortem interval is greater than 20 years. An alternative approach is to measure amino acid racemization in dental enamel, which is believed to be more resistant to change post-mortem. The extent of amino acid racemization in the acid soluble fraction of the enamel proteins was determined for modern known age teeth. A strong correlation was observed between the age of the tooth and the extent of racemization. No systematic bias in the direction of age estimation errors was detected. For the majority of teeth analyzed, the presence of dental caries did not affect the results obtained. In a minority of cases, carious teeth showed a higher level of racemization than would be expected given the age of the individual. These results indicate that amino acid racemization in enamel has the potential to be used in age estimation of skeletal remains.     

Age at death determinations for autopsied remains based on aspartic acid racemization in tooth dentin: importance of postmortem conditions

The extent of aspartic acid racemization in human dentin can be used to assess chronological age of the individual. This novel chemical method of aging human remains has potential applications in forensic sciences. Six autopsy cases have been analyzed for D/L aspartic acid contents, and their ages at death calculated. Independent age information came from known identity, tooth maturation, or tooth wear estimations. The six cases represented a range of postmortem fates: recent demise, burial, and ground surface exposure. Five samples, four recently deceased and one a burial, showed racemization ages in close agreement with other age data. One specimen, subjected to 51 days of open air exposure, exhibited a vastly inflated racemization age. Postmortem conditions are crucial to the accuracy of racemization age at death measurements.     

Age Estimation by Amino Acid Racemization in Human Teeth

Abstract: When an unidentified body is found, it is essential to establish the personal identity of the body in addition to investigating the cause of death. Identification is one of the most important functions of forensic dentistry. Fingerprint, dental, and DNA analysis can be used to accurately identify a body. However, if no information is available for identification, age estimation can contribute to the resolution of a case. The authors have been using aspartic acid racemization rates in dentin (D‐aspartic acid/L ‐ aspartic acid : D/L Asp) as an index for age estimation and have obtained satisfactory results. We report five cases of age estimation using the racemization method. In all five cases, estimated ages were accurate within a range ±3 years. We conclude that the racemization method is a reliable and practical method for estimating age.     

Age Estimation by Racemization Method in Teeth: Application of Aspartic Acid,Glutamate, and Alanine

Abstract: Here, we report on an experimental approach of simultaneous determination of various amino acids racemization (AAR) rates in teeth. We evaluated the measurements of aspartic acid (Asp), glutamate (Glu), and alanine (Ala) isolated from dentin. Asx D/L rates from total amino acid fraction, generally used for age estimation, showed high correlation (r = 0.98) with age. As Glx and Ala showed very slow racemization kinetics in TA, we performed further analysis of the acid‐soluble protein (SP) fraction. The results supported improved correlation between age and D/L rates for Glu (r = 0.84) and Ala (r = 0.85), as well as for Asp (r = 0.98). By providing further elucidation on dentin protein racemization, the technique offers a considerable opportunity to involve other amino acids in age estimation studies. As the process does not require additional separation steps, the method can be easily adapted to existing protocols.     

Comparison of Age Estimation in Japanese and Scandinavian Teeth Using Amino Acid Racemization*

Abstract: Racemization method is currently considered to be one of the most accurate methods for age estimation. There are many factors that affect racemization reaction velocities. We investigated ethnic differences in the racemization reaction velocities between Japanese and Scandinavian teeth. After a heating experiment, the Arrhenius equation was used to obtain a rate constant (k). Both groups presented almost identical velocities, indicating that there was no difference in the tooth racemization reaction between the two groups. However, because 14 of the 18 Scandinavian teeth had multiple roots, it is possible that accurate racemization rates of the teeth themselves were not reflected in the results. These findings reconfirm that the type of tooth selected is extremely important when evaluating age by racemization.     

A new approach to amino acid racemization in enamel: testing of a less destructive sampling methodology

Aspartic acid racemization has been found to be an accurate measure of age at death for recent forensic material. This paper examines the practicality of using acid etching of the tooth surface to extract amino acids from the enamel for racemization analysis. By serial etching of the tooth and contamination of the teeth with bovine serum albumin prior to etching, the ability of etching to remove contamination was assessed. The destructiveness of the method was visualized and quantified using micro-computed tomography (micro-CT). By bleaching the teeth and by deeper etching it was possible to obtain more consistent values. While etching had little effect on the enamel at the macroscale, it did have an impact at the microscale. The quantities of enamel removed varied depending upon the tooth morphology, but were not large. Acid etching of enamel thus appears to be a promising new method for extracting proteins for amino acid racemization age estimation noninvasively.     

Application of aspartic acid racemization to forensic odontology: post mortem designation of age at death

The extent of racemization of aspartic acid in the dentin of healthy, carious and impacted teeth was used to define the ages of individuals at the time of their death. These determinations were performed with an error of +/- 4 years. This method holds the promise of simplifying procedures in, and extending the application of forensic odontology. The current error range of this method may be reduced by analyzing teeth of the same positional types from deceased individuals of known age. Using this method, it appears that post mortem preservation of up to 10 years has a negligible effect on the values determined for the age of individuals at death.     

Strategy for the estimation of chronological age using the aspartic acid racemization method with special reference to coefficient of correlation between D/L ratios and ages

The estimation of chronological age has been performed by various methods in forensic science. Among these, racemization methods, which are based on the age-dependent non-enzymatic changes of L-form amino acids to D-form mainly using aspartic acid, are one of the most reliable and accurate methods to date. Separation of enantiomers is generally performed by gas chromatography or high performance liquid chromatography. Various tissues with low metabolic rates have been applied for this purpose. In addition, single proteins purified from these target tissues are also applicable. In this brief review we describe this method in detail, noting points of caution, as well as the advantages and disadvantages of the different target tissues. In addition, special attention is given to the correlation rates obtained between chronological age and enantiomer ratios. Currently, based on accuracy of estimated age, simplicity of the method, time required, and reproducibility, tooth dentin is considered one of the best target tissues. Alternatively, analysis of osteocalcin and elastin have also provided accurate and reproducible results.     

Racemization of aspartic acid from human dentin in the estimation of chronological age

The estimation of chronological age in cadavers, human remains and in living human beings by various methods is discussed. These methods, which are based on the age dependent non-enzymatic changes of l-form amino acids to d-form amino acids, mainly aspartic acid, are among the most reliable and accurate methods to date. Most of these methods use gas chromatography (GC). In this review, results of aspartic acid racemization in dentin at different targets are discussed. In addition, pre-considerations and guidelines are given for the selection of dentin from teeth. A pilot project was run to evaluate the efficiency of high performance liquid chromatography (HPLC) coupled with fluorescence detection. New buffer conditions were found to obtain stable derivatives of aspartic acid enantiomers for the estimation of racemization.     

法医骨组织学研究

在实际检案中 ,当现场发现的骨骼残片体积较小时 ,用解剖学观察无法进行骨骼残片的法医鉴定 ,需使用骨组织学的方法进行骨骼残片的个体识别。目前 ,这是法医人类学中一门较活跃的领域 ,即法医骨组织学。法医骨组织学的内容主要包括两个方面 :(1)骨骼残片是否属于人类骨骼 ,或属于何种动物骨骼。这方面的研究包括人类骨骼的组织学特征研究及不同动物的组织学特征研究。 (2 )人类骨骼个体识别的组织学研究。这方面的研究主要包括 ,人类骨骼的组织学特征的年龄判断 ,例如股骨、胫骨、肱骨、锁骨等 ,以及使用骨组织学方法 ,进行人类骨骼的年龄评价的准确性研究。本文对上述内容进行了综述。     

Postmortem assessment of fetal diaphyseal femoral length: validation of a radiographic methodology

Depending on the general condition of fetal remains, forensic specialists might face difficulties concerning age estimation. Reference tables and regression equations are helpful devices in this task, although they are generally applied for complete fetuses or fetal remains including soft tissues. However, the problem of age estimation stays for osseous remains, both for entire bones and ossified parts, since most of the reference tables come from ultrasonographic measurements, which are not easily reproducible on fetal osseous remains. Furthermore, the ultrasonographic measurements contain slight errors in comparison to the real anatomical ones. This study describes a radiographic protocol and a measurement technique that facilitate and improve bone measurements, and therefore, facilitate age estimation, too. A qualitative criterion, namely a clear-cut bony endplate, was defined and tested. Its reliability (repeatability and reproducibility) turned out to be good, showing nonsignificative differences to the threshold of 0.05, with average errors of 0.26 and 0.44 mm respectively. Moreover, concerning the test of eventual size differences between the right and left femurs showed a P value < 0.0001. The test of the qualitative criterion was based on the comparison of the radiographic in situ femur measurements and the radiographic measurements of the same bones after dissection. The results were satisfactory, since an average error of 0.58 mm was obtained, which did not give any significant differences to the threshold of 0.05. It was concluded that this methodology provides an easy and precise new measurement tool for forensic practice, and can allow us to establish some nonultrasonographic tables, which fit our population.     

Degradation of biomolecules in artificially and naturally aged teeth: implications for age estimation based on aspartic acid racemization and DNA analysis

Postmortem teeth are the most stable structures, and can be used to gain different information (age estimation, genetic data). Over long postmortem intervals (PMI), degradation processes may alter the molecular integrity and thus affect the reliability of applied molecular methods. Whereas some knowledge on the degradation of biomolecules in bone during the PMI exists, data for teeth are lacking. In particular, the impact of degradation processes in dentine on age estimation based on aspartic acid racemization (AAR) cannot be estimated yet. Hence, the molecular stability of both collagen and DNA was analyzed systematically, and their impact on the reliability of age estimation based on AAR and genetic analyses was checked. Two hundred and ten human and 59 porcine teeth were heated (90 degrees C in water) to simulate collagen and DNA diagenesis; 14 naturally aged teeth (PMI: 3 days to 1700 years) were analyzed comparatively. Peptide patterns of cyanogen bromide (CNBr)-cleaved collagen were employed as a new approach to check the collagen integrity. In the same samples, collagen yields, amino acid compositions, AAR in different protein fractions, and DNA integrity were analyzed. In heated human and porcine teeth the collagen content declined during the heating experiment. The amino acid composition in human samples was collagen-like until 12 days of heating. In naturally aged teeth, the collagen yielded from 9.5 to 15%, and no discrepancy of amino acid composition to that of modern collagen was observed. Electrophoresis of CNBr-peptides showed an altered pattern in experimentally degraded samples from day 10 on; naturally aged collagen displayed the typical collagen pattern. AAR increased in all protein fractions with increasing duration of the heating experiment; naturally aged samples displayed a slow accumulation of AAR. DNA degraded progressively, and after 32 h of heat exposure no more DNA was detectable, whereas the amplification of nuclear and mitochondrial DNA was successful up to 48 h. STR typing was reliable up to 16 h, and sex determination up to 40 h of heat exposure. In naturally aged samples of DNA quality, yield and typing success did not correlate with PMI. The data highlight a remarkable stability of collagen dental proteins. Within relevant forensic periods a postmortem rise of AAR under normal conditions is negligible, and analyses of dental DNA has a high chance to be successful. However, after large PMI and/or extreme postmortem conditions age estimation based on AAR and genetic analyses lose their reliability.     

身高推断的法医人类学研究进展

身高属于个体识别的重要指标之一,历来是法医人类学研究的重点和热点。近几年国内外对于身高推断的研究又有新进展。笔者主要对近六年来国内外学术界发表的相关文献进行查阅,并精选具有代表性的文献进行综述,发现主要有以下新的研究进展和趋势:(1)随着人类平均身高的不断增长,原有身高推断方程有不断更新修正的需要;(2)性别、年龄、人种、地域等因素对身高推断影响大,设计实验研究方案时应考虑加入上述因素;(3)医学影像学技术,特别是CT技术成为近年来法医人类学身高推断研究的新方法。     

端粒DNA在法医学年龄推断中的应用

法医学中年龄推断的常用方法是对齿龄和骨龄进行推断,但这二种方法都受地理环境、营养状况、生活习惯、民族等因素的影响,尤其对成年人而言准确性会下降。随着对端粒研究的不断深入,已证实端粒DNA长度可反映细胞的分裂程度,代表细胞的生长寿命并与供体年龄有负相关性,因此测定端粒DNA长度有望成为法医学推断年龄有价值的新方法。     

如何正确看待法医骨龄和刑事责任年龄

骨龄又叫骨骼测定年龄,刑法所规定的“刑事责任年龄”为某一个体的“生活年龄”,二者所代表的含义不同,且存在着较大的差异。司法机关直接利用骨龄作为对犯罪嫌疑人定罪量刑的证据判案时常引发不小争议。故此,实现“骨龄”推断到“生活年龄”推断是法医任重而道远的任务。本文分析了二者的差异及原因,并呼吁尽快出台综合评定其个体“生活年龄”的全新标准或规范。     

A more sensitive method for the quantitation of genomic DNA by Alu amplification

Current procedures for human DNA quantitation reach their limit at 150 pg DNA, which is above the limit of the PCR profiling range using Profiler-Plus (Applied Biosystems, CA). This study tested the potential for the use of primate specific Alu sequences in forensic science for the sensitive detection and quantitaion of DNA. A fluorescently labelled primer pair was designed enabling high efficiency amplification of the core Alu sequence within primate DNA. Quantitation was performed by measurement of fluorescence intensity and comparison to a series of standard template DNA amounts via the construction of a standard curve. The new Alu-based quantitation protocol developed has shown its feasibility in more sensitively quantitating (100-2.5 pg) unknown amounts of human DNA for forensic use. The method is compatible with the use and throughput of current forensic procedures.     

Discrimination between human and animal DNA: application of a duplex polymerase chain reaction to forensic identification

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.     

髂骨耳状面的人类学个体识别

本文综述了髂骨耳状面在人类学个体识别中的应用,髂骨耳状面对年龄、身高和性别的推断,具有简单、准确的特点,在人类学个体识别中具有重要意义。     

The extent of aspartic acid racemization in dentin: a possible method for a more accurate determination of age at death?

In the current study the objective was to find to what extent a reliable determination of age at death is made possible by establishing the degree of aspartic acid racemization in the dentin of teeth. The results of the investigation of 46 teeth are in agreement with the values found by other authors. The method presented makes a reproducible and accurate estimation of age possible. We intend to elaborate and improve this promising method for determination of age at death. The relevant points are presented here.