Stefanie Ritz-Timme

Age estimation: The state of the art in relation to the specific demands of forensic practise

Abstract Age estimation in cadavers, human remains and living individuals may clarify issues with significant legal and social ramifications for individuals as well as for the community. In such cases methods for estimating age should fulfil the following specific demands: (1) they must have been presented to the scientific community, as a rule by publication in peer-reviewed journals, (2) clear information concerning accuracy of age estimation by the method should be available, (3) the methods need to be sufficiently accurate and (4) in cases of age estimation in living individuals principles of medical ethics and legal regulations have to be considered. We have identified and summarized the methods that essentially fulfil these specific demands. In childhood and adolescence morphological methods based on the radiological examination of dental and skeletal development are to be recommended. In adulthood, the accuracy of most morphological methods is much reduced. Here a biochemical method based on aspartic acid racemization in dentine provides the most accurate estimates of age, followed by special morphological dental and skeletal methods. The choice of method has to take account of the individual circumstances of each case. Most methods require either the consultation of specialised and trained scientists or an adequate calibration by the “user”. Very few attempts have been made to find common standardisation, calibration and evaluation procedures or to develop means of quality assurance for methods of age estimation. Efforts in these directions are necessary to guarantee quality standards and adequate answers to the important legal and social issue of age estimation in forensic medicine.

Aspartic acid racemization : evidence for marked longevity of elastin in human skin

Background  In extracellular proteins, aspartic acid racemization (AAR) has the potential to identify long‐lived or permanent proteins.

Age estimation based on aspartic acid racemization in elastin from the yellow ligaments

The yellow ligaments of the spine are characterized by an exceptionally high content of elastin, a protein with a proved longevity in several human tissues. This unique biochemical composition suggested a suitability of yellow ligaments for age estimation based on aspartic acid racemization (AAR), which was tested by determination of AAR in total tissue specimens and in purified elastin from yellow ligaments of individuals of known age. AAR was found to increase with age in both sample sets. The purified elastin samples exhibited a much faster kinetics than the total tissue, with ca. 3.7–4.6-fold higher apparent rates. The relationship between AAR and age was much closer in the purified elastin samples ( r =0.96–0.99) and it can therefore be used as a basis for biochemical age estimation. The analysis of total tissue samples cannot be recommended since the AAR values can be strongly influenced even by slight, histologically non-detectable variations in the collagen content. Age estimation based on AAR in purified elastin from yellow ligaments may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g. no available teeth, body parts).

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.

Is amino acid racemization a useful tool for screening for ancient DNA in bone

Many rare and valuable ancient specimens now carry the scars of ancient DNA research, as questions of population genetics and phylogeography require larger sample sets. This fuels the demand for reliable techniques to screen for DNA preservation prior to destructive sampling. Only one such technique has been widely adopted: the extent of aspartic acid racemization (AAR). The kinetics of AAR are believed to be similar to the rate of DNA depurination and therefore a good measure of the likelihood of DNA survival. Moreover, AAR analysis is only minimally destructive. We report the first comprehensive test of AAR using 91 bone and teeth samples from temperate and high-latitude sites that were analysed for DNA. While the AAR range of all specimens was low (0.02–0.17), no correlation was found between the extent of AAR and DNA amplification success. Additional heating experiments and surveys of the literature indicated that d/l Asx is low in bones until almost all the collagen is lost. This is because aspartic acid is retained in the bone within the constrained environment of the collagen triple helix, where it cannot racemize for steric reasons. Only if the helix denatures to soluble gelatin can Asx racemize readily, but this soluble gelatine is readily lost in most burial environments. We conclude that Asx d/l is not a useful screening technique for ancient DNA from bone.

A review of the methodological aspects of aspartic acid racemization analysis for use in forensic science

Accurate age determination of adult cadavers and human remains is a key requirement in forensic practice. The current morphological methods lack accuracy and precision, require specialist training and are costly. The use of aspartic acid racemization (AAR) in human dentine provides a simple, cost-effective solution and the method can achieve accuracies of +/- 3 years at best. Currently, there are differences in AAR methodology between laboratories which produce different results on the rate of racemization in teeth. These inconsistencies must be resolved if the technique is to be successfully applied to age determinations in forensic cases. This paper reviews the differences in protocol which have been used, discusses how each method will affect the results obtained from AAR analysis and gives recommendations for optimization of the methological protocol as a first step towards international standardization.

Quality assurance in age estimation based on aspartic acid racemisation

Abstract Estimates of the age of living and dead individuals, obtained in order to answer legal or social questions, require minimum quality standards in order to guarantee data quality. We present an outline strategy (with recommendations) for the attainment of quality assurance in age estimation based on aspartic acid racemisation. The strategy is based on a definition of minimum standards for laboratories, including documentation of procedures, methodology and levels of expertise, and the formulation of guidelines for intralaboratory and interlaboratory quality control.

Sudden unexpected death under acute influence of cannabis.

The acute toxicity of cannabinoids is said to be low and there is little public awareness of the potentially hazardous cardiovascular effects of cannabis, e.g. marked increase in heart rate or supine blood pressure. We describe the cases of two young, putative healthy men who died unexpectedly under the acute influence of cannabinoids. To our knowledge, these are the first cases of suspected fatal cannabis intoxications where full postmortem investigations, including autopsy, toxicological, histological, immunohistochemical and genetical examinations, were carried out. The results of these examinations are presented. After exclusion of other causes of death we assume that the young men experienced fatal cardiovascular complications evoked by smoking cannabis.

DREEM on, dentists! Students’ perceptions of the educational environment in a German dental school as measured by the Dundee Ready Education Environment Measure

OBJECTIVES The educational climate in which future doctors are trained is an important aspect of medical education. In contrast to human medicine, it has been rather neglected in dental educational research. The aim of the study was to supplement this lack by applying and validating the Dundee Ready Education Environment Measure (DREEM) for the first time in a German-speaking sample of dental students. METHODS All dental students at the Medical Faculty of Heinrich-Heine-University Düsseldorf were asked to complete a German adaptation of the DREEM and the Düsseldorf Mission Statement Questionnaire (DMSQ) in a paper-pencil survey. Data from 205 participants were analysed. Psychometric validation included analysis of item homogeneity and discrimination, test reliability, criterion and construct validity (convergent, factorial). RESULTS DREEM item parameters were satisfactory, reliability (α = 0.87) and convergent validity (r = 0.66 with DMSQ) were also high. Factor analyses, however, yielded dimensions which did not fully correspond to the original DREEM subscales. Overall perception of the educational environment was positive (DREEM total score = 122.95 ± 15.52). Students in the clinical part of course rated the atmosphere more negatively, but their academic self-perception more positively than preclinical students. CONCLUSIONS Showing satisfactory psychometric properties, DREEM proved suitable for assessing educational environments among dental students. Given the right circumstances, e.g., small and early clinically oriented classes, traditional curricula can generate positive environments.

Aspartic acid racemisation in purified elastin from arteries as basis for age estimation

Aspartic acid racemisation (AAR) results in an age-dependent accumulation of d-aspartic acid in durable human proteins and can be used as a basis for age estimation. Routinely, age estimation based on AAR is performed by analysis of dentine. However, in forensic practise, teeth are not always available. Non-dental tissues for age estimation may be suitable for age estimation based on AAR if they contain durable proteins that can be purified and analysed. Elastin is such a durable protein. To clarify if purified elastin from arteries is a suitable sample for biochemical age estimation, AAR was determined in purified elastin from arteries from individuals of known age (n = 68 individuals, including n = 15 putrefied corpses), considering the influence of different stages of atherosclerosis and putrefaction on the AAR values. AAR was found to increase with age. The relationship between AAR and age was good enough to serve as basis for age estimation, but worse than known from dentinal proteins. Intravital and post-mortem degradation of elastin may have a moderate effect on the AAR values. Age estimation based on AAR in purified elastin from arteries may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g., no available teeth and body parts).