Postmortem estimation of age at death based on aspartic acid racemization in dentin: Its applicability for root dentin

Summary The extent of aspartic acid racemization in total dentin and in dentin protein fractions from theroots of third molars was determined. In several cases coronal dentin was also investigated. The results of other authors, according to which the racemization of aspartic acid in root dentin apparently proceeds differently than in coronal dentin, could be confirmed. Consequently, the data published so far on age determination based on the extent of aspartic acid racemization in coronal dentin and the entire dentin of longitudinal sections cannot be applied to root dentin. In total root dentin and the acid soluble protein of root dentin, a close relationship was observed between the extent of aspartic acid racemization and age. Accordingly, estimation of age at death based on aspartic acid racemization in dentin is also possible for root dentin, apparently with good results. This is important particularly in those cases where a large portion of the coronal dentin is absent, for instance following dental treatment. In the investigation of root dentin, regression equations specific for root dentin must be employed in the estimation of age at death. Corresponding equations for third molars were calculated.     

Age estimation using aspartic amino acid racemization from a femur

Age estimation is an important step in post-mortem investigation, and the aspartic acid racemization (AAR) of dentin is the best tool for age estimation from human remains. However, teeth may not be found or sufficiently preserved to analyse the Dextro/Levo (D/L) ratio, and so age estimation using AAR from other organs, such as bones, is required. The current study evaluated age estimation methods using aspartic amino acid racemization from a femur in a Thai population. D/L ratio analysis using high-performance liquid chromatography (HPLC) was performed on 40 femoral bones from 24 males and 16 females, and the correlation between D/L ratio and age was 0.8316. The correlation coefficient in the male sample was greater than the female samples – 0.912 and 0.716, respectively. The standard error of estimation of all samples was 11.01 years. The correlation between the D/L ratio and the age of the total amino acid fraction from a femur was not as strong as from dentin and single protein purification, such as elastin and osteocalcin, but this method was necessary because teeth may not be found with body remains, and this method is not too complicated for routine forensic work.     

Racemization in enamel among different types of teeth from the same individual

We measured the quantity of D-aspartic acid (degree of racemization of aspartic acid) in the enamel of different types of teeth from the same individual. We studied the correlation between the degree of racemization and the time of formation of each particular tooth, as well as the applicability of the degree of racemization to estimation of chronological age. If the environmental condition of the teeth is the same, the degree of racemization is expected to be highest in teeth that completed formation in the earliest period of time. Different degrees of racemization in enamel were found among different types of teeth, even in the same individual. The degree of racemization in enamel was found to be higher in molars than in incisors, and showed a tendency that did not necessarily coincide with the time of formation. This seemed to be due to the fact that the environmental temperature was higher in the molar region located deeper in the oral cavity than the front region, and that enamel was more affected by breathing air than dentin because the D/L ratios in enamel were lower than those in dentin. Using enamel, a better estimation of chronological age was obtained from calculations based on the degree of racemization of each type of tooth than from all the different teeth together. However, these estimated ages were not better than those from dentin.     

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

Summary 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.     

Quality assurance in age estimation based on aspartic acid racemisation

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. Received: 10 January 2000 / Accepted: 15 March 2000     

Estimation of chronologic age using the aspartic acid racemization method. I. On human rib cartilage

Determinations of chronologic age are of great importance in forensic science. At present the aspartic acid racemization method on teeth provides one of the best means in adult individuals. However, if teeth are not available, some other stable tissue has to be used. In this study, the applicability of cartilage from the ribs has been tested. Specimens of rib cartilage were obtained at autopsy from 24 individuals aged 0.2–96 years. An acid-soluble peptide (SP) fraction and an insoluble collagen-rich (IC) fraction were prepared, and the ratio of D/L aspartic acid was determined using the HPLC technique. The correlation coefficient between the D/L ratio and age was r = 0.91 in SP and r = 0.97 in IC. It thus seems as if cartilage from non-weight-bearing areas may be a useful source of tissue for the estimation of chronologic age with the aspartic acid racemization method when teeth are not available.This study was supported by grant # B93-241-10277-01 from the Swedish Medical Research Council and Deutscher Akademischer Austauschdienst grant # 517 023 502 3     

Estimation of chronologic age using the aspartic acid racemization method. II. On human cortical bone

Over the last 20 years a new chemical method, base on the racemization of aspartic acid, has been developed to be used for the estimation of chronologic age in adult individuals. The method has a good accuracy when used on dental enamel, dentine and cartilage. However, in forensic and archeological cases teeth and cartilage are not always available. Since preliminary studies have shown that there are some age-related changes of the D/L aspartic acid ratio also in bone, this study was carried out to further explore if the method could be used for age estimations of bone. Bone samples from 24 individuals, aged 0.2 to 95.6 years were analysed for the D/L ratios with HPLC-technique. Two different fractions of the bone were examined, an acid-soluble peptide fraction and an acid-insoluble collagen-rich fraction. The analyses showed age-related racemizations in both fractions, although of different rates. The correlation coefficients with age were 0.72 in the peptide fraction, and 0.84 in the collagen-rich fraction. It thus seems as if bone maybe used for age estimations when more stable tissues like dentine and cartilage are not available.This study was supported by grant # B93-241-10277-01 from the Swedish Medical Research Council and from Deutscher Akademischer Austauschdienst grant # 517 023 502 3     

Deoxypyridinoline crosslinks in human dentin and estimation of age

A new biochemical method for estimation of dental age from human dentin is presented. An adaptation and modification of a dentin protein extraction protocol was developed by which deoxypyridinoline crosslinks were measured using an enzyme immunoassay technique. Deoxypyridinoline, a non-reducible collagen crosslink, was measured in human dentin samples from permanent individual molars. The measurement of this protein in dentin samples from 22 patients with ages ranging from 15 to 73 years old was carried out to obtain an estimate of age at death. By linear regression between deoxypyridinoline values and age, equations for age calculations were computed. The error between the estimated age and the known age at extraction was calculated. Our results show that this method produces an expected associated error on calibration averaging 14.9 years about the mean estimated values, at a 65% level of confidence. Values of sensitivities and specificities were calculated for deoxypyridinoline at different ages. We conclude that determination of deoxypyridinoline in human dentin extracts is a new and reliable procedure, potentially suitable for most forensic laboratories and which could be a useful tool to estimate human dental age. This method could be considered as an alternative method to calculate dental age, contributing to a more accurate estimation of chronological age when used in addition to other morphological or biochemical dental methods. Received: 15 May 1998 / Received in revised form: 16 October 1998     

Age determination through DNA methylation patterns in fingernails and toenails

Over the past decade, age prediction based on DNA methylation has become a vastly investigated topic; many age prediction models have been developed based on different DNAm markers and using various tissues. However, the potential of using nails to this end has not yet been explored. Their inherent resistance to decay and ease of sampling would offer an advantage in cases where post-mortem degradation poses challenges concerning sample collection and DNA-extraction. In the current study, clippings from both fingernails and toenails were collected from 108 living test subjects (age range: 0–96 years). The methylation status of 15 CpGs located in 4 previously established age-related markers (ASPA, EDARADD, PDE4C, ELOVL2) was investigated through pyrosequencing of bisulphite converted DNA. Significant dissimilarities in methylation levels were observed between all four limbs, hence both limb-specific age prediction models and prediction models combining multiple sampling locations were developed. When applied to their respective test sets, these models yielded a mean absolute deviation between predicted and chronological age ranging from 5.48 to 9.36 years when using ordinary least squares regression. In addition, the assay was tested on methylation data derived from 5 nail samples collected from deceased individuals, demonstrating its feasibility for application in post-mortem cases. In conclusion, this study provides the first proof that chronological age can be assessed through DNA methylation patterns in nails.     

二层龋坏牙本质胶原蛋白中氨基酸含量测定及其临床意义

目的探讨牙本质龋损中胶原蛋白的变化特点,为临床牙本质龋的预防与治疗提供参考依据。方法实验采用氨基酸自动分析仪测量正常和二层龋坏牙本质龋胶原蛋白中18种氨基酸含量。结果(1)正常与内层龋坏牙本质胶原蛋白中18种氨基酸含量无明显差别。(2)外层龋坏牙本质胶原蛋白除甘氨酸明显增高外,其余氨基酸含量明显低于正常及龋坏内层牙本质中的胶原蛋白。结论说明牙本质发生龋损时,内层龋坏牙本质中的胶原蛋白,在其氨基酸含量及构成比方面无显著改变,同正常牙本质胶原蛋白-本质胶原蛋白一样为Ⅰ型胶原;而外层龋坏牙本质中的胶原蛋白则发生了明显变化,非Ⅰ型胶原。     

Relationship between thallium-201 myocardial SPECT and findings of endomyocardial biopsy specimens in dilated cardiomyopathy

The purpose of this study was to clarify which myocardial histological findings associated with dilated cardiomyopathy (DCM) are reflected in quantitative 201Tl myocardial SPECT. We obtained studied SPECT images from 21 patients with DCM 10 minutes and 2 hours after they received an injection of 111 MBq 201Tl at rest. We calculated the percent coefficient of variation of myocardial 201Tl counts [%CV(TI)], the washout rate (WR), standard deviation of WR [SD(WR)], extent score (ES) and severity score (SS). We used image analysis to measure % fibrosis, % myocytes, the ratio of fibrous tissue to myocyte tissue (F/My), myocyte size and standard deviation of myocyte size [SD(My)] in left ventricular endomyocardial biopsy specimens. The %CV(Tl) was correlated with % fibrosis and F/My. The ES and SS also correlated with F/My. The correlation between SD(WR) and SD(My) was significant. The present findings suggest that %CV(Tl), ES and SS of rest 201Tl SPECT reflect myocardial fibrosis and that the standard deviation of washout reflects the distribution of myocyte size.     

Age estimation via quantification of signal-joint T cell receptor excision circles in Koreans

The estimation of age from biological samples (i.e., remains) at crime scenes could provide useful information about both victims and other persons related to criminal activities. Signal-joint T cell receptor excision circle (sjTREC) levels in peripheral blood decline with age, and negative correlations between sjTREC levels and age have been demonstrated in several ethnic groups. To validate the utility of sjTREC for age estimation in Koreans, Taqman qPCR was used to quantify the sjTREC level in samples obtained from 172 individuals ranging from 16 to 65 years old. We modified the previously reported method by using a shorter amplicon and confirmed the efficiency and utility of this method in this report. Our results showed that the linear negative regression curve between sjTREC levels and age was characterized by r = −0.807 and a standard error of 8.49 years. These results indicate that sjTREC level is an effective age estimation method in Koreans. The value of the standard error of quantification was not different from previous reports for other population groups.     

Age estimation in dental pulp DNA based on human telomere shortening

Age estimation based on evidence found in teeth has received considerable attention within the field of forensic science. We determined the terminal restriction fragment (TRF) length, as telomere length, to estimate age. Using dental pulp DNA we found the average TRF length showed a tendency to shortening with aging. Our findings show that telomere shortening, based on dental pulp DNA is a new and useful approach to estimate age of the subject at the time of death.     

Touch-preparation cytologic examination of breast core biopsy specimens: Accuracy in predicting benign or malignant core histologic results

RATIONALE AND OBJECTIVES: The purpose of this study was to determine the accuracy of touch-preparation cytologic examination of breast core biopsy specimens in predicting benign or malignant core histologic results. MATERIALS AND METHODS: One hundred two core biopsies were performed on 88 women with stereotactic or ultrasonographic (US) guidance. Slides were prepared by smearing one core sample on each slide, spraying the slides with fixative, and staining them with the Papanicolaou technique. Slides were blindly reviewed by a cytopathologist. Cytologic results were categorized as positive for malignancy, not diagnostic for malignancy, or insufficient for diagnosis. Results were correlated with histologic results from all specimens obtained during the core biopsy. RESULTS: Imaging depicted the lesions sampled for biopsy as masses (n = 70), clustered calcifications (n = 29), focal asymmetries (n = 2), or architectural distortion (n = 1). Touch-preparation slides of 87 (85%) lesions contained sufficient material for diagnosis. Cytologic results correctly identified 12 of 16 (three of five intraductal and nine of 11 invasive) malignancies in 10 of 13 masses and two of three clusters of calcifications. Two false-positive results occurred, both with fibroadenomas. Overall, touch-preparation studies produced 69 true-negative and four false-negative results. Excluding slides with insufficient material, the sensitivity, specificity, and accuracy of touch-preparation results were 75%, 97%, and 93%, respectively. Including insufficient samples, accuracy was 79%. CONCLUSION: Although touch-preparation cytologic examination of breast core biopsy specimens is fairly accurate in prediction of benign or malignant core histologic results, its correlation with histologic results is not sufficient to justify routine use in immediate counseling and treatment planning.     

Soft tissue removal by maceration and feeding of <Emphasis Type="Italic">Dermestes</Emphasis> sp.: impact on morphological and biomolecular analyses of dental tissues in forensic medicine

Maceration techniques remove soft tissue by the destruction of biomolecules, but the applied techniques may also affect the morphology and the molecular integrity of the hard tissue itself. The impact of seven different techniques for soft tissue removal on morphological and biomolecular parameters of teeth and dental tissues was systematically examined. All methods tested showed significant changes in dental morphology and in the molecular integrity of DNA and the dental proteins, as revealed by aspartic acid racemisation (AAR). In forensic casework this may have severe impacts on the results of morphological methods (e.g. age estimation based on root translucency) and of biomolecular analyses (e.g. age estimation based on AAR and DNA analysis). Therefore, age estimation based on AAR should not be applied to tissue treated in such a manner, and it is recommended that teeth for analysis should be extracted before soft tissue removal. DNA in the hard tissue seems to be less susceptible to soft tissue removal than proteins, and several of the tested maceration techniques appear not to have a damaging effect on DNA. Generally, the indication for soft tissue removal demands a careful case management to avoid methodological collisions.     

Age estimation of decomposed bodies based on a combined arteriosclerotic index

Several methods exist for the estimation of the age at the time of death, ranging from mere visual inspection to costly laboratory examinations. The “combined arteriosclerotic index (CAI)” is considered to be a suitable low-budget tool for undecayed corpses. It defines the ratio between diameter and longitudinal pre-strain of the abdominal aorta. Its applicability in cases of decomposed corpses has not been studied yet. We examined whether it is a valid parameter in putrefied bodies as well and whether there is a correlation between CAI and the stage of decomposition. In conclusion the CAI becomes less accurate with increasing putrefaction. Nonetheless, even in case of high-grade putrefaction it remains a useful tool for instant age estimation which should be followed by the application of methods with higher accuracy.     

Age estimation in children by measurement of open apices in teeth: a European formula

The aim of the present paper was to improve and expand research with a larger number of children from various European countries and to provide a common formula useful for all these countries. Orthopantomographs taken from 2,652 European Caucasian children (1,382 boys, 1,270 girls) aged between 4 and 16 years were analyzed. The children came from Croatia, Germany, Kosovo, Italy, Slovenia, Spain, and the UK. Following the pilot study, subjects’ age was modeled as a function of gender (g), morphological variables (predictors) × 5(second premolar), s (sum of normalized open apices) N ₀, and the first-order interaction between s and N ₀. The results showed that all these variables contributed significantly to the fit, so that all were included in the regression model, yielding the following linear regression formula: Age = 8.387 + 0.282 g − 1.692 × 5 + 0.835 N ₀ − 0.116 s − 0.139 s × N ₀, where g is a variable, 1 for males and 0 for females. The equation explained 86.1% (R ² = 0.861) of total deviance. The median of the residuals (=observed age minus predicted age) was −0.114 years, with (RefB.2) interquartile range = 1.22 years.     

Age prediction in living: Forensic epigenetic age estimation based on blood samples

DNA methylation analysis in a variety of genes has brought promising results in age estimation. The main aim of this study was to evaluate DNA methylation levels from four age-correlated genes, ELOVL2, FHL2, EDARADD and PDE4C, in blood samples of healthy Portuguese individuals. Fifty-three samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for CpG dinucleotide methylation status. Linear regression models were used to analyze relationships between methylation levels and chronological age. The highest age-associated CpG in each locus was chosen to build a multi-locus age prediction model (APM), allowing to obtain a Mean Absolute Deviation (MAD) between chronological and predicted ages of 5.35 years, explaining 94.1% of age variation. Validation approaches demonstrated the accuracy and reproducibility of the proposed multi-locus APM. Testing the APM in 51 blood samples from deceased individuals a MAD of 9.72 years was obtained. Potential differences in methylation status between samples from living and deceased individuals could exist since the highest age-correlated CpGs were different in some genes between both groups. In conclusion, our study using the bisulfite PCR sequencing method is in accordance with the high age prediction accuracy of DNA methylation levels in four previously reported age-associated genes. DNA methylation pattern differences between blood samples from living and deceased individuals should be taken into account in forensic contexts.     

Age estimation from the IV rib by phase analysis in Indian males

The determination of age remains as an important tool towards establishment of identity of an individual. The Iscan's phase method for the estimation of adult age at death from the sternal extremity of the fourth rib was introduced in 1983. Over the years, numerous tests have confirmed the reliability of this technique on varied samples. Therefore, the purpose of this paper is to apply rib phase standards to an Indian sample to test if the progression of morphological changes follow the same age sequence. This study introduces the sternal end of the rib as a site for age estimation by direct observation. The sample consisted of 101 male ribs of verified age, sex, and race. The ribs were assigned to one of nine phases (0through 8) based on changes noted at the costochondral junction. These included the formation of a pit, its depth and shape, configuration of the walls and rim surrounding it, and the overall texture and quality of the bone. It was revealed that the mean ages per phase were nearly identical in both Indians and American whites.     

Age and sex estimation by metric measurements and fusion of hyoid bone in a Turkish population

The hyoid bone is of great importance in forensic medicine practice and it has been studied for various forensic purposes; however, there have been few studies dealing with age and sex estimation using the hyoid bone. Using discriminant function analysis this study attempts to determine the utility of metric measurements and non-metric variations of the hyoid bone in terms of estimation of sex and age in a Turkish population and to find out differences with other populations by comparison of obtained results with the literature. For this purpose, the hyoid bones of 85 cadavers of known sex and age were extracted and 33 measurements were made with a computer program following photography with a camera. In addition to these measurements, the degree of fusion of the greater cornua to the hyoid corpus was examined. Out of 33 measurements, 18 showed significant sex-related differences.There was a sharp rise in hyoid bone fusion in both males and females after the age of 60 years. However, no marked sex-related differences were observed regarding fusion. The unstandardised canonical discriminant function was estimated based on three variables, and the correct classification ratio was 77.4% for males and 81.3% for females. The obtained findings suggest that measurements of hyoid bone can be used for sex estimation in the Turkish population.