Assessment of histomorphological features of the sternal end of the fourth rib for age estimation in Koreans

The aim of this study was to assess the histomorphological features of the fourth rib and to develop age-predicting equations for Koreans. Sixty-four rib samples (36 males and 28 females) obtained from forensic cases were used for developing equations. Two thin sections (<100-microm thick) per sample were prepared by manual grinding. Multivariate analysis of covariance revealed statistically significant differences in age-adjusted histomorphological variables between sexes. Using stepwise regression analysis, osteon population density and average osteon area were correlated with unknown sex (r2 = 0.826), and sex plus two histomorphological variables provided the best results for an age-predicting equation given the assumption of knowing the sex of a specimen (r2 = 0.839). Average Haversian canal area had little influence on age estimation for male or female samples, and relative cortical area was not significantly related to age for any specimen.     

New Formulas to Estimate Age at Death in Maya Populations Using Histomorphological Changes in the Fourth Human Rib*

Abstract: This study develops new histomorphological algorithms for Maya populations’ human ribs and tests the applicability of published algorithms. Thin sections from the fourth rib of 36 individuals of known age were analyzed under polarized light microscopy. Osteon population density (OPD, the concentration of intact and fragmented osteons per mm²), cortical area (CA), and osteon size (OS) were recorded. Seven algorithms were calculated, using all combinations of variables, and compared to the performance of published formulas. The OPD‐based formulas deviate from the known age 8.7 years on average, while those from OS and CA deviate between 10.7 and 12.8 years. In comparison, our OPD‐based algorithms perform better than the one by Stout and Paine and much better than Cho et al. In conclusion, algorithms should be developed using OPD for different ethnic groups; although Stout and Paine’s can be used for Maya and maybe Mesoamerican individuals.     

Rib Variation at Multiple Locations and Implications for Histological Age Estimation*

Existing histological age estimation methods using the rib were developed mainly from the midshaft; however, in forensic practice, uncertainty of sampling location often arises due to fragmented or previously sampled ribs. The potential for error increases when sampling location is uncertain and utilizing a section beyond the midshaft (either anterior or posterior) may result in erroneous age estimates. Additionally, there is debate within the field regarding the minimum number of sections needed for accurate age estimation. The aim of this research is to determine the importance of the midshaft distinction for age-at-death assessment and the necessity of analyzing serial sections by evaluating histological variables at sampling locations along the length of the rib. Three seriated histological sections at three sampling locations (anterior, midshaft, and posterior) were obtained from sixth ribs of ten postmortem human subjects. Cortical area (Ct.Ar) and osteon population density (OPD) were collected from each section (n = 90). Significant differences were determined in Ct.Ar between sampling locations, demonstrating the variation present along the length of the rib. A comparison of OPD at sampling locations revealed significant differences, suggesting that sampling site is critical to accurate age estimates. When sampling location is uncertain, a more anterior section should be taken. Analysis of serial sections within locations revealed no significant differences in OPD or Ct.Ar, supporting the practice of collecting data from one section for age estimation. While an age estimate can be achieved through the analysis of one section, best practice suggests reading two sections to capture intraindividual variation.     

Estimation of age from the femur of Japanese cadavers

The purpose of this study is to estimate the age of cadavers by histomorphometry of the femur. Seventy-two Japanese males ranged from 43 days to 92 years old and 26 females ranged from 2 to 88 years old were used. The thickness of sections was adjusted at 50 to 70 microns by grinding with sand paper. The sections were not decalcificated. They were stained with Villanueva's bone staining powder and with thionin dye. Microradiographs of the sections were obtained by the soft X-ray apparatus. The area, maximum and minimum diameter, and perimeter of the perfect osteon and Haversian canal were measured. In addition, the type II osteon number, osteon fragment number, and area of triangle were also determined. All these parameters were examined by an image analyzer. The parameters of the osteon showed high correlation coefficient with age (magnitude of r > 0.77), while those of the Haversian canal were low (magnitude of r < 0.11). All parameters were subjected to multiple regression analysis for producing a multiple regression equation of age estimation. For the stepwise selecting method, the perimeter of osteon, maximum length of the Haversian canal and osteon fragment number were selected for the equation. Their multiple r2 and standard error of estimation were 0.8874 and 6.39, respectively. For the forward selection method, in addition to the above items, three parameters, the maximum length of Haversian canal, triangle area, osteon fragment number were selected. Their multiple r2 and standard error of estimation were 0.9484 and 4.884, respectively. Bone staining was useful to clarify the demarcation between osteon and fragment, leading to an increase in the accuracy of age estimation. However, the entire range from birth to 90 years was difficult to cover for precise age estimation.     

A Test of the Passalacqua Age at Death Estimation Method Using the Sacrum

A test of the accuracy of the Passalacqua (J Forensic Sci, 5, 2009, 255) sacrum method in a forensic context was performed on a sample of 153 individuals from the J.C.B. Grant Skeletal Collection. The Passalacqua (J Forensic Sci, 5, 2009, 255) method assesses seven traits of the sacrum using a 7‐digit coding system. An accuracy of 97.3% was achieved using the Passalacqua (J Forensic Sci, 5, 2009, 255) method to estimate adult skeletal age. On average each age estimate differed by 12.87 years from the known age. The method underestimated the age of individuals by an average of 4.3 years. An intra‐observer error of 6.6% suggests that the method can be performed with precision. Correlation and regression analysis found that the sacral traits used in the Passalacqua (J Forensic Sci, 5, 2009, 255) method did not have a strong relationship with age or an ability to strongly predict age. Overall, the method was not practical for use in a forensic context due to the broad age ranges, despite the high accuracy and low intra‐observer error.     

Forensic Dental Age Estimation by Measuring Root Dentin Translucency Area Using a New Digital Technique

Dentin translucency measurement is an easy yet relatively accurate approach to postmortem age estimation. Translucency area represents a two‐dimensional change and may reflect age variations better than length. Manually measuring area is challenging and this paper proposes a new digital method using commercially available computer hardware and software. Area and length were measured on 100 tooth sections (age range, 19–82 years) of 250 μm thickness. Regression analysis revealed lower standard error of estimate and higher correlation with age for length than for area (R = 0.62 vs. 0.60). However, test of regression formulae on a control sample (n = 33, 21–85 years) showed smaller mean absolute difference (8.3 vs. 8.8 years) and greater frequency of smaller errors (73% vs. 67% age estimates ≤ ±10 years) for area than for length. These suggest that digital area measurements of root translucency may be used as an alternative to length in forensic age estimation.     

Dietary health does affect histological age assessment: an evaluation of the Stout and Paine (1992) age estimation equation using secondary osteons from the rib

An age at death estimation equation that uses rib histological variables presented by Stout and Paine was used to evaluate a skeletal population of individuals with a known age at death and cause of death from either malnutrition or the niacin deficiency disease pellagra. The sample was comprised of 26 autopsied black South Africans. Histological analysis of mounted thin sections involved the microscopic measurement of cortical area and a count of the number of intact and fragmentary secondary osteons for the entire cross-section of the rib. Rib osteon population density values were then calculated for each case. It was found that this equation under-aged individuals on average by 29.2 years. Overall, secondary osteon size and Haversian canals tended to be larger than expected, while cortical bone area was less when compared with a control population. The implications of these findings are critical given that many of the skeletal remains examined by forensic anthropologists come from marginalized backgrounds, including malnutrition. This research suggests that measurements based on healthy cases may not be useful in an analysis of individuals with poor diet and health. It is argued that new standards for histological age assessment methods need to be created that account for variation in the health status of individuals examined by forensic anthropologists.     

Age at Death Estimation in Adults by Computer-Assisted Histomorphometry of Decalcified Femur Cortex

Abstract:  In 1965, Kerley pioneered histomorphometry of bone as an aging method. The technique has been modified by several authors, and some have used computer-assisted image analysis. Undecalcified bone sections used in these methods are obtained with a diamond wafer saw or by grinding the sections manually or automatically with abrasive paper. In the present study, we examined the application of histomorphometry to decalcified bone sections, routinely obtained in every pathology lab, from paraffin blocks cut with a standard microtome. This study was divided into two parts: in the first, we tested different decalcifying methods to determine the most appropriate for femoral bone; in the second part, we used computer-assisted histomorphometry to estimate age at death in 29 samples of femoral bone. We measured intact osteon density (N.On), fragmented osteon density (N.On.Fg) and percentage of lamellar bone surface per unit area (Lm.B.Ar) in the cortex of the femoral midshaft, on four or 20 fields per section. We found that 20% nitric acid solution at room temperature proved to be the best decalcifying method, with a mean decalcification duration of 1 week. Fragmented osteon density was found to be the morphometric feature most closely correlated with age, followed by intact osteon density; Lm.B.Ar. did not increase accuracy. The best accuracy (4.1 ± 3.5 years) was obtained for individuals under the age of 70 when measurements of 20 fields were used for the analysis. For all individuals, the inaccuracy was 6.1 ± 6.2 years and 8.1 ± 8 years, with 20 and four fields respectively. The present study shows that decalcification of bone sections can be used for age estimation at death. This procedure is particularly useful in case of mass disaster as it is easily done in any pathology department.     

A Reliable Regression‐Based Approach for Reassociating Human Skeletal Elements of the Lower Limbs from Commingled Assemblages

Accurate sorting of commingled human remains comprises a fundamental requirement for all further anthropological analyses. The lower limb bones are particularly important for reconstructing biological profiles. This study introduces a metric technique for sorting these elements using eight standard anthropological measurements and 222 adult individuals from Greece. The bones utilized were the os coxae, the femora, the tibiae and the tali. Simple regression analyses were used to develop functions for reassociating articulating bones, providing strong correlations (r = 0.74–0.95, p‐value <0.05) and high coefficients of determination (r²=0.54–0.91). Blind tests demonstrated that combining metric and morphoscopic techniques provides an excellent sorting accuracy for the hip and knee joints (ten of ten individuals), allowing for a reliable reassociation between a sex and age indicator (os coxae) and a body size indicator (femur). Overall, these results indicate the high value of metric methods in sorting commingled human remains.     

An Evaluation of Statistical Methods for the Determination of Age of Death Using Dental Root Translucency and Periodontosis

Abstract: Lamendin and colleagues (1992) proposed to assess age‐at‐death from root translucency and periodontosis. Several teeth from the same individual were included in their study. In our work, we evaluate the reliability of their formulas without introducing this bias. Our sample was constituted of 214 individuals (114 males and 100 women) selected from the Terry collection (U.S.A.). The R² between age and both indicators is equal to 0.33 and 0.08 (p < 0.001). Based on linear regression, the mean of standard error for individual age prediction was equal to 13.67 years, and the mean interval length is equal to 53.89 years. Multinomial logistic regression and Bayesian approach failed to give satisfactory results when classifying the individuals in age categories. Therefore, the use of root translucency and periodontosis may lead to incorrect age‐at‐death assessment, and it is thus necessary to complement this approach with other techniques to assess age‐at‐death.     

Histological age prediction from the femur in a contemporary Dutch sample. The decrease of nonremodeled bone in the anterior cortex

This paper presents an uncomplicated and minimally invasive method for age-at-death determination in a contemporary Dutch (West European) population, by modifying the approach of assessment based on the age-related remodeling of bone tissue. In contrast to the usual "osteon count," a "non-remodeled tissue count" is undertaken. To optimize the method, proper zeroing of the polarization filter set of the microscope is essential. Instructions for setting the filters are given. A sample of femoral shaft segments totaling 162 individuals with ages ranging from 15 to 96 years is analyzed. Subperiosteal quantitative assessments are recorded at the most anterior point of the femoral shaft and also at points 25 degrees to the left and to the right of that point. Interobserver agreement in the assessments shows an acceptable degree of correlation. Bone remodeling with age does not progress in a linear, but in a curvilinear manner. Dependence of predicted age on nonremodeled surface counts in the analyzed areas of the anterior cortex of the femur appears to be significant. A set of regression equations is given. Sex can be ignored in age prediction. The small but statistically significant dependence of predicted age on cadaver length corresponds with the present strong secular increase in stature in the Netherlands. A concise catalogue with micrograph examples for every 10-year period in life is available upon request.     

The Reliability of the Greulich and Pyle Atlas When Applied to a Modern Scottish Population

This study examines the reliability of age estimation utilizing the Greulich and Pyle atlas in relation to a modern Scottish population. A total of 406 left‐hand/wrist radiographs (157 females and 249 males) were age‐assessed using the Greulich and Pyle atlas. Analysis showed that there was a strong correlation between chronological age and estimated age (females R² = 0.939, males R² = 0.940). When age groups were broken down into year cohorts, the atlas over‐aged females from birth until 13 years of age. The pattern for males showed that the atlas under‐estimated age until 13 years of age after which point it consistently over‐aged boys between 13 and 17 years of age. This study showed that the Greulich and Pyle atlas can be applied to a modern population but would recommend that any analysis takes into account the potential for over‐ and under‐aging shown in this study.     

Stature Estimation from Various Foot Dimensions Among Slovak Population

The foot measurements are important in forensic field as they can be used as body height predictors for an individual. The morphology of human feet shows the variations and therefore the aim of this study was to find the relation between stature and foot measurements and to generate population‐specific equations. We measured the stature and bilateral foot measurements of 71 volunteers ranging in age between 18 and 27 years using standard measurements technique. The results revealed nonsignificant bilateral differences in all sex groups (p < 0.05). Sex differences were found to be significant in all foot measurements (p < 0.001). The highest significant and positive correlation coefficients with stature were observed for foot length in males (r = 0.759), in females (r = 0.722), and in the sex‐mixed group (r = 0.865). According to the nonsignificant sex effects on the relation between stature and foot dimensions, the regression equations were computed only for the pooled mixed‐sex group. Our study provides equations that can help to estimate stature from foot measurements among Slovaks.     

Qiagen's Investigator™ Quantiplex Kit as a Predictor of STR Amplification Success from Low‐Yield DNA Samples,

Qiagen's Investigator? Quantiplex kit, a total human DNA quantitation kit, has a 200‐base pair internal control, fast cycling time, and scorpion molecules containing a covalently linked primer, probe, fluorophore, and quencher. The Investigator? Quantiplex kit was evaluated to investigate a value under which complete short tandem repeat (STR) failure was consistently obtained. Buccal swabs were extracted using the Qiagen QIAamp^® DNA Blood Mini Kit, quantified with the Investigator? Quantiplex kit using a tested half‐volume reaction, amplified with the ABI AmpFlSTR^® Identifiler kit, separated on the 3100Avant Genetic Analyzer, and data analyzed with GeneMapper^® ID v.3.2. While undetected samples were unlikely to produce sufficient data for statistical calculations or CODIS upload (2.00 alleles and 0.82 complete loci on average), data may be useful for exclusionary purposes. Thus, the Investigator? Quantiplex kit may be useful for predicting STR success. These findings are comparable with previously reported data from the Quantifiler? Human kit.     

Bone Area Histomorphometry

Quantifying the amount of cortical bone loss is one variable used in histological methods of adult age estimation. Measurements of cortical area tend to be subjective and additional information regarding bone loss is not captured considering cancellous bone is disregarded. We describe whether measuring bone area (cancellous + cortical area) rather than cortical area may improve histological age estimation for the sixth rib. Mid‐shaft rib cross‐sections (n = 114) with a skewed sex distribution were analyzed. Ages range from 16 to 87 years. Variables included: total cross‐sectional area, cortical area, bone area, relative bone area, relative cortical area, and endosteal area. Males have larger mean total cross‐sectional area, bone area, and cortical area than females. Females display a larger mean endosteal area and greater mean relative measure values. Relative bone area significantly correlates with age. The relative bone area variable will provide researchers with a less subjective and more accurate measure than cortical area.     

Does Mirror Imaging a Radiograph Affect Reliability of Age Assessment Using the Greulich and Pyle Atlas?*

Abstract: Age estimation is routinely undertaken by comparing radiographs of the individual in question to published reference samples of individuals of known age. This study examines the reliability of age estimation utilizing the Greulich and Pyle atlas in relation to both left‐ and right‐hand/wrist radiographs and explores whether reversing right‐hand/wrist radiographs, so that they are in the same anatomical orientation as those images used in the atlas affects reliability. A total of 403 left‐hand/wrist radiographs and 415 right‐hand/wrist radiographs were age assessed using the Greulich and Pyle atlas. Analysis showed that there is no significant loss in reliability when radiographs of the right hand (women R² = 0.887 and men R² = 0.907) are utilized instead of the left (women R² = 0.939 and men R² = 0.940) or when they are assessed as mirror images to those printed in the reference atlas (reversed female left hand R² = 0.929 and reversed male left hand R² = 0.931).     

Quantification of Pubic Symphysis Metamorphosis Based on the Analysis of Clinical MDCT Scans in a Contemporary Malaysian Population

Forensic age estimation methods are biased to sex and population; in general, accuracy is reduced when applied to foreign populations. This study assessed the accuracy of the Suchey–Brooks method in contemporary Malaysian individuals and aimed to formulate population‐specific standards. Multi‐detector computed tomography scans of 355 individuals (165 male; 190 female) of 15–83 years of age were reconstructed using 3D‐volumetric rendering in RadiAnt. Pubic symphyseal phase, bias, inaccuracy, and percentage correct age classifications are examined. Transition analysis was used to develop age estimation standards. High observer agreement (κ = 0.763–0.832) and a positive relationship between age and pubic symphyseal phase (r = 0.884–0.90) were demonstrated. Mean inaccuracies were 8.62 and 8.95 years for males and females, respectively; overall correct classification was 97.8%. Transition ages between phases in males were 18.79, 23.29, 28.85, 43.64, and 61.15 years; in females, the corresponding data were 19.77, 22.53, 32.62, 41.85, and 57.39 years.     

Internal Morphological Analysis for Age Estimation of Blow Fly Pupae (Diptera: Calliphoridae) in Postmortem Interval Estimation*

Abstract: Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau‐Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot‐water‐killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot‐water‐killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.     

Identification Using Frontal Sinus by Three‐Dimensional Reconstruction from Computed Tomography*

Abstract: Radiographs and computed tomography (CT) images have been increasingly applied and accepted in forensic sciences. Along with radiographs and CT images, the frontal sinus is often used for the individual identification because of its unique and unchangeable characteristics. The purpose of this study is to define the usefulness of three‐dimensional (3‐D) images of the frontal sinus for identification. CT images from 119 Korean cadavers were built up for 3‐D reconstruction and surveyed with 15 measurements. The total volume of the sinus, some nonmetric characteristics, and the bilateral asymmetry index in men differed significantly from those in women (p < 0.05). The digit codes, six sections and 10‐digit number, were almost able to accurately identify individuals (98%). This study showed a statistical difference between the sexes and classified the fused and prominent middle sinuses for the first time. This proposed method for identification is more accurate than those used in other studies.     

Estimation of age at death by tibial osteon remodeling in an autopsy series

Estimation of age at death by histological methods in tibiae has been found to be inaccurate in individuals less than 55 years of age. This study describes reasons for the inaccurate age estimates and provides a new regression equation for age estimation. Bone cores were removed from tibiae in 53 individuals at autopsy ranging in age from 17 years to 53 years of age; 48 males and 5 females. The sample contained 41 whites and 12 blacks. Variables ascertained from each core were: cortical thickness, cortical bone density, secondary osteon number, area, and perimeter, and Haversian canal area and perimeter. The results showed significant differences in secondary osteon number, area, and perimeter between groups aged less than and greater than 35 years of age. Multiple linear regression analysis indicated osteon number to be the best predictor of age in this sample aged less than 55 years of age. Age at death was accurately estimated for 11 additional forensic cases using osteon number in tibiae. No significant histological differences were observed between males and females and between blacks and caucasians.