Microscopic Age Estimation from the Anterior Cortex of the Femur in Korean Adults*

Abstract: The purpose of this study was to develop age‐predicting equations from the anterior cortex of the femur of Korean adults. Seventy‐two femoral samples (44 male and 28 female) were obtained from Korean cadavers and used to develop the equations. The thin sections (<100‐μm thick) were prepared by manual grinding; the sections were not decalcified and were stained with Villanueva bone stain reagent. Analysis of covariance showed no significant differences in age‐adjusted histomorphological variables between sexes. In stepwise regression analysis, osteon population density, average osteon area, and the most anterior cortical width were selected for an age‐predicting equation which produced a high regression correlation (R² = 0.789). The average Haversian canal area was not significantly related to age for any specimen.     

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.     

Differences in osteon banding between human and nonhuman bone

The objective of this paper is to compare patterns of osteon organization in human and nonhuman bone. A linear organization of Haversian systems in nonhuman bone, where osteons line up in rows, has been reported but has not been quantified. The present research provides a quantitative examination of this observation through a comparative analysis of the femoral midshaft from human and nonhuman bone. Femoral midshaft thin sections from 60 humans were compared to femoral midshaft sections from nine sheep and six miniature swine. The presence or absence of osteon banding was recorded and, if present, described. Results indicate that 2 out of 60 human sections and 5 out of 15 nonhuman sections exhibit osteon banding (chi2 = 9.46; p < 0.01). Further, the type of banding present in the human and nonhuman samples is easily distinguished, indicating that human and nonhuman bone can be distinguished where handing is present in this study.     

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.     

The Effect of Decalcifying Solutions on Hemosiderin Staining

Abstract: To determine whether routine decalcification may reduce the amount of stainable iron that is visible on tissue sections, samples of liver and lung tissue with excessive iron stores were placed in three standard decalcifying solutions (i) formic acid [33%], formaldehyde [4%], and NaCl [0.85%]; (ii) formic acid [30%], formaldehyde [4%], and water; and (iii) nitric acid [5%] for 24, 48, 72, and 96 h. After exposure to the decalcifying solutions, the tissues were stained with Perls stain. The slides were examined blind and the intensity of iron staining was scored semiquantitatively from 0 to 3+. The trend in all samples over the course of the experiment (96 h) was for reduction in the intensity of hemosiderin staining. As the amount of stainable hemosiderin in tissues may be significantly altered by decalcification, the absence of hemosiderin in tissues adjacent to a fracture site does not necessarily indicate that the injury was acute.     

Population-specific histological age-estimating method: a model for known African-American and European-American skeletal remains

Previously developed histological age-estimating methods have been based on samples lacking interpopulation variability. A comparison of age-associated rib histomorphometrics between an European-American sample and an African-American sample indicates that ethnicity can have a significant effect on osteon population density (OPD), osteon cross-sectional area (On.Ar), and relative cortical area (Ct.Ar/Tt.Ar). Based upon these findings, new histological age-predicting formulae are presented that are recommended when estimating age for African-American or European-American skeletal remains. A general formula that is applicable to remains of unknown ethnicity is also provided.     

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.     

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.     

丛状骨和骨单位带的种属特征及法医学应用价值

目的 探讨丛状骨和骨单位带的种属特征及其法医学应用价值。方法 人及猪、牛、羊、狗长骨中段横切磨片35张，在光学显微镜下观察骨组织中丛状骨和骨单位带的形态学特征。结果 丛状骨和骨单位带在猪、牛、羊、狗的密质骨中广泛存在，人骨组织中几乎不见；同种动物不同个体之间骨组织学结构也存在差异。结论 观察丛状骨和骨单位带是根据人骨鉴别种属的重要排除指标。     

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.     

A Method to Interpolate Osteon Volume Designed for Histological Age Estimation Research

Aging adult skeletal material is a crucial component of building the biological profile of unknown skeletal remains, but many macro- and microscopic methods have challenges regarding accuracy, precision, and replicability. This study developed a volumetric method to visualize and quantify histological remodeling events in three dimensions, using a two-dimensional serialized approach that applied circular polarizing microscopy and geographic information systems protocols. This approach was designed as a tool to extend current histological aging methodologies. Three serial transverse sections were obtained from a human femoral midshaft. A total sample size of 6847 complete osteons from the three sections was identified; 1229 osteons connected between all sections. The volume of all connected osteons was interpolated using ArcGIS area calculations and truncated cone geometric functions. Each section was divided into octants, and two random samples of 100 and of 30 connected osteons from each octant were generated. Osteon volume was compared between the octants for each random sample using ANOVA. Results indicated that the medial aspect had relative uniformity in osteon volume, whereas the lateral aspect showed high variability. The anterolateral–lateral octant had significantly smaller osteon volume, whereas the posterior–posterolateral octant had significantly larger osteon volume. Results also indicated that a minimum of 100 osteons is statistically more robust and more representative of normal osteon distribution and volume; the use of 30 osteons is insufficient. This research has demonstrated that osteon volume can be interpolated using spatial geometry and GIS applications and may be a tool to incorporate into adult age-at-death estimation techniques.     

Chronic Alcohol Abuse Leads to Low Bone Mass with No General Loss of Bone Structure or Bone Mechanical Strength,

Chronic alcohol abuse (CAA) has deleterious effects on skeletal health. This study examined the impact of CAA on bone with regard to bone density, structure, and strength. Bone specimens from 42 individuals with CAA and 42 individuals without alcohol abuse were obtained at autopsy. Dual‐energy X‐ray absorptiometry (DEXA), compression testing, ashing, and bone histomorphometry were performed. Individuals with CAA had significantly lower bone mineral density (BMD) in the femoral neck and significantly lower bone volume demonstrated by thinner trabeculae, decreased extent of osteoid surfaces, and lower mean wall thickness of trabecular osteons compared to individuals without alcohol abuse. No significant difference was found for bone strength and structure. Conclusion: CAA leads to low bone mass due to a decrease in bone formation but with no destruction of bone architecture nor a decrease in bone strength. It is questionable whether this per se increases fracture risk.     

Analysis of histomorphometric variables: Proposal and validation of osteon definitions

Histological analysis of bone tissue has been used to explore a variety of questions relating to age-at-death, habitual behaviors, health, and nutritional stress. Identification of intact and fragmentary osteons is of key interest to many researchers in these studies, yet the definitions of these features vary between researchers making cross-study comparisons problematic. Furthermore, histological variable definitions are often ambiguous or require subjective classifications by the observer. As a result, and as indicated by previous studies, observer error and misclassification of certain variables, namely intact and fragmentary osteons, can be significant. This study proposes new definitions for intact and fragmentary osteons that are designed to limit observer subjectivity and also explore efficacy of combining osteon types into one variable. A sample of 30 6th rib cross-sections from a modern forensic population was used to test the validity of the proposed definitions. Observations of intact osteon population density (OPD(I)) and fragmentary osteon population density (OPD(F)) were made by three observers for each cross-section. These observations were used to explore the interobserver error associated with the proposed definitions and determine if combining variables into one variable (OPD) mitigates persisting classification difficulties. Results indicate that the proposed definitions significantly reduce interobserver error and misclassification of intact and fragmentary osteons. However, the interobserver error associated with fragmentary osteons is still high. Evaluation of the variables independently indicates that combining variables has potential to reduce the predictive strength of an age estimation model and the ability to interpret age-related bone remodeling.     

基于X线的活体骨骼年龄评定方法及其他技术

国内外通常利用X线片观察骨骼继发性骨化中心的出现和骨骺闭合程度的影像学特征来推断年龄。其中较常用的方法有计数法、计测法、图谱法、评分法及计算机辅助评定骨龄法等。目前国外普遍采用TW评分法。国内也有该评分法的应用报道。主要应用于体育和儿科临床实践中。法医临床学实践中常用六大关节判别方程法推断骨龄和活体年龄推断。其他还有利用牙齿内天冬氨酸的消旋化、骨密度检测以及端粒末端DNA片段长度的变化等年龄推断方法。     

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.     

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.     

Intra- and inter-observer variation in histological criteria used in age at death determination based on femoral cortical bone

The microscopic method of age at death determination was introduced by Kerley in 1965. The method, which relies on the quantification of selected elements in cortical bone tissue, has been widely used, and several other researchers have modified or added to the method. Yet, very few studies have been carried out dealing with the intra- and inter-observer error. Furthermore, when such studies have been completed, the statistical tools for assessing variability have not been adequate. This study presents the results of applying simple quantitative statistics on several counts of microscopic elements as observed on photographic images of cortical bone, in order to assess intra- and inter-observer error. Overall, substantial error was present at the level of identifying and counting secondary osteons, osteon fragments and Haversian canals. Only secondary osteons can be reliably identified, precluding the use of osteon fragments and Haversian canals. The observers in this study included experienced and inexperienced users of the microscopic method, yet the variability was uniformly large for all observers, suggesting fundamental problems in definition and identification of the structural elements. Until more rigorous definitions of such elements have been agreed upon, the use of microscopical methods must be discouraged as a sole or uncontrolled method of evaluating age at death.     

Differentiating fragmented human and nonhuman long bone using osteon circularity

Distinguishing between human and nonhuman bone is important in forensic anthropology and archeology when remains are fragmentary and DNA cannot be obtained. Histological examination of bone is affordable and practical in such situations. This study suggests using osteon circularity to distinguish human bone fragments and hypothesizes that osteons will more closely resemble a perfect circle in nonhumans than in humans. Standard histological methods were used, and circularity was determined using an image analysis program, where circularity was controlled for by Haversian canal measurements. Homogeneity was first tested for multiple variables within human and nonhuman samples. No significant differences were found between human sexes (p = 0.657) or among nonhuman species (p = 0.553). Significant differences were found among intraskeletal elements of both humans (p = 0.016) and nonhumans (p = 0.013) and between pooled samples of humans and nonhumans (p < 0.001). Results of this study indicate that osteon circularity can be used to distinguish between fragmented human and nonhuman long bone.     

Quantification of Osteon Morphology Using Geometric Histomorphometrics

Many histological methods in forensic anthropology utilize combinations of traditional histomorphometric parameters which may not accurately describe the morphology of microstructural features. Here, we report the novel application of a geometric morphometric method suitable when considering structures without anatomically homologous landmarks for the quantification of complete secondary osteon size and morphology. The method is tested for its suitability in the measurement of intact secondary osteons using osteons digitized from transverse femoral diaphyseal sections prepared from two human individuals. The results of methodological testing demonstrate the efficacy of the technique when applied to intact secondary osteons. In providing accurate characterization of micromorphology within the robust mathematical framework of geometric morphometrics, this method may surpass traditional histomorphometric variables currently employed in forensic research and practice. A preliminary study of the intersectional histomorphometric variation within the femoral diaphysis is made using this geometric histomorphometric method to demonstrate its potential.     

Histological Determination of the Human Origin of Bone Fragments

Abstract:  A frequently encountered task in the forensic scenario is verification of the human origin of severely degraded fragments of bone. In these cases histological methods which consider osteon size and morphology can prove to be useful. The authors in the present study verify the applicability of published algorithms to flat and subadult bones from human, dog, cat, cow, rabbit, sheep, pig, chicken, quail, and turkey samples. Metric analysis was performed on 2031 Haversian canals. Analyses carried out on human samples confirmed a success rate of around 70% on long adult bones; however the percentage of wrong answers was particularly high in the case of newborns and older subadults as well as on flat bones in general. Results therefore suggest that such regression equations should be limited only to bone fragments from long adult bones.