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.     

A Computational Framework for Age‐at‐Death Estimation from the Skeleton: Surface and Outline Analysis of 3D Laser Scans of the Adult Pubic Symphysis

In forensic anthropology, age‐at‐death estimation typically requires the macroscopic assessment of the skeletal indicator and its association with a phase or score. High subjectivity and error are the recognized disadvantages of this approach, creating a need for alternative tools that enable the objective and mathematically robust assessment of true chronological age. We describe, here, three fully computational, quantitative shape analysis methods and a combinatory approach that make use of three‐dimensional laser scans of the pubic symphysis. We report a novel age‐related shape measure, focusing on the changes observed in the ventral margin curvature, and refine two former methods, whose measures capture the flatness of the symphyseal surface. We show how we can decrease age‐estimation error and improve prior results by combining these outline and surface measures in two multivariate regression models. The presented models produce objective age‐estimates that are comparable to current practices with root‐mean‐square‐errors between 13.7 and 16.5 years.     

Full STR profile of a 67-year-old bone found in a fresh water lake

DNA extraction from and DNA typing of fresh water-exposed aged bone specimens poses a challenging task and is not very well examined. This study presents a new method to extract typable DNA from such problematic bone specimens. The procedure comprises low-heat drilling and cryogrinding, mild lysis conditions, and silica-column-based DNA cleaning. DNA quantity is assessed by quantitative PCR prior to short tandem repeat (STR) amplification. The procedure was employed with a 67-year-old tibia bone fragment recovered from a fresh water lake and succeeded to produce a full STR profile using the MPX-SP1 and MPX-SP2 mini-STR kits and a partial profile with 12 successfully amplified STRs using the Identifiler STR kit. The new method for the extraction of DNA from aged fresh water-exposed bone specimens presented herein was successfully applied to prepare DNA of sufficient quality and quantity to generate a full STR profile.     

Quantitative assessment of the percent fat in domestic animal bone marrow

Measurement of the amount of fat in femoral bone marrow can provide a quantitative assessment of the nutritional status of an individual animal. An analytical method is presented for quantitating the percent fat in bone marrow from three domestic species: bovine, canine, and equine. In this procedure, fat is extracted from bone marrow using pentane, and the percent fat recovered is determined gravimetrically. Based on analyses from adult animals (normal body condition scores), the average percentage of fat in the bone marrow was >80%. In cases in which animals have been diagnosed as emaciated or exhibit serous atrophy of fat (body scores of 1 or 2), the femoral bone marrow fat was less than 20%. In domestic animals, bone marrow fat analysis can be a useful, quantitative measure that, when used in conjunction with all other data available, can support a diagnosis of starvation or malnutrition.     

Evaluation of a Freezer Mill for Bone Pulverization prior to DNA Extraction: An Improved Workflow for STR Analysis

Traditional methods for bone pulverization typically generate heat, risking stability of DNA sample. SPEX? has developed cryogenic grinders which introduce liquid nitrogen to cool the sample and aid in the grinding process. In this study, the Freezer Mill 6970 EFM was used with two DNA extraction methods and routine downstream STR analysis procedures. DNA from as little as 0.1 g of bone powder was used to develop full STR profiles after freezer mill pulverization, and the method was reproducible. Further, no contamination was detected upon cleaning/reuse of the sample vials. There were no significant differences in DNA yield, STR alleles detected, or peak heights using the freezer mill as compared to traditional grinding, and successful DNA profiles were achieved from as low as 0.1 g of bone powder with this method. Overall, this work indicates that this cryogenic mill method may be used as a viable alternative to traditional tissue grinders.     

Scanning Electron Microscopy–Energy‐Dispersive X‐Ray (SEM/EDX): A Rapid Diagnostic Tool to Aid the Identification of Burnt Bone and Contested Cremains

This study investigates the use of Scanning electron microscopy–energy‐dispersive X‐ray (SEM‐EDX) as a diagnostic tool for the determination of the osseous origin of samples subjected to different temperatures. Sheep (Ovis aries) ribs of two experimental groups (fleshed and defleshed) were burned at temperatures of between 100°C and 1100°C in 100°C increments and subsequently analyzed with the SEM‐EDX to determine the atomic percentage of present elements. Three‐factor ANOVA analysis showed that neither the exposure temperature, nor whether the burning occurred with or without soft tissue present had any significant influence on the bone's overall elemental makeup (p > 0.05). The Ca/P ratio remained in the osseous typical range of between 1.6 and 2.58 in all analyzed samples. This demonstrates that even faced with high temperatures, the overall gross elemental content and atomic percentage of elements in bone remain stable, creating a unique “fingerprint” for osseous material, even after exposure to extreme conditions.     

The Survival of Gunshot Residues in Cremated Bone: An Inductively Coupled Plasma Optical Emission Spectrometry Study

Gunshot residue (GSR) has been sought and demonstrated on many types of material and with many techniques. Inductively coupled plasma optical emission spectrometry (ICP‐OES) could be a useful method on difficult substrates, but a systematic study on burnt material has never been performed. Hence, this study aims at evaluating the usefulness and reliability of this method on burnt samples. Sixteen adult bovine ribs (eight with soft tissues, eight totally skeletonized) were shot using two kinds of projectile (both 9 mm full metal‐jacketed or unjacketed). Then, every sample was led to complete calcination in an electric oven. The area of the gunshot entrance wound was swabbed and analyzed by ICP‐OES; the results were also correlated with a previously published parallel study by scanning electron microscopy (SEM) equipped with an SEM–energy dispersive X‐ray analyzer. ICP‐OES proved to be very sensitive and reliable even on degraded material and can be an appropriate nondestructive method for detecting residues on difficult and delicate substrates such as burnt bone.     

Application of the Iscan method to two- and three-dimensional imaging of the sternal end of the right fourth rib

Age determination is a major field of interest in physical and forensic anthropology. Among the different methods based on macroscopic skeletal study, the Iscan method, which analyzes the sternal end of the right fourth rib, is one of the most reliable. We applied the Iscan method to two- and three-dimensional multislice computed tomography (MSCT) reconstructions of the sternal end of the right fourth rib on 39 ribs. The intra-observer variability on MSCT reconstructions was good (gamma coefficient equal to 0.86; value of the Krippendorff's alpha reliability equal to 0.79); inter-observer variability on MSCT reconstructions was also good (gamma coefficient ranging from 0.82 to 0.88; value of the Krippendorff's alpha reliability ranging from 0.78 to 0.86). We demonstrated excellent agreement between the results of analysis of bone samples and those of the two- and three-dimensional images, in particular regarding bone projections, morphology of the pit and of its rim. The accuracy of age estimation did not significantly differ between the Iscan method applied to dry bones and the same method applied to MSCT images. Determination of the Krippendorff's alpha reliability coefficient for the inter-error method confirmed the agreement between phase estimations obtained with the two methods (ranging from 0.55 to 0.71). The real civil age was comprised in 21 cases out of 36 for assessment performed on dry bones and in 23 cases out of 36 on MSCT reconstructions, which represented 58.3% and 63.9%, respectively. Use of MSCT reconstructions in forensic anthropology offers many advantages: no bone preparation, no damage to bone material, and the possibility of application to living individuals.     

Utility of immunoassay in drug screening in skeletal tissues: sampling considerations in detection of ketamine exposure in femoral bone and bone marrow following acute administration using ELISA

Detection of ketamine exposure in skeletal tissues by automated enzyme-linked immunosorbent assay (ELISA) and gas chromatography with electron capture detection (GC-ECD) is described. Rats (n = 18) received 0, 15, 30, or 75 mg/kg ketamine hydrochloride acutely (i.p.), and were euthanized within 15 min or 1 h. Ketamine was extracted from ground femoral bone by methanolic incubation followed by liquid-liquid extraction (LLE), while marrow was homogenized in alkaline solution, and then underwent LLE. Extracts were analyzed by ELISA, and subsequently by GC-ECD following derivatization with trifluoroacetic acid anhydride. The effect of tissue type (i.e., diaphyseal bone vs. epiphyseal bone vs. bone marrow) on the immunoassay response was examined through determination of binary classification test sensitivity (S) and measurement of the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. The %DA varied significantly between different tissues examined under a given dose condition, and generally decreased in the order marrow > epiphyseal bone > diaphyseal bone, at all dose levels examined. Measured S values for marrow, epiphyseal bone, and diaphyseal bone were 100%, 77%, and 23%, respectively (75 mg/kg dose). These results suggest that the type of skeletal tissue sampled and position sampled within a given bone (diaphyses vs. epiphyses) are important parameters in drug screening of skeletal tissues.     

中国人四肢长骨骨性标志间各段长度与总长关系的分析

利用碎裂的长骨片段 ,计算长骨的总长。选择中国人长骨 5 0 0余例 ,取其骨性标志分段测量记录 ,将测得的数据输入计算机 ,用社会科学软件包 (SPSS)中全回归法进行统计学处理。在得出的 33个多元回归方程中 ,方程1、 9、 18、 2 6证明了骨性标志间各段长度与总长的正相关关系 ,其余 2 9个方程是在有选择的略去 1个变量和因长骨近端、远端或两端缺损使 1个或 1个以上变量缺失的情况下得出。根据碎裂长骨片段骨性标志间的长度测量值 ,应用多元回归方程 ,可计算出长骨的总长