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     

青少年骨关节X线片的骨龄研究

通过对11～16周岁女性及13～20周岁男性青少年左侧肩、肘、腕、髋、膝、踝关节拍摄X线片,进行了青少年的骨龄研究.根据骨骼生长发育的特点,提出了各大关节X线片判定骨龄的观察指标及分级标准,并使用SPSS软件包进行数据处理,建立了青少年利用6大关节骨骺愈合程度判定女性是否已满14周岁、男性是否已满16周岁或18周岁的判别分析方程.     

DNA analysis of the last Brazilian unknown soldier’s remains buried in Pistoia (Italy)

During World War II, many nations took part in the war. Among the supporters of the Alliance there was also Brazil. In August 1944, under the leadership of President Getúlio Vargas, Brazil declared war on Nazi Germany and took part in the Italian campaign by sending many troops to support the Allies in the Central Italy. Once the conflict was over, the deceased Brazilian soldiers were buried in Pistoia, a few kilometers from Florence. But only in 1960 the Brazilian government authorized the transfer of the dead soldiers to their homeland. Five years later, during the building of the Brazilian Military Votive Monument, still in the Pistoia cemetery, a last body was found but could not be identified: so he was buried as an “unknown soldier”. In December 2012, the Brazilian Embassy in Italy asked for performing forensic genetics analysis for identification purposes on the remains of this last unknown Brazilian soldier. After almost 70 years a complete short tandem repeats (STR) profile was obtained, useful for any relatives searching.Key points:

• Identification of the last Brazilian Unknown Soldier buried in Italy.
• DNA analysis on 70 years old skeletal remains.
• Brazilian soldier’s history during World War II.

     

头部受力后导致脑肿胀并发TNH的相互关系

目的探讨头部受力后导致脑肿胀并发TNH的相互关系。方法采用福尔马林固定的91例大脑标本,根据脑的损伤特点分三组分别作冠、矢及水平状切面,肉眼观察。结果发现脑损伤致脑肿胀后产生的TNH有三种类型,即前位疝65例(71.4％),后位疝5例(5.5％),及单、双侧全疝21例(23.1％)。其中16例全疝仍见以前位疝最显著。结论头部受力后易在额颞部发生前位疝。TNH虽与受力部位、受力机制有一定关系,但仍与受力强度、伤后存活时间关系密切。     

9例烧死的气管及肺的法医病理学组织学观察报告

<正> 实验性吸入性呼吸道损伤的组织学变化已有很多报告,烈火中烧死人体的气管及肺病理学变化的报告不多。我们对两起意外交通事故烧死的较完整的9例资料,其气管及肺脏尚可供组织学观察,现将结果报告如下。材料与方法 1986年2月,先后发生两起公共汽车燃烧事故,当场共烧死乘客12人,其中9例资料较