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.     

利用中国汉族成人股骨上端CR片特征判定年龄

目的探讨中国汉族成人股骨上端CR片的特征随年龄变化的规律,以推断年龄。方法 277例中国汉族成人骨盆正位CR片,其中男性139例,年龄范围18-83岁,女性138例,年龄范围18-85岁,选取股骨上端的股骨头、股骨颈、大转子内的骨小梁及股骨上端髓腔高度四项指标,观察不同年龄各指标的变化进行统计分析。结果成人股骨头、股骨颈骨小梁变化和股骨上端髓腔高度是影响45岁以下年龄变化的重要因素,股骨大转子影响较小。四项指标对45岁以上年龄组均是影响年龄变化的重要因素。利用以上指标建立的回归方程能很好的反映成人股骨上端的增龄性变化,预测骨龄较为准确。结论利用成人股骨头、股骨颈、股骨大转子内骨小梁的改变及股骨上端髓腔高度变化可推断年龄。     

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.     

How “Standardized” is Standardized? A Validation of Postcranial Landmark Locations

Since the mid‐1990s, osteometrics have been a major aspect in forensic anthropology. However, recent evidence has shown that practitioners have been using differing points to establish certain measurements; namely those involving the clavicle, scapula, ulna, femur, and tibia. Engaging 61 practitioners, this study examined and confirmed how extensive this discord is. The highest consistency rate among practitioners was the scapular breadth (62.3%), followed by the femoral anteroposterior subtrochanteric diameter (55%). All other levels fell below these, particularly the physiological length of the ulna which was the most inconsistently measured bone. Furthermore, these low consistency rates yielded percent mean differences between two and 20% of the measurement length, with ranges averaging 14 mm. In light of these results, it is recommended that the field take steps to improve the standardization of such problematic measurements, including reexamining all measurements currently listed in osteometric texts, reassessing the utility of each, and reissuing a comprehensive guide.     

Experimental adipocere formation: implications for adipocere formation on buried bone

Adipocere, or grave wax (adipo = fat, cere = wax), is a distinctive decomposition product composed primarily of fatty acids (FA) and their alkali salts. FA result from the bacterial enzymatic hydrolysis of body fats. Reactions with ammonia and alkali metals originating from body fluids and pore waters of the depositional environment produce alkali salts of FA (soap). Adipocere formation is generally associated with burial of corpses with ample adipose tissue available. No indications that adipocere can form on defleshed remains have been presented in the literature. At the termination of a long-term bone diagenesis experiment, several samples were found to possess growths of an unknown compound. Gas chromatography-mass spectrometry confirmed that the growths are adipocere. The results herein reveal that adipocere can indeed form on defleshed bones under the right conditions and that even residual adipose and lipids in defleshed bones are sufficient to produce adipocere growth on the surfaces of bone.     

中国汉族成人肱骨近端CR片特征与年龄的相关性

目的探讨中国汉族成人肱骨近端CR片的特征随年龄变化的规律,以推断年龄。方法 210例中国汉族成人肩关节CR片,其中男性124例,年龄范围18~83周岁,女性86例,年龄范围18~60周岁,选取与年龄变化关系密切的肱骨骨髓腔高度、肱骨近端骨骺骨小梁变化、肱骨大结节内骨小梁变化、锁骨骨髓腔肩峰侧骨小梁变化4项指标,观察不同年龄各指标的变化进行统计分析。结果四项指标均是影响年龄变化的重要因素。肱骨近端骨骺骨小梁变化、锁骨骨髓腔肩峰侧骨小梁变化对年龄的预测影响较大,肱骨骨髓腔高度、肱骨大结节内骨小梁变化稍小。利用以上指标建立的回归方程能很好的反映成人肱骨的增龄性变化,预测骨龄较为准确。结论利用成人肱骨骨髓腔高度、肱骨近端骨骺骨小梁变化、肱骨大结节内骨小梁变化、锁骨骨髓腔肩峰侧骨小梁变化可推断年龄。     

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.     

Use of a Gelatin‐based Consolidant to Preserve Thermally‐Altered Skeletal Remains,

Thermally altered skeletal remains can be very fragile and fragmented and are typically further fragmented or even destroyed when handled; recovery of such remains from a scene can therefore be extremely challenging. There are few recommendations and no generally accepted practices for preserving burned bone for recovery and transport. Here, we test whether the application of a gelatin‐based consolidant at the scene can preserve thermally altered bone in the condition and relative anatomical position in which it was discovered. A solution of Knox^® brand gelatin and water was applied to burned pig mandibles using a spray bottle. Qualitative and quantitative analysis indicates that the application of the consolidant significantly decreased fragmentation as compared to untreated controls (p < 0.05), with most of the treated mandibles remaining completely intact after recovery and transport to a secondary location. In addition to the effectiveness for preservation, the method is also easy to apply, inexpensive, and reversible.     

Plasma versus bone marrow desipramine: a comparative study

Correlation between plasma and bone marrow tricyclic antidepressants has not been studied before. Two groups of rabbits were given 10 and 20 mg of desipramine/kg body weight, respectively. Desipramine was administered to the animals once daily by mouth for 5 days. On the fifth day the animals were sacrificed and blood and bone marrow samples were collected and analyzed using a high performance liquid chromatographic (HPLC) method. Data showed that a correlation exists between bone marrow and blood desipramine. The bone marrow desipramine concentration increased as its blood levels increased. The average ratio of bone marrow to blood desipramine +/- S.D. (standard deviation) in both dosage groups was 37.2 +/- 4.46 with a range of 30.99-44.82. This investigation is promising and shows that bone marrow could be used as an alternative tissue in the absence of a suitable blood sample.     

A Case of Insect Colonization Before the Death

Forensic entomology is a branch of forensic science in which insects are used as evidence in legal investigations relating to humans, domestic animals and wildlife. One of the theoretical pillars on which the discipline is based concerns the fact that flies colonize a body after death. However in cases of myiasis, maggots are present before death, with consequences in the correct estimation of the minimum postmortem interval (mPMI). We report here the case of a woman, largely colonized by fly larvae, who has lain alive in her garden for four days prior to being rescued. Larvae were found on the conjunctivae, the bronchi, the rectum and vagina. The woman's death, two months later, was caused by tetanus. The consequences of myiasis on mPMI estimation are here discussed. In fact, despite she was still alive larvae, indicated and estimated age of 1.5–2.5 days, based on environmental and body temperature.     

Do bone elasticity and postmortem interval affect forensic fractographic analyses?

Forensic fractographic features of bone reliably establish crack propagation in perimortem injuries. We investigated if similar fracture surface features characterize postmortem fractures. Experimentally induced peri- and postmortem fractures were used to assess if fractographic features vary as bone elasticity decreases during the postmortem interval (PMI). Thirty-seven unembalmed, defleshed human femoral shafts from males and females aged 33–81 years were fractured at varying PMIs with a drop test frame using a three-point bending setup and recorded with a high-speed camera. Vital statistics, cause of death, PMI length, temperature, humidity, collagen percentage, water loss, fracture energy, and fractography scores were recorded for each sample. Results showed that fractographic features associated with perimortem fractures were expressed in PMIs up to 40,600 accumulated degree hours (ADH), or 60 warm weather days. Hackle was the most consistently expressed feature, occurring in all fractures regardless of ADH. The most variable characteristics were wake features (78.4%) and arrest ridges (70.3%). Collagen percentage did not correlate strongly with ADH (r = −0.04, p = 0.81); however, there was a strong significant correlation between ADH and water loss (r = 0.74, p < 0.001). Multinomial logistic regression showed no association between fractographic feature expression and ADH or collagen percentage. In conclusion, forensic fractographic features reliably determine initiation and directionality of crack propagation in experimentally induced PMIs up to 40,600 ADH, demonstrating the utility of this method into the recent postmortem interval. This expression of reliable fractographic features throughout the early PMI intimates these characteristics may not be useful standalone features for discerning peri- versus postmortem fractures.     

Detection of acute fentanyl exposure in fresh and decomposed skeletal tissues part II: The effect of dose–death interval

The effects of dose–death interval on the detection of acute fentanyl exposure in fresh and decomposed skeletal tissues (marrow and bone), by automated enzyme-linked immunosorbent assay (ELISA) are described. Rats (n = 14) were administered fentanyl acutely at a dose of 0 (n = 2) or 60 μg/kg (n = 12) by intraperitoneal injection, and euthanized within 20, 45, 135, or 225 min. Femora and tibiae were extracted from the fresh corpses and marrow was isolated from the femoral and tibial medullary cavities. The remains were then allowed to decompose outdoors to the point of complete skeletonization, and vertebrae, pelvi and miscellaneous (humeri and scapulae) were recovered for analysis. In all cases, bones were cleaned in alkaline solution and then ground into a fine powder. Marrow was homogenized in alkaline solution. Fentanyl was extracted from ground bone by methanolic extraction. Extracts were adjusted to pH 6 and analyzed by ELISA. Perimortem heart blood was also collected and diluted in phosphate buffer prior to screening by ELISA. The effect of tissue type on ELISA response was examined through determination of binary classification test sensitivity and the relative decrease in absorbance (%DA, drug-positive tissues vs. drug-free controls) in each tissue type. Overall, the %DA varied significantly between extracts from different skeletal tissues at a given dose–death interval, according to the general order of marrow > decomposed bone > fresh bone. Binary classification test sensitivity values for fentanyl in marrow, fresh epiphyseal (femoral and tibial) bone, fresh diaphyseal (femoral and tibial) bone, decomposed vertebrae, decomposed pelvic bone, and decomposed miscellaneous bone were 67–100%, 0–33%, 0–33%, 0–67%, 0–67% and 0–33%, respectively, over all dose–death intervals. Although group mean %DA values showed a strong negative correlation with dose–death interval in marrow, fresh epiphyseal bone, decomposed vertebrae, pelvic and miscellaneous bone (r = ?0.989, ?0.930, ?0.955, ?0.903, and ?0.974, respectively), the high variability in both fresh and decomposed bone precluded differentiation of the dose–death intervals based on %DA value alone. Overall, the results suggested that the type of skeletal tissue sampled may not be as important as the amount of residual marrow remaining in skeletonized remains.     

Comparative study of ethanol levels in blood versus bone marrow,vitreous humor,bile and urine

Post-mortem ethanol levels in blood were compared to corresponding levels in rib bone marrow, vitreous humor, urine and bile. In forensic toxicology, a good correlation between blood and a tissue or body fluid is needed to estimate a blood alcohol concentration when blood is unavailable or contaminated. In this study, direct injection and headspace gas-chromatographic techniques were employed to quantitate the ethanol concentrations. Comparable findings by these two techniques showed a reproducibility of results. When the determined bone marrow ethanol levels were corrected for the lipid fraction, a consistent correlation could be established between ethanol levels in blood and bone marrow. The relationship (linearity and ratio range) between ethanol levels in blood and corrected levels in bone marrow was better than that between blood and vitreous humor, bile or urine. This study showed that blood ethanol levels can be predicted by extrapolating the corrected rib bone marrow ethanol level.     

Determining the human origin of fragments of burnt bone: a comparative study of histological, immunological and DNA techniques.

In situations where badly burnt fragments of bone are found, identification of their human or non-human origin may be impossible by gross morphology alone and other techniques have to be employed. In order to determine whether histological methods were redundant and should be superseded by biomolecular analyses, small fragments of artificially burnt bone (human and non-human) were examined by quantitative and standard light microscopy, and the findings compared with newer biomolecular analyses based on identifying specific human albumin by ELISA and amplifying human mitochondrial DNA by PCR. For quantitative microscopy, reference data were first created using burnt bones from 15 human and 20 common domestic and farm animals. Measured osteon and Haversian canal parameters were analysed using multivariate statistical methods. Highly significant differences were found between values for human and non-human bone, and a canonical discriminant function equation was derived, giving a predicted correct classification of 79%. For the main study, samples of cortical bone were taken from three fresh cadavers, six human skeletons and ten freshly slaughtered animals and burnt by exposure to temperatures ranging from 800 to 1200 degrees C; charred fragments of human cortical bone from two forensic cases were also tested. Quantitative microscopy and canonical discriminant function gave the correct origin of every sample. Standard microscopy falsely assigned burnt bone from one human skeleton and one forensic case to a non-human source, but otherwise gave correct results. Human albumin was identified in five individuals, including one of the forensic cases, but mitochondrial DNA could not be amplified from any of the human bone. No false positive test results were seen with either biomolecular method; and human albumin and mitochondrial DNA were correctly identified in all unburnt control specimens. It was concluded that histological methods were not redundant and that quantitative microscopy provided an accurate and consistent means of determining the human or non-human origin of burnt bone and was more reliable than standard microscopy or the newer immunological and DNA techniques tested here.     

Sex determination using nonmetric characteristics of the mandible in Koreans

The mandible is the largest and hardest facial bone and retains its shape better than other bones in the forensic and physical anthropologic field. The mandible can be used to distinguish among ethnic groups and between sexes. We examined the morphological characteristics of the mandibles of 102 Koreans of either sex. Of 13 nonmetric items of the mandible, the characteristic that best allowed the sexes to be distinguished was the contour of the lower border of the mandible: rocker-shaped mandibles predominated in males (68.1%), whereas most females (84.6%) exhibited a straight mandible. In addition, the mental region was shaped differently between the sexes: the shape of the chin in most males was generally bilobate or square (91.7%), whereas the chin in females was either square (45.5%) or pointed (54.5%). In this study, the positive predict values of male and female were 92.5% and 73.7%, respectively. Therefore, the nonmetric method used to analyze the mandible in this study can be used for sex discrimination.     

Metric Assessment of the Pubic Bone Using Known and Novel Data Points for Sex Estimation,

Biological sex estimation of skeletal remains is essential in forensic and archaeological analyses. Anthropologists most often use the pelvis, which is the most sexually dimorphic element both morphologically and metrically. While nonmetric pubic bone features have been studied extensively, few metric studies have examined this individual bone for dimorphism. For this study, three observers examined three previously identified and ten novel measurements of the pubic body on a modern sample of isolated pubic bones from the Maricopa County Forensic Science Center (FSC), in Phoenix, Arizona (n = 400). A relationship between pubic body measurements and biological sex was demonstrated, with significant correlations. Discriminant function analyses found that five measurements, four of which were novel, discriminated between males (89%) and females (86%). Observer experience level did not significantly impact the results. These five measurements were reliable and show promise for inclusion in metric methods for assessment of sex.     

A simple technique for imaging the human skeleton using a flatbed scanner

A simple technique for imaging the human skeleton with a flatbed scanner is presented using the auricular surface of the ilium as an example. A flatbed scanner with resolution capabilities of 600 dpi or greater allows for images of human bones. The auricular surface of the ilium was selected to demonstrate this technique as it is a fairly three-dimensional area that can be difficult to record photographically. Fifty left ilia of various ages at death from the Athens Collection were selected from which three observers (SCF, CE, and IM) scored the morphology of the auricular surface using a well-established aging method. Observations were taken of the dry bone, of digital photographs of the bone, and of scanned images of the bone, and in that sequence. Results indicate that scores of scanned images are equivalent or better than digital images of the same ilia. This technique allows for sharing data electronically with ease.     

Fat Transfer and Fatal Macroembolization

Fat embolism is usually associated with long bone fractures or other trauma. The diagnosis is usually clinical, and in most cases, emboli are not fatal and not usually seen on gross examination. At the Los Angeles County Coroner's Office, we autopsied the victim of fatal macroscopic fat embolization to the lungs. The patient died during buttock enhancement surgery when fat from liposuction was injected into her buttocks. Fat embolism from liposuction and fat injection is reportedly rare, and macroscopic embolization is rarer still. Varicose veins can occur in the area of the sciatic notch and are known to cause painful sciatica symptoms. We suggest them as a potential conduit for macroscopic fat to reach the lungs. Simple pre‐operative questioning for sciatica symptoms and possible radiologic study to rule out sciatic varices seem prudent before undertaking buttock‐enhancing surgery. Careful fat injection with pre‐aspiration is always advised.     

Colonization of diatoms in and on porcine bone substrate and considerations of diatom ecology for forensic science

The presence of diatom algae in bone marrow has been used as forensic evidence of drowning for several decades; however, these studies are based on known or suspected recent drowning events. This study addresses the potential for diatoms to enter the bone marrow of skeletal remains, that is, de-fleshed long bones post-mortem. In laboratory and field experiments, bones were either inflicted with two access points by a cut and acid pitting or left intact. The bones were submerged in water for at least 1 week and up to 3 months. Samples of the bone surface and marrow were inspected for diatoms. The analysis considered the time required for diatoms to enter marrow and whether genus characteristics like size or mobility affect entry. The presence of an access point influenced diatom entry in that bones without an introduced access point had zero to one diatom present in the marrow, whereas a bone with an access point had >150 diatoms present in the marrow. The results of both laboratory and field phases suggest that diatoms will reliably colonize bone in as quickly as 1 week, establishing and maintaining communities for at least 3 months. However, the bone surface assemblages differ from the source community. Bone marrow displayed even more restrictive access to diatom colonization, resulting in communities dominated by small raphid diatoms. Based on these findings, we suggest some caveats on the use of diatoms as trace evidence in forensic science with recommendations for future avenues of research.     

溺死尸体器官硅藻检出率季节性差异

目的 研究溺水死亡者肺、肝、肾、骨髓等器官淡水硅藻检出率可能存在的季节性差异,为法医学解决水中尸体死因鉴定提供技术帮助与科学证据.方法 收集2009～2011年1、4、7、10月沈阳市浑河中溺死尸体每月(组)各5例;45只家兔分相应月份生前、死后入水组及对照组,分别于当月发现水中尸体后第2天入水,取人及兔肺、肝、肾、股骨骨髓及现场水样备检,应用酶消化法检测各组器官中硅藻数量、种属并与现场水样比对.结果 各组溺死尸体肺中均检出多量硅藻且种属与现场水样一致,春、秋及冬季溺死尸体肝、肾中仅可检出少量体积较小的小环藻等中心纲硅藻,夏季肝、肾及骨髓中均未检出硅藻;各组溺死家兔肺中均检出少量小环藻等中心纲硅藻,肝、肾及骨髓中未检出硅藻.结论 硅藻种群的季节性变化及不同种群硅藻体积的差异可能造成肝、肾等大循环器官中硅藻检出率的季节性差异;仅肺中检出硅藻,肝、肾中硅藻检验结果阴性并不能排除溺死可能.