In Situ Identification of Semen Stains on Common Substrates via Raman Spectroscopy,,

The spectroscopic identification of body fluids in situ is a major objective in forensic science. This approach offers the confirmatory, nondestructive, rapid, and on‐scene identification of various body fluids. Although Raman spectroscopy has shown tremendous promise toward this goal in prior proof‐of‐concept experiments, a significant challenge which still remains is substrate interference. Here, an approach for detecting semen stains in situ on various substrates using Raman spectroscopy is explored. Simulated semen evidence was prepared on skin, glass, and various fabrics. Raman data were accumulated from stains without any pretreatment using a common confocal mapping spectrometer using 785 nm laser excitation. The results demonstrate that the spectroscopic interferences encountered by substrates can be reduced and eliminated using a combination of existing subtraction techniques and chemometric models. Heterogeneous substrates proved most challenging, however, automatic subtraction treatment, and location of fluid hotspots was able to elucidate a clear spectroscopic signature of semen in every instance.     

Forensic Autopsies can Determine Latent Prostate Cancer Prevalence

To evaluate the usefulness of forensic autopsies in determining latent prostate cancer (PC) prevalence, we examined latent PC prevalence from autopsies and compared our findings between decedents with and without cancer. Data from forensic autopsies performed in Japan from 2004 to 2014 were obtained. For each prostate, histopathological examinations were performed in both the base and the apex sections. Three hundred and seventeen Japanese decedents were selected for analysis. The mean age of decedents was 56.4 ± 17.8 years (range, 14–94 years). Among this population, 39.4% died suddenly of disease and 60.6% died of external causes. Latent PC was identified in 45 (14.2%) decedents, who ranged from 27 to 93 years old (mean, 71.1 ± 12.9 years). The prevalence of clinically significant PC with a Gleason score of 7 or more was 8.8%, and the rate increased with age. Fifteen males had cancers other than PC. The prevalence of overall latent PC was significantly higher for those with cancer compared with those without (40.0% vs. 12.9%; P = 0.003). In this study, the use of forensic autopsy materials provided the opportunity to obtain a more accurate natural history of PC, as the decedents in this situation would have been more likely to have died suddenly while behaving as normal prior to death, and less likely to have been impacted by long-term medical interventions.     

The acid phosphatase test two minute cut-off: An insufficient time to detect some semen stains

The ability to detect semen in sexual offence cases is a crucial first step to locating stains which may be suitable for DNA profiling. Since the development of the acid phosphatase test in the late1950s by Stuart S. Kind, the process undertaken to perform the test has gone largely unchanged. The method currently accepted by operational forensic science laboratories allows 2 min for a reaction to be obtained, and until relatively recently, this has not been challenged. In this research, samples of semen were obtained from three donors and a range of dilutions for each sample were prepared. Each dilution was subjected to acid phosphatase testing using both direct testing and the ‘press test’ method. The results showed that semen could be detected in excess of 15 min in dilutions up to 1 in 400 using the press test method and in dilutions up to 1 in 1000 using the direct method. Of further significance was the observation that using the press test method, the two minute cut-off was insufficient to detect the majority of stains and in some cases, semen stains as strong as 1 in 20 dilutions. This research provides compelling evidence for protocols currently utilised in forensic practice to be reviewed in order that forensic scientists do not overlook potential evidential material that may prove suitable for body fluid identification such as DNA STR profiling.     

mRNA荧光复合扩增系统鉴别正常和异常精液初探

目的构建mRNA荧光复合扩增体系,实现对不同种类精液(尤其是无精症精液)的区分鉴别。方法收集正常、少精症及无精症的精液样本,制备精斑样本后提取细胞总RNA,利用逆转录PCR技术扩增2个精子特异mRNA标记(PRM1、PRM2)、2个精浆特异mRNA标记(TGM4、SEMG1)和2个管家基因mRNA标记(TEF、UCE)。结果正常精液样本可检测到全部精液mRNA标记表达;少精症精液样本虽然可检测到全部mRNA标记表达,但精子特异mRNA标记表达量较低;无精症精液样本不能检测到精子mRNA标记,只能检测到精浆特异mRNA标记。结论利用mRNA荧光复合扩增系统可以实现对正常和无精症精液的区分,而正常精液和少精症精液相比差异无统计学意义。     

A Comparative Assessment of the Chen et al. and Suchey‐Brooks Pubic Aging Methods on a North American Sample, ,

Accurately estimating the age‐at‐death of adult human skeletons is fundamental in forensic anthropology. This study evaluates the accuracy of two pubic bone age estimation methods—Chen et al. and Suchey‐Brooks. Specimens were obtained from a known collection of modern pubic bones curated at the Maricopa County Forensic Science Center in Phoenix, Arizona. A sample of 296 left male pubic bones of European ancestry was statistically evaluated via bias, absolute mean error, and intra‐ and inter‐observer error. Results indicate that the two methods are similar; the Suchey‐Brooks method is the most accurate for aging young adults (error c. 7 years), while the Revised Chen et al. method is most accurate for aging middle‐age adults (error c. 6 years). Thus, the Chen et al. method is an important contribution to forensic anthropology for aging older adult skeletal remains. There are, however, some limitations such as subjectivity and the intricate scoring system of Chen et al. method.     

Age Estimation Based on DNA Methylation Using Blood Samples From Deceased Individuals

Age estimation using DNA methylation levels has been widely investigated in recent years because of its potential application in forensic genetics. The main aim of this study was to develop an age predictor model (APM) for blood samples of deceased individuals based in five age-correlated genes. Fifty-one samples were analyzed through the bisulfite polymerase chain reaction (PCR) sequencing method for DNA methylation evaluation in genes ELOVL2, FHL2, EDARADD, PDE4C, and C1orf132. Linear regression was used to analyze relationships between methylation levels and age. The model using the highest age-correlated CpG from each locus revealed a correlation coefficient of 0.888, explaining 76.3% of age variation, with a mean absolute deviation from the chronological age (MAD) of 6.08 years. The model was validated in an independent test set of 19 samples producing a MAD of 8.84 years. The developed APM seems to be informative and could have potential application in forensic analysis.     

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.     

中国汉族女性青少年法医学活体骨龄推断数学模型的建立

目的建立推断中国汉族女性青少年活体骨龄的数学模型。方法摄取华中、华南及华东等地区的838名年龄介于11～20周岁正常女性青少年双侧锁骨胸骨端以及左侧肩、肘、腕、髋、膝、踝关节的X线片。依据青少年骨发育分级标准对24项骨骼发育指标进行阅片、分级,结合考虑身高、体质量及地区等影响因素．应用SAS8．1及SPSS11．0软件进行统计学处理,探索各指标与年龄的相关性。结果建立了我国汉族女性青少年利用锁骨胸骨端及6大关节骨骺闭合程度联合推断活体年龄的多元回归数学模型．推导出判定我国汉族女性青少年是否已满14、16和18周岁的Fisher’S两类判别分析方程。结论本研究所建立的判定活体年龄的数学模型丰富了活体年龄的法医学鉴定方法,有利于提高活体骨龄鉴定方法的科学性和结论的准确性。     

Age Estimation Based on Pulp Cavity to Tooth Volume Ratio Using Postmortem Computed Tomography Images

Pulp cavity size is known to decrease with age and can therefore serve as an indicator for age estimation. Here, we evaluated whether reconstructed images of multidetector‐row computed tomography (MDCT) acquired before forensic autopsy are useful for estimating age at death. Images of 136 mandibular first premolars obtained from bodies of known age at death were analyzed, and the volume of the regions corresponding to pulp cavity and that of the whole tooth were determined using a voxel counting function. The pulp cavity was clearly distinguishable from dental hard tissue on the reconstructed images when using a cutoff value of 1400 Hounsfield units. Regression analysis adjusted for sex showed that estimated age correlated significantly with the pulp cavity to tooth volume ratio (r = 0.76). MDCT is gaining more widespread use in forensic medicine, and analyzing dental images to obtain parameters for age prediction is a practical approach for postmortem identification.     

Bone Mineral Density Adult Age Estimation in Forensic Anthropology: A Test of the DXAGE Application

Estimating age‐at‐death of individuals represented only by skeletonized human remains is a fundamental aspect of forensic anthropological casework. Recently, several researchers have proposed that bone mineral density (BMD) is a useful predictor of age‐at‐death in forensic contexts. Navega et al. (JFS 63(2):497–503) developed an online application called DXAGE for calculating age‐at‐death from BMD parameters. This study tests the utility of DXAGE by utilizing data from the National Health and Nutrition Examination Survey (NHANES). BMD data from a female subsample (n = 470) of the NHANES 2007–2008 dataset were analyzed, and the relationship between predicted age and real age was examined. Inaccuracy was 14.25 years, and bias was ?7.20 years. Results show that there is a weak correlation between predicted and actual age (r = 0.47) using the DXAGE application. While BMD data are potentially useful for predicting age age‐at‐death, the DXAGE application should be used cautiously in forensic anthropological contexts.     

A Reappraisal of Developing Deciduous Tooth Length as an Estimate of Age in Human Immature Skeletal Remains

This study provides an update on a quantitative method for immature age estimation based on postnatal deciduous mandibular tooth length. Two known sex and age skeletal collections from Western Europe were sampled (n = 97). Linear regression models for age estimated were calculated for each individual tooth, each sex, and sex combined sample using classical calibration. Prediction errors, residuals, and percentage of individuals whose real age fell within the 95% prediction interval were calculated. The teeth which develop earlier in life, the incisors and the first molar, showed the greatest precision, while the canine showed the least. This method has greater applicability to archeological skeletons or to children in developing countries than for use in North American or European forensic contexts. The method can be applied to incomplete or poorly preserved remains of unknown sex, particularly when dental radiographs are not an option or when teeth have been removed from the alveolus or crypt.     

Detection of human seminal γ-glutamyl transpeptidase in stains using sandwich ELISA

A sensitive and specific sandwich ELISA for human seminal γ-glutamyl transpeptidase (γ-GTP) was developed using a combination of monoclonal antibodies, SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. γ-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal γ-GTP equivalent to 10⁷ times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal γ-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal γ-GTP was detectable even in 14-year-old stains.     

A New Robust Epigenetic Model for Forensic Age Prediction

Forensic DNA phenotyping refers to an emerging field of forensic sciences aimed at the prediction of externally visible characteristics of unknown sample donors directly from biological materials. The aging process significantly affects most of the above characteristics making the development of a reliable method of age prediction very important. Today, the so-called “epigenetic clocks” represent the most accurate models for age prediction. Since they are technically not achievable in a typical forensic laboratory, forensic DNA technology has triggered efforts toward the simplification of these models. The present study aimed to build an epigenetic clock using a set of methylation markers of five different genes in a sample of the Italian population of different ages covering the whole span of adult life. In a sample of 330 subjects, 42 selected markers were analyzed with a machine learning approach for building a prediction model for age prediction. A ridge linear regression model including eight of the proposed markers was identified as the best performing model across a plethora of candidates. This model was tested on an independent sample of 83 subjects providing a median error of 4.5 years. In the present study, an epigenetic model for age prediction was validated in a sample of the Italian population. However, its applicability to advanced ages still represents the main limitation in forensic caseworks.     

人类岩藻糖基转移酶5特异性分布及其在精细胞的表达与定位

目的研究人类岩藻糖基转移酶5(fucosyltransferase 5,FUT5)的特异性分布及在精细胞的表达与定位。方法收集健康志愿者的精液(分离精细胞并提取精细胞膜蛋白)、阴道拭子、唾液及静脉血,应用免疫印迹方法检测FUT5在人类精细胞膜、精浆、阴道液、唾液及血清中的表达量,采用免疫荧光技术检测FUT5在精细胞中的表达与定位。结果免疫印迹方法结果显示FUT5在精细胞膜及血清中有较高表达,但在精浆、阴道液及唾液中未被检测到。免疫荧光实验结果显示FUT5主要存在于精细胞头部。表明人类精细胞膜存在一定表达量的特异性FUT5,可采用抗原-抗体反应分离精液和阴道液混合斑中的精细胞。结论人类FUT5表现出分布特异性,可应用到法医学性犯罪案件中混合斑的鉴定。     

用等电聚焦酶免疫分析法检测人类精斑GC表现型

本文用IEF结合使用过氧化物酶标记第二抗体的酶免疫分析法检测了17名键康成年男性的精液(斑)和唾液斑及10名健康成年女性的阴道分泌液中的GC表现型。结果发现17份人类精斑均可测出三种GC蛋白普通表现型。在10份阴道液中测出一份样本的GC表现型,3份样本有不甚清楚的GC带,不能定型,其他样本均无GC带。17份唾液斑未测出GC。本法的灵敏度(0.675ng)比文献报道的用过氧化物酶标记第二抗体的酶免疫分析(5.6ng)高。     

Age Estimation from Radiographic Images of the Knee

This study examines the reliability of age estimation utilizing the Pyle and Hoerr atlas in relation to a modern Scottish population. The knee radiographs of 442 individuals (168 females, 274 males) were age assessed using the Pyle and Hoerr atlas. Analysis showed that there was a strong correlation between chronological age and estimated age (females R² = 0.968, males R² = 0.952). For females, the atlas method was most accurate between the ages of 9 and 15 years of age with an underage of 2.27 months and an overage of 2.38 months. For males, the atlas consistently overestimated age from the age of 9 years to the age of 16 years from between 0.14 and 8.81 months. The standard deviation for females was 9.86 months and for males was 10.75 months. This study showed that the Pyle and Hoerr atlas can be applied to a modern population with small modifications.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

Application of an immunological method to detect trace amounts of dried human semen

An immunological assay based on a monoclonal antibody was used for identification of trace amounts of dried human semen in forensic science evidence. The monoclonal antibody (Mab 4E6) produced recognizes a human sperm-coating antigen which is specific to human seminal plasma. This antigen seems to be a protein secreted by the epithelial cells of the ejaculatory duct, which is stable indefinitely at room temperature. Mab 4E6 reacts positively with semen samples from individuals independently to their ABO group or secretory status, but does not react with semen from bull, ram, boar, horse, rabbit and dog. In the assay system developed, Mab 4E6 can detect human seminal plasma at concentrations of 0.5 micrograms/ml total protein. A similar sensitivity is found when human semen stains are eluted from forensic science samples and tested by the same assay. This method shows a good correlation with the microscopic methods routinely used. The method described is very sensitive and reproducible, it is time saving and special laboratory equipment is not needed.     

Evaluating the use of hypoxia sensitive markers for body fluid stain age prediction

To augment DNA profiling and body fluid identification techniques efforts are being made to increase the amount of information available from a crime scene stain, which includes efforts to identify externally visible characteristics through phenotypic analysis. A key question surrounding crime scene stains is the length of time between deposition of the stain and its subsequent recovery, in that is the stain recovered related to the incident in question or from a previously deposited stain number of weeks earlier? The inability to answer this fundamental question has a detrimental effect upon the successful completion of a criminal investigation. Once a body fluid leaves the body, the oxygen concentration in the environment changes; therefore, it may be that this change could cause a change in the expression of hypoxia-sensitive biomarkers. Here, a range of bloodstains, liquid saliva and liquid semen samples were collected at 0 days, 7 days, 14 days, 21 days and 28 days of degrading at room temperature (19–22 °C), before undergoing total RNA extraction and cDNA synthesis. Blood was recovered from filter paper with 3 mm², with saliva and semen being left in their tubes and swabbed at the appropriate times. All samples then underwent quantitative PCR targeting Vascular Endothelial Growth Factor A (VEGFA) and Hypoxia-Inducible Factor 1 Alpha (HIF1A), with B-Actin (ACTB) as a reference gene. A range of linear and quadratic correlation values was obtained from the qPCR data and used to develop a predictive model with a mean absolute deviation (MAD) of 4.2, 2.1, and 5 days for blood, saliva, and semen respectively. Blind testing indicated that a stain age prediction model based upon VEGFA with ACTB as a reference gene could be used on samples up to four weeks old with a margin of error ranging from 2 days through to 5 days. While a sizeable potential time frame exists using this model; this represents a significant step towards the target of having an accurate stain age prediction model.