新生儿呼吸系统疾病死亡的法医病理学分析

目的分析和探讨呼吸系统疾病引起新生儿死亡的病理学特点,为法医学死亡原因鉴定和相关医疗纠纷鉴定提供科学依据。方法回顾性总结分析1993～2008年16年间攀枝花市公安局尸检档案中85例新生儿死亡案例。结果呼吸系统疾病是引起新生儿死亡的首要因素（共56例,占65.88%）,其中新生儿吸入性肺炎20例（23.53%）、新生儿感染性肺炎14例（16.47%）、新生儿窒息12例（14.12%）、新生儿肺透明膜病6例（7.59%）、新生儿肺出血4例（4.71%）。结论新生儿尸检应重视呼吸系统的检查,根据新生儿发病特点和肺部病理变化明确临床诊断和死亡原因。     

Virological analysis in the diagnosis of sudden children death: a medico-legal approach

Infections are considered to be an important cause of unexpected death in children. It has also been assumed that respiratory viruses are involved in the genesis of sudden infant death syndrome (SIDS). The Spanish National Institute of Toxicology and Forensic Sciences act as the forensic reference centre for Spain. We analyse the experience of this centre in the virological study of 64 cases of sudden children death where viral serology, virological cultures, herpesviruses polymerase chain reaction (PCR) and electron microscopy were performed. According to pathological findings, death could only be attributed to an adenovirus infection in one amygdalitis with upper airways stenosis and asphyxia. Human herpes virus 6 (HHV-6) was detected by PCR in one case with pathological findings characteristic of SIDS. Recent infection by respiratory syncytial virus (RSV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were also detected. Meanwhile, 85.9% of the cases yielded negative viral results. Twenty-eight infants were finally categorised as SIDS. Pathological findings of infection were detected in 12 patients despite the negativity of viral analyses. Although viral infection is an uncommon cause of sudden children death, a complete microbiological investigation will help to solve the puzzle of SIDS. Definitive guidelines for microbiological analyses need to be updated whilst new pathogens are discovered or new techniques are implemented in order to clarify unsolved cases.     

Validation of Real‐time PCR Assays for Bioforensic Detection of Model Plant Pathogens

The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real‐time PCR assays were developed for three model plant pathogens: Pseudomonas syringae pathovar tomato, Xylella fastidiosa, and Wheat streak mosaic virus. Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100 fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence.     

Evaluation of Tamm‐Horsfall Protein and Uroplakin III for Forensic Identification of Urine

Abstract: In this study, Tamm‐Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium‐specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT‐PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.     

Genotypic Polymorphisms of Hepatitis B Virus Provide Useful Information for Estimating Geographical Origin or Place of Long‐term Residence of Unidentified Cadavers

Increasing numbers of unidentified cadavers are a major problem. We have developed a new method for providing identification information that can determine the geographical origin or place of long‐term residence of unidentified cadavers based on genotypic polymorphisms of hepatitis B virus (HBV) known to correlate with their geographical distribution. PCR of serum samples detected HBV DNA from 4 (3.9%) of 102 randomly selected Japanese forensic cadavers. Multiplex PCR did not detect multiple HBV genotypes from any single cadaver, confirming the absence of coinfection. Phylogenetic tree analysis based on a 485‐bp mutant region of the HBV S gene successfully classified the HBV genotypes into A to J. Among 10 HBV‐infected cadavers, 8 had genotype Ce/C2, a genotype prevalent in East Asia, and 2 had genotype Bj/B1, a Japanese‐specific genotype. HBV genotypic polymorphisms correlate with the geographical distribution of the virus and thus provide important information for identifying unidentified cadavers infected with HBV.     

Pediatric toxicologic deaths: a 10-year retrospective study

A 10-year retrospective study of pediatric toxicologic deaths was performed at the Medical University of South Carolina (Charleston, SC) from January 1989 to December 1998. During this time, 709 pediatric forensic autopsies were performed on children younger than 18 years of age. Eleven deaths were determined to be secondary to toxic exposures (excluding carbon monoxide poisonings secondary to fires). The remaining deaths were reviewed for the presence of alcohol or illicit drugs. The 11 toxicologic deaths were analyzed for age, sex, race, type of toxic exposure, cause and manner of death, location of incident, witness, and, in the younger age group, the primary caregiver at the time of exposure. The deaths had a bimodal age distribution (6 deaths in victims ages 15 to 17 and 5 deaths in victims ages 4 or younger), involving a wide range of toxins. The teenage group was composed of five males and one female, all white. The preschool group had three females and three males, all black. The manner of death ranged from accidental to suicidal to homicidal. In addition, in eight neonatal and fetal deaths, the victims tested positive for maternal cocaine use, and five of these victims tested positive for cocaine or benzoylecgonine. However, the cause of death was not stated to be cocaine in any of these neonatal and fetal cases.     

Recovery of Environmental Human DNA by Insects*,†

Abstract: We tested the hypotheses that foraging insects can acquire human DNA from the environment and that insect‐delivered human DNA is of sufficient quantity and quality to permit standard forensic analyses. Houseflies, German cockroaches, and camel crickets were exposed to dusty surfaces and then assayed for human mitochondrial and nuclear loci by conventional and qPCR, and multiplex STR amplification. Over two experiments, 100% of insect groups and 94% of dust controls tested positive for human DNA. Of 177 individuals, 33–67% tested positive and 13 yielded quantifiable human DNA (mean = 0.022 ± 0.006 ng; mean dust control = 2.448 ± 0.960 ng); four had at least one positive allele call for one or more locus; eight others showed multiple peaks at some loci. Results imply that application to routine forensic casework is limited given current detection methodology yet demonstrate the potential use of insects as environmental samplers for human DNA.     

Mucosal Cell Isolation and Analysis from Cellular Mixtures of Three Contributors

In forensic genetic analyses, mixtures of various biological materials are common samples. Micromanipulation, which is performed based on differences in cellular morphology, is an effective method for the isolation of cells from mixtures. In this study, mucosal cell was isolated from somatic cellular mixtures (blood and saliva) based on micromanipulation and a low volume‐PCR (LV‐PCR) platform. One hundred and twenty‐six parallel LV‐PCR processes were performed using an Identifiler^® kit, with 107 reactions yielding single‐source DNA profiles. Among them, 54 full profiles (50%) and 37 partial profiles (13–15 loci) were obtained. Based on the above method, we obtained a single‐source DNA profile from a cigarette butt contaminated by two victims’ blood in a murder case. The generated genotype was used to query a DNA database, and a perfect match was found.     

Simultaneous Identification of Four “Legal High” Plant Species in a Multiplex PCR High‐Resolution Melt Assay,

The international prevalence of “legal high” drugs necessitates the development of a method for their detection and identification. Herein, we describe the development and validation of a tetraplex multiplex real‐time polymerase chain reaction (PCR) assay used to simultaneously identify morning glory, jimson weed, Hawaiian woodrose, and marijuana detected by high‐resolution melt using LCGreen Plus^®. The PCR assay was evaluated based on the following: (i) specificity and selectivity—primers were tested on DNA extracted from 30 species and simulated forensic samples, (ii) sensitivity—serial dilutions of the target DNA were prepared, and (iii) reproducibility and reliability—sample replicates were tested and remelted on different days. The assay is ideal for cases in which inexpensive assays are needed to quickly detect and identify trace biological material present on drug paraphernalia that is too compromised for botanical microscopic identification and for which analysts are unfamiliar with the morphology of the emerging “legal high” species.     

Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples

Forensic scientists are constantly searching for better, faster, and less expensive ways to increase the first-pass success rate of forensic sample analysis. Technological advances continue to increase the sensitivity of analysis methods to enable genotyping of samples containing minimal amounts of DNA, yet few tools are available that can simultaneously alert the analyst to both the presence of inhibition and level of degradation in samples prior to genotyping to allow analysts the opportunity to make appropriate modifications to their protocols and, consequently, to use less sample. Our laboratory developed a multiplex quantitative PCR assay that amplifies two human nuclear DNA target sequences of different length to assess DNA degradation and a third amplification target, a synthetic oligonucleotide internal PCR control (IPC), to allow for the assessment of PCR inhibition. We chose the two nuclear targets to provide quantity and fragment-length information relevant to the STR amplification targets commonly used for forensic genotyping. The long target (nuTH01, 170-190 bp) spans the TH01 STR locus and uses a FAM-labeled TaqMan probe for detection. The short nuclear target (nuCSF, 67 bp) is directed at the upstream flanking region of the CSF1PO STR locus and is detected using a VIC-labeled TaqManMGB probe. The IPC target sequence is detected using a NED-labeled TaqManMGB probe. The assay was validated on the Applied Biosystems 7500 Real-Time PCR system, which is optimized for NED detection. We report the results of a developmental validation in which the assay was rigorously tested, in accordance with the current SWGDAM guidelines, for precision, sensitivity, accuracy, reproducibility, species specificity, and stability.     

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.     

Natural death in the forensic setting: a study and approach to the autopsy

Although unexpected natural deaths comprise a large proportion of the cases seen in the forensic setting, the investigation of these cases remains a unique challenge. Some cases are straightforward; however, others require special examination and dissections, as well as the use of special dissections and ancillary studies. For the forensic investigator to accurately assign a cause and manner of death, one must be aware of the differential diagnoses, common scenarios, and typical victim. The authors reviewed all forensic cases referred to the Forensic Section of the Medical University of South Carolina over a 15-year period from January 1989-December 2003. The number of natural deaths occurring in adults (age 18 years and older) was 6932, compared with the overall number of 13,227 cases. The natural deaths were then analyzed as to autopsy findings, cause of death, cardiac versus noncardiac, age, race, and gender. Current techniques, special dissections, and ancillary studies that can assist in the classification of these deaths in the forensic setting are reviewed.     

Two Fatal Cases of Hidden Pneumonia in Young People*

Abstract: Acute respiratory distress syndrome (ARDS) is a severe lung disease characterized by inflammation of the lung parenchyma leading to impaired gas exchange. This condition is often lethal, usually requiring mechanical ventilation and admission to an intensive care unit. We present two fatal cases of hidden pneumonia in young people and discuss the pathophysiological mechanism of ARDS with reference to the histological pattern. A complete forensic approach by means of autopsy and histological, immunohistochemical, and microbiological, examination was carried out. In both cases the cause of death was cardio‐respiratory failure following an acute bilateral pneumonia with diffuse alveolar damage and ARDS associated with sepsis and disseminated intravascular coagulation. Our cases suggest on one side the importance of an early diagnosis to avoid unexpected death while on the other that the diagnosis of ARDS has to be confirmed on the basis of a careful postmortem examination and a complete microscopy and microbiological study.     

An Analysis of The Morbidity and Mortality of Diabetes Mellitus in a Forensic Context

To investigate the spectrum of diseases seen in diabetes mellitus in a forensic context, all autopsy reports of diabetic individuals who presented to Forensic Science, South Australia (FSSA), over a 5‐year period from 2005 to 2009 were studied. The leading cause of death was cardiovascular disease (55.0%), followed by unnatural deaths (15.4%) and infections (9.4%). In type 1 diabetics, principal causes of death included cardiovascular disease (44.7%), acute metabolic complications (18.7%), unnatural deaths (17.9%), and infections (8.9%). However, frequencies of these diseases differed in type 2 diabetics, with cardiovascular events responsible for 56.6% of cases, followed by unnatural deaths (15.0%) and infections (10.9%). A larger number of male deaths were seen in all disease categories, except respiratory and gastrointestinal where the frequencies were similar to females. Cardiovascular disease was the leading overall cause of death across all ages except in those under the age of 30 where metabolic complications were more common.     

A Three‐Locus,PCR‐based Method for Forensic Identification of Plant Material

Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant‐derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA‐based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad‐spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL‐F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species‐targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.     

Detection of microRNAs in DNA Extractions for Forensic Biological Source Identification

Molecular‐based approaches for biological source identification are of great interest in the forensic community because of a lack of sensitivity and specificity in current methods. MicroRNAs (miRNAs) have been considered due to their robust nature and tissue specificity; however, analysis requires a separate RNA extraction, requiring an additional step in the forensic analysis workflow. The purpose of this study was to evaluate miRNA detection in blood, semen, and saliva using DNA extraction methods commonly utilized for forensic casework. RT‐qPCR analysis revealed that the tested miRNAs were consistently detectable across most tested DNA extraction methods, but detection was significantly reduced compared to RNA extracts in some biological fluids. DNase treatment was not necessary to achieve miRNA‐specific results. A previously developed miRNA panel for forensic body fluid identification was evaluated using DNA extracts, and largely demonstrated concordance with results from samples deriving from RNA extracts of semen, blood, and saliva.     

The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amplification of low copy number DNA

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications.     

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.     

宝鸡市358例法医送检器官的组织病理学分析

目的 分析法医尸体检验后送检器官的病理学特征,总结此类案件的特点.方法 对宝鸡市法医送检358例尸体解剖器官标本进行常规检查并进行组织病理学诊断. 结果 358例中以青壮年男性为主,死亡原因主要为创伤、猝死、中毒.组织学能明确死亡原因250例,无典型组织学病变101例,组织自溶腐败7例.病理诊断以心血管疾病为主,其次为呼吸、神经、消化系统疾病. 结论 法医解剖具有专业特点,与病理解剖不尽相同.组织病理学检验进行死亡原因诊断时,应积极与法医沟通,以充分掌握案情、死亡经过及特定的法医病理学特征.     

A decade of pediatric homicide: a retrospective study at the Medical University of South Carolina.

More than 3 million children are abused and/or neglected each year in the United States. Unfortunately, a significant percentage of these cases result in homicide by child abuse or child neglect. Causes of death range from blunt force trauma and shaking to asphyxia to immolation. We retrospectively reviewed all pediatric forensic cases referred to the Medical University of South Carolina Forensic Pathology Section over the past 10 years, from January 1986 to December 1995. Of these, we looked only at children < or =5 years of age. The majority (342 cases, 69%) of these deaths were classified as natural, 96 (19%) as accident, and 60 (12%) as homicide. Of the homicides, we examined the cause of death; age, gender, and race of the victim; relationship to the perpetrator; time interval between injury and death; and the initial history given as to the cause of the injury. The cause of death fell into nine categories, the number one category being head trauma. Forty-five percent of the homicides were by head trauma, 12% by abdominal or body trauma, 25% by asphyxia (with half of these due to drowning), 10% by carbon monoxide poisoning or thermal injury, and the remaining 8% involving cases of neglect, stabbing, and poisoning. The majority of the homicide victims were male (67%) and black (67%). Forty-six percent were < or =1 year of age. Approximately 25% of the homicide cases were designated as shaken baby syndrome (SBS). In 97% of the cases, the assailant was known to the victim and was a family relative in 77%. Sixty-three percent of the assailants were female and 45% of the assailants were male; in 12%, the assailants were both parents, and in 1 case, the assailant remains unknown. Of the asphyxia deaths, 87% of the assailants were female. The time interval between injury and death ranged from minutes to hours in most cases to months in cases of repeated abuse and chronic injury and sequelae. The time interval between injury and the onset of symptoms remains unknown in most cases due to inconsistencies in the history and lack of credibility of the caretaker. The most common initial history given was "a fall" (20%). We report our findings of a decade of pediatric homicides to increase awareness of the common scenarios and case histories, demographics of the victims, causes of death, and perpetrators of pediatric homicide.