Microsatellite stability in human post-mortem tissues

Human identification and forensic criminal casework may involve DNA profiling of decomposed material. Somatic microsatellite (STR) instability may lead to false exclusions and theoretically to false inclusions, both in criminal cases and in human identification. Hence, the somatic and postmortal stability of the actual sequences is crucial to the reliability of such analyses. Somatic STR stability in human tissues has been documented in small series only and the effect of postmortal tissue decomposition on microsatellite stability remains to be elucidated. On this basis, we have systematically searched for somatic STR mutations in 26 deceased humans without signs of decomposition at autopsy and 25 autopsy cases with obvious signs of postmortal decomposition. A blood sample and six tissue samples were collected from each case.Seven STRs were chosen for study, the tetranucleotides HUMVWA31/A, HUMTH01, HUMF13A1, and HUMFES/FPS, and the hyperpolymorphic markers HUMAPOAI1, D11S554 and HUMACTBP2. Denaturing gel electrophoresis was performed on an ABD Prism 377 gene sequencer with Genescan 672 software (Applied Biosystems, Inc.).The bone DNA profile of each case was chosen as the standard DNA profile. All cases gave profiles from additional tissues. By intraindividual comparison of DNA profiles in the cases without signs of degradation we find that the short repetitive sequences under study are stable, that is without evidence of somatic mutations. The cases with varying degree of decomposition display postmortal microsatellite stability, we detect no somatic mutations or other possible postmortal changes that could lead to between-organ non-matches.In conclusion, PCR-based STR analyses are suitable in human identification and forensic casework dealing with different tissues, even when the substrate is heavily decomposed.     

Effect of blood stained soils and time period on DNA and allele drop out using Promega 16 Powerplex kit

Blood stained soils may be of great interest in forensic incidents. Amplification of DNA from soil is often inhibited by co-purified contaminants. Different soils types from Pakistan and Turkey were stained with blood and samples were collected systematically after specified intervals. Rapid, inexpensive, large-scale DNA extraction method involving minimal purification was developed. DNA was quantized using Spectrophotometer and Fluorometer and was confirmed by Agarose Gel Electrophoresis. DNA extracted from different soils in different periods showed a remarkable decrease in yield as well as degradation in every extraction. PCR amplification was performed using various DNA targets present in Promega 16 Powerplex^® System kit. Amplification could not carry out in all loci especially in degraded samples taken after 20 days. Allele n locus drop out was noticed which shows that DNA was degraded. For some loci more than 2 alleles were also noticed showing contamination while working with the blood stained soils.     

The Achilles tendon as a DNA source for STR typing of highly decayed corpses

Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradational processes can affect PCR-based Short Tandem Repeat (STR) analysis. Degradation of DNA is observed to vary among different tissues and with time. Therefore, the stability of DNA in Achilles tendon samples is compared to that in muscle and kidney specimens with a variety of postmortem histories. Tissue samples from 28 autopsy cases, including 15 decomposed corpses and a control group of 13 nondecayed corpses were analysed. DNA was isolated using the All-tissue DNA Kit (GEN-IAL, Troisdorf, Germany), quantified by spectrophotometric measurement, amplified by the multiplex PCR genRES MPX-2 (Serac, Bad Homburg, Germany), and analysed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Quantitative analysis of nondecomposed tissues revealed that the recovery of DNA was highest in kidney followed by muscle, whereas Achilles tendon tissue was the poorest source of isolated DNA. Only small amounts of DNA were present in both kidney and muscle samples from decomposed corpses. However, from decayed Achilles tendon samples twice as much DNA as from nondecayed samples could be isolated on average. These results suggest DNA to be better protected in Achilles tendons. Moreover, postmortem changes in Achilles tendons may even improve DNA isolation.     

Analysis of Forensic Autopsy in 120 Cases of Medical Disputes Among Different Levels of Institutional Settings

Despite advances in medical science, the causes of death can sometimes only be determined by pathologists after a complete autopsy. Few studies have investigated the importance of forensic autopsy in medically disputed cases among different levels of institutional settings. Our study aimed to analyze forensic autopsy in 120 cases of medical disputes among five levels of institutional settings between 2001 and 2012 in Wenzhou, China. The results showed an overall concordance rate of 55%. Of the 39% of clinically missed diagnosis, cardiovascular pathology comprises 55.32%, while respiratory pathology accounts for the remaining 44. 68%. Factors that increase the likelihood of missed diagnoses were private clinics, community settings, and county hospitals. These results support that autopsy remains an important tool in establishing causes of death in medically disputed case, which may directly determine or exclude the fault of medical care and therefore in helping in resolving these cases.     

Uncertainty of determining cause of death in a medicolegal material without autopsy. An autopsy study

In constrast to other studies, this investigation was made on cases of medicolegal deaths that would not normally be autopsied. 223 females and 322 males, whose deaths were found to be natural before as well as after autopsy, were studied. The cause of death was estimated by external medicolegal examination, and after autopsy.In 79 females and 109 males, i.e. 35% and 34% respectively, estimated cause of death was found to be different after the autopsy. This was mostly because ischaemic heart disease as a cause of death was overestimated at the external medicolegal examination. No constant relationship between differing causes of death and age group could be demonstrated. Underdiagnoses and overdiagnoses tended to outweigh each other. Pneumonia, pulmonary embolism, cor pulmonale and aortic stenosis were clearly underestimated before autopsy. In addition, a variety of diseases that were not even mentioned at the medicolegal examination was found (subarachnoid haemorrhage, uraemia, perforated and bleeding gastric ulcers, tuberculosis).The same unreliability in the estimated cause of death therefore exists among cases not normally autopsied as found in retrospective studies of cases where autopsy is performed under all circumstances at the request of the police.False information will thus be given to the mortality statistics among the approximately 5000 cases of medicolegal deaths not autopsied in Denmark per year, most of these being natural deaths. Besides, contagious and inherited diseases could be overlooked, relatives given false information and the value of scientific studies in causes of death diminished.The conclusion is that autopsy is still essential to ensure continuous control and correction of causes of death.     

The value of the hospital autopsy. A study of causes and modes of death estimated before and after autopsy

Among 312 consecutive deaths in a Danish Central Hospital autopsy was performed in the pathology department on 266 cases, i.e. 85%. Retrospectively, the underlying causes of death were estimated from the clinical information alone by an experienced clinician and subsequently compared with the autopsy report. The definite cause of death was determined jointly by the clinician and the pathologist. The clinician's diagnosis was thereby confirmed as incorrect in 18% of the cases if small differences in site and type of malignant tumours were not considered. This is less than in many other investigations, but it is stressed that this could partly be because formal errors in completing the death certificate were avoided. The main causes of death were ischaemic heart disease and neoplasia. Clinical diagnosis of malignant diseases was never found to be erroneous. There was a slight tendency to clinically overestimate ischaemic heart disease, but in general the different errors outweighed each other, so that the total number of different causes of death before and after autopsy was nearly the same. The original death certificate was investigated in 12 accidental cases. Hereby it was found that the mode of death was originally stated erroneously as natural in 7 cases, i.e. 4.5%. It is concluded that hospital autopsy is still needed for the control and correction of causes of death, and it is stressed that clinicians as well as pathologists should be more aware of cases with a trauma in the history to avoid errors in the mode of death. Such errors can imply legal as well as insurance problems.     

A study of ethyl glucuronide in post-mortem blood as a marker of ante-mortem ingestion of alcohol

The possibility of post-mortem production of ethanol makes correct interpretation of ethanol detection in forensic autopsy samples difficult. Even though the levels of ethanol formed post-mortem are generally low, this may be highly relevant in cases where intake of alcohol was forbidden, for instance for pilots, professional drivers and countries with low legal alcohol limits for driving. Different criteria are used to determine whether a finding of ethanol is of exogenous origin, but there is no marker for alcohol ingestion that has been studied in detail. In this study, we wanted to evaluate the sensitivity and specificity of ethyl glucuronide (EtG), a direct minor metabolite of ethanol, measured in blood, as a marker of ante-mortem alcohol ingestion. Forensic autopsy cases were divided into groups with and without ante-mortem alcohol ingestion, according to strict inclusion criteria. In 93 cases with information on ante-mortem alcohol ingestion, EtG was detected in blood in all cases, even when levels of ethanol were low. In another 53 cases where there were no indications of ante-mortem alcohol intake, EtG could not be detected in blood in a single case, also in 11 cases in which ethanol was detected and considered to be most probably formed post-mortem. In conclusion, blood EtG determination seems to be a reliable marker of ante-mortem ingestion of alcohol, and it could be considered in forensic autopsy cases when post-mortem formation of ethanol is questioned.     

An evaluation of the relevance of routine DNA typing of fingernail clippings for forensic casework

DNA extracted from fingernail clippings of victims in forensic cases is a possible source of DNA from the perpetrator in cases where victims struggled or defended themselves. The source of this DNA on a victim's fingernails could possibly originate from contact with the suspect's blood, saliva, semen or scratched skin. In this technical note we evaluate the relevance of routine DNA typing of fingernail clippings in the forensic biology laboratory when, in real casework, normally only small quantities of nail material is sent. This was carried out by extracting DNA from fingernail clippings from a number of volunteers, before and after aggressively scratching other volunteers. No blood was drawn from the scratching, but skin flakes were observed under the nails before cutting and subsequent DNA typing. The DNA extracted was then typed using the STR systems: HUMTHO1, HUMTPOX and HUMCSF1PO (CTT triplex) and the system of D1S80. These profiles were compared with profiles achieved by similar typing of buccal swabs as a reference from each volunteer. In this study, the profile detected from each volunteer's clippings was the same before and after scratching, and matched the profile of the corresponding volunteer as defined by typing each volunteer's reference buccal swab. Fingernail clippings that are sent to our lab in actual casework are usually so small that additional treatment by swabbing or removing debris from below the clipping is not possible. For this reason, in this simulation the entire clippings were used for DNA extraction, to maximize the possibility of finding an additional profile. In conclusion, the findings from this study show that although the profiles obtained when typing fingernail clippings are those of the donors themselves, we suggest that typing of fingernail clippings should be carried out in forensic cases only when relevant. We would suggest that fingernail clippings not be routinely sent to the biology laboratory as items of evidence to be tested.     

Bacterial Translocation and Sample Contamination in Postmortem Microbiological Analyses

The diagnostic value of postmortem bacteriology has been discussed controversially for decades. In the study herein, contamination during sampling procedures and postmortem translocation were investigated to interpret postmortem microbiology results. One hundred medicolegal autopsy cases in total were included. Radiology, histology, bacteriology, and biochemistry were performed in all cases. Based on all investigation findings, 4 groups of cases were identified: death unrelated to infection, true infections, false positive (contamination during sampling procedures, postmortem translocation and mixed situations), and undetermined. The results of this study indicate that postmortem bacteriology provides useful data supporting infection‐related deaths, especially when potentially significant observations are accompanied by consistent autopsy, histology, and biochemistry. Result interpretation requires careful evaluation of number and type of isolated microorganisms.     

Serum beta-glucuronidase activity in polytrauma patients and in centrifuged autopsy blood samples]

The serum activity of beta-glucuronidase was investigated in 58 patients after severe trauma as well as in 43 autopsy cases. In 10 cases the enzyme activities in postmortem blood samples from the femoral vein were compared to those present in the correspondent heart blood samples. An elevated activity of beta-glucuronidase was observed in 14% of the patients within the first 36 h after severe trauma increasing to 62% in blood samples collected later on. The activity of beta-glucuronidase in the heart blood samples was always higher than in the corresponding sample from the femoral vein. In cases of prolonged post-mortem interval an elevated activity might have been due to bacterial contamination. In postmortal blood samples from the femoral vein an elevated enzyme activity was found in 70% of the study material. The results of the preliminary study on the activity of beta-glucuronidase in blood samples frequent in forensic routine work indicated that an elevated enzyme activity might be present for the following scenery: after severe trauma, in alcohol/drug abuse, presence of putridity/autolysis, presence of inflammatory processes, in diabetes as well as in carcinoma diseases. The significance of elevated beta-glucuronidase activity concerning alterations of unconjugated drug concentration due to in vitro cleavage of O-glucuronides should be investigated.     

Female criminals—It's not always the offender!

Numerous crimes (including murder), all having a common denominator, occurred in Germany and Austria between 1993 and 2009. All of these cases presented with identical female DNA traces being found at the crime scene. The crimes committed differed markedly, as did the suspects involved, which were of varied origin. Many of these cases could be solved. However, none of the suspects could identify an involved female. Fourteen of these cases (including one murder) occurred in Upper Austria.A special task force of the Austrian police, together with the Institute of Legal Medicine in Salzburg, began systematically searching for errors in the investigative process after the cases became more and more incoherent and nebulous. In the end, the DNA trace evidence was shown to be contaminated. A woman involved in the manufacture of the cotton swabs turned out to be the source of the female DNA profile.Following this, several products of other manufacturers were tested for contamination with DNA. It was noted that cotton swabs which had been sterilised with radiation were often contaminated. As a result, it is recommended that the manufacturing process, as well as the products themselves used in collection of DNA trace evidence, should be re-evaluated with the emphasis on preventing contamination.     

Correlation of the incidence of cocaine and cocaethylene in hair and postmortem biologic samples

Hair samples are useful as a matrix for drug testing because drugs can be detected in hair for longer periods than in blood or urine. The authors report a prospective comparison of the detection of cocaine and cocaethylene in routine postmortem biologic specimens to the detection of cocaine and cocaethylene in hair. The authors collected hair samples from various areas of the head in 53 autopsy cases, prepared them, and analyzed them by gas chromatography/mass spectrometry (GC/MS) for cocaine and cocaethylene. The authors compared the results of hair analysis with the results of toxicologic analysis performed on routine postmortem samples by enzyme multiplied immunoassay technique and GC/MS. Cocaine was found in either biologic fluids or in hair in 16 of 53 samples tested. Nine samples were positive for cocaine in both biologic fluids and hair. Five samples contained cocaine only in biologic fluids, and two contained cocaine only in hair. Cocaethylene was present in two cases. Drug screening of hair provides additional information in some autopsy cases, but the authors have not made hair analysis a routine practice. It may prove useful to save hair samples in all cases for later analysis if warranted by additional history or autopsy findings.     

Postmortem diagnosis of sepsis

Human sepsis is a spectrum of pathophysiological changes in the host system resulting from a generalized activation and systemic expression of the host's inflammatory pathways in response to infection. Since autopsy findings and routine histology in cases of suspected fatal sepsis are most often unspecific and unconvincing, a number of studies has recently dealt with different methods and markers to better define criteria for the postmortem diagnosis of sepsis. Research carried out on specimens obtained postmortem from sepsis-associated fatalities is an important tool to improve our understanding of inflammatory organ changes and the associated underlying pathophysiological mechanisms. One pitfall the investigator has to be aware of is how to select appropriate case material that constitutes the basis for the setting-up of reference values that derive from such studies. Since no scientific studies have investigated the value of cardiac blood samples in the present context, autopsy blood samples for the determination of biochemical sepsis markers have to derive from the femoral vein. In both sepsis cases as well as controls, the time of death has to be well defined.     

Investigation of Postmortem Absorption and Redistribution After the Application of a Fentanyl Patch

Fentanyl deaths have increased with availability of transdermal patches. Interpretation of postmortem fentanyl levels may be complicated by postmortem redistribution and absorption of fentanyl from a patch. We applied an unused 100‐μg/h fentanyl patch onto the lower abdomen of a decedent with no premortem fentanyl exposure. Ocular fluid, blood, and urine were collected prior to placement, and the decedent was refrigerated for 23 h. Prior to the autopsy, urine, subcutaneous tissue under the patch, and samples from the same anatomic sites were obtained. We observed no fentanyl in any postpatch placement samples (LOD: 0.1 ng/mL for blood and vitreous fluid, 1.0 ng/mL urine, 2.0 ng/g for tissues). Although we observed no postmortem absorption of fentanyl, this was only a single case; therefore, we recommend that patches be removed after receipt of a cadaver before initiation of an autopsy, with the location of removed patch documented.     

Association between LDLR polymorphism and diseases in the Japanese population: aging and distribution of the polymorphism

A number of DNA polymorphisms have been found to be associated with the pathophysiology of some common disease. If the LDLR polymorphism is directly or indirectly related to some fatal disease, the distribution of the polymorphism may vary with age. We therefore investigated the aging-associated distribution of the LDLR polymorphism. Blood samples were collected from Japanese cadavers (aged 0-91) at autopsy. The LDLR polymorphism was detected using a AmpliType PM PCR Typing kit. When the LDLR genotype was examined in cadavers divided according to age into 0-29 year group, 30-59 year group, and 60-91 year group, there were significant differences in genotype among the three age groups and between the 0-29 year group and 60-91 year group. The LDLR-A genotype tended to be lower in the older cadavers. The present study revealed that there were aging-dependent differences in the distribution of the LDLR polymorphism in autopsy samples, suggesting that a common mutation involved in the occurrence of fatal diseases may be present near the LDLR-A polymorphism locus.     

再次法医解剖及组织病理学检查的案例分析

剖析再次法医解剖及组织病理学检查的发生原因、鉴定注意事项及预防措施等 ,妥善地处理该类复杂案件。对 18例再次法医解剖及组织病理学检查的案件进行回顾性研究 ,按首次尸检方式及印象、尸检次数、再次尸检原因及方式、间隔时间及结论等归类、分析。较多见死于医疗纠纷 3例 ( 16 7% )、案件审查过程中 4例 ( 2 2 2 % ) ;首次尸检中 13例 ( 72 2 % )仅进行尸表检查或不完全解剖 ;再次尸检结论与首次尸检结论完全不符者 5例( 2 7 8% )。首次解剖的标准化与规范化及继续教育 ,不断提高法医病理学诊断水平 ,改善法医尸检的工作环境等对预防再次尸检的发生很有必要     

Multiple interchanging of tissue samples in cases of breast cancer

Due to the suspicion of a gynaecologist, a pathologist was suspected of incorrect diagnoses in cases of breast cancer and the interchanging of tissue samples. Many women applied to the attorney's bureau to clarify the reproaches. The privately owned laboratory for pathology was searched and 926 histological slides, roughly the same number of paraffin blocks and about 20 formalin fixed tissue samples were confiscated. Together with other confiscated material, at least 1236 histological slides and additional 249 paraffin blocks had to be sorted. Histological slides and paraffin blocks were matched with patients as far as possible following the laboratory book. Many of the warranted samples which were diagnosed as containing the carcinoma by the pathologist were missing. A total of 160 samples were chosen and rediagnosed by two independent pathologists. The formalin fixed tissue was negative for DNA most likely due to storage in formalin for years. Most of the histological slides were positive for DNA. On the whole, 18 expertises about histological findings and the DNA results were given. In some cases only DNA results could be presented, as previous experts had only performed DNA examinations without controlling the histological diagnosis. In six cases a carcinoma could be confirmed and the DNA profile matched with patient's DNA; in seven cases a carcinoma was confirmed without match with the patient; in two cases the carcinoma could not be confirmed in the presented samples. A jurisdictional solution was impossible because the accused pathologist died during the investigation. In conclusion, it must be stated that a DNA examination of histological slides should never be performed without a rediagnosis of an independent pathologist and photographic documentation of the findings. Whenever possible, material should be left on the slide.     

Unexpected death due to infectious mononucleosis

A 14-year-old boy with infectious mononucleosis died unexpectedly in hospital. The most significant finding at autopsy was the presence of marked bilateral tonsillar enlargement with considerable narrowing of the upper airway. There were no other underlying organic diseases that could have caused or contributed to death. Narcotic analgesia had been administered less than 2 h before death and may have contributed to respiratory compromise. The blood morphine level was 0.08 mg/L. Toxicological evaluation of individuals with obstructive lesions of the upper airway may, therefore, be a useful adjunct to the autopsy assessment of such cases as it may reveal factors exacerbating mechanical blockage.     

法医DNA实验室的DNA污染和防范

DNA污染是产生DNA鉴定结论错误的重要因素,法医DNA实验室要努力去解决这一问题。DNA污染有自身污染、交叉污染、PCR污染3种。法医DNA实验室要采取实验室分区、严格检验操作步骤、对试剂及消耗材料进行质量控制等方法防止发生DNA污染,采取设置对照样本、核查DNA结果、建立DNA排查数据库等方法监测和发现DNA污染。