Immunochemical and chromatographic determination of ABH antigens in compact bone tissue

The identification of ABH antigens from compact bone tissue is known from many sources. The purpose of this study was to make a contribution to the localization of blood-group-active substances in compact bone tissue. A variety of preparation and identification methods were successfully used and compared. Samples were extracted from compact bone tissue, separated by HPTLC, and examined using the absorption-elution and PAP techniques. Additionally, the PAP technique was carried out on cryostat sections. Serologically active blood-group substances were consistently demonstrated in the organic components of the haversian canals.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

Prostaglandin E in vaginal smears--a possibility for sperm detection in azoospermia]

The identification of seminal traces is exceptionally difficult, if the semen of the assailant is azoospermic. The evident value of prostatic acid phosphatase (PAP) activity must be evaluated in such cases with caution. In a murder investigation of a 13 year old girl a positive PAP reaction was found in vaginal swabs and in her underpants. Spermatozoa could not be found. Using the gas-chromatographic method, described by Douse (1985) the presence of prostaglandin E could be demonstrated in the swabs as well as in the crotch of the underpants. The offender was found to be a man with azoospermia, who admitted intercourse but with the consent of the victim. The E prostaglandins are mainly synthesized in the vesiculae seminal and seen to be specific for semen. Swabs taken from mouth and rectum showed negative reactions for prostaglandins in this case. Prostaglandins could never be detected in vaginal swabs taken at least 7 days after intercourse. Conversely Douse could detect prostaglandins in swabs up to 58 hours after intercourse. Apparently the prostaglandin detection by Douse provides a suitable alternative besides to the quantitative and immunological PAP detection or the immunological detection of the protein p 30.     

Microbial contamination of ABO antigens in bone tissue

Fragments of human bones were stored in different media for two years and then expression of the ABO antigens was indicated. Simultaneously, microbiological investigations were performed. In almost all cases, different ABO substances were detected in putrefied and fresh bones taken from the same person. Blood group antigens found in putrefied bones were compared with serological activity of bacterium cultured from these tissues. Attempts were made to remove unspecific reactions. The authors assume that bacteria are responsible for nonspecific serological reactions, not only as a carrier of blood-group-like substances, but also as a source of enzymes responsible for changes in the structure of ABO antigens in putrefied bones.     

急性吗啡染毒大鼠脑内吗啡的形态学观察

用免疫组织化学法对急性吗啡染毒大鼠脑内吗啡的分布进行研究。结果表明，吗啡存在于人脑的前连合、外侧嗅束、海马伞、胼胀体、扣带、穹隆；间脑的丘脑终纹、丘脑网状核、乳头丘脑束；脑干的脑桥横纤维、锥体束、脚间核；小脑髓质和脊髓白质中。注射吗啡10分钟后即可在脑少数部位出现吗啡。本研究为吗啡研究提供了形态学基础，并为吗啡染毒者的诊断提供了一个新的依据。     

Immunoenzyme determination of ABO and secretor status in paraffin-embedded autopsy material

Using the indirect immunoperoxidase technique (PAP method), A, B, and H antigens were identified on formaldehyde-fixed, paraffin-embedded kidney tissue from 100 autopsies. Comparison with the serologic findings showed all our blood group determinations to be correct. The labeling of the collecting tubules was evaluated as characteristic of the secretor. The secretor status determined according to this parameter was unequivocally confirmed by the Lewis constellation in 78 of 82 cases; group Le(a-b-) could be differentiated with immunohistochemical methods in secretors and nonsecretors. Determination of ABO blood groups and secretion behavior with immunohistochemical methods was correct even in those cases where classic serology failed due to hemolysis and decomposition. Immunohistologic results obtained with monoclonal antibodies were better than those obtained with human sera.     

The mechanism of death in whole-body overheating

Data on anatomo-clinical analysis of 36 cases of death due to general overheating of a body are presented. On the basis of these data can be stated that in most cases the effect of high ambient temperature combined with great physical exercise and prolonged exposure to the caused death within short (up to 3 days) periods of time as a result of cessation of CNS functioning. Without physical exercise and direct insolation death usually occurred later (on the average of 5-9 days) from acute renal and renal-hepatic failure. Morphologic changes were unspecific.     

Immunohistochemical determination of maternal and child blood groups (ABO) in mature placental tissue

Using the indirect immunoperoxidase technique (PAP method), ABO characteristics of mother and child were correctly identified in tissue specimens from 10 mature human placentas. In one case, a weak infantile A reaction was overlooked in the agglutination test but correctly identified by immunohistochemistry. In accordance with the weak expression of ABO characteristics in cord blood, immunohistochemical labeling of infantile erythrocytes with monoclonal and human antibodies, as well as Ulex europeaeus agglutinin I (UEA I), was less pronounced than that of mature erythrocytes. Labeling of the chorionic vessel endothelium, in contrast to that of adult endothelial tissue, was negative with anti-A or anti-B but, regardless of the infantile blood group, pronounced with UEA I. Regular identification of the blood groups was possible in decomposed placental tissue stored at room temperature for 1 week, but not in tissue stored for 2 or more weeks.     

Genetic markers in human bone: II. Studies on ABO (and IGH) grouping.

A combination absorption-elution, two-dimensional absorption-inhibition procedure was used to determine the ABH antigen composition of a series of human bone specimens of known ABO type that had been aged up to nine months under dry and humid conditions at ambient temperature, 37 degrees C, and 56 degrees C; at ambient temperature in dry and wet soil; and buried in soil outdoors. Grouping data for the separate elution and inhibition testing, as well as for the combination procedure, are given. The combination method was found to be a highly reliable procedure for bone tissue ABH typing. Some data on microbial contaminants of human bone specimens aging in soil, and their effects on ABH typing results, are presented. No direct correlation between the properties of microbial contaminants and specific changes in the ABH antigenic composition of aging bone tissue specimens could be ascertained. Data on IGH antigen determination and on the quantitation of immunoglobulin G (IgG) in human bone tissue extracts indicated that immunoglobulin levels were typically too low to expect routinely successful Gm antigen testing results. However, these factors can sometimes be determined in fresh bone tissue extracts, particularly if the extracts are concentrated.     

Determining the age at death of females in the Chinese Han population: using quantitative variables and statistical analysis from pubic bones

Determining the age at death of females by skeletal features is extremely difficult and important, both in forensics and in physical anthropology. Our previous study of male pubic symphysis suggests that the indicators of morphological changes provide the best results for personal age identification. The indicators that help deduce of the age of females was approximately the same as males except for two specific indicators, which are bone density of the symphysial surface and viz. Viz are ridges and furrows on the symphysial surface, ossific nodules, lower extremities, ventral and ridge of the rampart, dorsal margin, ventral beveling and general macroscopic changes on the symphysial surface. Samples were drawn from 338 female individuals. The study procedures are as follows: Firstly, we examined the morphological features of pubic symphysis using the criteria similar to Hanihara's, Gilbert-McKern and Suchey-Brooks's methods. Secondly, we evaluated each stage with an appropriate score. Thirdly, we deduced four equations to assess the morphological features of the ages of females by statistic analysis. The results were discussed by comparing with Hanihara's, Gilbert-McKern and Suchey-Brooks's methods. The results were consistent and the developing methods for determining the age of death of females were produced.     

Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning

The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.     

Immunohistochemical antigen detection in dried tissue samples

Investigations were carried out on dried tissue samples for the identification of ABH antigens and human hemoglobin using the indirect immunoperoxidase technique (PAP). Samples from various organs were stored at room temperature over a period of 1 year and periodically examined immunohistochemically. By means of a rehydration medium, blood group and species identification were successfully demonstrated in the complete experimental series.     

Heterotopic ossification in unidentified skeletal remains

Heterotopic ossification is a benign, ectopic bone growth that develops in muscle and other soft tissue. The exact cause is poorly understood, but it is a rarely serious complication of soft tissue trauma. Its most common form, myositis ossifications traumatica, occurs as a secondary complication of direct muscle injury. However, other forms are less common and can result from specific pathologic conditions, such as spinal cord trauma and metabolic disorders. In patients who have had spinal cord injury and subsequent paraplegia, heterotopic ossification often results in ankylosis of the hip and a loss in range of motion. Ectopic ossification occurs below the injury site, and, although the specific muscle groups can vary, it usually involves those for which the origin and insertion involve the anterior pelvis and proximal femur. In dried bone, heterotopic ossification can appear as a smooth, irregularly shaped benign tumor of mature bone, extending from the surface but not invading the cortical bone. These tumors range in size from a few millimeters to several centimeters. Because heterotopic ossification is often associated with specific types of injuries, it has a unique anthropological use in forensic cases.     

New approaches to investigation of bone tissue according to the isoserological system ABO

To detect ABO antigens in human bone tissue, we offer to treat standard isohemagglutinating and heteroimmune sera with anti-A and anti-B raising their specificity; use of high-avid immunoreagents produced by novel technique; different modifications of absorption-elution reactions providing significant results.     

Genetic markers in human bone: I. Deoxyribonucleic acid (DNA) analysis.

Deoxyribonucleic acid (DNA) was isolated from a number of spongy and compact human bone tissue specimens, and the yield was estimated on a "per milligram of starting tissue" basis. DNA was, in addition, isolated from a number of corresponding blood and bone tissue specimens. Spectrophotofluorometry and ethidium bromide visualization on minigels were used to estimate the quantity and degree of degradation of DNA. The DNA from several blood-bone pairs is shown to give concordant restriction fragment length polymorphism (RFLP) typing results by two different typing protocols with five different single-locus probes. DNA from several additional blood-bone pairs is shown to give concordant results for human leucocyte antigen (HLA)-DQ alpha phenotypes following polymerase chain reaction (PCR) amplification and hybridization to specific allele-specific oligonucleotide (ASO) probes, and for the variable numbers of tandem repeats (VNTR) length polymorphisms 3' to the human apolipoprotein B (APOB) gene following PCR amplification with specific primers and analysis of the products by electrophoresis and ethidium bromide visualization.     

MRI detects hemorrhages in the muscles of the back in hypothermia

Morphological findings in death due to hypothermia are variable and predominantly unspecific. Goal of this study was to check the usefulness of post-mortem cross-sectional imaging methods in the diagnosis of externally invisible findings in death due to hypothermia. Three consecutive forensic cases that died due to hypothermia were examined using post-mortem multi-slice computed tomography (MSCT) and magnetic resonance imaging (MRI) prior to autopsy. MSCT excluded traumatic skeletal and fatty tissue injury. Using MRI, it was possible to detect hemorrhages within the muscles of the back in all three cases, a so far unknown finding in death due to hypothermia. MRI also allowed the detection of hemorrhages in the iliopsoas muscles. Wishnewsky spots remained radiologically undetected using the present examination techniques. In conclusion, hemorrhages of the muscles of the back might serve as a new sign of death due to hypothermia; however, additional studies on their specificity are necessary. Post-mortem MRI is considered as a good diagnosing tool for muscular hemorrhages, with a great potential for examination and documentation.     

Methods for detecting diazoline in chemicotoxicological studies]

New specific methods of diazolin detection by microcrystalline and chromogenic reactions as well as by thin-layer sorbent chromatography were developed. These methods were used to detect diazolin, isolated from biological object by water acidified with oxalic acid. It was stated that diazolin is detectable in chlorophorm extraction obtained both from acidic and from alkaline aqueous solutions.     

Evaluation of a Freezer Mill for Bone Pulverization prior to DNA Extraction: An Improved Workflow for STR Analysis

Traditional methods for bone pulverization typically generate heat, risking stability of DNA sample. SPEX? has developed cryogenic grinders which introduce liquid nitrogen to cool the sample and aid in the grinding process. In this study, the Freezer Mill 6970 EFM was used with two DNA extraction methods and routine downstream STR analysis procedures. DNA from as little as 0.1 g of bone powder was used to develop full STR profiles after freezer mill pulverization, and the method was reproducible. Further, no contamination was detected upon cleaning/reuse of the sample vials. There were no significant differences in DNA yield, STR alleles detected, or peak heights using the freezer mill as compared to traditional grinding, and successful DNA profiles were achieved from as low as 0.1 g of bone powder with this method. Overall, this work indicates that this cryogenic mill method may be used as a viable alternative to traditional tissue grinders.     

DNA extraction of burnt bone and teeth casework samples using bead-beating homogenization technique

Sample disruption was a necessary step for DNA isolation. Bone and teeth were useful biological sources particular in human remains and advance decomposed bodies. The compact bone and teeth required several preparation steps prior to analyzing process. However, the methods in standard protocol were laborious and time consuming. An alternating pulverization, bead beating homogenizer, was purposed in its effectiveness for forensic casework. (1) Here, we applied this technique to the burnt cracked bone and tooth that recovered from house fire for forensic DNA analysis. After cleansed an external surface, the eight multidirectional motion tissue homogenizer, Precellys® evolution, was utilized to pulverize bone and tooth followed by a DNA extraction and amplification. For detection with a capillary electrophoresis, full profiles of autosomal STRs and Y-chromosomal STRs were recovered from tooth sample but the partial profile STR was demonstrated in bone sample. The new technique in bone homogenization was less time consuming (around 30 s), less exposure to chemical agents (no need of liquid nitrogen), high efficiency, with high-throughput productivity.     

吗啡染毒大鼠死后体内吗啡的分布研究

作者采用免疫组织化学（PAP）法观察慢性吗啡染毒大鼠死后，在不同时间尸体内吗啡公布的变化。发现随着死后时间的延长，吗啡在大鼠CNS的分布无明显改变；其它内脏器官组织中吗啡分布则有明显改变；内脏脂肪组织原未查见吗啡，随死后时间延长，吗啡含量增加．证实大鼠染毒死后尸体内吗啡存在重分布现象。初步讨论了死后体内吗啡分布的变化与死后时间的关系，为吸毒致死后尸检取材提供依据。