Immunohistochemical C5b-9-complement complex demonstration in early stages of myocardial necroses using paraffin sections

With conventional staining techniques it was nearly impossible to differentiate with certainty between early ischemic myocardial lesions, autolytic changes and intact myocardium without a nuclear section. In 1986 it was introduced an immunohistochemical method, which presented C5b-9 Complement Complex as a necrosis marker in cryostat sections. Our further development of this process has resulted in a technique for application on routine paraffin sections: C5b-9 Complement Complex is particularly well-suited for the exact and autolytically insensitive presentation of disseminated necroses of fiber bundels and single fibers of myocardium. The application of the technique on paraffin sections will lead to the use of the same in forensic medicine routine diagnostics of early ischemic myocardial lesions, even in cases of "sudden" cardiac death.     

Effects of anoxia on the isolated perfused rat heart: histological, histochemical and electron-microscopic observations

A study to evaluate hematoxylin-basic fuchsin-picric acid (HBFP) and some other conventional stains in the demonstration of early anoxic lesions of the perfused rat heart has been undertaken. The extent of the anoxic injury was readily demonstrable electron-microscopically and in semithin sections by light microscopy, and at 1.5--2 hours practically all myocardial cells showed irreversible changes. The HBFP method was unreliable, giving false positive and false negative results both in the control and anoxic specimens. No clear changes were seen in the four histochemical dehydrogenase reactions applied, whereas damaged myocytes lost some of their affinity to eosin. Contraction bands were frequently seen in the anoxic hearts stained by the phosphotungstic acid-hematoxylin method. However, their presence was not in a linear accordance with the extent of the lesion. The perfusion model is not directly comparable to the myocardial infarct caused, for example, by ligating the coronary artery but, however, the present results suggest the difficulty of demonstrating the very early anoxic myocardial lesion by means other than electron microscopy.     

Postmortem pink teeth. Histochemical identification of the causative pigment

The phenomenon of postmortem pink teeth has been reported in subjects who have died suddenly and unnaturally, and whose bodies have been subsequently exposed to a wet or moist environment. Ground and EDTA-decalcified sections of teeth of 21 corpses exhibiting postmortem pink-stained teeth were investigated for the identification of the responsible pigment. With histochemical methods and ultraviolet microscopy, the causative pigment was identified as undegraded hemoglobin. Staining from hemosiderin, bile and bile-related pigments, and porphyrins was ruled out. However, histochemical techniques are unable to reveal the form in which the hemoglobin occurred. The cause of death or time of death had no observable effect on the staining reactions for hemoglobin in the teeth.     

A Method to Interpolate Osteon Volume Designed for Histological Age Estimation Research

Aging adult skeletal material is a crucial component of building the biological profile of unknown skeletal remains, but many macro- and microscopic methods have challenges regarding accuracy, precision, and replicability. This study developed a volumetric method to visualize and quantify histological remodeling events in three dimensions, using a two-dimensional serialized approach that applied circular polarizing microscopy and geographic information systems protocols. This approach was designed as a tool to extend current histological aging methodologies. Three serial transverse sections were obtained from a human femoral midshaft. A total sample size of 6847 complete osteons from the three sections was identified; 1229 osteons connected between all sections. The volume of all connected osteons was interpolated using ArcGIS area calculations and truncated cone geometric functions. Each section was divided into octants, and two random samples of 100 and of 30 connected osteons from each octant were generated. Osteon volume was compared between the octants for each random sample using ANOVA. Results indicated that the medial aspect had relative uniformity in osteon volume, whereas the lateral aspect showed high variability. The anterolateral–lateral octant had significantly smaller osteon volume, whereas the posterior–posterolateral octant had significantly larger osteon volume. Results also indicated that a minimum of 100 osteons is statistically more robust and more representative of normal osteon distribution and volume; the use of 30 osteons is insufficient. This research has demonstrated that osteon volume can be interpolated using spatial geometry and GIS applications and may be a tool to incorporate into adult age-at-death estimation techniques.     

实验性早期心肌缺血c-fos-mRNA原位杂交研究

用原位杂交和图像分析技术 ,观察大鼠实验性急性心肌缺血模型心肌细胞c fos mRNA的表达情况。结果显示 :正常心肌细胞表达少量c fos mRNA ;急性心肌缺血 15min ,心肌细胞表达c fos mRNA的阳性反应增强 ,心肌缺血后 30min ,c fos mRNA阳性反应达到最高峰 ,以后逐步下降。表明 :c fos mRNA的原位杂交检测可为一项灵敏的急性心肌缺血的死后诊断指标。     

失血即刻死亡心肌超微结构,组织化学及免疫组化研究

本研究应用电镜、组织化学、免疫组化及形态计量分析等方法和技术，观察失血即刻死亡时家兔心肌超微结构、琥珀酸脱氢酶（ＳＤＨ）活性、肌红蛋白（Ｍｂ）脱失的变化特点。结果发现，失血即刻死亡时家兔心肌细胞轻度水肿，肌原纤维普遍强度收缩，肌节缩短，Ｚ线增粗，心肌细胞内ＳＤＨ活性明显下降，Ｍｂ轻度局灶性脱失．     

Pathomorphology of the endocardium and myocardium in the heart injury

Light, polarization, luminescent microscopy, phase contrast, special staining methods and scanning electron microscopy were used for studying sections and fragments of the endocardium and myocardium from 123 patients who had died of heart contusion and ischemic heart disease and from 50 rats with experimental heart contusion. The morphological classification of heart contusion and differential-diagnostic table of myocardial alterations due to contusion of the heart and ischemic heart disease are proposed to facilitate pathomorphological diagnosis of heart contusion. The classification of pathomorphological signs in heart contusion is presented.     

Advances in the diagnosis of wound vitality: a review

The diagnosis of the vital origin of wounds in many cases remains an unsolved problem for the forensic pathologist. Practical experience enables the expert to diagnose the vital or postmortem origin of wounds on the basis of macroscopic examination. In some cases, optic microscopy is used to confirm the diagnosis. In many other cases, additional more sensitive and specific markers of vitality are required. In the past 50 years, comprehensive research on this topic has resulted in a better understanding of the acute inflammatory reaction. The development and application of sensitive and specific markers through research in the areas of histochemistry, enzymology, and biochemistry has provided a partial solution to the problems involved in wound vitality diagnosis. A review of this challenging area of forensic pathology, including an explanation of these methods and markers, is presented in this paper.     

Degeneration of Nuclei and Mitochondria in Human Hairs

Abstract:  It is generally accepted that nuclei degrade in developing hair shafts but the point at which such occurs has not been investigated. The fate of mitochondria in the keratinizing hair shaft has been less clear. This study uses transmission electron microscopy to investigate when nuclei and mitochondria are no longer visible in the developing hair shaft. Serial sections were obtained from anagen head hairs absent follicles in order to determine the sequence of degradation of nuclei and mitochondria in the hair shaft by starting at the root bulb and proceeding toward the hair tip. It was demonstrated that nuclei and mitochondria become invisible in the keratinizing hair shaft at about the same time. This was found to occur fairly early in the process at the level of the hair shaft where the hair cuticle becomes permanent.     

实验性心脏震荡的法医病理学研究

本研究应用可产生稳定打击速度并可同时测定瞬间冲击速度及冲击力大小的机械式弹性拉力打击器，分别以６．７ｍ／ｓ及８．０ｍ／ｓ速度冲击大胸骨部心前区建立了心脏震荡动物模型，在左右心室压力及心电图监测下冲击后６０分钟进行尸体剖验检查，并应用多种组织化学染色、透射电镜技术、免疫组织化学染色以及硝酸镧示踪电镜技术对心肌组织学行系统性研究。结果表明：１．心前区受冲击瞬间左右心室内压力急骤升高，随即可恢复至正常水平。心电图在冲击后即刻表现为持续短暂的异常心律，逐渐恢复至正常心律。２．心脏血管麻痹性扩张，但宏观及微观均无损伤性所见。３．心肌超微结构有一定程度损害，心肝细胞膜破裂或通透性增高。本研究提示：心脏震荡者心脏宏观、微观无特殊改变，但超结构及心肌细胞膜轻度损害。     

A rapid screening technique for the detection of spermatozoa

Phase contrast microscopy has been used for some time to search for and identify spermatozoa. An enhancement to the technique using xylene in conjuction with phase contrast microscopy is discussed. The method has been found to save time by allowing identification of spermatozoa with xylene-enhanced phase contrast microscopy in many cases that would have been unsuccessful using the normal "dry" phase contrast microscopy techniques. This eliminates a staining and reexamination step.     

Immunofluorescence detection of drugs in postmortem tissues: a new technique with potential for assessment of drug influence in cause of death

This report describes a new technique, immunofluorescence, for the detection and possible characterization of drug content in postmortem tissues. By using antisera generated against a drug-protein conjugate, the stabilization of tissue-sequestered drug is accomplished by incubation of fresh frozen sections of tissue with dilute solutions of rabbit anti-drug antibodies. Secondary incubation with a fluorescence-labeled anti-rabbit immunoglobulin labels these points of sequestration. Tissue sections so stained are examined by fluorescence microscopy. In studies with rats given graded doses of morphine sulfate, there were discernible differences in tissue binding of morphine in brain sections from animals treated "therapeutically," fatally, and chronically. Extension of these studies to human autopsy material is anticipated and potential problems are discussed. This technique offers the forensic toxicologist the potential for evaluating the drug content of tissues in situ.     

Is immunohistochemistry a useful tool in the postmortem recognition of myocardial hypoxia in human tissue with no morphological evidence of necrosis?

Myocytes in the border zone of myocardial infarction are under severe hypoxia without characteristic morphology of necrosis, and show ultrastructural features similar to those seen within the first hours after coronary occlusion. This study was carried out to evaluate the possibility that immunohistochemical methods could be used for the early diagnosis of myocardial infarction by detecting areas of hypoxia. Nineteen human sections of formalin-fixed paraffin-embedded myocardial samples showing a necrotic area and its border were submitted to immunohistochemical staining with the markers antimuscle actin, antimyoglobin, antitroponin T, antifibronectin, and anticomplement component C9. Sections were also subjected to azan trichrome and hematoxylin-basic fuchsin-picric (HBFP) staining techniques. Immunohistochemistry and azan trichrome showed that in the border zone there was a pattern of reaction intermediate between the infarcted area and the normal myocardium. The HBFP failed to distinguish these two areas. In conclusion, immunohistochemistry and azan trichrome can recognize myocardial hypoxia. Because hypoxia is an invariable condition in infarction, these techniques can be used to confirm suspected cases of myocardial infarction in which necrosis is not yet evident. However, considering that agonal states may be associated with generalized hypoxia, further studies are needed to confirm the reliability of this procedure in the earlier phases of myocardial infarction.     

傅里叶红外光谱技术在法医学脑挫裂伤鉴定中的应用展望

脑挫裂伤是常见的一类颅脑损伤,单纯的组织病理学技术可能难以早期发现及精确诊断。傅里叶变换红外面扫描成像技术(FTIR-mapping)是病理学研究领域的新技术,因其结合了傅里叶变换红外光谱、红外显微镜技术以及面扫描成像技术的特点,具有测定方法简便、对样本无损、灵敏度高、准确、直观等优点,突破了传统技术的局限,能检测出发生了物质数量、结构以及构象变化但缺乏明显形态学变化的脑挫裂伤,从而实现早期精准地诊断脑挫裂伤。FTIR-mapping虽已能绘制某些损伤组织的红外光谱病理图像,但其在法医病理学中的价值有待全面深入开发。本文综述了脑挫裂伤的法医病理学研究进展以及傅里叶变换红外面扫描成像技术在法医病理学中的应用。     

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae),

Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high‐content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti‐ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High‐content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.     

Applications of Blue Light‐curing Acrylic Resin to Forensic Sample Preparation and Microtomy

This study discusses the results of an evaluation of a one‐part blue light‐curing acrylic resin for embedding trace evidence prior to the preparation of thin sections with a microtome. Through a comparison to several epoxy resins, the physical properties relevant to both trace evidence examination and analytical microscopy in general, including as viscosity, clarity, color, hardness, and cure speed, were explored. Finally, thin sections from paint samples embedded in this acrylic resin were evaluated to determine if, through smearing or impregnation, the resin contributed to the infrared spectra. The results of this study show that blue light‐curing acrylic resins provide the desired properties of an embedding medium, generate high‐quality thin sections, and can significantly simplify the preparation of paint chips, fibers and a multitude of other types of microscopic samples in the forensic trace evidence laboratory.     

Interpreting the Effects of Burning on Pre-incineration Saw Marks in Bone

This study examined the effects of fire on the features associated with saw marks in bone. Both class and individual characteristics were examined using stereomicroscopy and scanning electron microscopy (SEM). Twenty-four semifleshed Sus scrofa L. tibiae were sawed into three sections with the middle section having deep and shallow false starts. Twelve saw blades of varying age and type were each used to cut two tibiae. In each case, the first tibia was burned in an outdoor open fire to the point of partial calcination. The second tibia, our control, was macerated using a heated enzyme solution. Controls and burned specimens were examined for the following characteristics: breakaway spur, tooth hop, false start, exit chipping, tooth imprint, breakaway notch, pull out striae, kerf flare, and blade drift. In general, there was parity in the observed characteristics in the burned samples using the SEM and the stereomicroscope. SEM observation, however, provided enhanced images, with the addition of observing individual tooth imprints, previously not visible. Therefore, this study recommends using an SEM for the examination of saw cuts in burnt bone.     

Diagnosing death from burn shock

Morphological changes were studied in the organs of 28 victims dead from burn shock at the age of 20-65 years and in rabbits in which burn shock was induced. Histological sections were stained with hematoxylin and eosin, Sudan III, by Regaud's method; brain sections were stained by Nissl's and Brachet's methods and examined by electron microscopy. Morphological criteria of burn shock were determined, underlying the specific features of thermal injury.     

Differentiation of black gel inks using optical and chemical techniques

Gel ink pens have become a common writing instrument in the United States. Questioned document examiners often attempt to optically differentiate gel inks from each other and from other non-ballpoint ink writings (e.g., those from roller-ball pens). Since early formulations were primarily pigment-based, they do not elute when analyzed by thin-layer chromatography. However, recent gel ink formulations (i.e., within the past five years) include dye-based inks that can be easily separated. This study differentiates black gel inks using optical and chemical techniques. The techniques include: microscopy, visible and near infrared reflectance, near infrared luminescence, thin-layer chromatography (TLC), spot tests, and gas chromatography/mass spectrometry (GC/MS). As a result of this study a flow chart has been developed allowing for a systematic determination of a questioned ink. In addition, an analysis of volatile compounds found in gel inks revealed that there are some unique ingredients that may be found in gel inks that are not typically found in other non-ballpoint inks.     

A method for the longitudinal sectioning of single hair samples

A simple technique is presented for the longitudinal sectioning of hair samples without the need for any embedding medium. The technique applies to single hair samples that are placed in a groove of a particular depth and can either be scraped or cut to the desired level. Planar sections are obtained that reveal the internal structure of the sample and are ideal for the application of surface analytical techniques for the study of internal transverse and longitudinal distributions of xenobiotics.