Immunohistochemical study of blood group activities in severely burned human tissue

The blood, livers and lungs obtained from donors or cadavers of known blood groups were experimentally burned, while the temperatures inside the tissue specimens were automatically measured. All the distinguishable portions with various thermo-changes in each burned tissue specimen were examined for their blood-group activities A, B, Lea, Leb and P1 by means of the immunofluorescence and immunoperoxidase techniques. In the layer immediately before charring of the blood masses (tissue temperature: ca. 200-250 degrees C), the activities A, B, Lea, Leb and P1 could be specifically demonstrated on the erythrocyte membranes. In case of the burned livers and lungs, the A- and B-activities could be detected in the small blood vessels in the layer immediately before charring (ca. 200-250 degrees C), though the layer was so severely thermo-changed, that their original tissue structures could be no more observed. In the severely thermo-coagulated, porous and hardened layer of both organ tissues, the A- and B-activities could be demonstrated on the endothelial cells of the hepatic sinus and small blood vessels (especially in the alveolar walls). The simply thermo-coagulated inner portions well retained their original tissue structures and the A- and B-activities remained on the epithelial cells of the alveoli and bronchioles of the lung as well as the cells described above. The Lewis blood-group activities could be demonstrated on the epithelial cells of the bronchioles in the simply thermo-coagulated inner portion of the lung. The P1-activity could not be demonstrated in the burned tissues of the livers and lungs.     

Human-type blood group activities on chimpanzee erythrocytes with special reference to M and N

Human-type blood group activities on the red blood cells (RBCs) of three chimpanzees were individually examined with commercial mouse monoclonal antibodies (anti-A, -B, -H, -M, -N, -Lea, and -Leb) as well as lectins (UEA-I and VGA) and conventional polyclonal antisera for the systems ABO, MN, Lewis, Rh-Hr, P, Kell, Kidd, Duffy, and Lutheran. For further analysis of the MN antigens, treatment of the RBCs with sialidase, trypsin, and chymotrypsin were employed. The activities recognized among the three chimpanzees were A, H, M, N, Leb, c, S, k, and Jka. The RBCs of the three individuals possessed the A antigen which showed the same serologic activity as the human A1. Those chimpanzee RBCs showed higher H-activity than the human A1 RBCs. The Lewis b activity was revealed by the absorption-elution method. The RBCs of the three individuals showed a reactivity to the polyclonal anti-M reagents, which was affected by both the sialidase and trypsin treatment. The RBCs of two individuals were agglutinated with the monoclonal anti-N. The receptor was sensitive to sialidase and chymotrypsin. The RBCs of the three individuals, however, did not react with the monoclonal anti-M or with one of the polyclonal anti-N. These results indicate structural differences in the glycophorins and MN antigens between the human and chimpanzee.     

Immunohistochemical study of the pattern of distribution of ABO blood group substances in male genitalia

The distribution of the ABH antigens was investigated in 11 different sections of the male genitalia in 53 autopsy cases; the peroxidase-antiperoxidase technique was used. Specific staining of the epithelia differed considerably among and between individuals. Nonsecretors showed a tendency toward less staining in the epithelia, whereas in the endothelia there was no difference. A2 cases could be recognized, as the endothelia were marked almost completely with anti-H. In A2B nonsecretor epithelia and endothelia, there was only a minimal reaction with anti-A and anti-H. Spermatozoa were irregularly stained in the ampulla of the vas deferens, whereas in the testis and epididymis no reaction could be found.     

Fatal intoxications in the age group 15-34 years in Denmark in 1984 and 1985. A forensic study with special reference to drug addicts

This survey deals with the Danish part of a study on fatal intoxications conducted in the Nordic countries in 1984 and 1985 with special reference to drug addicts. There were 315 cases of fatal intoxications in people 15-34 years of age. These were examined at the Forensic Institutes in Denmark and described with reference to cause of death, sex, age and drug addiction. Of the deceased, 194 were drug addicts according to a specific definition of this term. Women accounted for 28% of all the fatalities investigated in the study and 24% of those in addicts. More than 90% of the deaths were caused by drugs, with ethanol as a contributory factor in approximately 40% of cases. Deaths caused by heroin/morphine predominated, causing 50% of the deaths among drug addicts, but legal drugs, such as dextropropoxyphene, methadone and ketobemidone were also frequent causes of death in this group. In half the cases the concentration of morphine in blood following injection of heroin/morphine was found to be equal to or less than 0.5 mumol/kg, and in only about one-tenth of cases was the blood concentration above 2.0 mumol/kg.     

Determination of total hemoglobin in forensic blood samples with special reference to carboxyhemoglobin analysis

For the determination of total hemoglobin (Hb) in blood containing elevated carboxyhemoglobin (COHb), a newly developed reagent containing a 100-fold concentration of ferricyanide (20 g/l) and a 2-fold concentration of Sterox SE was compared with a standard reagent (0.2 g/l ferricyanide), the reagent of van Kampen and Zijlstra, using forensic blood samples and experimentally heated blood samples. There were no significant differences between the spectra of hemiglobincyanide (HiCN) solution produced with our reagent and the van Kampen and Zijlstra reagent using experimentally heated blood samples. Although the spectra of HiCN changed gradually with increased heating time and with the passage of time after mixing, the absorbance at 540 nm (A540) did not change until at least 120 min for both the reagents. When forensic blood samples containing elevated COHb were mixed with the van Kampen and Zijlstra reagent, total-Hb concentrations determined 5 min after mixing were 10-20% higher than those determined after 180 min. The overestimates of total Hb determined after 5 min resulted in comparable underestimates of percentage saturation of COHb (COHb%) when COHb% was obtained from the ratio of COHb content, determined by gas chromatogrpahy, to total-Hb concentration in blood. However, there was an extremely good correlation between the values of total Hb in forensic blood samples determined with the van Kampen and Zijlstra reagent after 180 min and those determined with our reagent after 5 min. From the results obtained, our reagent proved to be suitable for the determination of total Hb in forensic science practice.     

Immunohistochemical contribution to the study of morphine metabolism in Calliphoridae larvae and implications in forensic entomotoxicology

Morphine was detected by immunohistochemistry on sections of third stage larvae of Calliphora vomitoria (Diptera, Calliphoridae) reared on minced beef meat previously treated with morphine hydrochloride. The detection was performed with an avidin-biotin-peroxidase-complex method. Positive specimens showed specific staining of the haemolymph and a more intense immunoreaction in an area located at the limit between exocuticle and endocuticle. These results constitute an evidence of morphine accumulation inside the cuticle of Diptera larvae during their development. During the pupariation, the larval cuticle is transformed into the sclerotized puparium. This study consequently points out the possibilities of analyzing empty pupariae when suitable tissues or living necrophagous insects are absent.     

Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma

Our headspace gas chromatographic flame ionization detection (HS-GC-FID) method for ethanol determination showed slightly, but consistently, low ethanol concentrations in whole blood (blood) in proficiency testing programs (QC-samples). Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T degrees ) of 70 degrees C and 20 min equilibration time (HS-EqT). Full factorial designs were used to study the variables HS-T degrees (50 degrees -70 degrees C), HS-EqT (15-25 min), ethanol concentration (0.20-1.20 g/kg) and storage at room temperature (0-6 days) with three sample-sets; plasma, hemolyzed blood and non-hemolyzed blood. A decrease in the ethanol concentration in blood was seen as a nearly equivalent increase in the acetaldehyde concentration. This effect was not observed in plasma, indicating chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells. The variables showed different magnitude of effects in hemolyzed and non-hemolyzed blood. A decrease in ethanol concentration was seen even after a few days of storage and also when changing the HS-T degrees from 50 to 70 degrees C. The formation of acetaldehyde was dependent on all the variables and combinations of these (interactions) and HS-T degrees was involved in all the significant interaction effects. Favorable instrumental conditions were found to be HS-T degrees of 50 degrees C and HS-EqT of 15-25 min. The ethanol concentrations obtained for the range 0.04-2.5 g/kg after analyzing authentic forensic blood samples with a HS-T degrees of 50 degrees C were statistically significantly higher than at 70 degrees C (+0.0154 g/kg, p < 0.0001, n = 180). In conclusion, chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells has been shown to contribute to lowered ethanol concentrations in blood samples. Storage conditions before analysis and the headspace equilibration temperature during analysis were important for the determination of blood ethanol concentrations.     

Fatal intoxications in the Nordic countries. A forensic toxicological study with special reference to young drug addicts

Fatal intoxications in the 15-34 age group in the five Nordic countries during the years 1984 and 1985 (Sweden only in 1984) were investigated. The known drug addicts were studied separately. The highest incidence of intoxications, calculated per 10(5) population, was found in Finland (11.3), followed by Denmark (10.3), Sweden (8.5), Iceland (7.2) and Norway (6.6). The percentage of intoxications caused by drugs was 92 in Denmark, 71 in Norway, 66 in Sweden, 50 in Finland and 17 in Iceland. Ethanol intoxications were seen 5-7 and 2-3 times as frequently in Finland and in Iceland, respectively, than in the other three countries. Carbon monoxide intoxications accounted for two-thirds of all fatal intoxications in Iceland. Drug addicts accounted for 62% of all fatal intoxications in the Danish material. The corresponding figures were 33% in the Norwegian, 16% in the Swedish and 5% in the Finnish material. No deaths in drug addicts were found in Iceland. Most drug addicts in Denmark, Norway and Sweden died of hard drugs and most in Norway and Sweden, from heroin or morphine, whereas in Denmark other strong analgesics, such as methadone, dextropropoxyphene and ketobemidone, accounted for 40% of all hard-drug-related fatal intoxications. To a certain extent the results reflect differences in the legal autopsy routines in the various Nordic countries. However, the ascertainment of drug addicts is assumed to be near-complete in each country.     

Immunocytochemical study on the ultrastructural localization of human-type ABO (H)-blood group activities in a macaque (Macaca irus)

The immunocytochemical study on the ultrastructural localization of human-type ABO(H)-activities in a crab-eating macaque (Macaca irus) was carried out by using postembedding and immuno-gold staining method. The tissue specimens examined were the esophagus, stomach (St), small intestine (Si), large intestine, liver, kidney, and pancreas. The specimens from these organs and submandibular gland (Sg) of a human (O-group) were used as staining reaction controls. Primary and secondary antibodies were commercially obtained mouse monoclonal anti-A, -B, -H (IgM), and goat anti-mouse IgM labeled with colloidal gold particles (luminal diameter 20 nm), respectively. The results were as follows: (1) In macaque specimens, only A-activity could be observed as the location of gold particles on the peripheral rim of serous secretory granules (Sg) and of epithelial cells (esophagus), the mucous droplets in epithelial cells and brush border (St, Si), the intracellular secretory canaliculi [ISC (St)] and the zymogen granules and secretory ducts (pancreas). Gold particles could be also noted at the Golgi apparatus and nascent secretory granules. (2) By periodic acid-thiocarbohydrazide-osmium tetroxide (PA-TCH-OS) reaction, hexose-rich neutral mucopolysaccharides were noted on the peripheral rim of serous secretory granules (Sg), the mucous droplets (St, Si), the ISC (St), and the brush border (Si). Such a distributional pattern corresponded well with that of gold particles, indicating that the substances were responsible for ABO(H)-activities.     

过敏性休克死亡尸体咽喉及肺组织中P物质免疫组化染色观察

目的探讨过敏性休克死亡法医学鉴定的形态学依据。方法15例明确诊断为过敏性休克死亡的尸体(设为实验组),取其咽喉部及肺组织,石蜡切片,分别进行HE染色及免疫组化超敏SP法染色,观察P物质染色变化,并通过图像分析系统对其阳性结果进行分析;20例因交通事故致颅脑损伤死亡的尸体相同组织作为对照。两组所得数据采用SPSS10.0统计软件进行t检验分析。结果实验组咽喉组织充血、水肿,复层扁平上皮和假复层纤毛柱状上皮P物质免疫组化染色均呈棕黄色染色,固有层内腺体上皮细胞及肌层染色变浅;对照组咽喉复层扁平上皮和假复层纤毛柱状上皮几乎不着色,固有层内腺体上皮细胞及肌层仅有少量棕黄色染色(实验组平均灰度值113.78,阳性信号面积均值17795.75;对照组平均灰度值104.63,阳性信号面积均值8953.50)。实验组支气管粘膜上皮呈深棕黄色染色,粘膜肌层染色变浅;对照组支气管粘膜上皮几乎不着色。(实验组平均灰度值136.32,阳性信号面积均值17194.84;对照组平均灰度值96.52,阳性信号面积均值8416.75)。计算各组的阳性指数PI,咽喉分别为50.62±10.04和23.42±6.84;支气管分别为58.60±12.3和20.31±5.68。两组数据差异具有显著性意义(P<0.01)。结论P物质免疫组化染色可能为过敏性休克致猝死的法医学鉴定提供形态学依据。     

Significance of various analytical methods with reference to the causes and manners of death in alcoholics

It was the aim of the present investigation to apply a broad spectrum of analyses to forensic autopsies of alcoholics in order to estimate the significance of the various analytical methods with reference to the cause and manner of death. The analyses were performed on a consecutive series of 73 medico-legal autopsies in alcoholics. Both extensive histology as well as toxicology and microbiology were used. The microbiology did not contribute substantially to the determination of the cause of death, while histology was decisive in six cases. Toxicology analyses were necessary for determining the cause of death in 37 cases. The results of the investigation may help in the selection of analytical priorities.     

Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.     

急性心肌缺血再灌注HSP70及c-Fos的免疫组织化学研究

运用免疫组织化学方法 ,观察心肌缺血后不同时间再灌注HSP70及Fos蛋白在心肌组织不同区域表达的规律 ,为心肌缺血 /再灌注损伤所致心性猝死的法医学诊断提供形态学依据。结扎大鼠左冠状动脉前降支建立急性心肌缺血 /再灌注模型 ,取缺血区域 (左冠脉前降支供血区 )及非缺血区 (右心室 )心肌组织。结果显示 :对照组正常心肌组织无Fos蛋白及HSP70表达。Fos蛋白在心肌缺血 15min再灌注 0 5h后即在缺血区域表达 ,随着再灌注时间延长其表达增强 ;非缺血区域 1h始有表达 ,缺血区域表达明显强于非缺血区域。HSP70在心肌缺血区域再灌注 1h后始有表达 ,随着再灌注时间延长其表达增强 ;非缺血区域心肌 2h始有表达 ,且明显弱于缺血区域。同时发现再灌注早期HSP70及Fos均先于心肌内层表达 ,随着时间延长其表达向心肌外层扩展。Fos在再灌注 0 5h时主要在心肌内层表达 ,1h时已扩展到心肌全层 ,4h时其心肌外层表达明显强于心肌内层。HSP70在再灌注 1、 2h时主要在心肌内层表达 ,4、 6h时表达扩展至全层心肌。心肌缺血 /再灌注早期不同时间HSP70及Fos表达有不同的区域性及强度 ,此可为心肌缺血 /再灌注的早期诊断提供一项新的辅助诊断依据     

不同保存条件下呋喃丹及其代谢物在血液中的稳定性研究

目的研究呋喃丹及其代谢物呋喃酚在不同条件保存血液中的稳定性,为呋喃丹中毒案件的法医学鉴定提供实验依据。方法犬经口灌胃4LD50(13.5mg/kg)呋喃丹致死后,取血液分为五等份,分别为添加1%氟化钠(1%NaF)、添加2.5mg/m L枸橼酸钠(NC)、20℃、4℃和-20℃保存实验组。于保存当时(0d)、5d、7d、15d、40d、83d和150d取上述样品,多反应离子检测(MRM),气相色谱-质谱联用(GC-MS/MS)法检测其中呋喃丹及呋喃酚含量。结果血液中呋喃丹的含量随保存时间均呈下降趋势,7d显著下降(P0.05),之后下降缓慢。不同条件保存血液中呋喃酚的含量均呈先升高后下降趋势。枸橼酸钠和氟化钠可加快血液中呋喃丹的分解。结论呋喃丹及其代谢物呋喃酚在保存检材中均可发生分解,20℃保存分解较快,4℃和-20℃保存分解速度较慢,不适宜用枸橼酸钠或氟化钠作抗凝剂或防腐剂。在呋喃丹的相关案件的法医学鉴定中,生物检材应注意冷藏、冷冻保存,并尽快送检。     

Pulmonary surfactant-associated protein A levels in cadaveric sera with reference to the cause of death

Pulmonary surfactant-associated protein A (SP-A) is an exclusively lung specific protein, and is considered to leak into the blood stream in alveolar septal damage. In this study we examined the serum SP-A level in forensic autopsy materials using an enzyme immunoassay with monoclonal antibodies to assess the postmortem level in relation to the cause and mode of death. Although a gradual postmortem degradation should be taken into consideration, topological relationship of serum level seemed to be fairly stable (arterial> or =venous blood in most cases), indicating no evident influence of postmortem diffusion. Significant elevation of serum SP-A (76.7-250 ng/ml in left heart blood) was observed in hyaline membrane diseases from various causes independent of the postmortem intervals (<30 h). However, mean SP-A levels in postmortem heart blood were usually low in asphyxia including hanging, strangulation and choking (left, 25.5 ng/ml; right, 22.3 ng/ml), polytrauma (left, 13.1 ng/ml; right, 9.0 ng/ml) and stab wound to the neck (left, 34.1 ng/ml; right, 29.4 ng/ml). Prominent elevation was noted in a case of fatal strangulation with complication of idiopathic interstitial pneumonia, and also in some deaths due to drowning, burns in fires, blunt and gunshot chest injuries. These findings indicated that postmortem elevation of serum SP-A levels was associated with alveolar septal damage due to inflammation, mechanical and physical stresses, which caused leakage of SP-A into the bloodstream.     

The seromuscular tear and other intestinal lesions in the seatbelt syndrome: a clinical and pathologic study of 29 cases

The authors describe the clinical and pathologic findings in 29 patients with injuries from motor vehicle accidents. The seromuscular tear (SMT), the hallmark intestinal injury of the seatbelt syndrome, is an unambiguous lesion similar in all segments of bowel and is caused by a tear that separates the inner muscularis from the submucosa. It is characterized by (1) a wedge that strips the submucosa from the inner circular muscle; (2) a bending retraction of the torn muscularis toward the uninvolved bowel wall; (3) mucosal-submucosal fold effacement, causing the mucosa-submucosa bridge spanning the tear to become paper thin; and (4) the vulnerability of this bridge to ischemia that in 35% of the tears studied culminated in incipient or frank perforations and/or gangrene. Large SMTs, particularly the circumferential degloving type, are most prone to develop these complications. These findings militate against the idea that the SMT is a trivial lesion. The SMT occurred in 90% of patients in this report and accounted for 65% of all intestinal lesions. Seventy-three percent of the tears developed in the colon, and one third of all SMTs occurred in the sigmoid colon. Two thirds of all intestinal and mesenteric injuries clustered in three sites: the ileocecal region, the sigmoid colon, and the jejunum. Perforations were the principal lesion in the jejunum and SMTs at the other two locations. Ninety percent of patients experienced two or more intestinal lesions. This suggests the simultaneous action of different traumatic mechanisms on the bowel and its mesenteries in seatbelted persons who are in motor vehicle accidents.