Further evaluation of immunocytochemical staining in the diagnosis of early myocardial ischaemic/hypoxic damage

The pattern of immunocytochemical staining with antibodies to caeruloplasmin, myosin, myoglobin and C-reactive protein seen in myocardium taken from deaths with macroscopic evidence of myocardial infarction and/or significant coronary artery atherosclerosis and from deaths with neither of these lesions has been correlated with H&E, PTAH and HBFP staining of myocardium and circumstances of each death indicative of antemortem hypoxia and/or ischaemia. Loss of staining with these antibodies correlated well with fuchsinorrhagia and both techniques are more sensitive than H&E and PTAH staining in the detection of early ischaemic/hypoxic damage to myocardium. However, their sensitivity is such that they appear to detect agonal changes and, therefore, cannot be used for specific diagnosis of early myocardial infarction.     

Immunohistochemical study of fibronectin for postmortem diagnosis of early myocardial infarction

The postmortem diagnosis of early myocardial infarction has been a puzzling problem in forensic practice. In the present study, an immunohistochemical study of fibronectin (FN) was performed for the first time on 34 autopsy hearts to determine early myocardial infarction with streptavidin/biotin/peroxidase technique. Five cases of definite myocardial infarction showed positive FN staining of cardiomyocytes; of 18 cases where early myocardial infarction was suspected, positive FN staining of cardiomyocytes was found in 15 cases, but no such staining was seen in 11 non-cardiac death controls. The results led to the conclusion that positive FN staining in cardiomyocytes is a reliable marker of acute myocardial infarction and could be used as a new, sensitive method for the postmortem diagnosis of early myocardial infarction. It is worth noting that all cases in this study were autopsied between 8 h and 4 days after death and 5 cases had been fixed in 10% formalin for over 10 years. FN immunohistochemistry still gave satisfactory results in those cases. It seemed that FN was not affected by postmortem autolysis and formalin-fixation and could be used in routine forensic practice, especially for retrospective analysis of cases.     

Evaluation of a lactate dehydrogenase-nitro blue tetrazolium method of detecting unapparent myocardial infarction using a standardised sampling technique

Using a standardised block technique, the microscopic lactate dehydrogenase-nitro blue tetrazolium (LDH-nitro BT) and conventional haematoxylin and eosin staining methods were used to investigate the presence of myocardial infarction in a series of fifty forensic autopsies. The microscopic LDH-nitro BT method was found to be reliable; however, it did not reveal the presence of myocardial infarction in any of the cases where this had not been suspected.     

早期心肌缺血猝死心肌NF-κB p65的表达研究

目的探讨核因子NF-κBp65（NF-κB p65）在心肌早期缺血猝死后诊断中的法医学价值。方法将收集的案例心肌蜡块分为3组：正常对照组（3例）、早期心肌缺血组（14例）、心肌梗死组（8例）,采用免疫组织化学技术（SP法）,观察猝死心肌内NF-κBp65的表达情况,并对其结果进行半定量分析。结果早期心肌缺血组和心肌梗死组的心肌细胞胞浆内及细胞核内均出现NF-κBp65的表达,且早期心肌缺血组与心肌梗死组表达强度无差异。结论NF-κBp65可以作为早期心肌缺血猝死的一个辅助诊断指标。     

Heart fatty acid binding protein as a marker for postmortem detection of early myocardial damage

Depletion of heart fatty acid binding protein (H-FABP) from cardiomyocytes with varying post-ischemia intervals was studied in acute myocardial infarction (AMI) model of rat, and 22 human autopsy cases were studied with streptavidin-peroxidase conjugated method (S-P). It was observed that as early as 15 min after ischemia, the depletion of H-FABP could be detected in model rats. With the ischemic time prolonged, the depletion of H-FABP was more and more evident. In all human cases with myocardial infarction, absent H-FABP staining could be found in infarcted area. And in some suspected early myocardial infarction cases, depletion of H-FABP staining could be demonstrated in areas that showed normal hematoxylin-eosin (HE) staining. The blood samples from model rats before ligation, at varying post-ischemia intervals and various postmortem time were measured for plasma concentration of H-FABP with enzyme-linked immuno-sorbent assay (ELISA) method. At 15 min after myocardial ischemia, the concentration of H-FABP was 4 times higher (546.0+/-85.3 microg/l) than that of the baseline level (103.7+/-94.1 microg/l). With the continuation of ischemic time, the concentration of H-FABP increased and peaked at 4 h (1953.5+/-405.3 microg/l), then decreased. The plasma concentration of H-FABP decreased slightly with postmortem time, but was still significant higher at any postmortem intervals than that of baseline level within 48 h after death. The results suggest that H-FABP staining can detect very early ischemic damages in human myocardium and the elevated plasma concentration of H-FABP in rat was an indicator of AMI, which was not affected by autolysis.     

结蛋白诊断早期心肌梗死的特异性研究

目的　探讨结蛋白在早期心肌梗死死后诊断的特异性。方法　应用免疫组织化学S -P法检测梗死心肌和其他非梗死性的直接或间接心肌损害的心肌结蛋白染色的变化。结果梗死心肌均可见不同程度的结蛋白缺染 ,其他非梗死性的直接或间接心肌损害的心肌中 ,如心脏挫伤、心肌炎、出血性休克、电击死、机械性窒息、有机磷中毒等 ,也有不同程度的结蛋白缺染。结论　应用结蛋白免疫组织化学方法诊断早期心肌梗死需慎重     

VEGF在早期心肌梗死死后诊断中的应用

运用免疫组化方法和图像分析与统计学处理系统 ,对人体心脏标本VEGF的表达进行定量研究。结果显示 :心肌梗死组 2 4例心脏标本心肌细胞膜及血管内皮细胞膜均可见棕黄色VEGF强阳性染色 ,有些胞浆也呈阳性 ,尤以心肌梗死灶周围区域心肌细胞为甚 ;在可疑梗死组 2 1例中 ,19例心肌细胞膜及血管内皮细胞膜VEGF强阳性 ,2例为弱阳性 ,未见阴性 ;在正常心脏对照组 16例中 ,仅 1例见心肌细胞膜及血管内皮细胞膜散在棕黄色VEGF弱阳性 ,其余均为阴性。图像分析与统计学处理结果表明 ,对照组VEGF阳性指数 ( 0 3 0± 0 10 )明显低于心肌梗死组 ( 12 46± 3 2 6,P<0 0 1)和可疑梗死组 ( 11 70± 3 5 6,P <0 0 1) ,后两组之间阳性指数无明显差异 (P >0 0 5 )。提示运用免疫组化染色方法结合图像分析处理技术定量检测心肌局部VEGF的表达可望作为因早期心肌缺血导致心脏性猝死死后诊断较为客观的病理形态学指标之一。     

Immunostaining by complement C9: a tool for early diagnosis of myocardial infarction and application in forensic medicine

Before the first 12 hours, diagnosis of early myocardial infarctions is always difficult for forensic pathologists. We tested complement C9 expression in 121 formalin-fixed and paraffin-embedded heart samples by an immunohistochemical procedure. The heart specimens were separated into four groups: 33 cases in group 1 with typical ischemic damages histologically located, 20 cases in group 2 with death related to myocardial infarction on the basis of ischemic presentation on electrocardiogram but no obvious histological ischemic damage, 35 cases in group 3 with severe coronary disease without cause of death found at the autopsy, and 33 cases in group 4 without sign of myocardial infarction and without coronary disease. In the first group, all 33 heart samples showed a well-defined C9 expression in the necrotic areas. The second group in 17 of 20 cases showed positive areas for C9 expression. In the other three heart specimens, only few stained cells were observed whereas the painful symptoms had begun less than 1 h before death. The third group showed C9 immunopositive areas in six of 35 cases, few stained cells in 8 cases, and no C9 deposition in the 21 other cases. The last group showed no staining area. To avoid nonspecific C9 staining due to tissue autolysis, we studied C9 expression during a controlled putrefactive process in four cases included in group 1; staining was found only in infarcted myocardial areas, and was observed up to ten days. Specificity of C9 expression was evaluated to be 100% [89.4 to 100%] and sensitivity to be 85% [62.11 to 96.79%]. In conclusion, evaluation of immunohistochemical expression of C9 appears to be a highly sensitive and specific marker of early myocardial infarction, useful in forensic medicine if survival is more than 1 h after the beginning of myocyte damage.     

结蛋白诊断早期心肌梗死的特异性研究

目的 探讨结蛋白在早期心肌梗死死后诊断的特异性。方法 应用免疫组织化学Ｓ－Ｐ法检测梗死心肌和其他非梗死性的直接或间接心肌损害的心肌结蛋白染色的变化。结果 梗死心肌均可见不同程度的结蛋白缺染,其他非梗死性的直接或间接心肌损害的心肿,如心脏挫伤、心肌炎、出血性休克、电击死、机械性窒息、有机磷中毒等,也有不同程度的结蛋白缺染。 结论 应用结蛋白免疫组织化学方法诊断早期心肌梗死需慎重。     

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.     

急性心肌缺血猝死心肌中血红素加氧酶-1的表达

目的观察血红素加氧酶-1（HO-1）在急性心肌缺血猝死心肌细胞胞浆中的表达情况。方法应用免疫组织化学技术（SP法）,对27例可疑早期心肌缺血猝死（早期梗死组）、10例心肌梗死猝死（心肌梗死组）、11例冠心病非心性猝死（对照组1）以及10例正常心肌（对照组2）中HO-1的表达进行观察分析。结果早期梗死组与心肌梗死组在心肌细胞胞浆中HO-1均呈强阳性表达,对照组1和对照组2心肌细胞胞浆中HO-1弱阳性表达或无表达。早期梗死组和心肌梗死组与对照组1和对照组2之间心肌细胞内HO-1表达的差异有显著性意义（P（0.01）;镜下各组心肌细胞胞浆内HO-1表达的阳性面积率及平均光密度差别明显。结论 HO-1可以作为急性心肌缺血所致心性猝死的一个诊断指标。     

Death following spontaneous recovery from cardiopulmonary arrest in a hospital mortuary: 'Lazarus phenomenon' in a case of alleged medical negligence

We report a possibly first forensic autopsy case of death following a spontaneous recovery from cardiopulmonary arrest (CPA) after clinical declaration of death: 'Lazarus phenomenon'. A 65-year-old male with congenital deafness and dumbness was found unconscious in his room at a public home. During pre-hospital and clinical resuscitation including defibrillation and medications for about 35 min, CPA persisted under electrocardiographic (ECG) monitoring and therefore, his death was pronounced. However, about 20 min later, a police officer who had been called for the postmortem investigation found the patient moving in the mortuary. The patient subsequently showed typical ECG signs and laboratory findings of early inferior wall myocardial infarction and died 4 days later. The forensic autopsy, due to alleged medical negligence, revealed myocardial infarction with thrombotic occlusion of the right coronary artery and secondary hypoxic brain damage. The present case and the related clinical literature suggest that, especially in cases of acute myocardial infarction in elderly patients, a careful observation to confirm death after discontinuation of resuscitation is recommended to provide appropriate medical care, irrespective of the quality or duration of advanced life supporting efforts.     

抗肌动蛋白单克隆抗体在早期心肌缺血诊断中的应用

应用抗肌动蛋白单克隆抗体（HHF35），对10例正常心肌、6例已知心肌梗死、18例可疑早期心肌缺血组织进行石蜡切片免疫组化染色。结果：正常心肌阳性，梗死心肌阴性，18例可疑病例中14例呈不同程度弱阳性或阴性。表明心肌发生了缺血性改变，对比清晰，特异性强，对心肌缺血诊断有明显的应用价值。     

白细胞介素-18在早期心肌缺血和心肌梗死心肌的表达

目的探讨白细胞介素-18(IL-18)对早期心肌缺血(EMI)和心肌梗塞(MI)死后诊断的法医学意义。方法筛选出34例心脏标本,分EMI(11例)、MI(10例)和对照(13例)组,检测心肌IL-18免疫组化染色(SP法)反应,并经全自动图像分析系统分析得到平均光密度值(AOD),所得数据进行统计学分析。结果对照组心肌IL-18免疫组化染色呈阴性,EMI组心肌呈阳性,MI组心肌呈强阳性;其AOD值分别是0.1193±0.0649、0.3827±0.0868、0.4792±0.0884,3组之间差异均具有显著性意义(P<0.05)。结论作为判断EMI和MI的指标之一,IL-18对EMI和MI的死后诊断具有法医学意义。     

Sudden cardiac death and myocardial ischemia indicators: a comparative study of four immunohistochemical markers

The postmortem diagnosis of acute myocardial infarction represents a current challenge for forensic pathologists, particularly when death occurs within minutes to a few hours after the ischemic insult. Among the adult population the single most important cause of sudden cardiac death (SCD) is the well-known atherosclerotic coronary artery disease, commonly asymptomatic or unrecognized. The recognition of early myocardial damage using routine hematoxylin and eosin (H&E) staining is possible only if death has occurred at least 6 hours after the onset of the ischemic injury. The usefulness of immunohistochemical markers to the diagnosis of early myocardial damage has been recently suggested because most of them can be visible even serologically as early as few minutes after the beginning of the symptoms. To evaluate the usefulness of plasma and cellular antigens, their distribution patterns have been studied among a group of 18 SCD cases in which a myocardial ischemia was strongly suspected. For the present study, 4 markers have been selected on the basis of their different diagnostic potential as follows: among the plasma markers the C5b-9 and fibronectin, among the cellular markers the myoglobin and cardiac troponin. The results show that only the study of multiple markers such as those selected can provide enough evidence of myocardial ischemia and/or necrosis, supporting the final diagnosis of SCD. No single immunohistochemical staining is ideal for diagnosing early myocardial ischemia but a set of markers can improve the ability of forensic pathologists to detect ischemic areas when no macroscopic or microscopic evidence of necrosis is available. However, the interpretation of data obtained in each individual cannot be isolated from the overall assessment of the factors (cardiopulmonary resuscitation and/or agonal artifacts) that can affect the expression of each marker.     

Cx45、Cx40及TNF-α在梗死心肌中表达及作用

目的检测缝隙连接蛋白Cx45、Cx40及TNF-α在梗死心肌中的表达情况,探讨三者的作用及相关性。方法应用免疫组织化学染色(S-P免疫组化染色法)及q RT-PCR检测缝隙连接蛋白Cx45、Cx40及TNF-α分别在30例早期心肌缺血猝死组、30例晚期心肌梗死猝死组及20例正常心肌组中的表达情况,并分析三者的作用及相关性。结果免疫组化结果:在两个猝死组中,Cx45表达高于正常心肌组,Cx40表达较正常心肌组明显减少,TNF-α呈高表达,三者在三组中的表达存在显著差异,有统计学意义(P0.05)。q RT-PCR结果显示:Cx45、Cx40及TNF-α三者的mRNΑ表达与各自蛋白本身表达相一致,在三组中表达存在显著差异,差异性有统计学意义(P0.01)。Cx45、Cx40、TNF-α在三组心肌中存在相关性。结论缝隙连接蛋白Cx45、Cx40及TNF-α将可能成为心源性猝死死因的辅助诊断指标。     

早期心肌缺血心肌细胞凋亡的实验研究

探讨早期心肌缺血心肌细胞凋亡的意义。采用DNA缺口末端标记法 (TUNEL)对大鼠实验性心肌缺血早期 (6h内 )不同时间缺血损伤区心肌细胞凋亡的情况进行观察。结果 :缺血 1h在缺血区域发现少数散在的凋亡阳性细胞 ,3h达高峰 ,随后下降。正常区域未发现凋亡细胞。缺血边缘区域也在缺血 1h局部开始出现心肌细胞凋亡 ,并随缺血时间延长心肌细胞凋亡指数增加 ,缺血 5h达高峰。表明凋亡是早期缺血性心肌细胞损伤的主要方式 ,心肌凋亡检测可望为早期心肌梗死的死后诊断提供一个灵敏客观的新方法。     

肌红蛋白诊断早期心肌梗死的特异性研究

目的　探讨肌红蛋白在早期心肌梗死死后诊断的特异性。方法　应用免疫组织化学S -P法检测梗死心肌和其它非梗死性的直接或间接心肌损害的心肌肌红蛋白染色的变化。结果　梗死心肌均可见不同程度的肌红蛋白缺染 ,其它非梗死性的直接或间接心肌损害的心肌中 ,如心脏挫伤、心肌炎、出血性休克、电击死、机械性窒息、有机磷中毒等 ,也有不同程度的肌红蛋白缺染。结论　应用肌红蛋白免疫组织化学方法诊断早期心肌梗死需慎重     

Right ventricular damage due to pulmonary embolism: examination of the number of infiltrating macrophages

To investigate the pathological changes in the heart induced by pulmonary embolism, 20 autopsy cases of pulmonary embolism and 10 control cases of acute death from traumatic injury were examined. Adding to the routine hematoxylin-eosin (HE) staining, immunostaining with CD68 pan-macrophage marker was performed on the specimens obtained from both right and left ventricular walls. The number of macrophages was counted semi-quantitatively in 100 random high-power fields (HPF). Although typical pathological findings of myocardial infarction was not observed in any of the cases, 16 of the 20 pulmonary embolism cases showed an increase in the number of macrophages, mainly in the right ventricular wall. Four cases showed massive macrophage infiltration in the entire right ventricular wall. It is speculated that ischemia due to pulmonary embolism may be connected to its pathogenesis.