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Simultaneous assessment of a panel of protein markers is becoming essential in order to enhance biomarker research and improve diagnostics. Specifically, postmortem diagnostics of early myocardial ischemia in sudden cardiac death cases could benefit from a multiplex marker assessment in the same tissue section. Current analytical antibody-based techniques (immunohistochemistry and immunofluorescence) limit multiplex analysis usually to not more than three antibodies. In this study, mass spectrometry-immunohistochemistry (MS-IHC) was performed by combining laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) with rare-metal-isotope-tagged antibodies as a technique for multiplex analysis of human postmortem myocardial tissue samples. Tissue sections with myocardial infarction were simultaneously analyzed for seven primary, rare-metal-isotope-tagged antibodies (troponin T, myoglobin, fibronectin, C5b-9, unphosphorylated connexin 43, VEGF-B, and JunB). Comparison between the MS-IHC approach and chromogenic IHC showed similar patterns in ionic and optical images. In addition, absolute quantification was performed by MS-IHC, providing a proportional relationship between the signal intensity and the local marker concentration in tissue sections. These data demonstrated that LA-ICP-MS combined with rare-metal-isotope-tagged antibodies is an efficient strategy for simultaneous testing of multiple markers and allows not only visualization of molecules within the tissue but also quantification of the signal. Such imaging approach has a great potential in both diagnostics and pathology-related research.

Immunohistochemistry is a well-established technique used in many research laboratories as well as in clinical diagnostics. The method allows to visualize the expression of proteins in biological tissues, as well as to evaluate this expression semi-quantitatively. For diagnosis, an optimal staining, based on a straightforward protocol, is crucial. In many sudden cardiac death cases, immunohistochemistry is the only tool enabling the diagnosis of myocardial ischemia/infarction, thus the diagnosis of the cause of death. Improvements in immunoreactions are actually possible thanks to optimized detection systems. The recently introduced detection system EnVision Flex™ by Dako allows to dramatically improve (in terms of intensity of the signal and practically absence of background) the visualization of antigens in formalin-fixed paraffin-embedded (FFPE) tissue sections. We tested this method for the detection of myoglobin and troponin T in human postmortem cases of myocardial infarction, as the results obtained by using the « classical » ABC (avidin-biotin complex) method have proven to be sub-optimal, thus rendering any interpretation very difficult, if not impossible.

The determination of vitality of skin injuries is one of the most central research areas in forensic pathology for it is often necessary to discern antemortem from postmortem damage. Typical is the case of a hanging, which should be distinguished from the postmortem suspension of a body. In this study, 15 human skin samples from ligature marks (study group) of victims of suicidal hanging and 15 uninjured skin samples (negative control group) were analyzed. In addition, 15 skin samples from ecchymoses in homicide victims with short survival intervals were investigated as a positive control group. Sections were processed for immunohistochemistry in order to detect the expression of Fibronectin, P-Selectin, FVIII, HSP-70, and MRP8. Immunohistochemical reactions were classified semiquantitatively (mild - score 1, moderate - score 2, and intense - score 3). In the ligature marks, Fibronectin was significantly less expressed compared to ecchymoses. The expression was similar to hanging marks and uninjured skin. In both ligature marks and ecchymoses, the expression of P-Selectin was significantly increased compared to uninjured skin. In both ligature marks and ecchymoses, expression of HSP-70 was significantly decreased in the epidermis compared to uninjured skin. In both ligature marks and ecchymoses, the expression of FVIII and MRP8 was significantly increased in the dermis and hypodermis compared to uninjured skin. The results of this study show that the immunohistochemical study of early inflammatory and coagulation factors could help determine the vitality of ligature marks. The combined analysis of P-Selectin, FVIII, HSP-70, and MRP-8 can be considered for this purpose. • Determination of vitality of ligature marks is important. • Immunohistochemical study of early inflammatory factors could be valuable. • P-Selectine, FVIII, HSP-70 and MRP-8 could indicate vitality of ligature marks. • Fibronectin seems not to be useful for the purpose. [ABSTRACT FROM AUTHOR]

The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB. [ABSTRACT FROM AUTHOR]

Data from the literature indicate that the pulmonary pressure rises in cases of ethanol intake. We have recently proposed a method for the detection of prevalent right ventricular damage in cases of fatal pulmonary thromboembolism and pulmonary fat embolism. In the present study, we compared the expression of the antibodies against fibronectin and C5b-9 in 19 cases of lethal alcohol intoxications (study group: 5 females, 14 males, mean age 46 years, mean blood ethanol concentration 3.5‰, min. 2.11‰, max. 5.31‰) to a group of 26 cases of fatal pulmonary thromboembolism (PE; group 2: 16 females, 10 males, mean age 56 years). Moreover, a group of 15 cases of hanging (group 3: 5 females, 10 males, mean age 50 years) as well as a group of 18 cases of myocardial infarction (group 4: 5 females, 13 males, mean age 61 years) were investigated as examples of typical cardiac damage due to global hypoxia during agony and ischemic damage, respectively. The results of this study show that fresh cardiac damage can be detected at both ventricles in cases of fatal ethanol intoxication with the antibody against fibronectin. The damage is prevalently localised at the right ventricle (RV), as already observed in cases of acute pulmonary hypertension determining right heart failure. The degree of damage at the RV in cases of ethanol intoxications is lower than the one observed in cases of fatal PE. [ABSTRACT FROM AUTHOR]

The scope of this study was to evaluate the incidence and the eventual consequences of amniotic fluid aspiration (AFA) in cases of sudden infant death. Cases of sudden infant death syndrome (SIDS; n = 113: 39 females, 74 males; mean age 4.6 months) were compared to a control group of 39 cases of explained death (14 females, 25 males; mean age 5.6 months). In each case, sections of the lung stained with hematoxylin and eosin and with the immunohistochemical reaction 34BE12 specific for cytokeratins were available. The microscope slides were observed at ×200 magnification and semi-quantitatively classified into four categories(−, +, ++, and +++). In both groups, rests of amniotic fluid could be observed up to the fourth month of life. The comparison between the two groups did not show any significant difference. In the SIDS group, immunohistochemical reactions with the antibodies CD68, MRP8, MRP14, 27E10, 25F9, CD3, CD20Cy, and CD45R0 were available for the lungs. Twelve cases with AFA were compared to a group of SIDS cases without AFA with similar age and pathological distribution to evaluate whether the presence of amniotic remnants induced inflammatory changes in the lungs. No differences emerged. This study shows that AFA is not a rare event. Even moderate to severe AFA does not necessary cause death. A correlation between AFA and SIDS could not be shown. [ABSTRACT FROM AUTHOR]