早期心肌缺血死后诊断的法医学应用研究
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摘要
心源性猝死(sudden cardiac death，SCD)系指由于心脏原因所致的突然、意外的自然死亡，占成人猝死原因的首位。在引发SCD的原因中，急性心肌缺血（acute myocardial ischemia，AMI）是最主要的原因，由于短暂的（冠状动脉痉挛）或持续的（冠状动脉闭塞）的急性心肌缺血后，冠状动脉血流量降低，心肌氧等物质供应不足，代谢产物清除减少，引起心脏电生理活动紊乱和/或心肌机械收缩功能障碍，导致大多数病人常在发病后6小时内死亡，甚至1h内死亡。心肌缺血后至少需要6小时在HE染色下才会出现光镜的形态学改变（如凝固性坏死、多灶性心肌纤维波浪样变、核固缩、收缩带坏死、炎细胞浸润等）。而在法医学实践中，早期心肌缺血（early myocardial ischemia, EMI）猝死者多在心肌缺血发生后6小时内死亡，死后常规检查除可见冠状动脉及其分支存在不同程度的动脉粥样硬化外，由缺血性损伤因素所致的病理形态学改变极难检出，使早期心肌缺血的死后诊断成为法医病理学鉴定的难点。
多年来国内外学者都在寻找敏感、稳定、实用的诊断早期心肌缺血的方法和指标，从常规HE染色、特殊染色和酶组织化学、免疫组织化学、超微结构、荧光检查、离子含量改变、心包液和血液心肌损伤标志物检测、死后影像学检查等方面进行了积极的探索，所有这些方法都从不同方面、不同程度地丰富了EMI死后诊断的内容。但是上述方法或因为操作过于复杂，或实用性不强，或因为缺乏特异性、费用过高等因素的影响而限制了它们在法医病理学中的应用，实际应用价值各家说法不一，迄今仍没有敏感、实用的方法或指标能很好解决实际办案中的死因诊断问题。
在众多研究方法中，免疫组织化学因其具有特异性强、敏感性高、实用性好等优点，已应用到EMI的研究和应用中来。近年来，许多学者陆续报道了如肌动蛋白（actin）、肌钙蛋白、心型脂肪酸结合蛋白（heart typefatty acid binding protein, H-FABP）、抗肌萎缩蛋白等蛋白分子在急性心肌缺血时从心肌细胞内脱失的现象；血浆中某些蛋白成分如补体成分、纤维连接蛋白（fibronectin, Fn）等在缺血心肌的异常分布情况；缺血心肌诱导表达某些敏感蛋白分子，如缺氧诱导因子-1α（hypoxia-inducible factor-1alpha, HIF-1α）、血管内皮生长因子等。这些研究从多角度观察了急性心肌缺血时心肌细胞发生的各种变化，部分指标在EMI心脏标本进行了验证，但考虑到缺血心肌自身演变的特点和法医学实践中案例的复杂性和特殊性，单一方法和单一检测指标的应用缺乏说服力而难以有效发挥其诊断价值，而以往这些研究为多指标联合应用提供了理论依据。同时，考虑到法医检案的特殊性，如EMI发病时间、尸体保存情况、固定时间等因素的影响，需针对不同的情况进行检测指标的组合，以期达到最好的诊断效果。
据此，本课题以大鼠急性心肌缺血模型和法医检案心脏标本作为研究对象，研究：①对比观察铁钒-苏木素-伊红染色法（Heidenhain染色）、变色酸2R-亮绿染色和苏木素-碱性复红-苦味酸染色（HBFP染色）对缺血心肌的染色能力，观察不同保存条件及10%福尔马林固定时间对染色的影响；②从不同角度选取检测指标：心肌结构蛋白或胞浆蛋白：actin(HHF35)、H-FABP、S100A1；黏附和进入心肌细胞内的血浆成分：C5b-9、Fn；缺血心肌诱导表达的产物：聚集素蛋白（clusterin，CLU）、HIF-1α、HIF-2α、含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶（adisintegrin and metalloproteinase with thrombospondin type1motifs，ADAMTS-1），观察大鼠急性心肌缺血后血浆S100A1含量的变化及9个诊断指标在大鼠急性缺血心肌中的表达变化情况；③观察不同保存条件及10%福尔马林固定时间对上述9个免疫组织化学检测指标染色的影响；④以法医病理检案心脏标本进行印证，观察检测指标表达情况、阳性率及特异性，为EMI猝死案例的法医病理学诊断提供客观依据。
第一部分三种组织化学特殊染色在早期心肌缺血死后诊断中的应用和比较
目的：对比观察Heidenhain染色、变色酸2R-亮绿染色和HBFP染色对急性缺血心肌的染色能力，观察不同保存条件及10%福尔马林固定时间对染色结果的影响。
方法：
采用结扎大鼠左冠状动脉前降支的方法建立AMI模型，实验动物190只，雌雄不拘，体重250~300g，随机分为正常对照组（5只）、假手术组（5只）、心肌缺血组（根据缺血时间不同分为急性心肌缺血15min、30min、1h、2h、3h、4h、5h、6h组共8个亚组，每组5只）、-20℃保存组、4℃保存组、室温保存组（大鼠急性心肌缺血6h心脏标本根据在三个保存条件下保存时间不同又分为保存1d、3d、5d、7d、14d、21d、28d组共7个亚组，每组5只）和固定时间组（大鼠急性心肌缺血6h心脏标本根据10%福尔马林固定时间不同又分为固定3d、5d、7d、14d、21d、28d、42d组共7个亚组，每组5只）。建立Heidenhain染色、变色酸2R-亮绿染色及HBFP染色三种组织化学特殊染色方法观察各组染色结果。
结果：
1大鼠急性心肌缺血15min，Heidenhain染色、变色酸2R-亮绿染色及HBFP染色均可在缺血区左心室心内膜下及乳头肌处见小灶状心肌细胞阳性着色，随缺血时间延长，阳性着色面积进一步扩大，三种组织化学特殊染色阳性着色部位一致。
2缺血心脏标本-20℃、4℃和室温保存过程中，随保存时间的延长，三种组织化学特殊染色均出现染色能力下降，阳性面积缩小的趋势，-20℃和4℃保存28d、室温保存21d后，Heidenhain染色效果优于另两种组织化学特殊染色，尤其显示收缩带坏死效果突出。
3缺血心脏标本10%福尔马林固定42d后，Heidenhain染色效果好于另两种组织化学特殊染色，大片状缺血心肌细胞成阳性着色，与周边非缺血区边界清晰。
小结：Heidenhain染色较变色酸2R-亮绿染色和HBFP染色可操作性强、敏感性高、死后变化及长时间固定影响小，可作为法医学实践中EMI死后诊断首选的组织化学特殊染色方法。
第二部分大鼠急性心肌缺血多指标免疫组织化学检测
目的：观察大鼠急性心肌缺血后不同时间点血浆S100A1含量变化及心肌细胞内actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1的表达敏感性及变化规律。
方法：
1采用ELISA法检测大鼠急性心肌缺血后不同时间点血浆S100A1含量。
2采用Western blot检测大鼠急性心肌缺血后不同时间点缺血心肌ADAMTS-1蛋白的表达
3采用免疫组织化学方法观察大鼠急性缺血后不同时间点心肌细胞内actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1的表达敏感性及变化规律。
结果：
1大鼠急性心肌缺血15min后血浆S100A1含量与假手术组相比明显升高（P<0.05），且随着缺血时间的延长，血浆S100A1含量逐渐升高，至缺血6h时达到高峰。
2大鼠急性心肌缺血15min后缺血区心肌细胞内ADAMTS-1蛋白表达与假手术组相比明显升高（P<0.05），且随着缺血时间的延长，ADAMTS-1蛋白的表达逐步升高，至缺血6h时达到高峰。
3大鼠急性心肌缺血后不同时间点免疫组织化学结果
①actin(HHF35)、H-FABP、S100A1：对照组心肌细胞呈现较均匀一致的棕黄色阳性染色，急性心肌缺血15min后actin(HHF35)、H-FABP和S100A1三种蛋白在缺血区左心室心内膜下及乳头肌可见小灶性心肌细胞胞浆缺染，随缺血时间的延长，缺染范围逐渐扩大，自心内膜向心外膜发展，缺血4h三种蛋白缺染扩展至近心肌全层，与周边非缺血区阳性着色的心肌细胞界限清晰。在各缺血组三种蛋白缺染位置基本一致。
②ADAMTS-1、CLU：对照组心肌细胞阴性表达，急性心肌缺血15min缺血区心内膜浅层及乳头肌散在心肌细胞出现阳性表达，随着缺血时间的延长，阳性范围逐渐扩大，缺血4h缺血区心肌细胞片状阳性表达，阳性着色扩展至近心外膜，CLU缺血4h时表达达高峰，ADAMTS-1缺血6h时表达达高峰
③C5b-9、HIF-1α：对照组及急性心肌缺血15min组心肌细胞呈阴性表达，缺血30min时在缺血区心内膜浅层及乳头肌散在心肌细胞出现阳性表达，随着缺血时间的延长，阳性范围逐渐扩大，缺血4h缺血区心肌细胞片状阳性表达，阳性着色扩展至近心外膜，缺血6h时表达达高峰。
④HIF-2α：对照组、急性心肌缺血15min和30min组心肌细胞呈阴性表达，缺血1h后，缺血区心内膜浅层及乳头肌散在心肌细胞出现阳性表达，随着缺血时间的延长，阳性范围逐渐扩大，缺血5h缺血区大部分心肌细胞表达阳性，阳性着色扩展至近心外膜，表达达高峰。
⑤Fn：对照组、急性心肌缺血15min和30min组心肌细胞呈阴性着色，缺血1h缺血区心内膜下浅层及乳头肌毛细血管腔内血浆出现阳性反应，缺血2h后缺血区心内膜下浅层及乳头肌心肌细胞出现Fn阳性着色，血管腔内血浆及血管壁阳性着色，随着缺血时间的延长，阳性着色范围逐渐扩大，缺血6h达到高峰。
小结：大鼠急性心肌缺血15min actin(HHF35)、H-FABP和S100A1即可出现缺失，ADAMTS-1和CLU蛋白出现阳性表达；C5b-9和HIF-1α在大鼠急性心肌缺血30min出现阳性表达；HIF-2α在大鼠急性心肌缺血1h出现阳性表达；Fn在大鼠急性心肌缺血2h出现阳性着色。随缺血时间的延长，actin(HHF35)、H-FABP和S100A1蛋白染色缺失愈加明显，而C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1蛋白则阳性着色逐渐增强。
第三部分不同保存条件及10%福尔马林固定时间对急性心肌缺血免疫组织化学诊断指标的影响
目的：观察三种保存条件：-20℃、4℃和室温及10%福尔马林固定时间对第二部分观察的9个免疫组织化学诊断指标染色的影响。
方法：
采用免疫组织化学方法观察不同保存条件及10%福尔马林固定时间对急性缺血心肌细胞内actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1表达变化的影响。
结果：
1不同保存条件对免疫组织化学诊断指标染色的影响：
①室温保存对目的蛋白免疫组织化学染色的影响最大，4℃保存其次，-20℃保存影响最轻。
②大鼠急性心肌缺血心脏标本在-20℃、4℃及室温条件下保存，actin(HHF35)、H-FABP及S100A1抗原稳定性最差，只限于对-20℃和室温保存1天、4℃保存3天标本有诊断意义。
③C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1均显示出良好的抗原稳定性，其中CLU稳定性最好，可适用于三个保存条件下28d以内心脏标本的检测；C5b-9相对稳定性最差，适用于-20℃保存28d、4℃保存21d、室温保存14d以内标本，且保存不同时间后，非缺血区心肌细胞均未见出现假阳性干扰结果判断。
210%福尔马林固定时间对免疫组织化学诊断指标染色的影响：
随固定时间的延长，各指标免疫组织化学阳性染色强度有下降的趋势，尤以缺血区中央明显，固定42d时，各候选指标免疫组织化学阳性染色仍清晰可辨，缺血区与非缺血区免疫组织化学染色分界清晰。
小结：actin(HHF35)、H-FABP及S100A1抗原稳定性最差，只限于对-20℃和室温保存1天、4℃保存3天标本有诊断意义。C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1均显示出良好的抗原稳定性，其中CLU稳定性最好，可适用于三个保存条件下28d以内心脏标本的检测；C5b-9相对稳定性最差，适用于-20℃保存28d、4℃保存21d、室温保存14d以内标本。9个诊断指标均可用于10%福尔马林固定42d以内的心脏组织标本的检测。
第四部分免疫组织化学诊断指标在人体尸检心脏标本的检测
目的：将9个免疫组织化学诊断指标在法医病理检案心脏标本上进行印证，观察检测指标的诊断价值、阳性率及特异性。
方法：实验分为正常对照组（10例）、明确心肌梗死组（10例）、疑似心肌梗死组（10例）、其他案例组（23例，包括机械性窒息、电击死、失血性休克、一氧化碳中毒、病毒性心肌炎），采用免疫组织化学方法观察各组心脏标本actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1的表达情况。
结果：
1正常对照组心肌细胞actin(HHF35)、H-FABP和S100A1呈现较为均匀一致阳性表达，而明确心肌梗死组、疑似心肌梗死组和其他案例组均可见actin(HHF35)、H-FABP和S100A1呈片状或弥漫性、灶状染色缺失。明确心肌梗死组、疑似心肌梗死组和其他案例组actin(HHF35)、H-FABP和S100A1缺染阳性率高于正常对照组，但三组之间无明显差异。
2正常对照组和失血性休克案例心肌细胞内C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1均呈阴性表达，明确心肌梗死组和大部分疑似心肌梗死组案例心肌细胞可见C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1阳性表达。而机械性窒息部分案例可见HIF-1α和HIF-2α阳性表达，电击死部分案例可见Fn阳性表达，一氧化碳中毒部分案例可见C5b-9、CLU、HIF-1α、HIF-2α和ADAMTS-1阳性表达，病毒性心肌炎案例可见C5b-9、Fn、CLU和ADAMTS-1阳性表达。明确心肌梗死组和疑似心肌梗死组6个诊断指标的阳性率明显高于正常对照组和其他案例组。
小结：actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1共9个诊断指标在急性心肌梗死人体心脏标本上得到了印证，其中actin(HHF35)、H-FABP和S100A1诊断特异性最差，不宜单独使用进行EMI的死后诊断，C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1诊断价值良好，但在其他非梗死性的直接或间接引起心肌损害的案例，如机械性窒息、电击死、一氧化碳中毒、心肌炎等中也有表达，将多个诊断指标联合应用将提高EMI死后诊断正确率。
结论：本研究从法医病理学实用角度出发，采用大鼠急性心肌缺血模型和人体尸检心脏标本作为研究对象，系统地观察了三种组织化学特殊染色和9个免疫组织化学诊断指标在急性心肌缺血6小时内死后诊断的敏感性、实用性和特异性，得出以下结论：
1Heidenhain染色较变色酸2R-亮绿染色和HBFP染色可操作性强、敏感性高、死后变化及10%福尔马林固定时间影响小，可作为法医学实践中早期心肌梗死死后诊断首选的组织化学特殊染色方法。
2大鼠急性心肌缺血15min actin(HHF35)、H-FABP和S100A1即可出现缺失，ADAMTS-1和CLU蛋白出现阳性表达；C5b-9和HIF-1α在大鼠急性心肌缺血30min出现阳性表达；HIF-2α在大鼠急性心肌缺血1h出现阳性表达；Fn在大鼠急性心肌缺血2h出现阳性着色。随缺血时间的延长，actin(HHF35)、H-FABP和S100A1蛋白染色缺失愈加明显，而C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1蛋白则阳性着色逐渐增强。
3actin(HHF35)、H-FABP及S100A1稳定性最差，只限于对-20℃和室温保存1天、4℃保存3天标本有诊断意义。C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1均显示出良好的稳定性，其中CLU稳定性最好，可适用于三个保存条件下28d以内心脏标本的检测；C5b-9相对稳定性最差，适用于-20℃保存28d、4℃保存21d、室温保存14d以内标本。9个诊断指标可用于10%福尔马林固定42d以内的心脏组织标本的检测。
4actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-19个诊断指标在EMI人体心脏标本上得到了印证，其中actin(HHF35)、H-FABP和S100A1诊断特异性最差，不宜单独使用进行EMI的死后诊断，C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1特异性强、敏感性高，诊断价值良好，但在其他非梗死性的直接或间接引起心肌损害的案例中也有表达，将多个诊断指标联合应用将提高EMI死后诊断正确率。
综上，本研究阐明了组织化学特殊染色和9个免疫组化诊断指标在心肌缺血的染色变化规律、影响因素、相互关系和应用条件，为早期心肌梗死死后诊断确立了较完善的病理形态学指标系统。
Sudden cardiac death (SCD) is most commonly defined as unexpecteddeath from a cardiac cause within a limited time period, which ranks aboveother causes of adult sudden death. Among the causes of SCD, acutemyocardial ischemia (AMI) is the major one. Due to transitory(coronarospasm) or constant (occlusive coronary stenosis) acute myocardialischemia, blood flow in coronary artery decreases, such supplies asmyocardial oxygen reduces and metabolite clearance becomes slow, resultingin the disorder of cardiac electrophysiological activities and/or the dysfunctionmyocardial mechanical contraction, which can cause most of the patients todie within6h, even1hour, of onset. Morphological changes (acidophilicdegeneration necrotic clotting, multifocal patches of wavy fibers,karyopyknosis, contraction band necrosis, marginal rearrangement reactionswith early inflammatory infiltrate, etc.) under light microscope with HE stainshall occur at least6h after myocardial ischemia. However, in the practice offorensic medicine, most victims of early myocardial ischemia (EMI) diewithin6h of the onset of myocardial ischemia. In routine examination, exceptfor coronary atherosclerosis of various degrees, the myocardial morphologicalchanges resulting from ischemic injuries are extremely difficult to detect. Inthe absence of convictive objective evidence, forensic expertise may bechallenged of postmortem diagnosis of EMI.
Over many years, Chinese and foreign researchers have been seekingsensitive, stable and practical methods and indicators for diagnosis of EMI,ranging from HE stain, histochemical stain, immunohistochemistry (IHC),ultrastructure observation, change of ion content, myocardial injury marker inthe blood and liquor pericardii, postmortem imaging examination, etc. A greatmany of active explorations have been made and all of them enrich the diagnosis of myocardial ischemia from various perspectives and to variousextent. However, the application of above-mentioned techniques are quitelimited due to complicated operation, less practical utility, absence ofdistinctive characteristics or excessive cost. The experts hold various views totheir practical values. There haven't been a sensitive and practical methods orindicators to solve the diagnosis problems in reality.
Among the researching techniques, IHC has been employed in theresearch and application of EMI because it's high sensitive, practical anddistinctive. In recent years, many scholars have found the phenomenon thatprotein molecule, such as actin, cardiac troponin, heart type fatty acid bindingprotein (H-FABP), dystrophin, etc, disappear from cardiomyocytes duringacute myocardial ischemia; that such proteins as complements and fibronectin(Fn) in blood distribute abnormally in ischemic myocardia; that ischemiccardiomyocytes express some sensitive proteins, such as hypoxia-induciblefactor-1alpha (HIF-1α), vascular endothelial growth factor, etc. Theseresearches have observed various changes from multiple angels when acutemyocardial ischemia occurs. Some indicators are tested in the heart specimensof sudden death from EMI. Considering the development features of EMI andthe complexity and specificity of forensic medicine practice, the application ofsolo method and solo testing indicator can hardly show their diagnostic valueeffectively for being less convictive. However, these researches have laid atheoretical basis for the combined application of multiple indicators.Meanwhile, considering the specificity of forensic investigation, such as theonset time of EMI, preserving condition of body, fixed time, etc., thecombination of different testing indicators for different cases is expected toachieve most satisfactory results.
Therefore, this project will take the rat model of AMI and human autopsyhearts as researching objects and is to①observe comparatively the stainingabilities to ischemic cardiomyocytes of Heidenhain stain, chrometrope2R-brilliant green stain and HBFP stain, as well as the effects resulting fromdifferent preserving conditions and10%formalin at fixed time;②choose testing marker from various perspectives: structural protein or cytoplasmicprotein in the cardiomyocytes: actin (HHF35)、H-FABP、S100A1；abnormalaccunmlation of plasma proteins: C5b-9、Fn；inducible expression of proteinmolecule in the ischemic cardiomyocytes: clusterin (CLU)、HIF-1α、HIF-2α、a disintegrin and metalloproteinase with thrombospondin type1motifs(ADAMTS-1), and observe the expression changes in the acute ischemiccardiomyocytes of rat through IHC;③observe the effects to theimmunohistochemical testing marker resulting from different preservingconditions and fixed in10%formalin for different time;④to verify withhuman autopsy hearts, observe the testing marker expression and specificity,and provide objective foundation for the postmortem diagnosis of EMI.
Part I: Application and comparison of three histochemical stains inthe postmortem diagnosis of early myocardial ischemia
Objective: To observe comparatively the applications of Heidenhainstain, chrometrope2R-brilliant green stain and HBFP stain for ischemiccardiomyocytes, as well as the effects resulting from different preservingconditions and fixed in10%formalin for different time.
Methods:
The rat model of AMI was constructed through permanent ligation of theleft anterior descending coronary artery (LAD). One hundred and ninety SDrats either sexes weighing250-300g were randomly distributed into7groups:control group (n=5), sham operated control group(n=5), acute myocardialischemia group (15min,30min,1h,2h,3h,4h,5h,6h after LADocclusion respectively, n=5), preservation at-20℃group, preservation at4℃group, preservation at room temperature group (The rat hearts, which weresubjected to6hours’ period of myocardial ischemia, were preserved at-20℃,4℃and room temperature for1d,3d,5d,7d,14d,21d,28d, n=5), fixed timegroup (The rat hearts, which were subjected to6hours’ period of myocardialischemia, were fixed in10%formalin solution for3d,5d,7d,14d,21d,28d,42d, n=5). The three histochemical stainings, i.e. Heidenhain stain,chrometrope2R-brilliant green stain and HBFP stain were used to observe ischemic cardiomyocytes.
Results:
1The punctate positive staining was seen in the subendocardialmyocardium and papillary muscle of left ventricular15min after acutemyocardial ischemia with Heidenhain stain, chrometrope2R-brilliant greenstain and HBFP stain, and the positive stained areas expanded as the ischemiacontinued. The positively stained areas of three special stains were concordant.
2After the hearts, which suffered acute myocardial ischemia for6hours,were preserved at-20℃,4℃and room temperature, the coloration ability andpositive stained areas of the three histochemical stains tended to decrease withthe preservation time elapsed. Preserved for28days at-20℃and4℃,21daysat room temperature separately, the Heidenhain stain was the more stable thanchrometrope2R-brilliant green stain and HBFP stain, especially for displaycontraction band necrosis.
3For the heart fixed for42d in10%formalin, the Heidenhain stain wasthe more stable than chrometrope2R-brilliant green stain and HBFP stain. Theischemic cardiomyocytes were stained positively, and the borderlines betweenischemic areas and non-ischemic areas were distinctive.
Summary: Comparing with chrometrope2R-brilliant green stain andHBFP stain, Heidenhain stain is more operable, sensitive and less liable tochange after death and be affected by the fixation time in10%formalin.Heidenhain staining can be the first choice for the postmortem diagnosis ofEMI in the practice of forensic medicine.
Part II: Detection of multiple immunohistochemical markers in acuteischemic myocardium of rat.
Objective: To observe the change of plasma S100A1concentrations andthe expression and change pattern of actin (HHF35), H-FABP, S100A1, C5b-9,Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1in the cardiomyocytes at differenttime after LAD occlusion of rats.
Methods:
1The plasma S100A1concentrations in different groups (sham operated group,15min,30min,1h,2h,4h,6h after LAD occlusion) were tested byELISA.
2The protein levels of ADAMTS-1in different groups (sham-operatedgroup;15min,30min,1h,2h,4h,6h after LAD occlusion) were examinedby western blotting.
3. IHC was used to observe the expression and change pattern of actin(HHF35), H-FABP, S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1in the cardiomyocytes at different time after acute myocardialischemia of rat.
Results:
1At15min after LAD occlusion, the S100A1concentration wassignificantly higher than that of the sham-operated group (P<0.001). With thecontinuation of occlusion time, plasma S100A1concentrations furtherincreased, and peaked at6h after LAD occlusion.
2At15min after LAD occlusion, the protein level of ADAMTS-1detected by western blotting was significantly elevated than that of thesham-operated group (P<0.001). With the continuation of occlusion time, theprotein levels of ADAMTS-1further increased, and peaked at6h after LADocclusion.
3Immunohistochemical results at different time for rats after acutemyocardial ischemia
①Actin (HHF35), H-FABP, S100A1: All cardiomyocytes of rats incontrol group are evenly stained positively brown-yellow. At15min afterLAD occlusion, the depletion of actin (HHF35), H-FABP, S100A1occurred inthe cardiomyocytes of the subendocardial layer and papillary muscles inischemic area. With the ischemic time prolonged, the depletion of stained areaextended from the endocardial to the epicardium. By4h after LAD occlusion,this area extended to all myocardial tissue in the ischemic area. A cleardividing line is drawn between the stained area and unstained area. Thepositions of unstained area of three proteins are basically the same in the eachgroup.
②ADAMTS-1, CLU: Negative expression occurred in thecardiomyocytes of rats in control group, sham operated control group. At15min after LAD occlusion, the positive expression of ADAMTS-1and CLUoccurred in the cardiomyocytes of the subendocardial layer and papillarymuscles in ischemic area. With the ischemic time prolonged, the positive areaextended. By4h after LAD occlusion, the sheet positive expression occurredin the cardiomyocytes of ischemic area. The positively stained area extendedto epicardium. Expression of CLU and ADAMTS-1reached the peak by4hand6h after LAD occlusion respectively.
③C5b-9, HIF-1α：The negative expression occurred in cardiomyocytesof rats in control group, sham operated control group and15min after LADocclusion group. At30min after LAD occlusion, the positive expression ofC5b-9and HIF-1α occurred in the cardiomyocytes of the subendocardial layerand papillary muscles in ischemic area. With the ischemic time prolonged, thepositive area gradually extended. By4h after LAD occlusion, the sheetpositive expression occurred in cardiomyocytes in ischemic area and itspositive stained area extended to the epicardium. Expression of C5b-9andHIF-1α reached the peak by6h after LAD occlusion.
④HIF-2α: Negative expression occurred in the cardiomyocytes of rats incontrol group, sham operated control group,15min and30min after LADocclusion group. By1h after LAD occlusion, the positive expression ofHIF-2α occurred in the cardiomyocytes of the subendocardial layer andpapillary muscles in ischemic area. The positive area extended with theprolongation of ischemic time. By5h after LAD occlusion, the positiveexpression of HIF-2α occurred in the majority of cardiomyocytes in ischemicarea and extended to epicardium, reached the peak.
⑤Fn: Negative expression occurred in the cardiomyocytes of rats incontrol group,15min and30min after LAD occlusion group. The positiveexpression of Fn was shown in the serum of capillary vessel of thesubendocardial layer and papillary muscles in ischemic area. By2h LADocclusion, the positive expression occurred in the cardiomyocytes of the subendocardial layer and papillary muscles in ischemic area. The serum ofcapillary vessel and vascular wall are stained positively. With the ischemictime prolonged, the positive area extended gradually and reached the peak by6h after LAD occlusion.
Summary: The diminished or absent actin (HHF35), H-FABP andS100A1occurs in rats at15min after LAD occlusion, and the positiveexpression occurs in ADAMTS-1and CLU. The positive expression of C5b-9and HIF-1α occurs at30min after LAD occlusion. The positive expression ofHIF-2α occurs at1h after LAD occlusion. The positive expression of Fnoccurs at2h after LAD occlusion. With the ischemic time prolongs, thedepletion of actin (HHF35), H-FABP and S100A1becomes more evident,while the protein expression of C5b-9、Fn、CLU、HIF-1α、HIF-2α andADAMTS-1become strong gradually.
Part III: Influence of different preservation conditions and fixationtime in10%formalin on the immunohistochemistrical diagnosticmarkers of acute myocardial ischemia.
Objective: To observe the influence of three preservation conditions:-20℃,4℃and room temperature, and fixation time in10%formalin on thenine stained immunohistochemistrical diagnostic markers observed in thesecond part.
Methods:
Influence of three preservation conditions:-20℃,4℃and roomtemperature, and fixation time in10%formalin on the changed expression ofthe nine diagnostic markers was observed in the ischemic cardiomyocytes byIHC.
Results:
1Influence of three preservation conditions on the nineimmunohistochemistrical diagnostic markers:
①The target protein of IHC was most serious seriously affected in roomtemperature preservation, less seriously affected by4℃preservation, andmost slightly affected by-20℃preservation.
②When the rat heart, which was harvested at6h after LAD, preservedat-20℃,4℃and room temperature, actin (HHF35), H-FABP and S100A1were worst in terms stability. Only when they were preserved in-20℃, roomtemperature for1day and in4℃temperature for3days can be meaningful forpostmortem diagnosis.
③C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all showed aexcellent feature of stability, among which CLU was most stable and can beapplicable to detect ischemic heart for28days preserved under threetemperature conditions. C5b-9was most unstable and can be used to detectischemic heart in-20℃temperature for28days, in4℃temperature for21days, and in room temperature for14days. After preserved for a differentperiod of time, no false positive result appeared in the cardiomyocytes of thenon-ischemic area that interrupted the estimation.
2Influence of fixation time in10%formalin on the nineimmunohistochemistrical diagnostic markers:
With the fixed time being prolonged, IHC-positive stained intensityshowed a tendency of decline, especially in the central part of ischemic area.When fixed for42days, all the diagnostic markers of IHC were still legibleand clear, and ischemic area and non-ischemic area was clearly demarked.
Summary: Actin (HHF35), H-FABP, S100A1are worst in terms stability,and only when the ischemic heart is preserved in-20℃temperature, in roomtemperature for1day, and in4℃temperature for3days can be meaningfulfor postmortem diagnosis. C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all show a excellent feature of stability, among which CLU is most stable andcan be applicable to detect of ischemic heart for28days preserved under threetemperature conditions. C5b-9is most unstable and can be used to detect theischemic heart preserved in-20℃temperature for28days, in4℃temperaturefor21days, and in room temperature for14days. All the nine diagnosticmarkers can be used to detect the heart samples which are fixed in10%formalin for42days.
Part IV: An immunohistochemical study on the postmortemdiagnosis with human autopsy hearts.
Objective: The nine diagnostic markers are used to detect human autopsyhearts with the purpose of testing its application value and specificity.
Methods:
The human autopsy hearts were divided into four groups：①normalcontrol group (n=10);②definite myocardial infarction group (n=10);③suspected myocardial infarction group(n=10);④other cases group (n=23,including mechanical asphyxia, electrocution, hemorrhagic shock, carbonmonoxide poisoning, viral myocarditis). The expression of all the ninediagnostic markers was observed in human autopsy hearts by IHC.
Results:1Normal control group showed homogenous brown reactions for actin(HHF35), H-FABP and S100A1. Diffuse or focal depletion of actin (HHF35),H-FABP and S100A1could be found in the definite myocardial infarctiongroup, suspected myocardial infarction group and other cases group. Thepositive rate of actin (HHF35), H-FABP and S100A1in the definitemyocardial infarction group, suspected myocardial infarction group and othercases group was significantly higher than that in the other cases group, butthere was not significant difference between the three groups.
2Normal control group and hemorrhagic shock group showed negativestaining for C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1. The positiveexpression of C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1occurred inthe definite myocardial infarction group and most cases of suspectedmyocardial infarction group. The positive expression of HIF-1α and HIF-2αoccurred in some cases of mechanical asphyxia. The Fn positive expressionoccurred in some cases of electrocution. The positive expression of C5b-9,CLU, HIF-1α, HIF-2α and ADAMTS-1occurred in some cases of carbonmonoxide poisoning case. The positive expression of C5b-9, Fn, CLU andADAMTS-1occurred in viral myocarditis cases. The positive rate was higherin definite myocardial infarction group and suspected myocardial infarctiongroup than that in normal control group and other cases group.
Summary: The nine diagnostic markers, namely actin (HHF35), H-FABP,S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1, are verified onthe EMI human autopsy hearts. Among them, actin (HHF35), H-FABP andS100A1are least in diagnostic specificity, which are not suitable to be appliedalone to postmortem diagnosis of EMI. C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1are more useful in postmortem diagnosis of EMI, but theirexpressions are also found in some cases of non-infarctive direct or indirectmyocardial injury (mechanical asphyxia, electrocution, hemorrhagic shock,carbon monoxide poisoning, viral myocarditis). The combined application ofmultiple diagnostic markers will improve the accuracy of the postmortemdiagnosis of EMI.
Conclusion：From a practical perspective of forensic pathology, thisproject took the rat model of AMI and human autopsy hearts as researchingobjects and observed the sensitivity, practicability and specificity of threehistochemical stains and nine immunohistochemistrical diagnostic markers forthe postmortem diagnosis of EMI. Main conclusions are reached as follows:
①Comparing with chrometrope2R-brilliant green stain and HBFP stain,Heidenhain stain is more operable, sensitive and less liable to change afterdeath and be affected by the fixation time in10%formalin. Heidenhainstaining can be the first choice for the postmortem diagnosis of EMI in thepractice of forensic medicine.
②The diminished or absent actin (HHF35), H-FABP and S100A1occurs in rats at15min after LAD occlusion, and the positive expressionoccurs in ADAMTS-1and CLU. The positive expression of C5b-9and HIF-1αoccurs at30min after LAD occlusion. The positive expression of HIF-2αoccurs at1h after LAD occlusion. The positive expression of Fn occurs at2hafter LAD occlusion. With the ischemic time prolongs, the depletion of actin(HHF35), H-FABP and S100A1becomes more evident, while the proteinexpression of C5b-9、Fn、CLU、HIF-1α、HIF-2α and ADAMTS-1becomestrong gradually.
③Actin (HHF35), H-FABP, S100A1are worst in terms stability, and
only when the ischemic heart is preserved in-20℃temperature, in roomtemperature for1day, and in4℃temperature for3days can be meaningfulfor postmortem diagnosis. C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all show a excellent feature of stability, among which CLU is most stable andcan be applicable to detect of ischemic heart for28days preserved under threetemperature conditions. C5b-9is most unstable and can be used to detect theischemic heart preserved in-20℃temperature for28days, in4℃temperaturefor21days, and in room temperature for14days. All the nine diagnosticmarkers can be used to detect the heart samples which are fixed in10%formalin for42days.
④The nine diagnostic markers, namely actin (HHF35), H-FABP,S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1, are verified onthe EMI human autopsy hearts. Among them, actin (HHF35), H-FABP andS100A1are least in diagnostic specificity, which are not suitable to be appliedalone to postmortem diagnosis of EMI. C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1are more useful in postmortem diagnosis of EMI, but theirexpressions are also found in some cases of non-infarctive direct or indirectmyocardial injury (mechanical asphyxia, electrocution, hemorrhagic shock,carbon monoxide poisoning, viral myocarditis). The combined application ofmultiple diagnostic markers will improve the accuracy of the postmortemdiagnosis of EMI.
To sum up, this research studies systematically the application ofhistochemical staining and the nine diagnostic markers of IHC, whichprovides theoretical basis for the forensic experts of the postmortem diagnosisof EMI.
多年来国内外学者都在寻找敏感、稳定、实用的诊断早期心肌缺血的方法和指标，从常规HE染色、特殊染色和酶组织化学、免疫组织化学、超微结构、荧光检查、离子含量改变、心包液和血液心肌损伤标志物检测、死后影像学检查等方面进行了积极的探索，所有这些方法都从不同方面、不同程度地丰富了EMI死后诊断的内容。但是上述方法或因为操作过于复杂，或实用性不强，或因为缺乏特异性、费用过高等因素的影响而限制了它们在法医病理学中的应用，实际应用价值各家说法不一，迄今仍没有敏感、实用的方法或指标能很好解决实际办案中的死因诊断问题。
在众多研究方法中，免疫组织化学因其具有特异性强、敏感性高、实用性好等优点，已应用到EMI的研究和应用中来。近年来，许多学者陆续报道了如肌动蛋白（actin）、肌钙蛋白、心型脂肪酸结合蛋白（heart typefatty acid binding protein, H-FABP）、抗肌萎缩蛋白等蛋白分子在急性心肌缺血时从心肌细胞内脱失的现象；血浆中某些蛋白成分如补体成分、纤维连接蛋白（fibronectin, Fn）等在缺血心肌的异常分布情况；缺血心肌诱导表达某些敏感蛋白分子，如缺氧诱导因子-1α（hypoxia-inducible factor-1alpha, HIF-1α）、血管内皮生长因子等。这些研究从多角度观察了急性心肌缺血时心肌细胞发生的各种变化，部分指标在EMI心脏标本进行了验证，但考虑到缺血心肌自身演变的特点和法医学实践中案例的复杂性和特殊性，单一方法和单一检测指标的应用缺乏说服力而难以有效发挥其诊断价值，而以往这些研究为多指标联合应用提供了理论依据。同时，考虑到法医检案的特殊性，如EMI发病时间、尸体保存情况、固定时间等因素的影响，需针对不同的情况进行检测指标的组合，以期达到最好的诊断效果。
据此，本课题以大鼠急性心肌缺血模型和法医检案心脏标本作为研究对象，研究：①对比观察铁钒-苏木素-伊红染色法（Heidenhain染色）、变色酸2R-亮绿染色和苏木素-碱性复红-苦味酸染色（HBFP染色）对缺血心肌的染色能力，观察不同保存条件及10%福尔马林固定时间对染色的影响；②从不同角度选取检测指标：心肌结构蛋白或胞浆蛋白：actin(HHF35)、H-FABP、S100A1；黏附和进入心肌细胞内的血浆成分：C5b-9、Fn；缺血心肌诱导表达的产物：聚集素蛋白（clusterin，CLU）、HIF-1α、HIF-2α、含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶（adisintegrin and metalloproteinase with thrombospondin type1motifs，ADAMTS-1），观察大鼠急性心肌缺血后血浆S100A1含量的变化及9个诊断指标在大鼠急性缺血心肌中的表达变化情况；③观察不同保存条件及10%福尔马林固定时间对上述9个免疫组织化学检测指标染色的影响；④以法医病理检案心脏标本进行印证，观察检测指标表达情况、阳性率及特异性，为EMI猝死案例的法医病理学诊断提供客观依据。
第一部分三种组织化学特殊染色在早期心肌缺血死后诊断中的应用和比较
目的：对比观察Heidenhain染色、变色酸2R-亮绿染色和HBFP染色对急性缺血心肌的染色能力，观察不同保存条件及10%福尔马林固定时间对染色结果的影响。
方法：
采用结扎大鼠左冠状动脉前降支的方法建立AMI模型，实验动物190只，雌雄不拘，体重250~300g，随机分为正常对照组（5只）、假手术组（5只）、心肌缺血组（根据缺血时间不同分为急性心肌缺血15min、30min、1h、2h、3h、4h、5h、6h组共8个亚组，每组5只）、-20℃保存组、4℃保存组、室温保存组（大鼠急性心肌缺血6h心脏标本根据在三个保存条件下保存时间不同又分为保存1d、3d、5d、7d、14d、21d、28d组共7个亚组，每组5只）和固定时间组（大鼠急性心肌缺血6h心脏标本根据10%福尔马林固定时间不同又分为固定3d、5d、7d、14d、21d、28d、42d组共7个亚组，每组5只）。建立Heidenhain染色、变色酸2R-亮绿染色及HBFP染色三种组织化学特殊染色方法观察各组染色结果。
结果：
1大鼠急性心肌缺血15min，Heidenhain染色、变色酸2R-亮绿染色及HBFP染色均可在缺血区左心室心内膜下及乳头肌处见小灶状心肌细胞阳性着色，随缺血时间延长，阳性着色面积进一步扩大，三种组织化学特殊染色阳性着色部位一致。
2缺血心脏标本-20℃、4℃和室温保存过程中，随保存时间的延长，三种组织化学特殊染色均出现染色能力下降，阳性面积缩小的趋势，-20℃和4℃保存28d、室温保存21d后，Heidenhain染色效果优于另两种组织化学特殊染色，尤其显示收缩带坏死效果突出。
3缺血心脏标本10%福尔马林固定42d后，Heidenhain染色效果好于另两种组织化学特殊染色，大片状缺血心肌细胞成阳性着色，与周边非缺血区边界清晰。
小结：Heidenhain染色较变色酸2R-亮绿染色和HBFP染色可操作性强、敏感性高、死后变化及长时间固定影响小，可作为法医学实践中EMI死后诊断首选的组织化学特殊染色方法。
第二部分大鼠急性心肌缺血多指标免疫组织化学检测
目的：观察大鼠急性心肌缺血后不同时间点血浆S100A1含量变化及心肌细胞内actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1的表达敏感性及变化规律。
方法：
1采用ELISA法检测大鼠急性心肌缺血后不同时间点血浆S100A1含量。
2采用Western blot检测大鼠急性心肌缺血后不同时间点缺血心肌ADAMTS-1蛋白的表达
3采用免疫组织化学方法观察大鼠急性缺血后不同时间点心肌细胞内actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1的表达敏感性及变化规律。
结果：
1大鼠急性心肌缺血15min后血浆S100A1含量与假手术组相比明显升高（P<0.05），且随着缺血时间的延长，血浆S100A1含量逐渐升高，至缺血6h时达到高峰。
2大鼠急性心肌缺血15min后缺血区心肌细胞内ADAMTS-1蛋白表达与假手术组相比明显升高（P<0.05），且随着缺血时间的延长，ADAMTS-1蛋白的表达逐步升高，至缺血6h时达到高峰。
3大鼠急性心肌缺血后不同时间点免疫组织化学结果
①actin(HHF35)、H-FABP、S100A1：对照组心肌细胞呈现较均匀一致的棕黄色阳性染色，急性心肌缺血15min后actin(HHF35)、H-FABP和S100A1三种蛋白在缺血区左心室心内膜下及乳头肌可见小灶性心肌细胞胞浆缺染，随缺血时间的延长，缺染范围逐渐扩大，自心内膜向心外膜发展，缺血4h三种蛋白缺染扩展至近心肌全层，与周边非缺血区阳性着色的心肌细胞界限清晰。在各缺血组三种蛋白缺染位置基本一致。
②ADAMTS-1、CLU：对照组心肌细胞阴性表达，急性心肌缺血15min缺血区心内膜浅层及乳头肌散在心肌细胞出现阳性表达，随着缺血时间的延长，阳性范围逐渐扩大，缺血4h缺血区心肌细胞片状阳性表达，阳性着色扩展至近心外膜，CLU缺血4h时表达达高峰，ADAMTS-1缺血6h时表达达高峰
③C5b-9、HIF-1α：对照组及急性心肌缺血15min组心肌细胞呈阴性表达，缺血30min时在缺血区心内膜浅层及乳头肌散在心肌细胞出现阳性表达，随着缺血时间的延长，阳性范围逐渐扩大，缺血4h缺血区心肌细胞片状阳性表达，阳性着色扩展至近心外膜，缺血6h时表达达高峰。
④HIF-2α：对照组、急性心肌缺血15min和30min组心肌细胞呈阴性表达，缺血1h后，缺血区心内膜浅层及乳头肌散在心肌细胞出现阳性表达，随着缺血时间的延长，阳性范围逐渐扩大，缺血5h缺血区大部分心肌细胞表达阳性，阳性着色扩展至近心外膜，表达达高峰。
⑤Fn：对照组、急性心肌缺血15min和30min组心肌细胞呈阴性着色，缺血1h缺血区心内膜下浅层及乳头肌毛细血管腔内血浆出现阳性反应，缺血2h后缺血区心内膜下浅层及乳头肌心肌细胞出现Fn阳性着色，血管腔内血浆及血管壁阳性着色，随着缺血时间的延长，阳性着色范围逐渐扩大，缺血6h达到高峰。
小结：大鼠急性心肌缺血15min actin(HHF35)、H-FABP和S100A1即可出现缺失，ADAMTS-1和CLU蛋白出现阳性表达；C5b-9和HIF-1α在大鼠急性心肌缺血30min出现阳性表达；HIF-2α在大鼠急性心肌缺血1h出现阳性表达；Fn在大鼠急性心肌缺血2h出现阳性着色。随缺血时间的延长，actin(HHF35)、H-FABP和S100A1蛋白染色缺失愈加明显，而C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1蛋白则阳性着色逐渐增强。
第三部分不同保存条件及10%福尔马林固定时间对急性心肌缺血免疫组织化学诊断指标的影响
目的：观察三种保存条件：-20℃、4℃和室温及10%福尔马林固定时间对第二部分观察的9个免疫组织化学诊断指标染色的影响。
方法：
采用免疫组织化学方法观察不同保存条件及10%福尔马林固定时间对急性缺血心肌细胞内actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1表达变化的影响。
结果：
1不同保存条件对免疫组织化学诊断指标染色的影响：
①室温保存对目的蛋白免疫组织化学染色的影响最大，4℃保存其次，-20℃保存影响最轻。
②大鼠急性心肌缺血心脏标本在-20℃、4℃及室温条件下保存，actin(HHF35)、H-FABP及S100A1抗原稳定性最差，只限于对-20℃和室温保存1天、4℃保存3天标本有诊断意义。
③C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1均显示出良好的抗原稳定性，其中CLU稳定性最好，可适用于三个保存条件下28d以内心脏标本的检测；C5b-9相对稳定性最差，适用于-20℃保存28d、4℃保存21d、室温保存14d以内标本，且保存不同时间后，非缺血区心肌细胞均未见出现假阳性干扰结果判断。
210%福尔马林固定时间对免疫组织化学诊断指标染色的影响：
随固定时间的延长，各指标免疫组织化学阳性染色强度有下降的趋势，尤以缺血区中央明显，固定42d时，各候选指标免疫组织化学阳性染色仍清晰可辨，缺血区与非缺血区免疫组织化学染色分界清晰。
小结：actin(HHF35)、H-FABP及S100A1抗原稳定性最差，只限于对-20℃和室温保存1天、4℃保存3天标本有诊断意义。C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1均显示出良好的抗原稳定性，其中CLU稳定性最好，可适用于三个保存条件下28d以内心脏标本的检测；C5b-9相对稳定性最差，适用于-20℃保存28d、4℃保存21d、室温保存14d以内标本。9个诊断指标均可用于10%福尔马林固定42d以内的心脏组织标本的检测。
第四部分免疫组织化学诊断指标在人体尸检心脏标本的检测
目的：将9个免疫组织化学诊断指标在法医病理检案心脏标本上进行印证，观察检测指标的诊断价值、阳性率及特异性。
方法：实验分为正常对照组（10例）、明确心肌梗死组（10例）、疑似心肌梗死组（10例）、其他案例组（23例，包括机械性窒息、电击死、失血性休克、一氧化碳中毒、病毒性心肌炎），采用免疫组织化学方法观察各组心脏标本actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1的表达情况。
结果：
1正常对照组心肌细胞actin(HHF35)、H-FABP和S100A1呈现较为均匀一致阳性表达，而明确心肌梗死组、疑似心肌梗死组和其他案例组均可见actin(HHF35)、H-FABP和S100A1呈片状或弥漫性、灶状染色缺失。明确心肌梗死组、疑似心肌梗死组和其他案例组actin(HHF35)、H-FABP和S100A1缺染阳性率高于正常对照组，但三组之间无明显差异。
2正常对照组和失血性休克案例心肌细胞内C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1均呈阴性表达，明确心肌梗死组和大部分疑似心肌梗死组案例心肌细胞可见C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1阳性表达。而机械性窒息部分案例可见HIF-1α和HIF-2α阳性表达，电击死部分案例可见Fn阳性表达，一氧化碳中毒部分案例可见C5b-9、CLU、HIF-1α、HIF-2α和ADAMTS-1阳性表达，病毒性心肌炎案例可见C5b-9、Fn、CLU和ADAMTS-1阳性表达。明确心肌梗死组和疑似心肌梗死组6个诊断指标的阳性率明显高于正常对照组和其他案例组。
小结：actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1共9个诊断指标在急性心肌梗死人体心脏标本上得到了印证，其中actin(HHF35)、H-FABP和S100A1诊断特异性最差，不宜单独使用进行EMI的死后诊断，C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1诊断价值良好，但在其他非梗死性的直接或间接引起心肌损害的案例，如机械性窒息、电击死、一氧化碳中毒、心肌炎等中也有表达，将多个诊断指标联合应用将提高EMI死后诊断正确率。
结论：本研究从法医病理学实用角度出发，采用大鼠急性心肌缺血模型和人体尸检心脏标本作为研究对象，系统地观察了三种组织化学特殊染色和9个免疫组织化学诊断指标在急性心肌缺血6小时内死后诊断的敏感性、实用性和特异性，得出以下结论：
1Heidenhain染色较变色酸2R-亮绿染色和HBFP染色可操作性强、敏感性高、死后变化及10%福尔马林固定时间影响小，可作为法医学实践中早期心肌梗死死后诊断首选的组织化学特殊染色方法。
2大鼠急性心肌缺血15min actin(HHF35)、H-FABP和S100A1即可出现缺失，ADAMTS-1和CLU蛋白出现阳性表达；C5b-9和HIF-1α在大鼠急性心肌缺血30min出现阳性表达；HIF-2α在大鼠急性心肌缺血1h出现阳性表达；Fn在大鼠急性心肌缺血2h出现阳性着色。随缺血时间的延长，actin(HHF35)、H-FABP和S100A1蛋白染色缺失愈加明显，而C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1蛋白则阳性着色逐渐增强。
3actin(HHF35)、H-FABP及S100A1稳定性最差，只限于对-20℃和室温保存1天、4℃保存3天标本有诊断意义。C5b-9、Fn、CLU、HIF-1α、HIF-2α、ADAMTS-1均显示出良好的稳定性，其中CLU稳定性最好，可适用于三个保存条件下28d以内心脏标本的检测；C5b-9相对稳定性最差，适用于-20℃保存28d、4℃保存21d、室温保存14d以内标本。9个诊断指标可用于10%福尔马林固定42d以内的心脏组织标本的检测。
4actin(HHF35)、H-FABP、S100A1、C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-19个诊断指标在EMI人体心脏标本上得到了印证，其中actin(HHF35)、H-FABP和S100A1诊断特异性最差，不宜单独使用进行EMI的死后诊断，C5b-9、Fn、CLU、HIF-1α、HIF-2α和ADAMTS-1特异性强、敏感性高，诊断价值良好，但在其他非梗死性的直接或间接引起心肌损害的案例中也有表达，将多个诊断指标联合应用将提高EMI死后诊断正确率。
综上，本研究阐明了组织化学特殊染色和9个免疫组化诊断指标在心肌缺血的染色变化规律、影响因素、相互关系和应用条件，为早期心肌梗死死后诊断确立了较完善的病理形态学指标系统。
Sudden cardiac death (SCD) is most commonly defined as unexpecteddeath from a cardiac cause within a limited time period, which ranks aboveother causes of adult sudden death. Among the causes of SCD, acutemyocardial ischemia (AMI) is the major one. Due to transitory(coronarospasm) or constant (occlusive coronary stenosis) acute myocardialischemia, blood flow in coronary artery decreases, such supplies asmyocardial oxygen reduces and metabolite clearance becomes slow, resultingin the disorder of cardiac electrophysiological activities and/or the dysfunctionmyocardial mechanical contraction, which can cause most of the patients todie within6h, even1hour, of onset. Morphological changes (acidophilicdegeneration necrotic clotting, multifocal patches of wavy fibers,karyopyknosis, contraction band necrosis, marginal rearrangement reactionswith early inflammatory infiltrate, etc.) under light microscope with HE stainshall occur at least6h after myocardial ischemia. However, in the practice offorensic medicine, most victims of early myocardial ischemia (EMI) diewithin6h of the onset of myocardial ischemia. In routine examination, exceptfor coronary atherosclerosis of various degrees, the myocardial morphologicalchanges resulting from ischemic injuries are extremely difficult to detect. Inthe absence of convictive objective evidence, forensic expertise may bechallenged of postmortem diagnosis of EMI.
Over many years, Chinese and foreign researchers have been seekingsensitive, stable and practical methods and indicators for diagnosis of EMI,ranging from HE stain, histochemical stain, immunohistochemistry (IHC),ultrastructure observation, change of ion content, myocardial injury marker inthe blood and liquor pericardii, postmortem imaging examination, etc. A greatmany of active explorations have been made and all of them enrich the diagnosis of myocardial ischemia from various perspectives and to variousextent. However, the application of above-mentioned techniques are quitelimited due to complicated operation, less practical utility, absence ofdistinctive characteristics or excessive cost. The experts hold various views totheir practical values. There haven't been a sensitive and practical methods orindicators to solve the diagnosis problems in reality.
Among the researching techniques, IHC has been employed in theresearch and application of EMI because it's high sensitive, practical anddistinctive. In recent years, many scholars have found the phenomenon thatprotein molecule, such as actin, cardiac troponin, heart type fatty acid bindingprotein (H-FABP), dystrophin, etc, disappear from cardiomyocytes duringacute myocardial ischemia; that such proteins as complements and fibronectin(Fn) in blood distribute abnormally in ischemic myocardia; that ischemiccardiomyocytes express some sensitive proteins, such as hypoxia-induciblefactor-1alpha (HIF-1α), vascular endothelial growth factor, etc. Theseresearches have observed various changes from multiple angels when acutemyocardial ischemia occurs. Some indicators are tested in the heart specimensof sudden death from EMI. Considering the development features of EMI andthe complexity and specificity of forensic medicine practice, the application ofsolo method and solo testing indicator can hardly show their diagnostic valueeffectively for being less convictive. However, these researches have laid atheoretical basis for the combined application of multiple indicators.Meanwhile, considering the specificity of forensic investigation, such as theonset time of EMI, preserving condition of body, fixed time, etc., thecombination of different testing indicators for different cases is expected toachieve most satisfactory results.
Therefore, this project will take the rat model of AMI and human autopsyhearts as researching objects and is to①observe comparatively the stainingabilities to ischemic cardiomyocytes of Heidenhain stain, chrometrope2R-brilliant green stain and HBFP stain, as well as the effects resulting fromdifferent preserving conditions and10%formalin at fixed time;②choose testing marker from various perspectives: structural protein or cytoplasmicprotein in the cardiomyocytes: actin (HHF35)、H-FABP、S100A1；abnormalaccunmlation of plasma proteins: C5b-9、Fn；inducible expression of proteinmolecule in the ischemic cardiomyocytes: clusterin (CLU)、HIF-1α、HIF-2α、a disintegrin and metalloproteinase with thrombospondin type1motifs(ADAMTS-1), and observe the expression changes in the acute ischemiccardiomyocytes of rat through IHC;③observe the effects to theimmunohistochemical testing marker resulting from different preservingconditions and fixed in10%formalin for different time;④to verify withhuman autopsy hearts, observe the testing marker expression and specificity,and provide objective foundation for the postmortem diagnosis of EMI.
Part I: Application and comparison of three histochemical stains inthe postmortem diagnosis of early myocardial ischemia
Objective: To observe comparatively the applications of Heidenhainstain, chrometrope2R-brilliant green stain and HBFP stain for ischemiccardiomyocytes, as well as the effects resulting from different preservingconditions and fixed in10%formalin for different time.
Methods:
The rat model of AMI was constructed through permanent ligation of theleft anterior descending coronary artery (LAD). One hundred and ninety SDrats either sexes weighing250-300g were randomly distributed into7groups:control group (n=5), sham operated control group(n=5), acute myocardialischemia group (15min,30min,1h,2h,3h,4h,5h,6h after LADocclusion respectively, n=5), preservation at-20℃group, preservation at4℃group, preservation at room temperature group (The rat hearts, which weresubjected to6hours’ period of myocardial ischemia, were preserved at-20℃,4℃and room temperature for1d,3d,5d,7d,14d,21d,28d, n=5), fixed timegroup (The rat hearts, which were subjected to6hours’ period of myocardialischemia, were fixed in10%formalin solution for3d,5d,7d,14d,21d,28d,42d, n=5). The three histochemical stainings, i.e. Heidenhain stain,chrometrope2R-brilliant green stain and HBFP stain were used to observe ischemic cardiomyocytes.
Results:
1The punctate positive staining was seen in the subendocardialmyocardium and papillary muscle of left ventricular15min after acutemyocardial ischemia with Heidenhain stain, chrometrope2R-brilliant greenstain and HBFP stain, and the positive stained areas expanded as the ischemiacontinued. The positively stained areas of three special stains were concordant.
2After the hearts, which suffered acute myocardial ischemia for6hours,were preserved at-20℃,4℃and room temperature, the coloration ability andpositive stained areas of the three histochemical stains tended to decrease withthe preservation time elapsed. Preserved for28days at-20℃and4℃,21daysat room temperature separately, the Heidenhain stain was the more stable thanchrometrope2R-brilliant green stain and HBFP stain, especially for displaycontraction band necrosis.
3For the heart fixed for42d in10%formalin, the Heidenhain stain wasthe more stable than chrometrope2R-brilliant green stain and HBFP stain. Theischemic cardiomyocytes were stained positively, and the borderlines betweenischemic areas and non-ischemic areas were distinctive.
Summary: Comparing with chrometrope2R-brilliant green stain andHBFP stain, Heidenhain stain is more operable, sensitive and less liable tochange after death and be affected by the fixation time in10%formalin.Heidenhain staining can be the first choice for the postmortem diagnosis ofEMI in the practice of forensic medicine.
Part II: Detection of multiple immunohistochemical markers in acuteischemic myocardium of rat.
Objective: To observe the change of plasma S100A1concentrations andthe expression and change pattern of actin (HHF35), H-FABP, S100A1, C5b-9,Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1in the cardiomyocytes at differenttime after LAD occlusion of rats.
Methods:
1The plasma S100A1concentrations in different groups (sham operated group,15min,30min,1h,2h,4h,6h after LAD occlusion) were tested byELISA.
2The protein levels of ADAMTS-1in different groups (sham-operatedgroup;15min,30min,1h,2h,4h,6h after LAD occlusion) were examinedby western blotting.
3. IHC was used to observe the expression and change pattern of actin(HHF35), H-FABP, S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1in the cardiomyocytes at different time after acute myocardialischemia of rat.
Results:
1At15min after LAD occlusion, the S100A1concentration wassignificantly higher than that of the sham-operated group (P<0.001). With thecontinuation of occlusion time, plasma S100A1concentrations furtherincreased, and peaked at6h after LAD occlusion.
2At15min after LAD occlusion, the protein level of ADAMTS-1detected by western blotting was significantly elevated than that of thesham-operated group (P<0.001). With the continuation of occlusion time, theprotein levels of ADAMTS-1further increased, and peaked at6h after LADocclusion.
3Immunohistochemical results at different time for rats after acutemyocardial ischemia
①Actin (HHF35), H-FABP, S100A1: All cardiomyocytes of rats incontrol group are evenly stained positively brown-yellow. At15min afterLAD occlusion, the depletion of actin (HHF35), H-FABP, S100A1occurred inthe cardiomyocytes of the subendocardial layer and papillary muscles inischemic area. With the ischemic time prolonged, the depletion of stained areaextended from the endocardial to the epicardium. By4h after LAD occlusion,this area extended to all myocardial tissue in the ischemic area. A cleardividing line is drawn between the stained area and unstained area. Thepositions of unstained area of three proteins are basically the same in the eachgroup.
②ADAMTS-1, CLU: Negative expression occurred in thecardiomyocytes of rats in control group, sham operated control group. At15min after LAD occlusion, the positive expression of ADAMTS-1and CLUoccurred in the cardiomyocytes of the subendocardial layer and papillarymuscles in ischemic area. With the ischemic time prolonged, the positive areaextended. By4h after LAD occlusion, the sheet positive expression occurredin the cardiomyocytes of ischemic area. The positively stained area extendedto epicardium. Expression of CLU and ADAMTS-1reached the peak by4hand6h after LAD occlusion respectively.
③C5b-9, HIF-1α：The negative expression occurred in cardiomyocytesof rats in control group, sham operated control group and15min after LADocclusion group. At30min after LAD occlusion, the positive expression ofC5b-9and HIF-1α occurred in the cardiomyocytes of the subendocardial layerand papillary muscles in ischemic area. With the ischemic time prolonged, thepositive area gradually extended. By4h after LAD occlusion, the sheetpositive expression occurred in cardiomyocytes in ischemic area and itspositive stained area extended to the epicardium. Expression of C5b-9andHIF-1α reached the peak by6h after LAD occlusion.
④HIF-2α: Negative expression occurred in the cardiomyocytes of rats incontrol group, sham operated control group,15min and30min after LADocclusion group. By1h after LAD occlusion, the positive expression ofHIF-2α occurred in the cardiomyocytes of the subendocardial layer andpapillary muscles in ischemic area. The positive area extended with theprolongation of ischemic time. By5h after LAD occlusion, the positiveexpression of HIF-2α occurred in the majority of cardiomyocytes in ischemicarea and extended to epicardium, reached the peak.
⑤Fn: Negative expression occurred in the cardiomyocytes of rats incontrol group,15min and30min after LAD occlusion group. The positiveexpression of Fn was shown in the serum of capillary vessel of thesubendocardial layer and papillary muscles in ischemic area. By2h LADocclusion, the positive expression occurred in the cardiomyocytes of the subendocardial layer and papillary muscles in ischemic area. The serum ofcapillary vessel and vascular wall are stained positively. With the ischemictime prolonged, the positive area extended gradually and reached the peak by6h after LAD occlusion.
Summary: The diminished or absent actin (HHF35), H-FABP andS100A1occurs in rats at15min after LAD occlusion, and the positiveexpression occurs in ADAMTS-1and CLU. The positive expression of C5b-9and HIF-1α occurs at30min after LAD occlusion. The positive expression ofHIF-2α occurs at1h after LAD occlusion. The positive expression of Fnoccurs at2h after LAD occlusion. With the ischemic time prolongs, thedepletion of actin (HHF35), H-FABP and S100A1becomes more evident,while the protein expression of C5b-9、Fn、CLU、HIF-1α、HIF-2α andADAMTS-1become strong gradually.
Part III: Influence of different preservation conditions and fixationtime in10%formalin on the immunohistochemistrical diagnosticmarkers of acute myocardial ischemia.
Objective: To observe the influence of three preservation conditions:-20℃,4℃and room temperature, and fixation time in10%formalin on thenine stained immunohistochemistrical diagnostic markers observed in thesecond part.
Methods:
Influence of three preservation conditions:-20℃,4℃and roomtemperature, and fixation time in10%formalin on the changed expression ofthe nine diagnostic markers was observed in the ischemic cardiomyocytes byIHC.
Results:
1Influence of three preservation conditions on the nineimmunohistochemistrical diagnostic markers:
①The target protein of IHC was most serious seriously affected in roomtemperature preservation, less seriously affected by4℃preservation, andmost slightly affected by-20℃preservation.
②When the rat heart, which was harvested at6h after LAD, preservedat-20℃,4℃and room temperature, actin (HHF35), H-FABP and S100A1were worst in terms stability. Only when they were preserved in-20℃, roomtemperature for1day and in4℃temperature for3days can be meaningful forpostmortem diagnosis.
③C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all showed aexcellent feature of stability, among which CLU was most stable and can beapplicable to detect ischemic heart for28days preserved under threetemperature conditions. C5b-9was most unstable and can be used to detectischemic heart in-20℃temperature for28days, in4℃temperature for21days, and in room temperature for14days. After preserved for a differentperiod of time, no false positive result appeared in the cardiomyocytes of thenon-ischemic area that interrupted the estimation.
2Influence of fixation time in10%formalin on the nineimmunohistochemistrical diagnostic markers:
With the fixed time being prolonged, IHC-positive stained intensityshowed a tendency of decline, especially in the central part of ischemic area.When fixed for42days, all the diagnostic markers of IHC were still legibleand clear, and ischemic area and non-ischemic area was clearly demarked.
Summary: Actin (HHF35), H-FABP, S100A1are worst in terms stability,and only when the ischemic heart is preserved in-20℃temperature, in roomtemperature for1day, and in4℃temperature for3days can be meaningfulfor postmortem diagnosis. C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all show a excellent feature of stability, among which CLU is most stable andcan be applicable to detect of ischemic heart for28days preserved under threetemperature conditions. C5b-9is most unstable and can be used to detect theischemic heart preserved in-20℃temperature for28days, in4℃temperaturefor21days, and in room temperature for14days. All the nine diagnosticmarkers can be used to detect the heart samples which are fixed in10%formalin for42days.
Part IV: An immunohistochemical study on the postmortemdiagnosis with human autopsy hearts.
Objective: The nine diagnostic markers are used to detect human autopsyhearts with the purpose of testing its application value and specificity.
Methods:
The human autopsy hearts were divided into four groups：①normalcontrol group (n=10);②definite myocardial infarction group (n=10);③suspected myocardial infarction group(n=10);④other cases group (n=23,including mechanical asphyxia, electrocution, hemorrhagic shock, carbonmonoxide poisoning, viral myocarditis). The expression of all the ninediagnostic markers was observed in human autopsy hearts by IHC.
Results:1Normal control group showed homogenous brown reactions for actin(HHF35), H-FABP and S100A1. Diffuse or focal depletion of actin (HHF35),H-FABP and S100A1could be found in the definite myocardial infarctiongroup, suspected myocardial infarction group and other cases group. Thepositive rate of actin (HHF35), H-FABP and S100A1in the definitemyocardial infarction group, suspected myocardial infarction group and othercases group was significantly higher than that in the other cases group, butthere was not significant difference between the three groups.
2Normal control group and hemorrhagic shock group showed negativestaining for C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1. The positiveexpression of C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1occurred inthe definite myocardial infarction group and most cases of suspectedmyocardial infarction group. The positive expression of HIF-1α and HIF-2αoccurred in some cases of mechanical asphyxia. The Fn positive expressionoccurred in some cases of electrocution. The positive expression of C5b-9,CLU, HIF-1α, HIF-2α and ADAMTS-1occurred in some cases of carbonmonoxide poisoning case. The positive expression of C5b-9, Fn, CLU andADAMTS-1occurred in viral myocarditis cases. The positive rate was higherin definite myocardial infarction group and suspected myocardial infarctiongroup than that in normal control group and other cases group.
Summary: The nine diagnostic markers, namely actin (HHF35), H-FABP,S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1, are verified onthe EMI human autopsy hearts. Among them, actin (HHF35), H-FABP andS100A1are least in diagnostic specificity, which are not suitable to be appliedalone to postmortem diagnosis of EMI. C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1are more useful in postmortem diagnosis of EMI, but theirexpressions are also found in some cases of non-infarctive direct or indirectmyocardial injury (mechanical asphyxia, electrocution, hemorrhagic shock,carbon monoxide poisoning, viral myocarditis). The combined application ofmultiple diagnostic markers will improve the accuracy of the postmortemdiagnosis of EMI.
Conclusion：From a practical perspective of forensic pathology, thisproject took the rat model of AMI and human autopsy hearts as researchingobjects and observed the sensitivity, practicability and specificity of threehistochemical stains and nine immunohistochemistrical diagnostic markers forthe postmortem diagnosis of EMI. Main conclusions are reached as follows:
①Comparing with chrometrope2R-brilliant green stain and HBFP stain,Heidenhain stain is more operable, sensitive and less liable to change afterdeath and be affected by the fixation time in10%formalin. Heidenhainstaining can be the first choice for the postmortem diagnosis of EMI in thepractice of forensic medicine.
②The diminished or absent actin (HHF35), H-FABP and S100A1occurs in rats at15min after LAD occlusion, and the positive expressionoccurs in ADAMTS-1and CLU. The positive expression of C5b-9and HIF-1αoccurs at30min after LAD occlusion. The positive expression of HIF-2αoccurs at1h after LAD occlusion. The positive expression of Fn occurs at2hafter LAD occlusion. With the ischemic time prolongs, the depletion of actin(HHF35), H-FABP and S100A1becomes more evident, while the proteinexpression of C5b-9、Fn、CLU、HIF-1α、HIF-2α and ADAMTS-1becomestrong gradually.
③Actin (HHF35), H-FABP, S100A1are worst in terms stability, and
only when the ischemic heart is preserved in-20℃temperature, in roomtemperature for1day, and in4℃temperature for3days can be meaningfulfor postmortem diagnosis. C5b-9, Fn, CLU, HIF-1α、HIF-2α and ADAMTS-1all show a excellent feature of stability, among which CLU is most stable andcan be applicable to detect of ischemic heart for28days preserved under threetemperature conditions. C5b-9is most unstable and can be used to detect theischemic heart preserved in-20℃temperature for28days, in4℃temperaturefor21days, and in room temperature for14days. All the nine diagnosticmarkers can be used to detect the heart samples which are fixed in10%formalin for42days.
④The nine diagnostic markers, namely actin (HHF35), H-FABP,S100A1, C5b-9, Fn, CLU, HIF-1α, HIF-2α and ADAMTS-1, are verified onthe EMI human autopsy hearts. Among them, actin (HHF35), H-FABP andS100A1are least in diagnostic specificity, which are not suitable to be appliedalone to postmortem diagnosis of EMI. C5b-9, Fn, CLU, HIF-1α, HIF-2α andADAMTS-1are more useful in postmortem diagnosis of EMI, but theirexpressions are also found in some cases of non-infarctive direct or indirectmyocardial injury (mechanical asphyxia, electrocution, hemorrhagic shock,carbon monoxide poisoning, viral myocarditis). The combined application ofmultiple diagnostic markers will improve the accuracy of the postmortemdiagnosis of EMI.
To sum up, this research studies systematically the application ofhistochemical staining and the nine diagnostic markers of IHC, whichprovides theoretical basis for the forensic experts of the postmortem diagnosisof EMI.
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