豚鼠过敏性休克组织中相关介质的表达及法医学意义
|
摘要
前言过敏性休克(anaphylactic shock)属于速发型变态反应,是IgE介导的Ⅰ型变态反应中最为危重与急骤的一种。过敏性休克可以出现皮肤、呼吸系统、消化系统以及心血管系统的症状和体征,严重者可以导致患者死亡,经常引起医疗纠纷和民事诉讼。
过敏性休克死亡尸体常无特异性病理学改变,其法医学诊断也缺乏明确的客观指标。过敏性休克的发生是由于肥大细胞脱颗粒所释放的各种介质发挥其作用生物学所致。类胰蛋白酶(tryptase)和胃促胰酶(chymase)是肥大细胞中两种中性丝氨酸蛋白酶,其分子量大于其他致过敏性反应的介质,且其理化性质稳定,因此,类胰蛋白酶和胃促胰酶被认为是肥大细胞脱颗粒的重要标志。
白细胞介素5(interleukin-5,IL-5)是嗜酸性粒细胞(Eosinophil,EOS)趋化因子之一,是Ⅰ型变态反应中主要的效应因子。组织中IL-5的表达主要限于哮喘的研究,而关于过敏性休克发生后IL-5在体内的表达情况,尚缺乏深入的研究。
本研究在建立豚鼠过敏性休克的动物模型基础上,应用免疫组织化学技术,观察人混合血清致豚鼠过敏性休克死亡体内多脏器组织中类胰蛋白酶、胃促胰酶和IL-5的表达情况,通过病理学观察和统计学分析,并结合《医疗事故处理条例》所规定的保存条件,来评定这三种介质在过敏性休克诊断中的意义,为过敏性休克死亡的法医学鉴定提供形态学依据和指标。
材料和方法
一、动物模型的建立及分组
健康成年豚鼠24只,随机分为死亡即时组、冷藏48小时组和冷冻7天组,每组8只,每组又分实验组和对照组,每组4只。实验组应用人混合血清诱发豚鼠发生过敏性休克死亡;对照组豚鼠采用生理盐水代替血清,乙醚麻醉过量致死,提攘阶殡嗍笮难票秆灞讣?取喉头、气管、双肺、胃等器官,4%多聚甲醛固定,石蜡包埋。准备明确过敏性休克死亡和非过敏性休克死亡实际案例各5例进行初步探索性观察研究。
二、实验方法
1、应用HE染色法观察组织的形态学特点;
2、采用铬变素2R染色法观察组织中嗜酸性粒细胞浸润情况;
3、使用化学发光法检测血清中IgE水平;
4、应用免疫组织化学染色法观察类胰蛋白酶、胃促胰酶和IL-5在组织中的表达情况;
5、使用Motic Images Advanced 3.2图像分析系统计算免疫组织化学染色阳性细胞数及表达面积;
6、应用SPSS统计学软件对实验数据进行统计分析。
实验结果
1、HE染色见过敏性休克的豚鼠喉头、气管等器官水肿,肺泡扩张,肺泡壁变薄;
2、铬变素2R染色后见实验组各脏器组织内较多嗜酸性粒细胞浸润
3、实验组血清IgE水平明显高于对照组,P＜0.01;
4、对照组各组可见散在类胰蛋白酶和胃促胰酶免疫组织化学染色阳性的肥大细胞,细胞形态完整,染色产物分布于细胞胞浆内,呈棕黄色。实验组类胰蛋白酶和胃促胰酶染色阳性细胞增多,表达增强,而且染色产物部分弥散到肥大细胞周围及组织间隙;对照组可见散在IL-5阳性细胞,染色产物位于胞浆内。实验组IL-5阳性细胞形态不完整,染色产物大部分弥散到细胞周围和组织间隙。使用Motic Image Advanced 3.2图像分析系统计算各组免疫组织化学阳性染色面积,通过SPSS软件统计分析,发现实验组和对照组在相同保存条件下仍然存在较大差异。
5、实际案例中非过敏性休克死亡者类胰蛋白酶和胃促胰酶免疫组织化学染色阳性颗粒出现于肥大细胞胞浆内,肥大细胞形态完整;过敏性休克死亡者大部分免疫组织化学染色产物弥散至肥大细胞周围和组织间隙,肥大细胞形态不规则。
讨论
过敏性休克是临床上常见的一种速发型变态反应,主要是由IgE介导,为Ⅰ型变态反应中最为危重与急骤的一种,常可导致患者猝死,因此,在法医学鉴定工作中经常接触到。
过敏性休克的发生是由于个体在接受变应原刺激后,产生IgE类抗体,当相应的抗原再次进入机体时通过与致敏肥大细胞表面的两个或两个以上相邻IgE抗体特异结合,使膜表面的FcεRI(FC受体)交联活化,最终导致肥大细胞脱颗粒,从而引发过敏性休克的各种临床征象。
过敏性休克死亡者,心血中IgE的浓度往往增高,组织中嗜酸性粒细胞数量增多。但IgE受外界因素影响较大,嗜酸性粒细胞也不是过敏性休克所特有,缺乏一定的客观性。
过敏性休克发生的一个重要机制是肥大细胞脱颗粒,释放介质引起相应的症状和体征。如何证明肥大细胞脱颗粒是诊断过敏性休克的关键问题。类胰蛋白酶和胃促胰酶是肥大细胞内两种重要的丝氨酸蛋白酶,同时它们的性质非常稳定,肥大细胞发生脱颗粒时它们也会随之脱出到细胞外和组织间隙中。IL-5是嗜酸性粒细胞趋化因子,在嗜酸性粒细胞增多的疾病中,其表达将会增强。本实验通过对实验组和对照组不同保存条件下类胰蛋白酶、胃促胰酶和IL-5进行免疫组织化学染色观察,证实了这3种介质在过敏的豚鼠体内表达增强,类胰蛋白酶和胃促胰酶也明显地反映出肥大细胞脱颗粒现象。因此,类胰蛋白酶、胃促胰酶和IL-5的免疫组织化学染色可以为过敏性休克的诊断提供新的形态学依据。
本实验将所使用的类胰蛋白酶和胃促胰酶免疫组织化学染色法应用于实际案例中,结果表明非过敏性休克死亡的标本两种蛋白酶表达的情况反映出肥大细胞的数量和形态,而过敏性休克死亡的标本内两种酶表达增强,染色产物脱出于肥大细胞外及周围组织间隙,肥大细胞形态不规则,因此,类胰蛋白酶和胃促胰酶的免疫组织化学染色法有望应用于过敏性休克的法医学实际案例的诊断。
结论
1、HE染色见过敏性休克的豚鼠喉头、气管等器官水肿,肺泡扩张,肺泡壁变薄;
2、过敏性休克的豚鼠脏器经过铬变素2R染色后,各脏器内出现了明显的嗜酸性粒细胞浸润情况;
3、过敏性休克豚鼠血清IgE水平明显高于对照组;
4、过敏性休克的豚鼠体内类胰蛋白酶、胃促胰酶及IL-5的表达增强,可以为过敏性休克的诊断提供形态学依据;
5、按国务院颁布的《医疗事故处理条例》实施细则规定的死亡后冷藏48小时或冷冻7天内进行尸检的条件标准,在以上两种条件下进行这三种介质的免疫组织化学染色,不影响它们的表达以及对其结果的判定;
6、本实验类胰蛋白酶和胃促胰酶免疫组织化学染色方法有望应用于实际案例中,并可为实际案例提供可靠的诊断依据。
Introduction
Anaphylactic shock belongs to the IgE-mediated immediate allergy which is urgent and dangerous. Anaphylactic shock can show dermal, respiratory, digestive systems and cardiovascular system symptoms, and severe cases can died of anaphylactic shock. Some of the cases can cause civil procedure and medical tangle.
The bodies died of anaphylactic shock are often without specific pathological changes and lacking in objective diagnose indicators. According to the mechanism of anaphylactic shock, the various mediators which degranulate from mast cell play an important role in anaphylactic shock. The tryptase and chymase which belong to neutral serine protease are stable, and its molecular weight is bigger than other mediators. Thus, they are both the signs of mast cell degranulated.
IL-5 is one of the chemokines of eosinophil(EOS), which is the main effect factor in allergic type I . Its study is focus on the asthma. At present, there is no research in anaphylactic shock.
This study is on the basis of animal models of guinea pigs who died of anaphylactic shock, using immunohistochemistry(IHC) technology, observe the expression of these mediators. Then analysis is made to evalue medicolegal significance by using the methods of pathology and statistics, depending on Malpractice Bill. Finally, the aim of this study is providing the objective evidences to the diagnosis of anaphylactic shock.
Materials and Methods
1. Establishment of animal model and packet
A total of 24 healthy guinea pigs are divided into 0 hour group, 48 hours group in 4℃, and 7 days group in -20℃at random, 8 in each group. Each group is also divided into experimental group and control group, 4 in each group. The guinea pigs in experimental group evoke anaphylactic shock, then died after injecting human mixed serum. The guinea pigs in control group executed by ether poisoning after injecting NS. Blood was drawn from heart and the serum was purified. Larynx, trachea, lungs, and stomach of guinea pigs were taken out, then fixed in 4% polyoxymethylene and embedded in paraffin. Immunohitochemistry of tryptase and chymase was performed in 5 cases died of anaphylactic shock and 5 cases died of the other reasons.
2. Methods
(1) HE staining.
(2) Stain EOS by using the method of Carbol Chromotrope 2R staining.
(3) Detect of the IgE levels in serum by using chemiluminescent.
(4) Tryptase, chymase and IL-5 immunohistochemical staining.
(5) Calculate and analyze positive expression by Motic Image Advanced 3.2 system.
(6) Analyze the differences between groups using SPSS.
Result
(1) The guinea pigs' larynx and trachea were of edema, pulmonary alveolus were of expansion, and alveolar wall became thin with HE staining in anaphylactic shock.
(2) There was a number of eosinophils in tissue by Carbol Chromotrope 2R staining in experimental group.
(3) The IgE levels of serum in experimental group is higher than that in controlgroup, P<0.01.
(4) There are scattered tryptase and chymase positive mast cells by IHC method in control groups, and brown staining product in cytoplasm. The positive cells number of tryptase and chymase increased and the expression enhanced, and staining product partly diffused around mast cells and in tissue space in experimental groups. There were scattered positive cells of IL-5 in control group, staining product in cytoplasm. In experimental group, positive cells staining product mostly diffused around cells and in tissue space. There were differences between experimental and control group by SPSS.
(5) The tryptase and chymase positive granules of non-anaphylactic shock in actual cases were seen in cytoplasm of mast cells, and mast cells have complete form. The most staining product diffused around cells and in tissue space, and mast cells have incomplete form in anaphylactic shock cases.
Discussion
Anaphylactic shock belongs to the IgE-mediated immediate allergy which is urgent and fatal to the human, therefore, it is met in forensic identification ususlly.
Because of the stimulater by antigen, body can produce much IgE, when the same antigen come in the body again, the FcεRI on the face of mast cells which have been sensitized can activate by neighboring IgE. Fanally, mast cells degranulate, and show up clinical symptoms.
The people who died of anaphylactic shock, the IgE levels of serum usually higher than the normal, the number of eosinophils in tissue increased. But, the IgE levels usually effected by external factors, while the eosinophils increased is not the unique apparence, so both of them are lack of objectivity in medicolegal expertise.
There is a pathological phenomenon, that is the mast cells degranulate in anaphylactic shock. How to prove if mast cells degranulate can provide a strong evidence to the diagnosis of anaphylactic shock. This study just to find the evidence of mast cells degranulate. Tryptase and chymase are both neutral mediators in mast cell, which are very stable. They can spread to tissue space accompanied by mast cell degranulate. IL-5 is the chemokines of eosinophils. In some diseases which register as eosinophils increased, the expression of IL-5 will be stronger. This study by comparing the experimental groups with control groups'expression of these mediators, prove that they were stronger in the guinea pigs who died of anaphylactic shock. Further more, tryptase and chymase can reflect if mast cells degranulate. Therefore, it is can say that use the medthod of IHC to observe the expression of tryptase, chymase and IL-5 will provide new evidence to the diagnosis of anaphylactic shock.
When have this method application in the actual cases, discover that the expression of tryptase and chymase stronger in anaphylactic shock group than that in non-anaphylactic shock group, The tryptase and chymase positive granules of non-anaphylactic shock in actual cases were seen in cytoplasm of mast cells, and mast cells have complete form. The most staining product diffused around cells and in tissue space, and mast cells have incomplete form in anaphylactic shock cases. So, this method of IHC can be applied to the diagnosis of actual cases to distinguish the anaphylactic shock and others.
Conclusion
1. The guinea pigs' larynx and trachea were of edema, pulmonary alveolus were of expansion, and alveolar wall became thin with HE staining in anaphylactic shock.
2. There was a number of eosinophils in tissue by Carbol Chromotrope 2R staining in experimental group.
3. The IgE levels of serum in experimental group is higher than that in control group.
4. The expression of tryptase, chymase and IL-5 of guinea pigs in anaphylactic shock enhanced, which can provide diagnostic evidences of morphology to anaphylactic shock.
5. According to the condition set by Malpractice Bill, there is no evidently effection about using IHC to observe these three mediators.
6. The IHC staining of tryptase and chymase is expected to be applied in anaphylactic shock cases, providing diagnostic evidences.
过敏性休克死亡尸体常无特异性病理学改变,其法医学诊断也缺乏明确的客观指标。过敏性休克的发生是由于肥大细胞脱颗粒所释放的各种介质发挥其作用生物学所致。类胰蛋白酶(tryptase)和胃促胰酶(chymase)是肥大细胞中两种中性丝氨酸蛋白酶,其分子量大于其他致过敏性反应的介质,且其理化性质稳定,因此,类胰蛋白酶和胃促胰酶被认为是肥大细胞脱颗粒的重要标志。
白细胞介素5(interleukin-5,IL-5)是嗜酸性粒细胞(Eosinophil,EOS)趋化因子之一,是Ⅰ型变态反应中主要的效应因子。组织中IL-5的表达主要限于哮喘的研究,而关于过敏性休克发生后IL-5在体内的表达情况,尚缺乏深入的研究。
本研究在建立豚鼠过敏性休克的动物模型基础上,应用免疫组织化学技术,观察人混合血清致豚鼠过敏性休克死亡体内多脏器组织中类胰蛋白酶、胃促胰酶和IL-5的表达情况,通过病理学观察和统计学分析,并结合《医疗事故处理条例》所规定的保存条件,来评定这三种介质在过敏性休克诊断中的意义,为过敏性休克死亡的法医学鉴定提供形态学依据和指标。
材料和方法
一、动物模型的建立及分组
健康成年豚鼠24只,随机分为死亡即时组、冷藏48小时组和冷冻7天组,每组8只,每组又分实验组和对照组,每组4只。实验组应用人混合血清诱发豚鼠发生过敏性休克死亡;对照组豚鼠采用生理盐水代替血清,乙醚麻醉过量致死,提攘阶殡嗍笮难票秆灞讣?取喉头、气管、双肺、胃等器官,4%多聚甲醛固定,石蜡包埋。准备明确过敏性休克死亡和非过敏性休克死亡实际案例各5例进行初步探索性观察研究。
二、实验方法
1、应用HE染色法观察组织的形态学特点;
2、采用铬变素2R染色法观察组织中嗜酸性粒细胞浸润情况;
3、使用化学发光法检测血清中IgE水平;
4、应用免疫组织化学染色法观察类胰蛋白酶、胃促胰酶和IL-5在组织中的表达情况;
5、使用Motic Images Advanced 3.2图像分析系统计算免疫组织化学染色阳性细胞数及表达面积;
6、应用SPSS统计学软件对实验数据进行统计分析。
实验结果
1、HE染色见过敏性休克的豚鼠喉头、气管等器官水肿,肺泡扩张,肺泡壁变薄;
2、铬变素2R染色后见实验组各脏器组织内较多嗜酸性粒细胞浸润
3、实验组血清IgE水平明显高于对照组,P＜0.01;
4、对照组各组可见散在类胰蛋白酶和胃促胰酶免疫组织化学染色阳性的肥大细胞,细胞形态完整,染色产物分布于细胞胞浆内,呈棕黄色。实验组类胰蛋白酶和胃促胰酶染色阳性细胞增多,表达增强,而且染色产物部分弥散到肥大细胞周围及组织间隙;对照组可见散在IL-5阳性细胞,染色产物位于胞浆内。实验组IL-5阳性细胞形态不完整,染色产物大部分弥散到细胞周围和组织间隙。使用Motic Image Advanced 3.2图像分析系统计算各组免疫组织化学阳性染色面积,通过SPSS软件统计分析,发现实验组和对照组在相同保存条件下仍然存在较大差异。
5、实际案例中非过敏性休克死亡者类胰蛋白酶和胃促胰酶免疫组织化学染色阳性颗粒出现于肥大细胞胞浆内,肥大细胞形态完整;过敏性休克死亡者大部分免疫组织化学染色产物弥散至肥大细胞周围和组织间隙,肥大细胞形态不规则。
讨论
过敏性休克是临床上常见的一种速发型变态反应,主要是由IgE介导,为Ⅰ型变态反应中最为危重与急骤的一种,常可导致患者猝死,因此,在法医学鉴定工作中经常接触到。
过敏性休克的发生是由于个体在接受变应原刺激后,产生IgE类抗体,当相应的抗原再次进入机体时通过与致敏肥大细胞表面的两个或两个以上相邻IgE抗体特异结合,使膜表面的FcεRI(FC受体)交联活化,最终导致肥大细胞脱颗粒,从而引发过敏性休克的各种临床征象。
过敏性休克死亡者,心血中IgE的浓度往往增高,组织中嗜酸性粒细胞数量增多。但IgE受外界因素影响较大,嗜酸性粒细胞也不是过敏性休克所特有,缺乏一定的客观性。
过敏性休克发生的一个重要机制是肥大细胞脱颗粒,释放介质引起相应的症状和体征。如何证明肥大细胞脱颗粒是诊断过敏性休克的关键问题。类胰蛋白酶和胃促胰酶是肥大细胞内两种重要的丝氨酸蛋白酶,同时它们的性质非常稳定,肥大细胞发生脱颗粒时它们也会随之脱出到细胞外和组织间隙中。IL-5是嗜酸性粒细胞趋化因子,在嗜酸性粒细胞增多的疾病中,其表达将会增强。本实验通过对实验组和对照组不同保存条件下类胰蛋白酶、胃促胰酶和IL-5进行免疫组织化学染色观察,证实了这3种介质在过敏的豚鼠体内表达增强,类胰蛋白酶和胃促胰酶也明显地反映出肥大细胞脱颗粒现象。因此,类胰蛋白酶、胃促胰酶和IL-5的免疫组织化学染色可以为过敏性休克的诊断提供新的形态学依据。
本实验将所使用的类胰蛋白酶和胃促胰酶免疫组织化学染色法应用于实际案例中,结果表明非过敏性休克死亡的标本两种蛋白酶表达的情况反映出肥大细胞的数量和形态,而过敏性休克死亡的标本内两种酶表达增强,染色产物脱出于肥大细胞外及周围组织间隙,肥大细胞形态不规则,因此,类胰蛋白酶和胃促胰酶的免疫组织化学染色法有望应用于过敏性休克的法医学实际案例的诊断。
结论
1、HE染色见过敏性休克的豚鼠喉头、气管等器官水肿,肺泡扩张,肺泡壁变薄;
2、过敏性休克的豚鼠脏器经过铬变素2R染色后,各脏器内出现了明显的嗜酸性粒细胞浸润情况;
3、过敏性休克豚鼠血清IgE水平明显高于对照组;
4、过敏性休克的豚鼠体内类胰蛋白酶、胃促胰酶及IL-5的表达增强,可以为过敏性休克的诊断提供形态学依据;
5、按国务院颁布的《医疗事故处理条例》实施细则规定的死亡后冷藏48小时或冷冻7天内进行尸检的条件标准,在以上两种条件下进行这三种介质的免疫组织化学染色,不影响它们的表达以及对其结果的判定;
6、本实验类胰蛋白酶和胃促胰酶免疫组织化学染色方法有望应用于实际案例中,并可为实际案例提供可靠的诊断依据。
Introduction
Anaphylactic shock belongs to the IgE-mediated immediate allergy which is urgent and dangerous. Anaphylactic shock can show dermal, respiratory, digestive systems and cardiovascular system symptoms, and severe cases can died of anaphylactic shock. Some of the cases can cause civil procedure and medical tangle.
The bodies died of anaphylactic shock are often without specific pathological changes and lacking in objective diagnose indicators. According to the mechanism of anaphylactic shock, the various mediators which degranulate from mast cell play an important role in anaphylactic shock. The tryptase and chymase which belong to neutral serine protease are stable, and its molecular weight is bigger than other mediators. Thus, they are both the signs of mast cell degranulated.
IL-5 is one of the chemokines of eosinophil(EOS), which is the main effect factor in allergic type I . Its study is focus on the asthma. At present, there is no research in anaphylactic shock.
This study is on the basis of animal models of guinea pigs who died of anaphylactic shock, using immunohistochemistry(IHC) technology, observe the expression of these mediators. Then analysis is made to evalue medicolegal significance by using the methods of pathology and statistics, depending on Malpractice Bill. Finally, the aim of this study is providing the objective evidences to the diagnosis of anaphylactic shock.
Materials and Methods
1. Establishment of animal model and packet
A total of 24 healthy guinea pigs are divided into 0 hour group, 48 hours group in 4℃, and 7 days group in -20℃at random, 8 in each group. Each group is also divided into experimental group and control group, 4 in each group. The guinea pigs in experimental group evoke anaphylactic shock, then died after injecting human mixed serum. The guinea pigs in control group executed by ether poisoning after injecting NS. Blood was drawn from heart and the serum was purified. Larynx, trachea, lungs, and stomach of guinea pigs were taken out, then fixed in 4% polyoxymethylene and embedded in paraffin. Immunohitochemistry of tryptase and chymase was performed in 5 cases died of anaphylactic shock and 5 cases died of the other reasons.
2. Methods
(1) HE staining.
(2) Stain EOS by using the method of Carbol Chromotrope 2R staining.
(3) Detect of the IgE levels in serum by using chemiluminescent.
(4) Tryptase, chymase and IL-5 immunohistochemical staining.
(5) Calculate and analyze positive expression by Motic Image Advanced 3.2 system.
(6) Analyze the differences between groups using SPSS.
Result
(1) The guinea pigs' larynx and trachea were of edema, pulmonary alveolus were of expansion, and alveolar wall became thin with HE staining in anaphylactic shock.
(2) There was a number of eosinophils in tissue by Carbol Chromotrope 2R staining in experimental group.
(3) The IgE levels of serum in experimental group is higher than that in controlgroup, P<0.01.
(4) There are scattered tryptase and chymase positive mast cells by IHC method in control groups, and brown staining product in cytoplasm. The positive cells number of tryptase and chymase increased and the expression enhanced, and staining product partly diffused around mast cells and in tissue space in experimental groups. There were scattered positive cells of IL-5 in control group, staining product in cytoplasm. In experimental group, positive cells staining product mostly diffused around cells and in tissue space. There were differences between experimental and control group by SPSS.
(5) The tryptase and chymase positive granules of non-anaphylactic shock in actual cases were seen in cytoplasm of mast cells, and mast cells have complete form. The most staining product diffused around cells and in tissue space, and mast cells have incomplete form in anaphylactic shock cases.
Discussion
Anaphylactic shock belongs to the IgE-mediated immediate allergy which is urgent and fatal to the human, therefore, it is met in forensic identification ususlly.
Because of the stimulater by antigen, body can produce much IgE, when the same antigen come in the body again, the FcεRI on the face of mast cells which have been sensitized can activate by neighboring IgE. Fanally, mast cells degranulate, and show up clinical symptoms.
The people who died of anaphylactic shock, the IgE levels of serum usually higher than the normal, the number of eosinophils in tissue increased. But, the IgE levels usually effected by external factors, while the eosinophils increased is not the unique apparence, so both of them are lack of objectivity in medicolegal expertise.
There is a pathological phenomenon, that is the mast cells degranulate in anaphylactic shock. How to prove if mast cells degranulate can provide a strong evidence to the diagnosis of anaphylactic shock. This study just to find the evidence of mast cells degranulate. Tryptase and chymase are both neutral mediators in mast cell, which are very stable. They can spread to tissue space accompanied by mast cell degranulate. IL-5 is the chemokines of eosinophils. In some diseases which register as eosinophils increased, the expression of IL-5 will be stronger. This study by comparing the experimental groups with control groups'expression of these mediators, prove that they were stronger in the guinea pigs who died of anaphylactic shock. Further more, tryptase and chymase can reflect if mast cells degranulate. Therefore, it is can say that use the medthod of IHC to observe the expression of tryptase, chymase and IL-5 will provide new evidence to the diagnosis of anaphylactic shock.
When have this method application in the actual cases, discover that the expression of tryptase and chymase stronger in anaphylactic shock group than that in non-anaphylactic shock group, The tryptase and chymase positive granules of non-anaphylactic shock in actual cases were seen in cytoplasm of mast cells, and mast cells have complete form. The most staining product diffused around cells and in tissue space, and mast cells have incomplete form in anaphylactic shock cases. So, this method of IHC can be applied to the diagnosis of actual cases to distinguish the anaphylactic shock and others.
Conclusion
1. The guinea pigs' larynx and trachea were of edema, pulmonary alveolus were of expansion, and alveolar wall became thin with HE staining in anaphylactic shock.
2. There was a number of eosinophils in tissue by Carbol Chromotrope 2R staining in experimental group.
3. The IgE levels of serum in experimental group is higher than that in control group.
4. The expression of tryptase, chymase and IL-5 of guinea pigs in anaphylactic shock enhanced, which can provide diagnostic evidences of morphology to anaphylactic shock.
5. According to the condition set by Malpractice Bill, there is no evidently effection about using IHC to observe these three mediators.
6. The IHC staining of tryptase and chymase is expected to be applied in anaphylactic shock cases, providing diagnostic evidences.
引文
1 Schwartz HJ.Anaphylaxis:issues in diagnosis.Curr Opin Allergy Clin Immunol.2001;1(4):357-359.
2 Erik E,Marianne VH.Postmortem Diagnosis of Anaphylaxis.Forensic Pathology Reviews.2005;Volume 3:267-281.
3 Simon GA,Raymond JM,Michael SG.Anaphylaxis:diagnosis and management.MJA.2006;185:283-289.
4 Schwartz LB.Tryptase:a clinical indicator of mast cell-dependent events.Allergy Proc.1994;15(3):119-23.
5 Matsumoto K,Saito H.The role of eosinophils in asthma:Sarastro or the Queen of the Night?Int Arch Allergy Immunol,2001;125:290-296.
6 郭薇,陈玉川,刘水平等.豚鼠过敏性休克类胰蛋白酶活力测定.法医学杂志.2001;17(4),212-213.
7 张勇,杨冬梅,张盛忠等.标记嗜酸性粒细胞的组化染色方法.临床与实验病理学杂志.2002;18(4):464.
8 Greenberger PA,Rotskoff BD,Lifschultz B.Fatal anaphylaxis:postmortem findings and associated comorbid diseases.Ann Allergy Asthma Immunol.2007;98(3):252-257.
9 Costello PS,Turner M,Walters AE,et al.Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells.Oncogene.1996;13(12):2595-605.
10 HeidaY,Kaguera M,Hara K,et al.Postmortem changes in hapten-specific IgE antibody responses in mice.International Journal of Legal Medicine.1990;104(4):1437-1596.
11 江荣林.IL25、Eotaxin与支气管哮喘.国外医学呼吸系统分册.2004;(24):118-20.
12 Oliveira SH,Taub DD,Naget J,et al.Stem cell factor induces eosinophil activation and degranulation:mediator release and gene array analysis.Blood.2002;100(13):4291-7.
13 Schwartz LB,Metcalfe DD,Miller JS,et al.Tryptase levels as an indicator of mast cell activation in systemic anaphylaxis and mastocytosis.Int J Legal Med.1987;316:1622-1626.
14 Schwartz LB,Lewis RA,Austen KF.Tryptase from human pulmonary mast cell:purificationda and characterization.J Biol Chem.1981;256(22):11939-43.
15 Fiorucci L,Ascoli F.Mast cell tryptase,a still enigmatic enzyme.Cell Mol Life Sci.2004;61(11):1278-1295.
16 Edson E,Van HM.Beta-tryptase measurements post-mortem in anaphylactic death and in controls.Forensic Sci Int.1998,93(2-3):135-142.
17 Fineschi V,Gambassi R,Gherardi M,et al.The diagnosis of amniotic fluid embolism:an immunlaistomchemical study for the quantification of pulmonary mast tryptase.Int J Legal Med.1998;111:238-243.
18 Buckley MG,Variend S,Walls AF.Elevated serum concentrations of beta-tryptase,but not alpha-tryptase,in Sudden Infant Death Syndrome(SIDS).Clin Exp Allergy.2001 Nov;31(11):1696-1704.
19 Lindstedt KA,Kovanen PT.Isolation of mast cell granules.Curr Protoc Cell Biol.2006;Chapter 3:Unit 3.16
20 Preobrazhenskii DV,Batyraliev TA,Peresypko MK,et al.Antiatherogenic effects of Angiotensin converting enzyme inhibitors from the point of view of evidence based medicine.Kardiologiia.2007;47(5):73-86.
21 Tani K,Ogushi F,Kido H,et al.Chymase is a potent chemoattractant for human monocytes and neutrophilis.Shock.2001;16:21-24.
22 陈谱,何韶衡.变态反应性疾病中肥大细胞的特异激活标志物.实用儿科临床杂志.2004;2:141-142.
23 Craig.SS,Schwartz LB.Human MCTC type of mast cell granule:the uncommon occurrence of discrete scrolls associated with focal absence of chymase.Lab Invest.1990;63(4):581-5.
24 Nishio H,Takai S,Miyazaki M,et al.Usefulness of serum mast cell-specific chmase levels for postmortem diagnosis of anaphylaxis.Int J Legal Med.2005;119:331-334.
25 Wardlaw A J,Brightling C,Green R,et al.Eosinophils in asthma and other allergic diseases.Br Med Bull.2000;56(4):985-1003.
26 Lampinen M,Carlson M,Sangfelt P,et al.IL-5 and TNF-alpha participate in recruitment of eosinophils to intestinal mucosa in ulcerative colitis.Dig Dis Sci.2001;46(9):2004-2009.
1 Schwartz HJ.Anaphylaxis:issues in diagnosis.Curt Opin Allergy Clin Immunol.2001;1(4):357-359.
2 D'Amato G.Therapy of allergic bronchial asthma with omalizumab - an anti-IgE monoclonal antibody.Division of Pneumology.2003;3(2):371-6.
3 Basso G,Case C,Dell'orto MC.Diagnosis and genetic subtypes of leukemia combining gene expression and flow cytometry.Blood Cells Mol Dis.2007;22-25.
4 刘东方.肥大细胞脱颗粒机制研究进展.国外医学临床生物化学与检验分册.2004:137-139.
5 Yokoi H,Okayama Y,Niyonsaba F,et al.Comparison of human tonsillar mast cell localization and ultrastructural observations between IgE-mediated allergic and nonallergic donors.Allergy Asthma Proc.2006,27(5):415-21.
6 罗正翟.休克学[M].天津科学技术出版社.2001,789-795.
7 Schwartz LB,Lewis RA,Austen KF.Tryptase from human pulmonary mast cell:purificationda and characterization.Biol Chem.1981;256(22):11939-43.
8 Craig.SS,Schwartz LB.Human MCTC type of mast cell granule:the uncommon occurrence of discrete scrolls associated with focal absence of chymase.Lab Invest.1990;63(4):581-5.
9 Tani K,Ogushi F,Kido H,et al.Chymase is a potent chemoattractant for human monocytes and neutrophilis.Shock.2001;16:21-24.
10 陈谱,何韶衡.变态反应性疾病中肥大细胞的特异激活标志物.实用儿科临床杂志.2004;2:141-142.
11 Boyce JA.Mast cells and eicosanoid mediators:a system of reciprocal paracrine and autocrine regulation.Immunol Rev.2007;217:168-85.
12 Mitsumori S.Recent progress in work on PGD2 antagonists for drugs targeting allergic diseases.Curr Pharm Des.2004;(28):3533-3538.
13 Genovese A,Stellato C,Marsella CV,et al.Role of mast cells,basophils and their mediators in adverse reactions to general anesthetics and radiocontrast media.Int Arch Allergy Immunol.1996;110(1):13-22.
14 Greenberger PA.Anaphylactic and anaphylactoid causes of angioedema.Immunol Allergy Clin North Am.2006;26(4):753-67.
15 龚非力.医学免疫学.北京科学出版社,2004;34-41.
16 Yunginger JW,Nelson DR,Squillace DL,et al.Laboratory investigation of deaths due to anaphylaxis.J Forensic Sci.1991;36(3):857-865.
17 Hogan AD,Schwartz LB.Markers of mast cell degranulation.Methods.1997;343-521.
18 Fazekas IG,Viragos-kisE.Free histamine content in the groove caused by hanging as an in vivo reaction.Dtsch Z Gesamte Gerichtl Med.1965;56(4):250-268.
19 Schwartz LB,Metcalfe DD,Miller JS,et al.Tryptase levels as an indicator of mast cell activation in systemic anaphylaxis and mastocytosis.Int J Legal Meal.1987;316:1622-1626.
20 Shunlin ren,Kentaro Sakai,Lawrence B.Regulation of human mast cell betatryptase conversion of inactive monomer to active tetramer at acid PH.J Immunol.1998;160(9):4561-4569.
21 Hogan AD,SchwartzLB.Makers of mast celldegranulation.Methods,1997;13(1):43-52.
22 Caughey GH.Tryptase genetics andanaphylaxis.Allergy Clin Immunol 2006;117:1411-1414.
23 Laroche D,Vergnaud MC,Sillard B,et al.Biochemical markers of anaphylactoid reactions to drugs comparison of plasma histamine and tryptase.Anesthesiology.1991;75:945-949.
24 Edson E,Van,HM.Beta-tryptase measurements post-mortem in anaphylactic death and in controls.Forensic Sci Int.1998;93(2-3):135-142.
25 Fineschi V,Gambassi R,Gherardi M,et al.The diagnosis of amniotic fluid embolism:an immunhistomchemical study for the quantification of pulmonary mast tryptase.Int J Legal Med.1998;111:238-243.
26 高彩荣,任广睦,范厚生等.肥大细胞类胰蛋白酶的免疫组化染色观察.中国法医学杂志.2003:18:212-214.
27 Buckley MG,Variend S,Walls AF.Elevated serum concentrations of beta-tryptase,but not alpha-tryptase,in Sudden Infant Death Syndrome(SIDS).Clin Exp Allergy.2001 Nov;31(11):1696-1704.
28 Badertscher K,Br6nnimann M,Karlen S,et al.Mast cell chymase is increased in chronic atopic dermatitis but not in psoriasis. Arch Dermatol Res. 2005; 296:503-506.
29 Nishio H, Takai S, Miyazaki M, et al. Usefulness of serum mast cell-specific chmase levels for postmortem diagnosis of anaphylaxis. Int J Legal Med. 2005; 119:331-334.
2 Erik E,Marianne VH.Postmortem Diagnosis of Anaphylaxis.Forensic Pathology Reviews.2005;Volume 3:267-281.
3 Simon GA,Raymond JM,Michael SG.Anaphylaxis:diagnosis and management.MJA.2006;185:283-289.
4 Schwartz LB.Tryptase:a clinical indicator of mast cell-dependent events.Allergy Proc.1994;15(3):119-23.
5 Matsumoto K,Saito H.The role of eosinophils in asthma:Sarastro or the Queen of the Night?Int Arch Allergy Immunol,2001;125:290-296.
6 郭薇,陈玉川,刘水平等.豚鼠过敏性休克类胰蛋白酶活力测定.法医学杂志.2001;17(4),212-213.
7 张勇,杨冬梅,张盛忠等.标记嗜酸性粒细胞的组化染色方法.临床与实验病理学杂志.2002;18(4):464.
8 Greenberger PA,Rotskoff BD,Lifschultz B.Fatal anaphylaxis:postmortem findings and associated comorbid diseases.Ann Allergy Asthma Immunol.2007;98(3):252-257.
9 Costello PS,Turner M,Walters AE,et al.Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells.Oncogene.1996;13(12):2595-605.
10 HeidaY,Kaguera M,Hara K,et al.Postmortem changes in hapten-specific IgE antibody responses in mice.International Journal of Legal Medicine.1990;104(4):1437-1596.
11 江荣林.IL25、Eotaxin与支气管哮喘.国外医学呼吸系统分册.2004;(24):118-20.
12 Oliveira SH,Taub DD,Naget J,et al.Stem cell factor induces eosinophil activation and degranulation:mediator release and gene array analysis.Blood.2002;100(13):4291-7.
13 Schwartz LB,Metcalfe DD,Miller JS,et al.Tryptase levels as an indicator of mast cell activation in systemic anaphylaxis and mastocytosis.Int J Legal Med.1987;316:1622-1626.
14 Schwartz LB,Lewis RA,Austen KF.Tryptase from human pulmonary mast cell:purificationda and characterization.J Biol Chem.1981;256(22):11939-43.
15 Fiorucci L,Ascoli F.Mast cell tryptase,a still enigmatic enzyme.Cell Mol Life Sci.2004;61(11):1278-1295.
16 Edson E,Van HM.Beta-tryptase measurements post-mortem in anaphylactic death and in controls.Forensic Sci Int.1998,93(2-3):135-142.
17 Fineschi V,Gambassi R,Gherardi M,et al.The diagnosis of amniotic fluid embolism:an immunlaistomchemical study for the quantification of pulmonary mast tryptase.Int J Legal Med.1998;111:238-243.
18 Buckley MG,Variend S,Walls AF.Elevated serum concentrations of beta-tryptase,but not alpha-tryptase,in Sudden Infant Death Syndrome(SIDS).Clin Exp Allergy.2001 Nov;31(11):1696-1704.
19 Lindstedt KA,Kovanen PT.Isolation of mast cell granules.Curr Protoc Cell Biol.2006;Chapter 3:Unit 3.16
20 Preobrazhenskii DV,Batyraliev TA,Peresypko MK,et al.Antiatherogenic effects of Angiotensin converting enzyme inhibitors from the point of view of evidence based medicine.Kardiologiia.2007;47(5):73-86.
21 Tani K,Ogushi F,Kido H,et al.Chymase is a potent chemoattractant for human monocytes and neutrophilis.Shock.2001;16:21-24.
22 陈谱,何韶衡.变态反应性疾病中肥大细胞的特异激活标志物.实用儿科临床杂志.2004;2:141-142.
23 Craig.SS,Schwartz LB.Human MCTC type of mast cell granule:the uncommon occurrence of discrete scrolls associated with focal absence of chymase.Lab Invest.1990;63(4):581-5.
24 Nishio H,Takai S,Miyazaki M,et al.Usefulness of serum mast cell-specific chmase levels for postmortem diagnosis of anaphylaxis.Int J Legal Med.2005;119:331-334.
25 Wardlaw A J,Brightling C,Green R,et al.Eosinophils in asthma and other allergic diseases.Br Med Bull.2000;56(4):985-1003.
26 Lampinen M,Carlson M,Sangfelt P,et al.IL-5 and TNF-alpha participate in recruitment of eosinophils to intestinal mucosa in ulcerative colitis.Dig Dis Sci.2001;46(9):2004-2009.
1 Schwartz HJ.Anaphylaxis:issues in diagnosis.Curt Opin Allergy Clin Immunol.2001;1(4):357-359.
2 D'Amato G.Therapy of allergic bronchial asthma with omalizumab - an anti-IgE monoclonal antibody.Division of Pneumology.2003;3(2):371-6.
3 Basso G,Case C,Dell'orto MC.Diagnosis and genetic subtypes of leukemia combining gene expression and flow cytometry.Blood Cells Mol Dis.2007;22-25.
4 刘东方.肥大细胞脱颗粒机制研究进展.国外医学临床生物化学与检验分册.2004:137-139.
5 Yokoi H,Okayama Y,Niyonsaba F,et al.Comparison of human tonsillar mast cell localization and ultrastructural observations between IgE-mediated allergic and nonallergic donors.Allergy Asthma Proc.2006,27(5):415-21.
6 罗正翟.休克学[M].天津科学技术出版社.2001,789-795.
7 Schwartz LB,Lewis RA,Austen KF.Tryptase from human pulmonary mast cell:purificationda and characterization.Biol Chem.1981;256(22):11939-43.
8 Craig.SS,Schwartz LB.Human MCTC type of mast cell granule:the uncommon occurrence of discrete scrolls associated with focal absence of chymase.Lab Invest.1990;63(4):581-5.
9 Tani K,Ogushi F,Kido H,et al.Chymase is a potent chemoattractant for human monocytes and neutrophilis.Shock.2001;16:21-24.
10 陈谱,何韶衡.变态反应性疾病中肥大细胞的特异激活标志物.实用儿科临床杂志.2004;2:141-142.
11 Boyce JA.Mast cells and eicosanoid mediators:a system of reciprocal paracrine and autocrine regulation.Immunol Rev.2007;217:168-85.
12 Mitsumori S.Recent progress in work on PGD2 antagonists for drugs targeting allergic diseases.Curr Pharm Des.2004;(28):3533-3538.
13 Genovese A,Stellato C,Marsella CV,et al.Role of mast cells,basophils and their mediators in adverse reactions to general anesthetics and radiocontrast media.Int Arch Allergy Immunol.1996;110(1):13-22.
14 Greenberger PA.Anaphylactic and anaphylactoid causes of angioedema.Immunol Allergy Clin North Am.2006;26(4):753-67.
15 龚非力.医学免疫学.北京科学出版社,2004;34-41.
16 Yunginger JW,Nelson DR,Squillace DL,et al.Laboratory investigation of deaths due to anaphylaxis.J Forensic Sci.1991;36(3):857-865.
17 Hogan AD,Schwartz LB.Markers of mast cell degranulation.Methods.1997;343-521.
18 Fazekas IG,Viragos-kisE.Free histamine content in the groove caused by hanging as an in vivo reaction.Dtsch Z Gesamte Gerichtl Med.1965;56(4):250-268.
19 Schwartz LB,Metcalfe DD,Miller JS,et al.Tryptase levels as an indicator of mast cell activation in systemic anaphylaxis and mastocytosis.Int J Legal Meal.1987;316:1622-1626.
20 Shunlin ren,Kentaro Sakai,Lawrence B.Regulation of human mast cell betatryptase conversion of inactive monomer to active tetramer at acid PH.J Immunol.1998;160(9):4561-4569.
21 Hogan AD,SchwartzLB.Makers of mast celldegranulation.Methods,1997;13(1):43-52.
22 Caughey GH.Tryptase genetics andanaphylaxis.Allergy Clin Immunol 2006;117:1411-1414.
23 Laroche D,Vergnaud MC,Sillard B,et al.Biochemical markers of anaphylactoid reactions to drugs comparison of plasma histamine and tryptase.Anesthesiology.1991;75:945-949.
24 Edson E,Van,HM.Beta-tryptase measurements post-mortem in anaphylactic death and in controls.Forensic Sci Int.1998;93(2-3):135-142.
25 Fineschi V,Gambassi R,Gherardi M,et al.The diagnosis of amniotic fluid embolism:an immunhistomchemical study for the quantification of pulmonary mast tryptase.Int J Legal Med.1998;111:238-243.
26 高彩荣,任广睦,范厚生等.肥大细胞类胰蛋白酶的免疫组化染色观察.中国法医学杂志.2003:18:212-214.
27 Buckley MG,Variend S,Walls AF.Elevated serum concentrations of beta-tryptase,but not alpha-tryptase,in Sudden Infant Death Syndrome(SIDS).Clin Exp Allergy.2001 Nov;31(11):1696-1704.
28 Badertscher K,Br6nnimann M,Karlen S,et al.Mast cell chymase is increased in chronic atopic dermatitis but not in psoriasis. Arch Dermatol Res. 2005; 296:503-506.
29 Nishio H, Takai S, Miyazaki M, et al. Usefulness of serum mast cell-specific chmase levels for postmortem diagnosis of anaphylaxis. Int J Legal Med. 2005; 119:331-334.