8种染色方法对组织切片中虾肝肠胞虫染色效果的比较
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摘要
病理检测是一种诊断病原及观察机体组织病理变化的重要方法,特殊染色是在病变组织中寻找病原体的前提手段,良好的染色有助于将病原体与组织区别,从而增加病原在组织切片中的检出率。为筛选出一种适用于虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)病理切片检测的最佳染色方法,使用苏木精-伊红(HE)染色法、Masson染色法、爱显蓝-雪夫(AB-PAS)染色法、甲苯胺蓝(TB)染色法、伟郝夫范吉森(EVG)染色法、劳克坚牢蓝(LFB)染色法、天狼猩红(Sirius red)染色和普鲁士蓝(PB)等8种染色方法对凡纳滨对虾(Litopenaeus vannamei)感染了虾肝肠胞虫的肝胰腺进行组织病理切片染色,并结合对EHP检出率的统计分析,比较了这些染色方法的优缺点。结果表明:HE染色法、AB-PAS染色法、TB染色法、EVG染色法、天狼猩红Sirius red染色法和PB染色法对孢子的染色效果较差,与背景颜色对比不明显,单个视野检出率较低; LFB染色法虽然使孢子易被识别,但是有孢子丢失现象,且组织染色效果差; Masson染色结果显示孢子呈现品红色,与组织颜色对比鲜明,孢子颗粒清晰可见,对组织细胞结构染色效果较好,且检出率也最高。综合分析染色效果及检出率,Masson染色法是一种非常适用于组织内的虾肝肠胞虫孢子染色的方法,本研究为微孢子虫病的组织病理研究提供一种有效的基础手段。
Histopathological examination is an important method for diagnosing pathogens and observing histopathological changes of the organism. Special staining is the prerequisite for finding pathogens in infected tissues. Good staining resluts can help to distinguish pathogens from tissues,and increase the detection rate of pathogens in tissue sections. In order to screen out a suitable staining method for the detection of Enterocytozoon hepatopenaei( EHP) in histopathology,8 different staining methods,including: Hematoxylineosin staining,Masson staining,Alcian blue-Periodic acid schiff staining,Toluidine blue staining,VerhoeffVan Gieson staining,Luxol fast blue staining,Sirius red and Prussian blue staining method,were selected to compare the dyeing effects using the EHP infected hepatopancereas tissues of Penaeus vannamei. Combined with the detection rate of EHP,the advantages and disadvantages of these staining methods were compared.The dyeing effects of HE,AB-PAS,TB,EVG,Sirius red and PB methods were poor,the contrast between the spores and the background was not noticeable,the detection rates of a single visual field were low. Although LFB method made the spores easy to be identified,it had spores loss and bad staining effect on host tissues.Masson method showed that the spores color was clearly visible magenta,the staining effects of the tissue cell structure were better, and the detection rate of the single field was the highest in the 8 methods.Comprehensive analysis of the effects of staining and detection rates showed that Masson method was very well suitable for the staining of EHP spores in host tissues. This study provides an effective basic method for the histopathological study of microsporidiosis.
Histopathological examination is an important method for diagnosing pathogens and observing histopathological changes of the organism. Special staining is the prerequisite for finding pathogens in infected tissues. Good staining resluts can help to distinguish pathogens from tissues,and increase the detection rate of pathogens in tissue sections. In order to screen out a suitable staining method for the detection of Enterocytozoon hepatopenaei( EHP) in histopathology,8 different staining methods,including: Hematoxylineosin staining,Masson staining,Alcian blue-Periodic acid schiff staining,Toluidine blue staining,VerhoeffVan Gieson staining,Luxol fast blue staining,Sirius red and Prussian blue staining method,were selected to compare the dyeing effects using the EHP infected hepatopancereas tissues of Penaeus vannamei. Combined with the detection rate of EHP,the advantages and disadvantages of these staining methods were compared.The dyeing effects of HE,AB-PAS,TB,EVG,Sirius red and PB methods were poor,the contrast between the spores and the background was not noticeable,the detection rates of a single visual field were low. Although LFB method made the spores easy to be identified,it had spores loss and bad staining effect on host tissues.Masson method showed that the spores color was clearly visible magenta,the staining effects of the tissue cell structure were better, and the detection rate of the single field was the highest in the 8 methods.Comprehensive analysis of the effects of staining and detection rates showed that Masson method was very well suitable for the staining of EHP spores in host tissues. This study provides an effective basic method for the histopathological study of microsporidiosis.
引文
[1]CHAYABURAKUL K,NASH G,PRATANPIPAT P,et al.Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand[J].Diseases of Aquatic Organisms,2004,60(2):89-96.
[2]TOURTIP S,WONGTRIPOP S,STENTIFORD G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships[J].Journal of Invertebrate Pathology,2009,102(1):21-29.
[3]SALACHAN P V,JAROENLAK P,THITAMADEES,et al.Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis(HPM)caused by Enterocytozoon hepatopenaei(EHP)[J].BMC Veterinary Research,2017,13(1):1-7.
[4]FLEGEL T W.Historic emergence,impact and current status of shrimp pathogens in Asia[J].Journal of Invertebrate Pathology,2012,110(2):166-173.
[5]RAJENDRAN K V,SHIVAM S,PRAVEENA P E,et al.Emergence of Enterocytozoon hepatopenaei(EHP)in farmed Penaeus(Litopenaeus)vannamei in India[J].Aquaculture,2016(454):272-280.
[6]陈禄芝,余霞艳,胡一丞,等.粤西地区凡纳滨对虾虾肝肠胞虫、传染性皮下和造血组织坏死病毒感染情况的初步调查[J].渔业研究,2016,38(4):273-280.CHEN L Z,YU X Y,HU Y C.Preliminary investigation of Enterocytozoon hepatopenaei(EHP)and infections hypodermal and hematopoietic necrosis virus(IHHNV)from Litopenaeus vannamei in west Guangdong Province[J].Journal of Fisheries Research,2016,38(4):273-280.
[7]刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测[J].渔业科学进展,2016,37(2):119-126.LIU Z,ZHANG Q L,WAN X Y,et al.Development of real-time PCR assay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates[J].Progress in Fishery Sciences,2016,37(2):119-126.
[8]NGUYEN T H,HA T D,THUY N T,et al.Enterocytozoon hepatopenaei has been detected parasitizing tiger shrimp(Penaeus monodon)cultured in Vietnam and showing white feces syndrome[J].Agriculture and Rural Development:Science and Technology,2010(12):45-50.
[9]TANG K F J,HAN J E,ARANGUREN L F,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP)in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp[J].Journal of Invertebrate Pathology,2016(140):1-7.
[10]BIJU N,SATHIYARAJ G,RAJ M,et al.High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India[J].Diseases of Aquatic Organisms,2016,120(3):225.
[11]SANTHOSHKUMAR S,SIVAKUMAR S,VIMAL S,et al.Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei(EHP)in naturally and experimentally EHP-infected whiteleg shrimp,Litopenaeus vannamei(Boone,1931),in India[J].Journal of Fish Diseases,2017,40(4):529-539.
[12]郑杰.提高作为"金标准"的病理诊断的含金量[J].中华病理学杂志,2011,40(1):1-3.ZHENG J.Improving capabilities of pathologic diagnosis as“a gold standard”[J].Chinese Journal of Pathology,2011,40(1):1-3.
[13]BELL T A,LIGHTNER D V.A handbook of normal penaeid shrimp histology[M].Baton Rouge,LA:World Aquaculture Society,1988.
[14]GOLDNER J.A modification of the Masson trichrome technique for routine laboratory purposes[J].American Journal of Pathology,1938,14(2):237.
[15]黄云梅,陈文列,黄美雅,等.多种特殊染色法在骨关节炎组织形态学研究中的应用比较[J].中国比较医学杂志,2011,21(5):45-48.HUANG Y M,CHEN W L,HUAGN M Y,et al.Comparison of several special stainings on osteoarthritis morphology[J].Chinese Journal of Comparative Medicine,2011,21(5):45-48.
[16]吴敏,胡天寒,周鸿铭,等.甲苯胺蓝快速显示肥大细胞的制作方法探讨[J].四川解剖学杂志,2013,21(1):11-12.WU M,HU T H,ZHOU H M,et al.Method on toluidine blue staining for rapid presenting mast cells[J].Sichuan Journal of Anatomy,2013,21(1):11-12.
[17]胡晓磊.腹主动脉瘤小鼠模型的病理形态学比较及抗血管紧张素治疗在其发病过程中的研究[D].中南大学,2012.HU X L.Study of pathological Morphology comparison in different rodent models and the therapeutic effect of anti-angiotension in the development of AAAs[D].Changsha:Central South University,2010.
[18]张丽,尹琳琳,李林.两种髓鞘染色方法在Cuprizone诱导脱髓鞘小鼠模型中的比较[J].首都医科大学学报,2012,33(6):762-765.ZHANG L,YIN L L,LI L.A comparison between two methods of myelin staining in Cuprizone-induced demyelination model mice[J].Journal of Capital Medical University,2012,33(6):762-765.
[19]董鸿瑞,程虹,李泳,等.普鲁士蓝染色在肾活检病理诊断中的应用[J].中华临床医师杂志(电子版),2012(20):6392-6394.DONG H R,CHENG H,LI Y,et al.Using of Prussian blue staining in the pathological diagnosis of renal biopsy[J].Chinese Journal of Clinicians(Electronic Edition),2012(20):6392-6394.
[20]沈蔷,陈莉,李昊,等.苦味酸-天狼猩红染色和Masson染色评价心脏纤维化的比较[J].中国分子心脏病学杂志,2012,12(2):118-120.SHEN Q,CHEN L,LI H,et al.Comparison of Picric Sirius red staining and Masson staining in evaluation of cardiac fibrosis[J].Molecular Cardiology of China,2012,12(2):118-120.
[21]顾怡瑾,陆新元,付华辉,等.Masson染色在辅助诊断肝纤维化中应用[J].临床与实验病理学杂志,2012,28(1):101-102.GU Y J,LU X Y,FU H H,et al.Application of Masson staining in diagnosis of liver fibrosis[J].Chinese Journal of Clinical and Experimental Pathology,2012,28(1):101-102.
[22]黄利,秦魏笈,华晓萍.Bouin氏液与10%福尔马林固定组织对Masson氏三色染法染色结果的影响[J].中药药理与临床,2016(2):230-232.HUAGN L,QIN W J,HUA X P.Effects of Bouin’s solution and 10%formalin fixation on Masson’s trichrome staining[J].Pharmacology and Clinics of Chinese Materia Medica,2016(2):230-232.
[23]郑叶,曾东,童海涛,等.特殊染色技术在艾滋病合并真菌病理诊断中的应用[J].中华临床感染病杂志,2014,7(3):207-211.ZHENG Y,ZENG D,TONG H T,et al.Application of special staining techniques in pathological diagnosis of fungi infections in HIV/AIDS patients[J].Chinese Journal of Clinical Infectious Diseases,2014,7(3):207-211.
[24]陈书媛,张彦宁.EVG弹力纤维染色方法的改良和效果观察[J].临床和实验医学杂志,2016,15(7):716-718.CEHN S Y,ZHANG Y N.Study on the modification of EVG elastic fiber staining and its results[J].Journal of Clinical and Experimental Medicine,2016,15(7):716-718.
[25]张丽,尹琳琳,李林.两种髓鞘染色方法在Cuprizone诱导脱髓鞘小鼠模型中的比较[J].首都医科大学学报,2012,33(6):762-765.ZHANG L,YIN L L,LI L.A comparison between two methods of myelin staining in Cuprizone-induced demyelination model mice[J].Journal of Capital Medical University,2012,33(6):762-765.
[26]王卫卫,田野,赖日权.苦味酸-天狼星红偏振光法在早期肝纤维化诊断中的应用[J].诊断病理学杂志,2003,10(5):289-290.WANG W W,TIAN Y,LAI R Q.Application of picrosirius polarization method in diagnosis of early hepatic fibrosis[J].Chinese Journal of Diagnostic Pathology,2003,10(5):289-290.
[27]刘广珍.CD34和普鲁士蓝双染色法在肝穿刺标本中的应用[J].徐州医学院学报,2015(9):617-618.LIU G Z.Application of CD34 and Prussian blue staining in the pathological analysis of liver biopsy specimens[J].Acta Academiae Medicinae Xuzhou,2015(9):617-618.
[2]TOURTIP S,WONGTRIPOP S,STENTIFORD G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships[J].Journal of Invertebrate Pathology,2009,102(1):21-29.
[3]SALACHAN P V,JAROENLAK P,THITAMADEES,et al.Laboratory cohabitation challenge model for shrimp hepatopancreatic microsporidiosis(HPM)caused by Enterocytozoon hepatopenaei(EHP)[J].BMC Veterinary Research,2017,13(1):1-7.
[4]FLEGEL T W.Historic emergence,impact and current status of shrimp pathogens in Asia[J].Journal of Invertebrate Pathology,2012,110(2):166-173.
[5]RAJENDRAN K V,SHIVAM S,PRAVEENA P E,et al.Emergence of Enterocytozoon hepatopenaei(EHP)in farmed Penaeus(Litopenaeus)vannamei in India[J].Aquaculture,2016(454):272-280.
[6]陈禄芝,余霞艳,胡一丞,等.粤西地区凡纳滨对虾虾肝肠胞虫、传染性皮下和造血组织坏死病毒感染情况的初步调查[J].渔业研究,2016,38(4):273-280.CHEN L Z,YU X Y,HU Y C.Preliminary investigation of Enterocytozoon hepatopenaei(EHP)and infections hypodermal and hematopoietic necrosis virus(IHHNV)from Litopenaeus vannamei in west Guangdong Province[J].Journal of Fisheries Research,2016,38(4):273-280.
[7]刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测[J].渔业科学进展,2016,37(2):119-126.LIU Z,ZHANG Q L,WAN X Y,et al.Development of real-time PCR assay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates[J].Progress in Fishery Sciences,2016,37(2):119-126.
[8]NGUYEN T H,HA T D,THUY N T,et al.Enterocytozoon hepatopenaei has been detected parasitizing tiger shrimp(Penaeus monodon)cultured in Vietnam and showing white feces syndrome[J].Agriculture and Rural Development:Science and Technology,2010(12):45-50.
[9]TANG K F J,HAN J E,ARANGUREN L F,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP)in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp[J].Journal of Invertebrate Pathology,2016(140):1-7.
[10]BIJU N,SATHIYARAJ G,RAJ M,et al.High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India[J].Diseases of Aquatic Organisms,2016,120(3):225.
[11]SANTHOSHKUMAR S,SIVAKUMAR S,VIMAL S,et al.Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei(EHP)in naturally and experimentally EHP-infected whiteleg shrimp,Litopenaeus vannamei(Boone,1931),in India[J].Journal of Fish Diseases,2017,40(4):529-539.
[12]郑杰.提高作为"金标准"的病理诊断的含金量[J].中华病理学杂志,2011,40(1):1-3.ZHENG J.Improving capabilities of pathologic diagnosis as“a gold standard”[J].Chinese Journal of Pathology,2011,40(1):1-3.
[13]BELL T A,LIGHTNER D V.A handbook of normal penaeid shrimp histology[M].Baton Rouge,LA:World Aquaculture Society,1988.
[14]GOLDNER J.A modification of the Masson trichrome technique for routine laboratory purposes[J].American Journal of Pathology,1938,14(2):237.
[15]黄云梅,陈文列,黄美雅,等.多种特殊染色法在骨关节炎组织形态学研究中的应用比较[J].中国比较医学杂志,2011,21(5):45-48.HUANG Y M,CHEN W L,HUAGN M Y,et al.Comparison of several special stainings on osteoarthritis morphology[J].Chinese Journal of Comparative Medicine,2011,21(5):45-48.
[16]吴敏,胡天寒,周鸿铭,等.甲苯胺蓝快速显示肥大细胞的制作方法探讨[J].四川解剖学杂志,2013,21(1):11-12.WU M,HU T H,ZHOU H M,et al.Method on toluidine blue staining for rapid presenting mast cells[J].Sichuan Journal of Anatomy,2013,21(1):11-12.
[17]胡晓磊.腹主动脉瘤小鼠模型的病理形态学比较及抗血管紧张素治疗在其发病过程中的研究[D].中南大学,2012.HU X L.Study of pathological Morphology comparison in different rodent models and the therapeutic effect of anti-angiotension in the development of AAAs[D].Changsha:Central South University,2010.
[18]张丽,尹琳琳,李林.两种髓鞘染色方法在Cuprizone诱导脱髓鞘小鼠模型中的比较[J].首都医科大学学报,2012,33(6):762-765.ZHANG L,YIN L L,LI L.A comparison between two methods of myelin staining in Cuprizone-induced demyelination model mice[J].Journal of Capital Medical University,2012,33(6):762-765.
[19]董鸿瑞,程虹,李泳,等.普鲁士蓝染色在肾活检病理诊断中的应用[J].中华临床医师杂志(电子版),2012(20):6392-6394.DONG H R,CHENG H,LI Y,et al.Using of Prussian blue staining in the pathological diagnosis of renal biopsy[J].Chinese Journal of Clinicians(Electronic Edition),2012(20):6392-6394.
[20]沈蔷,陈莉,李昊,等.苦味酸-天狼猩红染色和Masson染色评价心脏纤维化的比较[J].中国分子心脏病学杂志,2012,12(2):118-120.SHEN Q,CHEN L,LI H,et al.Comparison of Picric Sirius red staining and Masson staining in evaluation of cardiac fibrosis[J].Molecular Cardiology of China,2012,12(2):118-120.
[21]顾怡瑾,陆新元,付华辉,等.Masson染色在辅助诊断肝纤维化中应用[J].临床与实验病理学杂志,2012,28(1):101-102.GU Y J,LU X Y,FU H H,et al.Application of Masson staining in diagnosis of liver fibrosis[J].Chinese Journal of Clinical and Experimental Pathology,2012,28(1):101-102.
[22]黄利,秦魏笈,华晓萍.Bouin氏液与10%福尔马林固定组织对Masson氏三色染法染色结果的影响[J].中药药理与临床,2016(2):230-232.HUAGN L,QIN W J,HUA X P.Effects of Bouin’s solution and 10%formalin fixation on Masson’s trichrome staining[J].Pharmacology and Clinics of Chinese Materia Medica,2016(2):230-232.
[23]郑叶,曾东,童海涛,等.特殊染色技术在艾滋病合并真菌病理诊断中的应用[J].中华临床感染病杂志,2014,7(3):207-211.ZHENG Y,ZENG D,TONG H T,et al.Application of special staining techniques in pathological diagnosis of fungi infections in HIV/AIDS patients[J].Chinese Journal of Clinical Infectious Diseases,2014,7(3):207-211.
[24]陈书媛,张彦宁.EVG弹力纤维染色方法的改良和效果观察[J].临床和实验医学杂志,2016,15(7):716-718.CEHN S Y,ZHANG Y N.Study on the modification of EVG elastic fiber staining and its results[J].Journal of Clinical and Experimental Medicine,2016,15(7):716-718.
[25]张丽,尹琳琳,李林.两种髓鞘染色方法在Cuprizone诱导脱髓鞘小鼠模型中的比较[J].首都医科大学学报,2012,33(6):762-765.ZHANG L,YIN L L,LI L.A comparison between two methods of myelin staining in Cuprizone-induced demyelination model mice[J].Journal of Capital Medical University,2012,33(6):762-765.
[26]王卫卫,田野,赖日权.苦味酸-天狼星红偏振光法在早期肝纤维化诊断中的应用[J].诊断病理学杂志,2003,10(5):289-290.WANG W W,TIAN Y,LAI R Q.Application of picrosirius polarization method in diagnosis of early hepatic fibrosis[J].Chinese Journal of Diagnostic Pathology,2003,10(5):289-290.
[27]刘广珍.CD34和普鲁士蓝双染色法在肝穿刺标本中的应用[J].徐州医学院学报,2015(9):617-618.LIU G Z.Application of CD34 and Prussian blue staining in the pathological analysis of liver biopsy specimens[J].Acta Academiae Medicinae Xuzhou,2015(9):617-618.