肾组织石蜡切片免疫荧光检测在诊断X连锁Alport综合征中的应用

Use of paraffin-embedded renal section for immunofluorescence staining in Alport syndrome

目的:建立X连锁Alport综合征(XLAS)肾组织石蜡切片型胶原A5链免疫荧光检测方法,探讨其临床应用价值。方法:将对照组及XLAS患儿各4例肾组织,分别行石蜡切片及冰冻切片型胶原A5链间接免疫荧光染色,比较分析染色结果。石蜡切片分别采用微波修复、胃蛋白酶消化及微波修复+胃蛋白酶消化三种抗原修复方法进行修复,比较三者的修复效果。结果:单独微波修复、胃蛋白酶消化均不能修复肾组织型胶原A5链,微波修复+胃蛋白酶消化法可修复A5链。组织经微波修复10min,胃蛋白酶消化10min后,肾小球基底膜型胶原A5链免疫荧光染色在对照组患儿为连续线状阳性表达,在X连锁遗传型女性患儿为间断线状阳性,男性患儿为阴性。结果均与冰冻切片免疫荧光染色结果一致。结论:经微波修复+胃蛋白酶消化双抗原修复法的石蜡切片免疫荧光检测肾组织型胶原A5链能较好地应用于XLAS诊断,可对既往临床不典型XLAS病例进行回顾性诊断,为XLAS的诊断和研究提供了一种可靠的技术手段。

Objective ：To establish the diagnostic technique of X -linked Alport syndrome （XLAS ） by immunofluorescence staining of type Ⅳ collagen α5 chains on paraffin -embedded renal sections .Methods 4 patients with X -linked dominant form of AS （3 males and 1 females） were retrospectively analyzed ,who were admitted between April ,2012 to January ,2013 .AS was diagnosed according to clinical symptoms ,family history ,pathology ,immunofluorescence staining of type Ⅳcollagen chains on renal .Normal portions of renal tissue from 4 patients with renal duplication were used as controls .Type Ⅳ collagen α5 chains were stained by indirect immunofluorescence staining method on paraffin -embedded renal sections .Three antigen retrieval methods ,including microwave treatment , pepsin digestion and microwave treatment plus pepsin digestion were first used in the controls ,to find the best antigen retrieval method for type Ⅳ collagen α5 chains .The results were compared with immunofluorescence staining on fresh frozen sections from the same set of cases to evaluate the methodology .Results The type Ⅳ collagen α5 chains immunofluorescence stain were negative on formaldehyde -fixed tissue and paraffin - embedded sections preceded by microwave treatment or pepsin digestion epitope retrieval alone ,but the paraffin -embedded renal sections preceded by microwave treatment after pepsin digest 10minutes , type Ⅳ collagen α5 chains showed continuous linear pattern along glomerular basement membrane on sections from the controls ,negative for X -linked dominant male AS patients ,intermittent linear pattern for X -linked dominant female AS patient .The same results as immunofluorescence on frozen tissue .Conclusions XLAS can be diagnosed by immunofluorescence staining of type Ⅳ collagen α5 chains on paraffin - embedded renal sections .Moreover ,it can make those Previously suspect XLAS diagnosis by clinicopathology or renal biopsy electron microscope to be definite ,which is a useful technique for diagnosis of XLAS .