肾组织石蜡切片免疫荧光染色的新方法

A new application on immunofluorescence staining for paraffin embedded human renal tissues

目的:探讨高温修复联合酶消化法在肾组织石蜡切片进行免疫荧光染色及其在病理诊断中应用的可行性。方法:选取南京军区南京总医院全军肾脏病研究所经肾活检明确诊断为肾小球疾病的患者:狼疮性肾炎、抗肾小球基膜肾炎、致密物沉积病、轻链沉积病(κ)及淀粉样变性(λ)各4例,感染后肾小球肾炎、膜性肾病及膜增生性肾小球肾炎各6例,IgA肾病12例。采用高温修复(EDTA8.0高压锅加热)联合酶消化(胃蛋白酶)法对肾组织石蜡切片分别进行免疫球蛋白(IgG、IgA、IgM)、补体(C3、C1q)、轻链(κ、λ)染色,并将石蜡切片免疫荧光染色与冰冻切片进行比较。结果:石蜡切片经高温修复联合酶消化法进行免疫荧光染色,IgG、IgA、C1q阳性率、分布部位和强度与冰冻切片免疫荧光染色结果相一致。石蜡切片IgM和C3染色分布部位与冰冻切片无差别,阳性率和染色强度略低于冰冻切片,但不影响诊断。κ、λ轻链沉积的阳性率、分布部位和染色强度与冰冻切片一致,轻链沉积病和淀粉样变性患者石蜡切片染色部位显示得更加清晰。石蜡切片的诊断符合率达100%,且结构更清晰,免疫复合物沉积部位较冰冻切片容易判断。结论:石蜡切片高温修复联合胃蛋白酶消化法进行免疫荧光染色为肾活检冰冻组织无肾小球提供可靠的替代方法。

Objective：The aim of this study is to explore the application of heat antigen retrieval combined with pepsin digestion on immunofluoreseenee （IF） staining for paraffin embedded human renal tissues. Methodology：Renal-biopsy proved 9 groups of glomerular diseases from Research Institute of Nephrology, Jinling Hospital were enrolled, they were membranous nephropathy （ n = 6） , membranoproliferative glomerulonephritis （ n = 5）, lupus nephritis including class Ⅳ（n =2） and class Ⅳ ＋Ⅴ （n =2）, IgA nephropathy（n = 12）, anti-GBM disease（ n = 4 ） , dense deposit disease （ n = 4 ） , acute poststreptoeoeeal glomerulonephritis （ n = 6 ） , light - chain deposition disease （kappa） （ n = 4） , and primary mnyloid （lamhda） （ n = 4）. Paraffin-embedded sections of renal biopsy tissues were processed by using heat antigen retrieval with pressure cooke （ EDTA, pH = 8.0 ） combined with pepsin digestion and immunofluorescenee staining of IgG, IgA, IgM, C3, C1q, κ, λ light chain. And then we compare the immunofluoresence staining of frozen sections （IF-F） with heat antigen retrieval combined with pepsin digestion paraffin-embedded sections （IF-P）. Results：IF-P of IgG, IgA, and C I q, was in accordance with that of frozen sections in positive rate, distribution pattern and staining intensity. IF-P of IgM and C3 was the same with IF-F in distribution pattern. However, the intensity and positive rate was inferior to IF-F, which was not influent on diagnosis. One hundred percent of diagnostic accordance rate was obtained in IF-P. Furthermore, the structure of paraffin sections were clearer than that of frozen sections, which led to more easier judgment of the location of immunecomplex deposits. Conclusion：IF-P is a good alternative for immunofluoresenee staining in the condition of frozen tissues without glomerula. This method is useful for retrospective study on paraffin-embedded sections of renal biopsy tissues.