石蜡切片免疫荧光法在肾病穿刺组织中的应用

Use of paraffin sections for immunofluorescence staining in renal biopsy

目的:探讨石蜡切片免疫荧光法检测肾病穿刺组织中免疫球蛋白等指标的最佳实验条件。方法:收集北京大学基础医学院病理学系的肾病穿刺组织标本45例,包括IgA肾病20例、膜性肾病15例、膜型乙肝病毒相关性肾炎10例,将组织标本用10%(体积分数)中性甲醛溶液固定、石蜡包埋、组织切片,在不同的抗原修复条件和抗体结合时间下,分别标记IgG、IgA、HBsAg、HBcAg,观察染色结果,并与其新鲜组织的免疫荧光染色结果相比较,评判石蜡切片免疫荧光法的应用价值。结果:热诱导抗原修复法修复后一步法和二步法染色均为阴性;蛋白水解酶消化法中胰蛋白酶优于胃蛋白酶,并以胰蛋白酶消化30 min、IgG和IgA 37℃温箱孵育90 min、HBsAg和HBcAg二抗37℃温箱孵育150 min染色结果最佳,与新鲜组织标本染色强度接近或略弱一些,阳性表达的方式也与后者相同,符合率100%。结论:经胰蛋白酶消化的石蜡切片免疫荧光法能够较好地应用于肾病穿刺组织诊断。

Objective：To explore the best method for detecting immunoglobulin,HBsAg and HBcAg in formalin-fixed paraffin-embedded renal biopsy tissues by immunofluorescence（IF）.Methods： In the study,45 cases of renal biopsy were collected,including 20 cases of immunoglobulin A（IgA） nephropathy,15 cases of membranous nephropathy and 10 cases of membranous type hepatitis B virus associated nephritis.IgG,IgA,HBsAg and HBcAg depositions were evaluated in these cases by IF with different antigen retrieval methods and different periods of antibody incubation time.The results were compared with IF on frozen tissues from the same set of cases to evaluate the methodology.Results： All the cases were negative with heat-induced epitope retrieval alone.Immunoglobulin,HBsAg and HBcAg were more readily identifiable after digestion with trypsin than with pepsin.IgG and IgA were best visualized after 30 min of digestion with trypsin and 90 min of incubation with primary antibody for IgG and IgA.HBsAg and HBcAg were best visualized after 30 min of incubation with primary antibody and 150 min of incubation with secondary antibody.Comparing immunoglobulins,HBsAg and HBcAg on fresh sections and paraffin sections,their visualizations were almost identical with both techniques although the signal on paraffin sections was slightly weaker than that on fresh theretsections.Conclusion： Trypsin-digested paraffin sections may be used for diagnosis of renal disease whether there are false negative results.