双重修复石蜡切片免疫荧光染色在肾活检组织中的应用

目的：探讨常规10%福尔马林固定的肾穿刺活检组织进行石蜡切片免疫荧光染色在病理诊断中应用的可行性。方法：采用隔水微波修复及胰蛋白酶双重修复抗原。结果：经过双重修复的石蜡切片免疫荧光染色与冰冻切片免疫荧光染色结果基本一致。结论：双重修复石蜡切片免疫荧光染色为病理诊断提供了多一种方法,且为使用石蜡切片进行回顾性免疫荧光诊断和研究提供了技术保障。     

微波石蜡切片免疫荧光多重标记技术在肾活检病理诊断中的应用

目的：肾活检组织免疫荧光检查常规是在冰冻切片上进行。本研究探讨了甲醛固定的肾活检组织进行石蜡切片免疫荧光多重染色在病理诊断中应用的可行性。方法：采用微波修复抗原法,对5例膜性肾病、5例狼疮性肾炎、5例IgA肾病和5例乙型肝炎病毒相关性肾炎患者肾组织石蜡切片分别进行IgG/Fn/Hoechst、C3/Fn/Hoechst、IgA/Fn/Hoechst、HBsAg/Fn/Hoechst免疫荧光多重染色。利用免疫荧光显微镜观察并照相。结果：微波修复抗原的石蜡切片进行免疫荧光多重染色,显示IgG、C3、IgA与HBsAg染为绿色,FN为红色,两者共表达的部位为橙黄色,细胞核为蓝色。石蜡切片的免疫荧光染色与用于病理诊断的冰冻切片免疫荧光染色结果一致。且石蜡切片较冰冻切片薄,结构清楚,免疫沉积物定位更准确,易判断结果。结论：微波石蜡切片免疫荧光多重染色,能在一张切片上同时显示两种以上抗原的共表达,为病理诊断提供更加直观详实的依据,可用于肾组织分子免疫病理检查,为使用石蜡切片进行回顾性免疫荧光诊断和研究提供了技术保障。     

微波石蜡切片免疫荧光染色在肾活检病理诊断中的应用

冰冻切片免疫荧光染色在肾活检病理诊断中起着重要作用.由于冰冻切片需要价格昂贵的冷冻切片机,在很大程度上限制了该技术的广泛应用,我们利用微波抗原修复技术,对肾活检组织石蜡切片进行免疫荧光染色,取得了良好的效果.     

肾组织石蜡切片免疫荧光技术中抗原修复方法的探讨

目的 比较多种抗原修复方法在肾组织石蜡切片免疫荧光技术中的应用,探讨最佳抗原修复法.方法 选取2018年6月至2019年6月期间郑州大学第一附属医院肾脏病理中心的45例肾活检组织标本为研究对象,其中狼疮肾炎、膜性肾病、IgA肾病各10例及淀粉样变性肾病15例.分别用5种抗原修复法处理各组肾组织石蜡切片.按照标本来源和抗原修复方法分为6组:对照组(冰冻切片标本)、高压热修复联合胰蛋白酶消化双修复(高压热联合胰酶)组、微波热修复联合胰蛋白酶消化双修复(微波热联合胰酶)组、高压热修复(高压热)组、微波热修复(微波热)组、胃蛋白酶消化(胃蛋白酶)组.分析和比较5种热修复抗原方法的石蜡切片与冰冻切片免疫荧光染色和半定量评分的差异.结果 高压热联合胰酶、微波热联合胰酶组肾组织石蜡切片的免疫荧光染色结果与对照组一致,与对照组比较,两组免疫荧光半定量评分的差异无统计学意义(均P> 0.05).高压热组、微波热组和胃蛋白酶组石蜡切片免疫荧光染色结果较对照组假阴性率高,免疫荧光半定量评分的差异有统计学意义(均P< 0.05).结论 高压热修复联合胰蛋白酶消化双修复法与微波热修复联合胰蛋白酶消化双修复法为石蜡切片的最佳抗原修复方法.     

微波石蜡切片免疫荧光双重染色在肾脏病理研究中的应用

我们尝试用石蜡切片进行免疫荧光双重染色，目的是希望它既有冰冻切片免疫荧光染色抗原特异性高，敏感性强，能同时显示表达于同一部位的两种抗原的特点，又具有石蜡切片免疫组化染色组织细胞结构清晰，抗原定位准确的优点。     

肾穿刺活检石蜡组织切片DNA的提取和PCR扩增

目的建立从肾穿刺活检石蜡组织切片提取DNA并进行PCR扩增的方法。方法肾脏病理诊断为乙肝病毒相关性肾小球肾炎、原发性IgA肾病和膜性肾病各2例的肾活检组织蜡块，包埋时间1个月-5年。提取方法包括清洁切片、融蜡、消化及DNA的提取。紫外分光光度计测定DNA浓度。应用聚合酶链反应（PCR）扩增血管紧张素转换酶（ACE）和乙型肝炎病毒核心抗原（HBcAg）基因片段。结果2张2Ixm厚石蜡切片获取的DNA总量为10-30μg。ACE基因的PCR扩增结果显示，6例标本均在490bp和（或）190bp处显示清晰条带。HBcAg的PCR扩增结果显示，2例乙肝病毒相关性肾小球肾炎标本在132bp处显示清晰条带，其余4例阴性。结论本方法可以从肾穿刺活检石蜡切片中获取足够高质量DNA并进行PCR扩增，方法简便，结果稳定，不受包埋时间长短的限制，为肾脏疾病的辅助诊断、肾活检组织的大宗遗传学分析提供了可能。     

谈电镜检查对确诊肾小球病的价值

众所周知，肾穿刺活组织检查，包括光镜、免疫荧光和电镜检查，对于肾脏疾病，尤其是肾小球疾病的组织病理学分类或病理诊断常起决定性作用。而其诊断的正确与否，又直接关系到对肾病患者的合理治疗及其预后判断。因此，对每一个肾穿刺病例，我们历来主张将上述3种检查做全、做好，进而有可能通过仔细、综合的判断和分析，对其做到正确诊断和随后的合理治疗。然而，在我日常遇到的对肾活检组织切片进行会诊的过程中．常发现国内有相当数量的医院，     

快速石蜡切片与冷冻切片在供器官组织病理学评估中的比较研究

目的探究快速石蜡切片较之冷冻切片在器官捐献供器官的组织病理学评估中的优劣。方法收集2017年至2021年间华中科技大学同济医学院附属同济医院器官移植研究所的5例供肝标本和8例弃用供肾标本, 对供器官活检组织分别采用冷冻切片、快速石蜡切片和常规石蜡切片方法制片, 经苏木精-伊红染色后, 从供肾和供肝镜下的基本形态以及相关供器官评分的差异方面进行比较。结果快速石蜡切片在反映肾小球硬化比例(18.6%±22.3%)、小动脉透明样变(43.7%±23.8%)和小动脉狭窄(47.9%±29%)方面与常规石蜡切片相似。冰冻切片中观察到的肾小球硬化比例(0.8%±2.2%)、小动脉透明样变比例(4.9%±7.4%)和小动脉狭窄(5.3%±7.5%)比例均低于快速石蜡切片。另外快速石蜡切片对肝细胞坏死和水变性的判断也更为准确。结论快速石蜡切片较冷冻切片镜下组织结构更清晰、完整。在评分方面能更准确地反映供器官的组织病理学改变。     

假体周围组织冰冻切片对诊断髋关节假体无菌性松动的特异性分析

[目的]评价冰冻切片对诊断髋关节假体无菌性松动的作用.[方法]回顾分析了57例(63髋)因髋关节无菌性松动行髋关节翻修的病例,将冰冻切片和石蜡切片结果进行病理学分级,将至少5个高倍镜视野、每个高倍镜视野的中性粒细胞数≥5个视为切片结果阳性；以之前文献报道的无菌性松动的诊断标准为参照标准,计算冰冻切片的特异性、符合率.[结果]冰冻切片的特异性为96.8％,冰冻切片和石蜡切片诊断假体无菌性松动时符合率为96.8％.[结论]在全髋关节翻修术中利用组织冰冻切片进行诊断性检查来确定是否存在感染时要注意冰冻切片和石蜡切片的偏差.     

非冷冻方法快速石蜡切片制作技术的研究

目的:使用非冷冻方法进行快速石蜡切片,以帮助临床及术中明确病理诊断。方法:试验八种常用的快速石蜡切片制作和细胞学涂片技术,观察比较切片质量及染色效果。结果:所有组织切片和细胞涂片染色鲜艳,细胞核、胞浆比例适中,能够达到优质切片标准。结论:超声波及微波在病理技术中应用广泛,对快速石蜡切片制作具有很大优势。     

Immunofluorescence on pronase-digested paraffin sections: a valuable salvage technique for renal biopsies

Direct immunofluorescence (IF) on frozen tissue is the method of choice for the study of medical renal diseases. When no glomeruli are available, IF can be performed on the formalin-fixed paraffin-embedded tissue allocated for light microscopy after antigen retrieval with proteases. In this study, the results of IF on frozen tissue (IF-F) and on deparaffinized, pronase-treated tissue (IF-P) were compared in 71 renal biopsies representing 12 major renal diseases. Using IF-P, diagnostic findings were obtained in 100% of cases of lupus nephritis, acute post-infectious glomerulonephritis, cryoglobulinemic glomerulonephritis, fibrillary glomerulonephritis, primary amyloidosis, myeloma cast nephropathy, and light-chain Fanconi syndrome (LCFS), 88% of cases of immunoglobulin (Ig)A nephropathy, 80% of cases of light-chain deposition disease, 60% of cases of membranoproliferative glomerulonephritis type 1, 50% of cases of idiopathic membranous glomerulopathy (MGN) and 20% of cases of anti-glomerular basement membrane (GBM) disease. IF-P was less sensitive than IF-F for the detection of C3 in all disease categories and for the detection of IgG in cases of MGN and anti-GBM disease. The diagnostic kappa light-chain staining was demonstrated in 100% of cases of LCFS by IF-P versus 40% by IF-F. We conclude that IF-P is a valuable salvage immunohistochemical technique for renal biopsies lacking adequate cortical sampling for IF-F, and is superior to IF-F for the diagnosis of LCFS.     

The immunophenotyping of extramedullary myeloid cell tumors in paraffin-embedded tissue sections

Extramedullary tissue infiltrates of acute myeloid leukemia are rare and often difficult to recognize in routine paraffin-embedded tissue sections. Since appropriate therapy for these tumors depends on their precise identification, we have studied a series of tissues infiltrated with primitive myeloid cells using monoclonal and polyclonal antibodies capable of labeling cells of the myeloid/monocytic system in paraffin-embedded tissue sections. The current retrospective study involved tissues from 15 patients (eight men and seven women) with a mean age of 51 years (range, 23-77). A diagnosis of extramedullary myeloid cell tumors had been made on the basis of routine histology, chloroacetate esterase cytochemical stain, and--in some cases--electron microscopy. Paraffin-embedded tissue sections were cut and stained employing the alkaline phosphatase antialkaline phosphatase (APAAP) immunocytochemical procedure with monoclonal antibodies against leukocyte-common antigen (PD7/26-2B11), restricted components of the leukocyte-common antigen (UCHL1, 4KB5), granulocytes (Mac-387, Leu-M1), leukocytes (MT1, MT2, LN1, LN2), HLA-DR (LN3), and elastase (NP57), as well as polyclonal antibodies against lactoferrin, lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin. Results indicate that antibodies against Mac-387, elastase, and lysozyme are most useful in the recognition of neoplastic myeloid cells. We conclude that tissues containing granulocytic tumors can be identified in paraffin-embedded tissue sections using a panel of antibodies and the APAAP procedure.     

There Is High Sensitive and Specific Correlation Between Frozen and Permanent Sections in Renal Transplant Biopsies

Introduction

Frozen sections have been used for evaluating tumors and margins during daily practice in pathology with high specificity and sensitivity (>90% for both indices both at national level and in our department). The correlation between frozen section tissue for immunofluorescent (IF) studies and permanent sections for light microscopy, along with electron microscopy, is critical for constructing a final renal pathology diagnosis.

Comparison of tissue culture cells and histological sections for use in screening monoclonal antibodies

Monoclonal antibodies have been screened using either tissue culture cells or histologic sections as a source of antigen. We evaluated and compared these two methods using six murine monoclonal antibodies on human kidney cultures and histologic sections of human normal kidney and renal carcinoma. Antibody binding demonstrated shared antigens on kidney culture cells, renal tubular cells and renal carcinoma cells. The patterns of antibody binding were generally similar with either cultured cells or histologic sections. However, certain antibodies demonstrated significant differences in antibody binding depending on the method used for screening.     

Predominant synthesis of IgA with lambda light chain in IgA nephropathy

The nature of the light chains in mesangial IgA deposits and serum IgA was studied in patients with IgA nephropathy. Immunofluorescence (IF) studies using murine monoclonal antibodies, rabbit and goat anti-human monospecific antisera were performed in kidney sections from 15 IgA nephritic patients with only IgA isotype detected in the renal biopsy. Lambda light chain IF was demonstrated in all biopsy specimens and kappa light chain IF in 11 renal biopsy specimens. The majority of renal biopsies showed a predominance of lambda light chain IF staining in the mesangial deposits. The concentration of individual immunoglobulins and their light chain fractions, and the kappa/lambda ratio were determined in the serum and the supernate from peripheral blood mononuclear cells culture of 30 IgA nephritic patients and 30 age-matched healthy controls. The IgA nephritic patients had a higher serum concentration of total IgA (P less than 0.001) and a significantly lower IgA kappa/lambda ratio (P less than 0.001) compared with the controls. The kappa/lambda ratio of supernatant IgA from IgA nephritic patients (N = 20) was also significantly lower than that of the normal subjects (N = 14), both in the unstimulated (P less than 0.01) and pokeweed mitogen stimulated, peripheral blood mononuclear-cell culture (P less than 0.05). Our results showed that patients with primary IgA nephropathy displayed a unique immunologic response characterized by a predominance of IgA with lambda light chain in circulation.     

Does endothelial chimerism correlate with renal allograft rejection?

BACKGROUND: The blood vessels of a transplanted organ are an interface between the donor and the recipient. The endothelium is believed to be a major target for graft rejection. After transplantation endothelial cells of a transplanted organ may be of recipient origin. OBJECTIVES: In this study we sought to determine whether endothelial chimerism correlates with graft rejection. METHODS: Biopsy samples from 34 renal transplants of female recipients who received kidneys from male donors were studied for the presence of endothelial cells of recipient origin. Formalin-fixed, paraffin-embedded tissue sections of renal biopsy samples were examined by fluorescence in situ hybridization (FISH) for the presence of endothelial cells containing two X chromosomes, using a biotinylated Y-chromosome probe and digoxigenin-labeled X-chromosome probe. RESULTS: The FISH methods identified endothelial cells of recipient origin. Endothelial chimerism was common, irrespective of rejection. Its presence was focal with these elements, coexisting in the biopsy. CONCLUSIONS: We observed no correlation between the percentage of recipient endothelial cells among vascular elements and the type of graft rejection (P > .05).     

C4d Immunohistochemistry in glomerulonephritis with different antibodies

Background The presence of C4d in the kidney is generally detected particularly for the diagnosis of antibody-mediated rejection in renal transplants. In frozen sections of immunofluorescence (IF) staining with anti-C4d monoclonal antibodies (mAbs), we noted intrinsic C4d deposition even in normal glomeruli though their pathogenic or an intrinsic role is unkown. An anti-C4d polyclonal antibody (C4dpAb), which is suitable for paraffin immunoperoxidase (IP) staining, is less used than mAbs, and it has demonstrated that intrinsic C4d is not evident. To establish a stable and reproducible procedure for C4d detection with the C4dpAb and to determine the staining characteristics of it, the present study aimed to test whether the method was comparable with IF with a mAb. Methods We compared the C4dpAb with the mAb in adjacent sections of human diseased kidneys, and then compared IP with IF of C4dpAb. Two ways of antigen retrieval was examined for IP. Results On comparing the two antibodies for glomerular staining with IF, we found that the pattern and intensity (C4dpAb showed intrinsic C4d with IF) were similar. In addition, C4dpAb staining with IP and IF demonstrated that the intrinsic staining in the normal glomerulus was mostly undetectable by IP, whereas IF showed distinct staining. Likewise, C4d deposition with IP in some cases was apparently weaker than that on IF, suggesting that this deposition is not intrinsic but indicates pathogenic complement activation. Conclusions The advantage of the C4dpAb for immunohistochemistry is of value for reconsidering the role of C4d in glomerular diseases.     

Immunohistochemical localization of laminin in the basement membranes of normal, hyperplastic, and neoplastic human prostate

We examined the effects of fixatives and antibody sources on the immunohistologic localization of laminin in normal and cancer-containing human prostates and studied the localization patterns in carcinomas of varying degrees of histologic differentiation. Two different polyclonal antibodies were localized in paraffin-embedded or cryostat sections of fixed (alcohol, formalin, and paraformaldehyde) or unfixed tissue, using the immunofluorescence (IF) or immunoperoxidase (IP) techniques, with positive and negative controls. We found that the IF reactions were more intense in unfixed or alcohol-fixed sections than in paraformaldehyde-fixed specimens. IP reactions were very weak or absent in fixed and paraffin-embedded sections, but pepsin treatment of these sections resulted in more intense and uniform IP reaction products, stronger than in unfixed or ethanol-fixed cryostat sections. With the IP technique, laminin localization was intense and uniform in the basement membranes (BM) of acini, blood vessels, smooth muscle, and nerve fibers in normal prostate, benign hyperplasia (BPH), and well-differentiated carcinomas. The BM of poorly differentiated carcinomas showed widespread absence of laminin reactivity. In normal BPH and well-differentiated tumors, occasional epithelial cells and their surface and acinar lumina had laminin reactivity. However, in higher grade tumors, numerous neoplastic cells had laminin reactivity in cytoplasm, their surface, and secretory material. Some macrophages and neutrophils also contained laminin reactivity, presumably of degraded laminin. In some moderately and poorly differentiated tumors, the BM of small capillaries did not contain laminin. The BM of larger vessels always had laminin reactivity, even in the higher grade tumors.