Analytical & Histopathology Core

分析型

• 批准号: 7327516
• 负责人: JOSEPH PETER GRANDE
• 金额: $ 19.82万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7327516
• 关键词: Analysis of Variance; Antibodies; Antigens; Area; Atrophic; Basement membrane; Bibliography; Biological Assay; Biopsy; Biotin; Blinded; Blood Vessels; Blood capillaries; Budgets; Buffers; CDKN1A gene; Caliber; Cell Count; Cell Cycle; Cell Nucleus; Cells; Cellularity; Clinical; Collagen; Collagen Type IV; Color; Complement; Computer Assisted; Contralateral; Data; Development; Dilatation - action; E-Cadherin; Edema; Ensure; Enzyme-Linked Immunosorbent Assay; Enzymes; Epithelial; Epithelial Cells; Family suidae; Fibrosis; Formalin; Heating; Hematoxylin; Histologic; Histopathology; Horseradish Peroxidase; Human; Hypertrophy; Image; Image Analysis; Immunoglobulin A; Immunoperoxidase Technics; Inflammation; Kidney; Kidney Diseases; Laboratories; Laboratory Research; Lead; Light; Link; Localized; Medial; Mesenchymal; Methodology; Methods; Modeling; Molecular Weight; Mus; Neuro-Oncological Ventral Antigen 2; Newly Diagnosed; Numbers; Paraffin; Pathologic; Pathological Dilatation; Patients; Pepsin A; Perchloric Acids; Plasma; Preparation; Principal Investigator; Proteins; Protocols documentation; Publications; Publishing; Qualifying; Reader; Reading; Reagent; Relative (related person); Renal Artery Stenosis; Renal Tissue; Reporting; Reproducibility; Research Infrastructure; Retrieval; Sampling; Sclerosis; Score; Slide; Smooth Muscle Actin Staining Method; Staining method; Stains; Standards of Weights and Measures; Students; Support of Research; Surface; Sus scrofa; System; Techniques; Testing; Thick; Tissue Stains; Tissues; Trichrome stain; Trypsin; Tubular formation; Tunica Media; Urine; Validation; Vegetables; Water; Work; base; capillary; cohort; day; digital; glomerulosclerosis; human tissue; immunopathology; indexing; interstitial; kidney allograft; kidney cortex; oncoprotein p21; p27 Cell Cycle Protein; p27 Enzyme Inhibitor; polarized light; programs; research and development; research study; size; tissue processing; vector

support for many collaborative studies, including antibody optimization and validation, pathologic interpretation, and semiquantitative and quantitative histopathologic analysis. Of direct relevance to this program project, Dr. Grande has worked closely with the Directors of Projects 1 and 2 in analyzing histologic sections obtained from the pig renal artery stenosis model (1-6). Dr. Grande has also collaborated with the Director of Project 4 in both clinical and experimental studies (4, 6-9).Dr. Grande has established an infrastructure for processing tissue sections, including a Dako Automated Stainer, which will minimize batch to batch variability in immunostaining protocols and a MetaVue Image analysis system which will facilitate quantitative analysis of markers of interstitial fibrosis, inflammation, and proliferation. Ms. Gina Warner has served as Lead Technician in Doctor Grande's laboratory for the past 8 years and is highly qualified to perform antibody development, tissue staining, and computer assisted quantitative image analysis. Ms. Warner will also be responsible for blinding the samples so they can be interpreted in an unbiased fashion. Sample Preparation. Renal tissue will be fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin, per standard techniques. Histologic sections, 4 jam, thick, will be prepared. Representative sections will be stained with H&E, PAS, and trichrpme stains. Additional unstained slides will be prepared for Sirius red staining, and immunohistochemical staining for a-smooth muscle actin (a-SMA), collagens III and IV, p-ERK, Mib-1, p21, p27, and TGF-p1. Immunostaining of renal tissue often cannot be performed by clinical immunopathology laboratories because the slides often have to be treated differently than other tissues. For example, high endogenous biotin content in renal tissue produces an extremely high background when protocols that work well in other tissues are employed. In addition, many of the antibodies that work on human tissues do not work well in murine or porcine tissue. The core will support the infrastructure necessary to systematically optimize and validate all of the antibodies which are an integral part of the experimental protocols outlined in Projects 1-4. Our laboratory has implemented a number of immunostains for clinical use and in support of research studies (see table for list of antibodies). Examples from studies published within the past two years are highlighted in references (6, 10-16) in the bibliography. Many of the antibodies (TGF-p1, matrix proteins, Mib-1, etc.) have already been validated in our research laboratory. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 327 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): As necessary, antigen retrieval will be performed by heat treatment in EOTA for 30 minutes, using a vegetable steamer. Enzyme treatment will be performed as required for various antibodies (collagen IV requires pepsin treatment, TGF(31 and collagen III require trypsin treatment). Commercially available kits (Vectastain ABC kit, Vector Laboratories, and Envision Plus HRP kit, DakoCytomation) will be used for many of the blocking, secondary antibody and amplification steps. As necessary, reagents to block endogenous biotin activity will be used. Color development will be performed using NovaRed (Vector Laboratories) followed by hematoxylin counterstain. To facilitate consistency between staining batches, most of the stains will be performed on the Dako Autostainer, an automated staining machine. Staining reagents used with the machine include TBS-T, distilled water and DakoCytomation Special Stains Wash Buffer. Controls will include irrelevant isotype-specific antibody. A critical feature of the core is support of a dedicated technologist (Ms. Warner) who will standardize the staining protocols to ensure day-to-day reproducibility of the stains. Semiauantitative Histopatholoaic Analysis: Interpretation of studies outlined in Projects 1-4 is heavily dependent upon histopathologic analysis, which requires standardized and reproducible staining protocols (see above) as well as semiquantitative and quantitative histopathologic analysis, both of which will be provided by the Histopathology Core. The Core Director will perform semiquantitative histologic scoring, in a blinded fashion, on H&E, PAS, and trichrome stained slides; see Figure 1. The scoring system assigns points (0-3) for glomerular, interstitial, tubular, and vascular features of the renal tissue. The scoring system is similar to that previously used in a cohort of 148 biopsies obtained from patients with IgA nephropathy (17) and modified to include Banff criteria for assessment of renal biopsies (7, 18). In brief, glomerulosclerosis will be reported as number of sclerotic glomeruli, total glomeruli, and will be semiquantitatively analyzed as absent (0), mild (1, involving <25% of glomeruli), moderate (2, involving 26-50% of glomeruli), and severe (3, involving >50% of glomeruli). The number of segmentally sclerotic glomeruli will also be reported as a percent of total glomeruli and reported on a 0-3+ scale (see above). Additional glomerular features, including matrix increase, capillary loop narrowing, ischemic changes (wrinkling and folding), and mesangial cellularity will also be reported, as previously described. For the tubulointerstitial compartment, the extent of interstitial fibrosis, tubular atrophy, and interstitial infiltrates are scored as absent (0), involving <25% of the cortical surface area (1), involving 26-50% of the cortical surface area (2), and involving >50% of the cortical surface area (3). The presence of tubular dilatation, epithelial cell vacuolization, tubular atrophy, casts, or edema will be scored as absent (0), isolated (1), present in <10% of tubules (2), and present in >10% of tubules (3). Reduction in vascular caliber due to sclerosis or hyalinization is scored as absent (0), <25% luminal diameter PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 328 ,J. CoreD Figure 1A. Digital analysis of MT-stalned and oSMA- immunostained sections from renal allografts with newly-diagnosed CAN. MT-stained sections representative sections of renal cortex from biopsies with high interstitial fibrosis score (32.17%) (A, B) and low interstitial fibrosis score (12.64%) (C, D). Images are show before (A,C) and after (B,D) creation of a blue color-based threshold. oSMA immunoperoxidase-stained section - a representative section is shown before (E) and after (F) creation of a brown color-based threshold. Original magnification (10XFD). Figure 1B, Digital analysis of non-polarized and polarized Sirius Red-stained sections from renal allografts with newly-diagnosed CAN. A representative section is shown under non-polarized (A,B,C) and polarized (D,E,F) light. For each, the section is shown prior to (A,D) and after (B,E) conversion to a black and white Image and after thresholding (C,F) for black (non-polarized) or white (polarized) material. Original magnification (10XFD). Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): ROIflOrO, J. CaHOS COfG D (1), 26-75% diameter (2), and >75% (3). Intimal thickening is assessed by comparing the thickness of the intima relative to the total medial thickness and quantitated as normal (0), <25% medial thickness (1), intimal thickness <50% medial thickness (2), and intimal thickness >50% medial thickness (3). Medial hypertrophy is assessed by comparing the thickness of the muscular wall relative to the vascular caliber and is semiquantitatively assessed as normal (0), mild (1), moderate (2), or severe (3). Mean glomerular size and vascular media to total thickness ratios will be assessed by quantitative analysis (see below). Quantitative Assessment of Histopathologic Features, including Interstitial Fibrosis and Matrix Proteins: Methodologies will be as described in our recent publication (18); see Figure 1. Proliferative activity will be assessed as the number of positively staining nuclei/surface area of sections stained for Mib-1 (Projects 1-4), The MetaVue Image Analysis System will be used to evaluate mean glomerular planar surface area (of glomeruli containing the vascular pole) to complement the semiquantitative analysis of glomerular size described above. Glomerular size is used as a marker of renal hypertrophy. The core will assist with studies to determine protein/DNA ratios as an additional index of hypertrophy (DNA as assayed by spectrophotometric analysis of perchloric acid digest of renal cortex, as previously reported by us (19). Similarly, the number of cells staining positively for cell cycle regulatory molecules (p-ERK, p21, p27) will be assessed as the number of cells positively staining/cortical surface area. Glomeruli, tubules, interstitium, and vessels will be assessed separately. Interstitial fibrosis will be assessed over the entire renal cortex of Sirius red-stained slides as the relative amount of red staining material (observed under non-polarized light), or birefringent material (observed under polarized light), relative to the entire cortical surface area, as assessed by the MetaVue Image Analysis System. The percent of cortical surface area staining positively for a-smooth muscle actin will be quantitatively assessed with the MetaVue Image Analysis System. In serial sections, epithelial to mesenchymal transformation will be assessed as decreased E-cadherin staining of tubular epithelial cells, with increase in a- smooth muscle actin staining. This will be semiquantitatively reported as absent, mild, moderate, and severe (>50% cortical surface area). In select cases, we will perform double immunohistochemical staining for E- cadherin and a-SMA to identify and localize cells undergoing epithelial to mesenchymal transformation. Immunohistochemical stains for collagens III (as a marker of interstitial fibrosis) and IV (as a marker of tubular! basement membrane thickening), and TGF-p1 will be expressed as percent cortical surface area staining) positively for these markers, as assessed by the MetaVue Image Analysis System. TGF-B ELISA. We have developed this method for use in clinical samples and will support Project 4 (20, 21). Human urine samples (20 ml each, if possible) will be concentrated using Centriplus centrifugal filters with a 10,000 molecular weight cutoff (Amicon YM-10). Urines are centrifuged at 5000 x g at 25¿ C until the concentrate volume is approximately 300-500 j^l. Urines will be concentrated from 10- to 40-fold, depending on the start volume. Plasma samples do not require concentration. The ELISA will be performed using Quantikine TGF-pl ELISA Kits (R&D Systems, Minneapolis, MN). Kits are available for human samples. The assay will be completed following the R&D protocol, and the plate will be read on a Spectramax Plus 384 plate reader. Results will be in pg/mL after adjusting each sample for fold dilution and fold concentration. Statistical Analysis: All histologic sections will be read in a blinded fashion. Semiquantitative and quantitative data will be entered into an Excel Spreadsheet linked to the MetaVue Image Analysis System. The histotechnologist will calculate mean +/- SEM for all data. Comparisons between multiple groups (stenotic vs contralateral kidney; sham vs contralateral kidney) will be made by ANOVA, using Bonferroni correction for multiple comparisons, followed by the unpaired Students' t-test. D. Budget for Core D (See attached) PHS 398/2590 (Rev. 09/04, Reissued4/2006) Page 329 Continuation Format Page

支持许多协作研究，包括抗体优化和验证，病理解释， 以及半定量和定量组织病理学分析。与此计划项目的直接相关性， Grande博士在获得的分析组织学部分中与项目1和2的董事密切合作 来自猪肾动脉狭窄模型（1-6）。格兰德博士还与项目4项目主任合作 临床和实验研究（4，6-9）.dr。格兰德已经建立了一个用于处理的基础设施 组织切片，包括Dako自动染色器，该染色器将使批处理可变性最小 免疫染色方案和Metavue图像分析系统，该系统将有助于定量分析 间质纤维化，炎症和增殖的标记。吉娜·华纳女士曾担任首席技术员 在过去的8年中，格兰德医生的实验室中，有资格进行抗体开发， 组织染色和计算机辅助定量图像分析。华纳女士也将负责 使样品蒙蔽，以便可以公正地解释它们。 样品制备。肾组织将固定在10％中性缓冲福尔马林中，脱水并嵌入 石蜡，根据标准技术。组织学切片将制备4个果酱，厚度为厚。代表性部分 将用H＆E，PA和Trichrpme污渍染色。 Sirius Red将准备其他未染色的幻灯片 染色和A-Smooth肌肉肌动蛋白（A-SMA），胶原蛋白III和IV，P-ERK，P-ERK的染色和免疫组织化学染色 MIB-1，P21，P27和TGF-P1。 肾脏组织的免疫染色通常不能由临床免疫病理学实验室进行，因为 幻灯片通常必须与其他组织不同。例如，高内源性生物素含量 在肾脏组织中，当其他时间运行良好时，会产生极高的背景 雇用。此外，许多在人体组织上起作用的抗体在鼠或 猪组织。核心将支持系统地优化和验证所有的基础架构 抗体是项目1-4中概述的实验协议不可或缺的一部分。我们的实验室 已经实施了许多用于临床使用和支持研究的免疫抑制剂（请参阅表 抗体清单）。参考文献中强调了过去两年中发表的研究的例子（6，6，， 10-16）在书目中。许多抗体（TGF-P1，基质蛋白，MIB-1等）已经是 在我们的研究实验室中进行了验证。 PHS 398/2590（Rev. 09/04，重新发行4/2006）Page 327延续格式页面 首席调查员/计划主管（最后，第一，中间）： 必要时，将通过热量进行抗原检索 使用蔬菜蒸锅在EOTA中进行30分钟的治疗。 酶处理将根据各种需要进行 抗体（胶原蛋白IV需要胃蛋白酶治疗，TGF（31和 胶原蛋白III需要胰蛋白酶治疗）。市售 套件（Vectastain ABC套件，矢量实验室和Envision 加上HRP套件，Dakocytomation）将用于许多 阻塞，二抗和扩增步骤。作为 必要的，阻断内源性生物素活性的试剂将是 用过的。颜色开发将使用Notared进行 （载体实验室），其次是苏木精的抗染色。到 促进批处理之间的一致性，大部分 污渍将在Dako Autostainer上进行 自动染色机。染色试剂与 机器包括TBS-T，蒸馏水和Dakocytomation 特殊污渍洗涤缓冲液。控件将包括无关紧要 同种型特异性抗体。核心的关键特征是 支持专门的技术（华纳女士）的支持 标准化染色协议以确保日常 菌株的可重复性。 半义组织疾病分析：解释 项目1-4中概述的研究很大程度上取决于 组织病理学分析，需要标准化和 可重复的染色方案（见上文）以及 半定量和定量组织病理学分析 其中将由组织病理学核心提供。核心 主任将在 盲目的时尚，在H＆E，PA和三色染色的幻灯片上； 见图1。评分系统分配了点（0-3） 肾小球，间质，管状和血管特征 肾组织。评分系统类似于以前 用于从患者中获得的148个活检的队列 IGA肾病（17），并修改为包括班夫标准 评估肾脏活检（7，18）。简而言 肾小球硬化将报告为硬化次数 Glomerulli，Total Glomerulli，将是半定量的 分析为不存在（0），轻度（1，涉及glomerulli的<25％）， 中度（2个，涉及肾小球26-50％）和严重（3， 涉及glomerulli的50％）。分段的数量 硬化性肾小球也将报告为总计的百分比 glomerulli并以0-3+刻度报告（见上文）。额外的 肾小球特征，包括矩阵增加，毛细管环 变窄，缺血性变化（皱纹和折叠），以及 弥赛亚细胞也将如前所述 描述。对于肾小管间隙室， 间质纤维化，管状萎缩和间质浸润是 得分为（0），涉及<25％的皮质表面 区域（1），涉及皮质表面积的26-50％（2），并且 涉及皮质表面积的50％（3）。存在 管状扩张，上皮细胞泡，管状 萎缩，铸造或水肿将被评分为不存在（0），孤立 （1），<10％的小管（2）中存在，以> 10％的 小管（3）。由于硬化症或 透明度评分为不存在（0），<25％的腔直径 PHS 398/2590（修订版09/04，重新发行4/2006）第328页 ，j。登记 图1A。 MT-Staled和Osma-的数字分析 来自同种异体移植的免疫染色切片 新诊断的罐子。 MT染色的部分 代表来自活检的肾皮质部分 高间质纤维化评分（32.17％）（a，b）和低 间质纤维化评分（12.64％）（C，D）。图像显示 （a，c）和之后（b，d）创建基于蓝色的 临界点。 OSMA免疫过氧化物酶染色的部分-A 在（e）和（f）之后显示了代表性部分 创建基于棕色的阈值。原来的 放大倍数（10xFD）。 图1B，非极化和的数字分析 肾脏的偏光小天狼星染色部分 具有新诊断的同种异体移植物。代表 截面显示在非偏振（a，b，c）和极化下 （d，e，f）光。对于每个部分，该部分在（a，d）之前显示 之后（b，e）转换为黑白图像， 阈值后（C，F）用于黑色（非极化）或白色 （极化）材料。原始放大倍数（10xFD）。 延续格式页面 首席研究员/计划主管（最后，第一，中间）：Roifliveoro，J。CahosCofg D （1），直径26-75％（2）和> 75％（3）。通过比较厚度来评估内膜增厚 内膜相对于总培养基厚度，并按正常（0）定量，<25％培养基厚度（1），内膜 厚度<50％的培养基厚度（2）和内膜厚度> 50％培养基厚度（3）。媒体肥大是 通过比较肌肉壁相对于血管口径的厚度进行评估，并且 半定量评估为正常（0），轻度（1），中度（2）或严重（3）。平均肾小球尺寸和 血管培养基与总厚度比将通过定量分析评估（见下文）。 组织病理学特征的定量评估，包括间质纤维化和基质蛋白： 方法论将如我们最近的出版物中所述（18）；参见图1。扩散剂的活动将是 评估为对MIB-1染色的部分的阳性染色核/表面积（项目1-4）， Metavue图像分析系统将用于评估平均肾小球平面表面积（OF 肾小球包含血管极），以补充肾小球尺寸的半定量分析 上面描述。肾小球大小用作肾脏肥大的标记。核心将有助于研究 确定蛋白质/DNA比作为肥大的附加指数（通过分光光度法分配的DNA 如我们先前报道的那样，肾皮质的高氯酸摘要分析（19）。同样，数量 细胞对细胞周期调节分子的积极染色（P-ERK，P21，P27）将被评估为数量 细胞积极染色/皮质表面积。将评估肾小球，小管，间质和血管 分别地。间质纤维化将在Sirius红染色载玻片的整个肾皮质上进行评估 相对量的红色染色材料（在非偏振光下观察到）或双折射材料（观察到 在偏振光下），相对于整个皮质表面积，如Metavue图像分析所评估 系统。对于A-Smooth肌肉肌动蛋白的皮质表面积染色的百分比将是定量的 通过Metavue图像分析系统评估。在串行截面中，上皮到间充质 转化将被评估为肾小管上皮细胞的E-钙粘蛋白染色减少，随着A-的增加 平滑肌肌动蛋白染色。这将被半定为缺乏，轻度，中度和严重 （> 50％皮质表面积）。在某些情况下，我们将对E-进行双重免疫组织化学染色 钙粘蛋白和A-SMA识别和定位在间充质转化的细胞。 胶原蛋白III的免疫组织化学染色（作为间质纤维化的标记）和IV（作为管状的标记！ 地下膜增厚）和TGF-P1将以皮质表面积染色百分比表示） 通过Metavue图像分析系统评估，对这些标记物的积极成果。 TGF-B ELISA。我们已经开发了这种用于临床样本的方法，并将支持项目4（20，21）。 人类尿液样品（如果可能的话，每个20毫升）将使用带有A的Centriplus离心滤器浓缩 10,000分子量截止（Amicon YM-10）。尿液在25°C下以5000 x g离心直至 浓缩量大约为300-500 j^l。尿液将从10倍到40倍，具体取决于 开始卷。血浆样品不需要浓度。 ELISA将使用Quantikine进行 TGF-PL ELISA套件（R＆D Systems，明尼阿波利斯，明尼苏达州）。套件可用于人类样品。测定将是 按照研发协议完成，将在Spectramax加384板读取器上读取板。 在调整每个样品以获得折叠稀释和折叠浓度后，结果将在PG/mL中。 统计分析：所有组织学部分将以盲目的方式阅读。半定量和定量 数据将输入到链接到METAVUE图像分析系统的Excel电子表格中。这 组织技术学家将计算所有数据的平均值+/- SEM。多组之间的比较（stenotic vs 对侧肾脏； Sham vs对侧肾脏将由ANOVA进行，使用Bonferroni校正 多次比较，其次是未配对的学生的t检验。 D.核心D的预算（请参阅附件） PHS 398/2590（Rev. 09/04，重新发射4/2006）第329页延续格式页面