肺癌石蜡包埋组织及新鲜标本组织芯片制备方法学探讨

目的：探讨和建立石蜡包埋组织和新鲜冷冻标本制备组织芯片的方法，以及在基因表达分析中的应用。方法：选取肺癌石蜡包埋组织90例，新鲜液氮冻存肺癌及癌旁组织20例，常规切片HE染色下确定穿刺部位，石蜡包埋组织芯片制作按Kononem等方法（1998），每例穿取0.6mm组织柱2条，制备180点阵组织芯片；而新鲜组织芯片参考Fejzo等方法（2001），并略作改良，制备50点阵组织芯片。所有组织芯片模块，经4цm切片、常规组织学染色和免疫组化染色，以评价其在形态学及基因表达分析中应用的可行性。结果：180点阵石蜡包埋肺癌组织芯片，组织排列整齐，HE染色后组织形态可观察率在98%以上；p53、c-myc、bcl-2、bax和Ki-67等免疫组化染色后，肺癌组织可见不同程度的抗原表达，组织点阵的形态可观测率在89%-95%之间。新鲜组织芯片经HE染色后，形态可观测率和基因表达可分析率在70%左右。结论：手工法组织芯片制备简便易行，使用组织少，不破坏原蜡块；实验均一性、可比性好，且节省试剂。与常规组织切片相比，2点0.6mm组织可以获得原蜡块95%以上的信息，完全可以用于组织形态学和蛋白水平上的基因表达分析；新鲜组织芯片技术已经初步建立，但尚有待于进一步完善制备技术和mRNA分析方法。     

密集型组织芯片手工制作方法的改进

组织芯片是将数十个甚至更多的临床组织标本芯按预先设计的顺序排列在一张玻片上,用于形态学、免疫组化及原位杂交等多种方法的实验研究.有机械和手工两类制作方法,原则上都是在原组织包埋蜡块上采集组织芯,再植入受体蜡块已打好的孔中,然后让组织芯与受体蜡融合、切片[1].     

介绍一种简便的组织蜡块修边法

在病理组织制片过程中,传统的组织蜡块修边法常常是技术人员用刀具或者其它工具对蜡块逐一刮削的方法,修掉蜡块周围余蜡(组织周围要留有余蜡)[1]。随着病理标本的增多,蜡块数量也不断增加。手工逐一刮削蜡块包埋盒的修边方法已满足不了日益增加的工作需要,严重影响制片效率,且人工成本较大,安全性低,影响切片的质量。     

组织学考试用组织芯片制作初探

目的实现组织芯片在组织学教学中的应用。方法取正常Wistar大鼠心、肝、肾、脾等器官,采用组织芯片仪制作组织微阵列蜡块,常规切片,经苏木精—伊红染色制成组织学考试用组织芯片。结果芯片中组织微阵列排列整齐,各样本组织染色清晰,组织定位良好,可判断性强。结论与传统组织切片相比,组织学考试用组织芯片具有低消耗、可比性强、简便易行、局限性小的特点,应用前景广阔。     

快速冷冻剂R-134a于石蜡切片制作中的应用

正石蜡切片技术广泛应用于组织学与病理学教学、科研与临床诊断~([1,2]),其制作需经固定、脱水透明、浸蜡、包埋、切片、展片、捞片、烤片、脱蜡、染色等一系列复杂的步骤~([3]),耗时较长。石蜡切片质量的好坏与诸多因素相关,其中切片尤为关键。具有一定硬度的组织蜡块是保障切片能顺利进行的条件,而蜡块硬度与环境温度呈负相关。研究者常使用冰块冷却蜡块切面,利用热传导降低蜡块温度以提高蜡块硬度~([4])。但该方法存在效率低、易产生水珠干扰操作等缺点。     

石蜡切片显示大脑神经细胞方法探讨

以片显示大脑各种神经细胞一般都是用含有重铬酸钾的固定液固定,用硝酸银浸染的方法进行,所以要想制作大批教学标本只能用传统的方法。而大脑神经细胞的特殊染色,只能用火棉胶切片或冰冻切片制作标本,操作步骤复杂、时间长。为此我们对石蜡切片、镀银染色显示大脑神经细胞的方法进行了探讨。本实验选用小白鼠大脑,组织块大小为2×2cm,勿超过3cm;固定后入1%～1.5%硝酸银水溶液浸染5～7天;蒸馏水洗2分钟;用还原液作用24小时;组织块脱水、透明、浸蜡、包埋、切片,因低浓度酒精易使组织块退色,因此组织块经还原后在酒精内的脱水时间一定要精确。…     

自动包埋技术在大体标本包埋中的应用

目的探讨病理组织通过使用全自动化的设备完成包埋工作。方法采用HT-Auto E150全自动快速病理组织包埋工作站, 对取材的病理大体标本在完成脱水后进行自动化包埋, 同时与人工包埋等量同类型的组织进行时间与质量上的比较。结果全自动包埋机与人工包埋同等量的组织所用时间相当, 全自动包埋蜡块平整、无边蜡, 组织与取材放入时状态完全一致, 但进行粗修步骤时需比平常稍稍细修。人工包埋组织平整, 部分蜡块有边蜡存在, 需将边蜡切除后再进行粗修蜡块的步骤, 包埋过程中部分小组织与取材放入时的状态不同, 易发生组织翻转扭曲, 需技术人员辨认包埋方向进行包埋。两种方法包埋的蜡块切片染色完成后经显微镜观察均核质对比鲜明, 核膜核仁清晰。结论组织包埋工作可以引入全自动化设备配合完成。大体标本可全部使用设备进行自动包埋, 特殊包埋需求的组织仍需医技人员包埋操作, 自动化技术已能减少医技人员的工作量和节省工作时间, 且包埋蜡块一致性更佳。     

组织芯片制孔器和受体蜡块的制作

组织芯片是将几十个以上的小组织有序地包埋在一个蜡块里而制成的组织切片,在科研和临床中加以应用。我们制作了组织芯片制孔器和取材针以代替组织芯片仪制作的组织芯片受体蜡块,取得良好的结果。[第一段]     

影响淋巴结HE制片因素的探讨

淋巴瘤因其组织结构致密、细胞丰富等特点,使组织在脱水、透明、浸蜡、切片、染色等方面都受到一定的影响,导致病理诊断困难。作为一名合格的病理医师,要求懂得组织的石蜡包埋和HE染色的机制,熟练掌握石蜡切片和HE染色技术的操作并能够制出一张优质的病理玻片标本。本实验从石蜡切片和HE染色的机制出发,重点探讨淋巴结HE制片的注意事项。     

石蜡切片浸银染色显示大脑锥体细胞的新方法

目的 :组织学包埋制作大脑组织切片的特殊染色方法 ,用火棉胶技术。此法操作时间长 ,步骤复杂 ,单张切片 ,不宜批量教学标本的制作。为此我们研究了一种操作简单 ,适于大批制作的方法。方法 :用石蜡包埋组织块浸银染色显示大脑锥体细胞。结果 :锥体细胞显示清晰。结论 :与传统方法比较 ,此实验方法比较简单、稳定 ,且可连续切片 ,有利于大批教学标本的制作。     

甲基丙烯酸羟乙酯塑料包埋切片技术在小鼠胎脑组织学分析中的应用

目的 探索塑料包埋切片法在小鼠胎脑组织学分析中的应用。 方法 取胚胎期13.5d（E13.5）小鼠,4% 多聚甲醛4℃固定过夜,分离胎鼠头部,甲基丙烯酸羟乙酯(HEMA)塑料包埋组织并切片;对切片进行HE 染色。 结果 与石蜡包埋相比,采用HEMA塑料包埋的方法进行胎鼠脑组织学分析,形态结构保存较完整、HE染色效果较清晰。 结论 利用塑料包埋技术进行小鼠胎脑组织学分析,优于石蜡包埋方法。     

正丁醇联合松节油替代二甲苯用于石蜡包埋

目的 寻找一种能够替代二甲苯透明剂的组织石蜡包埋方法。 方法 使用正丁醇和松节油的混合物替代常规石蜡包埋过程中无水乙醇、二甲苯的作用,收取卷茎蓼干预的多发性脑梗模型大鼠的脑、肾、胃、肝、十二指肠组织进行石蜡包埋,最终根据石蜡切片的效果、HE染色以及免疫组织化学结果对这种新型脱水程序做出评价。 结果 正丁醇和松节油的混合物可以替代常规石蜡包埋程序中无水乙醇的脱水、二甲苯的透明作用。经正丁醇、松节油混合物处理后的组织切片流畅,组织无变脆、变硬;HE染色后,新型脱水处理组织的细胞核、细胞质分明,组织的着色度、色泽、透明度与常规脱水程序无差异;通过大鼠不同组织做免疫组织化学染色,经对比结果与常规包埋组织免疫组织化学染色无差异。 结论 正丁醇联合松节油用于组织脱水既能够避免二甲苯对人的毒性作用,还可以减少无水酒精过度脱水对组织的破坏。     

Method of double embedding of tissue in paraplast-piccolyte

A histological method of double embedding of the tissue in paraplast-piccolyte used for light microscopy is described. The method requires no special equipment, is simple and suitable for all methods of staining of sections obtained upon paraffin embedding, including immunofluorescent and immunoperoxidase procedures.     

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.     

优化细小穿刺组织石蜡包埋质量的研究

目的　优化细小穿刺组织石蜡包埋方法,以期提高其包埋质量。 方法　通过实验,分别比较不同温度的包埋模具、冷台添加水与否、不同包埋角度、不同二次注蜡角度等条件对细小穿刺组织包埋合格率、包埋时间、切片组织结构完整性、折叠、皱褶、刀痕、细胞显示效果、网状纤维染色效果的影响。 结果　①使用预热至66 ℃的包埋模具包埋合格率显著高于常温包埋模具（χ2=5.26,P＜0.05）;切片组织结构完整性、细胞透明度、核质对比清晰度、网状纤维完整性均分别优于常温组（P均＜0.05）。②包埋机冷台添加少量水使包埋合格率与包埋效率均显著优于未添加水者（P均＜0.05）;切片组织结构完整性、细胞透明度、核质对比清晰度、网状纤维完整性均分别优于未添加水者（P均＜0.05）。③包埋时穿刺组织与包埋模具长轴呈30°~45°角者无折叠、无皱褶、细胞透明度、核质对比清晰度、纤维完整性、网状纤维与胶原纤维对比度均分别优于穿刺组织与包埋模具长轴垂直者（P均＜0.05）;切片组织结构完整性、无刀痕、细胞透明度、纤维完整性均分别优于穿刺组织与包埋模具长轴平行者（P均＜0.05）。④二次注蜡时包埋模具倾斜10°~25°角的包埋合格率显著高于未倾斜者（χ2=4.82,P＜0.05）;组织结构完整性、细胞透明度、核质对比清晰度、纤维完整性均分别优于未倾斜者（P均＜0.05）。 结论　优化包埋模具温度、包埋机冷台、包埋组织角度、二次注蜡角度等条件,可显著提高细小穿刺组织包埋合格率、包埋效率、后续切片质量、细胞显示效果、网状纤维染色效果。     

Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.     

Evaluation of the tissue microarray technique for immunohistochemical analysis in rectal cancer

BACKGROUND: Immunohistochemical staining for tumor-associated proteins is widely used for the identification of novel prognostic markers. Recently, a tissue-conserving, high-throughput technique, tissue microarray, has been introduced. This technique uses 0.6-mm tissue core biopsy specimens, 500 to 1000 of which are brought into a new paraffin array block, which can be sectioned up to 100 times. METHODS: We evaluated the tissue microarray technique for immunohistochemical analysis in 20 rectal cancers. Immunohistochemical staining was performed for the proliferation marker Ki-67 and the tumor suppressor protein p53 in whole tissue sections and in tissue core biopsy specimens. RESULTS: The whole tissue sections were assessed by counting all cells in 10 high-power fields (x40), which resulted in a mean fraction of Ki-67-expressing tumor cells of 0.81 (range, 0.54-1.0). p53 expression assessed in whole tissue sections showed nuclear staining in 15 (75%) of 20 rectal carcinomas. For the tissue microarray technique, a median of 3 (range, 3-5) 0.6-mm tissue core biopsy specimens were studied from each of the 20 tumor specimens. The tissue microarray method gave a mean Ki-67 expression of 0.85 (range, 0.50-1.0) in tumor cell nuclei and showed p53 protein expression in the same 15 of 20 tumors as in the whole tissue sections. CONCLUSION: We conclude that the tissue microarray technique for immunohistochemical staining in rectal cancer yields staining of good quality and expression data for Ki-67 and p53 comparable to those obtained with whole tissue staining. The feasibility of tissue microarray thus enables time- and tissue-preserving studies of multiple markers in large tumor series.     

槲皮素对脓毒症急性肺损伤大鼠肺组织NF-κB表达的影响

目的观察槲皮素对脓毒症相关急性肺损伤(ALI)大鼠的炎症信号转导通路NF-κB p65的影响。方法选用健康雄性Wistar大鼠36只,随机均分为4组:对照组、脂多糖(LPS)组、槲皮素低剂量组(30mg/kg),槲皮索高剂量组(50mg/kg),用LPS 10mg/kg溶于生理盐水2ml腹腔内注射,建立脓毒症ALI大鼠模型,对照组予等量生理盐水2ml腹腔注射,治疗组于造模前30min腹复腔注射槲皮素,所有实验动物于造模后24h应用10%水合氯醛4ml/kg腹腔注射进行麻醉,并处死。所有大鼠取肺组织进行苏木素-伊红(HE)染色,观察病理学改变并进行病理评分且对比,应用免疫组化及Western blot检测NF-κB p65在肺组织的表达。结果与对照组相比,LPS组的肺损伤明显,病理评分明显增加(P0.05);较LPS组,槲皮素治疗组肺组织HE病理染色明显改善,病理评分显著下降(P0.05),但两个治疗组之间病理评分无统计学差异。与对照组比较,LPS组的免疫组化及Western blot检测的NF-κB p65含量明显增高(P0.05),而槲皮素治疗组比LPS组NF-κB p65含量明显下降(P0.05),但两治疗组比较无明显差异。结论槲皮素对脓毒症ALI大鼠的肺脏保护作用可能与其抑制NF-κB p65的表达相关。     

Is it necessary to embed bone marrow biopsies in plastic for haematological diagnosis?

With the increase in the use of bone marrow trephines for diagnosis have come numerous reports that traditional methods of preparation (by decalcification and embedding in paraffin wax) should be replaced by plastic embedding to avoid decalcification. It has been argued that only by this means can the high quality preparations needed for accurate haematopathological diagnosis be achieved. The present study challenges this viewpoint and argues that with a little extra care and attention conventional paraffin embedding techniques can give equally high quality preparations. Sections prepared in this way meet the diagnostic needs of the haematologist, without requiring a separate technique to be established in the pathology laboratory solely for bone marrow trephines.     

Tissue microarrays: a new approach for quality control in immunohistochemistry

AIMS: To improve the interpretation of immunohistochemistry (IHC) staining results the use of a tissue microarray technique was established in a routine setting. METHODS: A tissue microarray was constructed by harvesting 600 microm tissue cores from paraffin wax embedded samples available in a routine pathology department. The punches originating from non-tumorous tissue were placed on host paraffin wax blocks. The microarray contained 12 different tissue samples, with a wide antigen profile and a dimension of 3.5 x 3 mm. One section of the multitissue array was placed as an "internal" positive control on each slide of the patient tissue to undergo identical immunohistochemical procedures. RESULTS: Using the tissue microarray technique as a tool for internal quality control, the interpretation of immunohistochemical staining of more than 20 different antigens in routine IHC was improved. The tissue microarray did not influence the staining results in conventional IHC or in different automated IHC settings. CONCLUSION: The regular use of an institution adapted tissue microarray would be useful for internal positive control in IHC to enable different laboratory demands. Furthermore, this technique improves the evaluation of staining results in IHC.