高温烤片对石蜡切片黏附、厚度与染色的影响

目的笔者实验室最近发现,高温烤片可有效解决甲基丙烯酸树脂包埋的组织切片的脱片问题,本文拟进一步研究确定高温烤片对石蜡包埋切片黏附、厚度及染色的影响。方法大鼠肾脏石蜡切片(切片机设定的切片厚度10μm)脱蜡后经90℃、140℃热板烤片30 min处理,然后用过碘酸-席夫试剂和苏木精染色,观察脱片情况、切片厚度及染色效果。大鼠脊髓石蜡切片(14μm厚)同样处理后分别进行小胶质细胞和突触素颗粒的免疫组化染色。结果与对照(未烤片)相比,90℃或140℃烤片都有效防止了肾脏石蜡切片从载玻片上脱落,但染色后的实际切片厚度减少了约50%(与设定切片厚度相比),染色变浅,结构清晰度欠佳(90℃烤片后),甚至有细胞消失(140℃烤片后);90℃烤片后免疫阳性小胶质细胞或突触素颗粒减少,非特异性染色加深,140℃烤片后几乎未见免疫阳性结构。结论高温烤片防脱片的方法可能不适用于石蜡切片。     

HE褪色后免疫组化染色防脱片方法的应用

在肿瘤病理诊断中 ,经常遇到临床送检的细胞学涂片或基层医院送来的ＨＥ会诊片 ,因确诊困难需要辅以免疫组化染色。然而 ,由于ＨＥ染片一般不涂粘胶剂 ,因此褪色后进行免疫组化染色容易脱片。针对这一弊病 ,笔者经过反复实践 ,探索出一种简便的防脱片方法 ,并广泛用于细胞学涂片和ＨＥ切片褪色后的免疫组化染色 ,效果满意 ,现介绍如下。１材料和方法１．１材料材料系我院病理科ＨＥ存档切片。选乳腺腺癌、食管鳞癌、胃腺癌、纤维肉瘤、恶性淋巴瘤切片和胸水、腹水涂片各５张 ,共３５张。褪色剂为０．２５ %高锰酸钾溶液和０．５ %草酸溶液［…     

四种人肺癌细胞株的滴片与石蜡切片HE染色色度学定量分析

目的从色度学角度定量分析不同人肺癌细胞株A549、GLC-82、H358及H2009的相似性和差异性,比较滴片与细胞石蜡切片HE染色的色度学分析结果的异同。方法用计算机图像分析技术分别测试上述四种肺癌细胞滴片HE染色和A549、GLC-82和H358肺癌细胞石蜡切片HE染色的色度学参数,包括Rp、Gp、Bp、Rn、Gn、Bn、rp、rn、gp、gn、bp、bn。结果细胞滴片A549、GLC-82、H358和H2009细胞间色度学参数差异具有显著性(P<0.05),石蜡切片A549、GLC-82和H358细胞间色度学参数差异具有显著性(P<0.05);细胞滴片与石蜡切片HE染色的色度学参数差异具有显著性(P<0.05),滴片与石蜡切片上不同肺癌细胞同一色度学参数具有一致性(P=0.048),但一致性不明显(Kappa=0.313)。结论不同类型肺癌细胞株滴片与石蜡切片HE染色的色度学参数差异显著,可用于不同肺癌细胞株的甄别;滴片与石蜡切片色度学参数间差异有显著性,两者间的一致性不明显,应区别应用。     

脱钙骨石蜡切片钙钴法碱性磷酸酶染色

碱性磷酸酶组化染色显示成骨细胞,可用于分析病变组织来源及发病机制,以往多用冰冻切片,而骨组织做冰冻切片几乎不可能。我们探讨用脱钙后石蜡切片钙钴法作碱性磷酸酶染色成功,介绍如下。一、方法及结果1.石蜡包埋的脱骨切成5μ厚组织片,用25％酒精滴裱片展平于载片上,待组织团着后放入37℃温箱24小时。2.常规脱蜡后令酒精自然挥发。3.入37℃孵育液(2％巴比妥钠10ml,3％B-甘油磷酸钠10ml,2％氯化钙20ml,5％硫酸镁1ml,蒸馏水5ml,调整PH为9.4)15～20分钟,对照片肝组织60分钟。4.流水洗3分钟。5.2％硝酸钴液5     

提高免疫组化反应的抗原修复方法

目的　探讨免疫组织化学反应不同预处理方法对反应结果的影响。方法　对5 0例经10 %中性福尔马林液固定过的组织,经脱水、石蜡包埋,制成切片,分别采用3种不同方法作预处理后,进行免疫组织化学(S P法)染色。结果　石蜡组织切片免疫组化反应预处理,采用中温水浴抗原修复法,其阳性表达率为5 2 .6% ,脱片率为2 .0 % ;采用高压锅抗原修复法,其阳性表达率为47.4% ,脱片率为2 0 .0 % ;采用水沸抗原修复法,其阳性表达率为3 9.5 % ,脱片率为12 .0 %。结论　免疫组化反应采用中温水浴抗原修复法作预处理,石蜡组织切片阳性表达率较高压锅抗原修复法提高了5 .2 % ,P <0 .0 5 ,较水沸抗原修复法提高了13 .1% ,P <0 .0 5 ;采用中温水浴抗原修复法石蜡组织切片脱片率较高压锅抗原修复法降低了18.0 % ,P <0 .0 1,较水沸抗原修复法降低了10 .0 % ,P <0 .0 1。预处理采用中温水浴抗原修复法,即提高了免疫组织化学反应阳性表达率,又提高了制片质量,操作方法易于掌握,是1种理想的免疫组化反应预处理方法,值得推广。     

A549细胞光镜及电镜制样收缩系数测试

目的测试A549细胞经过光镜及电镜制样处理后的收缩系数,为定量研究A549细胞及其超微结构奠定基础。方法对A549细胞分别进行涂片HE染色、涂片巴氏染色、石蜡包埋3μm切片HE染色及透射电镜制样半薄（3μm）切片HE染色处理。通过光学显微镜获取细胞图像,用Image-Pro Plus图像分析软件测试细胞的平均直径,然后以A549培养之等渗重悬细胞之平均直径为基准计算细胞的收缩系数。结果A549培养之等渗重悬细胞的平均直径为14.93μm。经涂片HE染色、涂片巴氏染色、石蜡切片HE染色和透射电镜制样半薄（3μm）切片HE染色处理后的A549细胞的平均直径分别9.88μm、9.98μm、10.29μm和10.59μm。以等渗重悬细胞之平均直径为基准,经涂片HE染色、涂片巴氏染色、石蜡切片HE染色和透射电镜半薄切片HE染色之细胞的收缩系数分别为0.66、0.67、0.69和0.71。结论经涂片HE染色、涂片巴氏染色、石蜡切片HE染色和透射电镜制样半薄切片HE染色处理后,A549细胞收缩明显,直径约收缩了1/3。因此,在实际测试中,欲知细胞收缩前之结构大小,须据细胞收缩程度进行必要的校正。     

乳腺肿块印片细胞快速病理诊断的应用价值

目的：探讨乳腺肿块印片细胞在快速病理诊断中的应用价值。方法：将120例乳腺肿块切除标本进行细胞印片、HE染色、光镜观察并与快速石蜡切片进行对照。结果：细胞印片与快速石蜡切片定性完全一致115例,占95.83%,定性不完全一致5例,占4.17%,本组良性病变92例,恶性27例,潜在恶性1例。结论：该方法在乳腺肿块病理诊断中具有应用价值。     

术中细胞学印片法诊断乳腺癌前哨淋巴结转移的准确性分析

目的探讨细胞学印片评估乳腺癌前哨淋巴结转移的准确性。方法 选择70例早期乳腺癌患者进行前哨淋巴结检查,前哨淋巴结切除后立即送检,沿前哨淋巴结短轴每隔2～3 mm剖开,每个剖面均进行印片细胞学检查,印片使用HE染色。印片后的淋巴结分别行石蜡包埋切片,将印片结果与石蜡切片的HE染色结果进行对比。结果 共检出134枚淋巴结。印片细胞学检查乳腺癌淋巴结转移的灵敏度为96.2％（25／26）,特异度为98.1％（106／108）,准确度为97.8％（131／134）。阳性预测值与阴性预测值分别为92.6％（25／27）、99.1％（106／107）。假阳性原因主要是组织细胞形态学与分化好的癌细胞相似;假阴性原因是转移灶微小。结论 印片细胞学对乳腺癌前哨淋巴结术中病理诊断有较高的价值,可以准确提供术中诊断信息,与石蜡切片有很高的一致性。     

乳腺肿块印片细胞快速病理诊断的应用价值

目的:探讨乳腺肿块印片细胞在快速病理诊断中的应用价值.方法:将120例乳腺肿块切除标本进行细胞印片、HE染色、光镜观察并与快速石蜡切片进行对照.结果:细胞印片与快速石蜡切片定性完全一致115例,占95.83%,定性不完全一致5例,占4.17%,本组良性病变92例,恶性27例,潜在恶性1例.结论:该方法在乳腺肿块病理诊断中具有应用价值.     

术中组织印片法快速诊断212例分析

目的探讨组织印片在术中快速诊断的可行性。方法将术中送检的212例新鲜标本，即刻切开，在不同病变切面上用载玻片轻轻触印，制成印片，快速HE染色，光镜下观察，报告诊断结果。所剩组织均做石蜡切片诊断作对照。结果212例印片诊断与石蜡切片诊断比较，印片阳性(恶性肿瘤)57例(26．9％)；阴性(非肿瘤性)146例(68．9％)，印片诊断准确率95．8％(203／212)。石蜡切片诊断阳性64例(30．2％)，阴性148例(69．8％)，准确率100．0％(212／212)。用印片诊断阳性57例与石蜡切片诊断作类型比较，有38例完全相符，诊断符合率66．7％(38／57)。7例误诊，误诊率仅为3．3％(7／212)。3例定性准确但分类不符合，9例病变交界难定。结论组织印片诊断不同于一般涂片单纯细胞学诊断，而是细胞学与组织学相互结合观察的结果，有时可进行组织类型的诊断。在冷冻切片与快速石蜡切片同时作快速印片，可有效纠正部分误诊。     

Immunostaining of human glioblastomas with various PCNA antibodies

The aim of the study was to compare various PCNA clones (PC10, 19A2, and 19F4) on frozen and paraffin wax sections of ten human glioblastomas. Standard immunohistochemical methods were used (avidin-biotin-peroxidase technique). Half of the paraffin sections were pretreated in a microwave oven. With few exceptions positive staining was achieved only on paraffin sections after microwave treatment. Staining with the 19A2 antibody revealed few (<1%) positively stained tumor nuclei in only three cases whereas staining with 19F4 was positive in eight cases with a labeling index (LI) in the range of <1-3.5%. All cases showed positive staining with the PC10 antibody with an LI of <1% in three cases and in the range of 2.0-17.2% in the remaining seven. However, most of the PC10 stained sections showed a disturbing unspecific background staining. Thus, in spite of the background staining we conclude that PC10 offered the best immunostaining of the antibodies tested.     

Immunohistochemical demonstration of estrogen receptors on routine paraffin sections of breast carcinomas: a comparison with frozen sections and an enzyme immunoassay.

In this study the monoclonal antibody ER-ICA (HSpy222) to human estrogen receptor (ER) protein and the peroxidase-antiperoxidase method was used to detect the presence of ER in 83 cryostat sections and in 68 paraffin sections pretreated with pronase in a total of 86 primary breast cancers. In 72 out of the 86 studied cases, a comparative evaluation was performed between the semiquantitative ER-ICA method and the quantitative enzyme immunoassay ER-EIA. A good correlation was found between the semiquantitative ER-ICA results in cryostat and paraffin sections (95.38%; p less than 0.01) in a total of 65 compared cases, concerning both the percentage of ER-positive or negative cells and the staining intensity. In addition, the overall appraisal of the lesion as ER-ICA-positive or ER-ICA-negative as well as the ER-ICA staining intensity and the proportion of ER-ICA stained cancer cells, in both cryostat and paraffin sections, correlated significantly with the mean values of fmol ER/mg determined by the enzyme immunoassay ER-EIA. The performance of the ER-ICA method on paraffin sections as used in the present study proved to be a reliable and reproducible immunohistochemical technique.     

Rapid immunohistochemical detection of tumor cells in gastric carcinoma

The detection of single tumor cells or tumor cell clusters represents an important issue in intraoperative frozen section analysis. For example, surgical margins may be evaluated in order to minimize the number of additional operations. Furthermore, intraoperative diagnosis of lymph node micrometastasis (LNM) may help to define the area of appropriate lymph node dissection. In addition to haematoxylin and eosin (H&E)-stained sections, immunohistochemical detection of single tumor cells or cell clusters may be helpful in this context. The aim of this study was to evaluate the clinical significance, reliability and sensitivity of intraoperative rapid immunostaining of frozen sections. Therefore, we compared the results of rapid immunohistochemical staining of frozen sections and paraffin sections applying the EnVision and Histofine(R) detection systems. In a prospective immunohistochemical study, paraffin and frozen sections of 20 gastric cancer specimens were analyzed. Paraffin as well as frozen sections were stained immunohistochemically applying the EnVision and Histofine detection systems. As primary antibodies, AE1/AE3 (anti-cytokeratin), EMA (anti-MUC1) and B lymphocyte marker anti-CD20 were applied. The rapid immunostaining procedure was able to be completed within 10-13 min. Rapid immunohistochemical staining of frozen and paraffin sections of the same tumors resulted in comparable immunoreactivity. The rapid EnVision and Histofine procedures allowed immunostaining of frozen sections in less than 13 min. These methods can represent useful additional tools in routine surgical pathology and research, enabling a more accurate frozen section diagnosis compared to staining with H&E alone. Intraoperative rapid immunostaining can be a simple and useful technique to detect LNM.     

Histochemical studies of human breast cancer using a monoclonal antibody against an oestrogen receptor-related antigen

The presence or absence of an oestrogen receptor-related antigen in breast tumours has been examined histochemically using a monoclonal antibody ('Ds' - Coffer & King, 1981). In frozen sections, fixed either by the method of Tamura et al. (1980) or in methanol, staining was apparent in 14/24 (58%) and 22/26 (85%) of the breast cancers respectively. In paraffin sections fixed in ethanol, staining was present in 25/33 breast cancers (76%). In either type of section, staining was predominantly in the cytoplasm of the epithelial cells. When staining was scored by independent observers (2 or 3) and related to the tumour oestrogen receptor activity, determined by a standard biochemical technique, antigen was present in both receptor-positive and receptor-negative tumours. No significant association was found between the presence of antigen and receptors in the frozen sections, but for the series of paraffin sections, there was a weak association (r = +0.48) between the presence of the two proteins. Histochemical processing of paraffin sections from 9 tumours under conditions of higher sensitivity increased the staining significantly in 2/9 tumours, but did not alter the relationship between staining and receptor status. Six tissues were stained after exposure to 'receptor-translocating' conditions (25 degrees C/2 nM oestradiol/both for 1 h): this did not consistently change the subcellular staining pattern, though all tissues tended to stain more after exposure to 25 degrees C. Staining was not blocked by absorption of the D5 antiserum with a variety of pure proteins or human serum but at higher concentrations (approx. 2-15 mg protein ml-1), extracts from human uterus, an oestrogen-receptor-positive breast cancer and an oestrogen-receptor-negative breast cancer all effectively abolished staining in sections from another breast cancer. These results are consistent with other reports suggesting that the D5 antibody detects an antigen which is not the oestrogen receptor, but which may be associated with the receptor in its tissue distribution.     

Absence of retinoblastoma protein expression in primary non-small cell lung carcinomas.

Retinoblastoma (RB) protein expression was examined in paraffin and frozen tissue sections of 36 primary non-small cell lung carcinomas (NSCLC) using immunohistochemistry with confirmation by direct Western blotting. A normal RB protein staining pattern was present in 24 and absent in 10 NSCLC. Two additional RB positive primary tumors have major foci in which all tumor cells showed no RB protein staining. Significantly more high-stage (stages III and IV) NSCLC had altered RB protein expression than those with low-stage (stages I and II) tumors (P less than 0.05). The results suggest that absence of the RB expression may be associated with the initiation and/or progression of many NSCLC. This is the first report of successful RB staining in paraffin sections.     

HNK-1-defined antigen detected in paraffin-embedded neuroectoderm tumors and those derived from cells of the amine precursor uptake and decarboxylation system

The reactivity of the HNK-1 monoclonal antibody that, besides a subset of hematopoietic cells with natural killer activity, also detects neuroectodermal cells, was screened on 153 human tumors. Immunoperoxidase staining of paraffin sections revealed that HNK-1 detects neuroectoderm-derived cancers and tumors with endocrine activity. Because the antigen detected by HNK-1 is well conserved in paraffin sections, this antibody is of potential diagnostic value in routine histopathology and might help to elucidate the histogenesis of some poorly understood tumors.     

Colocalization of estrogen and progesterone receptors with an estrogenregulated heat shock protein in paraffin sections of human breast and endometrial cancer tissue

Summary We have studied by immunocytochemistry and monoclonal antibodies the presence and localization of estrogen receptors, progesterone receptors, and a 24-kD estrogen-regulated heat shock protein in biopsies from breast and endometrial cancer patients. Three different tissue processing protocols were used to colocalize the antigens in the same tissue sections: a) frozen sections, b) formalin fixation with routine paraffin embedding, and c) picric acid-formaldehyde (PAF) fixation with a rapid embedding in paraffin. Frozen sections showed good receptor staining but poor 24-kD protein immunoreactivity, while routine paraffin sections (with or without DNase pretreatment) were inadequate to reveal the nuclear receptor proteins at the same level seen in frozen sections. On the other hand, all three proteins could be detected satisfactorily in PAF-fixed paraffin-embedded tissue. Using this procedure we were able to visualize 24-kD protein and estrogen receptor or progesterone receptor in individual cells in paraffin sections. The study revealed that in all of the estrogen receptor positive breast and endometrial tumor samples, almost 90% of the cells expressing the cytoplasmic 24-kD protein contained estrogen receptor in the cell nucleus. In contrast, 24-kD immunoreactive cells did not express progesterone receptors in almost 40% of the progesterone receptor positive tumor samples.     

Differential expression of isopeptide bonds N epsilon (gamma-glutamyl) lysine in benign and malignant human breast lesions: an immunohistochemical study.

N epsilon (gamma-glutamyl) lysine isopeptide bonds were detected in situ in histological sections from benign and malignant human breast tissue using the monoclonal antibody (MAb) 81D1c2. On cryostat sections of fresh-frozen mammary tissue post-fixed in acetone, or on paraffin sections also from mammary tissue fixed in Bouin's solution, the MAb reacted preferentially with glandular epithelial cells and staining was restricted to the nuclei. In 41 out of 44 benign lesions examined, staining was strong or moderate, while in 25 out of 33 malignant lesions no staining was observed. In the 8 remaining lesions of this group, staining was positive but weak. The difference in reactivity of this MAb with the 2 types of lesions is highly significant (p less than 0.0005) according to the chi 2 test.     

核仁形成区AgNORS染色法及其临床意义

在233例多种肿瘤的石蜡切片和细胞学涂片上进行银染核仁形成区(AgNoRs)染色,并取各种正常组织作对照,其染色结果和定量观察表明:该法有助于临床病理学鉴别诊断,是肿瘤病理学研究的有效方法。本文从方法学的角度探讨AgNoRs染色法的可行性及其临床意义。对应用该法时可能碰到的有关问题进行了实验对比,并提出了解决途径。     

Laminin immunodetection in tumorous and nontumorous disorders of human thyroid

Laminin distribution in tissues from surgically removed thyroids consisting in normal gland (5), Basedow's disease (5), thyroiditis (5), follicular adenomas (8), papillary carcinomas (8), follicular carcinomas (6) was studied using rabbit laminin antibody raised against murine laminin. Immunoperoxidase technique (Avidin-Biotin-Peroxidase Complex) was performed on (1) paraffin sections of fixed tissue (2) frozen sections for light microscopy examination. Vibratome thick sections (100 micron) and pre-embedding technique were used for electron microscopy study. Positive staining, was obtained only on frozen and vibratome sections and was found within basement membranes but never in epithelial cell cytoplasm. Laminin had a similar distribution in follicular adenomas, Basedow's disease and normal tissue. Nests of damaged cells in Hashimoto's thyroiditis lacked positive laminin immunostaining. In papillary carcinomas positive staining was found beneath the epithelial cells along the cores. In well differentiated follicular carcinomas the perifollicular laminin staining was preserved, whereas in poorly differentiated follicular carcinomas laminin staining was barely visible or absent.