三种Feulgen染色方法的比较

目的探索一种快速的Feulgen染色方法,并尝试其在细胞核DNA含量分析中的应用。方法选取十只健康小白鼠的肝细胞涂片,每只小白鼠的肝细胞涂片分成三组,采用快速Feulgen染色法、改良Feulgen染色法、传统Feulgen染色法对这三组分别染色,用TIGER细胞图像分析仪测量肝细胞涂片内单个完整肝细胞核的DNA含量,比较各组染色效果和肝细胞核DNA含量的测量结果。结果应用快速Feulgen染色法,获得了满意的效果,细胞核染色深,结构清晰,背景着色淡,而所需染色时间仅为15分钟;同一种方法染色的不同小鼠肝细胞涂片,相同DNA含量倍体肝细胞核的DNA含量均值的变异系数(CV)<10%,CV(快速)IOD(改良)>IOD(传统);各组2、4、8倍体肝细胞核DNA含量间的比值均接近2或4;结论此种快速Feulgen染色方法优于其他两种方法,可应用于图像分析系统对细胞核DNA含量与倍体的测量和分析。     

光衍射现象对细胞核DNA含量检测的影响

利用图像分析仪测量小鼠肝细胞涂片内单个肝细胞核的DNA含量,探讨图像分析仪在测量显微图像的光度时光衍射现象所导致的测量误差.本文选取10只成年健康雄性小鼠的肝组织涂片,Feulgen染色,TIGER细胞图像分析仪测量单个肝细胞核的DNA含量并分析其DNA含量倍体;结果显示,(1)涂片内肝细胞核分布均匀,轮廓清晰,呈紫红色;(2)不同小鼠同一DNA含量倍体的肝细胞核的DNA含量大致相同;(3)二、四、八倍体肝细胞核的DNA含量比值均大于/[Dept.1]等于2或4,二、四、八倍体肝细胞单个核DNA含量的CV值均小于10%;(4)同一小鼠不同DNA含量倍体肝细胞核的平均光密度值差异较小.结论光衍射现象可导致DNA含量的测量结果偏低,其偏低的程度随待测细胞核的面积增加而减小.     

乳腺癌细胞核DNA含量不同分析方法的比较

目的:比较组织原位分析乳腺癌细胞核DNA含量的不同方法。方法:收集30例乳腺癌标本的归档蜡块,4m和相邻8m切片各一张,Feulgen染色,采用TIGER细胞图像分析仪测量每个癌巢的以单个完整细胞核体积为单位计算的DNA指数和以细胞核切面面积为单位计算的DNA指数,它们均以同一病例正常上皮细胞核作内参照。结果:(1)同一病例的VIOD分布直方图比IOD更集中;(2)30例标本的90个样本中,以VIOD为单位所测得的DI值比以IOD为单位测得的DI值高。结论:使用细胞图像分析仪对组织切片内细胞核DNA含量进行定量分析时,应以单个完整细胞核体积的DNA含量为单位,计算DI值。     

石蜡包埋组织自动和手动切片对切片厚度的影响

目的观察手动与自动两种切片方法和组织种类对实测切片厚度的影响。方法选取5只成年健康雄性SD大鼠的肝、胰腺、胃及肺4种组织,各组织单独包埋制成蜡块,预设电动切片机的切片厚度刻度值为3、5、7、10μm,分别作自动和手动两种方法切片,所获得的薄组织片经二次包埋、切片、HE染色,TIGER图像分析仪测量切片的实际厚度。结果组织切片实测厚度受自动和手动两种切片方法的影响,切片机的切片厚度预设值低估了石蜡包埋组织切片的真实厚度,自动和手动实测切片厚度分别比切片机预设切片厚度要厚29％～50％和48％～91％。结论自动切片与手动切片的切片厚度相差25％,二者相对误差为61％,自动切片获得的组织切片的厚度精度较高,优于手动切片。     

滤光片对细胞核DNA含量检测的影响

目的探讨滤光片对图像分析仪测量DNA含量的影响。方法选取10只成年健康雄性小鼠,制备肝细胞涂片,Feulgen染色和天青B染色,显微镜与摄像机之间分别插入400 nm、560 nm、590 nm、600 nm、626 nm、670 nm带通滤光片和中灰滤光片,利用TIGER细胞图像分析仪测量肝细胞核的积分光密度（IOD）与平均光密度（AOD）和面积。结果肝细胞核在Feulgen染色法中呈紫红色,天青B染色法呈蓝色。Feulgen染色和天青B染色分别采用560 nm和600 nm的带通滤光片时,能够测得最大的IOD值,不同倍体肝细胞核间的比值接近2、4,CV值最低。与天青B染色方法相比,Feulgen染色法中不同倍体间的比值更接近2、4,CV值更低。结论适宜的滤光片可提高细胞核DNA含量测量结果的精确性和准确性。Feulgen染色测量结果精确性和准确性明显高于天青B染色,应作为首选的染色方法。     

厚、薄组织切片上细胞核体积测量结果的比较

目的: 研究组织切片厚度对细胞核体积测量结果的影响。方法: 收集鼻咽癌归档病例42例, 每个病例包括正常鼻咽上皮组织和3个癌巢或肿瘤区域, 制成4μm、8μm连续组织切片各一张。使用TIGER细胞图像分析仪分别测量4μm、8μm连续鼻咽癌组织切片上正常鼻咽上皮细胞核与肿瘤细胞核的体积。结果: 8μm组织切片上测得的细胞核体积明显大于4μm组织切片上测得的, 差异具有统计学意义(P<0 .05)。结论: 组织切片厚度影响细胞图像分析仪测量细胞核体积的结果; 采用图像分析仪测量细胞核内化学成分的含量应以完整细胞核为单位。     

人肝细胞癌DNA干系倍体异质性的组织原位分析

目的探讨人肝细胞癌DNA干系倍体异质性的临床意义。方法选取45例肝细胞癌患者的归档蜡块,分别制备4μm、10μm的连续组织学切片,利用细胞图像分析仪测量细胞核DNA干系倍体值及其形态学参数。在4μm切片上测量细胞核DNA的平均光密度,在10μm切片上测量单个完整细胞核的体积。经细胞图像分析仪计算获得以单个完整细胞核体积为单位的DNA总量,以同一切片内正常淋巴细胞作为内参照,计算其DNA干系倍体值和异质性率。统计学分析DNA干系倍体异质性率与组织学分级、瘤体大小、淋巴结转移以及AFP表达水平的关系。结果肝细胞癌DNA干系倍体异质性率与组织学分级、瘤体大小、AFP表达水平有关联,与淋巴结转移无关联。结论组织原位法分析肝癌DNA干系倍体异质性对肝细胞癌的诊断、恶性度判定及预测预后均具有重要的参考价值。     

应用肝细胞涂片评估DNA染色方法的可靠性

目的探讨应用肝细胞涂片评估DNA染色方法的可靠性。方法选取10只成年健康雄性小鼠，制备肝细胞涂片，Feulgen染色和天青蓝染色，TIGER细胞图像分析仪测量肝细胞核的DNA含量与倍体。结果涂片内肝细胞核分布均匀，轮廓清晰；Feulgen染色法中肝细胞核呈紫红色，天青蓝染色法呈蓝色；作为参比的Feulgen染色涂片中不同倍体间IOD的比值比天青蓝染色标本更接近倍体数的比值，其CV值也低于天青蓝染色法。结论小鼠肝细胞涂片可应用于评估显示DNA染色方法的可靠性。     

肝肿瘤细胞DNA倍体分析中选择参照细胞核的依据

目的　利用细胞图像分析仪测量小鼠肝细胞涂片的 DNA含量 ,探讨细胞图像光度术 (ICM)和流式细胞光度术 (FCM)分析肝肿瘤细胞核 DNA倍体时 ,选取参照细胞核的原则。方法　本文选取 10只成年健康雄性小白鼠的肝组织涂片 ,Feulgen染色 ,TIGER细胞图像分析仪测量单个完整肝细胞核的 DNA含量与倍体。结果　 (1)不同小鼠同一倍体的肝细胞核 DNA含量大致相同 ;(2 )二、四、八和十六倍体肝细胞单个核 DNA含量间的比值接近 2、4、8,它们的 CV<8.0 %;(3)当仅出现二倍体和四倍体时 ,以二倍体的细胞核为主 (80 .4%) ,伴随八倍体和十六倍体的出现 ,四倍体肝细胞核所占百分率大于二倍体。结论　由于肝细胞具有多核和多 DNA倍体的特点 ,分析和计算肝脏肿瘤细胞核 DNA倍体和其它指标时 ,不能简单地遵循以同种属、同个体、同源的正常二倍体肝细胞核作为参照细胞核 ,正常四倍体和八倍体甚至十六倍体肝细胞核作为参照细胞核也应该加以考虑。     

组织原位测算单个肝细胞核的DNA总量

目的再次探讨利用图像分析系统在组织切片原位测算以单个完整细胞（核）体积为单位的化学物质总量方法的可靠性。方法选取10只成年健康雄性昆明小鼠,每只小鼠按常规制片方法制作4μm和11μm两种厚度的肝组织切片各一张,改良Feulgen染色,应用细胞图像分析仪在组织原位测量和计算以单个完整二倍体、四倍体和八倍体肝细胞核体积为单位的DNA总量。结果组织原位测算的以单个完整二倍体、四倍体、八倍体肝细胞核体积为单位的DNA总量间的比值基本上呈2或4的倍数关系。结论应用细胞图像分析仪在组织切片原位通过合适的抽样和测量计算,可较准确地获得以单个完整细胞（核）体积为单位的化学物质总量。     

测量生物组织Epon薄切片厚度的研究

组织切片厚度的准确性对生物组织切片上的显微结构和化学成份定量分析结果的影响极大。至今，国内外已建立起的确定组织切片厚度的方法均有不足之处。本文将分光光度法的基本原理应用于组织切片厚度的测量，采用显微分光光度计和显微图像分析仪以及显微摄影测光表这些组织细胞形态和组织化学观察和定量分析中常用的仪器，对经锇酸固定的组织Ｅｐｏｎ薄切片的厚度进行测量。结果表明，这三种仪器均能用于测量Ｅｐｏｎ薄切片的厚度，而且经厚度测量的Ｅｐｏｎ薄切片尚能用于组织细胞形态和组织化学的定量分析；显微分光光度计和显微摄影测光表的测量灵敏度以及测量结果间没有显著差异，它们的测量结果比显微图像分析仪的测量结果更灵敏。本文讨论了这三种仪器在测量Ｅｐｏｎ薄切片厚度中的优缺点及其产生原因。     

不同器官石蜡包埋切片的切片压缩对比研究

目的比较研究不同器官石蜡包埋切片的切片压缩。方法从一成年雄性SD大鼠的肺、心、肝、睾丸和脊髓腰膨大各切取3个长、宽、高均约为2 mm的组织块,石蜡包埋后从每个组织块切取5～40μm厚(切片机设定厚度)的切片各3张进行苏木精染色,然后利用体视学图像系统的指针测微计测量每张切片染色后的实际厚度,以后一厚度(实际厚度)与前一厚度(切片设定厚度)之比作为切片压缩系数。结果与结论不同器官的平均压缩系数分别为84.4%(肺)、81.4%(心脏)、80.6%(肝脏)、77.9%(睾丸)和72.2%(脊髓),不同器官间有显著性差异。     

切片位置对兔睾丸组织体视学研究的影响

目的比较不同包埋介质内切片位置对成年新西兰大耳白兔睾丸组织体视学研究的影响。方法将成年雄性新西兰大耳白兔麻醉后,随机抽选一侧睾丸经Bouin液固定后从中央及两端（垂直于睾丸中心长轴）切取共三个平行组织薄片。将每个组织薄片平分为二,再随机将其中一块平分得到2个（1/4圆）组织块,分别随机采用羟乙基甲基丙烯酸树脂或石蜡包埋、切片和染色后,在低倍镜（10×）下分别对睾丸组织树脂切片（25μm）和石蜡切片（7μm）不同切片位置进行观察：紧贴白膜选择若干视野（所有视野间无任何重叠）,再向扇形切片中心连续选择3个视野,利用体视学方法估计各视野睾丸组织内生精小管的体积分数。结果石蜡切片较树脂切片估计的生精小管体积分数减少15.9%;树脂切片不同部位估计的生精小管体积分数无差异,而石蜡切片不同部位估计的结果存在差异：距离白膜愈远,体积分数愈小,近切片中心与近白膜相比显著减少了30.1%。结论石蜡包埋能引起睾丸组织不均匀皱缩,因此,当应用石蜡切片进行睾丸体积分数研究时,应考虑不均匀组织皱缩因素。     

Pitfalls in frozen section diagnosis of malignant melanoma

The importance of obtaining an intraoperative diagnosis and measurement of thickness of a malignant melanoma has emerged since the change in the surgical approach towards thin melanomas (0.75 mm). In experienced centers, both diagnosis and thickness, can be established on frozen section. Eighty-four pigmented lesions were examined on frozen section. Thirty of 31 were correctly diagnosed as malignant melanomas. The one not diagnosed was a regressing melanoma. The frozen section diagnosis of a regressing melanoma is difficult, and this should be deferred until paraffin sections are examined. In 29 consecutive skin melanomas thickness was measured on frozen section and was found to be 0.1-0.4 mm more than that measured on paraffin sections of the same specimens. It is therefore suggested that tumors measuring up to 0.85 mm on frozen section should be included in the group of thin melanoma.     

A simple inflation method for frozen section diagnosis of minute precancerous lesions of the lung

BACKGROUND: Recently, the demand for intraoperative pathology consultation for small pulmonary nodules including ground-glass opacity (GGO) has been increasing. Evaluation of minute precancerous lesions of the lung by frozen section is very difficult for the pathologist as uninflated lung tissue usually shows severe atelectasis and frozen artifact. We tried to inflate lung tissue with the embedding medium used for frozen section and to determine the appropriate dilution ratio of the embedding medium for optimization of frozen section morphology. METHODS: The lung specimens were derived from 10 patients who underwent video-assisted thoracoscopic surgery (VATS) due to pneumothorax (four patients) and GGO (six patients) detected on high-resolution computed tomography (HRCT) at Seoul National University Bundang Hospital. The pneumothorax specimens were divided into six groups-uninflated, inflated with saline, and inflated with embedding medium (not diluted, 1:1, 2:1 and 2:3). The qualities of the frozen sections were compared with corresponding permanent paraffin sections. Lung specimens obtained from the six people with GGO detected on HRCT were submitted for intraoperative pathology consultation. Frozen sections were made after inflation with optimally diluted embedding medium determined by the above experiment with pneumothorax specimens, and the frozen section diagnoses (FSD) were compared with the final pathologic diagnoses of corresponding permanent paraffin sections. RESULTS: The frozen section quality of lung tissue was excellent after simple inflation with diluted embedding medium (2:3). Minute precancerous foci such as atypical adenomatous hyperplasia (AAH) and bronchioloalveolar carcinoma (BAC) could be readily identified in frozen sections using this method. Of the six patients with solitary GGO, four were diagnosed as BAC, nonmucinous type and two were as AAH on the frozen sections. Intraoperative FSD corresponded well with final diagnoses obtained with paraffin sections. CONCLUSIONS: An inflation procedure using diluted embedding medium can make lung tissue expand well during frozen section. Minute and even nonpalpable GGO lesions could be detected more easily by this technique in frozen sections, which would be helpful in determining how extensive a surgical procedure needs to be. Application of this procedure appears to improve the accuracy of FSD of minute precancerous pulmonary nodules.     

组织芯片简易制作方法及免疫组化有效性分析

 目的 探索一种组织芯片的简易制作方法,并对它的有效性进行分析。方法 在实验中探索出组织芯片的制作方法,收集乳腺癌病例124例,制作成直径1.6cm的组织芯片,进行雌激素受体（ER）、孕激素受体（PR）及c-erBb2受体免疫组化染色,同时与普通切片的免疫组化染色结果对比,并进行统计学分析。结果 组织芯片与普通切片的ER、PR及c-erBb2染色积分（0～7分）显著相关,ER、PR及c-erBb2的表达（阳性/阴性）一致率分别达94.35%、93.55%和91.94%。比较组织芯片与普通切片的ER、PR及c-erBb2的表达,两者之间无统计学差异（P〉0.05）,即组织芯片的结果与普通切片的结果是一致的。结论 自制1.6mm直径的组织芯片在进行免疫组化染色时能够代表普通切片,自制组织芯片的方法可靠有效。     

Frozen Section Diagnosis of Breast Cancer

In Savonlinna Central Hospital, Southeastern Finland, 371 frozen section examinations were made from September 1981 to December 1986 to detect breast cancer. There was one false positive diagnosis (0.7%) among the 147 lesions interpreted as carcinoma. Among the 224 biopsies, which were benign on frozen section investigation, four showed carcinoma in paraffin sections (1.8%). Thus, the diagnostic sensitivity of frozen section method was 97.3% (146/150) and the clinical diagnostic specificity 99.5% (220/221) when the diagnosis in the paraffin sections was used as reference. In the four false negative cases the tumour was small and limited to the breast without any evidence of metastases. Two of them were ductal carcinomas, one was microinvasive lobular carcinoma, and one intraductal non-invasive papillary carcinoma. The false positive case had benign intraductal papillomatosis. Our results suggest that the probability of a false diagnosis in frozen section examination increases with diminishing size of the lesion. We suggest that small lesions (less than 1 cm in diameter, or non-palpable) should not be subjected to frozen section examination to avoid unnecessary loss of neoplastic tissue during the preparation process. Instead surgically radical lumpectomy and careful investigation of paraffin-embedded tissue are recommended.     

Intraoperative histologic assessment of surgical margins and lymph node metastasis in breast-conserving surgery

The diagnostic value of intraoperative histologic examination of frozen sections of surgical margins and axillary lymph nodes (AX) was investigated in 95 patients with breast cancer who underwent breast-conserving surgery. The periphery of the excised breast tissue was peeled like an orange and examined histologically by frozen section. The results were compared with examination by permanent section. Evaluation of surgical margins by frozen section resulted in a diagnostic accuracy of 87%, a sensitivity of 96%, and a specificity of 84%. Enlarged or hardened AXs were sampled from the axillary pad which was derived from a complete AX dissection. Histologic examination using frozen section was performed during surgery. After the operation, the remaining AXs were removed from the axillary pad by hand dissection and histologically examined on permanent section. A diagnostic accuracy of 97%, a sensitivity of 77%, and a specificity of 100% were achieved in the diagnosis of AX involvement on frozen section. It was therefore concluded that intraoperative histologic examination of frozen sections may be useful in the determination of involvement of the surgical margins and the AXs in patients with breast cancer. © 1995 Wiley-Liss, Inc.     

Histopathology of ventricles, coronary arteries and mast cell accumulation in transverse and longitudinal sections of the rat heart after irradiation

The aim of the study was to compare cross sections with longitudinal sections in histopathological examination of the rat heart after irradiation, to find the most optimal method for the detection of cardiac radiation injuries. For this purpose, rats were irradiated locally on the heart with a single dose of 0 or 20 Gy. At different time points after irradiation, hearts were perfused and cut into longitudinal or cross sections. In both sections, several histopathological changes were scored on a graded scale between 0 and 3. Mast cells, which are thought to play a role in tissue remodelling, were counted. After 20 Gy, frequently occurring lesions were most severe in the upper half of the ventricles and the septum. These lesions could only be detected when using longitudinal sections, resulting in a higher total histopathological score than the examination of a single cross section. From 3 months onwards, changes in coronary arteries of irradiated hearts included endothelial cell loss, a loss of smooth muscle cells and fibrosis in media and adventitia. Up to 1 month after irradiation, mast cell densities of the left and right ventricles were decreased after 15 and 20 Gy, compared to time-matched controls, followed by increases from 3 months onwards. In the left ventricle, mast cell densities correlated with myocardial degeneration and fibre loss. The results of this study show that the usage of a single longitudinal section in the histopathological examination of the irradiated rat heart leads to the recognition of more severe injuries, including myocardial degeneration and fibrosis, in ventricular tissue than the usage of a single midventricular cross section. Morphological changes observed in coronary arteries of irradiated hearts might lead to a decreased compliance of the coronary artery wall. Further investigation is needed to determine the role of mast cells in cardiac tissue remodelling after irradiation.     

免疫组化方法检测肿瘤标志在肺癌组织芯片中表达的可靠性研究

 目的　用免疫组化方法检测肺癌组织芯片肿瘤标志的可靠性和有效性进行了探讨。方法　建立含有 4 18例非小细胞肺癌标本的组织芯片 ,并随机选取了 5 0例与组织芯片标本相对应的全组织标本。用免疫组化方法研究并对比了Ki 6 7和p5 3在组织芯片和全组织标本中的表达情况及其符合程度和可靠性。结果　组织芯片和全组织切片中Ki 6 7和 p5 3表达的一致率分别为 98%和 96 %。非小细胞肺癌三位点组织芯片检测Ki 6 7和p5 3表达的可靠性好 ,Kappa值分别为0 .95和 0 .91。结论　三位点组织芯片可有效、可靠地检测肿瘤标志物在非小细胞肺癌中的表达。本研究表明组织芯片技术特别适用于大样本、回顾性的临床研究