肝细胞癌DNA干系倍体分析及其临床意义

目的:测量与分析肝细胞癌DNA干系倍体及其临床意义。方法:使用TIGER细胞图像分析仪测量 4 5例肝细胞癌组织 4 μm、10 μm切片上DNA干系倍体值。4 μm组织切片测量肝细胞癌细胞核DNA的光密度,10 μm组织切片测量单个完整肝细胞癌细胞核的体积,经TIGER细胞图像分析仪计算获得以单个完整肝细胞癌细胞核体积为单位的DNA总量(以体积积分光密度表述),以同一切片内正常淋巴细胞作为内对照,计算其DNA干系倍体值。结果:⑴无 1例DNA干系倍体为二倍体;DNA干系倍体值在 2～ 5范围者 11例;在 5～ 8范围者 2 8例;大于 8者 6例。⑵DNA干系倍体与瘤体大小、有无淋巴结转移、组织学分级、术后生存率等有相关性。结论:DNA干系倍体能较准确反映肝细胞癌的生物学特性,为认识肝细胞癌的病理学特征以及判断预后提供了有价值的客观依据。     

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。     

组织原位分析鼻咽癌细胞核DNA干系倍体及其异质性

组织原位分析鼻咽癌细胞核的DNA干系倍体及其异质性。收集42例鼻咽癌病例的归档蜡块,4μm、8μm连续组织学切片各一张。每个病例选取三个癌巢或肿瘤组织区域(共126个样本),使用TIGER细胞图像分析仪4μm组织切片测量鼻咽癌细胞核DNA的光密度等参数,8μm组织切片测量单个完整鼻咽癌细胞核的体积等参数,计算获得每个癌巢或肿瘤组织区域的以单个完整鼻咽癌细胞核的体积为单位的DNA含量(以体积积分光密度表述),以同一切片内正常鼻咽部上皮非基底细胞作为其内参照,计算其DNA干系倍体。结果显示:鼻咽癌细胞核的DNA干系倍体主要集中在1.76～3.00(DNA指数为0.88～1.50)之间,14个样本为DNA干系四倍体,仅2个样本为DNA干系亚二倍体;DNA干系倍体异质性率为66.67%。可得结论:(1)鼻咽癌DNA干系倍体主要在近二倍体与近四倍体之间;(2)鼻咽癌中存在DNA干系倍体异质性。     

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。     

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。     

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。     

不同实验室小鼠肝细胞核DNA总量图像分析结果的比较研究

目的了解图像分析仪测量组织细胞化学物质含量或总量结果的国内现状,为制定用于细胞化学物质量测定的图像分析系统的生产及使用过程中应达到的统一的技术性能标准提供参考。方法正常成年小鼠肝细胞悬液涂片,部分涂片统一进行Feulgen染色,未染色涂片与已染色涂片各一张寄给六个实验室,进行Feulgen染色,测量两种染色涂片的单个完整肝细胞核DNA的量,结果送回本实验室,按统一标准比较单个完整肝细胞核DNA总量的直方图。结果 1.从统一和不同实验室染色涂片测得的DNA总量直方图形状较相似,说明细胞核DNA的Feulgen染色法具有较广泛的适应性;2.各实验室表述DNA量的术语极不规范;3.不同的图像分析仪测得不同投影面积大小肝细胞核的平均光密度或平均光度或平均灰度的值相差较大,而同一图像分析仪测得的值的差异较小;4.不同的图像分析仪和不同的测量参数区分三种DNA总量细胞核群体的能力存在明显的差异,说明这些图像分析仪测量细胞化学物质量的可靠性不同;5.改变图像分析仪关键硬件的设置,摄取相同细胞核的黑白图像和彩色图像,测量结果显示,细胞核DNA的彩色图像与黑白图像在不同的图像分析仪测量软件上可得到不同的测量结果。结论测量细胞化学物质的量,不但应重视图像分析系统测量软件的性能,而且其图像形成和采集硬件的系统性能与功能状态同样不可忽视,建立用于细胞化学物质量测定的图像分析系统性能的评价标准是非常必要的、有益的和刻不容缓的。     

组织切片对细胞核DNA含量检测的影响

目的探讨组织切片对图像分析仪测量细胞核DNA含量的影响。方法选取10只成年健康雄性小鼠,制备肝细胞涂片和肝组织切片,肝组织切片分成两部分,分别用于Feulgen染色和石蜡包埋,包埋后的组织采用垂直切片,图像分析仪测量切片实际厚度,依据切片机标识厚度和测量厚度分别分组,TIGER细胞图像分析仪分别测量肝细胞涂片和组织切片内肝细胞核的积分光密度(IOD)。结果肝细胞涂片内各DNA含量倍体肝细胞核IOD间的比值接近2和4,IOD之变异系数(CV值)<3.5,肝组织切片IOD间比值明显偏离2和4,IOD之CV值均>6;切片机标识厚度为4、6、8和10μm组织切片平均测量厚度分别为6.75、7.18、6.96和7.59μm,测量厚度最大值为9.25μm,最小值为4.62μm;依据切片机标识厚度分组中不同切片厚度相同DNA含量倍体肝细胞核的IOD值差异均无统计学意义;依据测量厚度重新分组后5、6微米组与7、8、9微米组IOD值间的差异具有统计学意义(P<0.05)。结论组织切片的实际厚度与切片机标识厚度间存在明显差异,本实验方法可较准确地判断组织切片厚度;厚组织切片测量结果优于薄组织切片,但与细胞涂片相比,厚组织切片仍难...     

组织原位三维测量和分析计算几种正常细胞核的的DNA含量

目的　探讨在肿瘤组织切片上确定二倍体参考细胞核的选择依据 ;方法　五只健康兔的七种正常组织 ,每种组织包埋在同一包埋块内切片 ,Feulgen染色 ,TIGER细胞图像分析仪测量和分析计算其中五种组织细胞核的以完整细胞个体体积为单位的DNA含量 ;结果　脊神经节细胞核和小脑浦肯野氏细胞核都未能染上颜色 ,其余五种正常组织着色呈紫红色 ,其细胞核的DNA含量值存在一定程度的差异 ( 5 5～ 13 6% ) ;结论　在进行肿瘤细胞核的DNA倍体和DNA恶性度等指标的分析时 ,最好选择与肿瘤组织同一个体和和同源的正常组织细胞核作为二倍体参考细胞 ,采用同样的样品制备和同步的染色方法 ,才可获得准确的分析结果。     

Changes in ploidy distributions in human liver carcinogenesis

Cellular and nuclear DNA content was measured by flow cytometry and the fraction of binucleated cells by fluorescence microscopy in normal adult human livers, hepatocellular carcinomas, cirrhotic livers surrounding tumors, and in some benign liver conditions. In five normal livers about one-half of the hepatocytes were polyploid; the majority of these were binucleated tetraploids containing two diploid nuclei. Thus, polyploidization in human liver does not progress as far as, for example, in the rat, where 80%-90% of adult hepatocytes are polyploid, mostly with tetraploid or octoploid nuclei. In five human euploid hepatocellular carcinomas and one investigated case of focal nodular hyperplasia, the percentage of polyploid cells was significantly reduced. Four other carcinomas exhibited a prominent aneuploid (hypotetraploid) peak in addition to the diploid peak. An abnormally low fraction of binucleated cells was also indicated in these tumors. Liver tissue surrounding the tumors had a ploidy distribution similar to that of normal liver. The results suggest that, like in several models of experimental hepatocarcinogenesis, human hepatocellular tumor growth is associated with a decreased polyploidization tendency and a corresponding increase in diploid, divisional growth, which may give the tumors a growth advantage relative to the surrounding liver.     

Cytophotometric studies of the nuclear DNA content in cartilaginous tumors.

The histopathological differentiation between chondromas and low-grade malignant chondrosarcomas can be difficult. For this reason we studied in 37 different cartilaginous tumors the mitotic index and the Feulgen DNA content using a scanning-integration cytophotometric technique. In 23 chondromas the Feulgen DNA content was diploid and showed a unimodal normal distribution. The number of mitoses was 0--0, 5%. The nuclei of a chondroblastoma were also diploid and the Feulgen DNA content was normally distributed. The mitotic index was 1% and few tetraploid nuclei, which were probably G2 nuclei, were observed. In two chondromyxoid fibromas, the average Feulgen DNA content was diploid and normally distributed. Several tetraploid nuclei were noted. The mitotic index was respectively 0.25% and 1.75%. Recurrence was noted in the first case. The Feulgen DNA content and mitotic index were clearly different in the chondrosarcomas. The distribution of the DNA content was bimodal or unimodal in low-grade chondrosarcomas. The mitotic index was less than 3%. In high-grade malignant chondrosarcomas, the histograms were broad unimodal or aneuploid. The mitotic index was above 5%.     

染色质浓缩对细胞核DNA含量检测的影响

目的利用三种不同方法制备小鼠肝细胞涂片，探讨染色质浓缩对图像分析仪测量DNA含量的影响。方法本文选取10只成年健康雄性小鼠，采用传统涂片、液基制片法和甲醛固定后液基制片法制备小鼠肝细胞涂片。Feulgen染色。TIGER细胞图像分析仪分别测量三种涂片内肝细胞核的积分光密度、平均光密度和面积。结果三种涂片内肝细胞核分布均匀，轮廓清晰，呈紫红色。与传统涂片法相比。液基法内肝细胞核面积明显减小。类色质高度浓缩；相同倍体的肝细胞核在传统涂片中面积最大，平均光密度最低，各倍体间的比值最接近2和4，积分光密度CV值最低（〈3．5）。固定法和液基法平均光密度明显升高，各倍体间比值明显偏离2和4。积分光密度CV值〉6。结论不同制片方法可导致同一类型、处于相同功能状态的细胞核染色质浓缩程度产生明显差异，染色质浓缩可导致平均光密度值升高，积分光密度值降低，测量结果的精确性和准确性降低。     

Effect of tamoxifen upon cell DNA analysis by flow cytometry in primary carcinoma of the breast

The effect of tamoxifen upon cellular DNA ploidy in carcinoma of the breast was assessed by flow cytometry (FCM), in a prospective group of 77 patients with primary operable disease. Each had a needle biopsy at the outpatient visit for diagnosis and FCM analysis, and definitive surgery was performed a median of 8 days later. Forty received tamoxifen during this period - 40 mg qds loading dose for 24 h, followed by 20 mg daily until the day of operation: 37 patients received no therapy. The DNA histogram from the needle biopsy was compared with that obtained from the resected tumour for each individual. There was little change between the pair of histograms from tumours from the untreated patients. In those who had received tamoxifen the most consistent effect was a marked reduction in the magnitude of the 'tetraploid' peak in tetraploid or near-tetraploid tumours with DNA indices 1.8-2.0. There was little change in diploid or 'other DNA-aneuploid' tumours. In tetraploid tumours (DNA index of 2.0) the percentage of nuclei in the diploid S phase was significantly related to the percentage of nuclei in the diploid G2 + M/tetraploid G1 peak (P less than 0.003, unpaired t test). These data suggest that an effect of tamoxifen can be demonstrated by FCM upon tumours exhibiting a tetraploid or near-tetraploid DNA content. It is possible that tetraploid or near-tetraploid human mammary tumours may be a distinct group of endocrine responsive tumours within the overall group of aneuploid tumours, and that the majority are probably derived from the diploid population rather than being a true aneuploid population.     

两种染色方法在细胞核DNA含量检测中的应用及比较

目的 探讨改良、快速两种Feulgen染色方法达到最佳染色效果的水解时间段，为科研和临床的应用提供方法学指导。方法 选取5只成年健康雄性SD大鼠的肝组织，制成肝细胞涂片，分别在室温和60℃温度下水解不同时间，应用改良和快速两种方法染色，TIGER图像分析仪检测和分析单个肝细胞核的DNA含量。结果 （1）不同大鼠肝细胞涂片在同一水解温度、时间作用下，相同DNA倍体含量肝细胞的DNA含量大致相同，CV值均小于10％；（2）二、四、八倍体肝细胞核的DNA含量比值均接近2或4；（3）同一大鼠相同水解温度不同水解时间，同一倍体的肝细胞核DNA含量存在差异：60℃水解温度下，IOD（5-7min）〉IOD（9-15min）〉IOD（1min,20min），室温水解温度下，IOD（50min）〉IOD（20-30min,70-90min）〉IOD（5-1min,100min）。结论 HCL水解肝细胞涂片时间过长或过短均不能理想的染色，快速法和改良法Feulgen染色达较佳染色效果的时间段分别为：快速法5—7min，改良法20-90min。     

Urothelial dysplasia and carcinoma in situ of the bladder

Urothelial cells were pepsin-extracted from paraffin-embedded specimens taken from human nontumorous bladder mucosa, dysplasia, and carcinoma in situ. After Feulgen staining for DNA, nuclei were measured with an integrating microdensitometer. The measurements show that normal urothelium consists mostly of diploid nuclei. Dysplasia means that there is a predominance of tetraploid DNA values, whereas carcinoma in situ is characterized by a high percentage of aneuploid cells. In both dysplasia and carcinoma in situ there is a considerable percentage of diploid nuclei. Thus, DNA cytophotometry can be used for standardization of preneoplastic and early stages of tumor development in bladder cancer.     

Reduced proliferative activity of polyploid cells in primary hepatocellular carcinoma.

The proliferative activity of tumor cells differing in DNA content (ploidy) and nuclearity was investigated in primary hepatocellular carcinomas of the rat by flow cytometric analysis of collagenase-isolated cells immunostained after labelling with bromodeoxyuridine (BrdU) in vivo. The diploid cell fraction in these euploid tumours was higher than in normal liver, and the rate of binucleation as well as the proliferative activity of the binuclear cells was very low. The highest proliferative activity (BrdU labelling index) was found among the diploid tumour cells. The activity in mononuclear tetraploid and octoploid cells was reduced in inverse proportion to their increasing DNA content, possibly suggesting a loss of proliferative potential associated with polyploidization. There was a significant correlation between the proliferative activity of hepatocellular carcinoma cells and nonparenchymal liver cells in the different tumours, indicating that different cell types within a tumour may respond to common growth stimuli. Treatment of tumour-bearing rats with a promoting carcinogen (2-acetylaminofluorene) resulted in significant stimulation of tumour cell proliferation (all ploidy classes), whereas the proliferation of non-parenchymal (stromal) cells in the tumour was slightly inhibited.     

Feulgen-DNA cytofluorometry of the liver cell carcinomas

Twenty-four autopsy cases of liver cell carcinomas were investigated by Feulgen-DNA cytofluorometry (NIKON SPM-RF 1-D) on paraffin-embedded tissue materials. The methods employed for tissue preparation and cytofluorometry were previously reported by us. The ploidy pattern based on cytofluorometry of the liver cell carcinomas exhibited wide DNA content distributions, in which many S phase cells having intermediate DNA content values ranging from diploid level to various, higher ploidy levels were included. From the comparative studies on both the histopathological findings and the ploidy pattern of the liver cell carcinomas, it was indicated that the peak value of the DNA content distribution was shifted from the diploid to the tetraploid level with the increasing appearance of the polyploid cells, and also that the degree of polyploidization appeared to correspond to the extent of the histological malignancy.