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。     

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。     

成人正常肝和肝细胞癌DNA干系倍体的组织原位分析

目的分析成人正常肝和肝细胞癌DNA干系倍体及细胞核形态学参数的变化。方法：选取15例成人正常肝和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TIGER 920G1细胞图像分析仪测量DNA干系倍体值及细胞核形态学参数。结果（1）成人正常肝DNA干系倍体主要以二倍体（73%）为主。（2）肝细胞癌的DNA指数在0.86~9.32之间。45例肝细胞癌病例中DNA干系倍体异质性率为77.8%,其中,34例（75.6%）为DNA干系非整倍体肿瘤,11例（24.4%）为近四/八/十六倍体肿瘤,无1例为DNA干系二倍体肿瘤。结论细胞核DNA干系倍体和细胞核形态学参数分析可作为鉴别人正常肝和肝细胞癌的参考指标。     

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。     

乳腺癌细胞核体积的定量研究

目的: 探讨乳腺癌组织中细胞核体积与相关临床病理特征之间的关系; 方法: 收集55例乳腺癌标本的归档蜡块, 作连续切片, 8μm切片1张和相邻4μm切片4张, 行Feulgen染色;用细胞图像分析仪测定每例标本8μm切片的细胞核体积, 并比较与相关临床病理特征、雌孕激素受体及其DNA含量的关系; 结果: 8μm切片所测得的体积比4μm薄切片所测得的体积要大; 不同病理分型、不同组织学分级、不同临床分期、不同淋巴结转移状态、以及不同ER表达状态的样本细胞核体积有显著性差异; 165个样本的细胞核体积与DNA指数呈正的直线相关关系。结论: 8μm切片可以获得较为完整的细胞核体积; 细胞核体积可以作为判断肿瘤恶性度的较为客观的指标。     

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。     

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。     

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...     

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。     

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.     

DNA ploidy in primary testicular cancer

The DNA stemline ploidy was measured by flow cytometry (FCM) in 129 samples from paraffin-embedded primary testicular tumours (61 seminomas, 68 non-seminomas). Only one DNA stemline was found in 38 seminomas and 44 non-seminomas. Two seminomas and one non-seminoma were DNA diploid, the other tumours being non-diploid. Twenty-three seminomas and 24 non-seminomas displayed two or three DNA stemlines. The median minimal DNA index (DI) of all seminomas was significantly higher than that of all non-seminomas (1.58 vs 1.43; P: 0.008). Three seminomas removed from two monozygotic twins within 1 week had DIs of 1.66, 1.56 and 1.59. In this limited series there was no association between DNA ploidy of the primary tumour and the metastatic status for either seminomas or non-seminomas. The results support the pathogenetic model stating that at least some (if not all) non-seminomas develop from a seminoma by additional chromosomal aberration. The clinical relevance of DNA stemline ploidy has to be further evaluated in larger series.     

Prognostic value of DNA ploidy and S-phase fraction in relation to estrogen receptor content and clinicopathological variables in primary breast cancer

Tumors from 472 women with primary breast cancer were analyzed by flow cytometry. Divided into four categories, DNA ploidy showed significant association with disease recurrence and mortality. When allowance was made for its correlation with nodal status and estrogen receptor (ER) content, DNA ploidy did not add prognostic information. S-phase fraction was estimated in 290 DNA histograms. In contrast, it was significantly related to recurrence and mortality when controlling for nodal status, tumor size and ER content. When the follow-up was divided into two periods DNA ploidy and S-phase fraction showed association with disease recurrence in the first period only (<2.5 years), while the association with mortality was valid for both periods. Light scatter was measured in 234 samples. A low light scatter variability for the stemline nuclei was related to a high recurrence rate during the early follow-up period. In conclusion, DNA flow cytometry adds prognostic information concerning breast cancer patients.     

肿瘤细胞DNA干系倍体分析及其临床应用

DNA非整倍体是恶性肿瘤的特征性标志之一。测量和分析细胞核DNA含量与倍体对恶性肿瘤的病理诊断、恶性程度判定、疗效估价、预测预后具有重要价值。其主要测定方法有流式细胞术和细胞图像光度术。本文主要综述了肿瘤细胞DNA干系倍体分析及其临床应用的现状以及流式细胞术和细胞图像光度术两种检测方法的优缺点和相应的改进方法,希望对其应用于临床以提高诊断的准确性和预测肿瘤的发展起到积极的作用。     

Heterogeneity of colorectal adenocarcinomas evaluated by flow cytometry and histopathology

Flow cytometry and histopathology were utilised in evaluating 50 primary and 16 metastatic colorectal carcinomas to determine the influence of heterogeneity and proportion of dying cells on pathological assessments. A new procedure was developed for staining unfixed whole cells with acridine orange and ethidium bromide to quantify DNA and RNA content and number of dead and dying cells. Attempts were made to reduce interobserver variation in histological assessment and to determine whether flow cytometry could refine current grading and staging procedures. Interobserver variation in grading was not improved by estimating proportions of differing grades in multiple samples from individual tumours. Considerable heterogeneity was observed within tumours although this was less apparent when defining ploidy status than histological grade. No consistent differences were observed between superficial and deep parts of tumours or between primary and secondary tumours by either method of analysis. The proportion of dead and dying cells varied widely between tumours but there was no correlation with tumour grade or stage. Non-diploid tumours were not of more advanced stage or poorer histological grade than diploid tumours. Since ploidy status may be an important prognostic factor, analysis of colorectal carcinomas by flow cytometry could be of greater value than conventional grading and staging procedures.     

Regional DNA content heterogeneity in colonic adenocarcinoma: prognostic significance in patients with liver metastases.

We retrospectively examined by flow cytometry the DNA ploidy pattern in tissue blocks from 25 primary colon adenocarcinomas and their lymph node and liver metastases. Intratumoral heterogeneity was present in 22% of primary tumors and 21% of metastatic liver deposits. Intertumoral heterogeneity, measured between the primary tumor and its lymph node and liver metastases, was 0% and 20%, respectively. Of 24 patients who underwent successful resection of their liver metastases, 8 neoplasms had uniformly diploid DNA content, while 16 tumors had aneuploid DNA pattern in either the primary tumor, the metastases, or both. Five-year survival was better in the diploid group (38% vs. 7%, P = 0.10 by log rank analysis). Three of eight patients in the diploid group remain free of disease, while all 16 patients with aneuploid cell populations have died of recurrent disease.     

Common acute lymphoblastic leukemia characterized by four different DNA stemlines with heterogeneity in RNA content, antigen expression and sensitivity to chemotherapy

Four subpopulations of cells with different DNA content were present in the bone marrow of a pediatric patient with acute lymphoblastic leukemia. Flow cytometry of DNA/RNA and DNA/surface antigen expression enabled the identification and characterization of diploid, hypertriploid, tetraploid and hypertetraploid leukemic cells. This was not appreciated by cytogenetic analysis, which identified only some tetraploid cells (3/20 metaphases). Common acute lymphoblastic leukemia antigen was expressed in all aneuploid and also on 17% of diploid cells. Quantitative CALLA expression was unrelated to ploidy and cell size. Cellular RNA content paralleled ploidy, i.e. the more aneuploid cells had increased RNA content and there was pronounced RNA heterogeneity within each DNA stemline. The different subclones showed almost identical stages of early B-cell differentiation. The early pre B-cell antigens BL1 and BL2 were expressed in approx. 80 and 60%, respectively, of aneuploid leukemic cells. Cytoplasmic immunoglobulin heavy chain was also present. Clonal excess of lambda light chain immunoglobulin on the cell surface was present on less than 10% of hypertriploid, tetraploid and hypertetraploid leukemic cells indicating differentiation of a subpopulation of aneuploid leukemic cells to mature B cells. This was not seen for any diploid cells. The heterogeneity of the different subpopulations was also evident in the differences in response to chemotherapy: the hypertriploid and hypertetraploid subpopulations were most sensitive to initial induction chemotherapy.     

Prognostic implications of the ploidy pattern of the nuclear DNA in hepatocellular carcinomas

The nuclear DNA contents of paraffin-embedded specimens of 41 cases of a hepatocellular carcinoma have been measured by means of flow cytometry. Results have indicated that 25 cases (61%) were diploid and 16 cases (39%) were aneuploid. In the aneuploid cases, the serum AFP level was found to be higher and stage more advanced. We also found that patients with aneuploid tumors had a poorer prognosis than those with diploid tumors, this fact uncovered by means of a Cox's proportional hazard model. In conclusion, the ploidy pattern of the nuclear DNA may serve as a useful prognostic marker for a hepatocellular carcinoma.     

A case of pulmonary carcinoma presenting flow cytometrical heterogeneity of the nuclear DNA content between its primary focus and the metastatic foci

We have experienced case involving a 63-year-old patient with a pulmonary carcinoma, who was given an enterectomy following a lobectomy, due to minimal intestinal metastasis. In this case, using flow cytometry, the cancer cell nuclear DNA content was analyzed for the primary tumor focus, the mediastinal lymph node metastatic focus, and the small intestinal metastatic focus. For the primary focus, a cancer cellular population of polyploidy with 2 ploidies of DNA content was observed, while for both the metastatic foci, only a single cancer cellular population was observed, indicating the heterogeneity of the nuclear DNA content between the primary focus and metastatic foci. These 2 metastatic foci had DNA contents completely corresponding to that for a ploidy with a high DNA content in the primary focus, suggesting a metastasis of only the above population from the primary focus. The present case apparently formed metastatic foci in other organs than the lungs but only by cancer cells more susceptible to metastasis among the cancer cells found in the primary focus.     

Intratumoral heterogeneity of DNA content in renal cell carcinoma and its prognostic significance.

BACKGROUND: A multiple sampling study was performed on 124 specimens of renal cell carcinomas to analyze the consistency and reliability of DNA measurements. The authors investigated intratumoral DNA heterogeneity and its role as a adverse prognostic factor for disease progression. METHODS: DNA content was analyzed by flow cytometry on three different samples of the same tumor. The Cronbach alpha coefficient was used to assess the reliability and a Cox proportional hazards model was used to test the effect of DNA ploidy heterogeneity on time of disease progression. RESULTS: The agreement among the DNA ploidy samples was high. The number of aneuploid findings increased significantly with the number of samples analyzed. The presence of non-diploid cell populations was a significant adverse predictive value for disease progression. However, the authors were unable to demonstrate that intratumoral heterogeneity DNA content had any influence on the biological behavior of the tumor. CONCLUSIONS: Determination of DNA ploidy based on single samples may be inaccurate. Spatial variation in DNA ploidy is a feature of renal cell carcinoma; however, its biologic significance remains to be demonstrated.