Population-based study of DNA image cytometry as screening method for esophageal cancer

AIM: To explore the DNA image cytometry (DNA-ICM) technique as a primary screening method for esophageal squamous precancerous lesions.METHODS: This study was designed as a population-based screening study. A total of 582 local residents aged 40 years-69 years were recruited from Linzhou in Henan and Feicheng in Shandong. However, only 452 subjects had results of liquid-based cytology, DNA-ICM and pathology. The sensitivity and specificity of DNA-ICM were calculated and compared with liquid-based cytology in moderate dysplasia or worse.RESULTS: Sensitivities of DNA-ICM ranging from at least 1 to 4 aneuploid cells were 90.91%, 86.36%, 79.55% and 77.27%, respectively, which were better than that of liquid-based cytology (75%). Specificities of DNA-ICM were 70.83%, 84.07%, 92.65% and 96.81%, but the specificity of liquid-based cytology was 91.91%. The sensitivity and specificity of a combination of liquid-based cytology and DNA-ICM were 84.09% and 85.78%, respectively.CONCLUSION: It is possible to use DNA-ICM technique as a primary screening method for esophageal squamous precancerous lesions.     

DNA image cytometry in MDS bone marrow smears

The nuclear DNA content, defined as DNA index (DI) in blasts/promyelocytes (bla/pro), were determined on Feulgen-stained bone marrow smears from 39 patients with myelodysplastic syndromes (MDS) and eight control subjects by the use of image cytometry (ICM). The DI in patients was compared to that of corresponding normal cell types, and to cytogenetic data available in 32/39 patients. The mean DI in bla/pro of patients with MDS was significantly (P < 0.01) lower compared to corresponding cell types in control subjects. By ICM, a DNA aneuploidy in bla/pro was found in 67% of the MDS patients, and 59% expressed DNA hypodiploidy. By cytogenetics, an abnormal karyotype was found in 31%, and 6/9 MDS patients with a 'hypodiploid' abnormal karyotype showed DNA hypodiploidy. Of patients with a normal karyotype (69%), seven (32%) showed a normal, 12 (55%) a lower, and three (14%) a higher DI compared to controls. No difference between groups of MDS patients was found. DNA hypodiploidy is suggested to be a common feature in MDS without a relationship to cytogenetics.     

Flow cytometry of colorectal carcinoma with three-year follow-up

Flow cytometry DNA measurements were carried out on colorectal carcinomas from a series of 33 patients who have since been followed for three to four years. The tumors could be subclassified by this technique into 15 that had near-diploid DNA and 18 that were aneuploid. Prior reports had suggested that the near-diploid tumors carry a better prognosis than aneuploid tumors, but in this study the distribution by Dukes' stage and survival were the same in both groups. supported by NCI Grants No. CA-34134 and CA-31545.     

DNA cell-cycle analysis of cervical cancer by flow cytometry using simultaneous cytokeratin labelling for identification of tumour cells

DNA ploidy and cell-cycle distribution were determined by flow cytometry in fresh tumour tissue of 53 cervical carcinomas. Epithelial cells were labelled by a fluorescein-isothiocyanate-conjugated cytokeratin antibody (CK6, CK18) to study the influence of contaminating stromal and inflammatory cells on results of cell-cycle analysis of tumour cells. Without identification of cytokeratin-positive cells 30/53 (57%) tumours were found to be DNA-aneuploid compared to 43/53 (81%) after gating for cytokeratin. Only 7 of 15 DNA-multiploid tumours could be detected without cytokeratin staining. In addition, cytokeratin-negative cells, which are found in all tumours, can be used as an internal standard for the calculation of ploidy and for quality control (coefficient of variation, linearity) of each individual sample. Cell-cycle analysis revealed significantly higher S-phase and G₂M-phase fractions in cytokeratin-gated compared to ungated samples (13.1% versus 10.0% and 8.0% versus 5.4%;P<0.001). This difference was more pronounced in DNA-diploid than DNA-aneuploid tumours. In conclusion, about 30% of DNA-aneuploid tumours could only be detected after cytokeratin labelling of epithelial cells. Owing to the identification of cytokeratin-positive cells the influence of non-tumoural cell elements on cell-cycle analysis was reduced markedly. Therefore, in cervical cancer, cytokeratin labelling can optimize both the determination of DNA ploidy and cell-cycle analysis.     

Simultaneous analysis of cell surface antigens and DNA content by flow cytometry

Summary Cells obtained from the blood or bone marrow of patients with haematological malignancies can both be stained with fluorescent labelled monoclonal antibodies to determine an immunophenotype and permeabilized with 30% ethanol then stained with propidium iodide for simultaneous DNA analysis. In the technique described here, no evidence of selective depletion of the malignant cell population was demonstrated and decreases in the mean fluorescence intensity of the surface markers were insufficient to affect the sensitivity of flow cytometric analysis. The combined method is simple and robust enough to allow incorporation of DNA analysis into routine immunophenotyping of leukaemia and lymphoma cells.     

Prognostic significance of DNA cytometry in thymoma

 Purpose: The aim of this work was to evaluate the prognostic significance of DNA image cytometry in thymoma. Patients and methods: Image cytometric studies with an automatic video-based analysis system (LEYTAS) were carried out on 47 archival specimens from 36 patients with thymomas who underwent operation at a single institution from 1954 to 1992. The significance of aneuploidy DNA-content (5c-exceeding events), and nuclear size on stage and survival were evaluated. The median follow-up was 52.7 (6–164) months. Results: Masaoka's stage was predictive of aneuploidy (P < 0.01) and disease-free survival (P < 0.015). In stage I 18% of the tumors were aneuploid, in stage II 78%, in stage III 85% and in stage IV 100%. The occurrence of 5c-exceeding events was associated with both decreased disease-free survival (P < 0.01) and overall survival (P = 0.013). Nuclear size was not significantly correlated to stage. Under multivariate analysis, aneuploidy and DNA content failed to attain independent significance for stage, performance status, and histology. Conclusion: DNA image cytometry may provide additional information about the prognosis of resected thymoma. Received: 25 August 1999 / Accepted: 2 December 1999     

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL/AUL), were analysed for their cellular DNA und DNA/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB subtype, white blood cell count, lactate dehydrogenase, S-phase index and percentage of blasts in the bone marrow, was observed, cases with aneuploid DNA stemlines revealed a tendency towards longer remission duration. In ALL/AUL 21.8% of 280 patients expressed DNA aneuploidies, which were less frequently found in T-cell ALL (11.1%) as compared to common(C)-ALL (21.4%) or null-cell(null)-ALL (23.5%). DNA aneuploidy was not related with other clinically defined risk factors such as age, white blood cell count, and rapid achievement of remission. Patients with DNA indices <1.0, however, tended to have shorter remissions. A significant difference in RNA indices was observed between AML and ALL/AUL with median values of 14.4 and 10.1, respectively (P<0.05). These data indicate the usefulness of flow cytometric analyses of cellular DNA and RNA content for the characterization and classification of acute leukemias, complementing the identification of clinical risk factors, immuno-phenotyping and cytogenetics.Abbreviations AML acute myeloid leukemia - ALL acute lymphoblastic leukemia - AUL acute undifferentiated leukemia - T-ALL, C-ALL and Null-ALL T-cell, common and Null-cell ALL     

DNA ploidy study of resected hepatocellular carcinoma in cirrhotic liver

Background/Aims: Results of several studies on DNA ploidy as a prognostic indicator in hepatocellular carcinoma are contradictory. The present study analysed the correlations between DNA ploidy of resected hepatocellular carcinoma and tumour characteristics, tumour recurrence, risk factors and survival.Methods: Tumoural DNA ploidy of hepatocellular carcinomas from 37 patients with cirrhosis who underwent curative tumour resection was studied by flow cytometry.Results: A diploid pattern was found in 23 hepatocellular carcinomas (62.2%) and an aneuploid pattern in 14 (37.8%). The tumour recurrence rate did not differ statistically between diploid (69.6%) and aneuploid (50%) hepatocellular carcinomas. The only prognostic variable with significant difference in DNA pattern was the histologic tumour type; the majority of non-trabecular tumours were aneuploid while most trabecular hepatocellular carcinomas had a diploid DNA pattern. Actuarial survival at 1, 2, 3 and 4 years of patients with diploid and aneuploid tumours was 69.6%, 40.6%, 16.2% and 0%, and 69.3%, 59.4%, 49.5% and 32.9%, respectively (log rank p=0.1927).Conclusion: These results indicate that DNA ploidy has no prognostic value in hepatocellular carcinoma.     

流式细胞仪DNA倍体测定鉴别良恶性胸腔积液的诊断价值

目的通过流式细胞仪（FCM）技术测定胸水脱落细胞细胞核的脱氧核糖核酸（DNA）含量,并将此方法与癌胚抗原（CEA）相比较。方法病例选自2004～2006年5月,本院住院患者,留取新鲜的胸腔积液标本,用流式细胞仪检测细胞核DNA倍体,分析细胞的增殖能力。结果在恶性胸腔积液组中70％的标本可以检测到DNA异倍体,在良性胸腔积液中90％的标本表现为两倍体。结论流式细胞仪DNA倍体测定,与CEA联合测定可以提高恶性胸腔积液的检出率。     

The streaming liver V: Time and age-dependent changes of hepatocyte DNA content,following partial hepatectomy

Two processes contribute to the change in hepatocyte ploidy following partial hepatectomy: one is age-dependent, while the other depends on the time which has elapsed since hepatectomy. While in the normal liver hepatocyte DNA content increases with the age of the cell, following hepatectomy the average DNA content (or ploidy) for the entire population rises as well. Hepatocyte age was derived from the cell's distance from the portal tract. We have previously shown that hepatocytes are formed adjacent to the portal tract and stream toward the terminal hepatic vein, advancing 2 μm/day. This finding enables the estimation of hepatocyte age from its location, since the older a cell the more remote it is from the portal tract. Twenty-four young male random-bred rats were partially hepatectomized and killed in groups of four animals at the following times: 1 h and 1, 2, 3, 7 and 14 days. Liver sections and nuclear suspensions which were collected by fine needle aspiration were stained with Feulgen and measured with the aid of computerized image cytometry. Part of the suspension which was aspirated with a fine needle was stained with propidium iodide and measured by flow cytometry. Both nuclear area and the DNA content increase with the age of the cell. The older a cell, the higher its ploidy. Generally, young hepatocytes which synthesized DNA did also divide, while older cells tended to accumulate DNA and became polyploid. The average nuclear DNA content of the entire population increased by 14% in 3 days. Partial hepatectomy thus triggers cells to synthesize DNA and proliferate. Since not all cells which synthesize DNA divide, it is proposed to distinguish between two triggers, one initiating DNA synthesis and the other, proliferation.     

流式细胞仪DNA倍体测定鉴别良恶性胸腔积液的诊断价值

目的通过流式细胞仪（FCM）技术测定胸水脱落细胞细胞核的脱氧核糖核酸（DNA）含量，并将此方法与传统的病理学方法，肿瘤标记物癌胚抗原（CEA）的检测相比较，以鉴别胸水的良恶、性。方法病例选自2003-03～2004-03苏州大学附属第二医院呼吸内科的住院患者，留取新鲜的胸腔积液标本，以肝素抗凝，荧光染料染色后，用流式细胞仪检测细胞核DNA倍体，分析细胞的增殖能力。结果根据临床检查将胸水分为良性和恶性两组，在恶性胸腔积液组中，87．5％的标本可以检测到DNA异倍体，在良性胸腔积液中，90％的标本表现为两倍体。结论流式细胞仪DNA倍体测定使得病理诊断有了可依据的客观指标，是病理学检查的有益补充，与CEA联合测定可以提高恶性胸腔积液的检出率。     

Measurement of feto-maternal haemorrhage: a comparative study of three Kleihauer techniques and two flow cytometry methods

In the UK a Kleihauer test is routinely performed on all RhD-negative women after the birth of an RhD-positive child to ensure that an adequate dose of anti-D immunoglobulin is given. The results of Kleihauer testing are interpreted to assess the volume of any feto-maternal haemorrhage and additional anti-D immunoglobulin is administered if necessary. A similar procedure is followed ante-natally when incidents occur known to be associated with alloimmunization. The performance of Kleihauer tests is difficult to standardize and there can be problems in interpreting the volume of feto-maternal haemorrhage. The use of flow cytometry to measure feto-maternal haemorrhage is reported to give more accurate and reliable results. This study compared three different Kleihauer methods and two different flow cytometry techniques all performed using the same maternal sample and within a single laboratory. The results demonstrated variability between the Kleihauer tests used and also in the flow cytometry measurements. A well-performed Kleihauer test would still appear to be useful as a screening technique for detection of feto-maternal haemorrhage. However, accurate quantitation of size of feto-maternal haemorrhage is more reliably determined by flow cytometry.     

Alteration of DNA ploidy and cell nuclearity in human hepatocellular carcinoma associated with HBV infection

Background/Aims: Hepatocellular carcinoma usually arises in cirrhotic livers as a complication of chronic liver disease, and may show a variable trend towards increasing ploidy. The aim of this study was to investigate possible associations between different etiological factors, particularly hepatitis B virus and hepatitis C virus infection, and alteration of DNA-ploidy and nuclearity of neoplastic hepatocytes.Methods: DNA-ploidy, the percentage of binucleated cells in the total cell population and the fraction of mononucleated hepatocytes in the polyploid compartment were assessed by image cytometry on cellular suspensions obtained by fine-needle biopsy from 60 hepatocellular carcinomas in patients whose viral status had previously been assessed.Results: Significantly higher DNA-ploidy values (p=0.005), with a reduction in the percentage of binucleated hepatocytes (p=0.003) and an increase in the fraction of mononucleated hepatocytes in the polyploid compartment (p<0.0001), were found in hepatocellular carcinoma with actual or previous hepatitis B virus infection (including also HCV+ve patients) in comparison to those not associated with hepatitis B virus infection, but not when HCV+ve hepatocellular carcinomas were compared to HCV−ve ones. Statistically significant differences for ploidy values (p<0.05), percentage of binucleated hepatocytes (p<0.05) and fraction of mononucleated hepatocytes in the polyploid compartment (p=0.003) were also found between hepatocellular carcinoma associated only to hepatitis B virus infection (“pure” hepatitis B virus cases) and those associated only to hepatitis C virus infection (“pure” hepatitis C virus cases).Conclusions: Hepatocellular carcinoma associated with a previous or actual hepatitis B virus infection shows a peculiar phenotypical appearance, characterized by a trend towards increasing ploidy and reduction of binuclearity.     

胃癌组织甲基绿染色DNA/RNA图像分析

目的研究甲基绿＿派洛宁（ＭＧ＿Ｐ）染色法进行胃癌细胞的ＤＮＡ／ＲＮＡ双参数图像分析的可行性。方法选择２６例已用Ｆｅｕｌｇｅｎ染色法检测过ＤＮＡ倍体类型的胃癌石蜡组织，采用同一蜡块的连续切片进行ＭＧ＿Ｐ染色。提纯甲基绿并经反复摸索，找到了ＭＧ＿Ｐ染色的理想条件和最佳染色效果。细胞的染色质呈蓝绿色，核仁和胞浆呈红色，两种颜色对比明显；其他细胞结构不着色。经ＤＮＡ酶处理后染色质不着色，ＲＮＡ酶处理后核仁及胞浆不着色。用图像分析法同时测定胃癌细胞的ＤＮＡ／ＲＮＡ含量，并进行ＤＮＡ倍体分析。结果２６例胃癌患者Ｆｅｕｇｅｎ染色的异倍体率为１５／２６（５７８％），而ＭＧ＿Ｐ染色为１７／２６（６５４％），两者符合率为８８２％。采用退色的方法测定相同组织细胞的ＤＮＡ含量，Ｆｅｕｌｇｅｎ染色与ＭＧ＿Ｐ染色的相关系数的０９９。ＲＮＡ含量在肿瘤细胞中明显高于正常胃粘膜细胞。结论ＭＧ＿Ｐ染色法是可靠的同时测定肿瘤细胞ＤＮＡ／ＲＮＡ含量的方法。     

Priming study of human phagocytes oxidative burst by using flow cytometry

In response to a variety of stimuli, eg pathogens, phagocytes release reactive oxygen species which are essential for bacterial killing and also potentiate inflammatory reactions. We have used flow cytometry measurements to study the priming process of phagocyte oxidative burst in whole blood, in order to avoid introducing artefacts due to the purification process and to simulate the in vivo situation more closely. In these conditions, we examined the in vitro effects of proinflammatory cytokines (TNF alpha, IL-1 alpha, IL-1 beta, IL-6, IL-8 and GM-CSF) on the PMN oxidative burst. We found that none of the cytokine tested directly activated the PMN oxidative burst. In contrast, TNF, GM-CSF and IL-8 strongly primed PMN in HIV-infected patients. This impairment, which correlated with the clinical stage of the disease, could contribute to the increased susceptibility to bacterial infections in HIV-infected patients. In addition, we reported the case of a child with severe recurrent infections due to intracellular microorganisms which could be related to an impairment of the phagocyte priming process of the oxidative burst.     

Comparison of acute myeloid leukemia occurring de novo or preceded by a myelodysplastic stage: Differences in cellular DNA content

The DNA content of bone marrow cells in patients with acute leukemia preceded by a myelodysplastic stage (MDS-AML) was compared to that in patients with de novo AML. We studied granulocytes, lymphocytes, monocytes, and blasts/promyelocytes from Feul-gen-stained bone marrow smears of 11 patients with de novo AML, ten patients with MDS-AML, and 13 apparently healthy controls. The mean amount of DNA per cell (DNA index; Dl) in each cell population was determined using a digital video-based imageanalyzing system (CAS-100). Analysis of variance (F test) showed a significant difference in the DNA content between de novo AML on one hand and MDS-AML and controls on the other as regards to blasts/promyelocytes (P < 0.01), lymphocytes (P < 0.05), and monocytes (P < 0.01), respectively. In three of 11 (27%) patients with de novo AML, a lower than normal limit Dl was found both in immature and mature bone marrow cells. Patients with MDS-AML had those of Dl values similar to normal controls. In consequence, a significantly reduced mean Dl was found in patients with de novo AML in blasts/promyelocytes (P < 0.01), and monocytes (P < 0.05) compared to both normal controls and MDS-AML. Together with data published separately, suggesting differences in granulocyte morphology, clonality, and HLA-DR expression, these data suggest biological differences between the two diseases. © 1993 Wiley-Liss, Inc.     

甲状腺肿与甲状腺癌DNA倍体差异性研究

目的研究甲状腺肿与甲状腺癌DNA倍体差异的临床意义。方法采用流式细胞术对34例甲状腺肿(结节性甲状腺肿22例,弥漫性甲状腺肿12例)及29例甲状腺癌(分化型甲状腺癌20例、未分化型甲状腺癌9例)进行DNA倍体分析及细胞周期检测。结果甲状腺癌组(分化型、未分化型)DNA异倍体率明显高于甲状腺肿组(结节性、弥漫性)(P<0.05),甲状腺癌组(分化型、未分化型)DNA整倍体细胞S期百分率也明显高于甲状腺肿组(结节性、弥漫性)(P<0.05),未分化型与分化型甲状腺癌的DNA异倍体率之间无差异(P>0.05),未分化型与分化型甲状腺癌的DNA整倍体细胞S期百分率有差异(P<0.05)。结论DNA倍体分析及细胞周期检测有助于甲状腺癌的早期诊断。     

食管鳞癌组织中线粒体和细胞核DNA的提取和鉴定

目的:研究在同一组织中同时提取线粒体DNA(mtDNA)和核DNA(nDNA)的方法的有效性.方法:同一组织中既含有mtDNA又含有nDNA,利用试剂盒同时提取出mtDNA和nDNA,用琼脂糖凝胶电泳,聚合酶链反应(PCR)的方法,对所提取的mtDNA和nDNA进检测.结果:用同一组织从线粒体中得到mtDNA,从细胞核中得到nDNA.与用传统方法分别提取mtDNA和nDNA效果一样,并且两者相关性强,相比较更有说服力.结论:同一组织中能同时提取mtDNA和nDNA,既节省组织又节省时间和费用,为DNA的研究提供了较好的实验方法.     

结直肠癌组织中人乳头瘤病毒DNA的研究

目的研究１６、１８型人乳头瘤病毒（ＨＰＶ）是否与结直肠癌的发生有关．方法结直肠粘膜活检组织１２３例，其中结直肠癌３５例，乳头状腺瘤１７例，炎性息肉１１例，结肠炎３０例，以及正常结肠粘膜３０例，用对各型ＨＰＶ高度保守的通用引物和１６、１８型特异性引物作聚合酶链反应（ＰＣＲ）检测ＨＰＶＤＮＡ．结果ＨＰＶＤＮＡ总检出率１４６％，正常粘膜，结肠炎和炎性息肉组为３３％（２／７１），乳头状腺瘤为１７６％（３／１７），结直肠腺癌为３７１％（１３／３５）．在正常粘膜，结肠炎和炎性息肉组未发现１６、１８型ＨＰＶＤＮＡ，在乳头状腺瘤组有３例为１８型ＨＰＶＤＮＡ阳性，结直肠腺癌组１３例ＨＰＶ阳性病例中有３例为１６型，９例为１８型感染；在正常组织、癌旁组织和癌组织中ＨＰＶＤＮＡ检出率依次增高．ＨＰＶ在直肠、左半结肠，右半结肠感染率依次为２８６％，１４％，２６％．结论结肠癌的发生与ＨＰＶ１６、１８型有关，腺癌以１８型感染为主．ＨＰＶＤＮＡ检出率在右半结肠，左半结肠和直肠依次增高．