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。     

Flow DNA analysis of primary bone tumors. Relationship between cellular DNA content and histopathologic classification

The cellular DNA content of 15 benign and 34 malignant primary bone tumors was analyzed by means of flow cytophotometry. All benign tumors except one of questionable histologic type exhibited a normal DNA content (diploid), whereas 23 of 34 malignant tumors showed an abnormal DNA content (aneuploid). Closer analysis revealed that all supposedly highly malignant tumors, i.e., 16 osteosarcomas and 1 Ewing sarcoma were aneuploid, while 8 of 13 chondrosarcomas, 2 periosteal osteosarcomas, and 1 of 2 adamantinomas were diploid. Interestingly, these diploid malignant tumors represent tumor entities which are known to include variants of low-grade malignancy. Cell distribution analysis showed that the aneuploid tumors exhibited a higher proportion of S-phase and G2 + M cells than the diploid tumors, indicating differences in proliferative activity. However, no significant difference in this respect could be demonstrated between diploid benign and diploid malignant tumors. The current study clearly shows that flow DNA cytophotometry can be applied to most primary bone tumors despite a substantial content of hard tissue. The results also indicate that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy. Thus, regardless of histogenetic origin, it appears that benign bone tumors as well as malignant bone tumors of low-grade malignancy in general, are diploid, whereas highly malignant bone tumors in general are aneuploid.     

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.     

DNA aneuploidy and cell proliferation in breast tumors

The cellular DNA content of 30 benign and 180 malignant breast tumors was analyzed by means of flow cytometry (FCM). All benign tumors exhibited a normal DNA content (diploid), whereas 65% of the malignant tumors showed an abnormal DNA content (aneuploid). The ploidy distribution of malignant tumors was bimodal with an increasing frequency near diploid DNA index (DI), and a second group had a DI ranging from triploid to tetraploid. In estimating the degree of malignancy eight independent histomorphologic and cytologic criteria were introduced. A good correlation was observed between DNA content abnormalities and the grade of differentiation of breast carcinomas. The percentage of S-phase cells of DNA aneuploid cell lines was significantly higher than in the diploid ones. The highly differentiated breast carcinomas (Grade 1) indicated lower S-phase values as compared to the undifferentiated (Grade 3) ones. S-phase values estimated by FCM were about two times higher than the 3H-thymidine labeling index (LI) obtained by an in vitro procedure. The data estimated in this study showed that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy and prognosis of patients with breast carcinoma.     

Flow cytometry and Feulgen cytophotometry in evaluation of effusions

Fifty-eight effusions (42 pleural and 16 ascitic fluids) from patients with and without cancer were analyzed by conventional cytology and the results compared with DNA patterns generated by flow cytometry of 10(4) nuclei and several modes of Feulgen cytophotometry. In 31 patients (24 without evidence of cancer and seven with history of cancer and cytologically negative fluids), the fluids were diploid by flow cytometry. One fluid with atypical cells from a lymphoma suspect was also diploid. Flow cytometry of 26 cytologically cancerous fluids disclosed aneuploid DNA patterns in 16 and diploid patterns in ten. Feulgen cytophotometry of 11 of these fluids (three aneuploid, eight diploid) was performed on nuclear preparations identical to those used in flow cytometry and on restrained smears used for visual analysis. The analysis was performed in two modes: as a study of 500 sequential nuclei in an automated system, mimicking flow cytometry, and visually selected large, presumably malignant nuclei. In nine of the 11 cases, the DNA content of visually selected cancer cells was aneuploid, even though this DNA pattern was not evident in the analysis of 500 sequential cells. In two cases, both diploid by flow cytometry, the Feulgen analysis confirmed the presence of cancer cells in the diploid range. In samples of 10(4) nuclei representing a mixed population of cells occurring in effusions, the presence of aneuploid cancer cells may not be disclosed by conventional flow cytometry. A larger sample of cells, a detailed analysis of DNA histograms, and perhaps sorting of select cells in the hypertetraploid range, may prove essential before flow cytometry can be accepted as a diagnostic tool in the laboratory in the assessment of effusions.     

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 content in dysplasias and early invasive forms of cervical cancer

Cytological preparations of normal cervical epithelium and in different forms of the epithelial dysplasia and in early cervical cancer were studied in 8 healthy females and in 35 patients. To determine the content of DNA in these cells a fluorescent variant of the Feulgen reaction was employed, using the national drug rivanol as fluorochromium in the Shiff reagent. The amount of DNA in cell nuclei of the normal epithelium and in moderate dysplasia is shown to correspond to diploid, while in marked dysplasia the content of DNA varies from di-to tetraploid values. But there are some cases when in marked dysplasia the DNA content in cells is diploid, i. e. it is characteristic of moderate dysplasia, or considerably exceeds tetraploid level, and it is in such cases that histological assays reveal early forms of cancer. Invasive cancer is charcterized by a sharp increase in the DNA content in most cells and by a considerable number of polyploid cells. Fluorescence-microscopy may serve as a valuable diagnostic adjunct to recognize early forms of cervical cancer and be used clinically.     

Nuclear DNA in intestinal carcinoid tumors. A study before and after cytotoxin (streptozotocin and 5-fluorouracil) treatment

Imprint cytology specimens of metastases of intestinal carcinoids obtained by percutaneous biopsy were analysed cytofluorometrically with regard to nuclear DNA records. All untreated tumors (nine cases) exhibited diploid DNA values with a relatively low proliferative activity (less than 2% nuclei in S-phase region). The mean number of tetraploid cells was 5%. Cytofluorometry also was performed on five tumor metastases treated with the cytotoxin streptozotocin and 5-fluorouracil. After treatment, an increase in the number of tetraploid cells (mean value, 30%) was noted, indicating that the cytotoxin treatment (possibly streptozotocin) on the tumor cells in vivo blocked progression from G2 to M phase. The current cytofluorometric analyses show that diploid nuclear DNA records and a low proliferative activity is a characteristic of malignant carcinoid tumors of the intestine. Due to regular DNA histograms in the carcinoid tumors, it is suggested that reliable studies are permitted of the effect of cytotoxins on the different phases of the cell cycle in vivo.     

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。     

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。     

Ploidy and proliferative activity of human brain tumors. A flow cytofluorometric study

Ploidy and proliferative characteristics were estimated by flow cytometry of the nuclear DNA content of 92 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and single cell suspensions were obtained with a mechanical dissociation technique. Propidium iodide was employed for nuclear DNA staining. Human normal brain tissue was used as internal diploid reference standard. 86% of benign tumors had unimodal DNA distribution with a DNA index (DNA I = modal channel of the G0/1 peak of the studied population/modal channel of the G0/1 peak of the normal brain) usually within the diploid or near-diploid range. 14.0% had aneuploidy, with an additional cell peak having a median DNA I of 1.60. Among malignant tumors, these figures were 61.2 and 38.8% (p less than 0.001). The percentage of S phase cells was higher in malignant (median = 3.6) than in benign tumors (median = 2.0, p less than 0.01), without correlation to histological tumor subtype. Flow cytometry appears to be a useful method for evaluating differences in DNA distribution in tumors of the central nervous system.     

Some effects of acute and chronic dosing with aflatoxin B1 on rat liver nuclei.

A study has been made of changes occurring in rat liver nuclei as a result of the chronic and acute administration of aflatoxin B1. This has involved resolution of the nuclei into populations of differing ploidy by means of centrifugation in a zonal rotor. Chronic feeding of the toxin to weanling rats prevents the development of the predominant tetraploid hepatocyte nuclear population, which normally takes place during maturation. Increased populations of diploid and octaploid hepatocyte nuclei are observed. Chronic feeding of the toxin to adult animals also causes a reduction in the tetraploid population already established and, again, leads to increased numbers of diploid and octaploid nuclei. Once an abnormal nuclear population has been established by feeding the toxin, it persists until the development of hepatocarcinoma (if a 6-week carcinogenic feeding regimen has been used). The hepatomas have diploid nuclei. Labeling hepatic RNA and DNA in vivo has indicated that feeding the toxin causes a reversion to the immature distribution of DNA synthesis among the nuclear population, with little effect on the pattern of distribution of RNA synthesis. Acute administration of aflatoxin to adult rats also causes a reduction in the size of the tetraploid population, with increased proportions of diploid, octaploid, and higherploidy nuclei. These results are discussed in terms of a dual action of the toxin, antimitotic and necrogenic, and the possible relationship of these to the carcinogenic process.     

Prognostic relevance of DNA flow cytometry in oligodendroglioma.

In a retrospective study of 85 cases, the prognostic value of DNA flow cytometry in oligodendrogliomas was evaluated. Paraffin-embedded material was processed for flow cytometry, and the survival rates of the patients with DNA diploid, aneuploid, and tetraploid tumors were compared using analysis of variance. In addition, the mitotic index was correlated with the results of flow cytometry. Finally, the results of flow cytometry, histopathologic grading, and counting mitoses were tested for dependency. Thirty-one percent of the tumors were diploid, 39% were tetraploid, and 31% were aneuploid. The results of the DNA flow cytometry did not correlate with the survival times (P = 0.798) or with tumor degree. In contrast, the number of mitoses (P less than 0.05), and the grades of the grading system of Smith (P less than 0.003) had relevance for the prognosis. No correlation between flow cytometry, histopathologic grading, and mitotic index was found. It is concluded that flow cytometry has no value in predicting the biologic behavior of oligodendrogliomas, whereas the number of mitoses is a valuable prognostic parameter and thus is considered to be incorporated into the grading system for oligodendrogliomas.     

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.     

DNA flow analysis of soft tissue tumors

The cellular DNA content of 81 soft tissue tumors was determined by means of flow cytometry and related to conventional histologic classification of the same tumors. Comparison of histologic and cytometric analysis showed that all 23 benign tumors were diploid (normal DNA content), whereas the malignant group included both diploid and aneuploid (abnormal DNA content) lesions. There appeared to be a relationship between tumor grade and ploidy level in that 92% of Grade II, 28% of Grade III, and 11% of Grade IV lesions were diploid. Cell distribution analysis, feasible in 51 cases, disclosed that diploid lesions had a low proportion of S and G2 + M cells and most aneuploid lesions a high proportion, indicating a relationship between ploidy level and proliferative activity. The current study shows that solid mesenchymal tumors may be analyzed by DNA flow cytometry. Regardless of histogenetic type, it appears that benign and low-grade tumors are diploid and high-grade tumors, in general, are aneuploid. As to exceptions, DNA analysis may prove to give information beyond that obtained by subjective histologic interpretation. Thus, adequate follow-up might show that high-grade lesions with a diploid DNA content are associated with a better prognosis than expected from histologic classification.     

DNA contents in Wilms' tumors. A cytofluorometric study

Cytofluorometric measurement of nuclear DNA was performed on individual tumor cells isolated from paraffin sections of Wilms' tumors. The DNA distribution of one untreated primary tumor showed the first major peak near the diploid range and the second peak in the tetraploid range. There were many cells having amounts of DNA interspersed between the diploid and tetraploid range. Polyploid cells were not observed. Most of the Wilms' tumor cells had a higher diploid DNA value than the small lymphocytes of the control cells. The primary tumor and its metastases showed similar DNA distribution patterns. After treatment, the distribution pattern showed a reduction in number of cells between the diploid and tetraploid range and in the tetraploid range, with the greater number of cells being found in the diploid range. Though the phases of the cell cycle are not always clearly detectable by cytofluorometry, the cells of the untreated Wilms' tumor were distributed into the phases of the cell cycle as follows: 65.9% in G0 and G1 phases; 31.2% in S-phase; and 2.9% in G2 and M phases.     

DNA cytometry in diagnostic cytology of the prostate gland

BACKGROUND: Differential analysis and cytological grouping of fine needle aspiration biopsy (FNAB) samples of the prostate are important in practice. We used image analytical DNA cytometry to achieve this and also studied the best method of interpretation of the histograms. MATERIALS AND METHODS: Sixty-two FNAB samples of the prostate were stained with Feulgen stain and nuclear DNA histograms were produced by image cytometry. The most atypical cell groups were selected for measurements. Also, free epithelial cells between cell groups were studied. The cells presented in the histograms were grouped according to the nuclear DNA content and by application of different gates of observation for diploid status. RESULTS: Several DNA histogram features (histogram classification categories, benign and malignant histogram patterns, presence of >5c-7c cells) showed significant relationships to differential diagnosis. Highly aneuploid (>5c-7c) cells had the potential for distinguishing a progressive-type of prostate cancer. CONCLUSION: The fraction of tetraploid and aneuploid histograms increased from atypical but benign to definitely malignant samples. DNA histograms have potential in the differential diagnosis and evaluation of the progressive character of prostate cancer.     

DNA index of ovarian carcinomas from 56 patients: in vivo in vitro studies

Out of 130 ovarian cancer patients the DNA index of cells from ovarian carcinoma was studied in 56 cases in which cytospin preparations showed the presence of atypical cells. In 24 patients the population had a diploid DNA index (1.0) and in the others the DNA index ranged from 1.2 to 2.0 (tetraploid). No hypodiploid or hypertetraploid populations were detected. Repeated samples from the same patients did not show any significant differences and primary culture did not alter the DNA index. In contrast, cell cycle phase distribution differed greatly from sample to sample, as also the ratio between DNA diploid and DNA aneuploid populations. Primary culture was successful in 57% of the tumours, with a higher percentage of success in DNA aneuploid tumours. After primary culture the ratio between DNA aneuploid cells and DNA diploid cells increased. In relation to the histological gradings of malignancy, DNA aneuploid cells clustered in the highest grade of malignancy. The mean S-phase for tumours with a DNA index of 1.0 was 3.5 and 14.1% for those with DNA index greater than 1. Ovarian carcinomas show a large difference in DNA index between patients even after primary culture.     

Comparison of cytologic composition with microfluorometric DNA analysis of the glioblastoma multiforme and anaplastic astrocytoma

The DNA content of selected areas of 16 glioblastoma multiforme predominantly composed of small anaplastic cells was investigated. These findings were contrasted with those of five cases of anaplastic astrocytomas. The microfluorometric determination of DNA was performed on mechanically isolated single cells stained by the Feulgen method, obtained from selected areas of the neoplasms in paraffin blocks. Twelve of 16 cases of glioblastoma multiforme had a main population that was diploid-near diploid. Although four of the five anaplastic astrocytomas disclosed a diploid-near diploid main population there was a more complex DNA distribution than in the small cells in glioblastomas. These differences were evident using two numerical DNA indices obtained from the analysis of each tumor histogram which defined, respectively, the extent to which the main tumor cell population deviated from euploid values and the probabilities to have multiple stemlines with abnormal DNA content. The results suggest that small anaplastic cells which appear to represent the most aggressive population in glioblastomas have a DNA content diploid-near diploid, and in malignant gliomas the degree of aneuploidy of the main stemline is not related to the biologic behavior of the neoplasm.