The DNA-Content of nuclei in the meristem of onion roots

Summary The techniques of making and evaluating measurements of nuclear DNA-contents by the two wave length method, including wave length determination were worked out. The efficiency of the analysis increased by unbiased elimination of off-values. Correction for non-specific absorption was made but remained, with the present material (sections), necessarily imperfect. This seems to be the factor which limited the, otherwise apparently very high, optical accuracy of which instrument and methods are capable. Errors in the determination of the nuclear dye-content were, even so, small compared with the more extreme cases of proportionality errors in the determination of the DNA-content that resulted from disproportionality of Feulgen dye- and DNA-content.Direct evidence of proportionality errors entering the DNA determination was found when different mitotic stages were compared. The ratio of mean dye-contents of two stages varied in slides which were prepared differently in regard to fixation, hydrolysing agent, or duration of hydrolysis. In seven such samples the mean dye-content of metaphases ranged from 80 to 115% (with mean 100.2%) of the prophase mean, the mean dye-content of mid-anaphases from 92 to 112% (with mean 105.5%). Similarly the mean dye-contents of daughter nuclei in late anaphases and telophases bracketed 50% of the prophase value. It appears virtually certain that this variation is not caused by a corresponding natural DNA variation but is due to proportionality errors.The results strongly support the view that the DNA-content does not change at all during mitosis apart from its halving brought about by the anaphase separation of sister chromatids. The results are further fully compatible with the hypothesis that the DNA-content per complete genome is strictly constant except during the period of DNA doubling. The dye-contents of prophase nuclei (also of metaphases) of the same sample have a coefficient of variation (corrected for non-systematic reading errors) of about 5.5%. This appears to be well within the limits of errors due to imperfectly corrected non-specific absorption and of proportionality errors.The proportionality errors found in measurements of different mitotic stages reveal a pattern similar to that prevailing in published DNA measurements: apparent deviations from DNA constancy are usually small and often negligible, but are occasionally large enough to be misleading. None of the claimed disproofs of DNA constancy is in any way conclusive, as such claims necessarily rest upon the unwarranted assumption that proportionality errors are always very small or absent.The development of the DNA-content during interphase was studied by correlating DNA-content and nuclear volume. Earlier findings on roots of other plants are confirmed. An interphase I with the DNA-content 2C is followed by a period of DNA synthesis (during this period the nuclear volume seems to increase only slowly, if at all) and this by an interphase III with the DNA-content 40. Each phase lasts several hours. Mitosis and DNA synthesis are essentially independent processes.Measurements on nuclei which are technically more favourable than those in sectioned onion roots have yielded a smaller or even disappearing inter-nuclear variation of the Feulgen dye-content. A strict DNA constancy per complete genome appears more and more likely.Contribution from the programme in Cytology, Department of Botany, University of Wisconsin, Madison, supported by grants to the late Dr. C. Leonard Huskins, from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council, from the Rockefeller Foundation and from the Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.     

Correlation between preoperative magnetic resonance spectroscopic data on high grade gliomas and morphology of Ki-67-positive tumor cell nuclei

OBJECTIVE: To investigate possible statistical correlations between metabolic data from preoperative proton magnetic resonance spectroscopy (1HMRS) and morphology of proliferating tumor cell nuclei in anaplastic gliomas and glioblastomas. STUDY DESIGN: Ki-67-positive tumor cell nuclei in paraffin sections of surgical specimens from 36 patients (7 anaplastic gliomas, World Health Organization grade 3; 29 glioblastomas, World Health Organization grade 4) were investigated by means of a digital image analysis system. Stringent inclusion criteria were formulated for all cases with respect to histologic quality and spectroscopic examination. As morphometric variables, nuclear area, shape variables (roundness factor, size-invariate Fourier amplitudes) and density of Ki-67-positive tumor cell nuclei per reference area were determined. RESULTS: Correlation analysis according to Spearman revealed a significant positive correlation between the total creatine (TCR) peak and nuclear area (P = .005). This correlation was also found within the glioblastoma group (P = .019). There was also a significant negative correlation of nuclear area with the ratio between choline and TCR in all cases (P = .014) and within the glioblastoma group (P = .046). No significant correlation of spectroscopic data was found with nuclear shape or density of Ki-67-positive tumor cell nuclei. CONCLUSION: The results demonstrate a correlation between spectroscopic data and morphology of proliferating tumor cell nuclei (nuclear size) in high grade gliomas. This study is part of a detailed investigation of the interrelationship between preoperative 1HMRS and quantitative histomorphology of gliomas.     

Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma

H. Ohsaki, E. Hirakawa, K. Kagawa, M. Nakamura, H. Kiyomoto and R. Haba Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma Objective: To assess whether computer‐assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low‐grade urothelial carcinoma cells (LG‐UCs) in voided urine. Methods: Nuclear morphometry was performed by a computer‐assisted image analyser system on Papanicolaou‐stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG‐UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. Results: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG‐UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG‐UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG‐UCs. On the other hand, the linear factor showed a mean higher value among LG‐UCs than reactive renal tubular cells (P = 0.023). Conclusions: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG‐UCs.     

Grading of ductal breast carcinoma by cytomorphology and image morphometry with histologic correlation

OBJECTIVE: To investigate the relevance of image analysis for grading breast carcinoma. STUDY DESIGN: Twenty-five ductal breast carcinoma cases were chosen randomly from routine fine needle aspiration clinics. The results of cytomorphologic grading and image morphometry were correlated with those of histologic grading. The five image morphometric parameters studied were nuclear diameter, nuclear area, nuclear roundness, nuclear perimeter and grey level to compare with chromatin texture. RESULTS: Cytologic grading alone had a high correlation with histologic grading. The lowest correlation was found in grade 2 tumors. When cytologic grading was supplemented with image morphometric parameters, the correlation was higher than that of cytologic grading alone. CONCLUSION: Cytologic grading has a high correlation with histologic grading. The correlation improves further on supplementation with image morphometric parameters.     

Cytologic grading and DNA image cytometry of breast carcinoma on fine needle aspiration cytology smears

OBJECTIVE: To correlate the cytologic grade of breast carcinoma with DNA image cytometry (ICM) and nuclear area on fine needle aspiration cytology (FNAC) smears. STUDY DESIGN: In this prospective study, FNAC material from 28 breast carcinomas were studied for cytologic grade and DNA ICM. Breast carcinomas were classified as grade 1-3 (low to high). DNA histograms were classified by the modified Auer method. Degree of hyperploidy (DH), ploidy balance (PB) and nuclear area (NA) were measured on Feulgen-stained smears by a CAS 200 image cytometer. Cytologic grade was correlated with DNA ICM findings and NA. RESULTS: There were 3 cytologic grade 1, 13 grade 2 and 12 grade 3 breast carcinomas. Seven of eight cases of hypertetraploid aneuploidy were grade 3 tumors. All cytologic grade 1 tumors were diploid. There were significant differences in DH, PB and NA in different grades of breast carcinoma (one-way ANOVA). CONCLUSION: DNA image cytometry in combination with cytologic grading might offer additional information for the characterization of breast carcinomas diagnosed by FNAC. These observations are of particular interest with the introduction of preoperative chemotherapy.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Prognostic value of morphometry in low grade papillary urothelial bladder neoplasms

OBJECTIVE: To analyze the prognostic value of morphometry in low grade papillary urothelial bladder neoplasms (LGPUBNs). STUDY DESIGN: The primary (most common) and secondary (second most common) histologic grades were considered in accordance with the 1998 World Health Organization/International Society of Urological Pathology and the 1999 World Health Organization classifications. With the primary grade, 54 cases were papillary urothelial neoplasms of low malignant potential (PUNLMPs) and 66 low grade papillary urothelial carcinomas (LGPUCs), whereas the secondary grade consisted of 45 PUNLMPs and 75 LGPUCs. To assess the proliferative index, an immunohistochemical study was performed. Regarding nuclear morphometry, an image analysis system on Feulgen-stained sections was utilized in different tumor zones (Zs): Z 1, 100-150 cells from the outer layers of the papillae; Z 2, 100-150 cells from the inner layers; and Z 3, 10 largest nuclei. In univariate studies, a t test, and Mann-Whitney U test and Kaplan-Meier curves were applied, whereas a Cox regression model was used for multivariate study of the variables: size, multiplicity, maximum Ki-67 index, mean nuclear area (MNA) and SD, mean nuclear perimeter and SD, and roundness factor. RESULTS: All 120 cases were followed for a mean of 76.6 months (range, 36-168). In univariate studies, many variables showed a significant correlation (p < 0.05) with recurrence prediction, relapse-free interval and histologic grade regardless of adjuvant therapy. Otherwise, only the MNA of the 10 largest nuclei (threshold, 52 microm2) and the maximum proliferative index (threshold, 7.9%) appeared as independent prognostic markers in the multivariate study. CONCLUSION: In LGPUBNs, the independent prognostic value of MNA of the 10 largest nuclei as well as the maximum proliferative index indicates the importance of histologic grade assessment based on the secondary (second most common) grade.     

Absorption-Spectrophotometric Determination of DNA (Deoxyribonucleic Acid) in Milk Ad Modum Feulgen

The Feulgen reaction is examined by absorption photometric measurements at 565 nm with correction for varying background absorption at 485 nm. This correction is done to improve the accuracy of measuring. Examinations of the accuracy of analysis for the Feulgen reaction and the direct microscopic cell counts show that the former is of the same order, when the cell count is made at a working factor of 17,000. The standard deviations expressed in percentage of the reaction value are greater the lower the reaction is, whereas the absolute standard deviation is reduced. It has been demonstrated that addition of a few (< 5) µg of DNA per ml milk can give Feulgen reaction. The coefficient of correlation between log added amount of DNA and log Feulgen reaction is 0.98. The coefficient of correlation between log Feulgen reaction and log cell content depends on the accuracy at which both determinations are made. In a determination of cell content at a working factor of 550, and several determinations of the Feulgen reaction the coefficient of correlation (r) is found to be 0.98. A cell content determination at a working factor of 20,000 and a single determination of Feulgen reaction on each milk sample yields r = 0.83. An examination of foremilk samples of the same cell content reveals on an average the highest reaction in mastitis-affected quarters which have been infected within the last 4 weeks. Quarters that have been infected for more than 4 weeks, on an average show the second highest reaction, whereas quarters of physiological cell number show the lowest reaction. The DNA-content in most cases is found to be too high compared with the number of cells counted microscopically. The DNA-content in centrifugated cells corresponds to the one calculated theoretically. The surplus DNA-content can be demonstrated in the cell-free skim milk fraction, probably originating from destroyed cells. The studies performed suggest that determination of the content of 2-deoxyribose in milk by means of the Feulgen reaction is a more correct measure of the cell content in the milk than is the microscopic cell count. Studies are being continued for illustration of these conditions.     

Cytomorphologic and morphometric limitations of the assessment of atypia in fibroadenoma of the breast

OBJECTIVE: To investigate the relevance of nuclear morphometry in separating the categories of "fibroadenoma" and "fibroadenoma with atypia." STUDY DESIGN: Thirty consecutive breast lumps, on which a fine needle aspiration (FNA) diagnosis of fibroadenoma was followed by excision and histopathologic confirmation of the diagnosis, were included. Atypia on cytology was based on cell overlap, nuclear enlargement and cell dyscohesion. Nuclear morphometric comparison was carried out between the categories of fibroadenoma, fibroadenoma with atypia and grade 1 ductal carcinoma cases that formed part of an earlier study. The parameters employed were area, roundness, diameter, perimeter and grey level. RESULTS: Among the 30 cases of fibroadenoma reported on FNA, an additional component of atypia was noted in 5. On subsequent histopathology, atypia was not confirmed in any of the cases. On morphometric analysis, a significant difference was noted between fibroadenoma and fibroadenoma with atypia categories, as between fibroadenoma and grade 1 ductal carcinoma cases. However, no significant difference was noted between fibroadenoma with atypia and grade 1 ductal carcinoma cases. CONCLUSION: FNA assessment of atypia in cases of fibroadenoma is difficult. Even conventional nuclear morphometry, though supporting the initial impression of atypia, does not help with this assessment. Also, based on morphometry alone, there may be difficulty separating fibroadenomas with atypia and grade 1 ductal carcinomas. Larger studies, employing other morphometric parameters, such as chromatin texture and fractal dimension, may shed further light on the subject.     

Combined morphometrical and syntactic structure analysis as tools for histomorphological insight into human lung carcinoma growth

Histological sections of formalin-fixed, paraffin-embedded tissue comprising 60 surgical specimens of human lung carcinoma were Feulgen stained. The histomorphological images were transferred to an automated image analysing system (VISIAC) and analysed as follows. The geometrical centers of tumor cell nuclei were defined as vertices, and the minimum spanning tree (MST) was calculated based on the two-dimensional distance between the vertices. Segmentation of the images was performed semiautomatically by interactive definition of nuclei of interest and automated detection of nuclear boundaries. Several morphometric features of tumor cell nuclei were measured including size, DNA-content (extinction), and form factor, and were set in relation to parameters of the MST. The following results were obtained: DNA-content and tumor cell nucleus size ('center cell') of different microscopic tumor growth patterns are related to the number of nearest neighboring cells. No relation was found in the neighboring (surrounding) cells. The different cell types of lung carcinoma, i.e., the different microscopic tumor textures expressed the relation of center cell features to the parameters of MST. A high amount of DNA content in branching points of the MST for epidermoid carcinoma may be interpreted as carcinoma growing in epidermoid textures tend to proliferate from tumor cell nuclei related to at least one neighboring cell. The opposite was found for large cell anaplastic carcinoma (no perceptible microscopic textures of the tumors) which showed the highest DNA content in tumor cell nuclei but which was not related to any neighboring cells. This technique allows analysis of growth centers and microenvironment conditions in human lung cancer in relation to tumor texture at the light microscopy level.     

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.     

EGFR gene amplification in glioblastomas. Is there a relationship with morphology of tumor cell nuclei and proliferative activity?

OBJECTIVE: To confirm a relationship between histomorphology of glioblastomas and amplification of the gene for the epidermal growth factor receptor (EGFR) as the most important molecular biologic alteration in these tumors. STUDY DESIGN: In paraffin sections of surgical specimens from 71 primary resected glioblastomas, tumor cell nuclei in the region with the highest proliferative activity (Ki-67 immunostaining) were investigated morphometrically. Shape variables (roundness factor, Fourier amplitudes) and nuclear area were measured. Additionally, the numerical density of Ki-67-positive tumor cell nuclei was estimated. Differential polymerase chain reaction (PCR) was performed from paraffin sections of the same tumor area. The signals for the EGFR gene and IFN gamma reference gene were quantified densitometrically. RESULTS: Cases with distinct EGFR gene amplification (EGFR/IFN ratios > 5) revealed significantly lower mean values for several Fourier amplitudes, indicating a more regular nuclear shape when compared with cases without evidence of EGFR gene amplification (EGFR/IFN-ratios < or = 1). The Ki-67 index and nuclear area showed no significant differences between these groups. Although a large variation in nuclear morphology was observed for cases without evidence of EGFR gene amplification, discriminant analysis based on morphometric variables provided a good separation of these cases from cases with distinct EGFR gene amplification, with a high percentage of statistically correct reclassified cases. CONCLUSION: Our results provide evidence of a relationship between genetic alterations and histomorphology of glioblastomas.     

Prognostic significance of DNA image cytophotometry for osteosarcoma

OBJECTIVE: To investigate the prognostic significance of DNA image cytophotometric data. STUDY DESIGN: Twenty-six osteosarcomas in patients without lung metastases were investigated for several cytophotometric data. In 24 cases, these data were correlated with the clinical course of the patients to assess the prognostic value of nuclear DNA content in osteosarcomas. RESULTS: Of all osteosarcomas, 96% showed aneuploid DNA content. Patients with tumors having a 2c deviation index (2cDI) of 12.00, DNA malignancy grade (DNA-MG) of 2.0, a mean DNA content (MDC) of 4.95 c, DNA index (DI) of 1.75 or mean nuclear area (MNA) of 130 microns 2 had a significantly lower overall survival rate as compared to those with lower values (P < .05). CONCLUSION: Image cytophotometric features, such as 2cDI, DNA-MG, MDC, DI and MNA, are of prognostic value in patients with osteosarcoma and free of lung metastases.     

Application of different methods for nuclear shape analysis with special reference to the differentiation of brain tumors

OBJECTIVE: To study the discriminatory power of different methods designed for nuclear shape analysis with reference to the differentiation and grading of brain tumors and the differentiation between proliferating and nonproliferating nuclei. STUDY DESIGN: At least 300 tumor cell nuclei per case were measured by means of a digital image analysis system. Fourier amplitudes no. 1 to 15, moments no. 1 to 7 according to Hu, roundness factor, ellipse shape factor, concavity factor, Feret ratio, fractal dimension and bending energy were determined for each nucleus. The discriminatory power of these parameters was tested in three pairwise comparisons: (1) oligodendrogliomas WHO grade II (n = 13) vs. grade III (n = 11), (2) medulloblastomas WHO grade IV (n = 14) vs. anaplastic ependymomas WHO grade III (n = 12), (3) Ki-67-positive vs. Ki-67-negative tumor cell nuclei in the 14 medulloblastomas. RESULTS: When data from Fourier analysis were included in statistical analysis, cross-validated discriminant analysis led to a 100% correct reclassification for the first and for the second pairwise comparison and to a 75% correct reclassification when comparing Ki-67-positive and Ki-67-negative nucleifrom medulloblastomas. Different combinations of the other shape parameters led to a lower percentage of correctly reclassified cases for all three pairwise comparisons, especially when Fourier analysis was not included in the analysis. CONCLUSION: Fourier analysis provided an optimal statistical discrimination between different brain tumor entities and between data sets from proliferating and nonproliferating tumor cell nuclei. Since nuclear shape is an important criterion for the investigation of tumors, the application of Fourier analysis is therefore recommended for quantitative histologic investigations in neuro-oncology.     

Risk biomarker assessment for breast cancer progression: replication precision of nuclear morphometry.

Nuclear morphometry is a method for quantitative measurement of histopathologic changes in the appearance of stained cell nuclei. Numerous studies have indicated that these assessments may provide clinically relevant information related to the degree of progression and malignant potential of breast neoplasia. Nuclear features are derived from computerized analysis of digitized microscope images, and a quantitative Feulgen stain for DNA was used. Features analyzed included: (1) DNA content; (2) nuclear size and shape; and (3) texture features, describing spatial features of chromatin distribution. In this study replicated measurements are described on a series of 54 breast carcinoma specimens of differing pathologic grades. Duplicate measurements were performed using two serial sections, which were processed and analyzed separately. The value of a single feature measurement, the nuclear area profile, was shown to be the strongest indicator of progression. A quantitative nuclear grade was derived and shown to be strongly correlated with not only the pathologic nuclear grade, but also with tubule formation, mitotic grade, and with the overall histopathologic grade. Analysis of replication precision showed that the standard methods of the histopathology laboratory, if practiced in a uniform manner, are sufficient to ensure reproducibility of these assessments. We argue that nuclear morphometry provides a standardized and reproducible framework for quantitative pathologic assessments.     

Nuclear size of myocardial cells in end-stage cardiomyopathies

OBJECTIVE: To determine the alteration of nuclear size in myocardial cells and the relationship between nuclear size and DNA ploidy classes in normal and cardiomyopathic human hearts. STUDY DESIGN: The study group consisted of 46 hearts obtained at biopsy. These patients had undergone cardiac transplantation for intractable congestive heart failure (18 cases with ischemic cardiomyopathy and 28 cases with idiopathic dilated cardiomyopathy). Another 10 hearts were collected at autopsy and used as control hearts according to preautopsy, autopsy and histology criteria. One hundred fibroblasts and 200 myocytes were evaluated in each ventricle. The nuclear area and DNA content were estimated using image cytometry. RESULTS: End-stage ischemic and dilated cardiomyopathies were characterized by an increase in nuclear size of both the myocyte and nonmyocyte population. The nuclear area of interstitial cells increased about 30% in cardiomyopathic hearts. Augmentation of average nuclear area of myocytes was 1.2-fold in the ischemic group and about 1.5-fold in the dilated group as compared with the control group. Also, a tendency was found for the coefficient of variation of average nuclear area to decrease in the interstitial cell population and increased in the myocyte population in cardiomyopathic situations. Furthermore, the nuclear area of myocytes enlarged as augmentation of nuclear DNA content. The relative nuclear areas of myocytes can be presented as: 2c:4c:8c:16c :32c:64c = 1:1.65:2.75:4.60:7.25:9.18. CONCLUSION: The increase in nuclear size follows either one of two different processes: the first does not involve an increase in DNA content, whereas the second is concomitant with an incremental increase in DNA content. In the first instance, the enlargement of nuclear size is limited. In the second, augmentation of nuclear size can become very impressive. In end-stage ischemic and dilated cardiomyopathies, the nuclear growth of myocytes and interstitial cells may be due to different mechanisms. Enlargement of the nuclear area of myocytes represents a complex process, including simple nuclear hypertrophy, polyploidization and multinucleation. The main pattern of nuclear growth of interstitial cells is nuclear hypertrophy without an increase in DNA content.     

Automated nuclear image morphometry on fine needle aspiration smears of malignant round cell tumors

OBJECTIVE: To analyze nuclear image morphometry in fine needle aspiration cytology smears of different groups of malignant round cell tumors (MRCTs) to evaluate its diagnostic role. STUDY DESIGN: In this study there were 55 cases of MRCT, consisting of 18 Ewing's sarcoma (EW), 10 neuroblastoma (NB), 5 non-Hodgkin's lymphoma (NHL), 6 rhabdomyosarcoma (RMS), 4 peripheral neuroectodermal tumor (PNET), 8 Wilm's tumor (WT), 2 retinoblastoma (RB) and 2 undifferentiated round cell tumor (URCT). A Leica image cytometer with Quantimet 600 software (Leica, Cambridge, U.K) was used to measure nuclear area, nuclear diameter, nuclear perimeter, nuclear convex perimeter (CP), nuclear roundness and nuclear convex area on hematoxylin and eosin-stained cytologic smears. At least 100 cells were studied in each case. RESULTS: The RB group of tumors showed the highest mean nuclear area (NA), convex area (CA), CP, diameter (D), perimeter (P) and roundness (R). RMS had the highest mean CA, and URCT had the highest mean roundness. ANOVA was performed on the tumors and showed significant differences for all the variables in all the groups (P < .000). All the morphometric data (except roundness) were significantly different in RMS versus all other MRCTs except RB. Similarly, morphometric data on WT were also significantly different from that on NHL. Most of the morphometric data (except CA and R) showed significant differences between RB and all other MRCTs except RMS. PNET, EW and NB could not be differentiated with those variables. CONCLUSION: RMS and RB could successfully be differentiated from all other MRCTs with the help of morphometry. It was not possible to differentiate RMS and RB by image cytometry (ICM) since the ICM data overlapped in those two groups. It was possible to differentiate WT and NHL with ICM. Nuclear ICM was not significantly different in the NB, PNET and ES groups, and probably ICM would not be very helpful to differentiate these groups of MRCT.     

Der Zellzyklus in Fibroblastenkulturen vom Menschen

Summary Autoradiography using H³-Thymidin and Feulgen photometry of nuclear DNA-content were carried out on human euploid fibroblast cultures.The average durations of S- and G2-periods are about 9 hours and 5 hours respectively, with only minor variation. The duration of G1-period may vary from a few hours to more than 20 hours.A combined study of H³-thymidin uptake and Feulgen photometry on the same cell nuclei showed that all cells whose DNA-content places them into the S-period are labelled when they are fixed immediate after a H³-thymidin puls. After continuous H³-thymidin uptake for 5 hours, all S- and G2-nuclei are labelled, whereas the G1-nuclei remain unlabelled.The Feulgen histogramm as well as grain counts over labelled nuclei indicate a constant rate of DNA synthesis during S-period.     

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.     

Correlation of mean nuclear area with estrogen receptor content in aspiration cytology of breast carcinoma

Thin needle aspirates of 42 consecutive breast carcinomas were obtained at the time of excisional biopsy. Nuclear diameters of 100 cells from each case were measured, and the nuclear areas were calculated. The concomitantly acquired histologic sections were reviewed and assigned a histologic grade according to the National Surgical Adjuvant Breast Project protocol no. 4. Estrogen receptor (ER) content was analyzed by both the DCCA and SDGA techniques. The ER content of each case was then compared to both the mean nuclear area of the cells on the cytologic smears and the histologic grade. All 16 cases with mean nuclear areas of less than 60 sq micrometer contained significant levels of ER (greater than 10 fmol/mg protein), as did 6 of 11 cases with nuclei between 60 and 90 sq micrometer. Only 5 of 15 cases with nuclei larger than 90 sq micrometer contained significant ER levels. Comparison of the sensitivity, specificity and predictive values of both techniques suggests that a quantitative assessment of nuclear area in cytologic thin needle aspirates correlates more closely with ER content than does histologic grading.