Morphometric characterization of nuclei in non-Hodgkin's malignant lymphoma

In addition to the nuclear area and a form factor, four morphometric parameters of nuclear shape (ID, R1, R2 and ND), obtained by the application of the principles of mathematical morphology, were used to characterize the nuclear contours in non-Hodgkin's malignant lymphomas. The values for each parameter were determined in 58 cases of non-Hodgkin's lymphoma categorized according to the Kiel and National Cancer Institute classifications. Small-cell, mixed and large-cell lymphomas could be distinguished on the basis of the mean nuclear area. The shape parameters R1, R2 and ID were efficient discriminators of the large centrocytic (cleaved-cell) lymphomas. Neither size nor shape factors could distinguish between centroblastic and immunoblastic tumors. The good correlation between the morphometric findings and the histopathologic categories suggest that morphometry may provide a quantitative and objective method for grading lymphomas.     

Comparison between morphometry and immunostaining of malignant cells in non-small cell lung cancer

OBJECTIVE: To investigate the morphometric properties of tumor cells in combination with the expression of 5 immunomarkers, quantitatively evaluated by image analysis, in a series of 60 non-small cell lung carcinomas (NSCLCs) and to assess their prognostic significance. STUDY DESIGN: Tissue sections from 60 NSCLCs were assessed for the expression of p53, Ki-67, bcl-2, bax and FasL using immunohistochemistry and antibodies. Correlations between morphometric features and clinicopathologic variables, including overall survival and the expression of the above markers, were statistically investigated. RESULTS: Major and minor axis nuclear values, as well as nuclear area and perimeter values, were positively interrelated. No statistically significant correlations were observed between nuclear morphometry and any of the clinicopathologic parameters. p53 Accumulation, when quantitatively estimated, displayed a positive correlation with major axis (P = .008), minor axis (P = .06), nuclear perimeter (P = .006) and mainly nuclear area values (P = .003). Bcl-2- and bax-quantified immunostaining demonstrated a weak negative correlation with shape factor values (P = .039 and P = .025, respectively). Univariate and multivariate analysis showed that stage was the only significant predictor of survival in all patients. In univariate statistical analysis there was a trend between worse survival and shape factor values < 0.8 (P = .0791); this trend almost reached statistical significance in the subgroup of squamous cell carcinomas (P = .0570). CONCLUSION: p53 Accumulation, when quantified, appears to be positively linked with nuclear morphometric values. The prognostic significance of shape factor in NSCLC warrants further investigation.     

Stereologic correlates of steroid receptor concentration in invasive ductal breast cancer

The hypothesis was tested that morphometric parameters of tumor cell nuclei correlate with the steroid receptor concentration in mammary carcinoma. In 50 consecutive mastectomy specimens with a diagnosis of invasive ductal cancer in which estrogen receptor (ER) and progesterone receptor (PR) concentrations had been assayed quantitatively, morphometric measurements were performed on four visual fields of two sections per case. The fields were sampled from the most cellular regions of the tumor. The number of tumor cell nuclear profiles per tissue area, the nuclear profile area and the long and short nuclear profile axes and their ratios were measured with a semiautomatic image analysis system. Estimates of the number of tumor cell nuclei per tissue volume (Nv) and of the mean tumor cell nuclear volume (V) were obtained by standard stereologic techniques. Association between the morphometric and biochemical parameters was tested by Spearman's rank correlation coefficient. Nv correlated positively with the steroid receptor concentration whereas V correlated negatively with both ER and PR concentrations. A correlation of the receptor concentrations to the standard deviation of the nuclear area or the mean ratio of the nuclear axes could not be demonstrated. These results suggest that receptor-rich tumors have a large number of small tumor cell nuclei whereas receptor-poor tumors have a small number of large tumor cell nuclei per tissue volume in the actively proliferating, highly cellular regions. These differences are not accompanied by significant changes in nuclear size variability or nuclear shape.     

人鼻咽癌克隆株体外传代过程的群体变化

将人低分化鼻咽癌克隆株CNE-2 Z-5-2-B_7在体外连续传代培养,从群体的角度观察了不同代数的细胞形态定量、DNA含量和体外增殖能力的变化。结果:(1)各细胞形态参数、DNA含量出现异质性,为多种瘤细胞亚群所构成,而且随传代过程变化、消长。(2)41代以细胞面积大、核大、核浆比小、DNA含量高、异质性明显的大细胞群体占优势。其形态特征与鼻咽低分化鳞癌的大核型相似。(3)81代主要为胞、核面积小、核浆比大、DNA含量高的小细胞群体,其体外增殖能力较第1代明显为高,形态及生长特征与鼻咽未分化癌相似。提示随着肿瘤的演进,如不给予影响(治疗),瘤细胞有恶性发展的内在倾向。     

Predictive value of discriminant analysis of monocyte ultrastructure in malignant lymphoma

Discriminant analysis was applied to morphometric data obtained from ultrastructural studies of blood monocytes from 20 normal subjects, 23 patients with Hodgkin's disease and 12 patients with non-Hodgkin's lymphoma. The aim was to assess the efficiency of predicting subject groups from such data. The analysis, performed on a microcomputer using a standard statistical package, considered nuclear volume, nuclear surface area, nucleolar volume, nucleolar surface area, nucleolar volume fraction, number of nucleoli per section, cell surface area, mitochondrial surface area and subject age. The overall agreement between predicted and actual subject groups was 64%; considering only normality and disease, the agreement was 80%. While the predictive value of such data from circulating monocytes would appear insufficient for diagnostic purposes, discriminant analysis as used here might be of value in indicating the state of host defense in malignancy.     

Differentiating large cell lymphoma from indolent small B-cell lymphoma in fine needle aspirates using p53, PCNA and transformed lymphocyte count

OBJECTIVE: To determine the usefulness of proliferating cell nuclear antigen (PCNA), p53 protein expression and transformed lymphocyte count (TLC) as adjunctive tests to differentiate indolent small B-cell lymphoma from large cell lymphoma in fine needle aspiration biopsies. STUDY DESIGN: Aspirates of lymphoproliferative disorders from April 1993 to January 1997 were reviewed. The percentage of TLCs was determined on the Papanicolaou smear. The percentage and intensity of p53 and PCNA immunocytochemical staining was evaluated on cell block sections. These results were compared and correlated with the final diagnoses based on available morphology, flow cytometry and clinical history. RESULTS: There were 40 cases of non-Hodgkin's lymphoma and 12 reactive lymph nodes. Adequate cell blocks were available on 16 large cell lymphomas, 7 grade 1-2 follicular center cell lymphomas, 6 mucosal associated lymphoid tissue lymphomas, 2 small lymphocytic lymphomas and 2 mantle cell lymphomas. Average TLC and p53 nuclear staining was highest in large cell lymphomas (57% TLC and 24% p53), followed by grades 1 and 2 follicular lymphomas (14% TLC and 15% p53) and lowest in other indolent lymphomas (< 10% TLC and < 1% p53). Average PCNA staining was highest in large cell lymphomas (46%) and lowest in small lymphocytic lymphomas (7%); however, TLC was the best parameter for differentiating large cell lymphoma from indolent small B-cell lymphoma. CONCLUSION: TLC differentiated large cell lymphoma from indolent small B-cell lymphoma better than either p53 or PCNA alone or in combination. Significant overlap between categories limits usefulness of these immunocytochemical stains for differentiating these entities.     

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.     

Morphometric analysis of the toxic effect of ethanol on the structure of chromatin in interphase nuclei of rat hepatocytes

A method of automated morphometric analysis of hepatic cell interphase nuclear chromatin (CHIN) structure was elaborated using scanning microspectrophotometer SMP-05 ("Opton", FRG). Liver sections of rats were used to investigate hepatic cell CHIN structure. The animals were administered ethanol intraperitoneally once per day within a month period in doses 4, 6, 8, 10 g/kg (1/3-4/5 LD50). Morphometric indexes indicating dose-dependent changes in hepatic cell CHIN structure are demonstrated. Statistical analysis of morphometric data has led to the conclusion that hepatic cell CHIN structural modification depends on two factors: ethanol damaging effect and hepatic cell regeneration.     

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.     

Stage-dependent variation in the rat Sertoli cells nuclear volume

A morphometric study was carried out to investigate stage-dependent variation in sertoli cell nuclear volume in the rat testis. sertoli cell nuclei had the largest volumes in stages IX to X of the seminiferous epithelium cycle (746 microns3), and the smallest volumes in stage XIV (624 microns3). In the remaining stages, the nuclei presented intermediate values, without significant differences. The results were discussed in terms of a possible functional cyclic variation in the sertoli cell reflecting changes in their nuclear size.     

Nucleotype and cell size in vertebrates: a review

The relationships between genome size and various cell morphometric parameters have been assayed in 357 species of Vertebrates, in order to verify the existence and significance of the so-called "nucleotypic effect" in this subphylum. The results obtained clearly manifest a significant relationship between the increase in genome size and that in nuclear volume, nuclear surface, cell volume and cell surface. A precise correlation is also observed between the increase in DNA content and the decrease in the surface/volume ratios of the nucleus and the cell. Other parameters, such as the nucleoplasmic index and DNA concentration, though showing a slight increase with increasing genome size, have values rather homogeneous in each Vertebrate group. These results have allowed some interesting speculations on various problems; for example, the mechanisms through which genome size can influence the cell size; the influence of the DNA content and cell morphometric parameters on functional level of the cell and the organism; the importance of the nucleotypic effect in the adaptation to the environment of the various Vertebrate groups. From this study it seems possible to make the following conclusions: 1) in Vertebrates, genome size would exert a real nucleotypic influence on cell size; 2) genome sizes and cell morphometric parameters seem to be involved in the regulation of cell metabolism; 3) the regulation of some morphometric parameters depends strictly and automatically on the DNA amount or on other morphometric parameters. The regulation of others, instead, depends on the interaction of different factors, which do not always act synergically; 4) the nucleotypic effect seems to have different distribution and importance in Anamniotes and Amniotes.     

Morphometry of macrophage development in malignant lymphoma. Predictive value of discriminant analysis

Discriminant analysis of morphometric data on the ultrastructure of developing macrophages has been used to classify 62 individual subjects into one of the 3 groups of origin, namely normal, Hodgkin's disease and non-Hodgkin's lymphoma, each finding being compared with the known diagnosis. The data had been obtained from blood monocytes grown in suspension culture over a period of 6 days, and related to whole cell, nucleus, nucleoli and mitochondria. Over 80% of subjects were correctly classified as between the 3 groups and over 90% as to their normality or otherwise. Although the non-specific nature of changes in defence cells makes it unlikely that morphometric studies of macrophages will find a place in the diagnosis of specific malignancies, the present work indicates it could be useful in assessing host response and hence prognosis and response to treatment. Discriminant analysis of quantitative differences in cell structure could have wide clinico-pathological application.     

Morphometry and digital AgNOR analysis in cytological imprints of benign, borderline and malignant serous ovarian tumours

AIM: The aim of the study was to determine values of a quantitative morphometry analysis of nuclear characteristics and argyrophilic nucleolar organizer regions (AgNORs) in differential cytodiagnosis of benign, atypically proliferating (borderline) and malignant serous ovarian tumours. METHODS: Cytological imprints of benign (n = 20), borderline (n = 19) and malignant (n = 20) ovarian serous tumours were analysed. A computerized, digital analysis was used to determine morphometric nuclear features, the number and characteristics of single AgNORs, cluster AgNORs, total AgNOR and AgNOR area/nucleus (relative area) ratio. According to their size AgNORs were classified in three categories. A one-way variance analysis and post hoc test (Scheffé) were used for statistical analysis. RESULTS: The morphometric nuclear analysis showed that benign, borderline and malignant serous ovarian tumours are statistically different (P < 0.001) according to the area and outline, the values being highest in malignant tumours and lowest in the borderline group. Digital analysis of AgNORs in benign, borderline and malignant groups showed that the total AgNOR number increases with progression of the lesion (meaning tumour malignancy) significantly (P < 0.001) between benign and malignant as well as between borderline and malignant serous ovarian tumours (P < 0.001). The progression of the lesion malignancy was accompanied by a significant (P < 0.001) progressive increase of the total and relative AgNOR area per nucleus. The AgNOR size increases from benign to malignant tumours and a statistically significant difference (P < 0.001) was observed in all three groups regarding small and large AgNORs. CONCLUSION: Combining different markers of morphometric nuclear characteristics and AgNOR values could improve differential cytodiagnosis of benign, borderline and malignant serous ovarian tumours.     

Reproducibility of nuclear morphometric measurements in benign human epithelial cells. Simulation of variations in routine tissue processing.

The value of nuclear morphometric measurements in diagnostic pathology is determined largely by the reproducibility of the measurements. Although a variety of factors have been shown to affect tissues during processing, the regulation of fixative type and the avoidance of air drying in particular have been shown to avoid significant variations in nuclear measurements. The current study simulated routine tissue processing in order to identify any factors that may introduce variability of nuclear morphometric values in day-to-day processing if air drying is avoided and fixative type and pH are regulated. Samples of benign endometrium were collected from three uteri, fixed in phosphate-buffered formalin (PBF) from 2 hours to 15 days and dehydrated in an automated tissue processor on four occasions. In addition, tissue from one case was cut at 4, 6 and 8 microns, simulating the potential variations in section thickness that may occur during routine processing. Mean nuclear areas and shape factors of epithelial cells were then determined using computed planimetry. By analysis of variance, no significant differences were found in nuclear morphometric values in relation to time of fixation, dehydration runs or tissue section thickness; coefficients of variation for all variables were less than 7%. This study suggested that routinely processed tissues are adequate for morphometric analysis, including retrospective analysis, provided that tissues are fixed in a pH-regulated fixative such as PBF and air drying is avoided.     

Computerized morphometric discrimination between normal and tumoral cells in oral smears

The oral exfoliative cytology allows a quick and fairly accurate assessment of suspicious lesions of the oral cavity. Within this context, our paper proposes a quantitative approach, focusing on the construction of a classifier for detecting the presence of the tumoral cells on oral smears. The design of the classifier relies on a detailed computerized analysis of the individual morphometric features exhibited by two large known populations of normal and tumoral cells, respectively; the digital image processing was performed in the Zeiss KS400 environment. The classifier was implemented as a neural network with step activation function, whose parameters were obtained from an adequate training, based on the nuclear and cytoplasmic areas of the cells belonging to the two populations. Our procedure based on this classifier was meant to operate by identifying the tumoral or normal nature of any cell randomly selected from a smear. To identify the nature of an arbitrary cell, its nuclear and cytoplasmic areas are presented at the input of the classifier. The classification procedure was tested on several smears, and the results coincided with the pathological diagnosis in all the considered cases. The performances of our approach are discussed in comparison with other analytical methods previously reported in oral exfoliative cytology. These discussions emphasize the role of numerical information exploited for the classifier design, concluding that the individual morphometric features are more meaningful than the global characterization of smears by mean values.     

Nuclear morphometric features of epithelial cells lining keratocysts

OBJECTIVE: To investigate the nuclear morphometric features of epithelial cells lining keratocysts and some other odontogenic cysts. STUDY DESIGN: All cases were selected from the archives of the Department of Pathology, Gülhane Military Medical Academy, as follows: 20 keratocysts and 10 dentigerous and 10 radicular cysts. Nuclear morphometric variables were measured on hematoxylin and eosin-stained histologic slides. Basal and intermediate cells of the epithelium were evaluated separately. Nuclei of the cells were outlined interactively and measured using a specially written macro program. Area, feret ratio (ratio of the longest nuclear axis to the shortest one) and circularity (F circle) of the nuclei were calculated. Additionally, nuclear densitometric analysis was performed on the keratocyst cases. RESULTS: The number of cells in the basal layer (cell density) was higher in keratocysts than in other cysts. The mean nuclear area of basal cells was smaller than of intermediate cells in both keratocysts and other cysts (P < .001). The feret ratio values revealed that basal cell nuclei of keratocysts were more ovoid as compared to those of other cysts (P < .001). Nuclear densitometric findings showed that the DNA indices of all keratocyst cases were close to 1.0, and the cells were considered diploid. CONCLUSION: Increased cell density, a more ovoid nuclear shape and more variation in the size of basal layer cell nuclei in keratocysts were helpful in differentiating these lesions from other odontogenic cysts.     

Annotated proteome of a human T-cell lymphoma.

As the reliable identification of proteins by tandem mass spectrometry becomes increasingly common, the full characterization of large data sets of proteins remains a difficult challenge. Our goal was to survey the proteome of a human T-cell lymphoma-derived cell line in a single set of experiments and present an automated method for the annotation of lists of proteins. A downstream application of these data includes the identification of novel pathogenetic and candidate diagnostic markers of T-cell lymphoma. Total protein isolated from cytoplasmic, membrane, and nuclear fractions of the SUDHL-1 T-cell lymphoma cell line was resolved by SDS-PAGE, and the entire gel lanes digested and analyzed by tandem mass spectrometry. Acquired data files were searched against the UniProt protein database using the SEQUEST algorithm. Search results for each subcellular fraction were analyzed using INTERACT and ProteinProphet. All protein identifications with an error rate of less than 10% were directly exported into excel and analyzed using GOMiner (NIH/NCI). The Gene ontology molecular function and cell location data were summarized for the identified proteins and results exported as user-interactive directed acyclic graphs. A total of 1105 unique proteins were identified and fully annotated, including numerous proteins that had not been previously characterized in lymphoma, in functional categories such as cell adhesion, migration, signaling, and stress response. This study demonstrates the utility of currently available bioinformatics tools for the robust identification and annotation of large numbers of proteins in a batchwise fashion.     

A morphometric analysis of cytological features of tall cell variant and classical papillary carcinoma of the thyroid

In order to assess whether morphometric parameters could be of value in distinguishing between tall cell variant and classical pattern of thyroid papillary carcinoma, the fine needle aspiration cytology (FNAC) samples of 14 cases were analysed using Arcimage 5 software on an Acorn computer. Histological examination of the specimens allowe classification of nine of them as classical pattern and the remaining five as tall cell variants. The nuclear diameter (NDD) and standard deviation distribution (NDSDD), th nuclear area (NAD) and standard deviation distribution (NASDD), and the nuclear/cytoplasmic ratio (NCR) were assessed on May-Grunwald-Giemsa stained smears. Statistical analysis was performed by use of one-way analysis of variance (ANOVA) of the two groups as identified by histology. Whilst NDD (P = 0.007), NAD (P = 0.015) and NADSD (P = 0.026) all appeared statistically significant, NDSD (P = 0.06) and NCR (P = 0.71) were not. The cytological diagnosis of papillary carcinoma is established and reproducible, but morphometric data on the thyroid have so far focused on the differential diagnosis between benign and malignant nodules. The choice of simple morphometric parameters appears to be helpful in the preoperative distinction between the classical pattern and tall cell variant of papillary carcinoma.     

Relationship between cell size and nuclear volume in nucleated red blood cells of developmentally matched diploid and tetraploid mouse embryos.

Analysis of control diploid and polyploid amphibia indicated that cell and nuclear volumes were closely related to their ploidy, so that an increase in ploidy was generally associated with an increase in cell size. This relationship is also believed to occur in mammalian polyploids. However, since the latter are only rarely encountered spontaneously, or only occasionally following experimental manipulation, no detailed morphometric studies have been carried out to date to confirm whether such a relationship exists. In this study, the cellular and nuclear volume of primitive red blood cells was analyzed in carefully developmentally matched control diploid mouse embryos and tetraploid embryos produced by the technique of electrofusion. All of the cells and/or nuclei studied had a characteristic spherical shape which greatly simplified the morphometric analysis. A defined and predictable relationship between ploidy and cellular and/or nuclear volume was observed in the red blood cells between 8.25 and 14.5 days of gestation. During this period the primitive red blood cells are gradually replaced by the definitive erythrocytes. The ratio of control values to tetraploid values was found to be close to the theoretical value of 1:2 when comparable cells and/or their nuclei were analyzed in carefully developmentally matched material.     

Morphometric and functional study of apoptotic cell chromatin

Apoptosis is usually characterized by profound morphological nuclear changes. Chromatin undergoes a progressive condensation that eventually involves all the nucleus. At earlier stages chromatin appears as divided in compact and diffuse areas, while the nuclear pores disappear from the nuclear envelope that surrounds the compact areas, and cluster around diffuse chromatin. Here we have performed a morphometric study on the different chromatin areas of freeze-fractured apoptotic cell nuclei in order to investigate its morphometric and functional organization. We have found large portions of inactive chromatin aggregations corresponding to the dense cap-shaped patches, while domains of nucleosomic fibres have been identified in the diffuse chromatin areas. The correlation of the nucleosomic fibre/diffuse chromatin domain with the nuclear pore clusters is demonstrated, and its implications with a possible residual nuclear activity are discussed.