Nuclear Perimeter Research Articles

Dysplasia discrimination in intestinal-type neoplasia of the esophagus and colon via digital image analysis.

Determining gastrointestinal tract dysplasia level is clinically important but can be difficult, and given this challenge, we investigated colonic and esophageal dysplastic progression using digital image analysis (IA). Whole slide images were obtained for colonic normal mucosa (NCM), hyperplastic polyps (HP), conventional tubular adenomas (TA), and adenomas with high-grade dysplasia (HGD), and esophageal intestinal metaplasia negative for dysplasia (IM), indefinite for dysplasia (IFD), low-grade dysplasia (LGD), and HGD. Characteristic nuclei were circumscribed, and parameters discriminating groups included nuclear circumference (μm), area (μm(2)), and 15 positive pixel count (PPC) algorithm IA measurements. In colon polyps and esophageal lesions, average nuclear area and circumference ranged 30-108.6μm(2) and 27.5-48.9μm, respectively. Differences for average nuclear area and circumference met statistical significance (p < 0.05) between diagnostic groups in the esophagus and colon, except for IM versus IFD nuclear area. Pixel intensity (brightness) separated lesions within both groups with statistical significance except for colonic TAs versus HPs and esophageal LGD versus IM. HGD nuclei in both groups demonstrated more pixel staining heterogeneity than other lesions. Hierarchical clustering and principal component analysis demonstrated that lesions with similar diagnoses tended to cluster together on a low- to high-grade spectrum. Our results confirm that quantitative IA is an effective adjunct reflecting dysplasia in colon polyps and Barrett esophagus lesions. Nuclear area, circumference, and PPC algorithm findings distinguished lesions in a statistically significant manner. This suggests utility for future studies on similar methods, which may provide an adjunctive ancillary technique for pathologists and enhance patient care.

Morphometric Analysis of Thyroid Follicular Cells with Atypia of Undetermined Significance.

Background:Atypia of undetermined significance (AUS) is a category that encompasses a heterogeneous group of thyroid aspiration cytology. It has been reclassified into two subgroups based on the cytomorphologic features: AUS with cytologic atypia and AUS with architectural atypia. The nuclear characteristics of AUS with cytologic atypia need to be clarified by comparing to those observed in Hashimoto thyroiditis and benign follicular lesions.Methods:We selected 84 cases of AUS with histologic follow-up, 24 cases of Hashimoto thyroiditis, and 26 cases of benign follicular lesions. We also subcategorized the AUS group according to the follow-up biopsy results into a papillary carcinoma group and a nodular hyperplasia group. The differences in morphometric parameters, including the nuclear areas and perimeters, were compared between these groups.Results:The AUS group had significantly smaller nuclear areas than the Hashimoto thyroiditis group, but the nuclear perimeters were not statistically different. The AUS group also had significantly smaller nuclear areas than the benign follicular lesion group; however, the AUS group had significantly longer nuclear perimeters. The nuclear areas in the papillary carcinoma group were significantly smaller than those in the nodular hyperplasia group; however, the nuclear perimeters were not statistically different.Conclusions:We found the AUS group to be a heterogeneous entity, including histologic follow-up diagnoses of papillary carcinoma and nodular hyperplasia. The AUS group showed significantly greater nuclear irregularities than the other two groups. Utilizing these features, nuclear morphometry could lead to improvements in the accuracy of the subjective diagnoses made with thyroid aspiration cytology.

Morphometric and cytomorphologic characterization of EGFR-mutated cancer cells-comparison between cultured lung cancer cell lines and lung adenocarcinoma clinical samples.

Recently molecular targeting therapies such as inhibition of enzyme activities associated with gene mutations responsible for lung carcinogenesis have been demonstrating promising outcomes, increasing the importance of gene analysis using clinical samples. Cytomorphologic findings with predictive value toward specific gene mutation such as EGFR mutation could be a useful tool to select appropriate gene analyses using limited clinical samples. Morphometrical and cytomorphological evaluations were performed in 7 cultured lung cancer cell lines and 51 lung adenocarcinoma clinical samples to identify specific cytomorphologic characterization of EGFR-mutated cancer cells compared to the wild type. Morphometry demonstrated that the EGFR mutated cell lines had significantly smaller nuclear area and perimeter and more circular nuclei compared to the wild type. In contrast, EGFR-mutated clinical samples had significantly greater nuclear area and perimeter compared to the wild type EGFR samples. There were no clear differences in cytomorphologic parameters assessing nuclear atypicality between EGFR mutated cells and wild type EGFR cells in either cultured cell lines or clinical samples. Although our study suggested that EGFR mutation may have specific effects on nuclear morphology, no consistent characteristics of EGFR-mutated cells were identified in the clinical samples, probably due to various factors such as different pathologic stages and various incidences of lepidic growth. Further assessment of morphological characterization of EGFR-mutated cells in lung adenocarcinoma is warranted, increasing the number of samples and considering the effects of polyploidy, other gene mutations, pathology stage and tumor subtypes such as lepidic growth. Diagn. Cytopathol. 2016;44:717-724. © 2016 Wiley Periodicals, Inc.

Morphologic changes in the pineal gland of rats exposed to continuous darkness

We examined the pineal structure of rats exposed to constant darkness (DD) at light microscopic level. Two groups of rats were exposed to 12:12 light/dark cycle (LD) or DD from their prenatal ontogenesis and then for 3 months after birth. The gland structure of DD rats was observed to have an active appearance. Some of the observed pinealocytes with light nuclei from DD rats were determined to contain double nucleoli. Nuclear area and perimeter of both dark and light types were greater in rats kept in DD than in LD. Rats exposed to DD had more cells with light nuclei and lesser cells with dark ones than rats kept in LD. No significant differences in nuclear characteristics of intermediate type were found between rats kept in LD and those kept in DD. The activity of mammalian pineal can be altered by light conditions to which the animal is exposed.

Abstract 12665: Remodeling of Nuclear Ca 2+ Disposition in Atrial Fibrillation Associated Remodeling

Introduction: Atrial fibrillation (AF) induces substantial self-promoting remodeling related to the rapid atrial rate. There is evidence that changes in nuclear Ca 2+ content alter ion channel gene expression and thereby effect electrophysiological changes that increase AF vulnerability and maintenance. However, the ways in which AF affects atrial cardiomyocyte (ACM) nuclear Ca 2+ disposition are unknown. Here, we studied the effects of AF-related remodeling on ACM nuclear structure and Ca 2+ handling. Methods and Results: ACMs were isolated from control (CTL) dogs and dogs subjected to 1 week of AF maintained by atrial tachypacing at 600 bpm (n=7, 6 respectively). We used nuclear imaging with Fluo-5N AM and nucleoplasmic and cytoplasmic calcium transient (CaT) recordings from intact ACMs loaded with Fluo-4 AM. AF significantly decreased the number of nuclear invaginations (NIV) normalized to nuclear envelope (NE) circumference, and increased nuclear length and perimeter in ACMs (Figure A). Diastolic [Ca 2+ ] nuc and [Ca 2+ ] cyto increased in AF vs CTL (Figure B, by 110%*, 89%* respectively, *P&lt;0.05). Nucleoplasmic Ca 2+ transient amplitude decreased significantly with AF, but simultaneously measured cytoplasmic transient amplitude did not change (Figure C). Relaxation time from peak of the Ca 2+ transient to 50% decline (TD 50 ) of nucleoplasmic and cytoplasmic CaTs were significantly prolonged in AF. The addition of a physiological nuclear Ca 2+ transient enhancer, endothelin (200 nM), increased diastolic [Ca 2+ ] nuc and [Ca 2+ ] cyto in both CTL (by 25±6%, 21±6%) and AF (by 40±4%, 31±2 ) ACMs. The IP3 receptor blocker 2-aminoethoxydiphenyl borate (2-APB, 5 μM) abolished endothelin effects on [Ca 2+ ] nuc . Conclusions: AF-related remodeling alters atrial cardiomyocyte nuclear structure and nucleoplasmic [Ca 2+ ] handling. These alterations may play an important role in associated gene-expression changes that produce the AF substrate.

Acute exposure to bisphenol A and cadmium causes changes in the morphology of gerbil ventral prostates and promotes alterations in androgen-dependent proliferation and cell death.

Bisphenol A (BPA) and cadmium (Cd) are environmental pollutants that are implicated in potential reproductive effects, including damage to the prostate gland. Their action during puberty requires analysis to determine the relationship of these compounds with the testosterone peak that occurs during this phase. This study evaluated whether exposure to BPA and Cd during puberty can cause changes in the morphology, proliferation and cell death and androgen receptor (AR) immunostaining of the ventral prostates of normal and castrated male gerbils (Meriones unguiculatus), considering an acute exposure to the chemicals and evaluation after short (52d) and long (120d) periods. Generally, morphometric-stereological results demonstrated that administration of BPA and Cd (individually or in combination) increased epithelial height, smooth muscle layer (SML) thickness and nuclear area and perimeter, and that these parameters were reduced in castrated animals. In addition, these groups showed important inflammatory processes but not prostate lesions. The proliferation/death rates of prostatic cells obtained by PCNA and TUNEL immunostaining demonstrated increased cell death in the 52d groups; in contrast, the gland acquired a more proliferative nature in the 120d groups. AR immunostaining showed that BPA and Cd compounds interact with ARs in different ways depending on the evaluated period and the hormonal profile of the animal. We conclude that BPA and cadmium are important agents in changing the morphology, proliferation and death of prostatic cells, in addition to interacting with ARs in different patterns. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 48-61, 2017.

Nuclear morphometric study of prostatic atrophy

Abstract The prostatic atrophy is a lesion with small glands that can mimic adenocarcinoma, commonly diagnosed in the elderly and whose preneoplastic role is controversial. Over time several classifications have been developed for this lesions according to its architectural and cellular aspects. Materials and Methods: Aim of the study is morphometrically assessment of epithelial component nuclei in prostatic atrophy. We evaluated parameters represented by nuclear area (NA), nuclear perimeter P (μm) Elongation factor (E), average diameter (DEQ), maximum diameter (Dmax), Minim diameter (Dmin). The control group was represented by adenomatous hyperplasia component of benign hyperplasia of the prostate. Results: Morphometric analysis revealed significantly higher values of nuclear parameters in atrophy compared with adenomatous component in controls (p &lt;0.05) which can signify nucleomegaly and a potential preneoplastic role.

Morphometric Characterization of Marginal Zone Lymphoma

Abstract The morphometry in histopathology is used to characterize cell populations belonging to different tissues and to identify differences in their parameters with prognostic implications. To achieve morphometric examination were selected 6 of 8 cases identified as marginal zone lymphoma. For each case analysis was done on five fields, for each field measuring the parameters of 20 cells. The studied parameters were for cytoplasm: cytoplasmic area, maximum and minimum cytoplasmic diameter, cytoplasmic perimeter; for nucleus were measured: nuclear area, minimum and maximum nuclear diameter, nuclear perimeter, nuclear contour index, nuclear ellipticity index, nuclear irregularity index. Also the nucleo-cytoplasmic ratio was calculated in all studied cases. Marginal zone lymphoma is characterized in terms of morphometric parameters by small cytoplasmic and nuclear area, and small nucleo-cytoplasmatic ratio of about 1:1. Nuclear contour index is small, accompanied by a large ellipticity index and an small index of nuclear irregularity. Standard deviations for measured morphometric parameters are variable, having high values for cytoplasmic and nuclear area, highlighting the polymorphic nature of the cells. Morphometric aspects, with corresponding microscopic aspects of large and small lymphocyte proliferation with cleaved and uncleaved nuclei, fit this form of lymphoma in terms of clinical outcome in indolent lymphomas category.

Assessment of proliferative potential of tumor cells using KI-67 expression and morphometrical analysis for prognostication of oral squamous cell carcinomas

Background: Cancer cells exhibit a characteristic intrinsic ability of uncontrolled cell proliferation which plays a vital role in tumor development and progression. The Ki-67 antigen expression is strictly associated with cell proliferation which detects the cells entering the cell cycle. In addition, computer aided image analysis provides an objective and highly reproducible histological evaluation of OSCC. Assessment of Ki-67 expression along with morphometry may help in early and precise diagnosis and prognostication of OSCC. Aims: To correlate the ki-67 expression and the morphometrical parameters of oral squamous cell carcinomas and to assess the efficacy of the correlation in OSCC prognostication. Methods and material: OSCC cases (n=105) were examined immuno histochemically using Ki-67 antigen The nuclear area (NA), cell area (CA), nuclear perimeter (NP) and cell perimeter (CP) assessed using “image J 1.34 software”. The data was statistically analyzed. Results: Highly significant correlation was found between Ki-67 expression and the advancing grades of OSCC (p<.0001). NA (p=0.025), CA (p<.0001), NP (p=.027) and CP (p<.0001) of poorly differentiated OSCC were significantly lower than well differentiated and moderately differentiated OSCC. Follow up analysis revealed nuclear area and cell area to be higher in the recurrent cases than the non recurrent ones. Conclusion: Assessment of Ki- 67 expression and morphometry help in early and precise diagnosis and prognostication of OSCC. A correlation between Ki-67 expression and morphometrical analysis could not be ascertained in this study. Further studies with a larger sample number may provide more definitive results.

Diagnostic application of computerised nuclear morphometric image analysis in fine needle aspirates of breast lesions

Background: Alterations in nuclear structure are the indications of cancer diagnosis. This study has thus focused on variation in nuclear morphometry in breast lesions. Aim: The aim of the study is to compare morphometric characteristics of different types of breast lesions and evaluate its utility in differentiating benign against malignant. Study Design: Sixty fine needle aspiration cytology (FNAC) of ethanol fixed samples were obtained and stained with Papanicolou stain. All diagnoses reported were confirmed histopathologically. Nuclear morphometric parameters for free cells in smears were calculated using the ImageJ 1.47 morphometric computer software. Result: The nuclear morphometric parameters including mean nuclear area, perimeter, diameter, long axis, and short axis were found to be statistically significant (ANOVA, P < 0.0001) in differentiating benign and malignant breast aspirates. Conclusion: Nuclear morphometry has been found to be a valuable objective method in differentiating benign and malignant breast lesions, especially in the grey zones, when diagnostic dilemmas are encountered. This study results demonstrate that the size-related parameters (area, perimeter, diameter, long, and short axes) of the nucleus are the most appropriate nuclear morphometric parameters for differentiating between benign lesions and infiltrative ductal carcinoma of the breast.

Cytomorphometric analysis of buccal mucosa cells in tobacco chewers

Objective: The objective of the present study was to assess the effect of tobacco chewing on buccal mucosa by using cytomorphometry. Study Design: The study group consisted of 100 subjects divided into 50 tobacco chewer subjects (Group C) and 50 control subjects (Group N) aged between 31 and 80 years. We analyzed and compared the nuclear and cell morphometric features, that is, mean nuclear area (MNA), mean nuclear perimeter (MNP), mean of maximum nuclear diameter (Max-ND), mean of minimum nuclear diameter (Min-ND), mean cell area (MCA), mean cell perimeter (MCP), mean of maximum cell diameter (Max-CD), mean of minimum cell diameter (Min-CD) and nuclear to cell parameter ratio. Buccal epithelial cells of these individuals were collected with a brush and fixed smears were stained with Papanicolaou stain and cytomorphometric analysis performed using Image J image analysis software. Results: The mean of above nuclear and cell morphometric parameters and their ratio were found to be statistically significant by Univariate analysis of variance (ANOVA) being P value < 0.001. Conclusions: There is progressive increase in nuclear parameters, decrease in cellular parameters and increase in ratio of nuclear to cellular parameters in smears from tobacco chewers, as compared with normal subjects. It is possible to conclude that these adaptive changes in the cellular and nuclear parameters tend to be a progression toward dysplastic or premalignant change.

Applicability of preoperative nuclear morphometry to evaluating risk for cervical lymph node metastasis in oral squamous cell carcinoma.

BackgroundWe previously reported the utility of preoperative nuclear morphometry for evaluating risk for cervical lymph node metastases in tongue squamous cell carcinoma. The risk for lymph node metastasis in oral squamous cell carcinoma, however, is known to differ depending on the anatomical site of the primary tumor, such as the tongue, gingiva, mouth floor, and buccal mucosa. In this study, we evaluated the applicability of this morphometric technique to evaluating the risk for cervical lymph node metastasis in oral squamous cell carcinoma.MethodsA digital image system was used to measure the mean nuclear area, mean nuclear perimeter, nuclear circular rate, ratio of nuclear length to width (aspect ratio), and nuclear area coefficient of variation (NACV). Relationships between these parameters and nodal status were evaluated by t-test and logistic regression analysis.ResultsEighty-eight cases of squamous cell carcinoma (52 of the tongue, 25 of the gingiva, 4 of the buccal mucosa, and 7 of the mouth floor) were included: 46 with positive node classification and 42 with negative node classification. Nuclear area and perimeter were significantly larger in node-positive cases than in node-negative cases; however, there were no significant differences in circular rate, aspect ratio, or NACV. We derived two risk models based on the results of multivariate analysis: Model 1, which identified age and mean nuclear area and Model 2, which identified age and mean nuclear perimeter. It should be noted that primary tumor site was not associated the pN-positive status. There were no significant differences in pathological nodal status by aspect ratio, NACV, or primary tumor site.ConclusionOur method of preoperative nuclear morphometry may contribute valuable information to evaluations of the risk for lymph node metastasis in oral squamous cell carcinoma.

Morphometric Characterization of Small Cell Lymphocytic Lymphoma

Abstract The morphometry in histopathology is used to characterize cell populations belonging to different tissues and to identify differences in their parameters with prognostic implications. To achieve morphometric examination were selected 6 of 24 cases identified as small cell lymphocytic lymphoma. For each case analysis was done on five fields, for each field measuring the parameters of 20 cells. The studied parameters were for cytoplasm: cytoplasmic area, maximum and minimum cytoplasmic diameter, cytoplasmic perimeter; for nucleus were measured: nuclear area, minimum and maximum nuclear diameter, nuclear perimeter, nuclear contour index, nuclear ellipticity index, nuclear irregularity index. Also the nucleocytoplasmic ratio was calculated in all studied cases. Small cell lymphocytic lymphoma is characterized in morphometric terms having a small cytoplasmic area (average 29.206) and also a small nuclear area (mean 28.939) having a nucleo-cytoplasmic ratio appearance suggestive for adult lymphocyte. A nuclear contour index small value (3.946), ellipticity index value also small (3.521) and small nuclear irregularity index (3.965). Standard deviations, in any of the studied morphometric categories, is around or below 1 suggesting monomorphic cell appearance. These morphometric and microscopic features characterized mainly by a small population of adult lymphocytes, monomorphic, with rounded hipercromic nuclei, dense chromatin, support the framing into indolent lymphoma group in terms of clinical outcome.

Atypical cell populations associated with acquired resistance to cytostatics and cancer stem cell features: the role of mitochondria in nuclear encapsulation.

Until recently, acquired resistance to cytostatics had mostly been attributed to biochemical mechanisms such as decreased intake and/or increased efflux of therapeutics, enhanced DNA repair, and altered activity or deregulation of target proteins. Although these mechanisms have been widely investigated, little is known about membrane barriers responsible for the chemical imperviousness of cell compartments and cellular segregation in cytostatic-treated tumors. In highly heterogeneous cross-resistant and radiorefractory cell populations selected by exposure to anticancer agents, we found a number of atypical recurrent cell types in (1) tumor cell cultures of different embryonic origins, (2) mouse xenografts, and (3) paraffin sections from patient tumors. Alongside morphologic peculiarities, these populations presented cancer stem cell markers, aberrant signaling pathways, and a set of deregulated miRNAs known to confer both stem-cell phenotypes and highly aggressive tumor behavior. The first type, named spiral cells, is marked by a spiral arrangement of nuclei. The second type, monastery cells, is characterized by prominent walls inside which daughter cells can be seen maturing amid a rich mitochondrial environment. The third type, called pregnant cells, is a giant cell with a syncytium-like morphology, a main nucleus, and many endoreplicative functional progeny cells. A rare fourth cell type identified in leukemia was christened shepherd cells, as it was always associated with clusters of smaller cells. Furthermore, a portion of resistant tumor cells displayed nuclear encapsulation via mitochondrial aggregation in the nuclear perimeter in response to cytostatic insults, probably conferring imperviousness to drugs and long periods of dormancy until nuclear eclosion takes place. This phenomenon was correlated with an increase in both intracellular and intercellular mitochondrial traffic as well as with the uptake of free extracellular mitochondria. All these cellular disorders could, in fact, be found in untreated tumor cells but were more pronounced in resistant entities, suggesting a natural mechanism of cell survival triggered by chemical injury, or a primitive strategy to ensure stemming, self-renewal, and differentiation under adverse conditions, a fact that may play a significant role in chemotherapy outcomes.

CERVICAL NEOPLASIA AND VIRUS LOAD OF HPV 16 GENOTYPE

Abstract. The 83 biopsies from HPV16-positive cervical epithelial neoplasies were studied by cytological, histological, immunomorphological methods and PCR. At cervical ectopia virus loading changed from 2.49 up to 6.65 lg copies of DNA HPV/105. At CIN II and CIN III the quantity of a virus exceeded 4 lg copies of DNA HPV/105 of cells, at the CIN I, Ca in situ and SCC — 5 lg copies of DNA HPV/105 of cells. At progressing of neoplasia frequency of revealing of clinically significant parameters of virus loading correlated with increase in average values of nuclear perimeter of cells, associated with HPV. At ectopia and LSIL expression of capsid protein L1 testified about virus production. At HSIL and SCC at absence expression L1 HPV high parameters of virus load testified to infringement of life cycle papillomaviruses.

Quantitative morphology in canine cutaneous soft tissue sarcomas.

Stained cytological specimens from 24 dogs with spontaneous soft tissue sarcomas [fibrosarcoma (n = 8), liposarcoma (n = 8) and haemangiopericytoma (n = 8)], and 24 dogs with reactive connective tissue lesions [granulation tissue (n = 12) and dermal fibrosis (n = 12)] were analysed by computer-assisted nuclear morphometry. The studied morphometric parameters were: mean nuclear area (MNA; µm(2)), mean nuclear perimeter (MNP; µm), mean nuclear diameter (MND mean; µm), minimum nuclear diameter (Dmin; µm) and maximum nuclear diameter (Dmax; µm). The study aimed to evaluate (1) possibility for quantitative differentiation of soft tissue sarcomas from reactive connective tissue lesions and (2) by using cytomorphometry, to differentiate the various histopathological soft tissue sarcomas subtypes in dogs. The mean values of all nuclear cytomorphometric parameters (except for Dmax) were statistically significantly higher in reactive connective tissue processes than in soft tissue sarcomas. At the same time, however, there were no considerable differences among the different sarcoma subtypes. The results demonstrated that the quantitative differentiation of reactive connective tissue processes from soft tissue sarcomas in dogs is possible, but the same was not true for the different canine soft tissue sarcoma subtypes. Further investigations on this topic are necessary for thorough explication of the role of quantitative morphology in the diagnostics of mesenchymal neoplasms and tumour-like fibrous lesions in dogs.

A morphometric analysis of the postanatal development of the choroid plexus epithelium in the male and female rat

Abstract Morphometric parameters of the lateral ventricle choroid plexus epithelial cells (average area, perimeter, bounding rectangle area, average nuclear area, nuclear perimeter, nuclear circularity and average nucleocytoplasmic ratio) were studied in postnatal and juvenile (10th, 16th and 38th postnatal days) 15 male and 15 female rats. The results were statistically analyzed by factorial ANOVA. Mean values of epithelial cells area, bounding rectangle area and perimeter were significantly higher in 16 days old, than in 10 and 38 days old rats. Opposite to this, the nucleocytoplasmic ratio was lower in the 16 days old, than in 10 and 38 days old rats. Average nuclear area and perimeter showed similar trends, while nuclear circularity increased from the 10th to the 38th day. Significant sex differences were in the epithelial cells area, bounding rectangle area and perimeter, being higher in males than in females in both 16 and 38 days groups. Nucleocytoplasmic ratio was higher in 10 days old male rats, but lower in 16 and 38 days old male rats. Generally, choroid epithelial cells size increased on the 16th and then decreased on the 38th day, but still remained higher compared to the 10th day. Nuclear size after increasing on day 16, also decreased on day 38, but to values lower than on day 10. The general decrease of nucleocytoplasmic ratio which accompanied these changes indirectly suggests a functional decrease. In the investigated period the male rat choroid epithelial cells were larger, but their nucleocytoplasmic ratio, which suggests the functional status, was lower than in females, indicating sex differences in the growth dynamics of the rat choroid plexus.

Objective assessment of changes in nuclear morphology and cell distribution following induction of apoptosis.

BackgroundTo objectively measure changes in nuclear morphology and cell distribution following induction of apoptosis.MethodsA spontaneously immortalized retinal pigment epithelial cell line (ARPE-19) was cultured for three days in DMEM/F12 with 10% fetal bovine serum followed by 24 hours incubation in staurosporine to induce apoptosis. Cells that were not incubated in staurosporine served as control. Caspase-3 expression in apoptotic cells was demonstrated by quantitative immunofluorescence. Nuclei were counterstained with DAPI. Assessments of nuclear morphology and cell distribution were performed using ImageJ software. Statistical analyses included Student’s t-test and Pearson’s correlation coefficient. Nearest neighbor analysis was used to assess cell nuclei distribution.ResultsCaspase-3 expression in staurosporine-incubated cells increased by 471% ± 182% compared to control (P = 0.014). Relative to the control, cells in the staurosporine-incubated cultures had smaller average nuclear area (68% ± 5%; P < 0.001) and nuclear circumference (78 ± 3%; P < 0.001), while nuclear form factor was larger (110% ± 1%; P < 0.001). Cell nuclei from the staurosporine-group (R = 1.12 ± 0.04; P < 0.01) and the control (R = 1.28 ± 0.03; P < 0.01) were evenly spaced throughout the cultures, thereby demonstrating a non-clustered and non-random cell distribution. However, the staurosporine-incubated group had a significantly lower R-value compared to the control (P = 0.002), which indicated a move towards cell clustering following induction of apoptosis. Caspase-3 expression of each individual cell correlated significantly with the following morphological indicators: circumference of the nucleus divided by form factor (r = -0.475; P < 0.001), nuclear area divided by form factor (r = -0.470; P < 0.001), nuclear circumference (r = -0.469; P < 0.001), nuclear area (r = -0.445; P < 0.001), nuclear form factor (r = 0.410; P < 0.001) and the nuclear area multiplied by form factor) (r = -0.377; P < 0.001).ConclusionsCaspase-3 positive apoptotic cells demonstrate morphological features that can be objectively quantified using freely available ImageJ software. A novel morphological indicator, defined as the nuclear circumference divided by form factor, demonstrated the strongest correlation with caspase-3 expression.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3271993311662947

Morphometric study of nuclei and microvessels in gliomas and its correlation with grades

IntroductionModifications of nuclear morphology in conjunction with alteration in microvascular configuration are essential features encountered during the progression of glial tumors. In order to gain more insight into tumor biology of gliomas, objectives of the study were selected (a) to correlate morphometrically evaluated nuclear parameters [nuclear area (NA), nuclear perimeter (NP), nuclear density (ND), percentage of total nuclear area (%TNA)] and microvessel parameters [microvessel density (MVD), microvessel caliber (VC), microvessel cross sectional area (VCSA), total microvessel boundary density (TVBD), percentage of total VCSA (%TVCSA)] with WHO grading; (b) extend such correlations to the ratio parameters: ratio of MVD to ND (MDV/ND), ratio of TVBD to %TNA (TVBD/%TNA) and ratio of %TVCSA to %TNA (%TVCSA/%TNA); and (c) to correlate microvessel and ratio parameters with NP and ND. Materials and methodsA total of thirty gliomas managed at this institute during 2009–2012 were evaluated for various nuclear and microvessel parameters by image morphometry using a computerized digital photomicrograph system. For assessment of microvessel parameters CD34-immunostained sections were used while nuclear morphometry was performed on routine hematoxylin-eosin-stained sections. Appropriate statistical analysis was performed in correlation studies. ResultsAll nuclear morphometric parameters showed strong positive correlation with tumor grades (r>0.7). In contrast, though all microvessel parameters exhibited positive correlation with grades, the parameters TVBD and %TVCSA showed strong positive correlation. The ratio parameters (MVD/ND) and (TVBD/%TNA) showed negative correlation with grades, whereas (%TVCSA/%TNA) did not exhibit meaningful correlation with grades. Further, while all microvessel parameters showed positive correlation with NP and ND; ratio parameters showed negative correlation with them. ConclusionThis study indicates that the parameters related to tumor growth (NA, NP, ND, %TNA), and angiogenesis showed increasing trend with tumor grades simultaneously; whereas the parameters related to supply of nutrients per nucleus showed decreasing trends with tumor grades, nuclear size and nuclear density. Thus, the former accounts for increased cellularity, mitosis, and vascular proliferation, while the latter culminates in tumor necrosis, all of which are essential components for grading of gliomas. The present study will therefore have a vital role as surrogate markers of grading of tumor.

In vitro assessment of sperm quality from rams of high and low field fertility

The aim of the present study was to investigate whether differences in field fertility of rams are reflected in differences in several sperm quality parameters. Ejaculates from 8 adult rams, 4 with high and 4 with low field fertility, were collected weekly using an artificial vagina over 6 consecutive weeks. Analyses of sperm motility by computer-assisted sperm analysis (CASA), membrane integrity by acridine orange-propidium iodide combination and sperm DNA fragmentation using the sperm chromatin dispersion test (SCD) were performed at 0, 3, 6 and 24h of incubation at 37°C. Sperm nuclear morphometry was also determined at 0h by computer-assisted sperm morphometry-fluorescence (CASMA-F). Sperm viability and most CASA sperm motility parameters were higher at 0, 3 and 6h in the high fertility rams. These rams had also a higher sperm nuclear area, perimeter and length (P<0.05) determined by CASMA-F. Significant differences between high and low fertility groups were also found in the dynamics in DNA fragmentation, with significant differences at 6h (14.42±1.40 and 20.27±1.77, respectively, P<0.05) and at 24h (22.32±2.03 and 31.24±2.54, respectively, P<0.01). It was concluded that high and low fertility rams present clear differences in several sperm quality parameters. This opens up the possibility of selection of males for artificial insemination based on sperm quality data.