DNA image cytometry parameters to identify high-grade cervical lesions.

OBJECTIVE: Evaluate the performance of different DNA image cytometry (DNA-ICM) ploidy parameters in the categorisation of DNA-ICM results and identification of high-grade cervical intraepithelial neoplasia or worse (≥ CIN2). METHODS: Cervical samples from 232 women were collected for DNA-ICM analysis and biopsy confirmation. Five DNA parameters were used to define DNA aneuploidy: number of cells with exceeding events (EE) over 2.5cEE, 4cEE, 5cEE and 9cEE, and aneuploid stemlines. DNA-ICM results were categorised as normal, suspicious, and abnormal. RESULTS: For individual DNA ploidy parameters, sensitivity values for 50 cells with 2.5cEE, 45 cells with 4cEE, 1 cell with 9cEE and aneuploid stemline were 72.95%. 54.1%, 69.67% and 54.1%, while specificity values were 80.0%, 90.0%, 89.09% and 95.45%, respectively. For the 5cEE parameter, the sensitivity values for 1, 2, 3, 4 and 5 cells were 93.44%, 85.25%, 81.97%, 77.87% and 75.41%, while specificity values were 46.36%, 63.64%, 74.55%, 76.36% and 80.91%, respectively. For categorised DNA-ICM results, a suspicious result showed superior sensitivity than an abnormal result (87.70% vs 82.79%, P = 0.031), but lower specificity (54.55% vs 75.45%, P < 0.001). Both types of DNA-ICM result were statistically significantly different from a normal result (P < 0.05). CONCLUSION: For prognostic purposes, 1 cell with 9cEE, 45 cells with 4cEE and aneuploid stemline are the best parameters with which to categorise an abnormal DNA-ICM result, followed by 50 cells with 2.5cEE and 4 cells with 5cEE. For screening purposes, 10 cells with 2.5cEE, 10 cells with 4cEE, and 2 cells with 5cEE are suitable parameters with which to categorise a suspicious DNA-ICM result.

Histomorphometric analysis of liver biopsies of treated patients with Gaucher disease type 1

Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by a disturbance in the metabolism of glucocerebroside in the macrophages. Most of its manifestations - hepatosplenomegaly, anemia, thrombocytopenia, and bone pain - are amenable to a macrophage-target therapy such as enzyme replacement. However, there is increasing evidence that abnormalities of the liver persist despite the specific GD treatment. In this work, we adapted histomorphometry techniques to the study of hepatocytes in GD using liver tissue of treated patients, developing the first morphometrical method for canalicular quantification in immunohistochemistry-stained liver biopsies, and exploring histomorphometric characteristics of GD. This is the first histomorphometric technique developed for canalicular analysis on histological liver biopsy samples.

Dengue Fever, COVID-19 (SARS-CoV-2), and Antibody-Dependent Enhancement (ADE): A Perspective.

SARS-CoV-2 pandemic and recurrent dengue epidemics in tropical countries have turned into a global health threat. While both virus-caused infections may only reveal light symptoms, they can also cause severe diseases. Here, we review the possible antibody-dependent enhancement (ADE) occurrence, known for dengue infections, when there is a second infection with a different virus strain. Consequently, preexisting antibodies do not neutralize infection, but enhance it, possibly by triggering Fcγ receptor-mediated virus uptake. No clinical data exist indicating such mechanism for SARS-CoV-2, but previous coronavirus infections or infection of SARS-CoV-2 convalescent with different SARS-CoV-2 strains could promote ADE, as experimentally shown for antibodies against the MERS-CoV or SARS-CoV spike S protein. © 2020 International Society for Advancement of Cytometry.

Image cytometry DNA ploidy analysis: Correlation between two semi-automated methods.

BACKGROUND AND OBJECTIVES: Quantitation of cell DNA content, DNA ploidy, has been established as a research and prognostic technique for decades. A variety of instruments have been used although only a few commercially available systems have established quality assurance and published outcome data. The aim of this study was to compare two automated systems. METHODS: Nuclear monolayers were obtained from 112 oral biopsies by enzyme digestion and Feulgen staining. These were scanned on both the Fairfield and the Ploidy Work Station (PWS) systems. The overall ploidy diagnosis, number of epithelial nuclei, coefficient of variation (CV) and 5c exceeding rate (5CER) were compared by quantile-quantile plots, t test, Wilcoxon and Spearman's tests. RESULTS: The PWS system identified more nuclei (p < 0.0001) at a lower CV (p < 0.0001). Using the PWS system, fewer samples were classified as indeterminate. No difference between 5CER was found between systems (p > 0.54). There was complete concordance between the two systems in terms of DNA ploidy diagnosis. CONCLUSIONS: The PWS system is comparable to the Fairfield system for determination of DNA ploidy and has advantages that may lead to improved performance.

Applications of Aptamers in Flow and Imaging Cytometry.

Aptamers compete with antibodies in many applications, in which high-affinity and specificity ligands are needed. In this regard, fluorescence-tagged aptamers have gained applications in flow and imaging cytometry for detecting cells expressing distinct antigens. Here we present prospective methods, as a starting point, for using these high-affinity ligands for cytometry applications.

Adaptation of image cytometry methodology for DNA ploidy analysis of cervical epithelium samples: a pilot study.

OBJECTIVE: To determine DNA ploidy in the cervical specimens of patients revealing a suspicion of cancer by image analysis performed by using a combination of commercial analysis software, conventional microscopy, and certified filters. MATERIALS AND METHODS: This study followed a prospective design. Cervical samples were obtained from 20 patients undergoing routine screening in the Gynecologic-Oncology Unit of the University Hospital of the Federal University of Minas Gerais, Brazil. Three slides were prepared for each case and the DNA content was determined by image cytometry, post Feulgen staining. DNA ploidy, as well as events exceeding 5C and 9C, was assessed according to the guidelines and algorithms prescribed for diagnostic interpretation by the European Society for Analytical Cellular Pathology. RESULTS: By employing the adapted tool, identification of the lesions with euploid and aneuploid profiles was possible. Abnormal DNA content was found in 65% of the cases (13/20), with 45% (9/20) presenting nuclei with >5C content and 20% (4/20) with >9C content. In the analyses conducted in this study, the coefficient of variation with respect to DNA quantity was lower than the 5% threshold recommended by the European Society for Analytical Cellular Pathology. CONCLUSION: Image cytometry of the cervical specimens revealed DNA aneuploidy, most probably resulting from chromosomal alterations and appearing as precancerous lesions in 65% of the cases. The adaptations implemented in this study, enabled the DNA-image cytometry to become more accessible, enhancing its extended use as an adjuvant strategy for the early screening of the cervical epithelium samples during routine analyses.

Cytometry in the brain: studying differentiation to diagnostic applications in brain disease and regeneration therapy.

During brain development, a population of uniform embryonic cells migrates and differentiates into a large number of neural phenotypes - origin of the enormous complexity of the adult nervous system. Processes of cell proliferation, differentiation and programmed death of no longer required cells, do not occur only during embryogenesis, but are also maintained during adulthood and are affected in neurodegenerative and neuropsychiatric disease states. As neurogenesis is an endogenous response to brain injury, visible as proliferation (of to this moment silent stem or progenitor cells), its further stimulation can present a treatment strategy in addition to stem cell transfer for cell regeneration therapy. Concise techniques for studying such events in vitro and in vivo permit understanding of underlying mechanisms. Detection of subtle physiological alterations in brain cell proliferation and neurogenesis can be explored, that occur during environmental stimulation, exercise and ageing. Here, we have collected achievements in the field of basic research on applications of cytometry, including automated imaging for quantification of morphological or fluorescence-based parameters in cell cultures, towards imaging of three-dimensional brain architecture together with DNA content and proliferation data. Multi-parameter and more recently in vivo flow cytometry procedures, have been developed for quantification of phenotypic diversity and cell processes that occur during brain development as well as in adulthood, with importance for therapeutic approaches.

Evaluation epidermal p53 immunostaining by digital image analysis.

BACKGROUND/PURPOSE: Digital techniques have been developed and validated to assess semiquantitatively immunohistochemical nuclear staining. Currently visual classification is the standard for qualitative nuclear evaluation. Analysis of pixels that represents the immunohistochemical labeling can be more sensitive, reproducible and objective than visual grading. This study compared two semiquantitative techniques of digital image analysis with three techniques of visual analysis imaging to estimate the p53 nuclear immunostaining. METHODS: Sixty-three sun-exposed forearm-skin biopsies were photographed and submitted to three visual analyses of images: the qualitative visual evaluation method (0 to 4 + ), the percentage of labeled nuclei and HSCORE. Digital image analysis was performed using ImageJ 1.45p; the density of nuclei was scored per ephitelial area (DensNU) and the pixel density was established in marked suprabasal epithelium (DensPSB). RESULTS: Statistical significance was found in: the agreement and correlation among the visual estimates of evaluators, correlation among the median visual score of the evaluators, the HSCORE and the percentage of marked nuclei with the DensNU and DensPSB estimates. DensNU was strongly correlated to the percentage of p53-marked nuclei in the epidermis, and DensPSB with the HSCORE. CONCLUSION: The parameters presented herein can be applied in routine analysis of immunohistochemical nuclear staining of epidermis.

Using Cell-ID 1.4 with R for microscope-based cytometry.

This unit describes a method for quantifying various cellular features (e.g., volume, total and subcellular fluorescence localization) from sets of microscope images of individual cells. It includes procedures for tracking cells over time. One purposely defocused transmission image (sometimes referred to as bright-field or BF) is acquired to segment the image and locate each cell. Fluorescence images (one for each of the color channels to be analyzed) are then acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses the image-processing capabilities of Cell-ID and data analysis by the statistical programming framework R, which is supplemented with a package of routines for analyzing Cell-ID output. Both Cell-ID and the analysis package are open-source.

C-value reassessment of plant standards: an image cytometry approach.

Image cytometry (ICM) has been used to measure DNA 2C-values by evaluating the optical density of Feulgen-stained nuclei. This optical measurement is carried out using three basic tools: microscopy, digital video camera, and image analysis software. Because ICM has been applied to plants, some authors have remarked that studies should be performed before this technique can be accepted as an accurate method for determination of plant genome size. Based on this, the 2C-value of eight plants, which are widely used as standards in DNA quantifications, was reassessed in a cascade-like manner, from A. thaliana through R. sativus, S. lycopersicum, Glycine max, Z. mays, P. sativum, V. faba, to A. cepa. The mean 2C-values of all plants were statistically compared to the values reported by other authors using flow cytometry and/or ICM. These analyses demonstrated that ICM is an accurate and reliable method for 2C-value measurement, representing an attractive alternative to flow cytometry. Statistical comparison of the results also indicated Glycine max 'Polanka' as the most adequate primary standard. However, distinct authors have been advised that 2C DNA content of the reference standard should be close to that of the sample. As three further approaches also revisited the 2C-value of these eight plants, we have thus proposed a mean 2C-value for each eight species.

Direct concentration and viability measurement of yeast in corn mash using a novel imaging cytometry method.

Worldwide awareness of fossil-fuel depletion and global warming has been increasing over the last 30 years. Numerous countries, including the USA and Brazil, have introduced large-scale industrial fermentation facilities for bioethanol, biobutanol, or biodiesel production. Most of these biofuel facilities perform fermentation using standard baker's yeasts that ferment sugar present in corn mash, sugar cane, or other glucose media. In research and development in the biofuel industry, selection of yeast strains (for higher ethanol tolerance) and fermentation conditions (yeast concentration, temperature, pH, nutrients, etc.) can be studied to optimize fermentation performance. Yeast viability measurement is needed to identify higher ethanol-tolerant yeast strains, which may prolong the fermentation cycle and increase biofuel output. In addition, yeast concentration may be optimized to improve fermentation performance. Therefore, it is important to develop a simple method for concentration and viability measurement of fermenting yeast. In this work, we demonstrate an imaging cytometry method for concentration and viability measurements of yeast in corn mash directly from operating fermenters. It employs an automated cell counter, a dilution buffer, and staining solution from Nexcelom Bioscience to perform enumeration. The proposed method enables specific fluorescence detection of viable and nonviable yeasts, which can generate precise results for concentration and viability of yeast in corn mash. This method can provide an essential tool for research and development in the biofuel industry and may be incorporated into manufacturing to monitor yeast concentration and viability efficiently during the fermentation process.

Image cytometry: nuclear and chromosomal DNA quantification.

Image cytometry (ICM) associates microscopy, digital image and software technologies, and has been particularly useful in spatial and densitometric cytological analyses, such as DNA ploidy and DNA content measurements. Basically, ICM integrates methodologies of optical microscopy calibration, standard density filters, digital CCD camera, and image analysis softwares for quantitative applications. Apart from all system calibration and setup, cytological protocols must provide good slide preparations for efficient and reliable ICM analysis. In this chapter, procedures for ICM applications employed in our laboratory are described. Protocols shown here for human DNA ploidy determination and quantification of nuclear and chromosomal DNA content in plants could be used as described, or adapted for other studies.

Morphologic and morphometric evaluation of experimental acute crush injuries of the sciatic nerve of rats.

In order to qualify and quantify nerve fiber lesion following an acute crush injury, a morphologic and morphometric study was carried out in 25 Wistar rats divided into five groups of five animals each according to the crushing load applied, i.e., 500, 1,000, 5,000, 10,000, and 15,000 g. The injury was produced under general anesthesia on a 5mm-long intermediate segment of the right sciatic nerve for 10 min using a dead-weight machine. The animals were killed with an excessive dose of anesthetics 72 h later and submitted to perfusion with a fixing solution through the abdominal aorta immediately after death. Both the right and left sciatic nerves were removed and prepared for histologic and morphometric examinations; 5 microm-thick sections stained with 1% Toluidine blue were examined under a light microscope equipped with a video camera linked to a computer loaded with a graphic program (KS 400). The morphometric studies included measuring total number of fibers, fiber density, fiber diameter, myelin fiber area, axon diameter, axon area and G ratio. The results showed that damage to the nerve fibers began to appear as early as with the 500 g load and was similar in all groups despite the load applied, increasing with the 10,000 and 15,000 g loads, although the external supporting tissues and small diameter fibers were preserved. The predominant type of lesion produced was axonotmesis.

Análise da DNA-ploidia em carcinoma lobular de mama

O carcinoma mamário apresenta elevados números relacionados à incidência e prevalência nas mulheres em vários países. No Brasil, é a neoplasia com um dos maiores índices de mortalidade. O tipo de carcinoma mamário mais freqüente é o ductal invasor seguido do carcinoma lobular. Embora essas neoplasias tenham características próprias, há ainda muitas questões a serem dirimidas. Sabe-se que os carcinomas lobulares apresentam alto risco de desenvolvimento bilateral e recorrência; estão relacionadas à expressão hormonal e têm desfecho lento. Sabe-se que o carcinoma lobular não tem positividade para a proteína E-caderina, cuja ausência de expressão imuno-histoquímica tem sido usada para diferenciá-la do carcinoma ductal. O padrão do conteúdo de DNA no carcinoma lobular tem sido objeto de controvérsias, com trabalhos descrevendo-o como predominantemente diplóide e outros, ao contrário, aneuplóide. O objetivo deste estudo foi analisar os padrões da DNA-ploidia em carcinomas lobulares invasivos de mama, E-caderina negativos, e correlacioná-los com fatores prognósticos de notória importância: expressão da proteína p53, Cerb-B2, receptores de estrógeno, tamanho dos tumores, comprometimento linfonodal, metástases a distância e pós-cirúrgica. Para isso foi realizado um estudo retrospectivo, com 50 casos examinados no Departamento de Anatomia Patológica do Hospital do Câncer A. C. Camargo, São Paulo-SP. Essas mulheres foram tratadas com cirurgia e as que apresentaram comprometimento axilar, receberam terapia adjuvante com quimioterapia, radioterapia e hormônioterapia. As medianas dos tempos de seguimento, com início no tratamento inicial (cirurgia) até o evento foi: grupo que foram a óbito 50 ± 27,6 meses; grupo com perda de seguimento 33 ± 56,0 meses; grupo de censura por interrupção do tempo para análise 79 ± 45,3 meses. A idade média foi de 54 anos (variando de 34 a 80 anos). A análise ...

Análise da DNA-ploidia em carcinoma lobular de mama / DNA content analysis of breast lobular invasive carcinoma

O carcinoma mamário apresenta elevados números relacionados à incidência e prevalência nas mulheres em vários países. No Brasil, é a neoplasia com um dos maiores índices de mortalidade. O tipo de carcinoma mamário mais freqüente é o ductal invasor seguido do carcinoma lobular. Embora essas neoplasias tenham características próprias, há ainda muitas questões a serem dirimidas. Sabe-se que os carcinomas lobulares apresentam alto risco de desenvolvimento bilateral e recorrência; estão relacionadas à expressão hormonal e têm desfecho lento. Sabe-se que o carcinoma lobular não tem positividade para a proteína E-caderina, cuja ausência de expressão imuno-histoquímica tem sido usada para diferenciá-la do carcinoma ductal. O padrão do conteúdo de DNA no carcinoma lobular tem sido objeto de controvérsias, com trabalhos descrevendo-o como predominantemente diplóide e outros, ao contrário, aneuplóide. O objetivo deste estudo foi analisar os padrões da DNA-ploidia em carcinomas lobulares invasivos de mama, E-caderina negativos, e correlacioná-los com fatores prognósticos de notória importância: expressão da proteína p53, Cerb-B2, receptores de estrógeno, tamanho dos tumores, comprometimento linfonodal, metástases a distância e pós-cirúrgica. Para isso foi realizado um estudo retrospectivo, com 50 casos examinados no Departamento de Anatomia Patológica do Hospital do Câncer A. C. Camargo, São Paulo-SP. Essas mulheres foram tratadas com cirurgia e as que apresentaram comprometimento axilar, receberam terapia adjuvante com quimioterapia, radioterapia e hormônioterapia. As medianas dos tempos de seguimento, com início no tratamento inicial (cirurgia) até o evento foi: grupo que foram a óbito 50 ± 27,6 meses; grupo com perda de seguimento 33 ± 56,0 meses; grupo de censura por interrupção do tempo para análise 79 ± 45,3 meses. A idade média foi de 54 anos (variando de 34 a 80 anos). A análise do conteúdo de DNA mostrou-se predominantemente aneuplóide (63,16 % dos casos). Somente o comprometimento linfonodal mostrou-se significativo (p=0,043) em relação a análise de DNA-ploidia, os demais parâmetros não mostraram associação significativa. Concluímos que a análise do conteúdo de DNA não é um parâmetro relevante para avaliar a agressividade do carcinoma lobular.

Nucleus image properties assessed by video image analysis in mouse hepatocytes under a short lysis for extended chromatin fiber formation.

BACKGROUND: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. METHODS: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. RESULTS: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented 37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was evident in chromocenters of the fixed nuclei and in remnants and some ECF granules of the unfixed, lysed nuclei. CONCLUSIONS: The DNA content of the nuclear remnants was much more variable than that assumed from known variability in hepatocyte ploidy degrees. The variable constraint to chromatin extrusion from hepatocyte nuclei is hypothesized to depend on variable chromatin organization with possible involvement of nuclear matrix association, transcriptional activities, and AT-rich DNA-containing heterochromatin.

Immunohistochemistry quantification by a digital computer-assisted method compared to semiquantitative analysis.

PURPOSE: To compare immunostaining quantification obtained by a digital computer-assisted method with the well-established semiquantitative analysis. METHODS: Cytoplasmic staining of galectin-3 was obtained by standard immunohistochemical reactions in 25 cases of well-differentiated thyroid carcinoma. The expression index that associates the conventional area fraction of labeled cells with the immunostaining intensity score based on visual qualitative observation was used as the semiquantitative analysis. A digital computer-assisted method is described based on the use of an image processing program (ImageLab). Three parameters were obtained: (1) percentage of labeled cells; (2) digital immunostaining intensity, and (3) digital expression index. The proposed method allows numerical analysis of the immunostaining intensity. RESULTS: There was a strong correlation between the immunostaining intensity obtained by the two methods (Pearson correlation coefficient, r = 0.71, P = 0.0001). The same was observed between expression indexes (Pearson correlation coefficient, r = 0.66, P = 0.0001). CONCLUSION: Results obtained with our proposed digital computer-assisted method for immunoexpression analysis were concordant with the semiquantitative analysis. In addition, digital values can also resolve disagreement among different observers about the quality of staining intensity because the digital method does not classify the results into groups, but rather provides a numerical value for each individual case; thus, it increases the diagnostic and, more importantly, the prognostic sensitivity of the immunohistochemical analysis.

Immunohistochemistry quantification by a digital computer-assisted method compared to semiquantitative analysis

PURPOSE: To compare immunostaining quantification obtained by a digital computer-assisted method with the well-established semiquantitative analysis. METHODS: Cytoplasmic staining of galectin-3 was obtained by standard immunohistochemical reactions in 25 cases of well-differentiated thyroid carcinoma. The expression index that associates the conventional area fraction of labeled cells with the immunostaining intensity score based on visual qualitative observation was used as the semiquantitative analysis. A digital computer-assisted method is described based on the use of an image processing program (ImageLab®). Three parameters were obtained: (1) percentage of labeled cells; (2) digital immunostaining intensity, and (3) digital expression index. The proposed method allows numerical analysis of the immunostaining intensity. RESULTS: There was a strong correlation between the immunostaining intensity obtained by the two methods (Pearson correlation coefficient, r = 0.71, P = 0.0001). The same was observed between expression indexes (Pearson correlation coefficient, r = 0.66, P = 0.0001). CONCLUSION: Results obtained with our proposed digital computer-assisted method for immunoexpression analysis were concordant with the semiquantitative analysis. In addition, digital values can also resolve disagreement among different observers about the quality of staining intensity because the digital method does not classify the results into groups, but rather provides a numerical value for each individual case; thus, it increases the diagnostic and, more importantly, the prognostic sensitivity of the immunohistochemical analysis.

OBJETIVO: Comparar a quantificação da imunomarcação através de um método digital assistido por computador à bem estabelecida análise semiquantitativa. MÉTODO: A marcação citoplasmática de galectina-3 foi obtida por reações imunohistoquímicas em 25 casos de carcinoma bem-diferenciado da glândula tireóide. Determinou-se o índice de expressão da análise semiquantitativa que associa a convencional fração de área de células marcadas com os escores de intensidade da imunoexpressão, com base na observação visual qualitativa. O método digital assistido por computador foi desenvolvido com o uso de um programa de análise de imagem (ImageLab®). Três parâmetros foram obtidos: (1) porcentagem de células marcadas; (2) intensidade de imunoexpressão digital e (3) índice de expressão digital. O método proposto resulta na análise numérica da intensidade de imunoexpressão. RESULTADOS: Houve importante correlação entre as intensidades de imunoexpressão obtidas pelos dois métodos (coeficiente de correlação de Pearson, r=0,71, p=0,0001). O mesmo foi observado entre os índices de expressão (coeficiente de correlação de Pearson, r=0,66, p=0,0001). DISCUSSÃO: Os resultados de intensidade de imunoexpressão obtidos com o emprego do método digital assistido por computador foram concordantes com os escores da análise semiquantitativa. Entretanto, os resultados alcançados com o emprego do método digital podem resolver a discordância entre diferentes observadores com relação a esta intensidade de imunomarcação. Além disso, o método proposto não categoriza os resultados em grupos, o que torna a análise imunohistoquímica numericamente mensurável individualmente, aumentando seu poder diagnóstico e, sobretudo, prognóstico.

Digital image analysis and stereology of angiogenesis in polypoid and nonpolypoid colorectal adenomas.

OBJECTIVE: To evaluate 3-dimensional parameters and bidimensional microvascular quantification in the different morphologic presentations of colorectal adenomas. STUDY DESIGN: A study was carried out, including 102 neoplastic colorectal lesions obtained by endoscopy or surgical resection. For the analysis of angiogenesis, immunohistochemistry, digital image analysis, microvascular quantification and stereology were used. RESULTS: Microvascular quantification, volume and microvascular length estimate rose gradually with high grade dysplasia as compared to the low grade ones (P < .001). There was no significant difference in angiogenesis between polypoid and nonpolypoid colorectal adenomas in terms of quantification and microvascular length estimate. CONCLUSION: The use of digital image analysis and stereology added greater objectivity and effectiveness to angiogenic evaluation because they allowed accurate segmentation of hypervascular areas, representation of the characteristic 3-dimensional morphology of the vascular supply and identification of differences in microvascularization in the developmental stages of colorectal cancer. However, no significant relation could be found between macroscopic type and angiogenesis, suggesting that angiogenesis may contribute little to morphogenesis of colorectal adenomas.