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PubMed1998

Digitized image analysis reveals diffuse abnormalities in normal-appearing white matter during acute experimental autoimmune encephalomyelitis.

Demyelination of the central nervous system is a hallmark of multiple sclerosis and its widely used animal model, experimental autoimmune encephalomyelitis (EAE). Recent studies using magnetic resonance imaging and spectroscopy on multiple sclerosis patients have revealed abnormalities of central nervous system normal-appearing white matter suggesting that micro-demyelination and/or extensive membrane turnover accompanies and perhaps precedes the appearance of manifest inflammatory lesions. In the present study, we induced EAE in SWXJ mice and analyzed digitized images of immunocytochemically stained spinal cord for detection of myelin proteolipid protein (PLP). We found that digitized image analysis is a highly sensitive, objective methodology for measuring the extent of myelin loss during EAE. Our data show that two-thirds of the measured reduction of myelin PLP occurring in EAE spinal cord could be attributed to a loss of myelin in normal-appearing white matter. The marked decrease in detection of PLP was accompanied by a corresponding decrease in PLP mRNA in the central nervous system. Our results indicate that during acute EAE, diffuse myelin abnormalities extend far beyond visibly detectable inflammatory foci and are characterized by a global decrease in the expression of myelin genes and their encoded proteins.

Animals↗
PubMed1996

A generalized hermite pyramid for ultrasonic image analysis. I. Kidney and liver.

Ultrasonic images of the kidney and of the liver are subjected to a multiscale analysis in a generalized Hermite pyramid framework. The gradient images of the multiscale decompositions of the images of healthy and sick kidneys, and of the intraoperative and conventionally imaged livers, exhibit differences, in the structures of gray level regions, which can be interpreted by a medical doctor. These are used as inputs to an unsupervised classifier to automatically classify the images into homogeneous groups, which are found, in the case of the ultrasonic images examined, to correspond to the different physical characteristics of tissues of the organs under study. The main contribution of the paper is believed to be the multiscale tissue characterization along with its display in a manner that has utility as a diagnostic aid to the clinician.

Algorithms↗
PubMed1998

DNA ploidy determination of early molar pregnancies by image analysis: comparison to histologic classification.

BACKGROUND: To improve histologic diagnosis of molar pregnancies, updated guidelines have been proposed recently. These guidelines take into account that less developed molar gestations differ from their fully developed counterparts. OBJECTIVE: To test the validity of these criteria by correlating histologic diagnosis with ploidy determination accomplished by means of image analysis. DESIGN: Fifty archival cases of early molar pregnancy were reclassified according to the new criteria. The diagnosis had to be changed from partial to complete hydatidiform mole (PM to CM, respectively) in 9 cases and from CM to PM in 4 cases. DNA image cytometry could be performed in 40 cases (CM, n = 21; PM, n = 19). RESULTS: There was 100% agreement between histologic diagnosis and a diploid or polyploid histogram in CM and 79% agreement between triploidy and PM, when the updated diagnostic criteria were used. This represents an improvement compared with diagnoses made with former criteria. Nevertheless, problems of correct classification remain: In 3 cases classified as PMs, fetal remnants were accompanied by the histologic appearance of a CM. These 3 cases could represent either a true embryonic development in CM or a twin gestation with one normal pregnancy and one mole, or they could belong to a (very rare) third type of mole. All of them show the same risk of persistent trophoblastic disease observed in classic CM. CONCLUSIONS: As the groups at risk for developing persistent trophoblastic disease can be identified by their DNA histograms, ploidy analysis would be desirable in addition to histologic examination.

Adult↗
PubMed1995

[Importance of image analysis in immunoelectron microscopy. Visualization of heterogeneous reactivity of sera from patients with linear IgA dermatitis bullosa with structures of the dermo-epidermal junction].

Interpretation of electron microscopy pictures illustrating immunoperoxidase labelling is often difficult for non-specialists. We used an image analysis system to facilitate the comprehension of the results obtained with indirect immunoperoxidase technique on normal human skin. Sera from patients with linear IgA bullous dermatosis (LABD) were selected on the basis of an exclusive IgA class reactivity with the dermo-epidermal junction. Various patterns of epidermal labelling could be demonstrated with 82% of the immunoreactive sera. Only 2 out of 11 sera reacted with sub-lamina densa antigens. These results confirm the heterogeneous nature of diseases diagnosed, according to the present criteria, as LABD.

Epidermis↗
PubMed1991

Image analysis for quantitation of estrogen receptor in formalin-fixed paraffin-embedded sections of breast carcinoma.

Monoclonal antibody to human estrogen receptor (ER) provides a useful immunohistochemical tool for the evaluation of ER content in breast carcinoma, but visual interpretation is subjective. Computer-assisted image analysis has proved effective in immunohistochemical quantitation of ER in fresh tumor imprints and cryostat sections. We examined the usefulness of this technique in 5-microns-thick formalin-fixed paraffin-embedded tissue sections of 66 cases of primary breast carcinoma previously assayed by dextran-coated charcoal (DCC) analysis. Immunohistochemistry was automated and performed on a Code-on slide stainer (Instrumentation Laboratories, Lexington, MA) using Pronase predigestion, a monoclonal antibody (ER-ICA; Abbott, Chicago, IL), and a biotin-labeled secondary antibody. Detection was achieved with an avidin-alkaline phosphatase conjugate and nitroblue tetrazolium (NBT) bromochloroindoyl phosphate (BCIP) substrate. The immunohistochemical ER staining was analyzed visually and with the CAS/200 image analyzer (Elmhurst, IL). The visual semiquantitative histologic scores (HSCORE), the automated quantitative assays including the percentage of positive nuclear areas (PNA), and the quantitative immunocytochemical scores (QIC SCORE = PNA x % of positive stain/10) were compared with the corresponding DCC results. Linear correlations were demonstrated between all immunohistochemical assays and the logarithm of DCC, the strongest correlation seen with PNA (r = 0.91). Threshold points for positive HSCORE, QIC SCORE, and PNA assays were extrapolated using DCC as the reference. ER immunodetection by PNA as compared with visual examination alone was enhanced by 18% (up to 88%) in sensitivity and 34% (up to 94%) in specificity, and the DCC concordance rate increased by 26% (up to 91%). A comparative chart extrapolating DCC from PNA was thus established.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗
PubMed1984

Smooth-surfaced control and transformed C3H/10T-1/2 cells differ in cytology: a study by secondary electron, backscattered electron and image analysis.

7,12 dimethylbenz-a-anthracene (DMBA)-treated C3H/10T-1/2 mouse embryo fibroblasts have been characterized by the occurrence of pleomorphic microvilli (PMV) in contrast to the non-treated smooth-surfaced C3H/10T-1/2 cells. Recent studies by semiquantitative secondary electron image (SEI) have demonstrated a significant correlation between the number of cells with numerous PMV and the development of oncogenic potential. In these studies, so-called type III transformants scored highest in oncogenic potential and occurrence of cells with pleomorphic microvilli, but always contained a small fraction of cells displaying rather smooth surfaces. Quantitation of the size of nuclei and nucleoli and the number of intranucleolar silver granules after nucleolar organizer region (AgNOR) staining and back scattered electron image (BEI) with an automatic image analysis system (IBAS) revealed that these smooth-surfaced type III cells were significantly different (p less than 0.001) from the non-DMBA transformed smooth-surfaced control C3H/10T-1/2 cells. Compared to PMV transformants, the smooth transformants exhibited smaller nucleoli, a lesser nucleoli/nuclei fraction and fewer AgNOR sites, indicating reduced cellular activity. The biological significance of the smooth-surfaced fraction in PMV-covered type III transformants remains unclear.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

Regional variations of urokinase-type plasminogen activator in human colorectal cancer: a quantitative study by image analysis.

Qualitative histological studies in the distribution of urokinase-type plasminogen activator (uPA) in human colorectal carcinomas have been well documented. However, to our knowledge the histological distribution of this enzyme has not been quantified in any tumour. For the present image analysis study, uPA was demonstrated in sections of human colorectal cancer using immunoperoxidase technique. A total of 9 colorectal carcinoma cases were used, in which 132 regions were analysed. Within each region, staining intensity measurements were made at evenly spaced intervals. Samples of normal mucosa from 6 cases were also studied. Enzyme levels were assessed with staining intensity measurements. For each section, a negative control section was included, in which the primary antibody was omitted. Staining for uPA was quantified for each region in the test section, and the measurement for the corresponding region of the negative control was then subtracted. The enzyme uPA was localised more at the tumour edge than in the tumour centre or distant host tissue. These differences were highly significant (p < 0.0001). There was also a highly significant difference in staining intensity when tumour regions adjacent to pushing edge were compared with those adjacent to infiltrating edge (p < 0.0001). Infiltrating tumours showed stronger staining for uPA than tumours with pushing edges. Since invasive activity is thought to be maximal at the edge of the tumour, localisation of uPA at this site is consistent with the role of this enzyme in the process of tumour invasion.

Adenocarcinoma↗
PubMed1990

Relation between T cell number and epithelial HLA class II expression quantified by image analysis in normal and inflamed human gastric mucosa.

Epithelial expression of HLA class II determinants and the number of lamina propria and intraepithelial T cells were quantified in gastric body mucosa by means of paired immunofluorescence staining which was subjected to computerised image analysis. In normal mucosa, epithelial HLA-DR expression was virtually absent. A significantly increased expression was seen in simple chronic gastritis, most extensively in the isthmus zone, where a positive reaction was seen in 34% of the epithelial area when the gastritis was of low degree and in 85% when it was of moderate severity. The most extensive HLA-DR expression was found in moderate 'stump gastritis' 28 to 32 years after Billroth II resections. In these patients the epithelial area in the foveolar and isthmus zones showed 83% and 92% positive responses, respectively. The HLA subregion products were expressed in a differential manner (DR greater than DP greater than DQ). The number of both intraepithelial and lamina propria T cells increased significantly with increasing severity of gastritis, and the fraction of putative memory T cells was also raised. Correlation analyses showed a positive relation between the epithelial expression of HLA-DR and the intraepithelial as well as the lamina propria density of T cell. These results suggest a biological link between T cells, aberrant HLA-DR expression, and gastritis, although the pathogenic importance of this relation is unknown. Enhanced epithelial presentation of autoantigens or luminal antigens, or both leading to increased activation of T cells is one possible explanation.

Epithelium↗
PubMed1990

Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions.

Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40-45 degrees C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4 degrees C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9 degrees C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1 x SSC, 45 degrees C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4-11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Image analysis of microvessel surface area predicts radiosensitivity in early-stage laryngeal carcinoma treated with radiotherapy.

PURPOSE: The tissue oxygenation level, which is theoretically governed by distance from blood vessels, is one of the most important modulators of the radiosensitivity of carcinoma. A computed image analysis system for the detection of tissue oxygenation was developed to establish a method of predicting radiosensitivity in early-stage laryngeal carcinoma treated by curative radiotherapy. EXPERIMENTAL DESIGN: Microvessel structures labeled with CD31 antigen were investigated in 55 patients undergoing curative radiotherapy for T1 and T2 laryngeal carcinoma. We calculated (a) microvessel density [(MVD) vessels/field] under a microscope; (b) the ratio of the total microvessel number (TN):tumor area (TA) [TN:TA; vessels/mm2]; (c) the ratio of the total microvessel perimeter (TP):TA (TP:TA; mm/mm2); and (d) the ratio of tumor tissue area >150 microm from microvessels (hypoxic ratio; %) as parameters of tissue oxygenation in each whole biopsy specimen by using an image analyzer. We compared each of these factors with radiosensitivity. RESULTS: Mann-Whitney's U test revealed that tumors with a high MVD (median, 42 vessels/field), high TN:TA ratio (median=40.9 vessels/mm2), high TP:TA ratio (median, 2.92 mm/mm2), and low hypoxic ratio (median, 30.3%) had significantly greater radiosensitivity than tumors with a low MVD, low TN:TA ratio, low TP:TA ratio or high hypoxic ratio (P = 0.002, P = 0.0004, P < 0.0001, and P = 0.004, respectively). CONCLUSIONS: Prediction of radiosensitivity on the basis of the TP:TA ratio can be used as an efficient means of avoiding ineffective radiation, complications after salvage surgery, and prolonged hospital stays.

Adult↗
PubMed1994

Scanning microphotometry image analysis of Ha-ras-transformed human breast epithelial cells.

Nuclei of the human breast epithelial cells, MCF-10A, transfected with the c-Ha-ras oncogene, the ras proto-oncogene and the plasmid Homer 6 only, were studied by image analysis after Feulgen staining. This material had been previously used to demonstrate that the experimental insertion of the activated c-Ha-ras oncogene into the DNA of the MCF-10A cells induces their tumoural properties. The ras-transformed nuclei of the MCF-10A cells exhibited differences in chromatin supraorganization in comparison with the nuclei of human breast carcinoma MCF-7 cells, or c-Ha-ras-transformed NIH/3T3 cells and, to a much lesser extent, with those of other MCF-10A transfectants and the non-transfected MCF-10A cells. All MCF-10A transfectants exhibited unravelling of both condensed and non-condensed chromatin, which, however, was less drastic in the ras-transformed MCF-10A cells. It is hypothesized that simultaneous to a general chromatin loosening as a response to foreign transfected DNA, a reverse mechanism may be elicited by ras transformation in the chromatin of the MCF-10A cells. The result in terms of elicited chromatin condensation was not as strong as that promoted in ras-transformed NIH/3T3 cells. Considering that early steps of tumour progression in vitro have previously been assumed to be involved in the ras-transformed MCF-10A cells, the differences in chromatin supraorganization of the ras-transformed MCF-10A cells as compared with MCF-7 cells are probably due to their different tumoural stages plus the putative effect of the transfected DNA vector on the transfected MCF-10A cells.

Breast Neoplasms↗
PubMed1994

Determination of DNA ploidy, nuclear size, and proliferative activity by means of the computer-assisted image analysis of Feulgen-stained nuclei in 68 soft tissue tumors of adults.

The diagnostic values of the ploidy level, the proliferative activity, and the nuclear size in a series of 68 soft tissue tumors of adults were determined by digital cell image analysis of Feulgen-stained nuclei from formalin-fixed, paraffin-embedded tissues. The DNA ploidy level was characterized by calculating the DNA index (DI) and the percentage of the diploid and polyploid cells, and by typing the DNA histogram. Proliferative activity assessments were a function of the determination of the proliferation index (PI), ie, the percentage of cells engaged in the S phase of the cell cycle (SPF value). The present series included 19 benign and 49 malignant soft tissue tumors. The results show that DNA aneuploidy, as assessed by both the DI and the DNA histogram type, cannot be used as a discriminatory parameter for distinguishing between benign and malignant soft tissue tumors. Indeed, some benign cases may be highly aneuploid, whereas some highly malignant soft tissue tumors may be definitely diploid. In contrast, the determination of the percentage of polyploid cell nuclei seems to be a useful parameter in distinguishing between benign and malignant cases. In fact, the benign soft tissue tumors showed a very significantly lower mean percentage value of polyploid cell nuclei than the malignant cases. The determination of the proliferative activity also discriminated significantly between the benign and the malignant cases, the former proliferating more slowly than the latter. Lastly, the determination of nuclear size made it possible to differentiate the primary malignant soft tissue tumors, whether recurrent or not, that were associated with metastasis from those free of metastasis.

Adult↗
PubMed1994

Image analysis of the distribution of intraperitoneally administered fluids in patients with ovarian cancer.

Patients are selected for intraperitoneal chemotherapy based on the condition of the peritoneal cavity and the volume of residual disease, as determined by second-look surgery. Several techniques have been described to assess the distribution of intraperitoneal fluids. They rely on point, as opposed to area, measurements. We performed serial radionuclide scans of 15 patients undergoing intraperitoneal chemo for ovarian cancer. Scans were performed following intraperitoneal administration of 500-1000 ml of 99Tc-labeled human serum albumin in normal saline. Each patient underwent three scans at least 4 weeks apart. Radionuclide scans were digitized for computerized analysis. Area in picture elements (pixels) and optical density (mass) of the 99Tc-labeled regions were determined and analyzed for similarities in the pattern of distribution. Area analysis was used to divide the patients into two groups. Group 2 patients (n = 7) had more patients with < 0.5 cm of residual disease (P = .051) and no evidence of disease (P = 0.1) compared with group 1 patients (n = 8). No differences in overall survival were found (P = 0.21). No objective measurement of distribution of intraperitoneally administered fluids exists. Computer image analysis may be an improvement over static scoring techniques to assess non-invasive imaging studies.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1979

Computerised image analysis of split-drop micropuncture data.

A technique is described for rapid and reproducible analysis of split-drop micropuncture sequences recorded on film. Automatic identification of the droplet menisci in each cine frame, and subsequent evaluation is performed by a computerised T.V. image analysis system. In comparison with a more conventional technique, the improvement in reproducibility of the analysis is achieved without loss of accuracy and is accompanied by a three fold increase in speed.

Animals↗
PubMed1993

Malignant transformation of osteoblastoma: study using image analysis microdensitometry.

AIM: To determine if the malignant transformation, as perceived histologically, in a case of osteoblastoma from the right femur, was also expressed as a quantitative change in nuclear DNA during tumour progression over five months. METHODS: Nuclear DNA microdensitometry by computer image analysis was used to acquire relative DNA distribution patterns. Tissue had been removed on four separate occasions from a lesion in the right femur of an 18 year old man. Retrospective DNA analysis was performed on formalin fixed, paraffin wax-embedded tissue. RESULTS: The DNA profile of the initial biopsy specimen, which was histologically diagnosed as osteoblastoma, was euploid with a near diploid (2c) modal DNA. The second biopsy specimen taken one month later also resembled osteoblastoma but showed an aneuploid DNA profile with a diploid modal DNA and some nuclei with ploidy greater than 5c. The third biopsy specimen taken four months later showed histological evidence of osteosarcoma and a near pentaploid (5c) modal DNA with large number of nuclei exceeding 5c. CONCLUSIONS: DNA microdensitometry confirmed the initial and final diagnosis. The technique also seems to be capable of detecting aneuploidy before malignancy is morphologically evident.

Adolescent↗
PubMed2007

Image analysis by pulse coupled neural networks (PCNN)--a novel approach in granule size characterization.

A biologically inspired spiking neural network model, the pulse coupled neural network (PCNN), has been applied for the first time in bulk particle characterization, and specifically in the characterization of pharmaceutical granule size distributions. The PCNN was trained on surface images of pharmaceutical granule beds, and the adjustable parameters (radius neuron interconnection, r0, linking weight coefficient, beta, local threshold potential, VTheta, and number of iterations) were successfully optimized using design of experiments. As demonstrated with size fractions of granules, it was found that the PCNN produced granule size-dependent signals. In general, a first highest and relatively narrow peak located in the region of two to twelve iterations corresponded to smaller particle size, while larger particles resulted in wider peaks and in highest (not first) peak at a range between 13 and 25 iterations. Better predictions, i.e. lower RMSEP (root mean squared error of prediction) values, were obtained using high beta value, low r0 and VTheta values, while the number of iterations had to exceed 110 and the optimized model (RMSEP lower than 5) corresponded to PCNN variables: r0=1, beta=0.4, VTheta=2, and number of iterations=150. The coefficient of determination (R2) of the model was 0.94 and the predicted variation (Q2) was 0.91, while the Pearson correlation coefficient between the predicted and the measured mean particle size by sieving for eight test batches was 0.98. These findings could be characterized as promising and encouraging for the further use of image analysis by PCNNs in pharmaceutical bulk particle size and shape characterization.

Image Processing, Computer-Assisted↗
PubMed1983

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern.

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.

Animals↗
PubMed2005

Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis.

Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel). Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrome suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 mum thick sections could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrome without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions.

Animals↗