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PubMed2001

Nuclear/Nucleolar morphometry and DNA image cytometry as a combined diagnostic tool in pathology of prostatic carcinoma.

Paraffin tissue sections from 50 patients with prostate adenocarcinoma were used to study nuclear and nucleolar morphometric features by image analysis. The results were compared to DNA ploidy and Gleason grade. In the examined histological samples nuclear and nucleolar areas were positively interrelated. It was also noticed that the higher the percentage of nucleolated nuclei, the bigger the nuclear and nucleolar areas. The morphometric characteristics did not differ significantly among the four grades of the examined specimens. In well-differentiated carcinomas the DNA index was lower than in the rest at a statistically significant level. Hypodiploid carcinomas were found to possess significantly bigger nuclear areas than any other DNA index group. Morphonuclear evidence of anaplasia and DNA aneuploidy may be used as diagnostic tools in prostate cancer in addition to Gleason grade.

Adenocarcinoma↗
PubMed1997

In situ classification and image cytometry of pelagic bacteria from a high mountain lake (gossenkollesee, austria).

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently labeled oligonucleotide probes. Our approach is based on established image analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell size distribution and the morphological characterization of different bacterial taxa in plankton samples. The protocol was tested in a study of the bacterioplankton community of a high mountain lake during and after the ice break period. Cells that hybridized with a probe for the domain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4(prm1),6(prm1)-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution for members of the beta subclass of the Proteobacteria shifted toward larger cells and clearly distinguished this group from the total bacterial assemblage. In the surface water layer beneath the winter cover, bacteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgroup bacteria was significantly less than that of the beta subclass proteobacteria, and the beta 1 subgroup accounted for less than 30% of the total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup, and both the biovolume size distributions and cell morphologies of the beta Proteobacteria and the beta 1 subgroup were very similar. We could thus quantify the disappearance of large, morphologically distinct beta subclass proteobacteria which were not members of the beta 1 subgroup during the ice break period. Our results demonstrate that changes in biovolumes and cell size distributions of different bacterial taxa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspectives for the study of microbial food webs.

Journal Article↗
PubMed1995

[An immunohistochemical study of the relationship between p53 expression and the DNA ploidy pattern using image cytometry in colorectal carcinoma].

We studied p53 expression in 46 colorectal carcinomas by immunohistochemistry. We examined the relationship between p53 expression and the pathological findings, the prognosis and tumor proliferative activity by Ki-67 staining or DNA ploidy pattern using an image cytometer. Expression of p53 was observed in 41.3% of carcinomas. No correlation was found between p53 expression and the depth of invasion, lymph node metastasis or Dukes' stage. No difference in survival was found between p53 positive and negative patients after curative surgery. We also studied the DNA ploidy pattern using an image cytometer in 28 carcinomas. Aneuploidy was found more frequently in the p53 positive areas than in the negative ones, but there was no significant difference. The DNA index showed no difference between the p53 positive and negative areas. We examined proliferating activity using Ki-67. There was no difference in the Ki-67 labeling index between p53 positive and negative areas. In the present study, there was no correlation between p53 expression and pathological findings, prognosis, tumor proliferative activity using Ki-67 staining or DNA ploidy pattern. Thus, p53 expression was suggested to have little relationship to grade of malignancy.

Cell Division↗
PubMed1992

[Confocal laser scanning microscopy for image cytometry].

By using a focused laser beam as the light source of the microscope, to minimized flare, together with a pinhole in front of a photo detector to eliminate out-of-focus data, the confocal laser scanning microscope (CLSM) provides a depth-discriminated fluorescence image with high spatial resolution. The CLSM, therefore, has been widely used as a tool for accurate morphometric and densitometric analyses. We discussed here the biological applications of CLSM for demonstrating the three-dimensional features of the embryonic heart tube and chromosome and for imaging fast dynamic changes of [Ca2+]i in the living heart muscle cell pairs.

Animals↗
PubMed1994

Analysis of melanocytic lesions by DNA image cytometry.

BACKGROUND: There is increasing evidence that melanocytic nevi with architectural and/or cytologic atypia (dysplastic melanocytic nevi [DMN]) are lesions intermediate between banal nevi and malignant melanoma. Other studies have shown conflicting results regarding the DNA content in DMN. METHOD: In the present investigation, the authors measured DNA ploidy by image analysis (CAS-200 system, Cell Analysis Systems, Inc., Elmhurst, IL) using Feulgen staining in 54 melanocytic lesions. The lesions were categorized into 7 groups: compound nevus (CN) = 7; compound nevi with features of DMN (F) = 10; DMN with slight atypia = 8; DMN with moderate atypia = 9; DMN with severe atypia (S) = 8; melanoma in situ = 5; and malignant melanoma (CMM) = 7. The age distribution for these various lesions was also recorded. DNA histograms obtained by image analysis from these groups were examined for DNA aneuploidy by DNA index and classified according to Auer classifications (Auer I-IV). RESULTS: There was a progression of mean age for each group of melanocytic lesions, ranging from 39 years for ordinary nevi to 53 years for invasive melanoma. The Type I histogram, which is distinctly diploid, was seen in 4 of 7 cases of CN, 5 of 10 of nevi with features of dysplastic nevus, and 3 of 8 nevi with slight atypia. The Type I histogram was not observed at all in nevi with moderate atypia, severe atypia, melanoma in situ, and invasive melanoma. The Type IV histogram (aneuploid) was not identified in low grade lesions (CN, F, S), but was observed in two of nine nevi with moderate atypia, six of eight lesions with severe atypia, two of five melanomas in situ, and five of six invasive melanomas. Aneuploidy by DNA index was noted in one nevus with features of DMN, one DMN with slight atypia, one with moderate atypia, four of eight DMN with severe atypia, two of five in situ melanomas, and two of six invasive melanomas. CONCLUSION: The results show that DMN exhibit a spectrum of abnormal DNA content intermediate between banal nevi and CMM and that DNA content generally correlates with the age of patients and degree of atypia in melanocytic nevi.

Adult↗
PubMed1999

DNA ploidy and cell cycle distribution of breast cancer aspirate cells measured by image cytometry and analyzed by artificial neural networks for their prognostic significance.

Chromosomal abnormalities are commonly associated with cancer, and their importance in the pathogenesis of the disease has been well recognized. Also recognized in recent years is the possibility that, together with chromosomal abnormalities, DNA ploidy of breast cancer aspirate cells, measured by image cytometric techniques, may correlate with prognosis of the disease. Here, we have examined the use of an artificial neural network to predict: 1) subclinical metastatic disease in the regional lymph nodes and 2) histological assessment, through the analysis of data obtained by image cytometric techniques of fine needle aspirates of breast tumors. The cellular features considered were: 1) DNA ploidy measured in terms of nuclear DNA content as well as by cell cycle distribution; 2) size of the S-phase fraction; and 3) nuclear pleomorphism. A further objective of the study was to analyze individual markers in terms of impact significance on predicting outcome in both cases. DNA ploidy, indicated by cell cycle distribution, was found markedly to influence the prediction of nodal spread of breast cancer, and nuclear pleomorphism to a lesser degree. Furthermore, a comparison between histological assessment and artificial neural network prediction shows a closer correlation between the neural approach and the development of further metastases as indicated in subsequent follow-up, than does histological assessment. These data demonstrate that artificial neural networks are capable of providing powerful and reliable indicators of possible lymph node metastasis, using measurements of cellular features alone.

Breast Neoplasms↗
PubMed1990

Differentiation between lymphomas and pseudolymphomas of the skin by computerized DNA-image cytometry.

The histologic and immunohistologic differential diagnosis between pseudolymphomas (PL) and malignant lymphomas (ML) of the skin can be difficult. Since DNA cytometry has been found to be of both diagnostic and prognostic value in various neoplasms, its ability to discriminate between ML and PL in Feulgen-stained imprints of 17 PL and 49 ML skin biopsies was examined by high-resolution image analysis. The reliability of the following algorithms of DNA distribution was evaluated: 1) 2cDI (2c-deviation index), which reflects the variation of the nuclear DNA values around the diploid DNA peak; 2) percentage of cells having a DNA value greater than or equal to 5c (5cER; 5c-exceeding rate); 3) percentage of cells presenting with a DNA value greater than or equal to 4c (4cER). A 2cDI of 0.1 was found to be the most reliable marker for the differentiation between PL and ML. On the basis of this feature, 16 of 17 cases of PL and 46 of 49 cases of ML were correctly classified. The sensitivity, specificity, and efficiency of this feature were 94%. A 5cER greater than or equal to 1% had a specificity of 100%, but the sensitivity was only 43%. For the 4cER, a sensitivity of 61% and a specificity of 94% were found. In conclusion, the calculation of the 2cDI and the 5cER based on high-resolution image analysis provided additional helpful diagnostic features, and therefore should be included as a diagnostic tool. If the 5cER is at least 1%, the diagnosis of a ML can be confirmed with a specificity of 100%.

Cytophotometry↗
PubMed1991

DNA image cytometry of keratoacanthoma and squamous cell carcinoma.

The distinction between the keratoacanthoma (KA) and the squamous cell carcinoma (SCC) can sometimes be difficult on the basis of histologic and clinical criteria. The possible diagnostic significance of DNA ploidy initiated the present study evaluating the DNA ploidy in paraffin-embedded tissue sections of 7 KA and 15 SCC, and fresh frozen tissue touch preparations of 15 of the same cases using the CAS 200 Image Analyzer. In paraffin-embedded tissue sections the main peak DNA index was based on normal epidermis, and ranged from 1.03 to 1.59 in KA and from 1.47-2.71 in SCC. The DNA Index (DI) discriminated KA from SCC in 17 of 22 cases (p less than 0.0007). The highest DNA content of single nuclei ranged from 9.0-18.0 picograms (pg) (DI 2.9-6.03) in KA and 14.8-38.6 pg (DI 4.0-11.03) in SCC. The highest DNA content discriminated KA from SCC in 16 of 22 cases (p less than 0.003). In fresh frozen tissue touch preparations from 15 of the same lesions, there was considerable overlap in DNA indices of KA (0.534-1.39) and SCC (0.464-1.41). Abnormal DNA peaks seen in histograms from three SCC in paraffin-embedded tissue sections were lost in the touch preparation histograms, probably due to inadequate sampling. Therefore, image analysis of paraffin-embedded tissue sections is better able to distinguish KA from SCC than touch preparations.

Carcinoma, Squamous Cell↗
PubMed1994

DNA image cytometry of prostatic carcinoma: a comparison of needle core biopsy and subsequent prostatectomy specimens.

DNA ploidy has recently been identified as an objective prognostic factor in prostatic carcinoma. Although the diagnosis of prostatic carcinoma is increasingly being made with the use of needle core biopsies, the optimal method for the cytometric analysis of these specimens has yet to be determined. In addition, the degree to which the biopsy is representative of the subsequent prostatectomy specimen with respect to DNA heterogeneity has not been adequately addressed. In this study, image cytometric (ICM) DNA analysis was performed on tissue sections from 12 prostatic needle core biopsies and the results were compared with similar ICM analysis of the subsequent prostatectomy specimens. Multiple blocks (n = 48) of the prostatectomy specimens were utilized to prepare tissue sections and nuclear suspensions and each set of preparations were analyzed by ICM in a parallel comparison study. There was concordance of 0.80 in the classification of DNA diploid and aneuploid tumors by ICM analysis of tissue sections and nuclear suspensions from paraffin blocks. In all of the discordant cases, DNA aneuploid populations were identified by ICM analysis of tissue sections only. This is attributed to difficulties in obtaining a representative nuclear suspension from disaggregated paraffin-embedded prostatic tissue which often has a very desmoplastic stroma. ICM analysis of tissue sections seems to be an optimal method for DNA ploidy analysis of prostatic carcinoma and is well suited to small volume biopsy material. Determination of DNA ploidy status in prostatic biopsies was predictive of the subsequent prostatectomy specimens with a concordance of 0.92.

Aged↗
PubMed1995

Estimation of fetal hemoglobin levels in individual red cells via fluorescence image cytometry.

A method for estimating fetal hemoglobin (Hb F) levels in individual red blood cells was developed. Cell smears were prepared using a slide maker to ensure uniform thickness and were then stained with immunofluorescence. An antifading gel was applied to preserve a stable fluorescence. The total fluorescence intensities from the same number of red cells in different slide specimens correlated with their hemolysate Hb F levels, which were determined via column chromatography (R = 0.95). Hb F level in individual cells was estimated from fluorescence intensity and cell area, which were determined via image analysis techniques and the hemolysate Hb F level. Blood from a normal subject, a subject with hereditary persistence of fetal hemoglobin, and from sickle cell patients with varying Hb F levels was analyzed. Our analyses showed a wide distribution of Hb F among cells for the normal subject and a gaussian distribution with a peak at the hemolysate Hb F level for the subject with hereditary persistence of fetal hemoglobin. The Hb F distributions were unique to the patients with sickle cell disease. Because Hb F level in individual sickle cells is crucial to the inhibition of cell sickling, the unique hb F distribution may be important in determining the clinical course of this disease.

Anemia, Sickle Cell↗
PubMed1995

Invariance of textural features in image cytometry under variation of size and pixel magnitude.

Textural features of the granular structure of stained cell nuclei and nuclear sections derived from co-occurrence and run length matrices are often used for correlation with external (clinical) parameters. The representation of cell nuclei and nuclear sections vary considerably under changes of preparation and fixation conditions. Most obvious are changes in size e.g. by fixation as well as changes in the amount of bound stain. Computer-simulated variations in size and pixel magnitude of a set of images of cell nuclei stained with Feulgen were featured and compared. Resulting feature dependencies are used for the setting of parameters of feature extraction procedures as well as for the selection of invariant features for texture quantification of material with variations examined.

Cell Nucleus↗
PubMed2005

Simultaneous in situ profiling of DNA lesion endpoints based on image cytometry and a single cell database approach.

Analyzing the integrity of DNA is one of the most frequent used endpoints for risk assessment of chemical and physical agents. In the framework of a radiobiological space experiment, this work aimed at having (1) a histochemical tool for the in situ assessment of DNA damage in as long as 20 days old fixed cell cultures, (2) a comprehensive tool for the quantification of different types of DNA lesions, and (3) a methodology of sampling thousands of nuclei based on confocal microscopy, automated stage scanning and digital image processing. For this purpose several fixatives and permeabilization techniques were tested together with the combinatorial use of terminal dUTP transferase-mediated nick end-labeling (TUNEL) and the DNA polymerase I mediated in situ nick translation. These biochemical tools are useful for scoring DNA single and double breaks, and oxidative lesions. Ltk(-) cells were exposed either to hydrogen peroxide or heavy ion beam irradiation. Combination of paraformaldehyde fixation, sodium citrate permeabilization and heat gave the best staining results. A three-channel fluorescence methodology was established including a DNA counter stain for nucleus identification and normalization of DNA content. Communication between confocal imaging software, image analysis software and a relational database proved to be pivotal for a semi-automated high-end single cell analysis and storage of images. In this way, DNA damage data per nucleus can be traced back to the original image. As much as 2500 cells could be analyzed in situ within a day and correlations drawn between different DNA lesion endpoints.

Animals↗
PubMed1992

Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the methyl green-pyronin technique with the gallocyanin chromalum and Feulgen procedures using image cytometry.

For simultaneous cytophotometric measurement of DNA and RNA, the standardized Methyl Green-Pyronin Y technique is an obvious choice. It is, however, first necessary to correlate the uptake of Pyronin Y to the staining intensity of RNA. The material consisted of paraffin sections of formalin- or Carnoy-fixed rat liver. The sections were pretreated with water, buffer, deoxyribonuclease, ribonuclease, or both enzymes in sequence, and stained with the standardized Methyl Green-Pyronin Y procedure, Gallocyanin chromalum, or the Feulgen reaction. Sections stained directly without pretreatment served as controls. Staining intensities were measured with an image analyser for cell nuclei, nucleoli and cytoplasm. After deoxyribonuclease treatment, nuclear staining intensity with Methyl Green, Gallocyanin chromalum, and Schiff's reagent dropped nearly to zero. The same was seen for both nucleoli and cytoplasm with Pyronin Y and Gallocyanin chromalum after ribonuclease treatment. Staining intensity of Pyronin Y correlated directly with that of Gallocyanin chromalum for nucleoli and cytoplasm. After ribonuclease treatment, a direct correlation was found between the nuclear staining intensity of Methyl Green and nuclear absorption of Gallocyanin chromalum. We conclude that the standardized Methyl Green-Pyronin Y stain is reliable for the simultaneous quantitative assessment of both RNA and DNA. The simplicity of this technique makes it a valuable tool even for daily routine.

Animals↗
PubMed1989

Immunocytochemistry and image cytometry of progesterone receptors in breast carcinoma imprints. Expression of tissular and cellular heterogeneity: relations to biochemical assay, clinical and histological parameters and DNA analysis.

Breast cancer imprints from 93 patients were assayed for the presence of progesterone receptors (PgR) using a monoclonal antibody (Transbio) and an immunocytochemical assay (ICA) method which stains only the epithelial cell nuclei. Results were compared with conventional biochemical PgR determinations (DCCA) and were in qualitative agreement in 86% of the cases. Quantitative analyses were done on PgR-ICA- and Feulgen-stained imprints from 32 tumours using a SAMBA 2005 cell image processor. Results obtained showed a high correlation between DCCA values and the P product derived from the mean PgR concentration of marked tumour cells and the percentage of marked cells. Intra-tumoral and intra-cell heterogeneity were featured and showed relation to tumour differentiation and size.

Antibodies, Monoclonal↗
PubMed1988

Image cytometry of estrogen receptors in breast carcinomas.

A significant level of estrogen receptors (ER) in breast cancer cells is an indication of tumor differentiation and suggests that a homeostatic control of cell growth may persist in these cancers. In medical practice, the Dextran-coated charcoal assays (DCCA) are still the most frequently used test to characterize patients having ER-positive malignant breast tumors and for whom hormonal therapy is justified. Nevertheless, this routine biochemical technique is not satisfactory because it is a broad method unsuitable for revealing receptor tissue heterogeneity. However, immunocytochemical labeling, such as the ER-ICA method, which involves a monoclonal antibody linked to peroxidase, is a specific reaction for this purpose but which until now was not quantitative. The present study uses an original cell preparation technique combining the PAP reaction with toluidine blue counterstain for image analysis on the SAMBA system. Special software has been developed for the quantitative analysis of immunocytochemistry in cancers. Results obtained showed a high correlation between the DCCA values and the score derived from the mean ER concentration per positive tumor cell and the labeling index. In addition, intracell and intratumor heterogeneity can be displayed according to several parameters and were shown to vary according to tumor and to antiestrogen (Tamoxifen) presurgical therapy.

Breast Neoplasms↗
PubMed2001

Can semi-automated image cytometry on induced sputum become a screening tool for lung cancer? Evaluation of quantitative semi-automated sputum cytometry on radon- and uranium-exposed workers.

The correlations between semi-automated sputum cytometry (ASC), conventional cytology and the final diagnosis was investigated in industrially-exposed workers. Slides of sputum samples from 201 former uranium miners with silicosis, 100 patients with asbestosis, 103 workers resected for lung cancer, and 200 controls (50% smokers), were stained using the Papanicolaou (Pap) method and the Feulgen reaction with thionin. Cytometry was performed using the Cyto-Savant automated system. Atypical nuclei were found in 72 of 404 patient samples, 327 samples were normal and five were inadequate for ASC analysis. Thirteen tumours (Pap IV, Pap V) and 11 cases of severe dysplasia were identified by cytology. Lung cancer was confirmed in 20 patients. Compared to the final diagnosis of lung cancer, the sensitivity of ASC was 75% (15 out of 20) and specificity 89.8% (520 out of 579). The results represent a diagnostic efficiency of 89.3%. The combination of ASC with cytology increased sensitivity to 80% (16 out of 20) without significant loss of specificity (89.7% or 523 out of 581). In this investigation of a limited number of patients with occupational radon or asbestos exposure, semi-automated sputum cytometry appears to be sensitive and reliable for the detection of malignant changes in the tracheobronchial mucosa. Together with conventional cytology, it would be reasonable to test the validity of the combined methods in a large-scale feasibility study of early lung cancer detection.

Adult↗
PubMed1989

Image cytometry in automated cervical screening.

Severe restrictions with regard to false negative rates have played a major role in the development of the LEYden Television Analysis System (LEYTAS). The present paper describes a test with a continuous series of 1500 cervical samples illustrating the accuracy of LEYTAS in a fully automated screening procedure using cell selection transformations and artefact rejection procedures. Specimen classification with a cut-off at greater than 0.3% alarms (= percentage of automatically selected objects per epithelial cells) and greater than 10 alarms, results in a false negative rate (FNR) of 0.3% (1 case out of 321 cases with severe dysplasia or more serious lesions), a false positive rate (FPR) of 13% (663 negative cases) and a rejection rate of 2.7%. Besides a machine classification, LEYTAS offers a second, machine-interaction classification of those preparations which have been declared positive by the machine. Machine-interaction involves visual evaluation of the stored images of the detected objects (alarms) and reduces the FPR from 13 to 8%. Statistical tests further demonstrate the significance of the screening results. Presently the main drawback for routine use of automated screening with LEYTAS seems to be the time consuming preparation procedure, since instrumentation has now been updated to a new, fast and user-friendly version of LEYTAS.

Acriflavine↗