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PubMed1991

High-resolution three-dimensional images from confocal scanning laser microscopy. Quantitative study and mathematical correction of the effects from bleaching and fluorescence attenuation in depth.

Three-dimensional images can be assembled by piling up consecutive confocal fluorescent images obtained by confocal scanning laser microscopy. The present work was based on three-dimensional (50-microns-deep) images at high (x, y) resolution obtained with an MRC-500 after en bloc staining of thick slices of rat liver by chromomycin A3 for nuclear DNA. The results of studies on bleaching, fluorescence excitation and emission intensities at various depths of histologic preparations are described. These effects could be evaluated separately by acquiring piled-up ("brick-stepping") and non-piled-up ("side-stepping") (x, y) images at consecutive depths and also (x, z) images. Empirical equations allowed the fitting of experimental plots of bleaching versus time, at different laser intensities and at different depths, and of fluorescence emission intensity versus depth. The main conclusions were that under our experimental conditions: (1) there was no attenuation by depth of the fluorochrome penetration, (2) there was no attenuation of the exciting beam intensity up to at least 50 microns deep, (3) there was an attenuation of the fluorescence emission intensity by depth, (4) bleaching happened equally on all planes above and below any confocal plane being studied, and (5) the fluorescence bleaching half-life was independent of depth. A mathematical correction scheme designed to compensate for bleaching and for attenuation of fluorescence emission in depth is presented. This correction is required for obtaining three-dimensional images of better quality, for optimal three-dimensional image segmentation and for any quantitative analysis based upon voxel-discretized emission intensities (gray levels)--e.g., estimating, by confocal image cytometry, textural chromatin parameters and nuclear DNA amounts.

Animals↗
PubMed1989

Bowenoid papulosis. Classification as a low-grade in situ carcinoma of the epidermis on the basis of histomorphologic and DNA ploidy studies.

The nature of bowenoid papulosis was investigated by a comparative investigation of 12 biopsy specimens of this lesion, 19 biopsy specimens of Bowen's disease, 14 biopsy specimens of squamous cell carcinoma and 10 biopsy specimens of seborrheic keratosis. In addition to conventional histomorphologic and cytomorphologic studies, nuclear DNA measurements on single cells isolated from tissue blocks were performed using a TV image analysis system combined with an automatic microscope. Two parameters, the "5c exceeding rate" (5cER) and the "2c deviation index" (2cDI), were computed from the single-cell DNA values to arrive at a "DNA diagnosis" and a "DNA malignancy grade" (DNA-MG). All specimens of bowenoid papulosis and Bowen's disease were morphologically diagnosed as in situ carcinomas of the epidermis; a DNA diagnosis of malignant was rendered in all of these specimens due to the detection of aneuploid nuclei (5cER greater than or equal to 1). DNA diagnoses of malignant were also rendered on all specimens of squamous cell carcinoma (100% sensitivity) while DNA diagnoses of benign were rendered in all specimens of seborrheic keratosis (100% specificity). The mean DNA-MG for bowenoid papulosis (0.69) was significantly lower than that for Bowen's disease (1.04) and squamous cell carcinoma (1.15). The mean morphologic (Broder's) grade of malignancy was also lower for bowenoid papulosis than for Bowen's disease and squamous cell carcinoma. HPV 16 DNA was detected in 10 of 12 specimens of bowenoid papulosis. Thus, the results of DNA image cytometry and morphologic investigation suggest that bowenoid papulosis is a low-grade carcinoma in situ of the epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMedDate not supplied

Image analysis of cell proliferation in rat thymus throughout development.

A System for Analytical Microscopy in Biological Applications (SAMBA 200) was used to digitize, process and statistically analyze the cell proliferation of rat lymphoid cells throughout thymus development. This cytometric study shows that lymphoid cell proliferation starts in 14-day-old embryos, remains high throughout embryogenesis with a maximum around day 18, decreases after birth then rises again in 21-day-old rats, after which proliferation decreases gradually with age, this decrease appears even before the onset of the sexual maturation, and is very marked in 2-year-old animals. Image cytometry analysis makes it possible to distinguish 5 lymphoid cell subpopulations (lymphoid stem-cells, lymphoblasts, large lymphocytes, medium lymphocytes, and small lymphocytes), and to discriminate in each cycling and non-cycling cells. The S-fraction of the lymphoid stem-cells decreases in 16-day-old rats, while the S-fraction of the lymphoblasts increases rapidly during the embryonic period. The large lymphocytes show the highest S-fraction at the stage E18, while the medium lymphocytes show the more stable S-fraction. The S-fraction variation for small lymphocytes can be divided into three periods: 1. up to E16 (S-fraction increases rapidly); 2. between newborn and 21 days (S-fraction increases slowly and irregularly); 3. after 21 days (S-fraction decreases progressively).

Aging↗
PubMed1985

DNA grading of malignancy and tumor regression in prostatic carcinoma under hormone therapy.

The prognostic value of the DNA malignancy grade (DNA-MG) and of the DNA regression index (DNA-RI) for survival was tested in 19 patients with prostatic carcinoma under hormone therapy. Measurements were performed on repeated Feulgen-stained transrectal aspiration biopsy smears by DNA image cytometry. The DNA-MG was demonstrated to be an objective scalar index of high prognostic value for the individual patient. The prospective tumor response to hormone therapy could be predicted from the DNA-MG. Independently of the DNA-MG, the DNA-RI provided additional prognostic information concerning tumor regression and survival of prostatic cancer patients under conservative therapy. The determination of DNA-RI allows the early identification of an ineffective conservative therapy.

Aged↗
PubMed1995

ERBB-2 (HER2/neu) gene copy number, p185HER-2 overexpression, and intratumor heterogeneity in human breast cancer.

Amplification of the ERBB-2 (HER-2/neu) gene is accompanied by overexpression of its cell surface receptor product, p185HER-2. Heterogeneity has been observed for both the gene copy number and the level of overexpression of its protein product. To better understand their relationship, correlation between the level of cellular expression of p185HER-2 and ERBB-2 gene amplification was studied in four human breast cancer cell lines (BT-474, SK-BR-3, MDA-453, and MCF-7) and in a primary human breast tumor sample. The relative expression of p185HER-2 was measured by immunofluorescence by using flow and/or image cytometry while correlated DNA analysis was performed on the same cells by fluorescence in situ hybridization to determine ERBB-2 gene and chromosome 17 copy numbers. Marked heterogeneity was observed in both protein expression and ERBB-2 copy number. Despite this heterogeneity, and in accordance with previous studies, the average levels of p185HER-2 expression correlated well with average ERBB-2 gene copy numbers in the four lines examined (r = 0.99). When the relationship between copy number and protein expression was studied on a cell-by-cell basis, p185HER-2 expression correlated with both the absolute number of ERBB-2 gene copies/cell (r = 0.59-0.63) and chromosome 17 copy number (r = 0.45-0.61). It is of interest that there was weak or no correlation between p185HER-2 protein expression and the ERBB-2 copy number:chromosome 17 copy number ratio (r = 0.0-0.25). In more than one-half of cells expressing a high level of p185HER-2, the chromosome 17 copy number was high (two or three times the average copy number), whereas < 2% of an unselected population had a high chromosome 17 copy number. Bromodeoxyuridine incorporation indicated that the S-phase-labeling index was homogeneous across various p185HER-2-expressing subpopulations in the SK-BR-3 cell line. Analysis of the primary breast tumor sample showed results similar to the cell lines, supporting the strong possibility of a mechanistic link among p185HER-2 overexpression, ERBB-2 amplification, and high chromosome 17 copy number.

Breast Neoplasms↗
PubMed1993

DNA distribution in the prostate. Normal gland, benign and premalignant lesions, and subsequent adenocarcinomas.

DNA content in paraffin-embedded tissue from 25 normal prostates (NP), 49 specimens of benign prostatic hyperplasia (BPH), 37 specimens of prostatic intraepithelial neoplasia (PIN) and 22 prostatic adenocarcinomas was measured by static image cytometry. During an eight-year follow-up, 47% (23/49) of the males with BPH and 38% (14/37) of the males with PIN subsequently had histologically confirmed prostatic adenocarcinoma, of which 22 cases were analyzed. In 32% of the NPs, 1-3 of 200 measured cells were aneuploid. The mean age of males with aneuploid cells was 55.8 years; it was 33 years for those with only diploid cells (P = .03). In hyperplastic prostatic lesions the percentage of aneuploid specimens increased with the increasing frequency of histologic atypia and with a subsequent malignant outcome. Agreement between the primary benign and later malignant specimen regarding diploidy versus nondiploidy occurred in 17 of 22 patients (77%).

Adenocarcinoma↗
PubMed1993

A rapid and simple staining method, using toluidine blue, for analysing mitotic figures in tissue sections.

Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tissues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.

Animals↗
PubMed1995

Overexpression of human cyclin A advances entry into S phase.

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian cells. Using a genomic construction corresponding to the human cyclin A gene under the control of its own promoter, we have established stable transfectants overexpressing cyclin A protein. Experiments assisted by laser scanning image cytometry showed that this overexpression begins from late G1 phase onwards and is therefore cell cycle-regulated in this model. We demonstrated that this overexpression advances entry into S phase, leading to a contraction of the overall cell generation time. These results provide evidence that cyclin A can be a rate-limiting factor with respect to the control of the transition to S phase in mammalian cells.

Cyclins↗
PubMed1995

In vivo bromodeoxyuridine (BrdU)-labelling index of rat thymus: influence of different BrdU doses and exposure times as analyzed both in tissue sections and in single cell suspensions.

Tissue sections and single cell suspensions of rat thymus were analyzed by flow and image cytometry to study the effects of different doses of bromodeoxyuridine (BrdU) (10, 50 and 100 mg/kg and different labelling periods (15, 30 and 60 min) on the BrdU-labelling index. The proportions of BrdU-labelled cells, as analyzed by flow cytometry, were similar in the different groups of our study. However, the quality of the results was not exactly the same, since when the BrdU dose decreased, the separation between BrdU-positive S-phase cells and both the G0/G1 and the G2/M BrdU-negative cells on a DNA/BrdU histogram became less clear. On the other hand, the proportion of BrdU-labelled cells in tissue sections was greater in the groups of animals that received doses of 100 mg/kg and were killed 15, 30 or 60 min later than in the sections obtained from the remaining groups. Our results show that both dose and exposure time to BrdU may influence the final results when cell proliferation is assessed, the variations obtained clearly depending on the technique used for the immunological detection of BrdU-positive S-phase cells.

Animals↗
PubMed1995

[Ploidy analysis in a case of ovarian small cell carcinoma with hypercalcemia].

The authors studied DNA content in a case of small cell carcinoma with hypercalcemia. This tumor exhibited typical clinical, histological, immuno-histochemical, ultrastructural and biological patterns. DNA content was measured both by flow and image cytometry performed on unfixed tumoral samples. The proliferation index was 10%. These results are similar to those of the literature obtained retrospectively from 10% formalin fixed tissues. The DNA content is a clue to distinguish this entity from other small cell carcinomas of the ovary because immunohistochemical findings are not always informative. The diagnosis of small cell carcinoma of hypercalcemic type should be questioned if DNA content is abnormal.

Adult↗
PubMed1995

DNA cytometric abnormalities in human esophageal squamous intraepithelial and invasive carcinomas.

DNA ploidy abnormalities of 21 archival human esophageal intraepithelial neoplasia samples were assessed, using image cytometry of deparaffinized samples, with reference to invasive squamous cell carcinoma and corresponding uninvolved squamous epithelium. Cytometric parameters investigated were proportion of G0G1 aneuploid cell population, histogram typing, proportion of G0G1 diploid nuclei, coefficient of variation, mean DNA content, crude 5c exceeding proportion, 2c deviation index, malignancy index and grade, and entropy. The distributions of the above parameters were compared using the paired t test and Fisher's exact test. Among 10 parameters used, Auer typing of DNA histograms, crude 5c exceeding rate, 2c deviation index and malignancy grade according to Böcking allowed discrimination between uninvolved epithelium and invasive squamous cell carcinoma as well as intraepithelial neoplasia. In particular, the distribution of 2c deviation index in the uninvolved epithelium did not overlap that of intraepithelial and invasive carcinomas. The above four parameters, however, were unable to discriminate intraepithelial neoplasia from invasive carcinoma.

Aneuploidy↗
PubMedDate not supplied

Effects of vinblastine, colchicine, and verapamil on rhodamine 123 accumulation in human P-glycoprotein-positive leukemia cells.

Multidrug-resistant (MDR) cells have been characterized by reduced accumulation of rhodamine 123 (R123). We addressed the question of whether R123 could compete with substrates or inhibitors (vinblastine, colchicine, verapamil) of P-glycoprotein (Pgp) overexpressed in MDR cells, using fluorescence image cytometry. Verapamil caused a dose-dependent increase in R123 accumulation. R123 accumulation was increased by vinblastine only at high levels and colchicine had no effect on R123 accumulation. Treatments with two drugs altered R123 accumulation depending on drug concentration ratio. The results indicate that vinblastine, R123 and verapamil can compete for outward transport by Pgp. A dual effect of vinblastine suggests that vinblastine can activate Pgp at low concentrations and inhibit R123 transport at higher concentrations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Histopathology, morphometry, and nuclear DNA content of iris melanocytic lesions.

PURPOSE: To compare the histologic, morphometric and nuclear DNA content of a group of benign and malignant melanocytic lesions of the iris. METHODS: Forty-four surgically excised melanocytic lesions of the iris were histologically classified as nevus or melanoma. Morphometric analysis using a digital filar micrometer (LaSICO 1602N-10 and 5-4A) measured the mean size of the 10 largest nucleoli, and Feulgen staining and image cytometry (CAS 200 Cell Analysis Systems) analyzed the nuclear DNA ploidy in the lesions. Patient follow-up information was obtained whenever possible. RESULTS: Sixteen cases were histologically classified as nevi and twenty-eight cases as melanoma. The mean of the 10 largest nucleoli of the nevi was smaller than the mean among the melanomas (1.772 microns [SD = 0.366] and 2.773 microns [SD = 0.565], respectively). Feulgen staining revealed that all lesions were diploid, with the exception of two hyperdiploid and two hypodiploid melanomas. Of the patients with follow-up information available, none with nevi developed a metastasis and two with melanoma died of metastatic disease. CONCLUSIONS: The histologic classification of iris melanocytic lesions (i.e., nevus versus melanoma) correlates to nucleolar size (P < 0.001) but not to nuclear DNA ploidy.

Adolescent↗
PubMed1994

DNA ploidy analysis of pediatric germ cell tumors.

To determine the usefulness of DNA ploidy analysis in the biological assessment of pediatric germ cell tumors (GCTs), paraffin-embedded tissues from primary, recurrent, and metastatic neoplasms in 32 patients under age 18 were analyzed using static image cytometry. Primary sites included testis (eight), ovary (nine), sacrococcygeal region (10), central nervous system (three), and other extragonadal sites (two). The majority of the tumors were endodermal sinus tumors (ESTs) and teratomas. Of 13 teratomas, eight were diploid and five aneuploid. Immature ovarian teratomas of up to Grade II were diploid, whereas all of three Grade III immature teratomas were aneuploid. All sacrococcygeal teratomas, regardless of grade, were diploid; the development of EST in two sacrococcygeal teratomas was associated with the development of aneuploidy. Of 17 endodermal sinus tumors, five were diploid, five aneuploid, and seven tetraploid. All diploid ESTs occurred in the testis or extragonadally in patients 24 mo of age or younger. Infantile testicular ESTs were either tetraploid (one) or diploid (two), and ovarian ESTs were either aneuploid (two) or tetraploid (three). Comparing our data with that available for adult GCTs, we suggest that some pediatric GCTs may be pathogenetically distinct from adult GCTs. Adult testicular GCTs have been shown to be aneuploid, whereas infantile testicular ESTs are often tetraploid or diploid. This suggests a different pathogenesis for these groups of tumors and may explain their biological differences as well. The transformation of a sacrococcygeal teratoma into a malignant EST is poorly understood. Ploidy analysis presented suggests genetic alterations may accompany this transformation.

Adolescent↗
PubMed1994

[DNA ploidy of stem cells and prognosis of ductal adenocarcinoma of the pancreas].

The findings on DNA stem line ploidy of ductal adenocarcinomas of the pancreas, which have hitherto been published in the literature, are rather contradictory last but not least due to methodological problems. Thus, the DNA stem line ploidy and its prognostic significance were examined by Feulgen image cytometry on 10 microns sections of 100 paraffin-embedded radically resected ductal carcinomas of the pancreas from the records. In that process the parts of the tumour with the highest grade of malignancy were preferentially investigated. The aneuploidy rate (42%) was conspicuously low if compared with other adenocarcinomas. A highly significant positive correlation between DNA ploidy and histological grading according to Klöppel et al. could be observed, while there was only a tendency towards a positive correlation of DNA ploidy and post-surgical tumour stage. Regarding the 1-year survival rate, the DNA aneuploid tumour cases were significantly more unfavourable than the DNA peridiploid ones (p < 0.05). The prognostically decisive parameter, however, was the histological grading (p < 0.00005), which also showed the best stratification of the patient population. However, contrary to expectations the postsurgical stage of the primary tumour and the nodular state were only of low prognostic significance. The reason for that is on the one hand that the distribution over the pT-stages (in 81% pT2 tumours) is unbalanced, and on the other hand that the actual tumour cell dissemination is only insufficiently represented in the post-surgical staging.

Adenocarcinoma↗
PubMed1994

Can colony size be used to detect low-dose effects?

Many insults at low doses are defined as nontoxic, even though obvious effects do occur. Among these are changes in colony size when clonogenic survival is assessed. Early attempts to quantify radiation effects on colony size have been reported, but the time-consuming nature of these experiments did not encourage the use of this parameter as an end point. Recently, however, developments in image cytometry technology have provided alternative, less labor-intensive means of measuring colony size. These techniques have been used in our ongoing investigations of radiation effects at low doses. Data accumulated to date show a measurable dose dependence of colony size in clones classed as survivors. This dose dependence is characterized by fluctuations in the 0-1.5-Gy dose range, followed by a gradual decrease in colony size at higher doses. The fluctuations at low doses correspond qualitatively to the concavity, thought to be indicative of inducible repair phenomena, that has been observed in cell survival curves in the same dose range. This concavity was also seen in the current study, but its detection appeared to be dependent on the method used to score survivors.

Animals↗
PubMed1994

[DNA cytophotometry of oxyphilic thyroid tumors].

Paraffin-embedded surgical specimen from 47 Hürthle cell tumours of the thyroid gland were examined for DNA content by image cytometry to assess the diagnostic and prognostic utility of ploidy determination in oxyphilic tumors. Both adenomas (31 cases) and carcinomas (16 cases) were studied. As a control for the algorithm of DNA-interpretation, 10 randomly selected normal thyroid tissues were analysed. All control cases showed normal diploid DNA content. Among the Hürthle cell tumours, aneuploid peaks were present in 15 adenomas (48%) and in 13 carcinomas (81%). These findings demonstrate a limited value of aneuploidy as diagnostic feature for malignancy in Hürthle cell tumours of the thyroid. In view of prognosis, there is no unfavourable predictive prognostic value for abnormal DNA content in histologically benign Hürthle cell tumours. No metastases or recurrences occurred in this group during a mean follow up period of 4.4 years. Three patients with aneuploid carcinomas and local recurrences or metastases had higher levels of basic DNA indices. This suggests that aneuploidy has a prognostic value for histologically defined carcinomas.

Adenoma↗
PubMedDate not supplied

Detection of human leukemia cells with multidrug-resistance phenotype using multilabeling with fluorescent dyes.

Reduced accumulation of multiple drugs is a characteristic of cells overexpressing P-glycoprotein. This phenotype is referred to as multidrug-resistance (MDR). A protocol based on reduced accumulation of fluorescent dyes is proposed for discriminating MDR cells in cell populations. The combination of three fluorescent dyes, Hoechst 33342, rhodamine 123 and Nile red, with different intracellular targets, has been designed to characterize cells with different levels of resistance, using image cytometry. The fluorescence intensity of each dye was quantified in living cells. The protocol was applied to human leukemia cell lines, (K562, K562/ADR, CCRF-CEM, CEM/VLB100, CEM/VM-1). The effect of verapamil on dye accumulation is emphasized.

Antineoplastic Agents↗