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PubMed1994

Measurement of nuclear DNA content in histological and cytological specimens: principles and applications.

Nuclear DNA content can be measured in histological and cytological material by the techniques of image and flow cytometry. The measurement depends on the availability of stoichiometric markers for DNA. Results are expressed in DNA histograms from which tumour DNA ploidy and cell proliferation status can be estimated. Flow cytometry analyses large numbers of cells and gives the most meaningful cell cycle data. Image cytometry is more specifically targeted to the population of interest and can accurately measure rare events. A large body of literature exists relating DNA cytometry to clinical outcome, but there are considerable inconsistencies between clinical series which may be due, in many instances, to problems of histogram interpretation and tumour sampling. Rapid 'user-friendly' systems currently under development may increase the clinical acceptance of DNA cytometry.

Cell Nucleus↗

PubMed1997

Diagnosis and treatment of superficial transitional cell carcinoma of the bladder: facts and perspectives.

The detection of potentially highly curable low-grade bladder cancers by noninvasive techniques remains an unsolved problem. Conventional cytology detects such tumors with 50% sensitivity, the addition of DNA image analysis cytometry in patients with previous transitional cell superficial bladder carcinoma increases the chance to detect low-grade tumors. The specificity and sensitivity of detection of low-grade bladder tumors can be further improved by quantitative fluorescence image analysis using antibodies directed against tumor-associated antigens. The chance of subsequent tumor following initial complete resection is significantly lowered compared to resection alone by either intravesical adjuvant chemotherapy or immunotherapy. BCG is superior to chemotherapy in patients with high risk for recurrence, adjuvant intravesical chemotherapy does not prevent progression to > T1 when compared with resection alone, BCG may delay progression and the need for cystectomy in high-risk patients (T1, Cis or Cis associated with papillary Ta/T1, > 3 recurrences/year, grade 3 tumors). Oral medication with bropirimine (an interferon inducer) induced complete response in patients with carcinoma in situ, oral Lactobacillus casei prolonged the recurrence-free interval to 1.8 times that in the control group. New compounds for adjuvant treatment of superficial bladder tumors are best first tested on their ablative effects (marker lesion, Cis) prior to be used for prophylaxis.

Animals↗

PubMed2006

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.

Algorithms↗

PubMed2000

Routine DNA cytometry of benign and malignant pleural effusions by means of the remote quantitation server Euroquant: a prospective study.

AIM: To analyse the practicability and potential assistance of static DNA cytometry performed by means of the remote quantitation server Euroquant and the internet in routine diagnostic analysis of pleural effusions, and to outline the role of DNA cytometry on pleural effusions in distinguishing between benign and malignant (and herein primary versus metastatic) effusions. MATERIALS AND METHODS: Cytological smears of 294 pleural effusions were stained with the Feulgen method. The DNA content of a minimum of 300 randomly chosen analysis nuclei and 30 reference nuclei (lymphocytes) was measured by internet connection to the remote quantitation server Euroquant. Cytometric features were derived from the histograms, and the time needed for case evaluation, the reliability of staining and measurement procedures, and the contribution to the final diagnosis were evaluated. RESULTS: Only 120 of 294 pleural effusions could be measured. The total measurement time for each specimen was 60 minutes. The guidelines of the consensus report on DNA measurements were fulfilled. Seventy eight malignant (18 mesotheliomas, 60 metastatic tumours) and 42 benign effusions were measured. Seven of 78 malignant effusions were euploid and none of 42 benign effusions were aneuploid. The sensitivity and specificity were 91% and 100%, respectively, for distinguishing benign from malignant effusions, and 95% and 100%, respectively, for discriminating between benign and malignant effusions caused by metastatic malignant tumours. CONCLUSIONS: Static DNA cytometry using the remote quantitation server Euroquant can be performed reliably in the routine diagnosis of pleural effusions; however, only 40% of effusions meet the technical requirements for static DNA cytometry. Within the measurable cases, static DNA cytometry made an important contribution to the confirmation/exclusion of malignancy.

Aged↗

PubMed2005

7-Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation.

BACKGROUND: Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis. METHODS: Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry. RESULTS: During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures. CONCLUSIONS: During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures.

Apoptosis↗

PubMed1995

Some reflections on the history, and presence of quantitative pathology.

The quantitative approach has always been present in pathology, but this approach is now practiced more often and in more numerous fields than before. Today pathology is applying DNA cytometry, morphometry, stereology, and quantitative immunohistochemistry, all less useful or useless without the quantitative approach. The present fast development of computerized instrumentation has much helped in the spread and application of quantitative principles.

History, 19th Century↗

PubMed2000

Primary mucinous carcinoma of the skin.

Primary mucinous carcinomas of the skin are very rare. To date, 120 cases have been described in the literature. This tumor is a histologic subtype of sweat gland carcinoma. Because of the histopathologic appearance, primary mucinous carcinoma of the skin can be mistaken for metastasis from extracutaneous sites. We report on the cases of two elderly women with mucinous carcinomas arising in the scalp. Immunohistochemical staining of both tumors was positive for low-molecular-weight cytokeratin and epithelial membrane antigen. Carcinoembryonic antigen was positive in Case 2. Neuroendocrine features represented by neuron-enolase-specific positivity were also observed in both cases, and Grimelius and chromogranin A positivity were observed in Case 2. In both cases, there was strong positivity for estrogen receptor and progesterone receptor. Image analysis cytometry showed a diploid DNA content with a low rate of proliferative cells and negativity for p53 and c-erbB-2 proteins in agreement with the low aggressiveness of these neoplasms.

Adenocarcinoma, Mucinous↗

PubMedDate not supplied

DNA cytometry in diagnostic cytology of the prostate gland.

BACKGROUND: Differential analysis and cytological grouping of fine needle aspiration biopsy (FNAB) samples of the prostate are important in practice. We used image analytical DNA cytometry to achieve this and also studied the best method of interpretation of the histograms. MATERIALS AND METHODS: Sixty-two FNAB samples of the prostate were stained with Feulgen stain and nuclear DNA histograms were produced by image cytometry. The most atypical cell groups were selected for measurements. Also, free epithelial cells between cell groups were studied. The cells presented in the histograms were grouped according to the nuclear DNA content and by application of different gates of observation for diploid status. RESULTS: Several DNA histogram features (histogram classification categories, benign and malignant histogram patterns, presence of >5c-7c cells) showed significant relationships to differential diagnosis. Highly aneuploid (>5c-7c) cells had the potential for distinguishing a progressive-type of prostate cancer. CONCLUSION: The fraction of tetraploid and aneuploid histograms increased from atypical but benign to definitely malignant samples. DNA histograms have potential in the differential diagnosis and evaluation of the progressive character of prostate cancer.

Biopsy, Needle↗

PubMedDate not supplied

Laser scanning cytometry in pathology of solid tumors. A review.

OBJECTIVE: To review application of laser scanning cytometry (LSC) to analyze several different parameters of human solid tumors in relation to the cell cycle. STUDY DESIGN: Tissue sections of cytology specimens were stained for specific parameters and analyzed by LSC. RESULTS: Examples of LSC analysis of the expression of ER, Ki-67, cyclin B1, BrdUrd and ploidy are given. CONCLUSION: LSC provides rapid, high-precision measurement of the chosen parameters or constituents of each cell in a population of cells and correlates those measurements with the visual image of the corresponding cell.

Apoptosis↗

PubMed2001

Nuclear DNA content analysis by static cytometry in cervical intraepithelial lesions using retrospective series of previously stained PAP smears.

AIMS: To evaluate the nuclear DNA content in cytological smears of uterine cervix previously stained with Papanicolaou method, focusing the potential of the method in retrospective series. METHODS: Consecutive cases of Pap smears examined at the Adolfo Lutz Institute, a Public Health Laboratory of São Paulo State were selected. The diagnosis were: CIN 1 (n=20), CIN 2 (n=24), CIN 3 (n=20). Slides were previously stained with Papanicolaou method. The stain was removed with 5% hydrochloric alcohol-acid solution and the slides were stained with Thionin-Feulgen using a Becton & Dickinson kit. Ploidy evaluation was performed using the DNA Quantitative Measurement software 3.0 (version 8.1) from Becton & Dickinson and the CAS 200 system of image analysis. Cell ploidy was evaluated after analysis of atypical nuclei found in the selected cases. The DNA index was obtained using histograms for interpretation. MAIN RESULTS: CIN 1 cases showed the following DNA profile: 55% of diploid, 5% of tetraploid and 40% of aneuploid. CIN 2 cases showed 45.8% of diploid, 8.3% of tetraploid and 45.8% of aneuploid cells. CIN 3 cases showed 10% diploid, 15% of tetraploid and 75% of aneuploid cells. CONCLUSION: DNA analysis was useful to distinguish CIN 3 to CIN 1 and 2, but did not discriminate CIN 1 and 2 in these series. Aneuploidy was strongly associated to the CIN 3 cases.

Cell Nucleus↗

PubMed1999

Improving the prognostic value of histopathological grading and clinical staging in renal cell carcinomas by means of computer-assisted microscopy.

The present work aims to refine prognosis in cases of renal cell carcinoma (RCC) by integrating a variety of parameters with the prognostic information provided by histopathological grading and clinical staging, carried out on a series of 97 RCCs. To this end, Feulgen-stained RCC cell nuclei were characterized by means of 38 variables describing nuclear DNA ploidy levels and morphology. All of these data were subjected to a principal components analysis. On the basis of this multivariate analysis, Fuhrman grade II was subdivided into grades II- and II+, and Fuhrman grade III into grade III- and III+. The same kind of subcategorization was performed in the case of the T2 and T3 clinical stages. The results show that the classification into grade II- and III- RCCs correspond to a more favourable prognosis than grade II+ and III+, to which shorter survival periods were attributable. Similar results were obtained for the subcategorization of the T2 and T3 clinical stages. Very simple biological characterizations of these grade- or stage-related RCC groups were obtained by means of a decision tree approach applied to the cytometry-generated variables. The resulting classification rules were validated on a new series of 18 patients and enabled very accurate predictions of survival.

Adult↗

PubMed1997

Multiparameter analysis of DNA content and cytokeratin expression in breast carcinoma by laser scanning cytometry.

OBJECTIVE: The objective of this study was to test a new laboratory technology, laser scanning cytometry, for the purpose of performing multiparameter DNA content analysis of breast carcinomas. DESIGN: We developed a simplified method of multiparameter DNA content analysis using cytokeratin expression to positively gate epithelial cells. Over 300 consecutive cases of breast carcinoma were analyzed by multiparameter laser scanning cytometry. The first 73 cases were analyzed in parallel by single parameter flow cytometry. SETTING: The Department of Pathology, Christ Hospital and Medical Center, Oak Lawn, Ill. SPECIMENS: Three hundred eighteen consecutive cases of breast carcinoma presenting between March 1994 and December 1995. MAIN OUTCOME MEASURES: Outcome measures included the percentage of cases for which DNA content analysis could be successfully performed given the limitations of specimen size. Additionally, for the first 73 cases, laser scanning cytometry results were compared with flow cytometry results. RESULTS: All of the first 73 cases were successfully analyzed by laser scanning cytometry, but for 8 cases (11%) there was insufficient material for flow cytometry. Correlation of DNA content for the remaining 65 cases analyzed in parallel by the two methods was nearly perfect (p = .994). Five seemingly discrepant cases highlighted the importance of cytokeratin gating of epithelial cells by any technique, as well as other advantages specific to laser scanning cytometry, such as the ability to examine individual cells microscopically and correlate cytologic morphology with DNA content results. CONCLUSIONS: Laser scanning cytometry is a promising new technology for DNA content analysis of solid tissue tumors. Further work needs to be performed to validate the prognostic potential of the laser scanning cytometric assay results and to generate methodologies aimed at providing highly objective determinations of tumor cell S-phase fraction.

Biomarkers, Tumor↗

PubMed2006

3D parallel coordinate systems--a new data visualization method in the context of microscopy-based multicolor tissue cytometry.

BACKGROUND: Presentation of multiple interactions is of vital importance in the new field of cytomics. Quantitative analysis of multi- and polychromatic stained cells in tissue will serve as a basis for medical diagnosis and prediction of disease in forthcoming years. A major problem associated with huge interdependent data sets is visualization. Therefore, alternative and easy-to-handle strategies for data visualization as well as data meta-evaluation (population analysis, cross-correlation, co-expression analysis) were developed. METHODS: To facilitate human comprehension of complex data, 3D parallel coordinate systems have been developed and used in automated microscopy-based multicolor tissue cytometry (MMTC). Frozen sections of human skin were stained using the combination anti-CD45-PE, anti-CD14-APC, and SytoxGreen as well as the appropriate single and double negative controls. Stained sections were analyzed using automated confocal laser microscopy and semiquantitative MMTC-analysis with TissueQuest 2.0. The 3D parallel coordinate plots are generated from semiquantitative immunofluorescent data of single cells. The 2D and 3D parallel coordinate plots were produced by further processing using the Matlab environment (Mathworks, USA). RESULTS: Current techniques in data visualization primarily utilize scattergrams, where two parameters are plotted against each other on linear or logarithmic scales. However, data evaluation on cartesian x/y-scattergrams is, in general, only of limited value in multiparameter analysis. Dot plots suffer from serious problems, and in particular, do not meet the requirements of polychromatic high-context tissue cytometry of millions of cells. The 3D parallel coordinate plot replaces the vast amount of scattergrams that are usually needed for the cross-correlation analysis. As a result, the scientist is able to perform the data meta-evaluation by using one single plot. On the basis of 2D parallel coordinate systems, a density isosurface is created for representing the event population in an intuitive way. CONCLUSIONS: The proposed method opens new possibilities to represent and explore multidimensional data in the perspective of cytomics and other life sciences, e.g., DNA chip array technology. Current protocols in immunofluorescence permit simultaneous staining of up to 17 markers. Showing the cross-correlation between these markers requires 136 scattergrams, which is a prohibitively high number. The improved data visualization method allows the observation of such complex patterns in only one 3D plot and could take advantage of the latest developments in 3D imaging.

Humans↗

PubMedDate not supplied

Multiparameter immunophenotypic analysis of fine needle aspiration biopsies and other hematologic specimens by laser scanning cytometry.

OBJECTIVE: To test the new laboratory technology of laser scanning cytometry with respect to immunophenotyping of all types of hematologic and lymphoreticular specimens and particularly those of limited size, such as fine needle aspiration biopsies and hypocellular body fluids. STUDY DESIGN: Over the course of two years, 343 hematologic and lymphoreticular specimens of all types were immunophenotyped by laser scanning cytometry using methodologies modified from those of conventional flow cytometric immunophenotyping. Results for all cases were corroborated with histology and/or cytology and, for some cases, immunohistochemistry and/or flow cytometric immunophenotyping. RESULTS: Over 98% of the 343 cases were successfully immunophenotyped by laser scanning cytometry. These included many hypocellular specimens, such as 38 fine needle aspiration biopsies and 33 body fluid specimens. CONCLUSION: Laser scanning cytometry is a new laboratory technology with several significant advantages relative to flow cytometry for immunophenotypic analysis of hematologic malignancy. The laboratory techniques are simplified, and antibody usage is reduced by 80%. Even more important, full-panel immunophenotyping with multiple antibodies can be performed on specimens as small as 50,000 cells total, making the technology particularly relevant to cytopathology. After immunophenotypic analysis, specimens can be stained for light microscopic examination, and individual cells meeting user-defined antigenic or physical characteristics can be automatically relocalized.

Adult↗

PubMed2002

Determination of ploidy and steroid receptor status in breast cancer by laser scanning cytometry.

BACKGROUND: Measurements on DNA content and steroid receptor status in breast cancer are of great clinical interest. Objective determination of estrogen and progesterone receptor expression should help to define the lowest levels of positivity still responding to adjuvant antihormonal therapy. For this purpose, a simple protocol for laser scanning cytometry is presented. METHODS: Analysis of 54 routine breast cancer samples was performed by laser scanning cytometry (LSC). To obtain single cell preparations from fresh tumor tissue, slides were prepared using the Cervisoft cytological device. Exact determination of tumor cell DNA content was done by referring to the CD45-positive tissue leukocyte fraction as the internal diploid reference cell population. Steroid receptor-expressing cells were detected by indirect immunolabeling. RESULTS: Indirect immunofluorescence allowed the best quantification of both the estrogen and progesterone receptor-expressing cell fractions by LSC. The number of receptor-expressing cells could be given in percentage. For comparison, the 10% cutoff value was used to determine receptor positivity. CONCLUSION: LSC enabled a simple, reliable, and inexpensive determination of DNA index and steroid receptor expression in breast cancer specimens by objective criteria.

Breast Neoplasms↗

PubMed2005

Estrogen receptor-alpha is associated with the plasma membrane of astrocytes and coupled to the MAP/Src-kinase pathway.

Estrogens influence CNS development and a broad spectrum of neural functions. Several lines of evidence also suggest a neuroprotective role for estrogen. Different modes of estrogen action have been described at the cellular level involving classical nuclear estrogen receptor (ER)-dependent and nonclassical membrane ER-mediated rapid signaling. We have previously shown that nonclassical estrogen signaling is implicated in the control of dopamine cell function and protection. Since nonclassical interactions between estrogens and glia may contribute to these effects, our aim was to demonstrate the presence of membrane-associated ERs and their putative coupling to intracellular signaling pathways in astrocytes. Confocal image analysis and fluorescence-activated cell sorting (FACS) studies indicated the attachment of ER-alpha but not ER-beta to the plasma membrane of astrocytes. ERs were located in the cell soma region and glial processes. FACS analysis revealed that only a subpopulation of midbrain astrocytes possesses membrane ER-alpha. In FACS studies on ER-alpha knockout astrocytes, only a few membrane ER-positive cells were detected. The activation of membrane ERs appears to be coupled to the MAP-kinase/Src signaling pathway as shown by Western blotting. In conclusion, our data provide good evidence that nonclassical estrogen action in astrocytes is mediated by membrane ER-alpha. The physiological consequence of this phenomenon is not yet understood, but it might have a pivotal role in estrogen-mediated protective effects on midbrain dopamine neurons.

Animals↗

PubMed1997

Deoxyribonucleic acid ploidy enhances the cytological prediction of recurrent transitional cell carcinoma of the bladder.

PURPOSE: We determined whether deoxyribonucleic acid (DNA) ploidy analysis by image analysis cytometry enhances the cytological diagnosis of recurrent transitional cell carcinoma of the bladder. MATERIALS AND METHODS: A retrospective study was performed during a 5-year period to evaluate the cytological diagnosis and DNA ploidy analysis of 469 patients with previously diagnosed superficial transitional cell carcinoma. Cytological and DNA ploidy analysis was performed on 1,034 urine and bladder wash specimens, and the patients were monitored with cystoscopy and biopsies as clinically indicated. Cytology results were classified as normal, atypical, dysplastic or cancerous, and DNA ploidy was defined as normal if the diploid index was 1.2 or less, the S phase+G2M fraction was less than 21% or if there were 3% or less hyperploid cells, or abnormal if there was an increased S phase+G2M fraction, an aneuploid peak on the histogram or tetraploidy or hyperploidy was present. RESULTS: The majority of patients (85 of 88, 97%) with a cytological diagnosis of cancer had an abnormal DNA ploidy, and in 60 of 85 of these patients (71%) recurrence was diagnosed within 6 months. Only 5 of 284 specimens (2%) with normal cytology had abnormal DNA ploidy and 1 of these 5 (20%) heralded transitional cell carcinoma recurrence. However, in 145 patients with atypical cytological findings 29 (20%) with abnormal DNA ploidy had a recurrence, compared to 20 of 391 (5%) with normal DNA ploidy (p < 0.0001). Similarly, in 101 patients with dysplastic cytological findings 39 (39%) with abnormal DNA ploidy had transitional cell carcinoma recurrence compared to 4 of 25 with normal ploidy (p = 0.033). CONCLUSIONS: Abnormal DNA ploidy determined by image analysis significantly enhances the detection of bladder tumor recurrence in patients with atypical or dysplastic cytology but not in those with normal cytology or frank carcinoma on cytological findings.

Adult↗

PubMedDate not supplied

Slide-based laser scanning cytometry.

OBJECTIVE: To show that laser scanning cytometry (LSCM) can provide data equivalent to flow cytometry (FCM) data and furnish a number of benefits, including cell relocation for visualization and several additional measurement features that may make it more suitable than FCM for pathology laboratories. STUDY DESIGN: A laser scanning cytometer, the LSC, was developed. Several instruments, at sites in the United States and Japan during the last two years, provided data characterizing the instrument and its usefulness. RESULTS: Data describing the sensitivity, precision, accuracy, utility of added measurement features and cell relocation capabilities of the LSC are presented. The data illustrate the applicability of the LSC to multiparameter DNA ploidy studies, resolution of phases of the cell cycle and cytogenetics. CONCLUSION: Because it is microscope based and measures cells on a slide, not in a flow chamber; records the position of each cell on the slide; and has higher resolution, LSCM provides a number of benefits that may make it more suitable than FCM for pathology laboratories.

Animals↗