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PubMed1997

A novel image cytometric method for quantitation of immunohistochemical staining of cytoplasmic antigens.

Evaluation of molecular markers by immunohistochemical labelling of tissue sections has traditionally been performed by qualitative assessment by trained pathologists. For those markers with a staining component present outside of the nucleus, there has been no image histometric method available to reliably and consistently define cell interfaces within the tissue. We present a new method of approximating cellular boundaries to define cellular regions within which quantitative measurements of staining intensity may be made. The method is based upon Voronoi tessellation of a defined region of interest (ROI), and requires only the position of the nuclear centroids within the ROI. Here we describe the VORSTAIN software which has been developed based on the Oncometrics CytoSavant Automated Image Cytometry System. To demonstrate this technique, human breast cancer sections immunohistochemically stained for bcl-2 protein and counter-stained with nuclear methyl green stain were evaluated. Intra-observer variation in the measured values was between 1.5-2.6% and inter-observer variation was between 1.8-4.4%. The primary source of variability was due to difficulties in interpreting the exact position of the nuclear centroids. Analysis of mean staining densities for each slide correlated well with subjective scoring performed by two independent pathologists. Using VORSTAIN, significant variation of staining intensities between regions within the same slide was measured for some sections, indicating a large degree of heterogeneity within the tumours. The ability to accurately quantitate the degree of heterogeneity of molecular marker expression within tumours may be a valuable tool in prognostication.

Biomarkers, Tumor↗
PubMed1997

Morphological heterogeneity of p53 positive and p53 negative nuclei in breast cancers stratified by clinicopathological variables.

The study was aimed to detect differences in nuclear morphology between nuclear populations as well as between tumours with different p53 expression in breast cancers with different clinicopathological features, which also reflect the stage of tumour progression. The p53 immunohistochemistry was performed on paraffin sections from 88 tumour samples. After the cells had been localised by means of an image cytometry workstation and their immunostaining had been categorised visually, the sections were destained and stained by the Feulgen protocol. The nuclei were relocated and measured cytometrically by the workstation. There were significant differences in the nuclear features between tumours as well as between nuclear populations with different p53 expression in the most subgroups. The variability of nuclear shape in tumour groups, classified by the tumour size or the lymph node status, increase with the p53 immunoreactive score, whereas in tumours grouped by the Bloom-Richardson grade features of the chromatin distribution were different between the p53 staining categories. The nuclear subpopulations showed differences in the amount and distribution of chromatin in most subgroups. The results demonstrate the relationship between the nuclear morphology and the p53 expression in different stages of breast cancers. The p53 status is an important factor of the biological behaviour but not the only one.

Breast Neoplasms↗
PubMed2000

Discrimination between dysplastic and malignant epithelium of the ampulla of vater based on quantitative image cytometric data.

OBJECTIVE: To assess the ability to associate histopathologic grading with objective criteria obtained by nuclear image cytometry in epithelium of the ampulla of Vater. STUDY DESIGN: Forty-one resected ampullary specimens were studied, including 8 dysplastic ampullomas together with 22 well-differentiated and 11 poorly differentiated ampullary adenocarcinomas. The nuclei were Feulgen stained and analyzed using a computer-assisted microscope, which generated 38 quantitative variables describing chromatin texture and nuclear DNA content (DNA ploidy level). These variables were explored by discriminant analysis to determine the most stable and informative variables. Univariate analysis was performed on the four most informative ones. The whole set of variables was also subjected to principal component analysis in order to characterize intragroup and intergroup heterogeneity. RESULTS: The univariate analysis defined two morphonuclear variables (related to nuclear chromatin distribution) discriminating between dysplasia and well-differentiated cancers. Aneuploidy occurrence was associated with discrimination between well-differentiated and poorly differentiated cancers. CONCLUSION: While alterations in chromatin distribution may be an early event in the malignant degeneration of this epithelium, alterations in nuclear DNA content should correspond to a later phenomenon. Quantification of these features can be exploited to assist in diagnosis.

Ampulla of Vater↗
PubMed1995

Nuclear DNA content and chromatin texture in multidrug-resistant human leukemic cell lines.

Nuclear morphological alterations associated with multidrug resistance (MDR) were evaluated by image cytometry in various human leukemic cell sub-lines: 3 cell lines with P-gp-mediated resistance (CEM-VLB, HL60/Vinc, K562-Dox), the non-Pgp-mediated MDR HL60/AR leukemic cell line with over-expression of MRP, and the at-MDR CEM-VMI leukemic cell line with alteration of topoisomerase II. All these MDR cell sub-lines were obtained by drug selection and were compared with their sensitive counterparts and with the hamster LR73-R cell line obtained by transfection of mouse mdrl cDNA. All MDR cell sub-lines obtained by drug selection displayed decreased DNA Feulgen stainability as compared with their respective sensitive parental cell line, a phenomenon not observed in the transfected LR73-R cells. Nuclear texture analysis on G0/G1-selected cell nuclei revealed 2 types of textural phenotype. The first phenotype was characterized by chromatin decondensation with small but compact chromatin clumps, and was observed in drug-selected P-gp-mediated MDR cells (CEM-VLB, HL60-Vinc, K562-Dox) and in the non-P-gp-mediated MDR HL60/AR cell line. The second phenotype was characterized by a condensed and homogeneous chromatin pattern, and was observed in the at-MDR CEM-VMI cell line. LR73-R cells transfected with mdrl cDNA did not display any significant changes in textural phenotype as compared with sensitive LR73 cells, suggesting that P-gp over-expression alone cannot account for the cytological modifications observed in MDR cells. These data suggest that multidrug resistance could be associated with specific nuclear morphological changes which appeared to be a consequence of alterations occurring during selection by cytotoxic drugs rather than of P-gp over-expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMedDate not supplied

Follicular tumors of the thyroid gland: diagnosis, clinical aspects and nuclear DNA analysis.

From 1985 to 1990, 169 patients underwent surgery for follicular thyroid tumors at the Department of Surgery, Karolinska Hospital, Stockholm, Sweden. Nine of the patients had tumors which were diagnosed as follicular carcinoma, 4 of whom had recurrences in the neck region. In 6 of the patients the carcinomas were diagnosed as widely invasive and in 3 patients as minimally invasive. Of the 160 patients with follicular adenomas, 21 adenomas were of oxyphil type ("Hürthle" adenomas), 17 adenomas were diagnosed as "atypical", and 6 adenomas were classified as being both "atypical" and having oxyphil cell differentiation. The nuclear DNA content was measured with image cytometry and/or flow cytometry. Six of the 9 carcinomas were euploid and 3 were aneuploid. In the adenoma group, 32 (20%) were aneuploid. Thus 38% of all adenomas showed atypical cellular features, oxyphil cell differentiation, and/or aneuploid nuclear DNA pattern. None of the patients with adenomas have shown any sign of recurrence. In the present prospective study, the nuclear DNA content could not discriminate between a benign and a malignant follicular tumor, and was of limited value in predicting prognosis in the patients with follicular carcinoma. Still, the best way to establish a diagnosis and to predict prognosis is to surgically remove the follicular tumor for a proper histopathological examination.

Adenocarcinoma↗
PubMed1998

Progression from colorectal adenoma to carcinoma is associated with non-random chromosomal gains as detected by comparative genomic hybridisation.

AIMS: Chromosomal gains and losses were surveyed by comparative genomic hybridisation (CGH) in a series of colorectal adenomas and carcinomas, in search of high risk genomic changes involved in colorectal carcinogenesis. METHODS: Nine colorectal adenomas and 14 carcinomas were analysed by CGH, and DNA ploidy was assessed with both flow and image cytometry. RESULTS: In the nine adenomas analysed, an average of 6.6 (range 1 to 11) chromosomal aberrations were identified. In the 14 carcinomas an average of 11.9 (range 5 to 17) events were found per tumour. In the adenomas the number of gains and losses was in balance (3.6 v 3.0) while in carcinomas gains occurred more often than losses (8.2 v 3.7). Frequent gains involved 13q, 7p, 8q, and 20q, whereas losses most often occurred at 18q, 4q, and 8p. Gains of 13q, 8q, and 20q, and loss of 18q occurred more often in carcinomas than in adenomas (p = 0.005, p = 0.05, p = 0.05, and p = 0.02, respectively). Aneuploid tumours showed more gains than losses (mean 9.3 v 4.9, p = 0.02), in contrast to diploid tumours where gains and losses were nearly balanced (mean 3.1 v 4.1, p = 0.5). CONCLUSIONS: The most striking difference between chromosomal aberrations in colorectal adenomas and carcinomas, as detected by CGH, is an increased number of chromosomal gains that show a nonrandom distribution. Gains of 13q and also of 20q and 8q seem especially to be involved in the progression of adenomas to carcinomas, possibly owing to low level overexpression of oncogenes at these loci.

Adenoma↗
PubMed1999

[Tumor DNA content as a prognostic indicator in squamous cell carcinoma of the larynx].

Celluar DNA content has been found to be an important prognostic factor in many head and neck tumours, yet within the larynx few studies have examined its effect on survival. 31 patients with squamous carcinoma of the larynx treated in years 1983-85 by total laryngectomy were studied. The tumours were classified according to the TNM classification 1987 and graded histopathologically according to Jaccobson's classification. Quantitative DNA content was determined with the use of image cytometry performed by an image analyzer (CAS-200). Quantitative DNA analysis correlated with tumor grade and patient outcome within 6 years of follow-up.

Carcinoma, Squamous Cell↗
PubMed1990

Efficacy of combined image and flow cytometric DNA assessments in human breast cancer: a methodological study based on a routine histopathological material of 2024 excised tumour specimens.

In 2024 excised specimens of malignant tumours of the female mammary gland. the nuclear DNA distribution pattern of the neoplastic cells was assessed by means of two procedures. One was image cytometry (ICM); here, all the 2024 samples were assessed. The other was flow cytometry (FCM) where 1336 specimens were analysed. In 829 of the 2024 tumour nodules the results of ICM and FCM could be compared. The efficacy of both techniques alone was about 80%; that of the combination was about 60%. In the ICM procedure the main reason for the reduction of samples was the failure to obtain representative specimens. The losses in the FCM method were due to poor quality of the histograms (too much background noise and too broad coefficients of variation). In addition, in as much as one third of all the cases, no specimens were set aside for FCM assessments. In 16% of the samples, where the results of the ICM assessment could be compared with those of the FCM analyses, completely diverging DNA ploidy patterns were obtained. The discrepancy was caused by differences in the interpretation of the histograms. In addition, the calculations of so-called S-phase fractions from the diploid FCM histograms was found to be associated with methodological errors, further contributing to differences in the DNA assessments by means of ICM and FCM. Nevertheless, it was advantageous to use combined ICM and FCM assessments, particularly in the interpretation of DNA histograms of uncommon types.

Adult↗
PubMed1982

Automated cytology. The state of the art.

Analytical cytology is the science of automatically analyzing morphologic, biochemical, biophysical, and functional aspects of cells by machine. Two basic techniques are used: image cytometry, in which microscopic objects on a slide or in a photograph are analyzed; and flow cytometry, in which biologic particles in aqueous suspensions are forced to pass through a measuring device. The latter technique has the added power of sorting particles according to type for further study. Currently, analytic cytology is being studied for use in the following areas: WBC differentiation, cell-cycle kinetics, prescreening for uterine cancer, screening of high-risk groups, monitoring of tumor therapy, and microbial and chromosome analysis. In all these areas, analytic cytology has the potential to improve existing techniques and to make results more uniform; likewise, its sensitivity, accuracy, and speed may well allow a breadth and depth of analysis not presently allowed by human study.

Automation↗
PubMed1992

DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissue.

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non-tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue. Twenty-eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post-resection (group B). Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid by ICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months. The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade.

Adult↗
PubMed2001

Biopathological significance of single cell DNA aneuploidy measured by static cytometry in breast cancer.

DNA ploidy image analysis in breast carcinomas has occasionally revealed cases with a diploid DNA content but with a single hypertetraploid element, the so-called single cell aneuploidy (SCA). To identify the biologic significance of this little known phenomenon, we selected 40 cases with SCA from a series of 599 consecutive operable breast carcinomas. The clinical, pathological and biological characteristics of SCA cases were compared with those of a control group of 40 pure diploid breast carcinomas. Hormonal receptor status, proliferative indexes (Ki-67) and p53 overexpression were determined immunohistochemically and quantitatively evaluated by image analysis. The overexpression of c-erbB-2 was determined semiquantitatively. SCA was observed in 6.6% of cases (40 of 599) and in 17% of otherwise diploid cases (40 of 236). Breast cancers with SCA occur in younger women (mean age 54.75 y vs 61.12 y, P<0.05), are smaller (mean diameter 20.00 vs 21.62 mm), less differentiated (percentage of G3 cases 13.2 vs 2.9), more frequently hormone independent (ER positivity 70.0 vs 77.5%; PgR positivity 57.5 vs 77.5%) and have a greater cell proliferative activity (mean S-phase: 4.6 vs 2.8, P<0.05), (percentage of Ki-67 immunostaining: 24.1 vs 19.7%). There is also more overexpression of c-erbB-2 and P53, particularly in pT1 cases in which the percentage of c-erbB-2 positive cells is 54 vs 32 (P value is not significant) and the percentage of p53 is 29 vs 4 (P<0.05). According to these results SCA may be a reliable marker of genetic instability and of greater biologic aggressiveness. Image cytometry of DNA content may be a cost effective means of identifying breast cancer patients with an increased risk of tumour recurrences despite otherwise favourable prognostic parameters.

Journal Article↗
PubMed2001

Application of laser scanning cytometry for evaluation of DNA ploidy in routine cytologic specimens.

The laser scanning cytometer (LSC) is a relatively new instrument that combines the features of both flow and static image cytometry. The purpose of this study was to examine the application of the LSC for evaluation of DNA ploidy in routine cytologic specimens. The material for this study consisted of 60 routine cytologic specimens obtained from 33 males and 27 females ranging in age from 23-87 yr (mean, 58 yr). The specimens were simultaneously stained with propidium iodide and FITC-cytokeratin, either on Thin-Prep slide (35 cases) or in a concentrated cell suspension (25 cases). In each case a minimum of 500 cells was evaluated (range, 527-17,963; mean, 3,889). All abnormal cell populations were relocated for the presence of malignant cells. The results were defined as diploid and aneuploid/tetraploid. In 10 bladder washes, the results of LSC were compared to results of flow cytometry. Out of 60 specimens, 7 (11%: 6 bladder washes and 1 renal wash) were excluded due to low cellularity. Of the remaining 53 cases, 11 (20%) were aneuploid/tetraploid, and 42 (80%) were diploid. All but one cytologically diagnosed malignancy had abnormal DNA content. Additionally, two bladder washes diagnosed as suspicious and atypical were aneuploid. All abnormal LSC results were confirmed by relocation of the cells. The concordance between flow cytometry and LSC in the 10 control bladder washes was 100%. In conclusion, LSC proved to be a suitable instrument for the evaluation of DNA ploidy in routine cytologic specimens.

Adult↗
PubMed1996

Analysis of DNA content in multidrug-resistant cells by image and flow cytometry.

Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells.

Animals↗
PubMed1993

Image and flow cytometry: companion techniques for adherent and non-adherent cell analysis and sorting.

Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.

Cell Adhesion↗
PubMed2007

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening.

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cellos functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

Animals↗
PubMed1999

Laser-scanning cytometry: A new instrumentation with many applications.

The laser-scanning cytometer (LSC) is a microscope-based cytofluorometer which has attributes of both flow and image cytometry. Laser-excited fluorescence emitted from fluorochromed individual cells on a microscope slide is measured at multiple wavelengths rapidly with high sensitivity and accuracy. Though the instrument has been available commercially for only 3 years, it is already used in a variety of different applications in many laboratories. This review focuses on the following unique analytical capabilities of LSC which complement those of flow cytometry and fluorescence image analysis: (a) the cells are positioned on slides during measurement so they may be examined repeatedly over time, a feature useful for studies of enzyme kinetics and other time-resolved processes; (b) sequential analysis of the same cells can be carried out using different immuno- or cytochemical stains or genetic probes, merging information on cell immunophenotype, cell functions, expression of particular proteins, DNA ploidy and cell cycle position, and/or cytogenetic profile for each measured cell; (c) any of the cells measured can be relocated to correlate with visual examination by fluorescence or brightfield microscopy or with any other parameter; (d) topographic distribution of fluorescence measurements within the cell, in cytoplasm vs nucleus, permits analysis of the translocation of regulatory molecules such as NFkappaB, p53, etc., and is essential for FISH analysis; (e) hyperchromicity of nuclear DNA as measured by maximal pixel fluorescence intensity allows one to identify cell types differing in degree of chromatin condensation such as mitotic or apoptotic cells; (f) analysis of tissue section architecture and of the constituents in transected cells within tissue sections by ratiometric assays normalized to DNA content extends applications of LSC in clinical pathology; (g) because cell loss during sample preparation and staining is minimal, samples with a paucity of cells can be analyzed; and (h) analyzed cells can be stored indefinitely, e.g., for archival preservation or additional analysis. Potential future applications of LSC are discussed.

Animals↗
PubMed2000

Reliability of DNA cytometric S-phase analysis in surgical biopsies: assessment of systematic and sampling errors and comparison between results obtained by image and flow cytometry.

Three major parameters in DNA histograms that contribute to the reliability of S-phase analysis were evaluated. These parameters are (1) the extent of background in relation to the amount of S-phase cells (and the validity of its subtraction), (2) the size of the "free" S-phase range (S(free)), and (3) the sampling error of cell counting. Tests in histograms obtained from surgical biopsies by flow cytometry (FCM) showed that the background subtraction is reliable if the found S-phase fraction is higher than the fraction of background events in the histogram range of the cell population. The size of S(free) was determined in computer-generated test histograms as a function of variables such as the coefficient of variation (CV) and the DNA index (DI). To calculate the sampling error of cell counting above background and in S(free), a model was developed that was validated by experimental data. This error can serve as an indicator of the uncertainty in S-phase analysis. The poor correlation found between %S values measured by image cytometry (ICM) and FCM in surgical biopsies was assigned to high uncertainty by low cell numbers in ICM histograms. A method is proposed to estimate quantitatively the reliability of S-phase analysis that can facilitate the interpretation of results.

Biopsy↗
PubMed1999

[Value of Markovian nuclear texture to predict progression in surface transitional cell carcinoma of the bladder].

The aim of this work has been to determine if the DNA and nuclear Markovian textures of tissue sections evaluated by image cytometry correlate with the histologic grade and the progression probability of superficial transitional carcinoma of the bladder. In our study, DNA ploidy is related to histologic grade in that aneuploidy frequency increases with a higher grade of malignancy. Image analyses of superficial TCC tissue sections of the bladder, has allowed us to identify "sub-visual parameters" as well as nuclear textures that according to our results can be useful in clarifying the evolutive behaviour of these tumours. Two Markovian textures that identify entropy (TXI) and mean internal contrast (TXB) allow to discriminate between histologic grades as well as progression or non-progression. In conclusion, image analysis cytometry of paraffin embedded tissue sections of TCC of the bladder supplies densitometric parameters related to grade and provides valuable information for the prediction of progression. Quantification of chromatin pattern description in Feulgen-stained nuclei using the Markovian method can be useful in this context.

Carcinoma, Transitional Cell↗