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PubMed1990

Automated image cytometry in cytopathology.

Image cytometry is used more and more for the study of clinical cytology, notably for the determination of morphometrical and densitometrical values, the quantification of monoclonal antibody labelling and the detection of DNA probes after in situ hybridisation. Aspects of automated and interactive image cytometry are discussed, including a brief evaluation of limitations and advantages of the image technique in connection to flow cytometry. Some new technologies such as a sampling technique for paraffin embedded tissue and a new automated microscope, which are of special interest to the pathologist, are described in more detail. Applications in image cytometry include diagnostic and prognostic studies. Examples of diagnostic studies are the automated screening for cervical cancer and the detection of rare remaining cancer cells (minimal residual disease) in the peripheral blood. The use of archival material in image cytometry allows interesting retrospective studies with regard to the relation of the course of the disease with the ploidy characteristics of the tumor.

Autoanalysis↗
PubMed1992

[Quantitative DNA image cytometry by color image analyzer].

We developed a new method to analyze DNA quantitatively by a color image analyzer. A microscopic image of the nucleus stained with Feulgen's reaction, is taken by a CCD color camera and inputted in a color image analyzer. DNA quantity can be calculated directly from the RGB components of the color image instead of the monochrome image taken through band-pass filter, which is specific for the absorption of Feulgen's stain. It takes about 10 minutes to measure 500 nuclei by this method and its accuracy is almost the same as flow cytometry (CV = 3%). The nuclear color image can also be analyzed morphometrically. This method can select tumor cells in a usual cytologic specimen and measure their DNA contents.

Cytophotometry↗
PubMed1996

Effects of topoisomerase I inhibition on the expression of topoisomerase II alpha measured with fluorescence image cytometry.

A fluorescence image cytometry technique was developed to measure the effects of topotecan, a topoisomerase I inhibitor, on the nuclear expression of topoisomerase II alpha in a series of patients with refractory or relapsed acute myeloid leukemia (AML). We used a commercially available affinity-purified rabbit polyclonal antibody and a fluorescein-conjugated secondary antibody. By using DAPI as a DNA counterstain and dual wavelength excitation, it was possible to measure enzyme expression in the cell nucleus, and to examine its cell cycle phase distribution. In human acute leukemia cell lines, topoisomerase II alpha expression was greatest in late S and G2 phases, but in leukemia patient samples the enzyme expression appeared to be much less cell cycle dependent. There was considerable interpatient variation in the effects of topotecan on topoisomerase II alpha expression in the leukemia patients, with a threefold increase in the median value after 48 h followed by a decline to pretreatment levels after 5 days of treatment with the topoisomerase I inhibitor. Although these findings should be treated with caution because of the small number of cases studied, they support the prediction that topoisomerase I inhibitors might be capable of increasing sensitivity to topoisomerase II active drugs such as anthracyclines and epipodophyllotoxins by upregulating topoisomerase II expression. They also illustrate the potential value of fluorescence image cytometry for making sequential measurements of the effects of drug resistance modulating agents in cancer patients.

Animals↗
PubMed1993

Comparison of flow and static image cytometry in the determination of ploidy.

AIMS: To compare the efficiency of flow cytometry and computed image cytometry; and to see if a reliable set of guidelines regarding interpretation of histograms could be drawn up. METHODS: The two methods were applied to a series of 111 formalin fixed renal cell carcinomas. Data generated by both methods were compared. RESULTS: The methods agreed in 85 cases. Hypodiploidy was detected by computed image cytometry in seven cases in which flow cytometry had shown only an aneuploid peak. Aneuploidy in seven in which the corresponding flow cytometry histogram was diploid. There was an overlap in the second peak proportions on flow cytometry histograms between those classed as diploid or tetraploid by computed image cytometry. In six cases the flow cytometry histograms had unacceptably high coefficients of variation and in all of these cases computed image cytometry demonstrated aneuploidy. CONCLUSIONS: Computed image cytometry is particularly useful for clarifying difficult areas in flow histograms--specifically, high coefficients of variation, high G2M phase, as well as possible near diploid aneuploidy and hypodiploidy.

Carcinoma, Renal Cell↗
PubMed1995

Quantitative DNA analysis in renal cell carcinoma. Comparison of flow and image cytometry.

In 70 renal cell carcinomas, nuclear DNA content was determined by means of flow cytometry (FCM) and image cytometry (ICM). The two methods produced comparable results as to DNA tumor ploidy (DNA tumor stemlines, DNA index): 14 of the tumors were tetraploid or aneuploid and 56 diploid. Results with the two methods were also comparable in a comparison of DNA ploidy with degree of tumor malignancy (tumor grade G1-3) and local tumor spread stage (pT stage). As a consequence, both methods appear suitable as means of determining DNA tumor ploidy and thus of formulating a prognosis in renal cell carcinoma. Renal cell carcinomas with diploid stemlines tend to be characterized by local growth, whereas tetraploid or aneuploid tumors show a tendency toward perirenal spread and venous invasion.

Carcinoma, Renal Cell↗
PubMed1997

Quantitative measurement of estrogen and progesterone receptors in breast lumps using image cytometry.

OBJECTIVE: To evaluate image cytometry for detecting estrogen (ER) and progesterone receptors (PR) in breast lumps quantitatively. STUDY DESIGN: Fifty consecutive breast lumps were analyzed for ER and PR using the avidin-biotin complex on tissue sections. RESULTS: Mean ER-positive nuclear area percentage (PA%) for 25 invasive ductal carcinomas was 47.81% and for PR was 57.83%. Mean ER PA% was 40.78% and PR 58.91% for seven invasive lobular carcinomas. There were two papillary carcinomas and one in situ ductal carcinoma. Fourteen of 15 benign breast lumps were benign breast lesions. Mean ER PA% was 65.95% and PR 73.43% for 10 fibroadenomas and for 4 fibrocystic lesions was 51.57% and 44.13%, respectively. CONCLUSION: Quantitative detection of ER and PR can be achieved by image cytometry. We aim in the future to assess this method in prognostic studies and the preoperative cytology of breast cancers.

Adult↗
PubMed1994

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry.

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears.

Algorithms↗
PubMed1989

Automated detection and recognition of live cells in tissue culture using image cytometry.

An automated image cytometry device, the Cell Analyzer, was used to locate live V79 cells plated at low densities in a tissue culture flask. Cells and other objects were detected by moving the flask in steps across a linear solid-state image sensor. The step size was selected to be small enough to allow detection of all the cells in the area being scanned but sufficiently large so that most cells would be detected on only one image line. To distinguish cells from other detected objects, a recognition algorithm utilizing 18 characteristic cell signal features was developed. The algorithm first tests whether a set of feature values falls within specified upper and lower bounds, and then applies a linear discriminant function to the remaining data to further discriminate cells from debris. False-positive errors of 5% or less were achieved with this method, whereas 15-35% of cells were misclassified as debris.

Algorithms↗
PubMed1992

[The advancement of image cytometry in immunohistology].

An image processing system has been developed by the combination of recent electronic and computer technologies. The system may have a potential power for the quantitative analysis on the immunohistology. The image cytometry was applied to quantitative analysis on Ki-67-positive cells in lymphomas of Waldeyer's ring and nasal cavity. High grade lymphomas showed significantly larger number of Ki-67-positive cells than intermediate-grade lymphomas, even analyzed separately by immunophenotypes. A large mean area per Ki-67-positive cell was significantly associated with T-cell phenotype and unfavorable clinical outcome. Thus, Ki-67 immunostaining combined with image cytometry is a novel method for determining a tumor proliferative index which provides useful clinical data of non-Hodgkin's lymphomas.

Cytophotometry↗
PubMed1994

[Image cytometry].

A new system of image cytometry, a fully integrated optical microscope and computer workstation for the pathology laboratory, was reviewed. This system is useful in research in distribution of DNA mass, cell proliferation, hormone receptor, and cancer gene expression. The image cytometry has many improvements, allowing the investigator to use the microscope as a measuring instrument.

Aneuploidy↗
PubMed1997

Image cytometry for quantitative analysis of DNA in the testes of infertile men with varicocele: comparison with flow cytometry.

PURPOSE: The first aim of this study is to evaluate the usefulness of image cytometry comparing flow cytometry to analyze the spermatogenesis using testicular biopsy specimens obtained from infertile male. The second is to investigate the spermatogenesis of infertile men with varicocele by image cytometry. MATERIALS AND METHODS: A total of 76 biopsy specimens of testicular tissue were obtained from 38 infertile men with varicocele and 6 specimens of normal testicular tissue controls were obtained from 6 volunteers at vasectomy. They were evaluated by image cytometry and pathohistological examination. Out of 76 specimens, 21 biopsy tissues were also analyzed by flow cytometry, and the results by both image cytometry and flow cytometry were compared. Two specimens from Sertoli cell only testes were also examined by image and flow cytometry. RESULTS: In comparative study, image cytometry revealed a decrease in the proportion of haploid and diploid cells (18.8 +/- 6.6% and 32.4 +/- 7.4%), and an increase in that of tetraploid cells (21.8 +/- 5.6%) vs. the flow cytometry findings (haploid, 25.9 +/- 9.2%; diploid, 36.8 +/- 6.5% and tetraploid, 15.1 +/- 5.9%). Image cytometric analysis of the testes of varicocele patients showed 15.9 +/- 5.6% haploid, 31.7 +/- 7.9% diploid and 16.2 +/- 8.9% tetraploid cells in the left testis of infertile men, while their right testis showed 16.6 +/- 5.4% haploid, 30.0 +/- 7.6% diploid and 17.2 +/- 6.4% tetraploid cells. The testes of normal men showed 37.6 +/- 8.3% haploid, 19.4 +/- 4.8% diploid and 17.4 +/- 5.8% tetraploid cells. The left and the right testes of the patients with varicocele showed a lower proportion of haploid cells and a higher proportion of diploid cells vs. the testes from normal men. The proportions of the haploid and diploid cells are correlated with sperm concentration. CONCLUSION: Image cytometry could exclude interstitial cells, Sertoli cells, and sperm on the static image, so produced an accurate assessment of spermatogenesis. The alteration in the germ cell population in the testes of infertile men with varicocele suggested that the impairment mostly affected the cells in the meiotic stage.

Adult↗
PubMed1991

The role of DNA flow and image cytometry in the evaluation of body cavity fluids.

In addition to conventional cytomorphologic study, 50 body cavity fluid specimens (benign and malignant) were analyzed by both flow cytometry (FCM) and image cytometry (ICM) in order to evaluate the potential application of these techniques in the diagnosis of malignancy. While 88% of the fluids were similarly classified by FCM and ICM as being either diploid (66%) or nondiploid (22%), with similar DNA index values, nondiploid peaks were identified by ICM alone in 12% of the fluids. Aneuploid populations were present in 92% of the cytologically positive fluids and in 15% of the cytologically negative fluids. All of the latter fluids came from patients who clinically had tumors involving the respective body cavities. These results show that (1) both FCM and ICM are useful adjuvant techniques in the evaluation of body cavity fluid specimens and (2) ICM is superior to FCM in the identification of small aneuploid subpopulations. The identification of aneuploid populations by either method is highly suggestive of malignancy.

Adult↗
PubMed1993

Caveats in the estimation of DNA-ploidy in paraffin embedded specimens of primary prostate cancer and lymph node metastases by flow and image cytometry.

DNA ploidy assessments of 72 archival paraffin embedded specimens were made utilizing flow cytometry (FCM) and image cytometry (ICM). Forty four specimens were obtained from primary prostatic tumours and 28 from metastatic lymph nodes. Eighteen of 30 FCM diploid range tumours were aneuploid by ICM. Nineteen of 22 FCM tetraploid range tumours were ICM aneuploid. All FCM non-diploid range specimens were non-diploid range by ICM, and all ICM diploid range specimens were diploid range by FCM. ICM more easily identifies small numbers of aneuploid cells than FCM. Discrepancy between FCM and ICM measurements was particularly high in lymph node specimens. FCM diploid range distributions mostly occurred in lymph nodes with a small tumour fraction whereas the number of aneuploidy specimens increased with larger tumour volume. Lymphocytes are different from solid tumour cells since they are not fixed in tissue and will probably be more easily dissolved in cell suspension when sections are disintegrated. This may influence FCM measurements and has to be considered when DNA ploidy measurements of lymph node metastases are made.

Adenocarcinoma↗
PubMed1985

DNA image cytometry on machine-selected breast cancer cells and a comparison between flow cytometry and scanning cytophotometry.

A DNA image cytometry method, implemented on the LEYTAS image processing system, has been applied to acriflavine-Feulgen-stained breast cancer cytology specimens. An essential feature of the LEYTAS image cytometry method (LCM) is the automated selection of single nuclei according to predetermined specifications. Visual interaction has been used to reject remaining artefacts like overlapping nuclei. DNA profiles obtained with LCM have been compared with DNA profiles obtained by scanning cytophotometry (SCM) or flow cytometry (FCM). The resolution of DNA profiles obtained with LCM is similar to that from SCM but lower than that from FCM. However, a high correlation is found for the DNA indices measured with LCM and FCM (r = 0.97). The LCM profiles of aneuploid tumours generally showed lower accessory diploid fractions than FCM profiles due to the automated rejection of leukocyte nuclei. Also, LCM profiles frequently showed the presence of minor subpopulations of highly aneuploid/polyploid tumour cells that could not be identified by FCM. Therefore, LCM appears to be supplementary to FCM for studying tumour cell stemline heterogeneity.

Aneuploidy↗
PubMed1987

Use of DNA image cytometry in addition to flow cytometry for the study of patients with advanced ovarian cancer.

Forty-five patients with advanced ovarian cancer were studied with both DNA flow cytometry (FCM) and automatic DNA image cytometry carried out with the Leiden Television Analysis System (Leytas). There was a significant difference in survival between the diploid and nondiploid cases as determined by FCM. Furthermore, the presence of nuclei with a high DNA content (defined as a DNA content higher than 5C) as determined by Leytas indicated a poor prognosis. When the combined results of FCM and Leytas were taken into account, three different groups of patients could be distinguished. The group of patients with a diploid malignancy (n = 12) had a median survival of more than 60 months. The group of patients (n = 11) with a nondiploid tumor having fewer than 100 nuclei with a high DNA content per 1600 microscope fields formed an intermediate group (median survival, 42 months), whereas the median survival of the remaining patients (n = 22), who had a nondiploid malignancy combined with more than 100 of these nuclei per 1600 microscope fields, was only 15 months. In addition, comparison of the clinical parameters by means of a multivariate analysis (Cox regression model) showed that the combined results of FCM and DNA image cytometry had the largest influence on survival. It is concluded that DNA image cytometry appears to be supplementary to FCM for the study of DNA ploidy abnormalities and that the combined results of these methods have a major influence on the clinical outcome.

Aneuploidy↗
PubMed1994

Image cytometry: current applications and future trends.

Image cytometry has numerous clinical and research applications and is particularly useful in anatomic pathology for the study of malignant lesions. Modern image systems encompass morphometry, densitometry, neural networks, and expert systems. Rapid advances in technology and the development of user-friendly systems have provided pathologists with an alternative to flow cytometry, particularly useful in the evaluation of small or hypocellular specimens. The most common current application of image cytometry is for DNA analysis, followed by quantitation of immunohistochemical staining. Newer uses under active investigation include development of expert systems that may act as diagnostic consultants in the future. Beyond DNA analysis, image cytometry holds great promise for improved tumor classification, for screening and surveillance in high-risk populations, and as a tool to improve diagnostic ability. This article discusses types of image analysis systems, specimen preparation, data acquisition, current applications in specific organ sites, and possible future applications.

Cell Division↗
PubMedDate not supplied

An automated image cytometry system for monitoring DNA ploidy and other cell features of radiotherapy and chemotherapy patients.

DNA content and distribution in cell nuclei were studied in samples of fine-needle aspiration (FNA) from 27 locally advanced breast and head and neck cancers in two going randomized trials that compared accelerated fractionation to standard fractionation radiation in locally advanced breast cancer and head and neck cancer. Two image cytometry methods were compared: a new, fully automated DNA image cytometry system (AIC) and a conventional image cytometry (CIC) system with manual selection, focusing, and segmentation of cells. The results of both techniques were compared on the basis of DNA histogram parameters including DNA index (DI), mean DNA values (MDV), and Auer's DNA histogram patterns. An excellent correlation was achieved between the two imaging techniques in terms of DI (r=0.985, p<0.001) and MDV (r=0.951, p<0.001) as well as between Auer's histogram patterns, where both methods agreed completely. It was concluded in these analyses that the two image cytometry methods were equivalent. However, the AIC offered an advantage by scanning samples in a fully automated way, which represented significant time saving for cytopathologists working with the system, as well as a larger number of cells used in the automated analysis. With the automated image cytometer, 500 relevant cells were collected and analyzed in about 10 minutes, where with the interactive (manual) method, it took typically an hour to collect and analyze only about 250 cells. Seventeen samples were sufficient for flow analysis. Image cytometry and flow cytometry showed good agreement in DI determination; however, three cases reported as diploid by flow cytometry were found to be aneuploid by image cytometry techniques.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

Comparison of flow and image cytometry for DNA content analysis of fresh and formalin-fixed, paraffin-embedded tissue in breast carcinoma.

DNA ploidy and S-phase fraction are considered to be prognostic variables in breast carcinoma. DNA content of 35 cases of breast carcinoma of varying histologic types and nuclear grades was analyzed by flow cytometry and image analysis in both fresh and formalin-fixed, paraffin-embedded tissue. Fresh cell and deparaffinized nuclear suspensions were used for flow cytometry. Fresh and deparaffinized tumor tissue samples were used for image analysis. The results of analysis for DNA ploidy, DNA index of DNA aneuploid Go/G1 peaks, and S-phase fraction were compared in different tissue preparations for both techniques. The two techniques produced comparable DNA ploidy results with both fresh and formalin-fixed, paraffin-embedded tissue. Sensitivity for detection of DNA aneuploidy was somewhat greater by image analysis, particularly in deparaffinized tissue. There was 89% agreement in detection of DNA aneuploidy by flow cytometry in fresh and paraffin-embedded, formalin-fixed tissue; the coefficients of variation of the DNA diploid Go/G1 peaks were much wider in the latter. In image analysis there was 91% agreement between fresh and fixed specimens. Agreement between the flow cytometry and image analysis in fresh specimens was 91%; in deparaffinized nuclear suspensions it was 94%. There is a high degree of correlation between the values of DNA index of DNA aneuploid Go/G1 peaks; the estimates of S-phase fraction are much more variable. Results also show a good correlation of the DNA ploidy with the nuclear grades.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma, Mucinous↗