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PubMed2002

Diagnostic DNA-flow- vs. -image-cytometry in effusion cytology.

AIMS: To determine the sensitivity and specificity of flow- and image-cytometry for the detection of DNA-aneuploidy as a marker for malignant cells in effusions. METHODS: 200 effusions (80 tumor cell-positive, 74 negative and 46 cytologically equivocal) were stained with DAPI-SR for DNA-flow- and with Feulgen-Pararosaniline for -image-cytometry. They were measured using a PAS-flow-cytometer and an AutoCyte-QUIC-DNA-workstation according to the ESACP consensus reports for DNA-flow- and -image-cytometry, respectively [7,23,29,49]. RESULTS: Sensitivity of DNA-aneuploidy for the identification of malignant cells was 32.1% for DNA-flow- and 75.0% for -image-cytometry, specificity of -euploidy in benign cells was 100.0% for both methods. Positive predictive value of DNA-aneuploidy for the identification of malignant cells was 100.0% for both techniques, negative predictive value of DNA-euploidy was 48.6% for DNA-flow- and 72.0% for -image-cytometry. CONCLUSIONS: Searching for DNA-aneuploidy as a diagnostic marker for neoplastic cells in serous effusions image-cytometry revealed superior sensitivity as compared with monoparametric flow cytometry.

Algorithms↗
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↗
PubMed1991

Imaging cytometry by multiparameter fluorescence.

A system is described for performing multicolor fluorescence image cytometry of cell preparations. After the setting up stage, the operation is automatic: the microscope fields are found and focused; then images are acquired for each fluorophore, corrected and analyzed, without any operator interaction. Human peripheral blood lymphocytes on microscope slides were used as a test system. In these experiments, three fluorescent antibodies were used to identify lymphocyte sub-populations, and a DNA content probe was used to identify all nucleated cells. The cell subset percentages determined by image cytometry were comparable to percentages obtained when cells from the same preparation were analyzed by flow cytometry. Multicolor fluorescence imaging cytometry can potentially be extended to the analysis of cells in smears, fine needle biopsies, imprints, and tissue sections.

Algorithms↗
PubMed1990

Interrelation of formalin fixation, chromatin compactness and DNA values as measured by flow and image cytometry.

The severity and consistency of the effect of formalin fixation on the quantitation of DNA by flow cytometry (FCM) and image cytometry (ICM) were studied. As compared to ethanol, formalin fixation substantially decreased the propidium iodide fluorescence from mouse hepatocyte nuclei analyzed by FCM; it was also associated with an altered 4n-to-2n signal ratio and with false aneuploid peaks by FCM, but not by ICM (microspectrophotometry). ICM, on the other hand, suffered from a dependence of the DNA signal on nuclear size, which was not seen with FCM. The DNA signal variation was related to variations in the chromatin state, as shown by differences between monocytes and lymphocytes, and between RAJI cells fixed under various ionic strengths. The dependence of the DNA signal on the chromatin state indicates a need for caution in interpreting aneuploidy in formalin-fixed cells. For FCM, pseudoaneuploidy appears avoidable by using a Feulgen fluorescence staining technique. New imaging modes may be necessary to solve the problem of cell size dependence for ICM DNA determination.

Animals↗
PubMed1999

DNA image cytometry. An alternative method in osteoblast proliferation assays.

OBJECTIVE: To assess whether DNA image cytometry can be used as an alternative method to tritiated thymidine uptake quantification in osteoblast proliferation assays. STUD DESIGN: Proliferation of normal human osteoblasts incubated with normal human serum at 0%, 2.5%, 5%, 10%, 20% and 40% was quantified by tritiated thymidine uptake quantification and DNA image cytometry. RESULTS: Tritiated thymidine uptake quantification showed that normal human serum stimulated the proliferation of normal human osteoblasts and that the degree of stimulation was directly related to the concentration of serum in the culture medium. Similar results were obtained when the DNA image cytometry assay was used. A highly significant linear relationship between the ranks of both methods was found (Spearman's r = 1.00, P = .0253). CONCLUSION: DNA image cytometry may be a valuable alternative when the use of radioactive material is not desired and/or subsequent morphologic or immunocytochemical characterization of cells under study is required.

Aged↗
PubMed2000

Clinical applications of image cytometry to human tumour analysis.

Image cytometry (ICM) is widely applied to the automated screening, the detection, the diagnosis, the classification, the prognosis and the therapeutic follow-up of different types of cancers (breast, bladder, cervix,...). This review describes the analysis methods and the applications of nuclear image analysis, the determination of DNA content and the analysis of morphometry and of nuclear texture. DNA content analysis can contribute to a prognostic information in addition to other prognostic factors for breast, renal and prostate cancers. For ovarian cancer, aneuploidy seems to be related to prognosis. Bladder tumours with DNA aneuploidy were frequently of high malignancy while ploidy was significantly correlated to relapse risk. For digestive cancers, patients presenting DNA diploid tumours show a better survival than patients with aneuploid ones. Morphometry seems to be a more important criterion than other conventional prognostic factors of invasive breast and digestive carcinomas. A differential diagnosis between normal and neoplastic thyroids is more precise when based on a quantitative evaluation of texture associated to morphometry. Textural parameters permit the discrimination of two populations of patients having a different prognosis and could thus be an aid for prognosis in prostatic cancers. Morphonuclear parameters contribute to separate low and high grade bladder carcinomas. Although ICM was frequently reported, results from the reported examples were not always obvious. In conclusion, the measurements obtained with ICM could be helpful for a decision in several cancers but could not be a substitute for the classical approach of the pathologist.

Animals↗
PubMed2003

Clinical aspects for the use of DNA image cytometry in detection of bladder cancer: a valuable tool?

The employment of DNA flow or image cytometry for oncological diagnostic procedures is favored because of its high correlation to tumor biological behavior. Prognostic statements and therapeutic strategies therefore are based on the high validity of DNA cytometric measurements. Using 151 bladder washings from patients suspected of bladder cancer for this study, we examined the clinical value of various common methods of DNA single cell (SCI) and stemline interpretations (SLI). Comparing the specificity and sensitivity of DNA image cytometry in detection of bladder tumors, we found 81 and 52%, respectively, for SCI of Boecking, 84 and 45% for SLI of Boecking, 61 and 58% for SLI of Fu, and 82 and 40% for conventional stemline interpretation. To improve diagnostic and prognostic validity of DNA image cytometry, we designed our own method of interpretation. In consequence, we identified six single DNA parameters out of all recorded measurements that correlated most to histopathological grading (G1-G3). Creating reference values at random and rating by points, we used a cytometric grading system for ranking. In detection of bladder cancer specificity and sensitivity ultimately arrived at almost 70% in application of our method. Thus, by this study, we were able to show that sensitivity of DNA examination can be increased by combining various DNA parameters. Apart from our own scheme, the discrepancy in interpretation of DNA image cytometry does not allow us to recommend this procedure as the only diagnostic in detection of bladder cancer. However, in regard to prognostic statements, particularly tumor biological behavior, DNA image cytometry appears to be useful.

Aneuploidy↗
PubMed1994

DNA ploidy in carcinoma of the gallbladder. Prognostic significance and comparison of flow and image cytometry on archival tumor material.

The aim of this investigation was to determine whether the cytometrically assessed DNA ploidy is a parameter of prognostic significance in gallbladder carcinomas. For this purpose the DNA content of tumor cells from archival tumor material from 80 patients with cholecystic cancer was analysed retrospectively by using comparatively different cytometrical methods, namely flow cytometry (FCM) and image cytometry, using tissue sections (ICM-S) and nuclear suspensions (ICM-N). Conventional tumor classifications (according to pTNM and Nevin) were able to predict the further clinical course. DNA ploidy showed a statistically significant correlation only to histological grade (P < 0.001), but not to tumor stage and survival. Only a trend towards a poorer outcome for patients harboring non-diploid tumors became evident especially when no residual tumor was present postoperatively (R 0-stage; P = 0.08). The different cytometrical methods discussed in detail, showed a highly significant concordance in the determination of DNA index values (DI) and in the classification of the tumors as either diploid or non-diploid. In conclusion, our results indicate that cytometrical determination of DNA ploidy provides no further prognostic informations in gallbladder carcinomas as compared to conventional tumor staging.

Aged↗
PubMed1998

Image cytometry in early graft-versus-host disease.

Image cytometry was used to measure the size, shape, and texture of lymphocyte nuclei in 24 skin biopsy specimens retrospectively selected from patients with early, nonspecific, graft-versus-host disease (GVHD)-like eruptions. Half of these patients had progressed to overt GVHD, whereas half had never developed more than a mild nonspecific eruption. Significant differences were detected in the nuclear texture of the eruptions of the two groups, but the differences were not consistent enough to suggest that image cytometry might play a significant role in recognition of early GVHD.

Cell Nucleus↗
PubMed1998

Use of Lewis X antigen and deoxyribonucleic acid image cytometry to increase sensitivity of urinary cytology in transitional cell carcinoma of the bladder.

PURPOSE: To improve sensitivity and specificity of urinary cytology for bladder cancer cell detection we used deoxyribonucleic acid (DNA) image cytometry and the monoclonal anti-Lewis X antibody P12. MATERIALS AND METHODS: Spontaneously voided urine and additional barbotage bladder washings of 25 patients with transitional cell carcinomas and 25 patients with benign diseases of the bladder were analyzed by conventional cytology, immunocytology and DNA image cytometry. The DNA content was determined in specimens stained with Feulgen according to the European Society for Analytical Cellular Pathology consensus report on standardization of DNA image cytometry. For the immunocytological examination we used the avidin-biotin-complex immunoperoxidase method with the monoclonal antibody P12 directed against the Lewis X determinant. RESULTS: The cytological examination revealed a sensitivity of 72% and a specificity of 100%. By using DNA image cytometry with Kolmogoroff-Smirnow test sensitivity increased up to 84% with a specificity of 100%. The immunocytological examination with Lewis X showed a sensitivity of 84% and a specificity of 80%. CONCLUSIONS: Combining cytology and DNA cytometry the overall sensitivity increased to 92% and with the additional application of immunocytology for Lewis X antigen sensitivity it increased to 96% but was accompanied by a decrease in specificity to 80%. DNA image cytometry and Lewis X antigen detection are not suitable for screening but suspicious urothelial cells in urinary cytology specimens can be evaluated specifically by DNA measurements. In the future it needs to be clarified whether DNA image cytometry in combination with Lewis X antigen detection can help to signal relapse and progression of transitional cell carcinoma of the bladder.

Aged↗
PubMed2000

Diagnostic accuracy of DNA image cytometry and urinary cytology with cells from voided urine in the detection of bladder cancer.

OBJECTIVES: To assess the potential of DNA image cytometry in screening for bladder cancer, compare it with conventional urinary cytology, and evaluate its possible use in routine urinary evaluation. Urinary cytology is still the most common method for detection of bladder cancer in routine clinical use. The considerable shortcomings of urinary cytology include its low sensitivity in low-grade carcinomas and its poor reproducibility. METHODS: Spontaneously voided urine specimens from 40 patients with grade 1 (n = 27), grade 2 (n = 10), and grade 3 (n = 3) histologically proven transitional cell carcinoma and 40 patients with symptomatic urologic disease of the bladder were analyzed by cytology and DNA image cytometry. The DNA content was determined by use of the CM-1 Cytometer according to the guidelines in the ESACP Consensus Report on Standardization of DNA Image Cytometry. RESULTS: Urinary cytology yielded an overall sensitivity of 47.5%. Conventional analysis of DNA histograms measuring the presence of DNA stemline aneuploidy (1.8c > stemline ploidy [STP] > 2.2c) revealed a sensitivity of 62.5%; applying the stemline interpretation according to Böcking et al. increased the overall sensitivity to 75%. The specificity of both methods was 100%. DNA image cytometry demonstrated a high sensitivity in grade 1 tumors (70.4%) compared with cytology (26%). CONCLUSIONS: In light of its highly improved sensitivity compared with urinary cytology, DNA image cytometry should be used to evaluate suspect urothelial cells in urinary cytology specimens. Since the method provides more objective and reproducible results with a specificity comparable to that of cytology, we encourage its primary application in the screening for bladder cancer, provided these results can be confirmed in a multicenter evaluation study.

Adult↗
PubMed1990

Proposed standard for image cytometry data files.

A number of different types of computers running a variety of operating systems are presently used for the collection and analysis of image cytometry data. In order to facilitate the development of sharable data analysis programs, to allow for the transport of image cytometry data from one installation to another, and to provide a uniform and controlled means for including textual information in data files, this document describes a data storage format that is proposed as a standard for use in image cytometry. In this standard, data from an image measurement are stored in a minimum of two files. One file is written in ASCII to include information about the way the image data are written and optionally, information about the sample, experiment, equipment, etc. The image data are written separately into a binary file. This standard is proposed with the intention that it will be used internationally for the storage and handling of biomedical image cytometry data. The method of data storage described in this paper is similar to those methods published in American Association of Physicists in Medicine (AAPM) Report Number 10 and in ACR-NEMA Standards Publication Number 300-1985.

Electronic Data Processing↗
PubMed2002

The clinical value of image cytometry DNA analysis in distinguishing branchial cleft cysts from cystic metastases of head and neck cancer.

OBJECTIVES/HYPOTHESIS: A branchial cleft cyst presents as a lump in the neck that, generally, is easily cured by surgical excision. The preoperative diagnosis is based on clinical examination and, especially in the Scandinavian countries, fine-needle aspiration cytology. However, at times, the histopathological analysis of the excised cyst reveals a cystic metastasis of squamous cell carcinoma of the head and neck. If adequate diagnosis could be obtained preoperatively, patients would most likely fare better. The study was performed to investigate whether the diagnostic accuracy for these lesions could be improved preoperatively by image cytometry DNA analysis of the fine-needle aspiration cytology specimen. STUDY DESIGN: Image cytometry DNA analysis was performed on the preoperative fine-needle aspiration cytology specimen and the surgical specimens from 51 patients with solitary cysts in the lateral region of the neck. Thirty-six patients were selected because there was a discrepancy between findings on fine-needle aspiration cytology and the final histopathological diagnosis or an uncertain cytological diagnosis. There were 25 metastatic squamous cell carcinomas and 3 thyroid cancers, there was 1 lymphoma and 1 sialoadenitis, and there were 21 branchial cleft cysts. METHODS: The cytodiagnostic Giemsa-stained slides were destained in Methanol and then stained with Schiff's reagent. The paraffin-embedded material from excised cysts were cut and deparaffinized and then stained with Schiff's reagent. Ahrens image analysis was used for DNA analysis and lymphocytes were used as control cells. DNA valves exceeding 5c was regarded as aneuploid. RESULTS: Image cytometry DNA analysis of the preoperative cytological specimen was possible in 41 of 51 patients. We found that in 53% of the cases with cystic metastasis, image cytometry DNA analysis, when possible, revealed aneuploidy, thus indicating malignancy. DNA analysis showed diploidy in all benign cases. CONCLUSIONS: Aneuploidy is highly specific for malignancy. Image cytometry DNA analysis increases the diagnostic sensitivity for malignant cystic metastasis and therefore is a valuable supplement to conventional cytological study for these lesions.

Adult↗
PubMed2003

Toluidine blue test for sperm DNA integrity and elaboration of image cytometry algorithm.

BACKGROUND: Sperm DNA integrity is of paramount importance in the prognosis of fertility. We applied image cytometry to a toluidine blue (TB) test we recently proposed. METHODS: Sperm samples from 33 men were assayed for standard sperm parameters and classified as normal or abnormal. Sperm smears were subjected to the TB test, DNA denaturation testing with acridine orange (AO), and terminal deoxyuridine triphosphate biotin nick end labeling (TUNEL). In CCD image analysis, TB-stained sperm cell heads were microscopically assigned to one of four color groups (dark, blue, light violet, and light blue). The optical densities of 6,600 cells in green and red CCD images were used to elaborate an algorithm for discrimination of these groups. RESULTS: The proportions of sperm in TB color groups, as estimated with the developed image cytometry algorithm, correlated with microscopic features. The number of TB dark cells correlated with the number of AO-red and TUNEL(+) cells. The proportion of TB dark cells in normal samples did not exceed 35%. Light-blue sperm cell heads prevailed in normal samples, whereas dark and blue sperm cell heads dominated in abnormal samples. CONCLUSIONS: The TB test was suitable for the assessment of sperm cell DNA integrity. The elaborated image cytometry algorithm can be used for this purpose and for finer determination of sperm nucleus status.

Acridine Orange↗
PubMed1998

Application of confocal scanning laser microscopy to 3-D DNA image cytometry of prostatic lesions.

OBJECTIVE: To explore the potential of three-dimensional (3-D) image cytometry for the measurement of DNA content in prostatic specimens using confocal scanning laser microscopy (CSLM) and 3-D image analysis. STUDY DESIGN: Thick tissue slices (100 microns), stained for DNA with chromomycin A3, from four patients with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), well- and poorly differentiated adenocarcinoma of the prostate, were studied. Two different blocks from the same slice were studied for each case. Cell nuclei were segmented automatically. DNA content and nuclear volume were measured. RESULTS: DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, two peaks were observed, one in the diploid range and one in the tetraploid range. The two peaks were observed on two different blocks of the same tissue slice. Poorly differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinoma, we observed a considerable variation in nuclear volume. CONCLUSION: The results indicate the potential of 3-D image cytometry for the measurement of DNA content in prostatic specimens while preserving tissue architecture.

Cell Nucleus↗
PubMed2001

Image cytometry in nonproliferative fibrocystic breast changes. Ploidy evaluation and epidemiologic study.

OBJECTIVE: To determine the usefulness of image cytometry on fine needle aspirates from patients with fibrocystic changes (FCCs) to assess the subsequent risk of breast cancer. STUDY DESIGN: Thirty-five hundred archival cases with fine needle aspiration (FNA) biopsy were assessed to select nonproliferative FCCs of the breast (500 cases), also classified as type 1 (FCC I). DNA evaluation was analyzed by means of image cytometry on ductal epithelial cells and on apocrine metaplastic cells; 97 quantifications were performed. Cytometric variables investigated were: DNA ploidy, G0/G1 peak of diploid nuclei, S-phase fraction, 5cER, 2cDI and coefficient of variation. Two groups each with 15 years of follow-up were formed: Simple pathology (SP), fibrocystic changes alone; associated pathology (AP), FCCs plus breast cancer. Each was studied separately and compared. RESULTS: In SP cases the average ploidy was 2.2 for epithelial cells and 4.2 for apocrine cells. The proportion of G0G1 diploid nuclei was 100%. In the study of AP, the average ploidy was 2.2 for epithelial ductal cells and 4.1 for apocrine ones, for a slight increase in SPF. Ductal cells were diploid, while apocrine cells were tetraploid, with statistical significance of P < .05. For the epidemiologic study we calculated the proportion of patients with FCC I who developed cancer by referring to a historical cohort study, obtaining a relative risk of 0.7. CONCLUSION: Our results prove that DNA image cytometry applied on FNA cytology is a very useful, minimally invasive and reliable tool to determine higher activity and risk for development of breast cancer in FCC I and thus to establish the need for closer follow-up of these patients. In addition, apocrine metaplastic cells of the breast display a tetraploid DNA histogram, while the other karyometric variables remain in the range of normality.

Apocrine Glands↗