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PubMed1989

Predictive value of DNA measurements in bladder washings. Comparison of flow cytometry, image cytophotometry, and cytology in patients with a past history of urothelial tumors.

Comparative DNA ploidy measurements were carried out by flow cytometry and by image analysis on cells in 71 bladder washing specimens from 50 patients with past histories of bladder tumors. Among the specimens classified as diploid or questionable by flow cytometry, 14 showed the presence of aneuploid DNA values documented by image analysis. In 18 of the 50 patients, recurrent tumors were observed during a relatively brief period of follow-up. In 15 of them the DNA pattern was aneuploid and in three it was questionable. In nine of the 15 patients, both methods of DNA analysis disclosed aneuploidy, but in six patients aneuploidy was detected by image analysis only. A combination of DNA aneuploidy, whether observed by flow cytometry, image analysis, or both, and of positive or suspicious urine cytology is highly predictive of recurrence of high grade bladder tumors. Image analysis of DNA content in bladder washings adds information of clinical value above and beyond that obtained by flow cytometry.

Aged↗
PubMed1987

Nuclear DNA analysis of oral hyperplasia and dysplasia using image cytometry.

We investigated the value of image analysis in discriminating among oral white lesions with hyperplasia without dysplasia and oral white or white-and-red lesions with moderate or severe dysplasia. Normal oral epithelial tissue was used as a control. Image analysis was applied to 5-micron formalin-fixed sections stained with the azure A-Feulgen reaction for nuclear DNA. For 150-200 cells from each section, 5 nuclear variables were assessed: area, form factor, total stain, average stain and ellipticity. For each variable, 2 measurements were obtained, the mean and the interquartile range, and were used for stepwise discriminant analysis. Using this test, a model of 3 measurements with the most discriminating power was developed. When the jackknife classification test was applied to this model, we could discriminate with 81% accuracy between the 4 groups of tissue studied.

Carcinoma, Squamous Cell↗
PubMed1991

Fluorescent image cytometry: from qualitative to quantitative measurements.

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.

Analog-Digital Conversion↗
PubMed1992

Distribution of non-heme porphyrin content of individual erythrocytes by fluorescence image cytometry and its application to lead poisoning.

The quantitation of the non-heme porphyrin content of circulating erythrocytes is an important tool in the screening, diagnosis, and management of lead disease, iron deficiency anemia, and certain porphyrias. Useful information about these pathological conditions may be obtained from the distribution of the non-heme porphyrin content of individual red blood cells. An image-based cytometry system was developed and used to determine the cellular distributions of zinc protoporphyrin (ZPP) in the erythrocytes of normal and lead-exposed human subjects. The fluorescence cytometry system described here lends itself to a wide range of statistical studies of cell populations, in which extrinsic fluorescent probes or antibodies may be used instead of porphyrin. Image-based cytometry appears to offer a number of important advantages over the more conventional flow cytometry systems, including greater sensitivity, applicability to a wider range of cellular parameters, and the ability to retain images of each cell used in the survey for subsequent examination. Pairs of absorption and fluorescence images of many fields of dispersed immobilized red cells were acquired by means of a program which controls the microscope stage, focussing, shutters, filter wheels, and a cooled, slow-scan CCD camera. The observed marked differences in the ZPP distributions of erythrocytes from donors with chronic and acute lead exposure are discussed in terms of the metabolism of internalized lead.

Algorithms↗
PubMed1995

Methods for cell proliferation analysis by fluorescent image cytometry.

Methods were developed for multimodal microscopic image analysis in order to identify and analyze one cell type under various microscopic conditions. Our purpose was to quantify both total DNA content using propidium iodide (PI) stain and S-phase fraction using the bromodeoxyuridine (BrdUrd) incorporation technique in cell population subsets. The model chosen was plasma cells in bone marrow triply labelled with fluorescein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, with amino-methylcoumarin-acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recognized on FITC images, and the centroid positions were stored. Second, plasma cell nuclei were geodesically reconstructed from these stored positions using PI images in which DNA content was measured, and the nuclear mask outlines were stored. Third, BrdUrd incorporation level of plasma cells was measured on AMCA images inside PI nuclei masks and stored. Image DNA vs. BrdUrd scatterplots were obtained for cells selected according to the expression of intracytoplasmic immunoglobulin. Thus, both ploidy and proliferation could be independently evaluated on a subset of the cellular population.

Animals↗
PubMed1995

Improved prognostication in small (pT1) breast cancers by image cytometry.

Feulgen-stained samples from 460 small (pT1) primary breast cancers were investigated by means of an image analysis system. Several DNA, morphometrical and textural parameters were evaluated for each patient, and the prognostic meaning of these parameters was then investigated by the Cox regression analysis. As prognostic criterion a distant recurrence-free survival of five years was considered. All investigated DNA- and morphometrical parameters as well as several textural parameters showed a significant univariate correlation with the clinical course. In a multivariate approach the axillary nodal status was the most important prognostic parameter, followed by a morphometric parameter (anisokaryosis) and two textural parameters (runlength and co-occurrence). None of the DNA histogram derived parameters could add prognostic information in this multivariate approach. By the linear combination of the four selected variables, an individual prognostic factor was calculated. Using this factor the patients could be split into several groups according to their risk for distant metastases. Thus a low risk group of pT1 patients could be identified with a distant recurrence rate of only 2% after 5 years, and also a group of patients with a considerably worse prognosis and a 5-year distant recurrence rate of 53%. In contrast, using the nodal status as single parameter allows the identification of a low risk group of patients (pN0pT1) with a distant recurrence rate of 10.6%. Therefore, morphometrical and textural parameters can provide powerful prognostic information in small breast carcinomas and may allow a better selection of patients for adjuvant therapy.

Aged↗
PubMed1997

DNA analysis of squamous cell carcinoma in the oral cavity by image cytometry and its rating in the assessment of tumor prognosis.

Image cytometric measurements on the DNA content of squamous cell carcinoma of the oral cavity were carried out to evaluate its relationship with clinicopathological features. In the prospective study 52 patients with operable oral cavity carcinoma in stage T1-4 and N0-3 were studied. The tumors were classified according to the TNM classification (UICC 1987) and graded histopathologically. A positive correlation between tumor size and ploidy status was observed. There was a significant correlation between ploidy status and histologically confirmed cervical lymph node metastases (P < 0.03). A relationship between histopathological grading and ploidy status was not found. The cumulative 5-year survival rate was significantly higher (P < 0.03) among patients with diploid DNA findings than among those with nondiploid findings. When assessing the prognosis of squamous cell carcinoma, DNA cytometry analysis should be used as an additional method. Particularly the primarily existing, significantly higher N stage, a higher frequency of metastases, and a significantly lower survival rate with nondiploid tumors underline the importance of this examination method.

Adult↗
PubMedDate not supplied

Detection of high risk patients in chronic myelogenous leukemia by DNA-image cytometry.

Peripheral blood smears from the time of diagnosis of 64 patients with chronic myelogenous leukemia (CML) in chronic phase were restained according to Feulgen. Nuclear DNA content of at least 100 myeloblasts and promyelocytes was measured by interactive DNA-cytometry using a TV-image analysis system, combined with an automated microscope. From the resulting DNA values the DNA parameters stemline ploidy, stemline shoulder fraction and "proliferative" fraction, which reflect cytogenetic and cell kinetic characteristics of CML, were derived. Each of these DNA parameters identified subgroups with a significantly different survival time and duration of chronic phase. Indicators for a good prognosis were a near-diploid stemline ploidy (median survival time 49 v. 26 months), a low percentage of nuclei in the stemline shoulder fraction (45 v. 28 months) and a high "proliferative" fraction (45 v. 23 months). Independently of clinical and hematological features, the DNA-cytometric parameters yielded additional prognostic information both in the univariate and multivariate analysis.

Adult↗
PubMed1991

DNA ploidy of malignant melanoma determined by image cytometry of fresh frozen and paraffin-embedded tissue.

Image analysis of DNA content was performed from single nuclei of melanoma monolayer imprints made from fresh frozen tissue of 14 patients with primary malignant melanoma and 16 patients with local recurrences at the incision site and local or distant metastases. This procedure requires fewer cells and is an advantage when the quantity of tumor available is limited, especially in thin low Breslow depth cutaneous melanomas. Image analysis allowed reproducible measurement of DNA ploidy from 100 cells. The frequency of aneuploidy was similar in primary and metastatic melanomas. Three of 3 patients with euploid primary melanomas showed no evidence of recurrences or metastases, though one died of unrelated disease with short follow-up. The 4 patients with primary melanoma who developed metastases had aneuploid primaries; two of these patients died of metastatic disease. Three of 4 patients with euploid metastatic tumors were free of disease at last follow-up, and 1 patient died with stable disease. Nine of 12 patients with aneuploid tumors died of metastatic disease. The frequency of DNA ploidy in the present image analysis study correlated with previous flow cytometry studies. In 9 patients with primary tumors with a Breslow depth greater than 0.75 mm, the DNA content was also determined in nuclei obtained from formalin-fixed paraffin-embedded tissue. The frequency of aneuploidy was higher in fresh tissue (7 of 9) as compared with paraffin-embedded tissue of the same cases (4 of 9).

Aneuploidy↗
PubMedDate not supplied

In vivo integrated flow image cytometry and lymph/blood vessels dynamic microscopy.

The high spatial resolution (approximately 350 nm) transmission digital microscopy (TDM) was developed for real time in vivo imaging of microlymphatics of rat mesentery at a single cell level without any contrast agent. The main mesenteric microstructures (lymph-vessel diameter, valve geometry, cells, etc.) and their dynamics (wall motion, valve function, cell velocity, etc.) were monitored with TDM. Depending on structure size, different magnifications were used to image relatively large whole lymphangion (x4 to x10) as well as to image single cells (x40 to x100) in lymph and blood flow including estimation of their shape, size, and aggregation state. Various potential applications of the TDM for in vivo studies are discussed, including visualization of circulating cells in lymph and blood flows, studying the kinetics of platelets, leukocyte rolling, as well as imaging absorbing nonfluorescent mesentery structures and leukocytes with a high optical resolution.

Animals↗
PubMed1997

Signal amplification of FISH for automated detection using image cytometry.

The purpose of this study was to improve the detection of FISH signals, in order that spot counting by a fully automated image cytometer be comparable to that obtained visually under the microscope. Two systems of spot scoring, visual and automated counting, were investigated in parallel on stimulated human lymphocytes with FISH using a biotinylated centromeric probe for chromosome 3. Signal characteristics were first analyzed on images recorded with a coupled charge device (CCD) camera. Number of spots per nucleus were scored visually on these recorded images versus automatically with a DISCOVERY image analyzer. Several fluochromes, amplification and pretreatments were tested. Our results for both visual and automated scoring show that the tyramide amplification system (TSA) gives the best amplification of signal if pepsin treatment is applied prior to FISH. Accuracy of the automated scoring, however, remained low (58% of nuclei containing two spots) compared to the visual scoring because of the high intranuclear variation between FISH spots.

Chromosomes, Human, Pair 3↗
PubMed1995

Cytology, flow cytometry, image analysis, and interphase cytogenetics by fluorescence in situ hybridization in the diagnosis of transitional cell carcinoma in bladder washes: a comparative study.

The diagnosis of transitional cell carcinoma (TCC) in bladder washes is a diagnostic challenge to cytology. This study assessed the role of flow cytometry (FCM), image analysis (IA), and interphase cytogenetics by fluorescence in situ hybridization (FISH) as adjuncts in the cytodiagnosis of TCC in bladder washes. Forty separate samples of bladder washes were prospectively evaluated by conventional cytology (CY), FCM, IA, and FISH, and the results were compared with the subsequent surgical biopsy specimens which revealed 26 TCC (3 GR I, 6 GR II, 17 GR III) and 14 benign lesions. Using histology as the "gold standard" and following the previously published criteria for detection of TCC by CY, FCM, IA, and FISH, the concordance rates between histology and CY, FCM, IA, and FISH were 75, 74, 89, and 83%, respectively. CY, FCM, IA, FISH, and histology were concordant in 54% of the cases. The sensitivity of CY, FCM, IA, and FISH were 61, 72, 91, and 73%, respectively, while the specificity were 100, 80, 83, and 100%, respectively. The combined sensitivity of all the parameters was 96%. Interestingly, the false positive cases by FCM and IA showed cystitis. We conclude that IA has the highest sensitivity in detecting TCC in bladder washes followed by FISH, FCM, and CY, while CY and FISH have the highest specificity. This study indicates that FCM, IA, and FISH are useful adjuncts to cytology in the diagnosis of TCC in bladder washes. The finding of DNA-aneuploidy in cystitis warrants further investigation.

Carcinoma, Transitional Cell↗
PubMed1992

DNASK--a new image analysis module for TV image cytometry.

The DNASK is a PC-based image analysis module for quantitative cytological and histological examinations on Feulgen-Schiff preparations. The module consists of a frame grabber, a CCD black and white video camera and the DNASK software package which can be incorporated in an IBM compatible PC-AT joining to a standard pathological research photo microscope. The use of 386 IBM AT systems leads to significant decrease of the measurement's time. Mono- or color VGA graphics card should be used. The resolution of the images is 512 x 512 pixels and 256 gray values. The image caption is made in less than 0.5 second, the measurement of a cell nuclei (18 parameters) takes less, than 5 seconds (IBM AT 286). The camera is a standard CCD one. The microscope should be a good quality, modern microscope with built-in light source, however, the application of voltage stabiliser is strongly recommended. The software is able to measure the parameters mostly used and suggested in the international literature: 15 morpho- and densitometric parameters of the cell nuclei 6 DNA histogram parameters of the measured case, like DNA Index, 2c deviation Index, 5c exceeding rate, G1-S-G2 phase fraction ratio. The system supports the grouping of the measured cases and at the end of a study results of the single measurements can be collected and summarized in an ASCII file, which is readable by major statistical programpackages. The DNASK has a special graphical user interface for the control of the program.

Algorithms↗
PubMed2001

Comparative analysis of DNA content estimated by flow and image cytometry in breast tumor samples.

The DNA ploidy status of 186 fresh primary breast tumors was analyzed in a comparative study of flow cytometric (FCM) and image (IA) analyses. Tumor size, histology and nodal status were also taken into account. The same piece of fresh tissue was used for touch imprints (IA) and for DNA analysis by FCM. Both methods provided concordant DI values in 158 (85%) tumors (r = 0.7490). The tumor grade and lymph node status significantly correlate with ploidy estimated by FCM and IA. We conclude that FCM and IA provide comparable results of DNA content although occasional discrepancies occur. IA seems to be a more sensitive method especially for diploid cases detected by FCM.

Breast Neoplasms↗