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PubMed2000

Image cytometry detection of breast cancer cells with > 5C DNA content and minor DNA stemlines.

OBJECTIVE: To determine if the presence of cells having a DNA content > 5c and occurring at very low frequency is related to breast cancer outcome. STUDY DESIGN: Feulgen-stained imprints of fresh tumors used for routine standard DNA image cytometry were reanalyzed, with the aim of detecting hyperploid (> 5c) cells or minor stemlines. Specially adapted software was used. RESULTS: The new DNA analysis showed discordance of 47.3% with standard DNA cytometry. Minor stemline or rarely occurring 5c exceeding cells were found. These were not detected by the first DNA analysis. The presence of both DNA hyperploid cells occurring as rare events and a DNA hyperploid stemline was related to outcome. CONCLUSION: The detection of DNA hyperploid cells, even in very small numbers, appears essential to outcome, particularly in diploid or single DNA aneuploid breast cancers.

Adenocarcinoma, Mucinous↗
PubMedDate not supplied

Prenatal diagnosis of a (X;X) translocation by fluorescence in situ hybridization and laser scanning image cytometry.

A de novo structural abnormality of one X chromosome was prenatally detected in a female fetus. This chromosomal abnormality has been analyzed by conventional cytogenetic methods, fluorescence in situ hybridization, and laser scanning image cytometry. The association of these techniques has demonstrated that this anomaly corresponds to a (X;X) translocation. Analysis of hybridization signals by laser scanning image cytometry allowed to localize that the breakpoints were at the X-centromeric region and Xp11.3, respectively. These results show the usefulness of image analysis and fluorescence in situ hybridization for a rapid characterization of de novo structural chromosome anomalies in prenatal diagnosis.

Adult↗
PubMed2002

Differentiation between malignant and benign follicular adnexal tumours of the skin by DNA image cytometry.

BACKGROUND: We have demonstrated in previous studies that DNA image cytometry (DNA ICM) can be helpful in detecting malignancy in sebaceous tumours of the Muir-Torre syndrome and sweat gland tumours. However, little is known about DNA ICM in cutaneous adnexal tumours with follicular differentiation. OBJECTIVES: To study a larger series of benign and malignant follicular adnexal tumours with DNA ICM. METHODS: We studied 13 malignant follicular tumours (seven trichilemmal carcinomas, five malignant proliferating pilar tumours, one pilomatrix carcinoma) and 55 benign follicular tumours (four tumours of the follicular infundibulum, seven Winer's pores, eight trichilemmomas, two trichofolliculomas, 16 trichoepitheliomas, 13 pilomatrixomas, five trichoblastomas) by DNA ICM. All cases were clear-cut as malignant or benign, respectively, on histopathological criteria. The stemline interpretation according to Böcking et al. (DNA distribution in gastric cancer and dysplasia. In: Precancerous Conditions and Lesions of the Stomach, Zhang YC, Kawai K, eds. Berlin: Springer-Verlag, 1993: 103-20) was performed in all cases. In addition, 5[c]-exceeding events (5cEE) and the 2[c] deviation index (2cDI) were calculated, except in one histopathologically benign tumour, which revealed euploid polyploidization, as the analysis of 5cEE and 2cDI is not valid in that case. RESULTS: A 2cDI threshold of 0.24 proved to be the most reliable marker for the distinction between malignant and benign follicular tumours. On the basis of this feature, all malignant and benign tumours were correctly classified. A specificity of 100% was achieved by all three interpretation methods, but the sensitivity of 2cDI for the detection of malignant tumours was superior to the analysis of 5cEE (sensitivity 77%) and to the stemline interpretation (sensitivity 23%). CONCLUSIONS: DNA ICM may be helpful in distinguishing between malignant and benign follicular tumours.

Aneuploidy↗
PubMed1995

Comparison of DNA measurement performed by flow and image cytometry of embedded breast tissue sections.

Tissue sections are important for morphometric studies, such as the analysis of architectural pattern of tissues as well as nuclear morphology and chromatin texture describing DNA distribution in nuclei. Tissue sections are generally not recommended for use in DNA ploidy measurements due to possible errors based on sectioned and overlapped nuclei. However, image cytometry (IC) on tissue sections has the advantage of preserved architecture and selective sampling of nuclei. This is particularly important for the analysis of specimens with small areas of diseases tissue or with a variety of histologic patterns in the same region, such as premalignant changes or in situ tumors. To evaluate the potential of measurements on breast tissue sections in the estimation of DNA ploidy status, we compared IC and flow cytometry (FC) measurements on formalin-fixed breast cancer tissue from 51 paraffin-embedded blocks. For each specimen the DNA index and ploidy were determined by both methods. DNA indices of IC and FC corresponded roughly in 66% of cases. Agreement in ploidy was achieved in about 80% (40/51) of cases, where some of the discrepancies could be explained by the differences between invasive cancer and ductal carcinoma in situ (DCIS) illustrated by IC. Both methods agreed highly in aneuploid cases. However, about 50% of FC diploid cases were aneuploid by IC. Despite significant shortcomings of measurements obtained on archival tissues, meaningful data can be obtained not only by FC but also by IC performed on tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1999

Immunocytochemistry and DNA-image cytometry in diagnostic effusion cytology. II. Diagnostic accuracy in equivocal smears.

To determine sensitivity and specificity of different antibodies for the immunocytochemical detection of malignant cells in diagnostically equivocal effusions in comparison with those achieved by DNA-image cytometry. 65 cytologically doubtful effusions of the serous cavities were stained with twelve antibodies. Furthermore, DNA-image cytometry was performed. Data were correlated with patient follow-up. Sensitivity of cellular staining of Ber-EP4 for the identification of malignant cells was 77.8%, specificity of absent staining for benign cells was 100%. Positive predictive value for the identification of malignant cells was 100%, negative value 65.5%. Sensitivity of DNA-aneuploidy for the identification of malignancy was 82.9%, specificity of DNA-non-aneuploidy for benignity 94.7%. The positive predictive value of DNA-aneuploidy for the occurrence of malignant cells was 96.7%. Negative predictive value of DNA-non-aneuploidy was 72.0%. Combining immunocytochemistry applying Ber-EP4 only and DNA-cytometry in equivocal effusions resulted in a sensitivity of 88.9% for the identification of malignant cells associated with a 95.0% specificity. Positive predictive value was 97.7%, the negative one 79.2%.

Aneuploidy↗
PubMed2002

DNA ploidy in soft tissue sarcoma: comparison of flow and image cytometry with clinical follow-up in 93 patients.

In soft tissue sarcoma, the prognostic importance of DNA ploidy status is limited. One possible explanation may be technical; small non-diploid stemlines will be diluted in relation to the presence of normal diploid cells and may not be detected by flow cytometry (FCM). We assessed DNA ploidy status in 93 tumors with both FCM and image cytometry (ICM). ICM may permit the exclusion of non-relevant cells. The ability of the two methods to detect non-diploid stemlines was compared, as were the prognostic consequences. The patients (54 males) had a median age of 69 years. Surgical procedures were performed on all patients. None of the patients had received preoperative radiotherapy or chemotherapy. FCM and ICM were performed with standard methods. The prognostic value was assessed with univariate and multivariate analysis. In 82 of the 93 tumors, a concordant ploidy status by FCM and ICM was found. In 5 FCM type 1-2 tumors (diploid), the identification of non-diploid stemlines by ICM did not influence the metastatic rates. Increasing tumor size, histotype other than liposarcoma, increasing malignancy grade, tumor necrosis, and ICM non-diploidy were univariate prognostic factors for metastasis. In a multivariate analysis, only tumor size larger than 9 cm was a prognostic factor. In about 10% of the tumors, a discrepancy between FCM and ICM ploidy status was found, but we could not find a consistent prognostic consequence of this. Neither FCM nor ICM ploidy status was an independent prognostic factor.

Adolescent↗
PubMed1985

Early detection of precancerous lesions in dysplasias of the lung by rapid DNA image cytometry.

Hematoxylin-and-eosin-stained cytologic smears of sputum from 28 patients with dysplastic and suspicious cell findings were subjected to DNA image cytometry after Feulgen restaining. The nuclear DNA contents were measured with a TV-based image-analysis system, the Leitz TAS plus, combined with an automatic microscope. Computation of DNA data was performed according to an algorithm for the diagnosis and grading of malignancy. Of the 19 cases that were proven to be malignant in the follow-up, either by histologic examination, sputum cytology, fine needle aspiration biopsy or autopsy, the algorithm identified 17 as malignant in a stage (dysplasia) in which cytology was not yet able to present a definite diagnosis of malignancy. Only two cases of bronchial carcinoma were not detected in the state of dysplasia by this procedure. The periods between the DNA diagnosis of malignancy in dysplasia and the morphologic evidence of cancer varied from three days up to six months. Of the 11 cases that had been classified as benign by the algorithm, 9 were confirmed as benign during the clinical follow-up. Rapid DNA image cytometry appears able to separate squamous dysplasias of the lung into precancerous and nonprecancerous lesions.

Adenocarcinoma↗
PubMed1997

Comparison of fixation procedures for fluorescent quantitation of DNA content using image cytometry.

DNA quantitation using fluorescent dyes is of interest in image cytometry in that it is nondestructive in its mode of staining and is compatible with techniques such as FISH and immunofluorescence, allowing multicolor analysis of a wide range of cellular markers of interest. Optimal preparation techniques were sought using human peripheral blood lymphocytes and fine needle samples of breast carcinomas. Unfixed (air-dried only), ethanol, ethanol/acetic acid, and paraformaldehyde/ethanol fixations were tested. Unfixed or fixed cells were placed on slides as a drop or by cytocentrifugation, stained with 4',6-diamidino-2-phenylindole dihydrochloride and DNA content was measured using image analysis. Histogram quality was evaluated using G0G1 peak coefficient of variation, and compared to those generated by flow cytometry. Drop preparations of ethanol/acetic acid fixed and cytospin preparations of paraformaldehyde/ethanol fixed cells appeared to give the best histograms for image analysis, which were inferior in quality to those generated by flow cytometry. Comparison of breast carcinoma histograms generated by flow and image showed the same DNA aneuploid populations but with slightly higher DNA indices measured by image analysis.

Breast Neoplasms↗
PubMed1996

Image cytometry of early nuclear events during apoptosis induced by camptothecin in HL-60 leukemic cells.

The nuclear morphological alterations occurring during apoptosis induced in human HL-60 cells by camptothecin were analyzed quantitatively by image cytometry. Two separate phases were identified during the apoptotic process. The first phase, observed between 0-2 h of incubation, consisted in the appearance of cells with an apparently decondensed chromatin. This phenomenon was blocked by the inhibitors of DNA fragmentation, TLCK and H7. In contrast, staurosporine and cytochalasin B, which inhibit apoptosis without any effect on DNA fragmentation in this system, did not prevent this morphological change. The second phase, observed after 3 h of culture, corresponded to the appearance of cells with very homogeneous and condensed chromatin. This phenomenon correlated with the detection of typical apoptotic cells with fragmented nuclei and was inhibited by all drugs (TLCK, H7, staurosporine, and cytochalasin B). These observations suggest that image cytometry allows the detection of subvisual microscopic events within the first hour after the induction of an apoptotic process and that the dissection of this process into several different phases might be associated with DNA fragmentation.

Apoptosis↗
PubMed2000

A telepathology based Virtual Reference and Certification Centre for DNA image cytometry.

An increasing need for flexible consultation between pathologists, including the application of fast evolving supplementary technologies, has been identified during the last years. Although pathology is already one of the most advanced application of telemedicine there is more to come from the fast evolution towards computerized microscope image analysis: A reproducible quantification of measurable descriptors of the lesions in cells and tissues (so-called biological markers) is an indispensable adjunct to routine diagnostic application. Among such quantitative methods DNA image cytometry is increasingly applied by pathologists for assistance in diagnostics. As for other pathological issues, too, a reference center for the clinical application of DNA image cytometry might be therefore of utmost value for pathologists using that method. Based on advanced telematic technologies, a Virtual Reference and Certification Center (VRCC) could be installed for certifying the cytometry hardware and software, the analytical procedures, and the basic interpretation of the results. It will be designed to be operated as a non-attended service, based on quantification servers accessible via Internet round the clock. The VRCC will supply appropriate standardization and normalization materials and run a GroupWare platform for consensus making by experts.

Certification↗
PubMed1990

Flow and image cytometry for quality assessment of fresh and frozen human sperm samples.

A previous study reported that sperm mitochondrial activity and sperm motility can be evaluated by combined flow and image cytometry, suggesting their potential interest in fertility clinical applications and for studying the effect of physical and chemical agents that modify sperm motility and/or metabolism. This paper focuses on the effect of freezing sperm in liquid nitrogen, extensively used in artificial insemination (AI), combined with different sperm manipulations (washing, swim-up in capacitating medium, CM) using flow and image cytometry on sperm samples from three fertile donors. Rhodamine 123 (Rh123) uptake profiles were bimodal both for fresh and frozen/thawed samples. The mean value of fluorescence of active cells (m+) remained nearly similar after freezing while the percentage of active cells (%C+) was significantly decreased and the percentage of dead cells (%dc, revealed by propidium iodide uptake) significantly increased. In all experiments, the decrease of MOT (percentage of motile sperm) due to freezing was concomitant, to a smaller extent, with a drop in %C+ and an increase in %dc; there was a good relationship between velocity (VCL, VSL) or trajectory characteristics (ALH) and the mean fluorescence values of active cells (m+). Sperm immobilization was neither found related to a major decrease of Rh123 fluorescence nor to an increase in dead cells.

Cryopreservation↗
PubMed1992

Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis >> manual counting.

Benzothiazoles↗
PubMed1998

Image cytometry as a discriminatory tool for cytologic specimens obtained by endoscopic retrograde cholangiopancreatography.

BACKGROUND: Routine brush cytology is relatively insensitive for the diagnosis of biliary and pancreatic malignancy. Sensitivity can be improved by measuring DNA and proliferation. The goal of this study was to assess the discriminatory capacity of image cytometry using pancreaticobiliary brush cytology specimens obtained during endoscopic retrograde cholangiopancreatography (ERCP). Analysis included morphometry, DNA quantification, and characterization of nuclear chromatin distribution and condensation. METHODS: Brush cytology specimens were obtained during ERCP from 22 chronic pancreatitis specimens, 11 pancreatic adenocarcinoma specimens, 13 primary sclerosing cholangitis specimens, and 11 cholangiocarcinoma specimens and contrasted with 25 normal epithelia specimens. A SAMBA 2005 image processor was used to analyze Feulgen stained chromatin density and distribution. Discriminant analysis of 37 morphonuclear variables was performed to characterize differences between: 1) chronic pancreatitis and pancreatic adenocarcinoma, and 2) primary sclerosing cholangitis and cholangiocarcinoma. RESULTS: Chronic pancreatitis was distinguished from pancreatic adenocarcinoma (P < or = 0.001); sensitivity and specificity were both estimated to be 82%. Primary sclerosing cholangitis was distinguished from cholangiocarcinoma (P < or = 0.01); sensitivity and specificity were estimated to be 82% and 85%, respectively. CONCLUSIONS: Multiparameter image cytometry has potential as an adjuvant diagnostic technique in patients with pancreaticobiliary malignancy.

Adenocarcinoma↗
PubMed2000

Diagnosis and prediction of recurrence and progression in superficial bladder cancers with DNA image cytometry and urinary cytology.

OBJECTIVE: To investigate the roles of urinary cytology and image cytometric analysis of nuclear DNA ploidy pattern in the diagnosis and prediction of recurrence and/or progression of superficial bladder cancers. PATIENTS AND METHODS: Aliquots of catheterized urine from 92 patients with primary (23) or previous (69) superficial bladder cancers were assessed using urine cytology and image-analysis cytometry independently. RESULTS: Of the 23 primary superficial transitional cell carcinomas (TCCs), 11 (48%) were detected by urinary cytology while 12 (52%) were detected by image-analysis cytometry (P>0.05) and 13 (57%) were revealed by combined cytology and cytometry. Of 42 recurrent superficial TCCs, 29 (69%) were detected by urinary cytology, whilst 19 (45%) were diagnosed by cytometry (P<0.05) and 29 (69%) by combined cytology and cytometry. The degree of ploidy in relation to pathological stage and/or grade showed an increasing frequency of aneuploid pattern in more invasive and undifferentiated tumours, but with no statistical significance (P>0.05). The positivity of DNA image cytometry had no significant association (P>0.05) with tumour recurrence and/or progression. CONCLUSIONS: DNA image cytometry can provide a limited but not significant advantage over urinary cytology in the detection of primary superficial TCCs, but it does not seem to be indicated for the prediction of tumour recurrence and/or progression.

Aneuploidy↗
PubMed2004

Interobserver reproducibility of DNA-image-cytometry in ASCUS or higher cervical cytology.

In the present study, the aim has been to investigate the interobserver reproducibility of DNA-image-cytometry (DNA-ICM) applied to routine Pap smears classified as Atypical Squamous Cells of Undetermined Significance (ASCUS) or higher lesions (ASCUS+). 202 Pap smears diagnosed as ASCUS or higher were included in the study. After cytological assessment, smears underwent restaining according to Feulgen. First measurements were performed as routine workup. The second measurements were blinded to the result of the first and consecutively performed. DNA-ICM met the consensus statements of the European Society of Analytical Cellular Pathology (ESACP). Interobserver agreement was assessed by calculating Kappa statistics. The diagnosis of DNA-aneuploidy in the first measurements was confirmed in all cases. Second measurement detected 12 additional cases with aneuploidy. Nine out of these cases were classified as aneuploidy by detection of 9c Exceeding Events (9cEE). In three cases stemline-aneuploidy was disclosed. The overall proportion of observed agreement was 94.1%, kappa=0.87, 95% CI=0.74-0.99. Our study shows a good interobserver reproducibility of DNA-ICM performed on cervical smears with ASCUS or higher lesions. DNA-ICM thus represents a highly reproducible diagnostic procedure.

Adolescent↗
PubMed1992

Quantitative image cytometry of hepatocytes expressing gamma-glutamyl transpeptidase and glutathione S-transferase in diethylnitrosamine-initiated rats treated with phenobarbital and/or phthalate esters.

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHP's stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.

Animals↗
PubMed2000

Malignancy-associated changes in breast tissue detected by image cytometry.

In several tissues, nuclear differences have been described in normal-appearing cells from patients with invasive carcinomas compared to cases without invasive carcinoma, a phenomenon known as malignancy-associated changes (MACs). The aim of this study was to determine the presence of malignancy-associated changes in breast tissue. Image cytometry was performed on Feulgen stained tissue sections of patients with usual ductal hyperplasia with (n = 30) or without (n = 41) adjacent invasive breast carcinoma. Nuclear features of normal-appearing cells as well as of usual ductal hyperplastic cells were separately compared between the two groups. Many features of normal-appearing epithelial cells were significantly different between cases with and without invasive cancer. Significant differences were also found by measuring ductal hyperplastic nuclei instead of normal-appearing nuclei. Cases with or without cancer could be distinguished with a classification accuracy of 80% by discriminant analysis using 2 nuclear features derived from ductal hyperplastic cells. In conclusion, image cytometry on breast tissue sections shows that malignancy-associated changes can be found in normal as well as in usual ductal hyperplastic breast cells. This could be clinically relevant for the detection of occult breast cancer, for the prediction of risk in these lesions, and to monitor the effect of chemopreventive agents.

Breast↗
PubMed1988

Diagnostic and prognostic value of DNA image cytometry in myelodysplasia.

The DNA content of erythropoietic cells from 10 patients with refractory anaemia (RA) with megaloblastic changes, who subsequently developed acute non-lymphoblastic leukaemia (ANL), and from seven patients with megaloblastic marrow aspirates due to pernicious anaemia were compared by DNA image cytometry. The DNA distribution, the rate of aneuploid cells exceeding 5c (5cER), and the square deviation index of DNA values from the normal 2c-peak (2cDI) were recorded. Both variables were of diagnostic and prognostic importance for epithelial tumours, malignant lymphomas, and dysplastic lesions. A rate of 5cER greater than 0 was found in eight of 10 myelodysplastic, but in none of seven control cases. Hypodiploidy was equally pronounced in both groups of patients. The 5cE had the highest discriminative value of all variables calculated. The 2cDI was not significantly different in either group. In pernicious anaemia the 2cDI depended mainly on the percentage of S cells, reflecting the defect of DNA synthesis. In RA with megaloblastosis the 2cDI correlated with the percentage of G2 cells, reflecting G2 arrest. In the myelodysplastic group the 2cDI correlated positively with the length of time until ANL developed, indicating the prognostic relevance of 2cDI. Our findings show that in megaloblastic anaemia DNA image cytometry can distinguish myelodysplasia from pernicious anaemia and that it also provides prognostic information.

Acute Disease↗