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PubMed1996

Image cytometric comparison of proliferating cell nuclear antigen and MIB-1 staining in hepatocellular carcinoma and adjacent liver tissue.

Studies of cellular proliferation may aid in elucidation of mechanisms of hepatocarcinogenesis and provide potential prognostic information in cases of hepatocellular carcinoma. Increased proliferation has been reported in the liver tissue adjacent to hepatocellular carcinoma and may be a risk factor for recurrence after tumor resection. We quantitated proliferating cell nuclear antigen (PCNA) clone PC10 and MIB-1 nuclear immunostain by image cytometry in fixed, paraffin-embedded tissue sections of normal, cirrhotic, and dysplastic livers with and without co-existent hepatocellular carcinoma and in the hepatocellular carcinoma. Fifteen high power fields were analyzed in each case using the CAS 200 image analyzer and results were expressed as the percent positive nuclear area. Cirrhotic liver showed significantly less proliferation adjacent to hepatocellular carcinoma than without hepatocellular carcinoma. There was a similar, but not significant, tendency for dysplastic liver. Increasing mean proliferation was noted from normal to cirrhotic to dysplastic liver to low grade to high grade hepatocellular carcinoma. These findings were similar using both PC10 and MIB-1. There tended to be slightly more staining with PC10 than with MIB-1, but the mean percent positive nuclear area was significantly greater with PC10 only in high grade hepatocellular carcinoma. Comparison of individual cases, however, yielded significant correlation between the two antibodies in the low grade and high grade hepatocellular carcinomas, but not in the benign livers. These findings demonstrate that image cytometric quantitation of PC10 and MIB-1 immunostain may be comparable measures of cellular proliferation and that there is no increase in proliferation in the hepatic tissue adjacent to hepatocellular carcinoma.

Antibodies, Monoclonal↗
PubMed2002

Quantitative FISH analysis on interphase nuclei may improve diagnosis of DNA diploid breast cancers.

The detection of DNA aneuploid cells using flow cytometry is an indication for the presence of tumor cells, but when DNA diploid cells are found in 25-33% of the cases, the diagnostic and prognostic significance of DNA ploidy is more limited. We analyzed interphase nuclei after in situ hybridization and using image cytometry on 50 breast tumors with diploid DNA content to investigate whether early chromosome rearrangements were detectable and if their occurrence was clinically significant. Imbalances between the two arms of chromosome 1 were found in 55% of the cases and values ranged from 1.5-3.0. Comparison with histological data showed that Grade I tumors mainly have imbalances (67%) and that Grade III tumors were mainly without the imbalance (67%), whereas Grade II tumors were intermediate (50% imbalance). These data suggest that the diagnosis of DNA diploid cases may be improved by using interphase FISH. In addition, the data also indicates that early breast tumors may have different genetic origins, which is important in the comprehension of tumor malignancy in early stages, especially for preinvasive lesions.

Adult↗
PubMed2003

Rapid prenatal diagnosis of Down syndrome using quantitative fluorescence in situ hybridization on interphase nuclei.

OBJECTIVES: Presently, conventional cytogenetic analysis of metaphase chromosomes remains the reference approach in prenatal diagnosis. However, this method is labor-intensive and time-consuming. The first step toward the rapid identification of aneuploidies is achieved by interphase fluorescence in situ hybridization (FISH) with centromeric or locus-specific probes. Spot counting using this type of probes is a reliable approach, but is very time-consuming with some technical and biological limitations. In this study, we present a new FISH method using image cytometry for the detection of trisomy 21 within interphase nuclei. METHODS: The method is based on a comparative quantitation of the fluorescence signals emitted by whole chromosome 21 and 22 painting probes cohybridized on interphase nuclei. The chromosomal imbalance was determined with an automated image cytometer by detecting an abnormal ratio of both fluorescence emissions when compared with the ratio obtained in normal cells. RESULTS: Ten blood samples and twenty amniotic fluids were analyzed. Results from FISH and standard cytogenetics were compared and 100% correlation was achieved. CONCLUSIONS: This method, which enables an easy detection of chromosomal imbalances without a need for metaphase preparations, can be applied to the diagnosis of trisomy 21 and extended to other disorders with chromosomal imbalances. Compared to other interphase FISH techniques, it avoids spot-scoring difficulties.

Adult↗
PubMed1995

Histometric texture analysis of DNA in thin sections from breast biopsies. Application to the detection of malignancy-associated changes in carcinoma in situ.

OBJECTIVE: To evaluate histometric measurement of nuclear texture in breast biopsy sections in order to detect malignancy-associated changes in apparently normal tissue in the vicinity of carcinoma in situ. STUDY DESIGN: We previously showed that image cytometry measurements of nuclear features--foremost, texture features, describing the organization of Feulgenstained DNA in the cell--can be used to distinguish normal-appearing, diploid epithelial cells from patients with invasive carcinoma of the breast from those with benign biopsies. In that study, referred to as the "single cell analysis," images of at least 200 epithelial cells were acquired for each slide, and substantial user interaction was required to segment cells from each field. Location of isolated cells and interactive segmentation are both time-consuming procedures, particularly in breast tissue, where nuclei can be tightly clustered within a duct. With histometric texture analysis on the same specimens, segmentation of individual cells was ignored, and texture measurements were performed over the entire cluster of relevant cells. With this approach, ploidy information is not available, and touching and overlapping nuclei are included in the measurements. Measurement of histometric texture properties requires substantially less time (at least an order of magnitude) than individual cell measurement and, if ploidy information is not significant, may therefore provide a more practical means of analysis for tissue sections. RESULTS: Seventeen cases of invasive carcinoma and 17 cases of nonproliferative breast disease were examined. Using stepwise discriminant function analysis, slides were classified into one of the two groups with an accuracy of 88.6% in the case of single cell analysis and with an accuracy of 88.2% using histometric analysis. CONCLUSION: The existence of malignancy-associated changes in the breast was confirmed by an independent analysis of the same specimens. Although the two methods are not directly comparable, we found that histometric texture analysis performs at least as well as single-cell analysis for the detection of malignancy-associated changes in breast carcinoma.

Biopsy↗
PubMed1998

Proliferative fasciitis of the forearm: case report with immunohistochemical, ultrastructural and DNA ploidy studies and a review of the literature.

A case of proliferative fasciitis arising in the left forearm of a 56-year-old man was examined. The lesion was preceded by blunt trauma, measured 1.5 x 1.3 x 1.0 cm, was poorly circumscribed and appeared white to light gray on the cut surface. Light microscopic examinations revealed that spindle cells and giant cells with one or two nuclei and abundant basophilic cytoplasm were arranged without any organized patterns in collagenous stroma. Ultrastructurally, well-developed rough endoplasmic reticulum separated by varying amounts of fine to course fibrillar materials was detected in the giant cells. Only vimentin immunoreactivity was detected in both spindle and giant cells. The Ki-67 labeling index of spindle cells was 35% but that of giant cells was less than 5%, and this reflects the quiescent or slow-growing features of these giant cells in proliferative fasciitis. DNA content of the cells, which was examined by image cytometry, demonstrated diploidy in both spindle (DNA index=1.01) and giant (DNA index=1.09) cells.

Cell Division↗
PubMed1994

A method of correcting DNA ploidy measurements in tissue sections.

A new method of performing DNA ploidy measurements in tissue sections using image cytometry is described. The method involves an image processing "object" filtering operation to remove cut and overlapping nuclei, and DNA correction of larger nuclei for the part of the DNA cut away. The methodology of the technique is developed in detail, and the results of testing using sections of rat liver are presented. The results indicated reliable correction of DNA ploidy histograms to reconstruct the polyploid nature of this material. The sensitivity of the material to sectioning thickness errors and methods to overcome this were shown, along with an example of its use on prostate bioptic gun tissue sections.

Animals↗
PubMed1998

DNA cytometry and chromosome 9 aberrations by fluorescence in situ hybridization of irrigation specimens from bladder cancer patients.

OBJECTIVES: To determine the sensitivity and specificity of combining fluorescence in situ hybridization (FISH) measurement of chromosome 9 and DNA cytometry of bladder irrigation specimens in the detection of bladder cancer. METHODS: Bladder irrigation specimens were obtained from 37 normal control patients and 317 bladder cancer patients during cystoscopic examinations. Bladder cancer patients were sampled in the absence of observable tumor (256 specimens) and concurrently with tumor (204 specimens). Chromosome 9 copy number was determined on a cellular basis by FISH, and cellular DNA content was determined by Feulgen DNA staining and image cytometry. RESULTS: Sensitivity of chromosome 9 FISH was 42% for all tumors and was not correlated to transitional cell carcinoma tumor grade, while the sensitivity of DNA cytometry was 55% and improved with increasing grade from 38% for grade 1 to 90% for grade 3 tumors. The results of FISH and DNA cytometry were combined, resulting in specificity of 92% and sensitivity of 69% for grade 1, 76% for grade 2, and 97% for grade 3 tumors. CONCLUSIONS: The lack of increase with grade in the percentage of positive specimens by FISH supports the hypothesis that chromosome 9 aberrations are critical events in bladder tumorigenesis for many patients. These data demonstrate the presence of cells in irrigation specimens with specific genomic lesions of chromosome 9 and DNA content. Combining FISH on chromosome 9 and DNA cytometry provides an increase in sensitivity to transitional cell carcinoma over either test alone.

Carcinoma, Transitional Cell↗
PubMed1994

Fluorescence array detector for large-field quantitative fluorescence cytometry.

A prototypic fluorescence array detector (FAD) has been designed and constructed which is capable of quantifying single-cell fluorescence emissions from a statistically significant population of cell-sized objects (over 10(3)) on a solid substrate. The system is comprised of a cryogenically cooled CCD, 50 mW air-cooled argon ion laser, and optics that image a large (1 x 1 cm) field at 1:1 (no magnification). The CCD is effectively treated as a two-dimensional array of 2.7 x 10(5) independent 20 x 20 microns photodetectors, with each cell-sized object imaged across only a few CCD pixels. Algorithms have been developed for focusing, image segmentation, shading correction, and noise rejection; performance data for the FAD with fluorescent calibration beads are presented. The FAD is a simple alternative to microscope-based imaging cytometry, allowing large-field imaging without a scanning stage.

Flow Cytometry↗
PubMedDate not supplied

Radiation induced early nuclear changes in cytologic brush specimens from the upper aerodigestive tract.

OBJECTIVE: To evaluate the feasibility of DNA image cytometry to assess radiation induced early nuclear changes in cytologic smears from the upper aerodigestive tract. METHODS: 100 cytologic brush specimens from 50 postoperatively irradiated patients with upper aerodigestive tract carcinoma were examined Smears were taken from clinically inconspicuous mucosa from the former tumor site and from a control region (contralateral side). Cellular DNA was quantified using the CAS 200 image analysis system on Feulgen stained slides. DNA histograms were evaluated using the coefficient of variation (CV), the DNA index (DI), the 2c deviation index (2cDI) and the 2.5c exceeding rate (2.5cER). RESULTS: All specimens from the control region were diploid. Forty-six smears from the former tumor region were diploid and in four cases non-diploid cells were found. Single cells with a DNA content exceeding 5c ('5c exceeding events'), were identified in three non-diploid and six diploid smears from the former tumor region. CONCLUSION: DNA analysis is a useful tool in detecting early nuclear changes preceding morphologic changes.

Adult↗
PubMed1986

Uptake and accumulation of the vital dye hydroethidine in neoplastic cells.

Hydroethidine, a reduced form of ethidium bromide, was used as a vital dye in fluorescence assays that allowed visual and semiquantitative monitoring of dye uptake and accumulation by fluorescence microscopy, flow cytometry, image analysis, and microfluorimetry. The excitation and emission filters were chosen to detect hydroethidine and exclude ethidium. Microscopically, there were differences in fluorescence intensities and fluorescence patterns among various tumor cell lines. The fluorescence pattern varied from homogeneous blue in the cytoplasm to blue plus brilliant packets of bluish-white distributed in the cytoplasm. Nuclear staining varied from brown to reddish orange fluorescence. These differences were confirmed by flow cytometry and image analysis. A preliminary survey of various tumors indicated that uptake and accumulation of hydroethidine were dependent on concentration of the dye, duration of cell exposure to the dye, and metabolic state of the cells. Microfluorimetry made possible monitoring of 96 samples in a microculture plate in 30 seconds; thus, this method allows large numbers of samples to be read, with a tremendous savings in time and reagents. The results obtained from the different techniques used were corroborative; therefore, any one of the above techniques may be used in an assay.

Animals↗
PubMed2003

Prognostic significance of standardized AgNOR analysis and Ki-67 immunostaining in gastrointestinal stromal tumors.

OBJECTIVE: To evaluate the prognostic utility of argyrophilic nucleolar organizer region (AgNOR) protein parameters and Ki-67-immunostained growth fraction (Ki-67 labelling index) and to correlate AgNORs with Ki-67 LI and the main clinicopathologic parameters in gastrointestinal stromal tumors (GISTs). STUDY DESIGN: On 55 patients with surgically excised GISTs, visualization and quantification of AgNORs were performed as specified in the guidelines of the Committee on AgNOR Quantification. RESULTS: AgNOR protein area (NORA) > or = 5.28 microns 2 was statistically associated with mitotic rate > or = 5 x 10 high-power fields (hpfs) (P < .001) and presence of necrosis (P < .001); Ki-67 LI > or = 9.69% was significantly associated with mitotic rate > or = 5 x 10 hpfs (P < .001), size > or = 5 cm (P = .033) and presence of necrosis (P < .001). Ki-67 LI and NORA strongly correlated. Preliminary Kaplan-Meier survival analysis showed that an increased value of NORA, Ki-67 LI, mitotic rate, tumor size and presence of necrosis had a negative influence on patient survival. Multivariate regression analysis showed that only NORA and Ki-67 LI were independent parameters in predicting the clinical outcome for patients with GISTs. Mitotic rate and necrosis remained as independent prognostic factors when NORA and Ki-67 LI were not allowed to enter in models. CONCLUSION: AgNOR protein quantity, as determined by image cytometry, and Ki-67 immunostaining seem to represent reliable predictive parameters in GISTs and are independent of mitotic rate, tumor dimension and necrosis.

Adult↗
PubMed1998

Static DNA cytometry as a diagnostic aid in effusion cytology: I. DNA aneuploidy for identification and differentiation of primary and secondary tumors of the serous membranes.

OBJECTIVE: To determine whether DNA aneuploidy is a sensitive and specific marker for the identification of tumor cells in effusions and whether the pattern of DNA aneuploidy can provide important information for the differential diagnosis of primary and secondary tumors of the serous membranes. STUDY DESIGN: One hundred eight malignant mesotheliomas as well as 102 metastatic carcinomas of the serous membranes were obtained from routine cytologic and histologic material. One hundred reactive effusions were investigated as controls. Nuclear DNA contents were measured after Feulgen staining using a TV image analysis system. RESULTS: DNA aneuploidy was assumed if abnormal DNA stemlines, a coefficient of variation of the first DNA stemline > or = 10%, or cells > 9c were observed. On this basis the prevalence of DNA aneuploidy in mesotheliomas was 83% for cytologic and 84% for histologic material. In effusions of metastatic carcinomas it was 100%. None of the 100 reactive effusions revealed DNA aneuploidy (prevalence, 0%). Positive predictive value for mesotheliomas was 100%; negative predictive value was 88% for cytologic and 82% for histologic material. Positive predictive value for metastatic carcinomas was 100%; negative predictive value was 100%. Seventy-two percent of the mesotheliomas revealed their greatest stemline within the range 1.80c-2.20c, whereas none of the metastatic carcinomas showed this stemline position. CONCLUSION: DNA image cytometry might be a very sensitive and highly specific, additional tool for identification of neoplastic cells in effusions as well as for the differential diagnosis of mesothelioma vs. metastatic carcinoma of the serous membranes.

Aneuploidy↗
PubMed2003

DNA ploidy of bladder cancer using bladder biopsy supernate specimens.

OBJECTIVE: To perform DNA image cytometry on 119 bladder biopsy supernate (BBS) specimens of transitional cell carcinoma (TCC) bladder to: (1) test the suitability of this cytologic specimen for use in DNA ploidy analysis, and (2) assess the value of DNA ploidy measured on this specimen as to the risk of tumor recurrence and survival. STUDY DESIGN: The histologic grade and cytologic grade were correlated, and the DNA ploidy produced was determined by image analysis of Feulgen-stained nuclei. Kaplan-Meier curves related age, sex, grade and DNA ploidy to recurrence of tumor and survival. Log rank analyses were used to ascertain the difference between the curves for each categorical variable. RESULTS: Urothelial cells derived from the BBS specimen were demonstrated to be representative of the tumor. The tumor recurrence rate was significantly higher (P = .0001) and the survival rate significantly lower (P = .0002) for patients with aneuploid tumors compared to those with diploid tumors. Patients with TCC 2 tumors had a significantly shorter time to recurrence (P = .003), although the relationship between ploidy and survival in this group was of marginal significance. CONCLUSION: The specimen was free of many of the problems associate with the other specimen types used for measuring DNA ploidy. The results show that the BBS specimen is diagnostically useful and suitable for DNA analysis, providing prognostically relevant information.

Adult↗
PubMed2000

Nuclear image morphometry and cytologic grade of breast carcinoma.

OBJECTIVE: To correlate visual cytologic grade with automated nuclear morphometry of carcinoma of the breast. STUDY DESIGN: We randomly selected 24 histologically proven infiltrating ductal carcinomas of the breast and 10 benign breast lesions (fibroadenoma). Hematoxylin-eosin-stained fine needle aspiration cytology (FNAC) smears were selected for both cytologic grade and automated image morphometry. The same hematoxylin-eosin-stained FNAC smears were studied for area, convex area, standard deviation of nuclear area, diameter, perimeter and convex perimeters of nucleus. At least 100 cells from each case were measured with an image cytometer. RESULTS: Mean nuclear area, standard deviation of nuclear area, nuclear diameter, convex area, convex perimeter and perimeter were significantly increased from benign versus grade 1 carcinomas and grade 1 versus grade 2 and 3 carcinomas (one way ANOVA test). However, there was no significant difference in grade 2 versus grade 3 carcinomas. CONCLUSION: Automated image cytometry rapidly and successfully measures various nuclear parameters. The measurement of various nuclear parameters would be helpful in future applications of automated diagnosis and grading of breast carcinomas from cytologic material.

Biopsy, Needle↗
PubMed1998

Static DNA cytometry as a diagnostic aid in effusion cytology: II. DNA aneuploidy for identification of neoplastic cells in equivocal effusions.

OBJECTIVE: The sensitivity of conventional cytology for identification of neoplastic cells in effusions is unsatisfactory, about 58%. The rate of diagnostically equivocal effusions in routine cytology is about 6%. DNA aneuploidy has previously been proven to be a sensitive and specific marker for the identification of tumor cells in effusions. In the present study we determined if malignancy can be identified in cytologically equivocal cells in effusions using DNA aneuploidy as a marker, thus decreasing the rate of cytologically equivocal diagnoses in effusions. STUDY DESIGN: One hundred cytologically equivocal effusions of the serous cavities were obtained from routine diagnostic material. Nuclear DNA content was measured after Feulgen staining using a TV image analysis system. Data were correlated with patient follow-up. RESULTS: DNA aneuploidy was assumed if abnormal DNA stemlines, a coefficient of variation of the first DNA stemline > or = 10% or cells > 9c were observed. The sensitivity of DNA aneuploidy for the identification of malignancy was 55.9%. Specificity of DNA nonaneuploidy for benignity was 94.1%. The positive predictive value of the marker DNA aneuploidy for the occurrence of malignant cells was 97.9% since all but one DNA aneuploid case showed malignancy in follow-up. CONCLUSION: Image cytometry applying DNA aneuploidy as a parameter is able to detect the occurrence of malignant cells in cytologically equivocal effusions in about every second case. Thus, this method is able to increase diagnostic accuracy of conventional effusion cytology by decreasing the rate of diagnostically equivocal effusions.

Aneuploidy↗
PubMed2000

AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix.

In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Cell Line↗
PubMed2007

Human papillomavirus-related cellular changes measured by cytometric analysis of DNA ploidy and chromatin texture.

BACKGROUND: Image cytometry has provided two highly sensitive markers for the identification of the malignant potential of squamous lesions. Aneuploidy and chromatin texture have been investigated as quantitative measures of nuclear damage in premalignant lesions and carcinoma. Real-time PCR methods have evolved to yield highly specific measurements of mRNA expression in very sparse cellular samples. METHODS: Human papillomavirus (HPV) 16 and 18 E7 mRNA expression was measured using quantitative RT-PCR. DNA index and chromatin measures were taken from image cytology samples. The chromatin features, through discriminant analysis, were aggregated into a score, and both measurements were related to mRNA expression. RESULTS: mRNA level and DNA index show an increasing trend over increasing histological grades. However, DNA index and chromatin score were not correlated to mRNA levels in these samples. Chromatin score differed by mRNA type found with HPV 18 infected samples having a higher score than those with HPV 16. Samples infected with HPV 16 and HPV 18 had even higher chromatin scores. CONCLUSIONS: DNA index and chromatin score were not directly correlated with mRNA levels. However both mRNA and DNA index were related to histological grade, and chromatin score was associated with HPV type. Therefore, DNA index and mRNA levels could be independent predictors of cervical dysplasia, and chromatin score could be related to the viral integration process in cells infected with HPV 18 or dual infections.

Adult↗