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PubMedDate not supplied

Comparative cytophotometric analysis of reactive lymphoid hyperplasia and low malignant non-Hodgkin lymphoma.

In fine needle aspiration biopsy (FNAB) of lymph nodes, differential diagnosis between reactive lymphoid hyperplasia (RLH) and low grade non-Hodgkin lymphoma (NHL) may be sometimes difficult. Cytophotometric analysis of DNA content using image analysis has found practical application in the determination of malignancy in different tumours. In this study, image cytometry was applied to compare the ploidy status of the above mentioned lesions to obtain diagnostically helpful information. Thirty smears of histologically proven low grade NHL (10 follicular lymphomas of centroblastic-centrocytic type, 10 lympho-plasmocytoid immunocytomas, 10 chronic lymphocytic leukemias) and 15 smears of RLH were stained with Feulgen and examined by image analysis (CAS 200, Becton Dickinson). The 5c exceeding rate was determined according to the Boecking's definition of aneuploidy. Concerning the histogram types the Auer classification was used. The aneuploidy was found in 4/10 follicular lymphomas and 4/10 lymphoplasmocytoid immunocytomas. All chronic lymphocytic leukemias were diploid. In none RLH a 5c exceeding rate was found. The results verify the value of image cytophotometry as a helpful method in some diagnostically difficult cases.

Adolescent↗
PubMed1996

Assessment of germ-cell kinetics in the testes of patients with varicocele using image analysis of immunostained proliferating cell nuclear antigen.

OBJECTIVE: To examine the germ-cell kinetics of varicocele testis using proliferating cell nuclear antigen (PCNA) immunostaining and image cytometry to evaluate the staining intensity, and to compare the proliferative index (PI) of varicocele and normal testes. PATIENTS AND METHODS: Fifty-six testicular biopsy specimens were taken from 28 patients with varicocele during high ligation. The specimens were fixed and embedded in paraffin, and sections immunostained using an anti-PCNA antibody. The PI was measured using an image analyser and expressed as the percentage of the total nuclear area stained positively with PCNA monoclonal antibody, assessed from > 20 seminiferous tubules. Sections stained with haematoxylin and eosin were used to determine Johnsen's score using conventional microscopy. Normal control testicular biopsies were obtained from subjects undergoing vasectomy. RESULTS: In patients with varicocele, both testes had a significantly lower PI than normal testes; the mean (SD) PI of the right and left testes was 11.3% (4.1) and 11.3% (4.8), respectively, and the PI of normal testes was 21.7% (3.1). There was no significant correlation between PI and sperm concentration or Johnsen's score. CONCLUSION: PI is useful for assessing germ-cell kinetics; in patients with varicocele both testes showed a deterioration in DNA synthesis, suggesting that reduced DNA synthesis is one of the causes of spermatogenic dysfunction in the varicocele testes.

Adult↗
PubMed2005

Validity of combined cytology and human papillomavirus (HPV) genotyping with adjuvant DNA-cytometry in routine cervical screening: results from 31031 women from the Bonn-region in West Germany.

Our aim was to improve the accuracy of routine cervical screening by a risk-adapted multimodal protocol with special focus on possible reduction and prognostic assessment of false positive results. A cohort of 31031 women from the Bonn-region in West Germany, median age 36 years, were screened by cytology (conventional or liquid-based), followed by PCR-based HVP detection with genotyping and adjuvant DNA image cytometry, if indicated, in a sequential manner. The true prevalence of high-grade cervical intraepithelial neoplasia and carcinoma (>/=CIN2) was 0.32% in the population as projected from cervical biopsies of 123 women (0.4%), of whom 100 showed >/=CIN2. Sensitivity of the cytology screening program at PapIIID/HSIL threshold for detecting histologically confirmed >/=CIN2 cases was 81%, with specificity, positive predictive value (PPV) and negative predictive value (NPV) of 99, 20.9 and 99.9%, respectively. Of 38 women receiving the complete screening protocol, all the 31 >/=CIN2 cases were correctly detected by cytology alone, 30 by positive high-risk HPV genotype and 30 by aneuploid DNA profile. The combination of the three methods resulted in an up to 6.9% increase in PPV for >/=CIN2 at practically unchanged detection rate with the additional benefit of being able to predict the probable outcome of CIN1 lesions detected as false positives with any single test. Multimodal cervical screening might permit identification of those women with low-grade squamous intraepithelial lesions likely to progress at an earlier and curable stage of disease and lengthen the screening interval in those with transient minor lesions caused by productive HPV infection.

Adult↗
PubMedDate not supplied

Metanephric adenoma of the kidney: an electron microscopic and immunohistochemical study with quantitative DNA measurement by image analysis.

Metanephric adenoma of the kidney is a newly recognized entity of renal adenoma, and the electron microscopic and immunohistochemical features of the tumor have not yet been well elucidated. The authors experienced a typical case of renal metanephric adenoma and studied the electron microscopic and immunohistochemical features of the tumor together with the DNA content of the tumor cells. Electron microscopically, the tumor was composed of immature epithelial cells with relatively uniform size, poor nuclear polarity, and scant cytoplasmic organelles arranged in small tubular or rosette pattern. Immunohistochemically, the cytoplasm of the majority of the tumor cells was positive for S-100, and occasionally for alpha-1-antitrypsin and vimentin. The tumor cells were also positive for cytokeratin, lysozyme, and Leu 7, with predominantly cell membrane staining. Only tumor cells arranged in papillary or large tubular pattern were positive for epithelial membrane antigen (EMA). Tumor cells were negative for Leu M1 and HMB-45. These immunohistochemical characteristics are quite similar to those of S-shaped body of the fetal kidney. An analysis of the nuclear DNA of the tumor cells by image cytometry revealed that the tumor entirely consisted of diploid cells' and lacked aneuploid cells. These results indicate that renal metanephric adenoma is composed of immature renal epithelial cells.

Adenoma↗
PubMed1984

The DNA-based human karyotype.

Image cytometry and computer analysis are used to determine the relative DNA content and the DNA-based centromeric index of the 24 chromosomes of the human karyotype. A two-step procedure is used. Chromosomes of cells in metaphase first are stained with quinacrine and identified visually by their fluorescent Q-band patterns. They then are stained for DNA using gallocyanin-chrome alum. The chromosome images are scanned and recorded as digital values of optical density by an CYDAC image cytometric microscope system, CYDAC. The digital images are processed by computer to measure for each chromosome the relative DNA stain contents of the whole chromosome and of the p and q arms and the DNA-based centromeric index. About ten cells are analyzed for each of the donors, who are phenotypically normal men and women. The chromosome measurements are pooled by chromosome type for each donor and are compared among donors. The means of the chromosome measurements give the DNA-based human karyotype. Analysis of the DNA-based data shows that some chromosomes or portions of chromosomes vary significantly among donors. These variants do not correlate with detectable morphologic polymorphisms, such as Q- or C-band variants; thus they represent new and otherwise undetectable chromosome polymorphisms whose genetic basis and clinical significance are yet to be determined.

Adult↗
PubMed1991

Seasonal variation in the proliferation fraction of Australian common nevi.

Image cytometry was used to assess seasonal variation in the proliferation fraction of Australian common nevi. Twenty pairs of nevi were evaluated. One member of each pair had been excised during the Australian summer, and the other member of the pair had been excised during the winter. The nevi were matched for age, sex, and body site. From each nevus, 6-microns sections were Feulgen-stained and evaluated with a CAS 200 image analyzer, which was used to obtain DNA histograms. Proliferation fractions were calculated from the histograms. The proliferation fraction of the nevi removed during the winter was 1.65 +/- 0.32%, whereas the proliferation fraction of the nevi removed during the summer was 1.95 +/- 0.42% (p = 0.41). For nevi from sun-exposed sites only, the proliferation rate of nevi excised during the winter was 1.81 +/- 0.39%, and 2.58 +/- 0.39% for nevi excised during the summer (p = 0.11). For nevi from sun-protected sites, there was no difference between winter and summer. When nevi excised during the summer were compared by site, sun-exposed nevi had a proliferation rate of 2.60 +/- 0.48% (p = 0.04). For nevi excised during the winter, there was a much smaller difference between sun-exposed and sun-protected nevi.

Adolescent↗
PubMed1995

Cytometric analysis of ventricular myocyte nuclei in idiopathic dilated cardiomyopathy: a tool for evaluation of disease progression?

The progression of idiopathic dilated cardiomyopathy (IDC) is governed by factors that remain obscure. The disease pathway toward cell degeneration or death results in irreversible myocyte change including nuclear cytometric alterations which may be evaluable and ultimately correlated with other measures of disease evolution. Using a novel image cytometry system, we analysed differences in ventricular myocyte nuclear morphology and DNA content and distribution in right and left ventricular free wall myocardium and in ventricular septal myocardium from 11 normal and 13 IDC human autopsy hearts. Nine morphological features of IDC myocyte nuclei differed significantly (P < 0.001) from normal. These were used to establish a classification matrix and cytometry-based assessment and allowed correct categorization of left and right ventricular and ventricular septal myocyte nuclei in concordance with their respective pathological diagnosis (i.e. normal or IDC) 71%, 81% and 77% of the time. Additionally, four photometric features were significantly different (P < 0.005) in IDC versus normal hearts, as were three discrete texture features (P < 0.001). Thus, the spectrum of myocyte nuclei seen in IDC have highly characteristic and measurable morphologic, photometric and texture features. Our findings indicate the potential value of cytometry in the classification of myocytes with regards to a disease continuum and suggest its applicability in both clinical and experimental studies.

Adult↗
PubMed1993

Histopathological variables and DNA cytometry in parathyroid carcinoma.

To undertake an evaluation of histopathological variables in parathyroid carcinoma, 95 cases with this diagnosis were collected from 37 hospitals. Two tumor categories emerged from a review of tissue sections and follow-up information: 56 cases demonstrating extraglandular invasiveness or tumor recurrence were classified as definitive carcinomas, whereas 39 tumors lacking these criteria were classified as equivocal cases. Several morphological variables other than invasiveness differed between the two groups: Fibrosis, necrosis, nuclear atypia (especially macronucleoli), and mitotic figures were significantly more frequent in the carcinoma group. These variables also showed a positive correlation with an aberrant DNA pattern demonstrated by image cytometry. The triad macronucleoli, more than five mitoses per 50 high-power fields, and necrosis were associated with an aggressive behavior in terms of recurrent disease. A minority of the carcinomas had a bland cytologic appearance and differed from benign lesions only by their invasiveness. Certain patterns of fibrosis and necrosis were common but neither pathognomonic nor constant features of malignancy. Mitotic activity constituted a prognostic risk factor but was of limited diagnostic significance. In half of the carcinomas, the frequency of mitoses did not exceed values recorded in benign parathyroid lesions.

Adenoma↗
PubMed1993

DNA image analysis in childhood acute lymphoblastic leukemia.

DNA index (DI) and percentages of cells in S and G2/M phase were determined in Feulgen stained nuclei of blasts from 31 cases of childhood ALL at diagnosis. In 6 cases the results of DNA analysis and cytogenetics were concordant showing hyperdiploidy. Two other cases with normal karyotype were revealed as DNA aneuploid with image analysis. Cases with cytogenetic abnormalities like translocation, deletion or presence of single or double supernumerary chromosomes had DI within normal ranges. Nine ALL cases (29%) were found to be DNA aneuploid--8 hyperdiploid and 1 hypodiploid. The percentages of cells in S and G2/M phase for blasts from bone marrow (mean 17.6%) were significantly higher than those estimated in the peripheral blood (mean 1.57%). We conclude that analysis by image cytometry can detect aneuploid DNA content even in cases, which showed a normal karyotype and provides new information concerning the biological aspects of leukemic blasts.

Adolescent↗
PubMed1990

Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution.

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.

Animals↗
PubMed1999

Altered surface and cyst epithelium of ovaries removed prophylactically from women with a family history of ovarian cancer.

Despite intensive investigation, the nature of epithelial ovarian cancer precursors remains controversial. Because women with a strong family history of ovarian cancer have a high probability of developing ovarian cancer themselves, ovaries removed prophylactically from such patients provide an opportunity to identify early neoplastic changes. Ovaries removed from 64 consecutive patients undergoing prophylactic oophorectomy and from 30 women with normal ovaries and no known family history of ovarian cancer were examined by light microscopy for a number of histopathologic features and by image cytometry for abnormalities of the cyst and surface epithelium. All analyses were performed without knowledge of the family history. Seven benign, but no tumors of low malignant potential or malignant epithelial tumors were found in the prophylactic oophorectomy group. There were more cortical inclusion cysts in the prophylactically removed than controls ovaries (P = .016), but no other architectural features differed between the two groups. No abnormalities were found in the surface or cyst epithelium in either group by light microscopy. In contrast, image analysis identified differences in the nuclei between the two groups, indicating that those from the surface epithelium of prophylactically removed ovaries were larger and contained more heterogeneously dense chromatin than those of controls, and that nuclei of the cyst epithelium had more irregular outlines. Ovarian epithelium from prophylactically removed ovaries exhibit abnormalities that are only identified by image analysis, and which might represent early preneoplastic changes. Such ovaries may be useful for identifying early molecular changes in ovarian cancer.

Adult↗
PubMed1999

Nuclear chromatin texture analysis of nonmalignant tissue can detect adjacent prostatic adenocarcinoma.

BACKGROUND: The aim of this study was to investigate the possibility of identifying prostatic adenocarcinoma by nuclear chromatin texture feature analysis of adjacent histologically benign-looking tissue. METHODS: Two hundred and forty prostatectomy specimens were selected from the archives of the Department of Pathology, University of Innsbruck. These consisted of 67 cases of benign prostatic hyperplasia (BPH) and 173 cases of prostatic adenocarcinoma (PAC). The specimen collection was divided randomly into a training set and test set. Cytospin preparations of disaggregated cells prepared from paraffin-embedded material were stained specifically for DNA by the Feulgen method. For the cancer cases, only tissue that histologically appeared nonmalignant, from the vicinity of the lesion, was used in the sample preparation. Only normal-appearing diploid cell nuclei were analyzed from both the BPH and PAC groups. A discriminator comprised of three nuclear texture features to separate BPH from PAC cases was derived from the training set of cases, and then applied to the independent test set cases. RESULTS: PAC cases were separated from BPH cases with a sensitivity of 90% and a specificity of 97% on the independent test set of cases. CONCLUSIONS: This retrospective investigation demonstrates that by high-resolution image cytometry it is possible to detect the presence of prostatic adenocarcinoma with very high reliability when examining prostate samples that only contain histologically normal-looking cells. This method could become clinically relevant in identification of cancers missed by histologically negative core needle biopsies.

Adenocarcinoma↗
PubMed2005

The significance of tumor heterogeneity for prediction of DNA ploidy of prostate cancer.

OBJECTIVE: In a previous study, we mapped the ploidy heterogeneity of prostate cancer using flow cytometry in 676 tumor samples from 50 radical prostatectomy specimens. Ploidy heterogeneity was common (42% of tumors) and was found in all non-diploid tumors. The volume of non-diploid tumor was estimated and found to predict extra-prostatic extension and seminal vesicle invasion. The aim of this study was to evaluate the impact of tumor heterogeneity on preoperative ploidy assessment. MATERIAL AND METHODS: In 50 men at least six core biopsies were taken before prostatectomy. Sections from biopsies with cancer were Feulgen-stained for image cytometry. After exclusion of biopsies with insufficient material, 123 histograms from 48 men (mean 2.6; range 1-7) remained for analysis. RESULTS: In 32 men, biopsies were diploid. In 16 men, at least one biopsy was non-diploid (14 tetraploid, two aneuploid) and 10 of them also had diploid biopsies. In 34 men (71%), the prostatectomy specimens were correctly predicted as being either diploid (48%) or non-diploid (23%). The sensitivity and specificity of biopsies for predicting non-diploid cancer were 55% and 82%, respectively, and the positive and negative predictive values were 69% and 72%, respectively. The ploidy status of tumors with and without ploidy heterogeneity was correctly predicted in 55% and 82% of cases, respectively (p=0.04). Biopsies underestimated ploidy in 9/20 tumors (45%) with heterogeneous ploidy status. Underestimation mainly occurred when one or two cores were analyzed. CONCLUSIONS: Preoperative prediction of the ploidy status of prostate cancer is hampered by tumor heterogeneity. Analysis of multiple biopsies is important for correct preoperative ploidy estimation.

Aged↗
PubMed1998

Automated fluorescence microscopic measurement of apoptosis frequency following ionizing radiation exposure in cultured mammalian cells.

PURPOSE: To develop and assess an automated image cytometric method of apoptotic cell classification for use under conditions in which apoptosis is a rare event (e.g. fibroblastoid cell lines or low-dose irradiation). METHOD: Image acquisition software was adapted to gather double-stained cell images from slides prepared using cell fixation and staining methods that emphasized apoptotic morphology. Chinese hamster ovary cells (CHO) were classified individually by discriminant analysis of morphological and nuclear texture features calculated for each image. Discriminant functions were constructed from a manually classified set of over 60000 cell images categorized as 'normal', 'apoptotic', 'cell doublets' or 'debris' and all subsequent cell images collected were classified using these functions. RESULTS: Application of this technique resulted in a 99.8% accuracy in classification of the normal cell population, and 81.7% classification accuracy for apoptotic cells. This method was then applied to study the time course of the apoptotic response of CHO cells following X-irradiation. Following irradiation with 5 Gy no increase above control levels of apoptosis was noted until 18 h post-irradiation, which corresponded with the release of the G2 block as determined by DNA-content analysis. Apoptotic frequency increased to a peak level of 12.1 +/- 4.6% at 42 h post-irradiation. CONCLUSIONS: Automated image cytometry provides an efficient and consistent method of apoptosis measurement. This study represents the first detailed characterization of the time course and the role of cell division in CHO cell apoptosis.

Animals↗
PubMed1997

Nuclear image analysis of p53-positive and -negative cells in breast carcinoma.

OBJECTIVE: To detect differences in nuclear morphology in breast carcinoma cells with different p53 expression. STUDY DESIGN: Immunohistochemical reactions were performed on paraffin sections from 88 breast cancers with the monoclonal antibody DO-1. By means of an image cytometry workstation, 500 nuclei were localized and categorized in an immunohistochemical staining scoring system in each case. After destaining and Feulgen staining, the nuclei were relocated and measured by means of the workstation. RESULTS: P53-positive nuclei showed greater irregularity of the chromatin pattern and stronger granularity of the chromatin. There were also differences in the chromatin texture and nuclear shape between tumors different in their p53 expression. The p53-negative nuclear population from the p53-positive tumors and the whole nuclear population of the p53-negative tumors were morphologically different, also. CONCLUSION: Nuclear image analysis of immunohistochemically characterized cells allows further insight into the relationship of structure and function in malignant tumor cells.

Adult↗
PubMed2004

Quantitative histopathology and chromosome 9 polysomy in a clinical trial of 4-HPR.

OBJECTIVE: This trial examined the use of 4-hydroxyphenyl-retinamide (4-HPR), demonstrated to be a potent inhibitor of carcinogenesis in vitro and in animal models, in patients with cervical intraepithelial neoplasia (CIN) grades 2 to 3. Quantitative pathology and chromosome 9 polysomy were used to understand the biology and quantify the clinical histopathologic changes observed. METHODS: Patients were randomized to 4-HPR or placebo for 6 months and followed for six more months. Cervical biopsies were obtained at baseline, 6 months, and 12 months; the biopsies were read blinded three times by the study pathologist. Feulgen-stained sections were also obtained and analyzed using computer-assisted image cytometry. Chromosome 9 polysomy was performed on tissue slices using in situ hybridization and measured quantitatively. Statistical analyses were carried out in S-Plus (Insightful Corporation, Seattle, WA) and R. RESULTS: The interim analysis, planned for 40 patients, was carried out on 39. The 6- and 12-month analyses showed a statistically significant difference between the two study arms. When code was broken, the 4-HPR-treatment arm was found to have fared less well than placebo. Analyses of Feulgen-stained sections provided a quantitative measure of the increase of DNA content and texture features. Chromosome 9 polysomy was also measured using image analysis. The changes observed were consistent with those of cells displaying cancerous changes, indicating a lack of response. CONCLUSION: 4-HPR is not active at 200 mg/day. The interim analysis was helpful in directing the study; and, in this case, ending it. The intermediate endpoint biomarkers of quantitative histomorphometry and chromosome 9 polysomy yielded quantitative and repeatable results consistent with the findings of the clinical pathologist.

Anticarcinogenic Agents↗
PubMed2003

Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays.

PURPOSE: Exposure to ionizing radiation results in phosphorylation of histone H2AX (gammaH2AX) at sites of DNA double-strand breaks. To determine the relationship between gammaH2AX formation and radiosensitivity, the rate of formation and loss of gammaH2AX were examined in several cultured cell lines following exposure to 253 kV X-rays. MATERIALS AND METHODS: Flow and image cytometry were both performed using a mouse monoclonal antibody against gammaH2AX. Immunoblotting was used to confirm cell line-dependent differences in antibody staining. Cell lines examined included V79 and CHO-K1 hamster cells, the human tumour cell lines SiHa, WiDr, DU145, WIL-2NS, HT144, HCC1937 and U87, and the normal cell strain HFL1. Radiosensitivity was measured using a standard clonogenic assay. RESULTS: Using flow cytometry, gammaH2AX formation was detected 1 h after doses as low as 20 cGy. Peak levels of gammaH2AX were observed within 15-30 min after irradiation and both the rate of radiation-induced gammaH2AX formation and loss were cell type dependent. Maximum levels of gammaH2AX formation were lower for HT144 cells mutant for the ataxia telangiectasia gene. Half-times of loss after irradiation ranged from 1.6 to 7.2 h and were associated with a decrease in the total number of foci per cell. The half-time of loss of gammaH2AX was correlated with clonogenic survival for 10 cell lines (r2=0.66). CONCLUSIONS: GammaH2AX can be detected with excellent sensitivity using both flow and image analysis. The rate of gammaH2AX loss may be an important factor in the response of cells to ionizing radiation, with more rapid loss and less retention associated with more radioresistant cell lines.

Animals↗
PubMedDate not supplied

Laminar-flow-based separations at the microscale.

The natural separation maintained by microfluidic flows is employed as the basis of a particle/cell sorting device. This method of separating particulate suspensions exploits the inherent laminar nature of microscale fluid dynamics and incorporates applied fields and image cytometry to enable sorting based upon any visually identifiable difference between colloid-sized cells or particles. This technique may be used to easily isolate, separate, sort, or enrich virtually any suspension of microscale biological or colloidal particles within a microfluidic system. The entire footprint of the device described here is less than 0.01 mm(2), allowing it to be readily incorporated within highly integrated micro total analysis systems (microTAS).

Cell Separation↗