Digital Image Cytometry Research Articles

Spatial distribution of centromeres and telomeres at interphase varies among Brachypodium species.

In this study the 3-D distribution of centromeres and telomeres was analysed in the interphase nuclei of three Brachypodium species, i.e. B. distachyon (2n=10), B. stacei (2n=20) and B. hybridum (2n=30), which is presumably a hybrid between the first two species. Using fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes, it was observed that the majority of B. distachyon nuclei in the root tip cells displayed the Rabl configuration while both B. stacei and B. hybridum mostly lacked the centromere-telomere polarization. In addition, differentiated leaf cells of B. distachyon did not display the Rabl pattern. In order to analyse the possible connection between the occurrence of the Rabl pattern and the phase of cell cycle or DNA content, FISH was combined with digital image cytometry. The results revealed that the frequency of nuclei with the Rabl configuration in the root tip nuclei was positively correlated with an increase in DNA content, which resulted from DNA replication. Also, the analysis of the influence of the nuclear shape on the nuclear architecture indicated that an increasing elongation of the nuclei negatively affected the occurrence of the Rabl pattern. Some possible explanations of these phenomena are discussed.

Benefit of baseline cytometry for surveillance of patients with Barrett’s esophagus

The current gold standard for the surveillance of Barrett's esophagus is the Seattle four-quadrant biopsies protocol (4-QB). Using endoscopic brush cytology, this study prospectively investigated whether digital image cytometry (DICM) is of additional benefit over regular histology as a predictor for progression to high-grade dysplasia or cancer during a surveillance of at least 3 years. The prospective cohort in this study included 93 patients (72% male) with Barrett's esophagus, baseline endoscopies, and at least one DICM in addition to 4-QB who had been followed up a minimum of 3 years at the time of analysis. High-grade dysplasia (HGD) and adenocarcinoma were defined as primary end points. The DICM was performed on Feulgen-restained cytology smears with a continuous collision detection (CCD) three-chip color video camera (Sony) and an AutoCyte QUIC DNA workstation. Of the 93 patients, 11 presented with the diagnosis of HGD and adenocarcinoma at baseline endoscopy. The remaining 82 patients were analyzed after a median follow-up time of 44 months (range, 36-65 months). Of these 82 patients, 9 (11%) had low-grade dysplasia (LGD) at baseline histology: One of two patients with LGD and aneuploid DICM showed HGD at follow-up assessment, whereas none of seven patients with LGD and diploid DICM had development of HGD. Of the 82 patients, 73 (89%) had either specialized intestinal metaplasia (SIM) without dyplasia or indefinite findings for dysplasia at baseline histology. Of the eight patients with SIM and intermediate/aneuploid DICM, two had development of HGD. None of those with negative or indefinite findings for dysplasia and diploid DICM had HGD at the follow-up evaluation. In summary, the three patients who had development of HGD showed a pathologic DICM at baseline, and no patient with diploid DICM had HGD. Cytometry from brush cytology as an add-on to histology appears to be of additional benefit during surveillance of Barrett's esophagus. Whereas an aneuploid/intermediate DICM warrants an early re-endoscopy, a diploid DICM underscores the low-risk status especially of patients with low-grade dysplasia.

Lugol chromoendoscopy combined with brush cytology in patients at risk for esophageal squamous cell carcinoma

Patients with achalasia or malignancies of the head and neck are at increased risk for esophageal squamous cell carcinoma. The discussion of a screening and surveillance program is controversial. The aim of the present study was to determine the diagnostic potential of Lugol chromoendoscopy combined with brush cytology to diagnose esophageal squamous cell carcinoma and high-grade dysplasia. Secondly, the benefit of additional biomarkers was investigated. A total of 61 patients (21 patients with achalasia and 40 patients with malignancies of the head and neck) were included. Chromoendoscopy with 1.2% Lugol iodine solution with targeted biopsies and brush cytology processed by digital image cytometry (DICM) and fluorescence in situ hybridization (FISH) from unstained lesions (USLs) and stained mucosa were performed. Six of the 61 patients had USLs ≥2 cm. Four patients had high-grade dysplasia (HGD) or carcinoma in situ (CIS). One patient with HGD and one patient with CIS were detected only after Lugol chromoendoscopy. The sensitivity and specificity for detected HGD or CIS in USLs ≥2 cm were 100% and 96.5%. No dysplasia was found in USLs <2 cm. DNA ploidy by DNA cytometry and p53 loss of heterozygosity (LOH) by fluorescence in situ hybridization showed no additional impact on diagnostic accuracy. Lugol chromoendoscopy enhances the detection rate of high-risk lesions with dysplasia or carcinoma in situ in large unstained lesions. Biomarkers such as aneuploidy and p53 LOH from brush cytology were not of additional benefit in this setting.

Is there an advantage to be gained from adding digital image cytometry of brush cytology to a standard biopsy protocol in patients with Barrett’s esophagus?

The current gold standard in Barrett's esophagus monitoring consists of four-quadrant biopsies every 1-2 cm in accordance with the Seattle protocol. Adding brush cytology processed by digital image cytometry (DICM) may further increase the detection of patients with Barrett's esophagus who are at risk of neoplasia. The aim of the present study was to assess the additional diagnostic value and accuracy of DICM when added to the standard histological analysis in a cross-sectional multicenter study of patients with Barrett's esophagus in Switzerland. One hundred sixty-four patients with Barrett's esophagus underwent 239 endoscopies with biopsy and brush cytology. DICM was carried out on 239 cytology specimens. Measures of the test accuracy of DICM (relative risk, sensitivity, specificity, likelihood ratios) were obtained by dichotomizing the histopathology results (high-grade dysplasia or adenocarcinoma vs. all others) and DICM results (aneuploidy/intermediate pattern vs. diploidy). DICM revealed diploidy in 83% of 239 endoscopies, an intermediate pattern in 8.8%, and aneuploidy in 8.4%. An intermediate DICM result carried a relative risk (RR) of 12 and aneuploidy a RR of 27 for high-grade dysplasia/adenocarcinoma. Adding DICM to the standard biopsy protocol, a pathological cytometry result (aneuploid or intermediate) was found in 25 of 239 endoscopies (11%; 18 patients) with low-risk histology (no high-grade dysplasia or adenocarcinoma). During follow-up of 14 of these 18 patients, histological deterioration was seen in 3 (21%). DICM from brush cytology may add important information to a standard biopsy protocol by identifying a subgroup of BE-patients with high-risk cellular abnormalities.

Three-dimensional Texture Analysis of Renal Cell Carcinoma Cell Nuclei for Computerized Automatic Grading

The extraction of important features in cancer cell image analysis is a key process in grading renal cell carcinoma. In this study, we analyzed the three-dimensional chromatin texture of cell nuclei based on digital image cytometry. Individual images of 2,423 cell nuclei were extracted from 80 renal cell carcinomas (RCCs) using confocal laser scanning microscopy (CLSM). First, we applied the 3D texture mapping method to render the volume of entire tissue sections. Then, we determined the chromatin texture quantitatively by calculating 3D gray level co-occurrence matrices and 3D run length matrices. Finally, to demonstrate the suitability of 3D texture features for classification, we performed a discriminant analysis. In addition, we conducted a principal component analysis to obtain optimized texture features. Automatic grading of cell nuclei using 3D texture features had an accuracy of 78.30%. Combining 3D textural and 3D morphological features improved the accuracy to 82.19%.

BlobFinder, a tool for fluorescence microscopy image cytometry

Images can be acquired at high rates with modern fluorescence microscopy hardware, giving rise to a demand for high-speed analysis of image data. Digital image cytometry, i.e., automated measurements and extraction of quantitative data from images of cells, provides valuable information for many types of biomedical analysis. There exists a number of different image analysis software packages that can be programmed to perform a wide array of useful measurements. However, the multi-application capability often compromises the simplicity of the tool. Also, the gain in speed of analysis is often compromised by time spent learning complicated software. We provide a free software called BlobFinder that is intended for a limited type of application, making it easy to use, easy to learn and optimized for its particular task. BlobFinder can perform batch processing of image data and quantify as well as localize cells and point like source signals in fluorescence microscopy images, e.g., from FISH, in situ PLA and padlock probing, in a fast and easy way.

Benefit of Baseline Cytometry As Add-On to Standard Histology On Surveillance in Patients with Barrett' Esophagus: Preliminary Results

Background and Aims: The current gold standard in the surveillance of Barrett's esophagus (BE) is the Seattle four-quadrant biopsies protocol (4-QB) every 1 to 2 cm. Using endoscopic brush cytology we studied prospectively whether digital image cytometry (DICM) is of additional benefit to regular histology as a predictor for progression to high-grade dysplasia (HGD) or cancer during a surveillance of at least 3 years. Methods: In this prospective cohort we included 74 patients (65% male) with BE and baseline endoscopies with at least one DICM in addition to 4-QB.

Affordable CD4 T-cell enumeration for resource-limited regions: A status report for 2008

The global struggle with human immunodeficiency virus (HIV) and the battle to develop affordable CD4 T-cell counting technology are both unfulfilled goals in 2008. The need for such instrumentation is more critical now as implementation of antiretroviral therapy (ART) is in progress in many resource limited regions. Major scaling-up efforts in rural situations are difficult to implement without laboratory infrastructure. CD4 T-cell counting is especially critical when trying to reach individuals with HIV to have them enrolled in ART as soon as they qualify for treatment based on CD4 count. This review covers both the chronological evolution and the scientific milestones of technological development of affordable immunophenotyping. It is more focused on flow cytometry but does consider the potential contribution by digital image cytometry. Thus far flow cytometry offered only modest progress toward affordable immunophenotyping. A list with desirable features is offered for side by side comparison. Digital image cytometry has yet to show its enormous affordable market potential. It is possible to develop truly affordable, portable flow cytometry but it is not here yet. There are some hopeful signs as there are innovative and practical technical components appearing at regular intervals. However, so far the technical breakthroughs have been fragmented efforts without any attempts to consider intercorporate collaboration to optimize critical mass and synergy. The smaller players in the industry have made some progress toward meeting the monumental needs in Africa and Asia. Digital image cytometry may well be the ultimate winner in the affordable technology race.

Development of a multistage classifier for a monitoring system of cell activity based on imaging of chromosomal dynamics

Cell-based assays utilizing digital image cytometry yield multivariate sets of information measuring the efficacy of medicines/chemicals. The use of a HeLa cell line that expresses a GFP-Histone-H1 fusion protein further enhances the performance of these systems, avoiding the use of dyes that may have detrimental influence on cells. Aside from the mitotic index, the distribution of the cell-cycle phases during mitosis can be used as measures of drug/treatment efficacy. Quantification of these parameters, however, requires skill and is time consuming. The purpose of this research was therefore to create a classifier to be incorporated into a system that can automatically identify the cell-cycle phases in a given image. Features based on the shape and texture of the chromosomal regions in images of live HeLa cells were measured and analyzed. Linear discriminant functions were calculated for the eight cell-cycle phases: interphase, prophase, prometaphase, metaphase, early anaphase, anaphase, telophase and cytokinesis. The multistage linear discriminant classifier developed had an average classification efficiency of 87.30%. We demonstrated the possibility of creating a classifier to discriminate between cell-cycle phases using shape and texture features of chromosomal regions. The classifier can be fused to an algorithm for image segmentation, forming a system to automatically and rapidly measure the aforementioned parameters. The results can then be collated to constitute an assay assessing the effects of a drug or treatment on mammalian cells.

Combined Digital Image Cytometry with Fluorescence in Situ Hybridization of Brush Cytology Identifies Patients with Barrett's Esophagus at Increased Risk for Neoplastic Progression

Background: We have previously shown that Digital Image Cytometry (DICM) of brush cytology in Barrett's Esophagus (BE) identifies additional high risk patients with aneuploid and intermediate DNA-pattern. Similarly, flowcytometry of biopsy specimens with aneuploidy or increased 4N combined with p53 loss of heterozygosity (LOH) may identify patients at risk of progression to cancer. Methods: This prospective cohort study aimed to assess specific genetic changes of p53 by Fluorescence in situ Hybridization (FISH) as compared to DICM in cytologic specimens of patients with BE: a) no dysplasia (No D) b) low-grade dysplasia (LGD), c) high-grade dysplasia (HGD) and d) early cancer (Ca). We examined 58 brush specimens from 43 patients (27 male; mean age 64 yrs) with BE by DICM on Feulgen restained cytology smears. DNA patterns were defined as diploid, intermediate and aneuploid. Fifty cytologies (94%) were further processed by two-color FISH using a locus-specific probe for 17p13.1 (p53 gene) with an enumeration probe. A pathological FISH probe was defined as loss of p53 (LOH) in more than 6.7% of cells (> mean + 3 SD of diploid controls). Results: 15/58 (26%) of DICM examinations showed aneuploidy with p53 LOH by FISH in 10/11 (91%). 13/58 (22%) intermediate DNA-patterns were diagnosed with p53 LOH in 12/13 (92%). 30/58 (52%) brush cytologies were diploid with p53 LOH by FISH in 5/30 (17%). Thus, pathological DICM (aneuploid or intermediate pattern) correlated with p53 LOH by FISH in 23/25 (92%) of brush cytologies, yielding good agreement beyond chance (Cohen's kappa of 0.74). The combined analysis selected a high-risk constellation with p53 LOH in 21/42 (50%) of patients negative for HGD or Ca. Conclusion: Combined DICM with p53 LOH by FISH detects patients with BE without HGD or Ca who may be at increased risk for neoplastic progression according to current literature. Provided that prospective studies support an high risk prognosis of FISH positive individuals with non-dysplastic histology, brush cytology may be a promising additional diagnostic tool guiding surveillance patterns for BE.

Digital Image DNA Cytometry: A Useful Tool for the Evaluation of Malignancy in Biliary Strictures

Background: Cytologic evaluation of the biliary tract strictures is nowadays widely used for distinguishing between benign and malignant lesions but remains a challenge for some problematic cases. Digital Image cytometry (DNA-cytometry) helps cytopathologists to resolve some unclear situations. Methods: We have analysed 41 specimens of bile duct brushings obtained from patients during ERCP (11 benign cases, 7 suspicious for malignancy cases and 23 malignant cases) by DNA-cytometry and correlated them with the histological biopsy counterpart. Results: All eleven cytological and histological benign cases were DNA-diploid and among 22 patients with malignant cytological and histological diagnosis 21 were DNA-aneuploid. One case considered malignant by the cytopathologist revealed DNA-aneuploid but malignancy could not be confirmed by histology. The analysis of the suspicious for malignancy cases revealed that all DNA-aneuploid cases were malignant and all DNA-diploid cases were benign referring to the follow-up of the patients.The comparison between cytology alone and cytology combined with DNA-cytometry related to the histological diagnosis (gold standard) resulted in a sensitivity of 100% and a specificity of 79% for cytology alone; a specificity of 94% and a sensitivity 92% for DNA-cytometry and a specificity of 93% and a sensitivity of 100% with combined analyses. The positive predictive value was 90% for cytology, 96% for DNA-cytometry and for both analyses. The negative predictive value showed 100% for cytology, 89% for DNA-cytometry and 100% for combined studies. Conclusions: Despite the limited number of patients involved in the study, the Results obtained indicate an increased of specificity and of positive predictive value using DNA-cytometry. These Results confirm the pertinence of these method for challenging cases, in conjunction with other available diagnostic tools.

A multivarite model for differentiation of Spitz nevu and malignant melanomas using digital image cytometry

A multivarite model for differentiation of Spitz nevu and malignant melanomas using digital image cytometry