Triantafyllos Tafas

Chronic pain is a manifestation of the Ehlers-Danlos syndrome

The Ehlers-Danlos syndrome (EDS) is a group of heritable systemic disorders of connective tissue manifesting joint hypermobility, skin extensibility, and tissue fragility. Although the presence of pain has been documented in the various types of the EDS, its natural history, distribution, and management have not been defined. We conducted a structured interview in 51 individuals affected with different types of EDS. Affected individuals reported chronic pain of early onset involving most frequently the shoulders, hands, and knees. Pain was generally refractory to a variety of pharmacologic and physical interventions. Chronic pain is a common manifestation of EDS.

Detection of circulating tumor cells in peripheral blood with an automated scanning fluorescence microscope

We have developed an automated, highly sensitive and specific method for identifying and enumerating circulating tumour cells (CTCs) in the blood. Blood samples from 10 prostate, 25 colorectal and 4 ovarian cancer patients were analysed. Eleven healthy donors and seven men with elevated serum prostate-specific antigen (PSA) levels but no evidence of malignancy served as controls. Spiking experiments with cancer cell lines were performed to estimate recovery yield. Isolation was performed either by density gradient centrifugation or by filtration, and the CTCs were labelled with monoclonal antibodies against cytokeratins 7/8 and either AUA1 (against EpCam) or anti-PSA. The slides were analysed with the Ikoniscope® robotic fluorescence microscope imaging system. Spiking experiments showed that less than one epithelial cell per millilitre of blood could be detected, and that fluorescence in situ hybridisation (FISH) could identify chromosomal abnormalities in these cells. No positive cells were detected in the 11 healthy control samples. Circulating tumour cells were detected in 23 out of 25 colorectal, 10 out of 10 prostate and 4 out of 4 ovarian cancer patients. Five samples (three colorectal and two ovarian) were analysed by FISH for chromosomes 7 and 8 combined and all had significantly more than four dots per cell. We have demonstrated an Ikoniscope® based relatively simple and rapid procedure for the clear-cut identification of CTCs. The method has considerable promise for screening, early detection of recurrence and evaluation of treatment response for a wide variety of carcinomas.

Gain of 3q26: A genetic marker in low-grade squamous intraepithelial lesions (LSIL) of the uterine cervix

OBJECTIVE Physicians have few resources for determining which LSIL will progress to HSIL or regress. Recently the chromosome 3q26 region was found to be amplified in patients with cervical cancer. The frequency of this 3q gain increased with severity of dysplasia. The primary objective of this study was to evaluate an automated FISH assay for detection of 3q gain in liquid cytology samples as a potential tool for risk stratification and triaging. METHODS Slides prepared from 257 liquid cytology specimens (97 Negative, 135 LSIL 25 HSIL) were hybridized with a single-copy probe for the chromosome 3q26 region and a probe for the centromeric alpha-repeat sequence of chromosome 7, using standard FISH methods. Using automated analysis, the total number of nuclei and the number of nuclei with >2 signals for 3q26 were determined, using a 20x objective. The nuclei were rank ordered based on number of 3q26 FISH signals. The 800 nuclei with the highest number of signals were scored using both FISH probes and nuclei with increased numbers of 3q signals were enumerated. RESULTS AND CONCLUSIONS Analysis of 257 specimens demonstrated that a fully automated FISH scoring system can detect 3q gain in liquid cytology samples. A fully automated method for determination of 3q gain in liquid cytology may be the assay necessary to implement routine testing. Additional studies to validate the utility of this technology are needed.

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliellasalina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO4-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 106 L−1 with typical densities of 1.5–4.5 × 106 L−1. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L−1 within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO3-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO4-P applications (4.0 and 8.0 mg L−1) at 60 ppt salinity and 23 °C. The lower PO4-P applications (0.6–2.0 mg L−1) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.

Detection of circulating fetal cells utilizing automated microscopy: potential for noninvasive prenatal diagnosis of chromosomal aneuploidies

As fetal cells can be indisputably identified through detection of Y FISH signals, we utilized an automated microscopy system developed to identify and enumerate cells bearing X and Y FISH signals. We further investigated the potential of fetal hemoglobin expression as a gender independent marker for automated identification of fetal cells.

Automated Microscopy of Amniotic Fluid Cells: Detection of FISH Signals Using the FastFISH® Imaging System

Objective: FISH (fluorescence in situ hybridization) analysis is a valuable adjunct to cytogenetics that provides a rapid screen for common abnormalities. However, FISH is expensive, labor-intensive, and requires a high skill level and subjective signal interpretation. A fully automated system for FISH analysis could improve laboratory efficiency and potentially reduce errors and costs. Methods: In this study we blindly compared automated FISH signal acquisition and display against standard FISH analysis. A total of 62 amniocentesis samples were prepared using the AneuVysion® multicolor DNA probe kit and probed for chromosomes 13, 18, 21, X, and Y. Two sets of slides were produced from each sample. Fifty cells were scored in each slide. One set was evaluated using standard manual microscopy and the other using the automated image acquisition and display capabilities of the Ikoniscope® fastFISH® amnio Test System. This system uses epifluorescence optics, along with optimized slide management to process slides automatically. Results: A 100% concordance was observed between the results obtained using manual microscopy and the automated system. There was also 100% concordance between the FISH results and those obtained by conventional karyotyping. Conclusion: Our data suggest that the automated system is capable of providing accurate and rapid identification and display of cells and FISH signals.

Limnological survey of the warm monomictic lake Trichonis (central western Greece); II. Seasonal phytoplankton periodicity – a community approach

Phytoplankton assemblages of the warm monomictic lakeTrichoniswere studied during the period April 1985 to February 1986.Speciescomposition and biomass data are presented along withinformationon the seasonal periodicity of dominant taxa of microalgae.Multivariate methods were used to analyze community structureandannual succession. Population succession patterns correspondtochanges in environmental variables. According to thegeneralizedphytoplankton sequences Trichonis is classified as anoligotrophiclake.

Limnological survey of the warm monomictic lake Trichonis (central western Greece); I. The physical and chemical environment

The physical and chemical status of Trichonis – the largestanddeepest natural lake in Greece – is examined over two annualcycles (1985–86 and 1988–89). A correlation between lakenutrientpatterns and phytoplankton biomass is attempted. Limnologicalfeatures are compared with data from other warm and temperatelakes.With regard to its thermal regime, Trichonis is classified asawarm monomictic lake. The lakes stratification pattern andannualheat budget resemble those of other temperate lakes. Trichonisisa carbonate type, low conductivity lake (‘class II, lowsalinity’warm lake). Nitrogen and phosphorus concentrations were ratherlow.The inorganic nitrogen content fluctuated widely over the twoannual cycles examined. On the contrary, phosphorusconcentrationsshowed no significant changes. The limiting factor during1985–86was P, while N was limiting during stratification in 1988–89.Aweak correlation was found between the plankton communityfeatures(species abundance, biomass, chlorophyll-a) and lightpenetration. At present the eutrophication process fromoligotrophytowards mesotrophy has not been essentiallyaccelerated.

An Automated Image Analysis Method for the Measurement of Neutrophil Alkaline Phosphatase in the Prenatal Screening of Down Syndrome

OBJECTIVES (1) To develop an image analysis method for the measurement of neutrophil alkaline phosphatase (NAP); (2) To establish a correlation of urea-resistant fraction of NAP (URNAP)/NAP scoring between the manual and automated methods, and (3) to assess the value of URNAP/NAP in the prenatal screening of Down syndrome. STUDY DESIGN Slides from 15 unaffected controls were blindly scored by both methods. The Pearson test was used for correlation analysis. Slides from 15 Down syndrome pregnancies and 25 unaffected controls were scored manually. RESULTS A coefficient r = 0.93 was obtained comparing the URNAP/NAP scores generated by the two methods. Average time for scoring by the automated method was 8 min. The median URNAP/NAP values for Down syndrome and unaffected controls were 112/86.1 and 51/51.5, respectively. CONCLUSIONS Scores obtained by both methods highly correlate. Automated scoring is threefold faster. Down syndrome cases have higher URNAP/NAP scores compared to unaffected controls, which suggests that URNAP/NAP is an extremely useful marker for mid-trimester prenatal screening.

Amplification of the 3q chromosomal region as a specific marker in cervical cancer

OBJECTIVE Chromosome 3q gain has been consistently observed in cervical intraepithelial neoplasia grades 2 and 3 (CIN 2,3) and squamous cell carcinomas of the cervix. There are a number of potential clinical uses of testing for 3q gain in liquid cytology specimens, including the identification of subsets of women with atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion cytology who are at greatest risk of having CIN 2,3 and would thus benefit most from immediate colposcopy. The objective of this study was to establish the sensitivity and specificity of 3q gain for discriminating between CIN 2,3 and normal. STUDY DESIGN Residual cytology specimens were collected from 199 women. Liquid-based cytology (LBC) was used for the selection of subjects, with women with high-grade squamous intraepithelial lesion or high-grade squamous intraepithelial lesion who had colposcopy and adjudicated biopsy-confirmed CIN 2,3 forming the disease-positive group (n = 28) and women doubly negative for both cytology and high-risk human papillomavirus (hrHPV) testing forming the disease-negative group (n = 171). A single slide was prepared from each residual LBC specimen and analyzed for 3q gain by fluorescent in situ hybridization, using a probe specific for the 3q26 region and a control probe for the chromosome 7 centromere. Two approaches were compared for the determination of 3q gain. The first was based on the analysis of an entire cervical cytology slide for the presence of rare cells with a high copy number (>4 copies) for the 3q locus. The second approach was based on the analysis of 400 cells to determine the percentage with 3 or more copies of the 3q locus. RESULTS Using the approach based on the detection of rare cells with a high copy number (>4 copies) for the 3q locus, 26 of the specimens from women with CIN 2,3 and none of the 171 specimens from women who were both hrHPV and cytology negative was positive for 3q gain. This translates to a sensitivity of 92.9% (95% confidence interval [CI], 76.5-98.9%), a specificity of 100% (95% CI, 97.8-100%), a positive predictive value of 100% (95% CI, 86.7-100%), and a negative predictive value of 98.8% (95% CI, 95.9-99.8), for distinguishing CIN 2,3 from normal. CONCLUSION These data support the potential clinical use of 3q gain for the evaluation of women in a number of clinical situations, including women with atypical squamous cells of undetermined significance, low-grade squamous intraepithelial lesion, and those who are hrHPV positive.