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Undetermined thyroid cytology precludes any definitive distinction between malignant and benign lesions. Recently several classifications have been proposed to split this category into two or more cytological subcategories related to different malignancy risk rates. The current study was performed retrospectively to investigate the results obtained separating "undetermined" cytologic reports into two categories: "follicular lesion" (FL) and "atypia of undetermined significance" (AUS). Biochemical, clinical, and echographic features of each category were also retrospectively analyzed. Altogether, 316 undetermined fine-needle aspirated cytologies (FNACs) were reclassified as 74 FL and 242 AUS. Histological control leads to a diagnosis of carcinomas, adenomas, and nonneoplastic lesions, respectively, in 42.2%, 20%, and 37.8% of AUS and in 8.3%, 69.4%, and 22.2% of FL. Among biochemical, clinical, cytological, and echographic outcomes, altered thyroid autoantibodies, multiple versus single nodule, AUS versus FL, and presence of intranodular vascular flow were statistically significant to differentiate adenoma from carcinoma and from nonneoplastic lesions, whereas no significant differences were found between carcinomas and nonneoplastic lesions for these parameters. The results of this retrospective study show that undetermined FNAC category can further be subclassified in AUS and FL, the former showing higher malignancy rate. Further prospective studies are needed to confirm our results.

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Goal of this study was to asses the performance of Aperio computer-assisted analysis for HER2 immunohistochemical measurement in 292 equivocally (score2+) HercepTest immunoreactive breast cancer cases, evaluated by an experienced pathologist and analyzed with fluorescent in situ hybridization (FISH). The automatic Aperio categorization and the percentage of immunoreactive cells as evaluated by the computer and by the pathologist were recorded. Computer-assisted analysis classified 7 (2.4%) cases as negative (0), 136 (46.6%) as faintly positive (1+), 134 (40.5%) as moderately positive (2+), and 15 (5.1%) as strongly positive (3+). Correlative component analysis (CCA) classification is associated with Her2 amplification (P<0.0001). Compared with the human evaluation, automated CCA classification would save 157 (58%) FISH analyses, while not identifying 15 amplified cases (6% false-negative rate). The mean computer percentage value (CPV) is 18.44% standard deviation ±19.00 (range, 0.01 to 76.10). CPV and the pathologist percentage value are significantly associated and correlated (P<0.001) and have similar sensitivity and specificity in identifying Her2 FISH-amplified cases. CPV has a very low interobserver variation. The difference in CPV in amplified and nonamplified subgroups is statistically significant (P<0.001). Receiver operating characteristic analysis indicates that CPV is good at separating FISH nonamplified from amplified cases (P<0.001). The optimal cut-off value maximizing both sensitivity and specificity is 17.6% (sensitivity=73.3%, specificity=71.6%). Using a different cut-off value (2% of positive cells) we would have missed only 3 amplified cases (1% false-negative rate) while not submitting to FISH 52 cases (18% of the whole series). This false-negative rate is well below the expected false-negative rate usually observed in score 1 cases, supporting the use of CCA with a modified cut-off value in routine diagnostics for equivocally stained HER2 cases.

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Accurate classification of nonsmall cell lung cancers is of paramount clinical relevance, as novel chemotherapeutic agents show different efficacy in adenocarcinomas (ADCs) compared with squamous cell carcinomas (SQCCs). Cyto and histomorphology may sometimes be insufficient for this distinction and immunohistochemistry may improve diagnostic accuracy. The measurement of miR-205 may be another tool for the distinction between ADC and SQCC. The aim of our study was to compare morphologic and immunohistochemical classification with the relative quantification of miR-205 and miR-21 insurgically resected and well-characterized lung tumors (25 ADCs, 24 SQCCs, 1 adenosquamous). The miR-21 relative levels were similar in SQCC and ADC, whereas the miR-205 relative levels were lower in ADC (P<0.0001). The miR-205 sample score value, determined according to Lebanony et al, was higher in ADC (range, 2.8 to 9.08) compared with SQCC (range, -4.17 to 2.445) (P<0.0001). Accordingly, 22 tumors were classified as ADC and 28 tumors as SQCC, although 8 cases (2 SQCCs and 6 ADCs) were in the range of "near cutoff values." Four cases classified as SQCC (according to the sample score method) corresponded to cases classified as ADC on the basis of morphoimmunohistochemical evaluation. In conclusion, the relative quantification of miR-205 and miR-21 seems to be a promising diagnostic tool. However, the molecular approach is still not completely satisfactory as it may misclassify a non-negligible percentage of cases. Therefore, it cannot be used as a substitute of accurate morphologic and immunophenotypical characterization of tumors, but could be used as an adjunctive diagnostic criterion in selected cases.

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BACKGROUND AND OBJECTIVES: Increased plasma homocysteine, a sulphur amino acid closely related to methionine metabolism, is considered an independent risk factor for cardiovascular diseases. Over the last years, the request to clinical laboratories for homocysteine measurement is constantly increased and, for this reason, several new methods have been developed, mainly with the aim of using them on completely automated instruments for routine analyses. In this paper, we evaluated a new immunonephelometric method for homocysteine determination on the Dade Behring BNII nephelometer. METHODS: Linearity, recovery, limit of detection (LOD) and total imprecision and were assessed; moreover, the method was compared with a HPLC reference method and with an automated immunoassay method (AxSYM Abbott). RESULTS: Recovery range was 96.4-104.2%, LOD was 0.5 micromol/L and total imprecision ranged from 5.0% to 7.6%. For the comparison study, the immunonephelometric method showed a good correlation both with HPLC (Y=1.02X-0.79, R(2)=0.99) and with the AxSYM method (Y=1.003X+0.06, R(2)=0.98). The Bland-Altman plot analysis shows that the immunonephelometric method has a slight positive bias with both HPLC (mean: 1.03 micromol/L, 95% confidence interval: 0.28-1.79 micromol/L) and AxSYM methods (mean: 0.45 micromol/L, 95% confidence interval: -0.03-0.94 micromol/L). CONCLUSION: The new nephelometric method from Dade Behring, for its analytical performance, can be easily considered a suitable method for homocysteine routine measurement; moreover, it cannot be ruled out that the widespread availability of nephelometers in clinical laboratories play a leading role in the choice of this method.

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BACKGROUND AND OBJECTIVES: The increased need in clinical chemistry laboratories for methods of homocysteine determination, in correlation with cardiovascular diseases and nutritional deficient status, has led to the development of different analytical methods; fluorescent immunoenzymatic assays and, recently, new fully automated spectrophotometric methods are commercially available. In this paper, we compared data obtained from a new enzymatic method for homocysteine assay (Carolina Liquid Chemistries), with data obtained from a HPLC reference method and an immunoenzymatic method (Abbott AxSYM immunoassay). RESULTS: The enzymatic method shows a good correlation with both the HPLC (Y = -1.3 + 1.02X; R2 = 0.93) and the immunoenzymatic method (Y = 0.7 + 1.02X; R2 = 0.92), although a bias enhancement was present in some samples. However, the enzymatic method shows a superior analytical feasibility because it needs only common laboratory instruments (UV-visible spectrophotometer) and can be easily adapted to large automatic clinical chemistry analyzers. Moreover, it lowers the laboratory cost of the analysis in comparison to both HPLC and immunoenzymatic methods. CONCLUSIONS: The enzymatic Carolina Liquid Chemistries method for homocysteine assay shows acceptable analytical performance and undoubtedly possesses technical and cost advantages.

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HL-60 leukemia cells, Rat-1 fibroblasts and WI-38 diploid fibroblasts were exposed for 24-72 h to 0.5-1.0-mT 50-Hz extremely low frequency electromagnetic field (ELF-EMF). This treatment induced a dose-dependent increase in the proliferation rate of all cell types, namely about 30% increase of cell proliferation after 72-h exposure to 1.0 mT. This was accompanied by increased percentage of cells in the S-phase after 12- and 48-h exposure. The ability of ELF-EMF to induce DNA damage was also investigated by measuring DNA strand breaks. A dose-dependent increase in DNA damage was observed in all cell lines, with two peaks occurring at 24 and 72 h. A similar pattern of DNA damage was observed by measuring formation of 8-OHdG adducts. The effects of ELF-EMF on cell proliferation and DNA damage were prevented by pretreatment of cells with an antioxidant like alpha-tocopherol, suggesting that redox reactions were involved. Accordingly, Rat-1 fibroblasts that had been exposed to ELF-EMF for 3 or 24 h exhibited a significant increase in dichlorofluorescein-detectable reactive oxygen species, which was blunted by alpha-tocopherol pretreatment. Cells exposed to ELF-EMF and examined as early as 6 h after treatment initiation also exhibited modifications of NF kappa B-related proteins (p65-p50 and I kappa B alpha), which were suggestive of increased formation of p65-p50 or p65-p65 active forms, a process usually attributed to redox reactions. These results suggest that ELF-EMF influence proliferation and DNA damage in both normal and tumor cells through the action of free radical species. This information may be of value for appraising the pathophysiologic consequences of an exposure to ELF-EMF.

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8-hydroxy-deoxyguanosine adducts (8-OHdG), indices of oxidative DNA damage, were measured by immunohystochemistry with diaminobenzidine detection in the brain, skeletal muscle, heart, liver, tenuum mucosa and lymphocytes from young (4 months) and aged (24 months) Sprague-Dawley rats fed ad libitum or held on two different caloric restriction diets (alternate day ad libitum feeding or daily feeding with 40% reduced calories). In the absence of caloric restriction the levels of oxidative DNA damage increased as a function of age in all tissues examined, with a maximum approximately 3-fold increase being detected in the peripheral lymphocytes and the heart and a minimum approximately 2-fold increase being detected in the liver and brain tissues. Caloric restriction regimens effectively reduced age-dependent increase of oxidative DNA damage in all tissues examined; in particular, the brain and small intestine did not exhibit any age-related increase of oxidative DNA damage. We propose that the levels of 8-OHdG in peripheral lymphocytes may serve a biochemical index of age-related whole organism oxidative DNA damage. Immunohistochemistry might be exploited as a rapid and simple techniques for measuring lymphocytes oxidative DNA damage in large scale studies.

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To study the role of Magnesium in the regulation of cell proliferation we characterized the proliferation behaviour of HC-11 mammary epithelial cells that were grown in media containing low to high Mg concentrations. Cells grown under control conditions (0.5 mM Mg in the medium) or in the presence of high (H) Mg (45 mM) displayed similar log-phases and reached confluence in 72h. In the presence of low (L) Mg (0.025 mM) the cells exhibited a reduced growth rate and did not reach confluence at 72h. Intra cellular total Mg increased from 12 to 36h of culture in all cells examined but returned to basal levels in those cells which reached confluence (i.e., control and H-Mg cells). Intra cellular Mg increased independent of mitosis-induced changes of volume and adenine nucleotides pools but correlated with an increased percentage of cells in the S phase and with total nucleic acid contents. These bell-shaped changes of intra cellular Mg were less evident in L-Mg cells, likely due to a combination of low Mg levels in the medium and decreased growth rate. Changes in membrane potential and pH were important factors that contributed to maintaining intra cellular Mg at physiologic levels in the face of increased or decreased availability of extra cellular Mg. H-Mg cells were depolarised and more acidic than control cells; conversely, L-Mg cells showed a pattern of hyperpolarization and alkalinization. These results lend support to the concept that Mg may be involved in regulating cell proliferation, and show that cells maintain adequate levels of intra cellular Mg, and hence their proliferation potential, even under conditions of extreme changes of extra cellular Mg.

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Cause-effect relationships between oxidative stress, DNA damage and aging were investigated in WI-38 human diploid fibroblasts at 21, 41 or 58 population doublings (PDs), corresponding to young, middle age or old fibroblasts, respectively. Oxidative DNA damage was evaluated by immunohistochemical detection of 8-hydroxy-2'deoxyguanosine (8-OHdG) adducts or by single cell microgel electrophoresis (COMET assay). Aging was evaluated by growth rate, senescence-associated-beta-galactosidase (SA-beta galactosidase) activity, cell cycle distribution, and expression of p21. Our results demonstrate that (i) oxidative DNA damage is proportional to the age of cells (ii) DNA damage in old/58 PDs cells reflects both an increased susceptibility to oxidative stress, induced by acute exposure to sub-lethal concentrations of hydrogen peroxide (H(2)O(2)), and a reduced efficiency of repair mechanisms. We also show that mild chronic oxidative stress, induced by prolonged exposure to 5 microM H(2)O(2), accelerates aging in fibroblasts. In fact, this treatment increased 8-OHdG levels, SA-beta-galactosidase activity, and G0/G1 cell cycle arrest in middle age/41 PDs, making them similar to H(2)O(2)-untreated old/58 PDs cells. Although other mechanisms may concur in mediating the effects of H(2)O(2), these results lend support to the concept that oxidative stress may be a key determinant of aging. Measurements of oxidative DNA damage might therefore be exploited as reliable marker of cellular aging.

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