LC-MS/MS identification of the one-carbon cycle metabolites in human plasma.

The one-carbon cycle is composed of four major biologically important molecules: methionine (L-Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy). In addition to these key metabolites, there are multiple enzymes, vitamins, and cofactors that play essential roles in the cascade of the biochemical reactions that convert one metabolite into another in the cycle. Simultaneous quantitative measurement of four major metabolites can be used to detect possible aberrations in this vital cycle. Abnormalities in the one-carbon cycle might lead to hyper- or hypomethylation, homocystinemia, liver dysfunction, and accumulation of white-matter hyperintensities in the human brain. Previously published methods describe evaluation of several components of the one-carbon cycle, but none to our knowledge demonstrated simultaneous measurement of all four key molecules (L-Met, SAM, SAH, and Hcy). We describe a novel analytical method suitable for simultaneous identification and quantification of L-Met, SAM, SAH, and Hcy with LC-MS/MS. Moreover, we tested this method to identify these metabolites in human plasma collected from patients with multiple sclerosis and healthy individuals. In a pilot feasibility study, our results indicate that patients with multiple sclerosis showed abnormalities in the one-carbon cycle.

Human papillomavirus distribution in vulvar intraepithelial neoplasia.

This pilot study investigated the prevalence and distribution of human papillomavirus (HPV) type in vulvar lesions in women with a history of vulvar intraepithelial neoplasia. Fifty-two specimens were collected. Uncommon HPV subtypes were found among the specimens, which may have implications for HPV vaccination coverage.

Salivary cortisol levels in students challenged with a testing stressor.

OBJECTIVE: The objective was twofold. The focus of the study was primarily to determine if the stress of a particularly difficult exam could cause students to lose the normal diurnal variation seen in human cortisol levels and secondarily, to validate the use of a competitive enzyme immunoassay for salivary cortisol. DESIGN: Physical therapy students enrolled in Research Design were asked to participate in the study by collecting baseline evening and morning salivary cortisols during what was regarded as a relatively stress free time in the Fall of 2009. The following spring, the same students were asked for samples the evening before and morning of their first Kinesiology test, traditionally a stressful time. Method validation was accomplished using instrumentation owned by the Medical Laboratory Science (MLS) Program and analysis was performed by MLS faculty and a second year MLS student. SETTING: Participants were enrolled in the College of Health Sciences at the University of Tennessee in Memphis. Sample collection and testing was performed in the student laboratory of the Medical Laboratory Science Program. PARTICIPANTS: Physical therapy students in their first year of a three-year entry level doctorate program, DPT. RESULTS: This group of students did not lose their diurnal variation of cortisol. However, an unexpected finding was noted: the students' salivary cortisol specimen collected in the morning of the fall semester was significantly higher than the salivary cortisol specimen collected the morning of the test in the spring semester (p = .019). Method validation was successful demonstrating a strong correlation ( r = 0.915) when compared to the reference laboratory. CONCLUSIONS: Cortisol diurnal variation was not lost in the study participants, but further studies should be performed due to the low percentage of students completing the study and the lack of demographic diversity. Even though the method validation in the student laboratory setting demonstrates that it is indeed possible to obtain the same excellent correlation as is seen in a clinical setting, the student laboratory is not CLIA certified, so assays can be performed for research use only.

Cytomorphologic features of poorly differentiated thyroid carcinoma: a multi-institutional analysis of 40 cases.

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) is an uncommon and aggressive malignancy. Despite the significant clinical implications of a diagnosis of PDTC, its cytomorphologic features have not been well defined. Statistical analysis was applied to a series of 40 PDTCs to identify a specific set of cytomorphologic features that characterized these tumors on fine-needle aspiration biopsy (FNAB). METHODS: In total, 40 thyroid FNABs that were highly diagnosed histologically as PDTC (19 insular carcinomas and 21 noninsular carcinomas) comprised the study group. A control group of 40 well differentiated thyroid neoplasms were selected for comparison. All FNABs were reviewed and scored for a series of 32 cytomorphologic features. The results were evaluated using univariate and stepwise logistic regression (SLR) analyses. RESULTS: In univariate analysis, 17 cytomorphologic features were identified that characterized the 40 PDTCs: insular, solid, or trabecular cytoarchitecture (P < .001); high cellularity (P = .007); necrosis (P = .025) or background debris (P = .025); plasmacytoid appearance (P = .0007); single cells (P < .0001); high nuclear/cytoplasmic ratio (P < .0001); scant cytoplasm (P = .03); nuclear atypia (P < .0001), including nuclear pleomorphism (P = .0052) and anisokaryosis (P < .0001); granular/coarse chromatin (P = .026); naked nuclei (P = .01); mitotic activity (P = .0001) and apoptosis (P < .0001); endothelial wrapping (P = .0053); and severe crowding (P < .0001). In logistic regression analysis, severe crowding (P = .0008) and cytoarchitecture (P < .0001) were identified as the most significant cytomorphologic features of PDTCs, and the combination of cytoarchitecture, severe crowding, single cells, and high nuclear/cytoplasmic ratio was the most predictive of PDTC. CONCLUSIONS: PDTCs have characteristic cytomorphologic features. By using logistic regression analysis, the features that were identified as the most predictive of PDTC were severe crowding, insular/solid/trabecular morphology, single cells, and high nuclear/cytoplasmic ratio.

Fine-needle aspiration biopsy of Hurthle cell lesions of the thyroid gland: A cytomorphologic study of 139 cases with statistical analysis.

BACKGROUND: Lesions of the thyroid gland composed of Hurthle cells encompass pathologic entities ranging from hyperplastic nodules with Hurthle cell metaplasia to Hurthle cell carcinomas. The cytologic distinction between these entities can be diagnostically challenging. Many cytologic features of Hurthle cell lesions that distinguish neoplastic Hurthle cell lesions requiring surgery from those that are benign and nonneoplastic have been described, but with variable usefulness. This is due, in part, to the small numbers of cases examined in previous studies and the limited application of statistical analysis. A morphologic study was made of 139 Hurthle cell lesions of the thyroid gland and statistical analysis applied to identify a set of cytomorphologic features that distinguish benign Hurthle cell lesions (BHCL) from Hurthle cell neoplasms (HCN). METHODS: Fine-needle aspiration biopsies (FNABs) of thyroid nodules with a predominant Hurthle cell component and corresponding histologic followup were included in the study. Cases were divided into BHCL and HCN groups on the basis of the histologic diagnosis. All cases were reviewed to assess the following 14 cytologic features: overall cellularity, cytoarchitecture, percentage of Hurthle cells, percentage of single cells, percentage of follicular cells observed as naked Hurthle cell nuclei, background colloid, chronic inflammation, cystic change, transgressing blood vessels (TBV), intracytoplasmic lumina, presence of multinucleated Hurthle cells, nuclear to cytoplasmic ratio, nuclear pleomorphism/atypia, and nucleolar prominence. The results were evaluated by using univariate and stepwise logistic regression (SLR) analysis; statistical significance was achieved at P-values < 0.05. RESULTS: One hundred thirty-nine FNAB specimens, corresponding to 56 HCN and 83 BHCL, fulfilled the study criteria. Six of the 14 cytologic features evaluated were shown by univariate analysis to be statistically significant in predicting HCN: nonmacrofollicular architecture (P < 0.001), absence of background colloid (P < 0.001), absence of chronic inflammation (P < 0.001), presence of TBV (P < 0.001), > 90% Hurthle cells (P < 0.001), and >10% single Hurthle cells (P = 0.014). The first four of these features were also shown to be statistically significant in the SLR analysis (P = 0.005, 0.010, 0.016, and 0.045, respectively), and when all four of these features were present HCN was correctly identified 86% of the time. CONCLUSIONS: In the current study of 139 FNAB specimens of thyroid Hurthle cell nodules, 14 cytologic features were examined and 6 were found to be statistically significant in identifying HCN. The following four features, when found in combination, were found to be highly predictive of HCN: nonmacrofollicular architecture, absence of colloid, absence of inflammation, and presence of TBV.

Hormonal effects of Depo-Provera in cervical smears: a comparison with Triphasil and postmenopausal effects.

BACKGROUND: Long-acting injectable contraceptive agents may cause changes in cervical smears that could impair the detection of epithelial abnormalities. The objectives of the current study were to 1) compare the hormonal effects of depot-medroxyprogesterone acetate (Depo-Provera) (DP) in cervical smears with those of levonorgestrel and ethinyl estradiol (Triphasil) (TP) and postmenopausal (PM) changes; and 2) determine whether the duration of DP use affects squamous maturation. METHODS: Satisfactory cervical smears from 50 DP users, 55 TP users, and 51 PM patients were evaluated blindly for: 1) squamous cell curling, crowding, cytolysis, and navicular cell formation; 2) pseudoparakeratosis, blue blobs, and histiocytes; 3) endometrial cells and blood; 4) single or enlarged endocervical nuclei and mucin-depleted endocervical cells; 5) lactobacilli and coccobacilli amounts; and 6) squamous maturation (ratio of parabasal:intermediate:superficial cells). RESULTS: No statistically significant differences were observed for blue blobs, histiocytes, blood, endometrial cells, or single or enlarged endocervical nuclei among smears from the three groups. More smears from DP and TP users demonstrated squamous cell curling, crowding, and cytolysis as well as navicular cells and abundant lactobacilli compared with smears from PM patients. There were more PM smears with pseudoparakeratosis and mucin-depleted endocervical cells compared with the other groups. The majority of PM smears (98%) demonstrated predominantly parabasal cells with some intermediate cells. The majority of DP (86%) and TP (93%) smears demonstrated mostly intermediate and some superficial cells, regardless of the duration of DP use. CONCLUSIONS: Certain progestational-dependent effects (i.e., curling, crowding, navicular cells, and abundant lactobacilli) were identified more often in TP users compared with DP users and less often in PM patients. The mostly parabasal pattern observed in smears from PM patients contrasted with the predominantly intermediate pattern found in smears from DP and TP users. The duration of DP use did not appear to have any effect on squamous maturation.