Citations

Abstract Background: Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays. Methods: We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias. Results: Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose–response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms. Conclusions: Multiplex measurements present difficult challenges that require further analytical and statistical developments.

Purpose: Efficiency, anatomic, and histopathologic outcomes of GreenLight™ HPS™ 120-W, 532-nm lithium triborate (LBO) laser photoselective vaporization of the prostate (PVP) in a survival model of living canines were studied and compared with the outcomes of the only benchmarked survival study of 60-W 532-nm potassium-titanyl-phosphate (KTP) laser PVP in living canines. Materials and Methods: Twelve dogs underwent anterograde PVP with the 120-W LBO laser and, 4 each, were euthanized 3 hours (acute), 3 days (early), or 8 weeks (chronic) postoperatively. Laser energy and time were recorded. Prostates were sectioned, measured, and histologically analyzed after hematoxylin and eosin (H&E), triphenyltetrazolium chloride (TTC), or Gomori trichrome (GT) staining and compared with a normal control. Results: LBO laser PVP at 120 W acutely created a 6.7 ± 3.2 cm 3 cavity, hemostatically, and vaporized tissue 160% more efficiently (mean 1.3 cm 3 /min vs 0.5 cm 3 /min), 500% faster (mean 4.9 vs 29.1 min), and needed 121% less energy (mean 28.8 vs 63.6 kJ) than the 60-W KTP laser. Histologic staining with H&E and TTC demonstrated a coagulation zone of 1.5 ± 0.3 mm for the 120-W LBO laser, comparable to the 1 to 2 mm for the 60-W KTP laser. H&E- and GT-stained, healed prostates at 8-weeks postoperatively showed reepithelialized cavities with minimal submucosal fibrosis compared with an identically stained normal and the benchmarked KTP laser PVP-treated prostates. Conclusion: Our in vivo canine survival study demonstrates GreenLight HPS 120-W, 532-nm LBO laser PVP has substantially more vaporization efficiency and speed, with equally favorable tissue interaction and healing vis-à-vis those benchmarked for the 532-nm wavelength by KTP laser PVP.

Abstract The t(2;14)(p16;q32) has been reported previously in only 12 cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). The clinicopathologic features have been incompletely described. We describe 6 new cases of CLL/SLL with t(2;14)(p16;q32). All had marrow involvement, 4 had absolute lymphocytosis, 4 had lymphadenopathy, and 3 had hepatosplenomegaly. All showed atypical lymphocyte morphologic features with plasmacytoid differentiation and irregular nuclei; 3 had increased prolymphocytes. Flow cytometry demonstrated typical immunophenotypes in 5 and an atypical immunophenotype in 1. All expressed ZAP70; 5 assessed showed unmutated IgVH genes. Karyotyping identified t(2;14)(p16;q32) as the sole abnormality in 1, primary abnormality in 2, and part of a complex karyotype in 3. Fluorescence in situ hybridization analysis revealed BCL11A/IgH rearrangement in all. After chemotherapy, 3 patients died of disease and 3 were alive with disease (median follow-up, 80 months). We conclude that CLL/SLL with t(2;14) (p16;q32) and BCL11A/IgH rearrangement is characterized by atypical morphologic features and unmutated IgVH genes.

Abstract Performance on specialized diagnostic tests for platelet disorders, including dense-granule deficiency, is rarely evaluated by external quality assessment (EQA). Members of the North American Specialized Coagulation Laboratory Association that evaluate platelet dense-granule deficiency commonly use whole-mount electron microscopy (EM) methods. This observation led us to develop a pilot EQA survey with standardized EM images and clinical samples on grids from a healthy control subject and a subject with dense-granule deficiency. The survey participants were 8 centers, including 2 with no experience in platelet whole mount EM. All participants, including inexperienced sites, correctly interpreted findings for the normal and dense-granule-deficient platelets. Among experienced sites, agreement was excellent (>82%) on platelet structures to count or not count as dense granules. Participants indicated that future EQA challenges should include clinical samples on grids and standardized images. This is the first report that platelet EM can be assessed by EQA.

OBJECTIVE To determine whether deuterium enrichment on carbons 5 and 3 (C5/C3) in plasma glucose is influenced by processes other than gluconeogenesis and, if so, whether these processes are altered by type 2 diabetes. RESEARCH DESIGN AND METHODS In this study, 10 obese diabetic and 10 obese nondiabetic subjects were infused intravenously with [3,5-2H2] galactose enriched at a C5-to-C3 ratio of 1.0 as well as the enrichment of deuterium on C5 and C3 of plasma glucose, measured with nuclear magnetic resonance using the acetaminophen glucuronide method. RESULTS The ratio of deuterium enrichment on C5 and C3 of glucose was <1 (P < 0.001) in all of the diabetic and nondiabetic subjects, resulting in a means ± SE C5-to-C3 ratio that did not differ between groups (0.81 ± 0.01 vs. 0.79 ± 0.01, respectively). CONCLUSIONS That the C5-to-C3 glucose ratio is <1 indicates that transaldolase exchange, selective retention of deuterium at the level of the triose-isomerase reaction, or both occur in humans. This also indicates that the net effect of these processes on the C5-to-C3 ratio is the same in people with and without type 2 diabetes. The possible effects of transaldolase exchange or selective retention of deuterium (or tritium) at the level of the triose-isomerase reaction on tracee labeling and tracer metabolism should be considered when the deuterated water method is used to measure gluconeogenesis or [3-3H] glucose is used to measure glucose turnover in humans.

Context: Stress-mediated hypothalamic-pituitary-adrenal axis activation, regulated by arginine vasopressin (AVP), may have a role in the pathophysiology of metabolic syndrome (MetSyn). Objective: The objective of the study was to investigate whether plasma C-terminal provasopressin fragment (copeptin), a surrogate for circulating AVP, was associated with measures of insulin resistance and presence of MetSyn. Design, Setting, and Participants: This was a multicenter, community-based study, investigating novel biomarkers for vascular disease. Participants included 1293 African-Americans (AA) (64 ± 9 yr) and 1197 non-Hispanic whites (NHW) (59 ± 10 yr) belonging to hypertensive sibships. Main Outcome Measures: Plasma copeptin levels were measured by an immunoluminometric assay. MetSyn was defined per Adult Treatment Panel III criteria. Generalized estimating equations were used to assess whether plasma copeptin was associated with measures of insulin resistance and MetSyn. Results: The prevalence of MetSyn was 50% in AA and 49% in NHW. In each group, after adjustment for age and sex, plasma copeptin levels significantly correlated with body mass index, fasting plasma glucose and insulin, homeostasis model assessment of insulin resistance, triglycerides, and (inversely) high-density lipoprotein cholesterol (P < 0.05 for each variable). In multivariable logistic regression models that adjusted for age, sex, smoking, statin use, serum creatinine, education, physical activity, and diuretic use, plasma copeptin levels in the highest quartile were associated with an increased odds ratio of having MetSyn compared with bottom quartile: odds ratio (95% confidence interval) in AA, 2.07 (1.45–2.95); in NHW, 1.74 (1.21–2.5). Conclusions: Our findings indicate a novel cross-sectional association between plasma copeptin and measures of insulin resistance and MetSyn.

The protein deacetylase SIRT1 has been implicated in a variety of cellular functions, including development, cellular stress responses, and metabolism. Increasing evidence suggests that similar to its counterpart, Sir2, in yeast, Caenorhabditis elegans, and Drosophila melanogaster, SIRT1 may function to regulate life span in mammals. However, SIRT1's role in cancer is unclear. During our investigation of SIRT1, we found that c-Myc binds to the SIRT1 promoter and induces SIRT1 expression. However, SIRT1 interacts with and deacetylates c-Myc, resulting in decreased c-Myc stability. As a consequence, c-Myc's transformational capability is compromised in the presence of SIRT1. Overall, our experiments identify a c-Myc–SIRT1 feedback loop in the regulation of c-Myc activity and cellular transformation, supporting/suggesting a role of SIRT1 in tumor suppression.

BackgroundStrokes, vascular risk factors, and apolipoprotein E (APOE) genotype are associated with cognitive decline in the elderly, but definitive evidence that these affect cognition as early as middle age is limited.ObjectiveWe describe the relationships of APOE genotype, stroke, and vascular risk factors with cognitive change over a 14‐year follow‐up in the Atherosclerosis Risk in Communities (ARIC) Study cohort recruited while in middle age.MethodsParticipants included a subset of the ARIC Study who underwent assessments of cognitive function and vascular risk factors. Four cognitive assessments were performed between 1990–1992 and 2004–2006. Cognitive assessments included the Delayed Word Recall (DWR) Test, the Digit Symbol Substitution (DSS) Test, and the Word Fluency (WF) Test. Vascular risk factors were assessed during the baseline visit in 1990–1992. Incident stroke was recorded over the 14 years of follow‐up.ResultsThere were 1130 participants (mean age, 59 ± 4.3 [SD] years; 62% women; 52% African‐American) with longitudinal data. In multivariate, random‐effects linear models adjusted for age, education, gender, and race, the risk factors diabetes and APOE ɛ4 genotype were independently associated with a decline in performance on the DSS test (both P < .005), whereas hypertension and stroke were not. For DWR, stroke and APOE ɛ4 genotype were independent predictors of decline (both P < .001). For the WF test, metabolic syndrome, hypertension, and stroke were independently associated with decline (all P < .005). No evidence of differential effects of risk factors on cognitive decline by race, gender, or interactions between risk factors was found.ConclusionsThe vascular risk factors diabetes and hypertension, a history of stroke itself, and APOE ɛ4 genotype independently contribute to cognitive decline in late middle age and early elderly years.