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Background Immunoassays provide a rapid tool for the screening of drugs-of-abuse (DOA). However, results are presumptive and confirmatory testing is warranted. To reduce associated cost and delay, laboratories should employ assays with high positive and negative predictive values (PPVs and NPVs). Here, we compared the results of urine drug screens on cobas 6000 (cobas) and ARCHITECTi2000 (ARCHITECT) platforms for six drugs against LC-MS/MS to assess the analytical performance of these assays. Methods Eighty nine residual urine specimens, which tested positive for amphetamine , THC-COOH, benzoylecgonine , EDDP, opiates and/or oxycodone during routine drug testing, were stored frozen until later confirmation by LC-MS/MS. Immunoassays were performed on cobas and ARCHITECT using a split sample. A third aliquot from these samples was tested by LC-MS/MS to assess the percentage of false positive, false negative , true positive and true negative results and calculate the PPVs and NPVs for each immunoassay. Results The PPVs of THC-COOH and EDDP assays were 100% on both platforms. Suboptimal PPVs were achieved for oxycodone (cobas, 57.1% vs ARCHITECT, 66.7%), amphetamine (77.8 vs. 100%), opiates (80.0 vs. 84.6%) and benzoylecgonine (88.9 vs. 84.2%) assays. The NPV was 100% for cobas and ARCHITECT oxycodone assays. Lower NPVs were achieved for THC-COOH (cobas, 28.6% vs ARCHITECT, 25.0%), EDDP (72.7% for both assays), benzoylecgonine (74.4% vs 73.8%), amphetamine (83.3% vs 82.8%) and opiates (100% vs 85.3%). Conclusion Overall, cobas and ARCHITECT urine drug screens have comparable analytical performance. Confirmatory testing is warranted for positive test results especially for oxycodone, amphetamine, opiates and cocaine. Negative drug screen results must be interpreted with caution especially for THC-COOH, EDDP, benzoylecgonine, amphetamine and opiates.

Background Core laboratory (CL), as a new business model, facilitates consolidation and integration of laboratory services to enhance efficiency and reduce costs. This study evaluates the impact of total laboratory automation system (TLA), electric track vehicle (ETV) system and auto-verification (AV) of results on overall turnaround time (TAT) (phlebotomy to reporting TAT: PR-TAT) within a CL setting. Methods Mean, median and percentage of outlier (OP) for PR-TAT were compared for pre- and post-CL eras using five representative tests based on different request priorities. Comparison studies were also carried out on the intra-laboratory TAT (in-lab to reporting TAT: IR-TAT) and the delivery TAT (phlebotomy to in-lab TAT: PI-TAT) to reflect the efficiency of the TLA (both before and after introducing result AV) and ETV systems respectively. Results Median PR-TATs for the urgent samples were reduced on average by 16% across all representative analytes. Median PR-TATs for the routine samples were curtailed by 51%, 50%, 49%, 34% and 22% for urea, potassium, thyroid stimulating hormone (TSH), complete blood count (CBC) and prothrombin time (PT) respectively. The shorter PR-TAT was attributed to a significant reduction of IR-TAT through the TLA. However, the median PI-TAT was delayed when the ETV was used. Application of various AV rules shortened the median IR-TATs for potassium and urea. However, the OP of PR-TAT for the STAT requests exceeding 60 min were all higher than those from the pre-CL era. Conclusions TLA and auto-verification rules help to efficiently manage substantial volumes of urgent and routine samples. However, the ETV application as it stands shows a negative impact on the PR-TAT.

Background Growing financial and workload pressures on laboratories coupled with user demands for faster turnaround time (TAT) has steered the implementation of total laboratory automation (TLA). The current study evaluates the impact of a complex TLA on core laboratory efficiency through the analysis of the In-lab to Report TAT (IR-TAT) for five representative tests based on the different requested priorities. Methods Mean, median and outlier percentages (OP) for IR-TAT were determined following TLA implementation and where possible, compared to the pre-TLA era. Results The shortest mean IR-TAT via the priority lanes of the TLA was 22 min for Complete Blood Count (CBC), followed by 34 min, 39 min and 40 min for Prothrombin time (PT), urea and potassium testing respectively. The mean IR-TAT for STAT CBC loaded directly on to the analyzers was 5 min shorter than that processed via the TLA. The mean IR-TATs for both STAT potassium and urea via offline centrifugation were comparable to that processed by the TLA. The longest mean IR-TAT via regular lanes of the TLA was 62 min for Thyroid-Stimulating Hormone (TSH) while the shortest was 17 min for CBC. All parameters for IR-TAT for CBC and PT tests decreased significantly post- TLA across all requested priorities in particular the outlier percentage (OP) at 30 and 60 min. Conclusions TLA helps to efficiently manage substantial volumes of samples across all requested priorities. Manual processing for small STAT volumes, at both the initial centrifugation stage and front loading directly on to analyzers, is however likely to yield the shortest IR-TAT.

Background With the recent outbreak in West Africa, hospitals worldwide have been developing protocols for suspect of cases of Ebola virus disease. Patients with Ebola virus disease present with a severe gastroenteritis leading to dehydration and electrolyte abnormalities and as such, routine chemistry analysis is essential for patient management. While point-of-care testing can be used with additional precautions for rapid chemistry analyses in a laboratory setting, significant delays could ensue before specimens arrive to the laboratory. This study evaluated the stability of eight chemistry analytes up to 4 h post-collection. Methods Blood was collected by venipuncture from 20 healthy volunteers and tested at times 0, 30, 60, 90, 120 and 240 h. Approximately 100 µl of blood was dispensed into a CHEM 8+Cartridge and processed on a model 300 i-STAT 1 Analyzer (Abbott Point of Care Inc.) and ANOVA was used to assess statistical significant difference from the initial time point. Results While the manufacturer recommends testing within 30 min of collection, no significant variation was observed for most analytes with time points extending up to 4 h. In contrast, glucose concentrations decreased significantly ( P Conclusions This study provides supporting data suggesting that delays up to 4 h can be tolerated, giving ample time for collection and transport of specimens to the clinical laboratory. For glucose, POC testing could still be used, taking into account the collection time and the average rate of decrease.

Introduction: Recent reports suggest that up to 30% of medical interventions provide no benefit to patients. In a response to ED over-crowding, guidelines commonly exist to guide blood test ordering in patients waiting to see a physician. In many cases, this increases the use of tests without benefiting patients. We describe a quality improvement project designed to reduce the number of laboratory tests considered routine for waiting patients. Methods: A multidisciplinary group reviewed existing symptom-prompted nursing blood test guidelines for serum electrolytes and glucose, renal function tests, liver tests, lipase, toxicological tests and beta Human Chorionic gonadotrophin levels. Order sets were revised with tests eliminated from the routine panels that were not felt to routinely contribute to patient care. The new guidelines were communicated to nursing staff in a series of educational sessions, and the revised guidelines were posted at nursing stations. Physician ordering practice was not changed. A pre-post evaluation compared the period 1 December 2014, - 30 November 2015 with 1 December 2015 - 30 November 2016. Clinical outcomes and patient wait times were not evaluated. Results: The use of tests in these categories decreased 32% between the two periods, at a net saving of $210, 246c. The largest savings came from total protein (73% decrease), Creatine kinase (68%), chloride (64%), glucose (49%), and albumin (47%). Sodium/Potassium testing decreased by only 13%. The only increase in test ordering recorded was AST (3% increase). Conclusion: Simply changing symptom driven order sets resulted in significant savings to the system. In the era of Choosing wisely regular review of lab order sets is indicated. Further study is needed to assess the effect of these changes on patient flow and on clinical outcome.

Although apolipoprotein E (ApoE) polymorphism is associated with variable risks of several illnesses, and with mortality, no persuasive relationship has been demonstrated with frailty. Here, the clinical examination cohort (n= 1452 older adults, aged 70+ years at baseline) of the Canadian Study of Health and Aging was evaluated, with 5-year follow-up data. Frailty was defined using both the phenotypic definition from the Cardiovascular Health Study (Frailty-CHS) and the ‘Frailty Index’, from which age-specific trajectories of deficit accumulation can be estimated. In age-sex adjusted analyses, people with ApoE 4 allele had a higher risk of death (hazard ratio [HR]= 1.20; 95% confidence interval: 1.01–1.45), but this relationship was not significant when adjusted for cognitive impairment (1.06; 95% confidence interval: 0.88–1.27). There was no association between frailty and ApoE polymorphism, defined in age-sex adjusted models either as Frailty-CHS (ApoE4 HR 1.17; 95% confidence interval: 0.98–1.40, frailty HR 1.37; 95% confidence interval: 1.28–1.46) or by the Frailty Index (ApoE4 HR 1.07; 95% confidence interval: 0.90–1.29, frailty HR 35.3; 95% confidence interval: 20.4–61.1). The data do not support an association between ApoE polymorphism and frailty. This result did not depend on how frailty was defined.

Background: Increased total homocysteine (tHcy) may be associated with placental-mediated adverse pregnancy outcomes, but few prospective studies have measured tHcy before pregnancy outcome. This study was undertaken to determine whether increased tHcy measured in early pregnancy is associated with pregnancy loss, gestational hypertension (GH), preeclampsia, or small for gestational age (SGA) infants. Methods: We conducted a prospective cohort study between 2002 and 2005. We measured tHcy and serum folate in blood samples from pregnant women ( Results: Of the 2119 women included in the study, 103 had a pregnancy loss, 115 had gestational hypertension, 65 had preeclampsia, and 129 had an SGA infant. Subjects with increased tHcy concentrations were at increased risk of pregnancy loss [relative risk (RR) 2.1, 95% CI 1.2–3.6] or preeclampsia (RR 2.7, 95% CI 1.4–5.0) than subjects with lower tHcy concentrations, but increased tHcy concentration was not associated with increased risk of developing GH or having an SGA infant. Conclusion: The finding of high tHcy in early pregnancy as a risk factor for pregnancy loss and preeclampsia is consistent with a hypothesis that increased tHcy results in abnormalities of the placental vasculature.

Objectives There is increasing recognition of the importance of appropriate laboratory test utilization. We investigate the effect of a multifaceted educational approach that includes physician feedback on individual test ordering, in conjunction with targeted restriction, on the utilization of selected laboratory tests. Design and methods Scientific evidence was compiled on the usefulness and limitations of tests suspected of being over utilized in our laboratories. A variety of approaches were used to deliver education on each of the targeted tests, with greater focus on primary care physicians (PCPs). Feedback on requesting behavior of these tests was also communicated to the latter group which included an educational component. Laboratory based restriction of testing was also exercised, including the unbundling of our electrolyte panel. Results PCP requesting patterns for the selected tests were found to be markedly skewed. The interventions implemented over the study period resulted in a substantial 51% reduction in overall ordering of five of the targeted tests equating to an annual marginal cost saving of $60,124. Unbundling of the electrolyte panel resulted in marginal cost savings that equated annually to $42,500 on chloride and $48,000 on total CO2. Conclusions A multifaceted educational approach combined with feedback on utilization and laboratory driven gate-keeping significantly reduced the number of laboratory tests suspected of being redundant or unjustifiably requested. Laboratory professionals are well positioned to manage demand on laboratory tests by utilizing evidence base in developing specific test ordering directives and gate-keeping rules.

Diabetes is associated with endothelial dysfunction, which in part may be related to uncoupling of the endothelial nitric oxide (NO) synthase enzyme, thus reducing the availability of NO. As folates may potentially reverse the uncoupling of NO synthase, we wanted to determine whether folic acid supplementation could modulate endothelial function and markers of inflammation in patients with type 2 diabetes without vascular disease. Nineteen patients with type 2 diabetes were treated with folic acid (10 mg/day for 2 weeks) versus placebo in a randomized, placebo-controlled, cross-over study with an 8-week washout period between treatments. Fasting endothelium-dependent flow-mediated dilatation (FMD) of the brachial artery, endothelium-independent nitroglycerin-mediated dilatation (NMD), plasma homocysteine, serum lipids, folate, and inflammatory markers (high-sensitivity C-reactive protein, soluble intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, interleukin-18, tumor necrosis factor-alpha) were assessed after each 2-week treatment period. Folic acid supplementation significantly increased folate levels and lowered plasma homocysteine levels. Folic acid significantly improved FMD compared to placebo (5.8 4.8% vs 3.2 2.7%, p 0.02). There were no significant effects of folic acid supplementation on lipids, NMD, or the inflammatory markers. There was no relationship between the change in homocysteine and the improvement in FMD. Thus, 2 weeks of folic acid supplementation can improve endothelial dysfunction in type 2 diabetics independent of homocysteine-lowering, but does not modulate markers of inflammation.

Objectives: Coronary artery disease (CAD) is often polygenic due to multiple mutations that contribute small effects to susceptibility. Since most prior studies only evaluated the contribution of single candidate genes, we therefore looked at a combination of genes in predicting early-onset CAD [apolipoprotein E ( APOE ) e4, butyrylcholinesterase ( BChE ) K, peroxisome proliferator-activated receptor γ2 (PPARγ2) Pro12Ala and endothelial nitric oxide synthase ( ENOS ) T-786C]. Design and methods: We examined the frequencies, individually and in combination, of all four alleles among patients with early-onset CAD ( n = 150; n = 150; >65 years) and healthy controls ( n = 150, age range 47–93 years). Differences in the proportion of subjects in each group with the given gene combination were assessed and likelihood ratios (LR) were calculated using logistic regression to combine the results of multiple genes. Results: Early-onset CAD patients had increased, but non-significant, frequencies of PPARγ2 Pro12/Pro12 ( P = 0.39) and ENOS T-786C ( P = 0.72), while BChE -K was only significantly higher in early-onset CAD patients compared to controls ( P = 0.03). There were significantly more APOE e4 alleles alone ( P = 0.02) or in combination with BChE -K ( P = 0.02) among early-onset CAD patients compared to late-onset CAD ones or controls. When combined, there was a higher prevalence of all four alleles in early-onset CAD (early-onset CAD patients: 10.7%, late-onset CAD patients: 3.3% and controls: 2.7%, P = 0.01). LR for early-onset CAD for a single allele was relatively small (1.08 for PPARγ2 to 1.70 for APOE e4). This increased to 2.78 (1.44–5.37) when combining all four alleles, therefore increasing the pre-test probability of CAD from 5% to a post-test probability of 12.7%. Conclusions: While any single mutation causes only a mildly increased LR (none > 1.7), in combination, the likelihood of early-onset CAD increased to 2.78 with four mutations. The genetics of early-onset CAD appear to be multifactorial, requiring polygenic models to elucidate risk.

Objectives The aim of this study was to determine whether selective cyclooxygenase-2 (COX-2) inhibition with rofecoxib can modulate endothelial dysfunction and levels of circulating inflammatory markers in patients with established coronary artery disease (CAD). Background Expression of COX-2 is upregulated in atherosclerosis. Thus, it has been hypothesized that COX-2 may contribute to atherogenesis by producing eicosanoids, which mediate vascular inflammation and endothelial dysfunction. Methods In a randomized, double-blind, placebo-controlled, parallel-design trial, we studied the vascular effects of rofecoxib on brachial artery vasoreactivity and inflammatory markers in 60 patients with angiographically proven CAD who were taking concomitant low-dose aspirin. Patients were randomly assigned to receive either rofecoxib (25 mg/day; n = 30) or placebo (n = 30) for eight weeks. Brachial artery endothelium-dependent flow-mediated dilation (FMD), endothelium-independent nitroglycerin-mediated dilation (NMD), and inflammatory markers (i.e., high-sensitivity C-reactive protein [CRP], soluble intercellular adhesion molecule-1 [sICAM-1], and soluble interleukin-6 receptor [sIL-6r]) were measured at baseline and after eight-week follow-up. Results Baseline clinical characteristics were similar in the two groups. After eight weeks of treatment, FMD did not significantly change in either the rofecoxib or placebo group (4.0 ± 3.0% to 4.0 ± 3.8% vs. 2.7 ± 2.7% to 3.1 ± 2.7%, respectively; p = 0.6 by two-way analysis of variance). Similarly, NMD remained unchanged in both groups. Levels of CRP, sICAM-1, and sIL-6r were not significantly altered in either the rofecoxib or placebo group. Conclusions The addition of selective COX-2 inhibition with rofecoxib did not appear to have any favorable or adverse effects on endothelial dysfunction or vascular inflammation in patients with CAD using concomitant low-dose aspirin.

The common K variant of butyrylcholinesterase (BChE-K), an enzyme which metabolizes acetylcholine and organophosphates, has been associated with Alzheimer's disease, especially in the presence of the apolipoprotein E epsilon 4 allele (APOE-epsilon 4). Although APOE-epsilon 4 has been associated with the development of coronary artery disease (CAD), an association between the BChE-K variant and CAD has not been explored. Paraoxonase 1 (PON1), located within HDL, is an enzyme which also metabolizes organophosphates and may be antiatherogenic. The R192 variant of PON1 (PON1-R) has been associated with CAD.To determine whether BChE-K is also associated with premature CAD, we examined the frequency of BChE-K among patients with early-onset CAD (n = 150;50 yr) vs. late-onset CAD (n = 150;65 yr) by molecular analysis. We also examined the frequency of the PON1-R allele in both groups, and explored whether there was synergism between BChE-K and APOE-epsilon 4, BChE-K and PON1-R or PON1-R and APOE-epsilon 4.The frequency of the BChE-K allele tended to be greater among early-onset CAD patients compared to late-onset CAD patients (41.3% vs. 31.3%; p = 0.07), but without any significant difference between males and females. There was no difference in the prevalence of the PON1-R allele between those with early- or late-onset CAD (46.0% vs. 52.7%; p = 0.25). Twenty-two patients with early-onset CAD had both the BChE-K plus APOE-epsilon 4 alleles (14.7%) compared to 11 late-onset CAD patients (7.3%) (p = 0.04). There was no such association between BChE-K and PON1-R, nor PON1-R and APOE-epsilon 4.Our study suggests that there is a minor association between BChE-K and early-onset CAD, especially in the presence of the APOE-epsilon 4 allele.

OBJECTIVESThe goal of this study was to determine whether postprandial hyperglycemia, induced by oral glucose loading, attenuates endothelial function in healthy subjects without diabetes and whether coadministration of vitamins C and E could prevent these postprandial changes.BACKGROUNDEpidemiologic evidence suggests that postprandial hyperglycemia, below diabetic levels, is a risk factor for cardiovascular disease. Postprandial hyperglycemia may promote atherosclerosis through endothelial dysfunction and oxidative stress.METHODSWe evaluated the acute effects of oral glucose loading (75 g), alone and with vitamins C (2 g) and E (800 IU), on endothelium-dependent flow-mediated dilation (FMD) of the brachial artery, in a randomized, double-blind, placebo-controlled, crossover study of 10 healthy volunteers. Changes in the levels of markers of oxidative stress (plasma malondialdehyde and erythrocyte glutathione, glutathione peroxidase and superoxide dismutase) were also assessed.RESULTSIncreases in plasma glucose and insulin after glucose loading were unaffected by vitamin coadministration. With glucose loading alone, FMD fell from 6.5 ± 2.2 at baseline to 5.4 ± 1.7, 3.7 ± 2.1∗, 4.1 ± 3.5∗ and 5.7 ± 1.9% at 1, 2, 3 and 4 h (∗p < 0.05 vs. 0 h). In contrast, FMD did not change significantly after glucose plus vitamins (6.4 ± 1.3, 7.6 ± 1.8, 7.9 ± 2.7, 6.9 ± 2.3, 6.9 ± 1.9% at 0, 1, 2, 3 and 4 h). By two-way repeated measures analysis of variance we found a significant interaction between vitamin treatment and time (p = 0.0003), indicating that vitamins prevented the glucose-induced attenuation of FMD. Oxidative stress markers did not significantly change with glucose loading alone or with vitamins.CONCLUSIONSOral glucose loading causes an acute, transient decrease of FMD in healthy subjects without diabetes, which is prevented by vitamins C and E.

OBJECTIVESThe purpose of this study was to determine whether lowering homocysteine levels with folic acid, with or without antioxidants, will improve endothelial dysfunction in patients with coronary artery disease (CAD).BACKGROUNDElevated plasma homocysteine levels are a risk factor for atherosclerosis. Homocysteine may promote atherogenesis through endothelial dysfunction and oxidative stress.METHODSIn a double-blind, placebo-controlled, randomized trial, we used vascular ultrasound to assess the effect of folic acid alone or with antioxidants on brachial artery endothelium-dependent flow-mediated dilation (FMD). Seventy-five patients with CAD (screening homocysteine level ≥9 μmol/liter) were randomized equally to one of three groups: placebo, folic acid alone or folic acid plus antioxidant vitamins C and E. Patients were treated for four months. Plasma folate, homocysteine, FMD and nitroglycerin-mediated dilation were measured before and after four months of treatment.RESULTSPlasma folate, homocysteine and FMD were unchanged in the placebo group. Compared with placebo, folic acid alone increased plasma folate by 475% (p < 0.001), reduced plasma homocysteine by 11% (p = 0.23) and significantly improved FMD from 3.2 ± 3.6% to 5.2 ± 3.9% (p = 0.04). The improvement in FMD correlated with the reduction in homocysteine (r = 0.5, p = 0.01). Folic acid plus antioxidants increased plasma folate by 438% (p < 0.001), reduced plasma homocysteine by 9% (p = 0.56) and insignificantly improved FMD from 2.6 ± 2.4% to 4.0 ± 3.7% (p = 0.45), as compared with placebo. Nitroglycerin-mediated dilation did not change significantly in any group.CONCLUSIONSFolic acid supplementation significantly improved endothelial dysfunction in patients with coronary atherosclerosis. Further clinical trials are required to determine whether folic acid supplementation may reduce cardiovascular events.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low density lipoprotein (LDL) receptor gene. Currently, diagnosis of heterozygous FH relies on clinical phenotype; however, the use of clinical criteria for the diagnosis of heterozygous FH does not always permit unequivocable diagnosis of the disease. Molecular diagnosis of FH is clinically valuable especially in regions where founder mutations exist or where polygenic hypercholesterolemia is prevalent. In this paper we report the identification of a novel mutation, a cytosine to guanine substitution, at codon 152 in exon 4 of the LDL receptor gene in a Nova Scotian family clinically diagnosed with heterozygous FH. The mutation creates a recognition sequence for the restriction endonuclease BsrI, and can be readily detected by BsrI restriction analysis of a 160 bp amplicon spanning the mutation. This analysis was used to show that the mutation segregated with the disease in this family and is the probable cause of FH in this kindred.

We used heteroduplex analysis to screen for mutations in the porphobilinogen deaminase gene in 21 patients with acute intermittent porphyria (AIP). Unique banding patterns were investigated by direct sequencing of polymerase chain reaction products and, when indicated, sequencing of cloned DNA containing the exon of interest. Two frameshift mutations were found, a 2-bp deletion in exon 5 and a 1-bp insertion in exon 7. Both mutations generate a premature stop codon. Two point mutations, in exons 10 and 14, were also observed. The C--T mutation in exon 10 codes for an Arg173 to Trp substitution, while a G--A mutation in exon 14 changes Trp283 into a premature stop codon. This study extends the spectrum of mutations that cause AIP and demonstrates the utility of heteroduplex analysis as a screening technique.

Familial defective apolipoprotein B-100 (FDB) is a genetic disorder resulting from a mutation in the apolipoprotein B-100 (apo B-100) gene, most frequently at position 3500, in which arginine is substituted for glutamine in the mature protein. This mutation drastically decreases the affinity of the mutant apo B-100 particle for the low-density lipoprotein (LDL) receptor, and hence decreases the clearance of cholesterol from the circulation. Familial hypercholesterolemia (FH), also a disorder of lipid metabolism, results from mutations in the gene for the LDL receptor. Both FDB and heterozygous FH occur at approximately the same frequency (1 in 500) among Caucasians and both produce clinical symptoms and signs that can be indistinguishable. Polymerase chain reaction (PCR) amplification and subsequent restriction analysis have been used to detect the substitution at codon 3500 in the apo B-100 gene using mutagenic PCR primers. At least one proband from 10 unrelated families with a history of hypercholesterolemia was screened by mutagenic PCR for FDB. Only one of 10 patients demonstrated the mutation for FDB. The mutant apo B-100 allele was shown to segregate with other clinically affected family members. These results demonstrate that molecular analysis is essential to distinguish between FDB and heterozygous FH in hypercholesterolemic families.

Context.—Two recent studies have identified a high rate of microsatellite instability (MSI) in pancreatic neuroendocrine tumors (pNETs). Microsatellite instability is rare in small intestinal neuroendocrine tumors (NETs). It is unclear why there is discordance in the frequency of MSI in the 2 studies of pNETs and why this mechanism is comparatively rare in small intestinal tumors. Loss of expression of DNA mismatch repair (MMR) proteins, which is known to correlate strongly with MSI, is not well studied in pancreatic or small intestinal NETs.Objective.—To determine if there is loss of expression of MMR protein expression in pancreatic or small intestinal NETs.Design.—Sixty-nine patients (31 male, 38 female; mean age, 59.2 years) were identified who had a resection for a primary pancreatic (n = 35) or primary small intestinal (n = 34) NET during an 18-year period. Immunohistochemical stains for MLH1, MSH2, MSH6, and PMS2 were applied to archived tissue from all cases. All pNETs with adequate tissue (n = 32) were also assessed by MSI analysis.Results.—There was preserved expression of MLH1, MSH2, MSH6, and PMS2 in all 35 pNETs. Of 32 pNETs tested by polymerase chain reaction, 28 were microsatellite stable and DNA did not amplify in 4. In 34 small intestinal NETs, 2 cases had indeterminate MLH1 and 1 case had indeterminate PMS2 expression. The remainder had intact MMR protein expression.Conclusion.—Defects in DNA MMR proteins are rare in pancreatic and small intestinal NETs, raising doubt that MSI plays a significant role in the pathogenesis of these tumors.

Osteoporosis is the most common cause of fragility fractures. Bone remodelling is essential for repairing damaged areas within bone to preserve bone strength and for assisting in mineral homeostases. In young adults, bone remodelling is usually balanced with approximately as much bone replaced as is removed during each remodelling cycle. However, when remodelling becomes accelerated in combination with an imbalance that favours bone resorption over formation, such as during menopause, precipitous losses in bone mass occur. Bone turnover markers (BTMs) measure the rate of bone remodelling allowing for a dynamic assessment of skeletal status and hold promise in identifying those at highest risk of rapid bone loss and subsequent fracture. Further, the use of BTMs to monitor individuals administered osteoporosis therapy is attractive as monitoring anti-fracture efficacy with bone mineral density has significant limitations. This review details remodelling biology, pre-analytical and analytical sources of variability for BTMs, describes the most commonly used resorption and formation markers, and offers some guidelines for their use and interpretation in the laboratory and the clinic.

CX3CR1 is a monocyte chemokine receptor and adhesion molecule. Two CX3CR1 mutations, V249I and T280M, reportedly decrease coronary artery disease (CAD) risk independent of established risk factors. An I249 protective effect is attributed to reducing CX3CR1 binding to fractalkine, its ligand.We examined the frequencies of V249I and T280M among early-onset CAD patients (G1; n = 149;50 years), late-onset CAD patients (G2; n = 150;65 years) and healthy controls (HC; n = 149, 47-93 years) without known CAD risk factors. We compared plasma total cholesterol (TC)/high density lipoprotein-C (HDL-C) and apolipoprotein B (APOB)/apolipoprotein AI (APOAI) ratios among the groups and mutation carriers and non-carriers, and the prevalence of the mutations in G1 and G2 patients with multiple coronary vessel disease (MVD) and myocardial infarction (MI).G1 patients had non-significantly lower frequencies of I249 versus (vs.) G2 or controls (G1; 51 %, G2: 61 %, controls: 58 %, p = 0.19), with no difference in T280M (p = 0.8). TC/HDL-C and APOB/APOAI ratios were significantly higher in G1 patients vs. G2 and controls (p0.0001) independently of either mutation. More G2 patients had MVD than younger ones (p0.0001); however, more G1 patients were homozygous for V249 compared to G2 patients, who more often had the I249 allele (p0.02). There was no such association with T280M (p = 0.38). Although more G1 patients had MI, this was not mutation related.There were significantly higher lipid ratios in G1 compared to G2 and HC (G1G2HC), but not in mutation prevalence. I249 mutation was associated with MVD in older patients, while V249 homozygosity was associated with the early-onset CAD. Neither allele affected MI or lipid levels.

Urothelial carcinomas have a synchronous or metachronous multifocal pattern of occurrence, questioning their clonal origin. Genetic alterations such as microsatellite instability (MSI) affect various tumors including urothelial cancers. These alterations can affect repeat sequences and cause mutations in coding regions of genes involved in transformation, tumor suppression and apoptosis. Recently, the eight-guanine (G8) and the seven-guanine (G7) repeat sequences of the BAX and AXIN2 genes respectively, were shown altered in different cancers. Since BAX is involved in apoptosis while the AXIN2 is involved in beta-catenin metabolism, a protein involved in cell adhesion and DNA transcription, and due to the multifocal nature of urothelial cancer, we investigated these two genes for alterations in repeat sequences in patients with this cancer.The eight microsatellites BAT25, BAT26, D2S123, D3S1029, D5S346, D17S588, D17S261, MYCL1 were used to screen 25 tumors from seven patients with eight upper and 17 lower urinary tract carcinomas and compare them to DNA from normal tissue. Regions spanning the G8 and G7 repeat sequences of BAX and AXIN2 were sequenced for mutations including expansion and deletion abnormalities.Six microsatellites were seen altered in one patient with kidney and bladder cancer affecting both tissues when compared to normal DNA albeit not similarly except for MYCL1. There was no change in the BAX G8 or AXIN2 G7 microsatellites. There was no MSI seen in any of the remaining six patients.MSI occurs in urothelial cancer, but was not seen to affect the BAX G8 or AXIN2 G7 repeats in this study. However, to determine if MSI affects these genes in these tumors will require a larger study. Moreover, our results suggest that these tumors may have a monoclonal origin with further genetic changes resulting in oligoclonality, or could suggest a similar initiating event leading to a similar initial genetic alteration at different sites with subsequent varying events due to a genetically unstable malignant phenotype.

Background The Glu298Asp polymorphism of endothelial nitric oxide synthase (eNOS) gene has been associated with coronary artery disease (CAD) in some but not all studies. To determine the impact of the mutant Asp298 eNOS allele on the development of premature CAD, we examined the prevalence of this mutation in patients with early-onset CAD compared with those manifesting CAD later in life. If this mutation confers an increased risk of premature CAD, we hypothesized that the frequency of the homozygous mutation (Asp298Asp298) would be greater among the younger patient group. Methods A total of 299 patients with a history of myocardial infarction (MI) or angina pectoris plus angiographically documented CAD were studied. Patients were divided into 2 groups: group 1 (149 patients) included patients with CAD before the age of 50 years and group 2 (150 patients) included patients with a first presentation of CAD at >65 years old. Prevalence of eNOS Glu298 and Asp298 alleles was assessed by molecular analysis and compared for the 2 groups. Results There was no significant difference in the frequency of the mutant Asp298 allele between the 2 groups (G1: 42% vs G2: 42.7%, P =.79). The frequencies of the Glu298Glu298, Glu298Asp298, and Asp298Asp298 genotypes were similar in both groups (34.9%, 46.3%, and 18.8% for G1 and 29.3%, 56%, and 14.7% for G2, respectively, P =.29). Conclusions Our study does not support the conclusion that the eNOS Asp298 allele contributes to the development of premature CAD. (Am Heart J 2001;142:586-9.)

OBJECTIVES: Microsatellite instability has been found in a variety of tumors including prostate cancer. Bax, a pro-apoptotic protein from the Bcl-2 family of proteins, has a microsatellite composed of an eight deoxyguanine [(G)8] tract located in exon 3. Prostate carcinoma cells have increased proliferation indices and lower levels of apoptosis when compared to benign tissue. We investigated whether instability in the Bax (G)8 microsatellite contributes to loss of apoptotic control in localized prostate cancer. PATIENTS AND METHODS: Thirty-eight patients undergoing radical prostatectomy for localized prostate carcinoma participated in this study. Prostate carcinoma was microdissected, and polymerase chain reaction amplification of a region containing the (G)8 microsatellite was performed on DNA from peripheral blood leukocytes and tumors, followed by single strand conformational polymorphism (SSCP) analysis and direct DNA sequencing. RESULTS: SSCP analysis showed no alteration in the number of bands detected upon comparison of tumor tissue to leukocytes, suggesting no alterations in the microsatellite. This was confirmed by direct sequencing, which demonstrated a normal (G)8 sequence in each case. CONCLUSION: We conclude that the Bax (G)8 microsatellite is stable in localized stage T2 and T3 prostate cancer. Our findings argue against a mutator phenotype pathway leading to loss of apoptotic control in localized prostate cancer.

Objective: Apolipoprotein E (APOE) E4, apolipoprotein B-100 (APOB) Q3611 allele, the angiotensin converting enzyme (ACE) deletion (D) allele and glycoprotein IIIa (GP3A) P33 mutant allele are reported to predispose to early-onset coronary heart disease (CHD). These associations were not all confirmed in more recent studies. To determine the impact of these alleles on CHD, we examined the prevalence of these mutations in patients presenting with early-onset CHD and compared them to those manifesting CHD later in life. The delayed-onset was considered a sign of longevity and would serve as a comparative group to assess prevalence of the biochemical and genetic risk factors. Methods: 300 patients with a history of myocardial infarction or angina pectoris and angiographically documented CHD were studied. Patients were divided into two groups: group 1 (G1 = 150 patients) presenting with these findings under the age of 50 years; while group 2 (G2 = 150 patients) were patients presenting for the first time over the age of 65 years. Prevalence of the alleles of APOE, APOB, ACE and GP3A was assessed by molecular analysis. An association of any of these genotypes with early onset CHD could lead to a higher prevalence in the younger age group. Results and Conclusions: None of the suspected alleles namely APOB Q3611 [G1: 10.7% vs. G2: 9.0%, p = 0.57], ACE D (G1: 52.0% vs. G2: 49.7%, p = 0.57), or the GP3A P33 (G1: 17.3% vs. G2: 15.7%; p = 0.58) showed any significant difference between the two groups. Subjects with APOE E4 were more frequent in the younger age group (G1: 18.3% vs. G2: 13.7%; p = 0.047), while APOE E2 was more frequent in G2 (G2: 10.0% vs. G1: 2.7%; p = 0.0002). Multivariate analysis showed an odds ratio of APOE E2 allele in G1 of 0.27 with a confidence interval of 0.10–0.73.

Objective: In the presence of low serum folate, mutant 5,10-methylenetetrahydrofolate reductase (MTHFR+ [A223V/C677T]) in the homozygous state (+/+), may predispose to higher plasma homocysteine (tHct) levels and coronary artery disease (CAD). To determine the impact of this relationship on predisposition to early-onset CAD, we examined the prevalence of the mutation and plasma tHct in patients with early-onset CAD and compared them to patients manifesting CAD later in life. Methods: Three hundred patients with history of acute myocardial infarction or angina pectoris and angiographically documented CAD were studied. Patients consisted of two groups: group 1 (G1 = 150 patients) presenting with these findings under age 50; while group 2 (G2 = 150) presented for the first time over age 65 years. Prevalence of the MTHFR+ mutation was assessed by molecular analysis, and plasma tHct and folate were measured. An association of the +/+ genotype with early onset CAD could lead to its higher prevalence in the younger age group. Results: There was no significant difference in the frequency of the (+/+) genotype between the two groups (G1: 11.3% vs. G2: 11.3%). However, patients with the (+/+) genotype in both groups had higher tHct when plasma folate was below the mean value (G1: p < 0.0001 while G2: p < 0.01). Conclusion: The mutant MTHFR genotype was not found to be a determining factor in early-onset CAD. Higher tHct values were obtained in the older age group, which is expected because other studies have shown that tHct levels increase with age. A significant relation was shown between MTHFR genotype and low folate status yielding high tHct levels in those with the (+/+) genotype. As this relation was seen in both groups, although to a lesser extent in the older G2, it does not explain the underlying cause of early-onset CAD.

Objectives: Acute intermittent porphyria (AIP) is caused by mutations in the porphobilinogen deaminase (PBGD) gene that disrupt the function of the enzyme. Many mutations that lead to decreased PBGD activity have been described. An Arg to Trp substitution at codon 173 (CGG→TGG in exon 10) and designated R173W, which leads to a CRIM-negative phenotype, has been reported in Swedish, Finnish, Scottish, and South African kindreds, and in a Nova Scotian proband with fatal AIP. In this work, we investigated the presence of this mutation in a Nova Scotian patient population presenting with AIP. Design and Methods: Single-strand conformation polymorphism analysis and DNA sequencing by TA cloning and Sanger’s dideoxy chain termination method, were used to confirm the maternal transmission of this mutation to the proband. The mutation also eliminates an NciI (also MspI) endonuclease restriction site, which allows for detection of the mutant allele by polymerase chain reaction amplification and restriction enzyme digestion. Results: The family of the Nova Scotian proband and four other AIP kindreds showed the presence of the same mutation. These five families are descendants of German, Swiss, and French immigrants historically known as the “Foreign Protestants,” who were recruited to Nova Scotia in the 1750s. Conclusion: In all these families, descent from one couple that settled in Nova Scotia in 1751 has been identified by genealogy research, consistent with a founder effect within this population. This is the first identified mutation in PBGD causing AIP that has been linked to a founder effect in descendants of an immigrant population to North America, and which could be traced to such a distant background, similar to the South African variegate porphyria mutation.

The rat mesenteric vascular bed releases prostaglandins when perfused in vitro. The present study evaluated the effect of perfusion of the rat mesenteric vascular bed in vitro with a buffer containing 0, 3, 6, or 9 nM of added zinc on the release of essential fatty acids over a 150-min period. Long chain fatty acids in the mesenteric lipids and in total lipid of the perfusion effluent were assayed by gas liquid chromatography. The presence of 6 nM zinc in the perfusing buffer almost completely prevented the change in 16–22 carbon long chain fatty acids in the mesenteric phospholipids and decreased the release of free fatty acids in comparison to that occurring in the absence of additional zinc. The results sugest that physiological amounts of zinc in the perfusion medium reduce the release of essential fatty acids from rat mesenteric lipids.Key words: zinc, phospholipid, linoleic acid, arachidonic acid, prostaglandin.

We investigated the effects of phenelzine and tranylcypromine on the release of prostacyclin, thromboxane A2, prostaglandin E2, and prostaglandin E1 from the isolated perfused rat mesenteric vascular bed. Perfusion of the preparation with phenelzine in concentrations of 15, 45, and 135 μM for 150 min led to attenuated release of all four prostaglandins measured. Inhibition generally occurred with the lowest dose used and was most prominent with the highest concentration. Tranylcypromine also decreased prostaglandin formation. However, low doses were not effective in the suppression of prostacyclin release. Both drugs had an inhibitory effect on production of prostaglandin E1, which is a metabolite of dihomo-γ-linolenic acid, the precursor of arachidonic acid, but this was only shown to be significant with phenelzine. In this work we demonstrate that phenelzine and tranylcypromine have an inhibitory effect on the production of 2-series prostaglandins derived from arachidonic acid, and possibly a similar effect on prostaglandins of the 1-series derived from dihomo-γ-linolenic acid.

The rat mesenteric vascular bed when perfused in vitro has been shown to release prostaglandins. The vasconstrictor effects of nerepinephrine in this preparation are sensitive to the presence of exogenous prostaglandins as well as to the presence of lnM-luM zinc (1). The effects of zinc appear to be related in part to modulating release of endogenous prostaglandins or their precursor fatty acids (1). In view of the known inhibitory effect of zinc on triglyceride lipase (2) and phospholipase A2 (3), we decided to investigate the lipid composition and metabolism of long chain fatty acids in the rat mesenteric vascular bed when perfused in vitro with physiological concentrations of zinc.