Baseline plasma concentrations of troponin T were measured with a high-sensitivity assay (hs-cTnT) (Roche Elecsys) in a cohort of 1050 CHD patients from 30 to 70 years of age. The prognostic value of hs-cTnT on a combined cardiovascular disease (CVD) end point after adjustment for covariates was determined with Cox proportional hazards modeling.

The median hs-cTnT concentration was 10.9 ng/L (interquartile range, 5.1–18.9 ng/L). Increased hs-cTnT concentrations were associated with an older age, history of hypertension and diabetes, more advanced coronary artery disease, and other CHD risk factors. Furthermore, hs-cTnT concentration was strongly correlated with N-terminal pro–B-type natriuretic peptide (NT-proBNP) and cystatin C ( = 0.61, and = 0.32, respectively; both P values <0.0001). During a median follow-up of 8.1 years, 150 patients (14.3%) experienced a secondary CVD event. In a multivariate model, hs-cTnT was associated with a hazard ratio (HR) for secondary events of 2.83 (95% CI, 1.68–4.79) when the extreme quartiles were compared. Further adjustment for cystatin C, NT-proBNP, and C-reactive protein attenuated this association only slightly (HR, 2.27; 95% CI, 1.31–3.95); P for trend < 0.002). ROC curve analysis of a clinical model that added hs-cTnT to a baseline model showed nonsignificant improvement in the area under the curve (0.69 vs 0.67), whereas the net reclassification improvement was 17.2% (P = 0.029).

Slightly increased hs-cTnT concentrations in stable CHD patients are associated with several cardiovascular disorders and predict long-term CVD events.

Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS.

We separated and quantified acarbose, M₄, and Glc₄ using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc₄ was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 μmol/L Glc₄), and 10.5% and 16.2% (200 μmol/L Glc₄). Glc₄ in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M₄ was present in 5% of urine samples. Mean Glc₄ concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001–0.005). The diagnostic sensitivity of Glc₄ for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc₄ was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients.

The UPLC-MS/MS assay of Glc₄ in urine was discriminative between Glc₄ and M₄ and confirmed the diagnosis in >98% of GSD-II cases.

We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR are described. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6–9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA.

A single thermocycling program with a denaturation-temperature window of 2.5–3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T_ms. Mutation enrichments of 6–9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR.

Low-level mutations in diverse amplicons with different T_ms can be mutation enriched via TT-COLD-PCR provided that their T_ms fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.

We sequenced ccf DNA isolated from maternal plasma samples obtained from 2 patients with confirmed 22q11.2 deletion syndrome and from 14 women at low risk for fetal chromosomal abnormalities. The latter samples were used as controls, and the mean genomic coverage was 3.83-fold. Data were aligned to the human genome, repetitive regions were removed, the remaining data were normalized for GC content, and z scores were calculated for the affected region.

The median fetal DNA contribution for all samples was 18%, with the affected samples containing 17%–18% fetal DNA. Using a technique similar to that used for sequencing-based fetal aneuploidy detection from maternal plasma, we detected a statistically significant loss of representation of a portion of chromosome 22q11.2 in both of the affected fetal samples. No such loss was detected in any of the control samples.

Noninvasive prenatal diagnosis of subchromosomal fetal genomic anomalies is feasible with next-generation sequencing.

We designed a microscale flow device with a functionalized surface of E-selectin and antibody molecules against epithelial markers. The device was additionally enhanced with a halloysite nanotube coating. We created model samples in which a known number of labeled cancer cells were suspended in healthy whole blood to determine device capture efficiency. We then isolated and cultured primary CTCs from buffy coat samples of patients diagnosed with metastatic cancer.

Approximately 50% of CTCs were captured from model samples. Samples from 12 metastatic cancer patients and 8 healthy participants were processed in nanotube-coated or smooth devices to isolate CTCs. We isolated 20–704 viable CTCs per 3.75-mL sample, achieving purities of 18%–80% CTCs. The nanotube-coated surface significantly improved capture purities (P = 0.0004). Experiments suggested that this increase in purity was due to suppression of leukocyte spreading.

The device successfully isolates viable CTCs from both blood and buffy coat samples. The approximately 50% capture rate with purities >50% with the nanotube coating demonstrates the functionality of this device in a clinical setting and opens the door for personalized cancer therapies.

We examined Hb A_1c requests (519 664 requests from 115 730 patients, January 2001 to March 2011) processed by the Clinical Biochemistry Department, University Hospital of North Staffordshire, and prevalence of requesting outside guidance from intervals between requests was calculated. Requests were classified as "appropriate," "too soon," or "too late." We also assessed the effect of demographic factors and publication of guidance, along with between-practice variability, on prevalence.

Only 49% of requests conformed to guidance; 21% were too soon and 30% were too late. Underrequesting was more common in primary care, in female patients, in younger patients, and in patients with generally poorer control (all P < 0.001); the reverse generally was true for overrequesting. Publication of guidance (e.g., American Diabetes Association, UK National Institute for Health and Clinical Excellence) had no significant impact on under- or overrequesting rates. Prevalence of inappropriate requests varied approximately 6-fold between general practices.

Although overrequesting was common, underrequesting was more prevalent, potentially affecting longer-term health outcomes. National guidance appears to be an ineffective approach to changing request behavior, supporting the need for a multisystem approach to reducing variability.

Only immunoassays are useful for measurement of gastrin in plasma. The original assays were RIAs developed in research laboratories that used antibodies directed against the C terminus of gastrin peptides. Because the C-terminal tetrapeptide amide sequence constitutes the active site of gastrin peptides, these assays were well suited for gastrinoma diagnosis. More recently, however, most clinical chemistry laboratories have switched to commercial kits. Because of recent cases of kit-measured normogastrinemia in patients with ZES symptoms, the diagnostic sensitivity and analytical specificity of the available kits have been examined. The results show that gastrin kits frequently measure falsely low concentrations because they measure only a single gastrin form. Falsely high concentrations were also encountered, owing to overreactivity with O-sulfated gastrins or plasma proteins. Thus, more than half of the gastrin kits on the market are unsuited for diagnostics.

Gastrinomas are neuroendocrine tumors, some of which become malignant. A delay in diagnosis leads to fulminant ZES, with major, even lethal, complications. Consequently, it is necessary that the diagnostic sensitivity of gastrin kits be adequate. This diagnostic sensitivity requires antibodies that bind the C-terminal epitope of bioactive gastrins without the influence of O-sulfation.

Our method uses a mutated fatty acid binding protein labeled with the fluorescent molecule acrylodan (BL22P1B11), whose fluorescence is quenched upon binding bilirubin. Another configuration (BL22P1B11-Rh) was developed that uses BL22P1B11 together with the fluorophore rhodamine B, which responds by a change in the ratio of its fluorescence.

The "B_f probes" were calibrated with aqueous solutions of bilirubin and yielded similar bilirubin dissociation constants [K_d = 16 (1.5) nmol/L]. We used the probes to determine B_f concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent B_f values in both systems, and the B_f probe results were in agreement with the peroxidase assay. B_f measurements revealed that bilirubin–HSA binding was well described by 2 sites with K_d values of 15.4 (1) nmol/L and 748 (14) nmol/L. We measured B_f concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%.

The BL22P1B11-Rh probe provides accurate plasma sample B_f concentrations with a single measurement, in 1 min with either a handheld B_f meter or a laboratory fluorometer.

We computed power functions mathematically and by computer simulation for 4 different 1:2s repeat-sampling strategies, as well as the 1:2s rule, the 1:3s rule, and 2 common QC multirules.

The false-rejection rates for the repeat-sampling strategies were similarly low to those of the 1:3s QC rule. The error detection rates for the repeat-sampling strategies approached those of the 1:2s QC rule for moderate to large out-of-control error conditions. In most cases, the power of the repeat-sampling strategies was superior to the power of the QC multirules we evaluated. The increase in QC utilization rate ranged from 4% to 13% for the repeat-sampling strategies investigated.

The repeat-sampling strategies provide an effective tactic to take advantage of the desirable properties of both the 1:2s and 1:3s QC rules. Additionally, the power of the repeat-sampling strategies compares favorably with the power of 2 common QC multirules. These improvements come with a modest increase in the average number of controls tested.

Only immunoassays are useful for measurement of gastrin in plasma. The original assays were RIAs developed in research laboratories that used antibodies directed against the C terminus of gastrin peptides. Because the C-terminal tetrapeptide amide sequence constitutes the active site of gastrin peptides, these assays were well suited for gastrinoma diagnosis. More recently, however, most clinical chemistry laboratories have switched to commercial kits. Because of recent cases of kit-measured normogastrinemia in patients with ZES symptoms, the diagnostic sensitivity and analytical specificity of the available kits have been examined. The results show that gastrin kits frequently measure falsely low concentrations because they measure only a single gastrin form. Falsely high concentrations were also encountered, owing to overreactivity with O-sulfated gastrins or plasma proteins. Thus, more than half of the gastrin kits on the market are unsuited for diagnostics.

Gastrinomas are neuroendocrine tumors, some of which become malignant. A delay in diagnosis leads to fulminant ZES, with major, even lethal, complications. Consequently, it is necessary that the diagnostic sensitivity of gastrin kits be adequate. This diagnostic sensitivity requires antibodies that bind the C-terminal epitope of bioactive gastrins without the influence of O-sulfation.

We enrolled 53 patients with benign colon diseases (e.g., diverticulosis, benign polyps, Crohn disease, ulcerative rectocolitis, colonic endometriosis) and analyzed their peripheral blood with 2 previously validated CTC assays: the epithelial immunospot (EPISPOT) assay and the CellSearch system. The EPISPOT assay detects only viable, CK19-releasing CTCs that were enriched by depletion of CD45⁺ leukocytes, whereas the CellSearch system detects CK-positive CTCs after positive EpCAM-based immunomagnetic enrichment.

In patients with benign colon diseases, positive events that met the criteria for "tumor cells" were detected with both the CellSearch system (11.3%) and the CK19-EPISPOT assay (18.9%), whereas no positive events were detected in samples from healthy volunteers. Positive events were detected most frequently in patients with diverticulosis and Crohn disease. All positive events lacked expression of CD45, a common leukocyte antigen.

These results indicate that patients with benign inflammatory colon diseases in particular can harbor viable circulating epithelial cells that are detectable with current CTC assays. This finding points to the need for further molecular characterization of circulating epithelial cells and has important implications for the use of CTC testing.

Several studies have demonstrated a strong association between altered circulating angiogenic factors and preeclampsia. These factors include circulating antiangiogenic proteins such as soluble fms-like tyrosine kinase 1 and soluble endoglin and proangiogenic protein such as placental growth factor. Abnormalities in these circulating angiogenic factors are not only present during clinical disease, but also antedate clinical signs and symptoms by several weeks. These alterations are particularly prominent in patients who present with preeclamptic signs and symptoms prematurely and/or in patients with severe preeclampsia. The availability of automated platforms for the rapid measurement of circulating angiogenic proteins in blood samples has now allowed researchers and clinicians to evaluate the utility of these assays in the diagnosis of the disease, in the stratification of patients in clinical trials, or in the monitoring of therapies. In this review we highlight the various studies that have been performed, with a focus on large validation studies.

Measurement of circulating angiogenic proteins for the diagnosis and prediction of preeclampsia is still at an early stage but is rapidly evolving. Standardization across the various automated platforms and prospective studies that demonstrate clinical utility are needed.

Several studies have demonstrated a strong association between altered circulating angiogenic factors and preeclampsia. These factors include circulating antiangiogenic proteins such as soluble fms-like tyrosine kinase 1 and soluble endoglin and proangiogenic protein such as placental growth factor. Abnormalities in these circulating angiogenic factors are not only present during clinical disease, but also antedate clinical signs and symptoms by several weeks. These alterations are particularly prominent in patients who present with preeclamptic signs and symptoms prematurely and/or in patients with severe preeclampsia. The availability of automated platforms for the rapid measurement of circulating angiogenic proteins in blood samples has now allowed researchers and clinicians to evaluate the utility of these assays in the diagnosis of the disease, in the stratification of patients in clinical trials, or in the monitoring of therapies. In this review we highlight the various studies that have been performed, with a focus on large validation studies.

Measurement of circulating angiogenic proteins for the diagnosis and prediction of preeclampsia is still at an early stage but is rapidly evolving. Standardization across the various automated platforms and prospective studies that demonstrate clinical utility are needed.

We designed a microscale flow device with a functionalized surface of E-selectin and antibody molecules against epithelial markers. The device was additionally enhanced with a halloysite nanotube coating. We created model samples in which a known number of labeled cancer cells were suspended in healthy whole blood to determine device capture efficiency. We then isolated and cultured primary CTCs from buffy coat samples of patients diagnosed with metastatic cancer.

Approximately 50% of CTCs were captured from model samples. Samples from 12 metastatic cancer patients and 8 healthy participants were processed in nanotube-coated or smooth devices to isolate CTCs. We isolated 20–704 viable CTCs per 3.75-mL sample, achieving purities of 18%–80% CTCs. The nanotube-coated surface significantly improved capture purities (P = 0.0004). Experiments suggested that this increase in purity was due to suppression of leukocyte spreading.

The device successfully isolates viable CTCs from both blood and buffy coat samples. The approximately 50% capture rate with purities >50% with the nanotube coating demonstrates the functionality of this device in a clinical setting and opens the door for personalized cancer therapies.

Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer.

Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers.

The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

Lp-PLA₂ mass concentration and activity were evaluated at baseline and after treatment in the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial comparing rosuvastatin 20 mg to placebo among 17 802 men and women without cardiovascular disease or diabetes at study entry. The relationships of Lp-PLA₂ mass and activity with risk of future vascular events were evaluated in the placebo and rosuvastatin groups.

Before randomization, levels of Lp-PLA₂ mass and activity correlated moderately with each other and with LDL-C. The magnitude of these correlations increased after statin therapy. Rosuvastatin reduced Lp-PLA₂ mass by 33.8%, Lp-PLA₂ activity by 33.2%, and LDL-C by 48.7% (all P < 0.0001). Among those study participants allocated to placebo, increasing quartiles of Lp-PLA₂ activity (P_trend = 0.04) but not Lp-PLA₂ mass (P_trend = 0.92) were associated with incident cardiovascular events after adjustment for LDL-C and conventional risk factors. Comparable analyses conducted among those allocated to rosuvastatin revealed no significant relationship between Lp-PLA₂ levels and subsequent vascular events. The ability of rosuvastatin to reduce vascular events was not significantly modified by baseline Lp-PLA₂ level.

Among JUPITER trial participants allocated to placebo, levels of Lp-PLA₂ activity, but not mass, were associated with cardiovascular risk. However, Lp-PLA₂ no longer predicted risk or modified clinical outcomes when participants were treated with rosuvastatin.

Healthy children and adolescents (n = 2188, newborn to 18 years of age) were recruited from a multiethnic population with informed parental consent and were assessed from completed questionnaires and according to defined exclusion criteria. Whole-blood samples were collected for establishing age- and sex-stratified reference intervals for 40 serum biochemical markers (serum chemistry, enzymes, lipids, proteins) on the Abbott ARCHITECT c8000 analyzer.

Reference intervals were generated according to CLSI C28-A3 statistical guidelines. Caucasians, East Asians, and South Asian participants were evaluated with respect to the influence of ethnicity, and statistically significant differences were observed for 7 specific biomarkers.

The establishment of a new comprehensive database of pediatric reference intervals is part of the Canadian Laboratory Initiative in Pediatric Reference Intervals (CALIPER). It should assist laboratorians and pediatricians in interpreting test results more accurately and thereby lead to improved diagnosis of childhood diseases and reduced patient risk. The database will also be of global benefit once reference intervals are validated in transference studies with other analytical platforms and local populations, as recommended by the CLSI.

Our method uses a mutated fatty acid binding protein labeled with the fluorescent molecule acrylodan (BL22P1B11), whose fluorescence is quenched upon binding bilirubin. Another configuration (BL22P1B11-Rh) was developed that uses BL22P1B11 together with the fluorophore rhodamine B, which responds by a change in the ratio of its fluorescence.

The "B_f probes" were calibrated with aqueous solutions of bilirubin and yielded similar bilirubin dissociation constants [K_d = 16 (1.5) nmol/L]. We used the probes to determine B_f concentrations in equilibrium with human serum albumin (HSA) and in human plasma samples supplemented with bilirubin. We obtained equivalent B_f values in both systems, and the B_f probe results were in agreement with the peroxidase assay. B_f measurements revealed that bilirubin–HSA binding was well described by 2 sites with K_d values of 15.4 (1) nmol/L and 748 (14) nmol/L. We measured B_f concentrations in the range expected in jaundiced neonates with a mean CV of approximately 3%.

The BL22P1B11-Rh probe provides accurate plasma sample B_f concentrations with a single measurement, in 1 min with either a handheld B_f meter or a laboratory fluorometer.

We computed power functions mathematically and by computer simulation for 4 different 1:2s repeat-sampling strategies, as well as the 1:2s rule, the 1:3s rule, and 2 common QC multirules.

The false-rejection rates for the repeat-sampling strategies were similarly low to those of the 1:3s QC rule. The error detection rates for the repeat-sampling strategies approached those of the 1:2s QC rule for moderate to large out-of-control error conditions. In most cases, the power of the repeat-sampling strategies was superior to the power of the QC multirules we evaluated. The increase in QC utilization rate ranged from 4% to 13% for the repeat-sampling strategies investigated.

The repeat-sampling strategies provide an effective tactic to take advantage of the desirable properties of both the 1:2s and 1:3s QC rules. Additionally, the power of the repeat-sampling strategies compares favorably with the power of 2 common QC multirules. These improvements come with a modest increase in the average number of controls tested.

Lp-PLA₂ mass concentration and activity were evaluated at baseline and after treatment in the Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin (JUPITER) trial comparing rosuvastatin 20 mg to placebo among 17 802 men and women without cardiovascular disease or diabetes at study entry. The relationships of Lp-PLA₂ mass and activity with risk of future vascular events were evaluated in the placebo and rosuvastatin groups.

Before randomization, levels of Lp-PLA₂ mass and activity correlated moderately with each other and with LDL-C. The magnitude of these correlations increased after statin therapy. Rosuvastatin reduced Lp-PLA₂ mass by 33.8%, Lp-PLA₂ activity by 33.2%, and LDL-C by 48.7% (all P < 0.0001). Among those study participants allocated to placebo, increasing quartiles of Lp-PLA₂ activity (P_trend = 0.04) but not Lp-PLA₂ mass (P_trend = 0.92) were associated with incident cardiovascular events after adjustment for LDL-C and conventional risk factors. Comparable analyses conducted among those allocated to rosuvastatin revealed no significant relationship between Lp-PLA₂ levels and subsequent vascular events. The ability of rosuvastatin to reduce vascular events was not significantly modified by baseline Lp-PLA₂ level.

Among JUPITER trial participants allocated to placebo, levels of Lp-PLA₂ activity, but not mass, were associated with cardiovascular risk. However, Lp-PLA₂ no longer predicted risk or modified clinical outcomes when participants were treated with rosuvastatin.

Among 2457 community-living individuals without cardiovascular disease (CVD), we measured serum fetuin-A concentrations by ELISA and evaluated the cross-sectional association of fetuin-A with CAC prevalence (any vs none) and severity; on follow-up 3.2 years (median) later, we evaluated the association of fetuin-A with CAC incidence and progression.

The mean age was 62 (SD 10) years, and the mean fetuin-A concentration was 0.48 (0.10) g/L. At baseline, 1200 individuals (49%) had CAC, and 272 individuals developed CAC during follow-up. At baseline, there was a threshold effect at the lowest fetuin-A quartile with CAC prevalence. In models adjusted for demographics, traditional cardiovascular disease (CVD) risk factors and kidney function, the lowest fetuin-A quartile had 7% (95% CI 1%–13%; P = 0.04) greater CAC prevalence compared with quartiles 2–4. Similar associations were observed with CAC severity at baseline, but the association was more linear. Each SD (0.10 g/L) lower fetuin-A was associated with a 12% (95% CI 3%–21%; P = 0.01) greater CAC severity in adjusted models. There was no significant association of fetuin-A with CAC incidence or progression.

Fetuin-A is inversely associated with CAC severity among community-living individuals without CVD. Whether fetuin-A concentrations are associated with incident CVD event in the general population requires future study.

This study was a retrospective case–control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping and received a diagnosis of viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were measured by immunoassays. The serum concentrations of miRNAs miR-323-3p, miR-517a, miR-519d, and miR-525-3p were measured with TaqMan real-time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers, both as single markers and as multimarker panels for EP.

Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA cases than in VIP cases (P < 0.01). In contrast, the concentration of miR-323-3p was significantly increased in EP cases, compared with SA and VIP cases (P < 0.01). As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed specificity of 90%). In comparison, the combined panel of hCG, progesterone, and miR-323-3p yielded the highest sensitivity (77.8%, at a fixed specificity of 90%). A stepwise analysis that used hCG first, added progesterone, and then added miR-323-3p yielded a 96.3% sensitivity and a 72.6% specificity.

Pregnancy-associated miRNAs, especially miR-323-3p, added substantial diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.

We investigated 643 consecutive ED patients with acute chest pain who had been discharged for outpatient management after acute myocardial infarction (AMI) had been ruled out by serial measurements of conventional cardiac troponin. hs-cTnT was measured blindly, and we calculated the rates of all-cause mortality (primary endpoint) and subsequent AMI (secondary endpoint) at 30, 90, and 360 days.

hs-cTnT concentrations were increased (>14 ng/L) in 114 patients (18%) but <30 ng/L in 95% of these patients. Of those 114 patients, 96 (84%) had an adjudicated noncoronary cause of chest pain. Thirty-day mortality (95% CI) was 0.9% (0.1%–6.1%), 90-day mortality was 2.7% (0.9%–8.1%), and 360-day mortality was 5.2% (2.2%–11.9%) in patients with increased hs-cTnT; respective rates (95% CI) of AMI were 0.0%, 1.9% (0.5%–7.2%), and 7.6% (3.7%–15.3%). Increased hs-cTnT was associated with increased mortality and AMI at 90 days (P = 0.006 and P = 0.081, respectively) and 360 days (P = 0.001 for both).

hs-cTnT is a strong prognosticator of intermediate and long-term mortality and AMI in low-risk patients discharged from the ED after AMI has been ruled out. The relatively low rate of 30-day events may suggest that patients without acute coronary syndrome and small increases in cardiac troponin are in need of further investigations and treatments, but not necessarily immediate hospitalization.

In a nested case-control study within MHS, 7 biomarkers were assayed in serum samples from 211 patients identified after 8–15 years of follow-up who died of cardiovascular causes (cardiovascular heart disease, stroke, congestive heart failure) and 253 controls matched on age, sex, and study year. Logistic regression analysis, adjusted for age, race, sex, education, study year, smoking, abdominal obesity, diabetes, serum total cholesterol, systolic blood pressure, previous hospitalization for a CVD event, and other significant biomarkers, was used to evaluate the relations of biomarkers relative to the odds of CVD mortality.

Cases survived a median of 7.2 years after enrollment. Increased N-terminal pro-B type natriuretic peptide (NT-proBNP) (19% vs 4.3%), increased high-sensitivity C-reactive protein (hs-CRP) (71% vs 51%), and increased high-sensitivity cardiac troponin I (hs-cTnI) (8.7% vs 1.0%) were more common among cases than among controls (all P < 0.001 in unadjusted analyses). The adjusted odds of death were greater among cases compared to controls for increased NT-proBNP [odds ratio (OR) 5.67, 95% CI 2.17–15], hs-CRP (OR 1.73, 95% CI 1.03–2.89), and hs-cTnI (OR 8.53, 95% CI 1.68–43), and decreased ST2 (OR 1.92, 95% CI 1.05–3.48).

When measured by an hs-cTnI assay, cTnI is a key biomarker associated with increased cardiovascular death in a community sample when evaluated in a multiple biomarker analysis.

We enrolled 53 patients with benign colon diseases (e.g., diverticulosis, benign polyps, Crohn disease, ulcerative rectocolitis, colonic endometriosis) and analyzed their peripheral blood with 2 previously validated CTC assays: the epithelial immunospot (EPISPOT) assay and the CellSearch system. The EPISPOT assay detects only viable, CK19-releasing CTCs that were enriched by depletion of CD45⁺ leukocytes, whereas the CellSearch system detects CK-positive CTCs after positive EpCAM-based immunomagnetic enrichment.

In patients with benign colon diseases, positive events that met the criteria for "tumor cells" were detected with both the CellSearch system (11.3%) and the CK19-EPISPOT assay (18.9%), whereas no positive events were detected in samples from healthy volunteers. Positive events were detected most frequently in patients with diverticulosis and Crohn disease. All positive events lacked expression of CD45, a common leukocyte antigen.

These results indicate that patients with benign inflammatory colon diseases in particular can harbor viable circulating epithelial cells that are detectable with current CTC assays. This finding points to the need for further molecular characterization of circulating epithelial cells and has important implications for the use of CTC testing.

Among 2457 community-living individuals without cardiovascular disease (CVD), we measured serum fetuin-A concentrations by ELISA and evaluated the cross-sectional association of fetuin-A with CAC prevalence (any vs none) and severity; on follow-up 3.2 years (median) later, we evaluated the association of fetuin-A with CAC incidence and progression.

The mean age was 62 (SD 10) years, and the mean fetuin-A concentration was 0.48 (0.10) g/L. At baseline, 1200 individuals (49%) had CAC, and 272 individuals developed CAC during follow-up. At baseline, there was a threshold effect at the lowest fetuin-A quartile with CAC prevalence. In models adjusted for demographics, traditional cardiovascular disease (CVD) risk factors and kidney function, the lowest fetuin-A quartile had 7% (95% CI 1%–13%; P = 0.04) greater CAC prevalence compared with quartiles 2–4. Similar associations were observed with CAC severity at baseline, but the association was more linear. Each SD (0.10 g/L) lower fetuin-A was associated with a 12% (95% CI 3%–21%; P = 0.01) greater CAC severity in adjusted models. There was no significant association of fetuin-A with CAC incidence or progression.

Fetuin-A is inversely associated with CAC severity among community-living individuals without CVD. Whether fetuin-A concentrations are associated with incident CVD event in the general population requires future study.

We investigated 643 consecutive ED patients with acute chest pain who had been discharged for outpatient management after acute myocardial infarction (AMI) had been ruled out by serial measurements of conventional cardiac troponin. hs-cTnT was measured blindly, and we calculated the rates of all-cause mortality (primary endpoint) and subsequent AMI (secondary endpoint) at 30, 90, and 360 days.

hs-cTnT concentrations were increased (>14 ng/L) in 114 patients (18%) but <30 ng/L in 95% of these patients. Of those 114 patients, 96 (84%) had an adjudicated noncoronary cause of chest pain. Thirty-day mortality (95% CI) was 0.9% (0.1%–6.1%), 90-day mortality was 2.7% (0.9%–8.1%), and 360-day mortality was 5.2% (2.2%–11.9%) in patients with increased hs-cTnT; respective rates (95% CI) of AMI were 0.0%, 1.9% (0.5%–7.2%), and 7.6% (3.7%–15.3%). Increased hs-cTnT was associated with increased mortality and AMI at 90 days (P = 0.006 and P = 0.081, respectively) and 360 days (P = 0.001 for both).

hs-cTnT is a strong prognosticator of intermediate and long-term mortality and AMI in low-risk patients discharged from the ED after AMI has been ruled out. The relatively low rate of 30-day events may suggest that patients without acute coronary syndrome and small increases in cardiac troponin are in need of further investigations and treatments, but not necessarily immediate hospitalization.

We examined Hb A_1c requests (519 664 requests from 115 730 patients, January 2001 to March 2011) processed by the Clinical Biochemistry Department, University Hospital of North Staffordshire, and prevalence of requesting outside guidance from intervals between requests was calculated. Requests were classified as "appropriate," "too soon," or "too late." We also assessed the effect of demographic factors and publication of guidance, along with between-practice variability, on prevalence.

Only 49% of requests conformed to guidance; 21% were too soon and 30% were too late. Underrequesting was more common in primary care, in female patients, in younger patients, and in patients with generally poorer control (all P < 0.001); the reverse generally was true for overrequesting. Publication of guidance (e.g., American Diabetes Association, UK National Institute for Health and Clinical Excellence) had no significant impact on under- or overrequesting rates. Prevalence of inappropriate requests varied approximately 6-fold between general practices.

Although overrequesting was common, underrequesting was more prevalent, potentially affecting longer-term health outcomes. National guidance appears to be an ineffective approach to changing request behavior, supporting the need for a multisystem approach to reducing variability.

In a nested case-control study within MHS, 7 biomarkers were assayed in serum samples from 211 patients identified after 8–15 years of follow-up who died of cardiovascular causes (cardiovascular heart disease, stroke, congestive heart failure) and 253 controls matched on age, sex, and study year. Logistic regression analysis, adjusted for age, race, sex, education, study year, smoking, abdominal obesity, diabetes, serum total cholesterol, systolic blood pressure, previous hospitalization for a CVD event, and other significant biomarkers, was used to evaluate the relations of biomarkers relative to the odds of CVD mortality.

Cases survived a median of 7.2 years after enrollment. Increased N-terminal pro-B type natriuretic peptide (NT-proBNP) (19% vs 4.3%), increased high-sensitivity C-reactive protein (hs-CRP) (71% vs 51%), and increased high-sensitivity cardiac troponin I (hs-cTnI) (8.7% vs 1.0%) were more common among cases than among controls (all P < 0.001 in unadjusted analyses). The adjusted odds of death were greater among cases compared to controls for increased NT-proBNP [odds ratio (OR) 5.67, 95% CI 2.17–15], hs-CRP (OR 1.73, 95% CI 1.03–2.89), and hs-cTnI (OR 8.53, 95% CI 1.68–43), and decreased ST2 (OR 1.92, 95% CI 1.05–3.48).

When measured by an hs-cTnI assay, cTnI is a key biomarker associated with increased cardiovascular death in a community sample when evaluated in a multiple biomarker analysis.

This study was a retrospective case–control analysis of 89 women who presented to the ED with vaginal bleeding and/or abdominal pain/cramping and received a diagnosis of viable intrauterine pregnancy (VIP), spontaneous abortion (SA), or EP. Serum hCG and progesterone concentrations were measured by immunoassays. The serum concentrations of miRNAs miR-323-3p, miR-517a, miR-519d, and miR-525-3p were measured with TaqMan real-time PCR. Statistical analysis was performed to determine the clinical utility of these biomarkers, both as single markers and as multimarker panels for EP.

Concentrations of serum hCG, progesterone, miR-517a, miR-519d, and miR-525-3p were significantly lower in EP and SA cases than in VIP cases (P < 0.01). In contrast, the concentration of miR-323-3p was significantly increased in EP cases, compared with SA and VIP cases (P < 0.01). As a single marker, miR-323-3p had the highest sensitivity of 37.0% (at a fixed specificity of 90%). In comparison, the combined panel of hCG, progesterone, and miR-323-3p yielded the highest sensitivity (77.8%, at a fixed specificity of 90%). A stepwise analysis that used hCG first, added progesterone, and then added miR-323-3p yielded a 96.3% sensitivity and a 72.6% specificity.

Pregnancy-associated miRNAs, especially miR-323-3p, added substantial diagnostic accuracy to a panel including hCG and progesterone for the diagnosis of EP.

Purification of PRCP from human neutrophils and establishment of a sensitive ELISA was carried out with the use of samples from study participants. Three cohorts were studied: healthy individuals (n = 40); a chest pain cohort (Fast Assessment of Thoracic Pain by Neural Networks) (n = 165); and a community-based cohort [Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS)] (n = 1004).

PRCP was purified to homogeneity. Mean (SD) plasma concentrations in healthy individuals were 12.9 (3.2) μg/L and were increased in patients with chest pain and in patients with obesity and/or diabetes mellitus (P < 0.0001). In the PIVUS cohort the concentrations were related to several measures of arterial plaque formation, thickness of arterial intima media and posterior wall of the heart (P = 0.04–0.000005); the Framingham score (r = 0.14, P < 0.0001); and concentrations of C-reactive protein (r = 0.16, P < 0.0001) and N-terminal pro B-type natriuretic peptide (r = –0.13, P < 0.0001).

Plasma concentrations of PRCP may be used to reflect metabolic conditions in individuals with obesity and diabetes mellitus. The associations of PRCP concentrations with signs of cardiovascular dysfunction and cardiovascular abnormalities suggest a pivotal role of the enzyme in disease.

An expectorated OF pool and a pool of OF collected with Quantisal™ devices were prepared for each of 10 participants. 9-Tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THCCOOH), cannabidiol (CBD), and cannabinol (CBN) stability in each of 10 authentic expectorated and Quantisal-collected OF pools were determined after storage at 4°C for 1 and 4 weeks and at –20°C for 4 and 24 weeks. Results within ±20% of baseline concentrations analyzed within 24 h of collection were considered stable.

All Quantisal OF cannabinoid concentrations were stable for 1 week at 4°C. After 4 weeks at 4°C, as well as 4 and 24 weeks at –20°C, THC was stable in 90%, 80%, and 80% and THCCOOH in 89%, 40%, and 50% of Quantisal samples, respectively. Cannabinoids in expectorated OF were less stable than in Quantisal samples when refrigerated or frozen. After 4 weeks at 4 and –20°C, CBD and CBN were stable in 33%–100% of Quantisal and expectorated samples; by 24 weeks at –20°C, CBD and CBN were stable in ≤44%.

Cannabinoid OF stability varied by analyte, collection method, and storage duration and temperature, and across participants. OF collection with a device containing an elution/stabilization buffer, sample storage at 4°C, and analysis within 4 weeks is preferred to maximize result accuracy.

We determined the serum concentrations of hCG-h, hCG, and the free β subunit of hCG (hCGβ) by time-resolved immunofluorometric assays in 176 serum samples (preoperative n = 67, relapse n = 20, follow-up n = 89) obtained from 84 testicular cancer patients. We analyzed the association between preoperative serum concentrations of hCG, hCG-h, and hCGβ with known prognostic factors and progression-free survival time.

A major proportion of hCG was hyperglycosylated preoperatively, at relapse, and shortly after treatment. The serum concentrations of hCG-h and hCG correlated strongly with each other and had similar diagnostic value. The preoperative serum concentration of hCG-h correlated with prognostic factors and outcome in the same way as hCG. Increased preoperative hCGβ concentration predicted shorter progression-free survival.

Most of the hCG expressed by testicular cancers is hyperglycosylated and therefore it is important that hCG assays used for management of testicular cancer recognize hCG-h.

Children younger than 16 years presenting at a pediatric emergency department within 3 h after TBI were enrolled prospectively for blood sampling to determine serum S100B concentrations. The following information was collected: TBI severity determined by using the Masters classification [1: minimal or Glasgow Coma Scale (GCS) 15, 2: mild or GCS 13–15, and 3: severe or GCS <13]; whether hospitalized or not; good or bad clinical evolution (CE); whether cranial computed tomography (CCT) was prescribed; and related presence (CCT+) or absence (CCT–) of lesions.

For the 446 children enrolled, the median concentrations of S100B were 0.21, 0.31, and 0.44 μg/L in Masters groups 1, 2, and 3, respectively, with a statistically significant difference between these groups (P < 0.05). In Masters group 2, 65 CCT scans were carried out. Measurement of S100B identified patients as CCT+ with 100% (95% CI 85–100) sensitivity and 33% (95% CI 20–50) specificity. Of the 424 children scored Masters 1 or 2, 21 presented "bad CE." S100B identified bad CE patients with 100% (95% CI 84–100) sensitivity and 36% (95% CI 31–41) specificity. Of the 242 children hospitalized, 81 presented an S100B concentration within the reference interval.

Serum S100B determination during the first 3 h of management of children with mTBI has the potential to reduce the number of CCT scans, thereby avoiding unnecessary irradiation, and to save hospitalization costs.

We analyzed the release kinetics of cTnT measured by fourth-generation and high-sensitivity assays, creatine kinase-MB (CK-MB), and myoglobin in patients with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH), a model of AMI. Consecutive patients (n = 21) undergoing TASH were included. Serum and EDTA-plasma samples were collected before and at 15, 30, 45, 60, 75, 90, and 105 min, and 2, 4, 8, and 24 h after TASH.

cTnT concentrations measured by the hs assay were significantly increased at 15 min [21.4 ng/L, interquartile range (IQR) 13.3–39.7 ng/L vs 11.3 ng/L, IQR 6.0–18.8 ng/L at baseline; P = 0.031]. In comparison, cTnT concentrations measured by the conventional fourth-generation assay increased significantly at 60 min (30.0 ng/L, IQR 20.0–30.0 ng/L vs <10.0 ng/L, IQR <10.0–10.0 ng/L; P < 0.01), CK-MB at 90 min (8.4 μg/L, IQR 6.9–14.4 μg/L vs 0.9 μg/L, IQR 0.4–1.1 μg/L; P < 0.01), and myoglobin at 30 min (188.0 μg/L, IQR 154.0–233.0 μg/L vs 38.0 μg/L, IQR 28.0–56.0; P < 0.01).

cTnT concentrations measured by the hs assay were significantly increased after TASH at all of the time points, with a doubling at 15 min after induction of AMI, confirming earlier evidence of myocardial injury compared to the fourth-generation cTnT assay and CK-MB and myoglobin.

We developed primers that amplify a 52-bp PCR product from homologous regions in the SMN1 and SMN2 (survival of motor neuron 2, centromeric) genes that flank a divergent site at site c.840. Post-PCR high-resolution melt profiling assessed the amplification product, and we used a unique means of melt calibration to normalize profiles. Samples that we had previously characterized for the numbers of SMN1 and SMN2 copies established genotypes associated with particular profiles. The system was evaluated with approximately 1000 purified DNA samples, 100 self-created dried blood spots, and >1200 dried blood spots from newborn-screening tests.

Homozygous deletion of SMN1 exon 7 produced a distinctive melt profile that identified SMA patients. Samples with different numbers of SMN1 and SMN2 copies were resolved by their profiles. All samples with homozygous deletions were unambiguously recognized, and no normal sample was misidentified as a positive.

This assay has characteristics suitable for population-based screening. A reliable screening test will facilitate the identification of an SMA-affected cohort to receive early intervention to maximize the benefit from treatment. A prospective screening trial will allow the efficacy of treatment options to be assessed, which may justify the inclusion of SMA as a target for population screening.

Ternary troponin complex was added into samples (serum or plasma, n = 132, 68% cTnAAb positive) from individuals without known cardiac conditions. The recovery of cTnI was then measured with 6 investigational cTnI assays (2, 3, or 4 antibodies per assay). Three of these assays were then selected for further comparison by use of samples (n = 210, 33% cTnAAb positive) from non–ST-elevation acute coronary syndrome patients in the FRISC-II (FRagmin/Fast Revascularisation during InStability in Coronary artery disease) cohort. Finally, these results were compared to those obtained with 3 commercial cTnI assays.

Analytical recoveries varied widely among the 6 investigational assays. Notably the low recoveries (median 9%) of the midfragment-targeting reference assay were normalized (median 103%) with the use of the 4-antibody assay construct (3 capture, 1 tracer antibody) with only 1 antibody against a midfragment epitope. Reduced analytical recoveries correlated closely with measured autoantibody amounts. cTnI concentrations from cTnAAb-positive patient samples determined with 3 investigational assays confirmed the reduced concentrations expected from the low analytical recoveries. The results from the commercial cTnI assays with antibody selections representative for contemporary assay constructs revealed a similar underestimation (up to 20-fold) of cTnI in cTnAAb-positive samples.

A novel cTnI assay deviating from the conventional IFCC-recommended midfragment approach substantially improves cTnI detection in samples containing cTnAAbs.

We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution LC-MRM/MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and LC-MRM/MS assays were then compared with commercially available immunoassays for each of the 6 analytes.

Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution LC-MRM/MS approaches showed correlations with immunoassays of r = 0.61–0.96. The LC-MRM/MS approaches had acceptable reproducibility (<13% CV) and linearity (r ≥0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards.

Because peak area ratios measured in multiplexed LC-MRM/MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that LC-MRM/MS could be used to replace immunoassays in a variety of settings.

We diluted serum samples with Tris-HCl buffer solution and spiked 200-μL aliquots with [¹³C₃]-glycerol. These samples were incubated and hydrolyzed under basic conditions. The samples were dried, derivatized with acetic anhydride and pyridine, extracted with ethyl acetate, and analyzed by ID-GC-MS. Linearity, imprecision, and accuracy were evaluated by analyzing calibrator solutions, 10 serum pools, and a standard reference material (SRM 1951b).

The calibration response was linear for the range of calibrator concentrations examined (0–1.24 mmol/L) with a slope and intercept of 0.717 (95% CI, 0.7123–0.7225) and 0.3122 (95% CI, 0.3096–0.3140), respectively. The limit of detection was 14.8 μmol/L. The mean %CV for the sample set (serum pools and SRM) was 1.2%. The mean %bias from NIST isotope dilution MS values for SRM 1951b was 0.7%.

This ID-GC-MS RMP has the specificity and ruggedness to accurately quantify total glycerides in the serum pools used in the CDC's Lipid Standardization Program and demonstrates sufficiently acceptable agreement with the NIST primary RMP for total glyceride measurement.

We developed a novel immunoassay for the signal peptide of preproANP (ANPsp) and used it to document cardiac tissue levels of ANPsp in explant human hearts (n = 9), circulating venous concentrations of ANPsp in healthy volunteers (n = 65), temporal ANPsp concentrations in patients with ST-elevation myocardial infarction (STEMI) <4 h after chest pain onset (n = 23), and regional plasma ANPsp concentrations in patients undergoing clinically indicated catheterization (n = 10). We analyzed the structure and sequence of circulating ANPsp by tandem mass spectrometry (MS/MS).

ANPsp levels in human heart tissue were 50–1000 times lower than those of ANP/NT-proANP. ANPsp was detectable in control human plasma at concentrations comparable with ANP itself (approximately 20 ng/L). In STEMI patients, plasma concentrations of ANPsp rose to peak values at 5 h after symptom onset, significantly earlier than myoglobin, creatine kinase-MB, and troponin (P < 0.001). There were significant arteriovenous increases in ANPsp concentrations (P < 0.05) across the heart and kidney; arterial and coronary sinus concentrations of ANPsp both negatively correlated with systolic and mean arterial blood pressures (both P < 0.01). MS/MS verified circulating ANPsp to be preproANP(16–25) and preproANP(18–25).

ANPsp is a novel circulating natriuretic peptide with potential to act as a cardiovascular biomarker. The rapid increase of plasma ANPsp in STEMI and its significant relationship with blood pressure encourage further study of its potential clinical utility.

The 2-site immunometric assay for total PSA employed 2 monoclonal antibodies, one conjugated to a double-stranded DNA label and the other bound to paramagnetic microparticles. After several washing steps, quantification cycles were determined and values were converted to PSA concentrations. We characterized analytical performance and compared accuracy with a commercially available total PSA assay.

The limit of quantification was 0.65 ng/L and the assay was linear in the range of 0.25–152.0 ng/L. Total imprecision estimates at PSA concentrations of 3.8, 24.1, and 69.1 ng/L were <15.2%, <9.4%, and <10.6%, respectively. Recovery of supplemented PSA ranged from 87.5% to 119.2% (mean 100.3%). Dilution recovery ranged from 96.4% to 115.3% (mean 102.3%). There was no high-dose hook effect up to 50 000 ng/L of PSA. Comparison with the commercial PSA assay showed a regression slope of 1.06 and a correlation coefficient of 0.996.

The analytical characteristics of the assay support the use of this assay for the accurate and precise measurement of serum PSA, even at sub–nanogram-per-liter concentrations.

We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products.

Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run).

This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

For the 3 genes, 38 primer pairs were designed and loaded on the Fluidigm Access Array, a microfluidic array in which a PCR was performed. We amplified 144 DNA samples (73 positive controls and 71 patient samples) and performed 3 sequencing runs on a GS FLX Titanium system from Roche 454, using pyrosequencing. Data were analyzed with the SeqNext module of the Sequence Pilot software.

From the 38 amplicons, 37 were amplified successfully, without any further optimization. Sequencing resulted in a mean coverage of the individual amplicons of 71-fold, 74-fold, and 117-fold for the 3 runs, respectively. In the positive controls, all known mutations were identified. In 29% of the patient samples, a pathogenic point mutation or small deletion/insertion was found. Large rearrangements were not detectable with NGS, but were picked up by multiplex ligation-dependent probe amplification.

Combining a microfluidic amplification system with massive parallel sequencing is an effective method for mutation scanning in FH patients, which can be implemented in diagnostics. For data analysis, we propose a minimum variant frequency threshold of 20% and a minimum coverage of 25-fold.

We have used a sensitive, specific, quantitative, and cost-effective homogeneous SMD method that has high single-well multiplexing potential and uses alternating-laser excitation (ALEX) fluorescence-aided molecule sorting extended to 4 colors (4c-ALEX). Recognition molecules are tagged with different-color fluorescence dyes, and coincident confocal detection of ≥2 colors constitutes a positive target-detection event. The virtual exclusion of the majority of sources of background noise eliminates washing steps. Sorting molecules with multidimensional probe stoichiometries (S) and single-molecule fluorescence resonance energy transfer efficiencies (E) allows differentiation of numerous targets simultaneously.

We show detection, differentiation, and quantification—in a single well—of (a) 25 different fluorescently labeled DNAs; (b) 8 bacterial genetic markers, including 3 antibiotic drug–resistance determinants found in 11 septicemia-causing Staphylococcus and Enterococcus strains; and (c) 6 tumor markers present in blood.

The results demonstrate assay utility for clinical molecular diagnostic applications by means of multiplexed detection of nucleic acids and proteins and suggest potential uses for early diagnosis of cancer and infectious and other diseases, as well as for personalized medicine. Future integration of additional technology components to minimize preanalytical sample manipulation while maximizing throughput should allow development of a user-friendly ("sample in, answer out") point-of-care platform for next-generation medical diagnostic tests that offer considerable savings in costs and patient sample.

For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform.

Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses.

This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

Cannabis smokers provided written informed consent for this institutional review board–approved study. OF was collected with the Quantisal™ device following ad libitum smoking of one 6.8% THC cigarette. Cannabinoids were quantified by 2-dimensional GC-MS. We evaluated 8 alternative cutoffs based on different drug testing program needs.

10 participants provided 86 OF samples –0.5 h before and 0.25, 0.5, 1, 2, 3, 4, 6, and 22 h after initiation of smoking. Before smoking, OF samples of 4 and 9 participants were positive for THC and THCCOOH, respectively, but none were positive for CBD and CBN. Maximum THC, CBD, and CBN concentrations occurred within 0.5 h, with medians of 644, 30.4, and 49.0 μg/L, respectively. All samples were THC positive at 6 h (2.1–44.4 μg/L), and 4 of 6 were positive at 22 h. CBD and CBN were positive only up to 6 h in 3 (0.6–2.1 μg/L) and 4 (1.0–4.4 μg/L) participants, respectively. The median maximum THCCOOH OF concentration was 115 ng/L, with all samples positive to 6 h (14.8–263 ng/L) and 5 of 6 positive at 22 h.

By quantifying multiple cannabinoids and evaluating different analytical cutoffs after controlled cannabis smoking, we determined windows of drug detection, found suggested markers of recent smoking, and minimized the potential for passive contamination.

This review provides a look at technology trends that could have applicability to high-sensitivity multiplexed immunoassays in resource-limited settings. Various technologies are explained and assessed according to potential for reaching relevant limits of cost, sensitivity, and multiplex capability. Frequently, such work is reported in technical journals not normally read by clinical scientists, and the authors make enthusiastic claims for the potential of their technology while ignoring potential pitfalls. Thus it is important to draw attention to technical hurdles that authors may not be publicizing.

Immunochromatographic assays, optical methods including those involving waveguides, electrochemical methods, magnetorestrictive methods, and field-effect transistor methods based on nanotubes, nanowires, and nanoribbons reveal possibilities as next-generation technologies.

Plasma creatinine concentration is the most widely used measure for estimation of GFR. Plasma cystatin C and β-trace protein may eventually prove to be superior to creatinine. GFR may be measured directly by use of exogenous filtration markers, although their role is primarily limited to the research setting. Real-time, noninvasive measurement of GFR by using fluorescently labeled markers may be available in the future. Novel biomarkers of tubular injury such as neutrophil gelatinase-associated lipocalin, kidney injury molecule-1, liver-type fatty acid binding protein, N-acetyl-β-(D)-glucosaminidase, and interleukin-18 may enable the early detection of acute kidney injury before or in the absence of a change in GFR.

A variety of methods are available to assist clinicians in the assessment of kidney function and injury. Ongoing investigation will help determine the utility of several new markers and clarify their role in the care of patients with and at risk for kidney disease.

We recruited 29 infants with C21OHD, 9 with PORD, and 67 with TH17OHP, and 1341 control infants. All were Japanese and between 0 and 180 days old; none received glucocorticoid treatment before urine sampling. We measured urinary pregnanetriolone (Ptl), the cortisol metabolites 5α- and 5β-tetrahydrocortisone (sum of these metabolites termed THEs), and metabolites of 3 steroids, namely dehydroepiandrosterone, androstenedione (AD4), and 11β-hydroxyandrostenedione (11OHAD4) by GC-MS.

At a cutoff of 0.020, the ratio of Ptl to THEs differentiated C21OHD and PORD from TH17OHP and controls with no overlap. Among metabolites of DHEA, AD4, and 11OHAD4, only 11β-hydroxyandrosterone (11HA), a metabolite of 11OHAD4, showed no overlap between C21OHD and PORD at a cutoff of 0.35 mg/g creatinine.

A specific cutoff for the ratio of Ptl to THEs can differentiate C21OHD and PORD from TH17OHP and controls. Additionally, the use of a specific cutoff of 11HA can distinguish between C21OHD and PORD.

Minor-groove binder (MGB) TaqMan probes were designed to discriminate between wild-type hemoglobin A and mutant (hemoglobin S) alleles encoded by the HBB (hemoglobin, beta) gene in cffDNA isolated from maternal plasma samples obtained from pregnancies at risk of sickle cell anemia. The fractional fetal DNA concentration was assessed in male-bearing pregnancies with a digital PCR assay for the Y chromosome–specific marker DYS14. In pregnancies with a female fetus, a panel of biallelic insertion/deletion polymorphism (indel) markers was developed for the quantification of the fetal DNA fraction. We used digital real-time PCR to analyze the dosage of the variant encoding hemoglobin S relative to that encoding wild-type hemoglobin A.

The sickle cell genotype was correctly determined in 82% (37 of 45) of male fetuses and 75% (15 of 20) of female fetuses. Mutation status was determined correctly in 100% of the cases (25 samples) with fractional fetal DNA concentrations >7%. The panel of indels was informative in 65% of the female-bearing pregnancies.

Digital PCR can be used to determine the genotype of fetuses at risk for sickle cell anemia. Optimization of the fractional fetal DNA concentration is essential. More-informative indel markers are needed for this assay's comprehensive use in cases of a female fetus.

We analyzed plasma from 12 patients with type 1 von Willebrand disease (vWD), 14 patients with type 2 vWD, and 10 healthy controls using a fluctuation-based immunoassay approach.

FCS enabled identification and proper classification of type 1 and type 2 vWD, producing quantitative results that correspond to qualitative gel multimer patterns. FCS required minimal sample preparation and only a 5-min analysis time.

This study represents the first implementation of FCS for clinical diagnostics directly on human plasma. The technique shows potential for further vWF studies and as a generally applicable laboratory test method.

In this method, similar sequences located on different chromosomes are amplified simultaneously with a single primer set; the PCR products are then analyzed by high-resolution melting. Aneuploidy-associated dosage abnormalities produce different ratios of similar amplicons, which produce melting curves that are detectably different from those of samples from unaffected individuals. We applied this method to DNA samples isolated from individuals with trisomy 21 (n = 48), trisomy 18 (n = 10), trisomy 13 (n = 3), 45,X (n = 8), and 47,XXY (n = 14), and from unaffected controls (n = 48).

As judged by the karyotyping results, our method attained 100% diagnostic sensitivity and 99.6% diagnostic specificity. Moreover, our method was able to detect a change in chromosome dosage as low as 1.05-fold.

This novel method clearly differentiates samples of patients with common aneuploidies from those of unaffected controls, while markedly simplifying the assays and reducing time and costs. The assay has sufficient throughput to meet the demands of large-scale testing, such as population-based postnatal screening, and is thus suitable for routine use.

We applied a nontargeted GC-MS metabolomic approach to plasma samples from 36 patients with PCOS and 39 control women without androgen excess, matched for age, body mass index, and frequency of obesity.

Patients with PCOS were hyperinsulinemic and insulin resistant compared with the controls. The increase in plasma long-chain fatty acids, such as linoleic and oleic acid, and glycerol in the obese patients with PCOS suggests increased lipolysis, possibly secondary to impaired insulin action at adipose tissue. Conversely, nonobese patients with PCOS showed a metabolic profile consisting of suppression of lipolysis and increased glucose utilization (increased lactic acid concentrations) in peripheral tissues, and PCOS patients as a whole showed decreased 2-ketoisocaproic and alanine concentrations, suggesting utilization of branched-chain amino acids for protein synthesis and not for gluconeogenesis. These metabolic processes required effective insulin signaling; therefore, insulin resistance was not universal in all tissues of these women, and different mechanisms possibly contributed to their hyperinsulinemia. PCOS was also associated with decreased α-tocopherol and cholesterol concentrations irrespective of obesity.

Substantial metabolic heterogeneity, strongly influenced by obesity, underlies PCOS. The possibility that hyperinsulinemia may occur in the absence of universal insulin resistance in nonobese women with PCOS should be considered when designing diagnostic and therapeutic strategies for the management of this prevalent disorder.

We performed biomarker tests on fecal samples from 386 patients with lower-abdomen complaints suggestive for OBD. Endoscopic and histological diagnosis served as reference.

OBD was diagnosed in 99 patients (prevalence 25.9%); 19 had adenocarcinoma, 53 adenoma, and 27 inflammatory bowel disease. Sensitivity for OBD was 0.64 (95% CI 0.54–0.72) for calprotectin POC, 0.56 (0.46–0.66) for iFOBT POC, and 0.74 (0.65–0.82) for calprotectin ELISA; specificities were 0.53 (0.48–0.59), 0.83 (0.78–0.87), and 0.47 (0.41–0.53), respectively. Negative predictive values (NPVs) were 0.81 (0.74–0.86), 0.85 (0.80–0.88), and 0.84 (0.78–0.89); positive predictive values (PPVs) varied from 0.32 (0.26–0.39) and 0.33 (0.27–0.39) (calprotectin tests) to 0.53 (0.44–0.63) (iFOBT POC). Combining the 2 POC tests improved sensitivity [0.79 (0.69–0.86)] and NPV [0.87 (0.81–0.91)] but lowered specificity [0.49 (0.44–0.55)] and PPV [0.35 (0.29–0.42)]. When adenomas ≤1 cm were considered non-OBD, the NPV of all tests improved to >0.90 [combined POC tests, 0.97 (0.93–0.99)].

Diagnostic accuracy of the tests alone or combined was insufficient when all adenomas were considered OBD. When only adenomas >1 cm were considered OBD, all tests could rule out OBD to a reasonable extent, particularly the combined POC tests. The tests were less useful for inclusion of OBD.

We prospectively included 720 patients in our study: 293 patients with stable CAD and 427 patients with acute coronary syndrome. During a median follow-up of 11.3 years corresponding to 6469 overall person-years, 278 deaths (38.6%) were recorded. We detected a significant and independent protective effect of butyrylcholinesterase on all-cause mortality [adjusted hazard ratio (HR) for a 1-SD increase, 0.62; 95% CI, 0.54–0.71; P < 0.001] and cardiovascular mortality (adjusted HR, 0.64; 95% CI, 0.54–0.76; P < 0.001) in a Cox proportional hazards regression analysis. The 10-year survival rates were 42%, 74%, and 87% in the first, second, and third tertiles of butyrylcholinesterase activity. The presentation of CAD affected the effect of butyrylcholinesterase on mortality (P for interaction = 0.012), with a stronger association found in patients with stable CAD (adjusted HR, 0.56; 95% CI, 0.45–0.70; P < 0.001).

Our study demonstrates a strong inverse association between butyrylcholinesterase activity and long-term outcome in patients with known CAD. Because butyrylcholinesterase added predictive information after adjustment for established cardiovascular risk factors, additional underlying pathophysiological mechanisms and the potential applicability of butyrylcholinesterase activity for secondary risk prediction needs to be addressed in future studies.