h-hydrogen

Hyperbilirubinemia in jaundiced neonates is routinely assessed by use of total serum bilirubin. However, the unbound or free form (B_f), not total bilirubin, crosses the blood–brain barrier and can be neurotoxic. Although the peroxidase-mediated oxidation of bilirubin can be used to measure plasma concentrations of B_f, this measurement is relatively complex and the assay is not routinely used. We describe a fluorescence sensor for quantifying B_f in plasma.

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.

Although lipoprotein-associated phospholipase A₂ (Lp-PLA₂) levels are associated with cardiovascular events, Lp-PLA₂ is physically linked to LDL cholesterol (LDL-C). Whether measures of Lp-PLA₂ mass or activity continue to predict risk after LDL-C reduction by statin therapy is uncertain.

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.

Fetuin-A is a hepatic secretory protein that inhibits arterial calcification in vitro. The association of fetuin-A with coronary arterial calcification (CAC) in the general population is uncertain.

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.

The use of serum human chorionic gonadotropin (hCG) and progesterone to identify patients with ectopic pregnancy (EP) has been shown to have poor clinical utility. Pregnancy-associated circulating microRNAs (miRNAs) have been proposed as potential biomarkers for the diagnosis of pregnancy-associated complications. This proof-of-concept study examined the diagnostic accuracy of various miRNAs to detect EP in an emergency department (ED) setting.

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.

Estimates suggest that approximately 25% of requests for pathology tests are unnecessary. Even in diabetes, for which international guidance provides recommended testing frequency, considerable variability in requesting practice exists. Using the diabetes marker, Hb A_1c, we examined (a) the prevalence of under- and overrequesting, (b) the impact of international guidance on prevalence, and (c) practice-to-practice variability.

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.

The appropriate management of patients discharged from the emergency department (ED) with increased high-sensitivity cardiac troponin T (hs-cTnT) but normal or borderline-high conventional cardiac troponin concentrations is unknown.

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.

Repeating a QC that is outside 2SD from the mean (1:2s rule) appears to be a common practice. Although this form of repeat sampling is frowned on by many, the comparative power of the approach has not been formally evaluated.

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.

We examined several novel biomarkers of different pathophysiologic pathways as predictors of cardiovascular mortality in participants enrolled in the Minnesota Heart Survey (MHS), a population-based study of cardiovascular disease (CVD) risk factors.

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.

Detection of circulating tumor cells (CTCs) in the peripheral blood is a rapidly developing research field with clear clinical implications for the staging and monitoring of cancer patients. Current CTC assays, including the US Food and Drug Administration–cleared CellSearch® system, typically use markers [e.g., cytokeratins (CKs), the transmembrane protein EpCAM (epithelial cell adhesion molecule)] that are expressed on normal and malignant epithelial cells but not on the surrounding normal leukocytes.

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.