Volume 54, Issue 10

Kounis syndrome has been established as a hypersensitivity coronary disorder induced by various conditions, drugs, environmental exposures, foods and coronary stents. Allergic, hypersensitivity, anaphylactic and anaphylactoid reactions are associated with this syndrome. Vasospastic allergic angina, allergic myocardial infarction and stent thrombosis with occluding thrombus infiltrated by eosinophils and/or mast cells constitute are the three reported, so far, variants of this syndrome. Apart from coronary arteries, it affects the cerebral and mesenteric arteries. Its manifestations are broadening and its etiology is continuously increasing. Kounis syndrome is a ubiquitous disease which represents a magnificent natural paradigm and nature’s own experiment in a final trigger pathway implicated in cases of coronary artery spasm and plaque rupture. Kounis syndrome seems to be not a rare disease but an infrequently diagnosed clinical entity which has revealed that the same mediators released from the same inflammatory cells are also present and in acute coronary events of non allergic etiology. These cells are not only present in the culprit region before plaque erosion or rupture but they release their contents just before an actual coronary event. Therefore, awareness of etiology, epidemiology, pathogenesis and clinical manifestations seems to be important for its prognosis, diagnosis, treatment, prevention.

High total homocysteine (tHcy) concentrations contribute to an increased risk of cardiovascular diseases and neurodegenerative disorders. Several investigations have focused on the effect of exercise on tHcy concentrations, but results remain controversial. The differences among the methodologies in the investigations make difficult the interpretation of results. This review differentiates the effects of exercise on tHcy and establishes the relation with the implicated biomarkers on tHcy metabolism related to exercise. The electronic database MEDLINE (http://www.ncbi.nlm.nih.gov) was used for searching studies published between years 2002 and 2015. ‘Homocysteine’, ‘Training ’, ‘Exercise’, ‘Physical Activity’ as well as combinations out of these terms were entered in the database. Articles were grouped in: 1) Acute effect of exercise on tHcy, 2) chronic exercise and tHcy, 3) relationship of physical activity (PA) level and cardiorespiratory fitness with tHcy, and 4) biomarkers related to tHcy and exercise. From a total of 30 articles, most of the studies analyzing the acute effect of exercise showed an increase on tHcy concentrations. Studies analyzing the chronic effect on tHcy concentrations showed contradictory results and no consensus exists probably due to the differences in the methodology, exercise interventions and participants characteristics. Low cardiorespiratory fitness seems to be associated with high tHcy; in contrast, the relation of PA levels and tHcy needs further research. Regarding biomarkers related to tHcy and exercise, some studies showed an increase of folate, vitamin B12, and creatine after acute exercise that could to be due to requirement of protein turnover and an increased metabolic demand of vitamin-B.

Clear evidence supports a role for circulating and locally-produced osteocalcin (OC) in the pathophysiology of cardiovascular (CV) lesions and CV risk, also in combination with metabolic changes, including type 2 diabetes mellitus (T2DM). Reduced plasma OC levels are associated with greater incidence of pathological CV changes, like arterial and valvular calcification, coronary and carotid atherosclerosis and increased carotid intima-media thickness. The actual relationship between OC levels and incidence of major CV events is, however, still unclear. Moreover, reduced circulating OC levels have been mostly associated with insulin resistance, metabolic syndrome or T2DM, indicating relevant OC actions on pancreatic β-cells and insulin secretion and activity. Based on these observations, this review article will attempt to summarize the current evidence on the potential usefulness of circulating OC as a biomarker for CV and metabolic risk, also evaluating the currently open issues in this area of research.

Management of viral diagnostic quality is based on external quality assurance (EQA), where laboratories involved in diagnostics of a targeted virus are offered to analyze a panel of blinded samples. The utility of EQAs is compromised because of the absence of an approach to EQA design which upfront defines acceptance criteria and associated statistical analysis ensuring fair and consistent interpretation. We offer a rigorous statistically based approach for EQA planning. Instead of a conventional performance characteristic (the score) which is calculated as the sum of the points for correctly identified samples in a blinded test panel, Youden index is used as the performance measure. Unlike the score, Youden index requires an estimate of sensitivity and specificity and incorporates the relationship of these performance parameters. Based on the assumption that the coordinator is a reputable expert of viral diagnostics, the performance of the coordinator’s laboratory is defined as a proficiency standard for performance evaluation. The immediate goal of EQA is defined as to obtain a statistically reliable estimation for every laboratory whether its performance meets the proficiency standard, while the overall goal is to match every laboratory to its specific performance level. Dependence of informational capacities of test panel from the panel size and content is quantitatively analyzed and the optimal design and informational capacities of both idealized panels (whose size is not restricted by financial factors) and currently feasible panels are considered. Our approach provides the basis both for rational design of currently feasible EQA test panels and for an increased panel size.

Background: Biological variation (BV) data have many fundamental applications in laboratory medicine. At the 1st Strategic Conference of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) the reliability and limitations of current BV data were discussed. The EFLM Working Group on Biological Variation is working to increase the quality of BV data by developing a European project to establish a biobank of samples from healthy subjects to be used to produce high quality BV data. Methods: The project involved six European laboratories (Milan, Italy; Bergen, Norway; Madrid, Spain; Padua, Italy; Istanbul, Turkey; Assen, The Netherlands). Blood samples were collected from 97 volunteers (44 men, aged 20–60 years; 43 women, aged 20–50 years; 10 women, aged 55–69 years). Initial subject inclusion required that participants completed an enrolment questionnaire to verify their health status. The volunteers provided blood specimens once per week for 10 weeks. A short questionnaire was completed and some laboratory tests were performed at each sampling consisting of blood collected under controlled conditions to provide serum, K 2 EDTA-plasma and citrated-plasma samples. Results: Samples from six out of the 97 enroled subjects were discarded as a consequence of abnormal laboratory measurements. A biobank of 18,000 aliquots was established consisting of 120 aliquots of serum, 40 of EDTA-plasma, and 40 of citrated-plasma from each subject. The samples were stored at –80 °C. Conclusions: A biobank of well-characterised samples collected under controlled conditions has been established delivering a European resource to enable production of contemporary BV data.

Background: There are limited evidence based studies demonstrating the stability of fat-soluble vitamins (FSV) measured in blood. This study aimed to examine the effects of light, temperature and time on vitamins A, D and E throughout the total testing process. Methods: Four experiments were conducted. Three investigated the sample matrix, of whole blood, serum and the extracted sample, against the variables of temperature and light; and the fourth experiment investigated the sample during the extraction process against the variable of light. All samples were analysed via our simultaneous FSV method using liquid chromatography-tandem mass spectrometry technology. The allowable clinical percentage change was calculated based on biological variation and desirable method imprecision for each analyte. The total change limit was ±7.3% for 25-OH-vitamin D3, ±11.8% for retinol and ±10.8% for α-tocopherol. Results: Vitamins D and E were stable in the investigated conditions (concentration changes <4%) in the pre-analytical and analytical stages. Vitamin A showed photosensitivity in times >48 h with concentration changes of −6.8% (blood) and −6.5% (serum), both are within the allowable clinical percentage change. By contrast, the extracted retinol sample demonstrated a concentration change of −18.4% after 48 h of light exposure. However, vitamin A in the serum and extracted solution was stable for one month when stored at −20°C. Conclusions: Blood samples for vitamins D and E analyses can be processed in normal laboratory conditions of lighting and temperature. The required conditions for vitamin A analysis are similar when performed within 48 h. For longer-term storage, serum and vitamin A extracts should be stored at −20°C.

Background: The Intermountain Mortality Risk Score (IMRS), a sex-specific mortality-prediction metric, has proven to be effective in various populations. IMRS is comprised of the complete blood count (CBC), basic metabolic panel (BMP), and age. Whether the addition of factors from the comprehensive metabolic panel (CMP) and white blood cell (WBC) differential count improves risk stratification is unknown. Methods: Patients with baseline complete metabolic panel (CMP) and IMRS measurements were randomly assigned (60%/40%) to independent derivation (n=84,913) and validation (n=56,584) populations. A sex-specific risk score based on IMRS methods was computed in the derivation population using adjusted multivariable regression weights from all significant and noncollinear CMP [expanded IMRS (eIMRS)] and, when available, WBC differential components (eIMRS+diff). Results: Age averaged 67±16 years for females and 67±15 years for males. Receiver operator characteristic (ROC) c-statistics for 30-day death showed marked improvement for the eIMRS compared to the IMRS in both females [0.895 (0.882, 0.908) vs. 0.865 (0.850, 0.880)] and males [0.861 (0.847, 0.876) vs. 0.824 (0.807, 0.841)]. These results persisted for 1-year death: females [0.854 (0.847, 0.862) vs. 0.828 (0.819, 0.836)] and males [0.835 (0.826, 0.844) vs. 0.796 (0.789, 0.808)]. In addition, the eIMRS significantly improved risk reclassification. Further precision was seen when WBC differential components were included. Conclusions: The addition of the CMP components to the IMRS improved risk prediction. WBC differential also improved risk score predictive ability. These results suggest that the eIMRS may function even better than IMRS as a tool in patient care, risk-adjustment, and clinical research settings for predicting outcomes.

Background: We evaluated the effect of kidney glomerular function on serum concentrations of human epididymis protein 4 (HE4) using creatinine (Cr), cystatin C (CysC) and related chronic kidney disease epidemiology collaboration (CKD-EPI) equations. Methods: We enrolled 101 women aged ≤56 years with a glomerular filtration rate (GFR) (estimated by CKD-EPI eGFR Cr ) ranging from 60 to 120 mL/min/1.73 m 2 , free of any disease and biological and life-style factors known to influence serum HE4 concentrations, and we measured serum Cr, CysC and HE4 concentrations. Cr and CysC values were included in the three CKD-EPI equations to obtain GFR estimates. Results: A statistically significant increase in HE4 median concentrations was detected in subjects with an eGFR Cr between 60 and 74 mL/min/1.73 m 2 when compared with those with an eGFR >90 mL/min/1.73 m 2 (54.2 vs. 42.2 pmol/L, p=0.003). Regression models showed that CysC measurement per se and eGFR CysC were the most sensitive markers to catch HE4 increases due to a mild decrease in renal function [adjusted r 2 , 0.38 (p=0.00003) and 0.37 (p=0.0004), respectively]. By assuming baseline CysC and eGFR CysC at 0.80 mg/L and 101.5 mL/min/1.73 m 2 , an increase of 0.10 mg/L in CysC concentrations and a decrease of 10 mL/min of eGFR CysC implied an average (±SE) increase in serum HE4 concentrations of 9.2 (±1.2) and 8.8 (±1.1) pmol/L, respectively. Conclusions: Our study shows that a better estimate of the effect of GFR on serum HE4 is obtained by measuring CysC in serum or using CKD-EPI eGFR CysC equation.

Background: Serum human epididymis protein 4 (HE4) has gained relevance as an ovarian cancer (OC) biomarker and new automated methods have replaced the first released manual EIA by tracing results to it. We verified agreement and bias of automated methods vs. EIA as well as possible effects on patients’ management. Methods: One hundred and fifteen serum samples were measured by Abbott Architect i 2000, Fujirebio Lumipulse G 1200, Roche Modular E170, and Fujirebio EIA. Passing-Bablok regression was used to compare automated assays to EIA and agreement between methods was estimated by Lin’s concordance correlation coefficient (CCC). The bias vs. EIA was estimated and compared to specifications derived from HE4 biological variation. Results: Median (25th–75th percentiles) HE4 concentrations (pmol/L) were 84.5 (60.1–148.8) for EIA, 82.7 (50.3–153.9) for Abbott, 89.1 (55.2–154.9) for Roche, and 112.2 (67.8–194.2) for Fujirebio. Estimated regressions and agreements (95% confidence interval) were: Abbott=1.01(0.98–1.03) EIA–4.8(–7.5/–2.6), CCC=0.99(0.99–1.00); Roche=0.91(0.89–0.93) EIA+5.7(4.2/8.0), CCC=0.98(0.98–0.99); Fujirebio=1.20(1.17–1.24) EIA+ 2.4(–0.6/4.9), CCC=0.97(0.96–0.98). The average bias vs. EIA resulted within the desirable goal for Abbott [–3.3% (–6.1/–0.5)] and Roche [–0.2% (–3.0/2.5)]. However, while for Abbott the bias was constant and acceptable along the measurement concentration range, Roche bias increased up to –28% for HE4 values >250 pmol/L. Lumipulse showed a markedly positive bias [25.3% (21.8/28.8)]. Conclusions: Abbott and Roche assays exhibited a good comparability in the range of HE4 values around the previously recommended 140 pmol/L cut-off. For patient monitoring, however, the assay used for determining serial HE4 must not be changed as results from different systems in lower and higher concentration ranges can markedly differ.

Background: Urinary liver-type fatty acid binding protein (L-FABP) measured by enzyme-linked immunosorbent assay method (ELISA) was approved as a clinical biomarker of tubular damage by the Japanese Ministry of Health, Labor and Welfare (MHLW) in 2011. We evaluated a new latex-enhanced immunoturbidimetric assay (LTIA) to evaluate the clinical utility of urinary L-FABP measured by LTIA versus an ELISA assay. Methods: LTIA with anti-human L-FABP mouse monoclonal antibodies was performed using an automated clinical chemistry analyzer. Five positive samples with low, medium and high L-FABP concentrations were analyzed to determine the within-run precision. In patients with chronic kidney disease (CKD) (n=91), urinary L-FABP levels were measured by ELISA and LTIA. Results: Measurement of urinary L-FABP revealed urinary L-FABP levels within 30 min. The within-run coefficient of variation was 10.0% for 1.4 ng/mL, 4.4% for 2.5 ng/mL, 3.2% for 9.8 ng/mL, 1.5% for 50.1 ng/mL, and 1.2% for 102.7 ng/mL. Concentrations of urinary L-FABP measured by LTIA were significantly correlated with those measured by ELISA (ρ=0.932). Proportional systematic error was almost within limits of agreement (LOA). Urinary L-FABP levels measured by LTIA were significantly correlated with urinary albumin (ρ=0.634), urinary NAG (ρ=0.688) and eGFR (ρ=–0.561). Conclusions: Measurement of urinary L-FABP by LITA was simple, speedy, and similar in quality to ELISA results. Therefore, this method was approved as external body diagnosing medicines by the Japanese MHLW in 2014. Urinary L-FABP is expected to be widely used in various pathophysiological conditions by measuring urinary L-FABP using LTIA.

Background: Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are specific cytoskeletal proteins of neurons and their quantification has shown encouraging results as a biomarker for axonal injury. Methods: We aimed at comparing a widely used conventional ELISA for Nf light chain (NfL) with an electrochemiluminescence-based method (ECL assay) and a newly developed single-molecule array (Simoa) method in clinically relevant cerebrospinal fluid (CSF) and serum samples. Results: Analytical sensitivity was 0.62 pg/mL for Simoa, 15.6 pg/mL for the ECL assay, and 78.0 pg/mL for the ELISA. Correlations between paired CSF and serum samples were strongest for Simoa (r=0.88, p<0.001) and the ECL assay (r=0.78, p<0.001) and weaker for ELISA measurements (r=0.38, p=0.030). CSF NfL measurements between the platforms were highly correlated (r=1.0, p<0.001). Serum NfL levels were highly related between ECL assay and Simoa (r=0.86, p<0.001), and this was less visible between ELISA-ECL assay (r=0.41, p=0.018) and ELISA-Simoa (r=0.43, p=0.013). Multiple sclerosis (MS) patients had significantly higher serum NfL levels than controls when measured with Simoa (p=0.001) but not with the other platforms. Conclusions: We found Simoa to be more sensitive than ELISA or the ECL assay. Our results support the feasibility of quantifying NfL in serum; the results correlate with the more-established CSF NfL test. The highly sensitive Simoa technology deserves further studies in larger patient cohorts to clarify whether serum NfL could be used in the future to measure disease severity and determine prognosis or response to treatment interventions in neurological diseases.

Background: Antiphospholipid syndrome (APS) is the most common acquired thrombophilia. Diagnosis is based on clinical criteria and the presence of antiphospholipid antibodies (aPLs) above the 99th percentile of a reference group. Data on the distribution of aPL in the population are limited. The distribution of aPL including diagnostic cutoffs should be determined in a population-based cohort. Methods: The Gutenberg Health Study (GHS) is a population-based cohort aged 35–74 years. We determined the presence of antibodies against cardiolipin (aCL, IgG, and IgM), β 2 -glycoprotein I (anti-β2GPI, IgG, and IgM), and domain 1 of β 2 -glycoprotein I (anti-domain 1, IgG) in a sample of 4979 participants. Results: aPL titers were similar in the whole sample and in an apparently healthy subgroup of 1049 individuals. There was a strong age-dependent increase of both aCL and anti-β2GPI IgM, while aPL IgG titers were stable or tended to decrease with age. A relevant decrease was observed for aCL IgG in women and anti-domain 1 IgG in both sexes. There was no association of aPL titers with a history of venous thromboembolism (VTE). Conclusions: Our data show that for IgM aPL, age-dependent reference ranges should be used. In fact, the controversy regarding the clinical utility of IgM aPL might be related to the use of inappropriate reference ranges among other causes. In our population, aPLs were not associated with a history of VTE.

Background: To investigate the predictive value of S100 (biochemical marker of neuroglial injury) for the occurrence of postoperative delirium (POD) in patients undergoing off-pump coronary artery bypass (OPCAB)-surgery. Methods: We enrolled 92 patients older than 18 years undergoing elective OPCAB-surgery. Serum-levels of S100 were determined at baseline (BL), end of surgery (EOS) and on the first postoperative day (PD1). Postoperatively, all-patients were evaluated daily until PD5 for the presence of POD using the confusion assessment method (CAM) or the confusion assessment method for the intensive care unit (CAM-ICU) for patients in the intensive care unit (ICU). Results: The overall incidence of POD was 21%. S100-values on PD1 significantly predicted the occurrence of POD during the later hospital stay [area under the curve (AUC)=0.724 (95% confidence interval (CI): 0.619–0.814); p=0.0001] with an optimal cut-off level of 123 pg mL −1 (sensitivity 100%, specificity 44%). Below this value, the absence of POD was predicted correctly in 43.66% of patients without POD [negative predictive value (NPV) of 100% (95%CI: 88.8%–100.0%) – positive predictive value of 29.8% (95%CI: 18.4%–43.4%) and likelihood ratio (LR) of the negative result of 0.0]. Conclusions: S100-levels <123 pg mL −1 measured on PD1 reliably rule out the development of POD after elective OPCAB-surgery. This finding warrants testing whether S100-levels could be used for a risk stratification of cardiac surgical patients and for the initiation of preventive measures against POD in patients with high postoperative S100-levels.

Background: Copeptin, the C-terminal part of the arginine vasopressin (AVP) precursor peptide, is secreted in response to stress and correlates with adverse clinical outcomes in the acute-care hospital setting. There are no comprehensive data regarding its prognostic value in the community. We evaluated associations of copeptin levels with 10-year mortality in patients visiting their general practitioner (GP) for a respiratory infection included in a previous trial. Methods: This is a post hoc analysis including data from 359 patients included in the PARTI trial. Copeptin was measured in batch-analysis on admission and after 7 days. We calculated Cox regression models and area under the receiver operating characteristic curve (AUC) to assess an association of copeptin with mortality and adverse outcome. Follow-up data were collected by GP, patient and relative tracing through phone interviews 10 years after trial inclusion. Results: After a median follow-up of 10.0 years, mortality was 9.8%. Median admission copeptin levels (pmol/L) were significantly elevated in non-survivors compared to survivors (13.8, IQR 5.9–27.8; vs. 6.3 IQR 4.1–11.5; p<0.001). Admission copeptin levels were associated with 10-year all-cause mortality [age-adjusted hazard ratio 1.7 (95% CI, 1.2–2.5); p<0.001, AUC 0.68]. Results were similar for discharge copeptin levels. Copeptin also predicted adverse outcomes defined as death, pulmonary embolism and major adverse cardiac and cerebrovascular events. Conclusions: In a sample of community-dwelling patients visiting their GP for a respiratory infection, copeptin levels were associated with 10-year all-cause mortality. In conjunction with traditional risk factors, this marker may help to better direct preventive measures in this population.

Background: The current research on acute appendicitis aims to improve the diagnostics and to clarify to whom antibiotic treatment might be the treatment of choice. Methods: The present study is a retrospective analysis of a prospectively collected data in our randomized multicenter trial comparing surgery and antibiotic treatment for acute uncomplicated appendicitis (APPAC trial, NCTO1022567). We evaluated 1321 patients with a clinical suspicion of acute appendicitis, who underwent computed tomography (CT). Age, gender, body temperature, pain scores, the duration of symptoms, white blood cell count (WBC) and C-reactive protein (CRP) were recorded on admission. Results: CT confirmed the diagnosis of acute appendicitis in 73% (n=970) and in 27% (n=351) it revealed no or other diagnosis. Acute appendicitis patients had significantly higher WBC levels than patients without appendicitis (median 12.2 and 10.0, respectively, p<0.0001), whereas CRP levels did not differ between the two groups. Ideal cut-off points were assessed with receiver operating characteristic (ROC) curves, but neither these markers or neither their combination nor any clinical characteristic could accurately differentiate between patients with acute appendicitis and those without. The proportion of patients with normal WBC count and CRP was significantly (p=0.0007) lower in patients with acute appendicitis than in patients without appendicitis. Conclusions: Both clinical findings and laboratory tests are unable to reliably distinguish between patients with acute appendicitis and those without. If both WBC count and CRP are normal, acute appendicitis is very unlikely. The current results emphasize the role of CT imaging in patients with suspected acute appendicitis.