Band 53, Heft 11

Aging is a complex biological process characterized by a progressive decline of organ functions leading to an increased risk of age-associated diseases and death. Decades of intensive research have identified a range of molecular and biochemical pathways contributing to aging. However, many aspects regarding the regulation and interplay of these pathways are insufficiently understood. Telomere dysfunction and genomic instability appear to be of critical importance for aging at a cellular level. For example, age-related diseases and premature aging syndromes are frequently associated with telomere shortening. Telomeres are repetitive nucleotide sequences that together with the associated sheltrin complex protect the ends of chromosomes and maintain genomic stability. Recent studies suggest that micronutrients, such as vitamin D, folate and vitamin B12, are involved in telomere biology and cellular aging. In particular, vitamin D is important for a range of vital cellular processes including cellular differentiation, proliferation and apoptosis. As a result of the multiple functions of vitamin D it has been speculated that vitamin D might play a role in telomere biology and genomic stability. Here we review existing knowledge about the link between telomere biology and cellular aging with a focus on the role of vitamin D. We searched the literature up to November 2014 for human studies, animal models and in vitro experiments that addressed this topic.

Thrombotic microangiopathy (TMA) is a relatively rare condition but a medical urgency requiring immediate intervention to avoid irreversible organ damage or death. Symptoms on presentation include microangiopathic haemolytic anaemia, thrombocytopenia and organ damage. The most frequent direct causes of TMA are thrombotic thrombocytopenic purpura (TTP) and haemolytic uremic syndrome (HUS). The most common form of HUS is related to Shiga toxin producing Escherichia coli (STEC) infection while approximately 10% of cases are due to dysregulation of the complement pathway (atypical haemolytic uremic syndrome, aHUS). Optimal treatment regimens differ depending on the underlying cause; however, differential diagnosis may be difficult. The most accurate method of diagnosis is based on exclusion and should consider, beyond the symptoms common to TMA, ADAMTS13 activity levels and STEC infection status. For the management of TTP, plasma exchange (PE) is the most important acute intervention and is associated with lower mortality and better outcomes than plasma infusion. In most patients with STEC-HUS, the course of disease is self-limiting although management of acute kidney injury is often required. Until recently, the management of aHUS consisted of early and intensive PE, although this was mostly ineffective in protecting from subsequent organ damage. Eculizumab, an inhibitor of the alternative complement pathway, produces a rapid and sustained inhibition of the TMA process, with significant improvements in long-term clinical outcomes. Due to the significant improvement achieved, eculizumab has subsequently been approved as first-line therapy when an unequivocal diagnosis of aHUS has been made.

In many inflammatory diseases, the cellular components in body fluids [cerebrospinal fluid (CSF), serous fluids] are increased, rendering essential diagnostic information. The diagnostic value of the total white blood cell count (WBC) and differential count has been evaluated extensively over the years, and a remarkable amount of knowledge has been gained; yet, there is a great deal of clinical uncertainty whether the diagnosis should be based solely on these variables. In some diseases, such as peritonitis, the total WBC and differential count has high sensitivity; whereas, in differentiating pleural effusions, it lacks the sensitivity required to be clinically useful. Nevertheless, many guidelines consider these tests as cornerstone parameters, and in combination with clinical variables, they can successfully guide clinical decision making in initiating or postponing a treatment course for infection and/or inflammatory diseases while awaiting culture results. Although other methods are available for detecting and differentiating WBCs in body fluids, manual microscopy is still considered the gold standard despite its many limitations. During the last decade, automated analyzers have become a popular method for first line screening. Continued progress in their design has led to major improvements including their speed, improved accuracy and lower variability compared with microscopy. Disadvantages of this method include high imprecision in low ranges (depending on the method) and interfering factors. In a time where automation is at the front line in clinical laboratories, it is essential the results obtained are precise, accurate and reproducible. This review provides an overview of the relevance for cell counting in a variety of diagnostic body fluids, and highlights the current technologies used.

Laboratory medicine is amongst the fastest growing fields in medicine, crucial in diagnosis, support of prevention and in the monitoring of disease for individual patients and for the evaluation of treatment for populations of patients. Therefore, high quality and safety in laboratory testing has a prominent role in high-quality healthcare. Applied knowledge and competencies of professionals in laboratory medicine increases the clinical value of laboratory results by decreasing laboratory errors, increasing appropriate utilization of tests, and increasing cost effectiveness. This collective paper provides insights into how to validate the laboratory assays and assess the quality of methods. It is a synopsis of the lectures at the 15th European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Continuing Postgraduate Course in Clinical Chemistry and Laboratory Medicine entitled “How to assess the quality of your method?” (Zagreb, Croatia, 24–25 October 2015). The leading topics to be discussed include who, what and when to do in validation/verification of methods, verification of imprecision and bias, verification of reference intervals, verification of qualitative test procedures, verification of blood collection systems, comparability of results among methods and analytical systems, limit of detection, limit of quantification and limit of decision, how to assess the measurement uncertainty, the optimal use of Internal Quality Control and External Quality Assessment data, Six Sigma metrics, performance specifications, as well as biological variation. This article, which continues the annual tradition of collective papers from the EFLM continuing postgraduate courses in clinical chemistry and laboratory medicine, aims to provide further contributions by discussing the quality of laboratory methods and measurements and, at the same time, to offer continuing professional development to the attendees.

Background: Cystic fibrosis, caused by mutations of the CFTR gene, is the most common autosomal recessive condition in the European population and there are specific screening programs aimed at investigating healthy carriers. They are usually articulated in two steps: initially individuals are screened with a panel of the 20–50 most common CFTR mutations; the second step is offered to partners of carriers who were found negative at the first test and consists in the analysis of the entire CFTR gene. This strategy provides high sensitivity, however, it often identifies novel variants (especially in introns) of unknown significance. Establishing the pathogenicity of these variants of the CFTR gene is not a simple task. Methods: We have examined five CFTR intronic variants of unclear significance (c.274-6T>C, c.744-6T>G, c.1117-64G>A, c.2620-26A>G, and c.3468+51C>A) using a functional splicing assay based on hybrid minigenes. Results: Four out of five variants (including c.2620-26A>G which was previously reported as a possible splice-site mutation) did not alter the correct splicing of the minigene and are likely to be neutral polymorphisms, whereas c.744-6T>G caused complete skipping of CFTR exon 7 and should be therefore regarded as a pathogenic CFTR mutation. Conclusions: Hybrid minigenes assay are a simple and rapid tool to evaluate the effects of intronic variants without the need of analyzing patient’s mRNA, and are particularly suited to analyze variants identified during population screenings.

Background: International organizations require from medical laboratories a quantitative statement of the uncertainty in measurement (UM) to help interpret patient results. The French accreditation body (COFRAC) recommends an approach (SH GTA 14 IQC/EQA method) using both internal quality control (IQC) and external quality assessment (EQA) data. The aim of this work was to validate an alternative way to quantify UM using only EQA results without any need for IQC data. This simple and practical method, which has already been described as the long-term evaluation of the UM (LTUM), is based on linear regression between data obtained by participants in EQA schemes and target values. We used it for 43 routine analytes covering biochemistry, immunoassay, and hemostasis fields. Methods: Data from 50 laboratories participating in ProBioQual (PBQ) EQA schemes over 25 months were used to obtain estimates of the median and 90th percentile LTUM and to compare them to the usual analytical goals. Then, the two UM estimation methods were compared using data from 20 laboratories participating in both IQC and EQA schemes. Results: Median LTUMs ranged from 2.9% (sodium) to 16.3% (bicarbonates) for biochemistry analytes, from 12.6% (prothrombin time) to 18.4% (factor V) for hemostasis analytes when using the mean of all participants, and were around 10% for immunoassays when using the peer-group mean. Median LTUMs were, in most cases, slightly lower than those obtained with the SH GTA 14 method, whatever the concentration level. Conclusions: LTUM is a simple and convenient method that gives UM estimates that are reliable and comparable to those of recommended methods. Therefore, proficiency testing (PT) organizers are allowed to provide participants with an additional UM estimate using only EQA data and which could be updated at the end of each survey.

Background: The objective of the study was to examine the bias of albumin and albumin/creatinine (ACR) measurements in urine. Methods: Pools of normal human urine were augmented with purified human serum albumin to generate a series of 12 samples covering the clinical range of interest for the measurement of ACR. Albumin and creatinine concentrations in these samples were analyzed three times on each of 3 days by 24 accredited laboratories in Canada and the USA. Reference values (RV) for albumin measurements were assigned by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) comparative method and gravimetrically. Ten random urine samples (check samples) were analyzed as singlets and albumin and ACR values reported according to the routine practices of each laboratory. Results: Augmented urine pools were shown to be commutable. Gravimetrically assigned target values were corrected for the presence of endogenous albumin using the LC-MS/MS comparative method. There was excellent agreement between the RVs as assigned by these two methods. All laboratory medians demonstrated a negative bias for the measurement of albumin in urine over the concentration range examined. The magnitude of this bias tended to decrease with increasing albumin concentrations. At baseline, only 10% of the patient ACR values met a performance limit of RV ±15%. This increased to 84% and 86% following post-analytical correction for albumin and creatinine calibration bias, respectively. Conclusions: International organizations should take a leading role in the standardization of albumin measurements in urine. In the interim, accuracy based urine quality control samples may be used by clinical laboratories for monitoring the accuracy of their urinary albumin measurements.

Background: Accurate and reliable testing reports play an important role in the prevention, diagnosis, treatment and prognosis of disease. However, little is known about the appropriateness of laboratory testing reporting in China. This national survey takes clinical biochemistry as an example to investigate the state of reporting appropriateness in our country. Methods: An electronic questionnaire was sent to 1209 laboratories. The participants were asked to retrospectively evaluate the error rates of the following quality indicators: report template integrity, report content filling integrity, report delay, report recall, non-conformities between instrument and laboratory information system (LIS) data, non-conformities between report and request, report notification error, and report modification. Mann-Whitney and Kruskal-Wallis tests were used to identify the potential impacts of reporting appropriateness. Results: A total of 662 of the 1209 laboratories (55%) submitted the survey results, with three returning incomplete data. For the integrity of the report, only 31% of the laboratories had a complete report template that contained all of 21 elements. In addition, the overall error rate of content filling integrity was 45.9% for 19,770 pieces of reports. The overall σ-values of other six quality indicators were all >4, and no significant difference was found among different departments. Group comparison suggested that reporting electronically had a better performance. Conclusions: The laboratory reporting system in China needs to improve, particularly the integrity of the report. Strengthening information technology will not only promote reporting appropriateness, but also guarantee accurate, standardized and traceable data collection and long-term monitoring.

Background: Extensive application of measurement of urinary iodine concentration (UIC) in several benign and malignant thyroid diseases could profit by the availability of rapid and inexpensive measuring techniques. Aim of this study was to apply a simple and inexpensive commercially available potentiometric method for the quantification of UIC based on iodine-specific ion-selective electrodes (ISE) in patients with thyroid diseases. Methods: This retrospective study included patients with differentiated thyroid cancer (n=286) and patients with hyperthyroidism of different etiologies (n=203). Within the whole sample (n=489) 20 patients had previously (1 week–6 months) been exposed to iodine overload, either from contrast media (n=8) or amiodarone (n=12). Results : In patients not exposed to iodine, the histogram showed that the distribution of UIC violated normality. The peak of the curve occurred between 5.0 μmol/L and 6.0 μmol/L. Variability was sizeable (percent coefficient of variation, %CV: 66%, 95% confidence interval: 1.48–18.72 μmol/L). The group of exposed patients could be easily distinguished from not exposed patients (median UIC: 47.5 μmol/L vs. 5.42 μmol/L). UIC was significantly correlated to urinary creatinine concentration, but normalization to urinary creatinine increased the inter-subject variability of UIC (%CV=96% vs. 66%). In test-retest studies (n=25) the intra-class correlation coefficient was 0.73 for UIC, 0.82 for creatinine and 0.64 for the UIC: creatinine ratio. Conclusions: Iodine-specific ISE-based potentiometric methods can be successfully applied as an alternative to existing methods in patients with thyroid diseases. The promising characteristics of the method need to be confirmed in future larger prospective studies.

Background: 21-Hydroxylase autoantibodies (21OHAb) are markers of an adrenal autoimmune process that identifies individuals with autoimmune Addison’s disease (AAD). Quality and inter-laboratory agreement of various 21OHAb tests are incompletely known. The objective of the study was to determine inter-laboratory concordance for 21OHAb determinations. Methods: Sixty-nine sera from 51 patients with AAD and 51 sera from 51 healthy subjects were blindly coded by a randomization center and distributed to 14 laboratories that determined 21OHAb, either by an “in-house” assay (n=9) using in vitro-translated 35 S-21OH or luciferase-labeled 21OH or a commercial kit with 125 I-21OH (n=5). Main outcome measures were diagnostic accuracy of each participating laboratory and inter-laboratory agreement of 21OHAb assays. Results: Intra-assay coefficient of variation ranged from 2.6% to 5.3% for laboratories using the commercial kit and from 5.1% to 23% for laboratories using “in-house” assays. Diagnostic accuracy, expressed as area under ROC curve (AUC), varied from 0.625 to 0.947 with the commercial kit and from 0.562 to 0.978 with “in-house” methods. Cohen’s κ of inter-rater agreement was 0.603 among all 14 laboratories, 0.691 among “in-house” laboratories, and 0.502 among commercial kit users. Optimized cutoff levels, calculated on the basis of AUCs, increased the diagnostic accuracy of every laboratory (AUC >0.9 for 11/14 laboratories) and increased the Cohen’s κ of inter-rater agreement. Discrepancies in quantitation of 21OHAb levels among different laboratories increased with increasing autoantibody levels. Conclusions: The quality of 21OHAb analytical procedures is mainly influenced by selection of cutoff value and correct handling of assay materials. A standardization program is needed to identify common standard sera and common measuring units.

Background: The association of anti-C1q antibodies (anti-C1q) with the renal activity of lupus nephritis (LN) and the methods for their determination is still a matter of debate. Methods: In 116 serum samples of 66 patients with biopsy proven LN, we aimed: 1) to compare the results of the determination of anti-C1q obtained by a commercial kit with a clinically validated in-house ELISA; 2) to evaluate the correlation of anti-C1q with the most important immunological and clinical parameters employed in LN, i.e., antibodies to dsDNA (anti-dsDNA), C3 and C4 complement component, haemoglobin and haematuria. Results: Good correlation and agreement between the two methods (r=0.81, p<0.0001; contingency coefficient=0.70, p<0.0001, respectively) were demonstrated. No differences were observed between the two assays by ROC curves comparison. Anti-C1q levels were significantly higher in patients with active LN [44 arbitrary units (AUs)] in comparison to those with inactive LN (23 AUs, p=0.047) and significantly correlated with anti-dsDNA (r=0.44, p<0.0001), complement fractions (C3: r=−0.33, p=0.001; C4: r=−0.29, p=0.003), haemoglobin levels (r=−0.34, p=0.0004) and the number of urinary red blood cells (r=0.26, p=0.01). Conclusions: Our results suggest the validity of this commercial assay in detecting anti-C1q and confirm the association of anti-C1q with renal involvement of LN and the importance of introducing this parameter in the analytical panel for the evaluation of LN activity.

Background: The identification of platelet alloantibodies is indispensible for the diagnoses of fetal/neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusions. Methods: We compared the results obtained with the gold-standard assay for the detection and specification of platelet antibodies, the monoclonal antibody-specific immobilization of platelet antigen assay (MAIPA), with those from two bead-based assays, simultaneous analysis of specific platelet antibodies assay (SASPA), and the recently commercialized bead-based assay Pak Lx. Results: For the detection of alloantibodies by SASPA, the sensitivity was 94.6%, the specificity was 99.5% The respective results for Pak Lx were 98% and 99.8%. Some antibody specificities were revealed by SASPA or Pak Lx but not by MAIPA. However, there were also antibodies detected by MAIPA that were not seen by SASPA or Pak Lx. Antibody dilution experiments suggest that SASPA or Pak Lx are more sensitive than MAIPA. Conclusions: All three assays have limitations, but Pak Lx is suited for screening. However, other assays are necessary if results are not conclusive or for evaluations beyond the commercial assay’s design.

Background: The aim of this study was to identify an inflammatory marker with fast and predictable kinetics to enable future discrimination between normal postoperative inflammation and potential infection after total hip and knee arthroplasty cases. Methods: Changes in serum levels of C-reactive protein (CRP), interleukins (IL) 1β, 6 and 8 and NT-proCNP peptide were measured before and during first 5 postoperative days in 100 patients undergoing total hip or knee arthroplasty. We also compared two methods to describe the magnitude of inflammation – applying separate two sample tests at each time point, and summary measures – area under the curve (AUC). Results: IL-6 showed a similar kinetics pattern to the CRP in response to surgical trauma. Significantly greater level changes in all markers but NT-proCNP were observed in knee patients. Persisting high levels of CRP, but not other markers, were observed in obese hip patients. IL-6 was found to be an adjunct to routine CRP use. Conclusions: IL-6 has faster kinetics and is less influenced by patient weight, therefore it seems to be more useful in clinical practice. Summary measures describe the inflammatory response well and are easier to analyze than multiple analyses of single time points.

Background: Preeclampsia (PE) is a pregnancy complication characterized by high blood pressure and significant amounts of protein in the urine. Various coagulation abnormalities have been described in pregnant women with PE. The aim of the present case-control study was to evaluate whole blood thromboelastometry profiles, performed by ROTEM ® , in women with PE in order to better characterize the PE-related discoagulopathy. Methods: Standard ROTEM ® (Tem International GmbH, Munich, Germany) parameters evaluating clot initiation [clotting time (CT)], propagation [clot formation time (CFT); α-angle], stability [maximum clot firmness (MCF)] and lysis [maximum lysis (ML)] in INTEM, EXTEM, NATEM, and FIBTEM assays were performed in 30 consecutive pregnant women with PE at diagnosis. Sixty (1:2 ratio with cases) healthy pregnant women, matched for gestational age (±2 weeks) with the cases, acted as controls. Platelet count, prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, antithrombin and D-Dimer were also evaluated. Results: Preeclamptic women showed a significantly more rapid propagation phase in EXTEM assay than controls (CFT 62±15 vs. 75±15 s and α-angle 78±4 vs. 75±4°, p<0.01 in both cases). Moreover, MCF was significantly higher and ML significantly lower in women with PE than in healthy pregnant women (p=0.001 for all comparisons). Conclusions: ROTEM ® profiles in women with PE were characterized by an increased tissue factor driven clot propagation capability. In addition, higher clot stability due both to the increase in clot firmness and the decrease in blood fibrinolysis was observed. Larger studies are needed to identify the clinical relevance of ROTEM ® alterations in women with PE.

Background: Urokinase plasminogen activator receptor (uPAR) is a key component of the fibrinolytic system involved in extracellular matrix remodeling and angiogenesis. Novel animal models supported the key role of uPAR not only in fibrosis but also in systemic sclerosis (SSc)-related microvascular abnormalities. The aim of this study was to investigate plasma soluble uPAR (suPAR) levels in SSc, and their association with organ-specific involvement. Methods: suPAR concentrations were measured by ELISA in SSc patient (n=83) and in healthy controls (n=29). Simultaneously, CRP and ESR were assessed. Detailed clinical data including skin, lung, heart and microvascular characteristics were evaluated at sampling. Results: suPAR values were higher in SSc patients than in controls. Subgroup analysis showed higher suPAR values in diffuse cutaneous- than in limited cutaneous SSc and correlated with anti-Scl-70+. suPAR levels also associated with pulmonary function test parameters of fibrosis, presence of microvascular lesions (e.g., Raynaud phenomenon, naifold capillaroscopic abnormalities and digital ulcers) and arthritis. Conclusions: Our data indicate that suPAR might be a valuable early diagnostic marker of SSc which also correlates with disease severity.

Background: Bile acids (BA) are found predominantly in bile but also in serum, where they can be used as markers for inborn and acquired hepatobiliary disorders. We measured serum BA levels by mass spectrometry to determine reference ranges for healthy children and adolescents in different age groups. Methods: In 194 healthy children and adolescents (0–19 years) concentrations of serum BA and BA composition were determined using high-performance liquid chromatography high-resolution mass spectrometry. Individuals were classified by ages into five groups: 0–5 months, 6–24 months, 3–5 years, 6–11 years, and >11 years. Results: The 95% confidence interval of serum total BA values in newborns was 3.85–6.32 μmol/L. In the cohort aged 6–24 months total BA values were significantly higher (6.61–9.43 μmol/L; p<0.001). During growth, values decreased (6–11 years; 3.61–5.41 μmol/L), and after 11 years (3.09–4.12 μmol/L) resembled those in adults (0.28–6.50 μmol/L). With respect to conjugation patterns, in neonates BA were primarily conjugated with taurine; however, after 6 months glycine conjugates clearly predominated. Conclusions : Our data show that serum BA values vary substantially during the first years of life and that reference ranges for BA are age-dependent. The physiologic mechanisms underlying these variations remain to be determined.

Background: Early diagnosis and treatment of neonatal infection is important to prevent morbidity and mortality. The gastrointestinal tract-derived hormones ghrelin and peptide YY (PYY), which participate in the regulation of food intake and energy balance, may also play roles in the inflammatory response. Their involvement in neonatal infection is not known. Methods: Plasma ghrelin and PYY(3-36) levels were serially measured (by ELISA) on Days 0, 1, 2, 3 and 7 following admission in 36-term neonates with febrile infection (22 of them were septic) and once in 20 healthy term neonates of similar postnatal age and gender distribution, as controls. Associations of ghrelin and PYY(3-36) levels with clinical and laboratory parameters, including anthropometrics, fever, leukocyte and platelet counts, serum glucose, C-reactive protein (CRP) and serum amyloid A levels, were assessed. Results: Plasma ghrelin levels were significantly higher in infected neonates than in controls at each study day (p=0.009), whereas PYY(3-36) levels did not differ significantly between patients and controls at any day. In infected neonates, ghrelin levels on admission correlated negatively with serum glucose levels (p=0.003), whereas fever change during the course of infection was significantly associated with change of ghrelin levels (p=0.01). Receiver operating characteristic analysis of ghrelin levels resulted in significant areas under the curve (AUC) for detecting infected neonates on admission (AUC=0.728, p=0.005). Conclusions: Circulating ghrelin, but not PYY(3-36), levels are increased in neonates with infection, possibly reflecting and/or participating in the inflammatory process.

Background: The aim of the study was to compare NT-proBNP and BNP levels in fresh samples from heart failure (HF) patients measured using 10 immunoassays and to assess their agreement. Methods: NT-proBNP (CobasH232 ® , Elecsys ® , Vidas ® , Vista ® , XPand ® , Vitros ® ) and BNP (Triage ® , Access ® , CentaurXP ® , Architect ® ) levels were measured in 39 heparin and 19 EDTA samples, respectively. Results: The Pearson correlation coefficient ranged between 0.929 (Triage ® -Centaur ® ) and 0.994 (Access ® -Architect ® ) for BNP assays and between 0.972 (Vidas ® -Cobas H232 ® ) and 0.999 (Vitros ® -Vidas ® ) for NT-proBNP assays. Passing Bablok regression analyses showed a significant difference in the slopes [0.80 (Centaur ® -Triage ® ) to 1.84 (Architect ® -Centaur ® )] and intercepts [–55 ng/L (Architect ® -Centaur ® ) to 48 ng/L (Access ® -Triage ® )] for BNP assays, and a lower heterogeneity between NT-proBNP assays [0.83 (Vidas ® -Elecsys ® ) to 1.20 (Vitros ® -Vidas ® ) and –97 ng/L (XPand ® -CobasH232 ® ) to 51 ng/L (CobasH232 ® -Elecsys ® ) for slopes and intercepts, respectively]. The concordance correlation coefficient revealed a poor (ρ c <0.90) to moderate (ρ c= 0.90–0.95) agreement in 4/6 pairs of BNP assays and an almost perfect (ρ c >0.99) agreement in 5/15 pairs of NT-proBNP assays. The acceptable difference limit reflecting the number of individual discrepant results between two assays, ranged between 15.1% (Access ® -CentaurXP ® ) and 34.5% (Architect ® -Triage ® ) for BNP assays, and between 10.9% (Vidas ® -Vitros ® ) and 55% (CobasH232 ® -Xpand ® ) for NT-proBNP assays. Conclusions: This study stresses the lack of transferability of the results obtained using different techniques to measure BNP and NT-proBNP levels in fresh samples. Individual reference ranges and HF diagnostic cut-offs should be assessed for each commercial NP immunoassay. We recommend to systematically monitoring HF patients using the same assay (BNP or NT-proBNP) over the time.

Background: The routine use of brain natriuretic peptide (BNP) in pediatric cardiac surgery remains controversial. Our aim was to test whether BNP adds information to predict risk in pediatric cardiac surgery. Methods: In all, 587 children undergoing cardiac surgery (median age 6.3 months; 1.2–35.9 months) were prospectively enrolled at a single institution. BNP was measured pre-operatively, on every post-operative day in the intensive care unit, and before discharge. The primary outcome was major complications and length ventilator stay >15 days. A first risk prediction model was fitted using Cox proportional hazards model with age, body surface area and Aristotle score as continuous predictors. A second model was built adding cardiopulmonary bypass time and arterial lactate at the end of operation to the first model. Then, peak post-operative log-BNP was added to both models. Analysis to test discrimination, calibration, and reclassification were performed. Results: BNP increased after surgery (p<0.001), peaking at a mean of 63.7 h (median 36 h, interquartile range 12–84 h) post-operatively and decreased thereafter. The hazard ratios (HR) for peak-BNP were highly significant (first model HR=1.40, p=0.006, second model HR=1.44, p=0.008), and the log-likelihood improved with the addition of BNP at 12 h (p=0.006; p=0.009). The adjunction of peak-BNP significantly improved the area under the ROC curve (first model p<0.001; second model p<0.001). The adjunction of peak-BNP also resulted in a net gain in reclassification proportion (first model NRI=0.089, p<0.001; second model NRI=0.139, p=0.003). Conclusions: Our data indicates that BNP may improve the risk prediction in pediatric cardiac surgery, supporting its routine use in this setting.

Background: A systemic inflammatory response is observed after cardiopulmonary resuscitation. We investigated two novel inflammatory markers, pentraxin 3 (PTX3) and soluble suppression of tumorigenicity 2 (sST2), in comparison with the classic high-sensitivity C-reactive protein (hsCRP), for prediction of early multiple organ dysfunction syndrome (MODS), early death, and long-term outcome after out-of-hospital cardiac arrest. Methods: PTX3, sST2, and hsCRP were assayed at ICU admission and 48 h later in 278 patients. MODS was defined as the 24 h non-neurological Sequential Organ Failure Assessment (SOFA) score ≥12. Intensive care unit (ICU) death and 12-month Cerebral Performance Category (CPC) were evaluated. Results: In total, 82% of patients survived to ICU discharge and 48% had favorable neurological outcome at 1 year (CPC 1 or 2). At ICU admission, median plasma levels of hsCRP (2.8 mg/L) were normal, while levels of PTX3 (19.1 ng/mL) and sST2 (117 ng/mL) were markedly elevated. PTX3 and sST2 were higher in patients who developed MODS (p<0.0001). Admission levels of PTX3 and sST2 were also higher in patients who died in ICU and in those with an unfavorable 12-month neurological outcome (p<0.01). Admission levels of PTX3 and sST2 were independently associated with subsequent MODS [OR: 1.717 (1.221–2.414) and 1.340, (1.001–1.792), respectively] and with ICU death [OR: 1.536 (1.078–2.187) and 1.452 (1.064–1.981), respectively]. At 48 h, only sST2 and hsCRP were independently associated with ICU death. Conclusions: Higher plasma levels of PTX3 and sST2, but not of hsCRP, at ICU admission were associated with higher risk of MODS and early death.

Background: The role of triglycerides carried in the triglyceride-rich lipoproteins (TRL) in the progression of atherosclerosis is uncertain. Identification of oxidized triglycerides and its possible association with atherosclerosis were largely ignored. Here we applied mass spectrometric approach to detect and identify triglyceride hydroperoxides (TGOOH) in human plasma and lipoproteins. Methods: EDTA plasma was collected from healthy human volunteers (n=9) after 14–16 h of fasting. Very low-density lipoprotein (VLDL) (d <1.006) and intermediate-density lipoprotein (IDL) ( d =1.006–1.019) were isolated from the plasma (n=6) by sequential ultracentrifugation in KBr, followed by the isolation of LDL and high-density lipoprotein (HDL) using size-exclusion high-performance liquid chromatography (HPLC). Total lipids from the plasma and isolated lipoproteins were extracted, and analyzed for the detection and identification of TGOOH using liquid chromatography/LTQ ion trap Orbitrap mass spectrometry. All the processes, from specimen collection to the mass spectrometric analysis, were carried out at 4 °C in the presence of antioxidant to prevent oxidation of lipoproteins. Results: We identified 11 molecular species of TGOOH in either plasma or VLDL and IDL, of which TGOOH-18:1/18:2/16:0, TGOOH-18:1/18:1/16:0, TGOOH-16:0/18:2/16:0, TGOOH-18:1/18:1/18:1, and TGOOH-16:0/20:4/16:0 were most dominant. These TGOOH molecules are carried by TRL but not by LDL and HDL. Mean concentration of TGOOH in plasma, VLDL and IDL were, respectively, 56.1±25.6, 349.8±253.6 and 512.5±173.2 μmol/mol of triglycerides. Conclusions: This is the first report to identify several molecular species of oxidized triglycerides in TRL. Presence of oxidized triglyceride may contribute to the atherogenicity of TRL. Further work is needed to elucidate the association of the oxidized triglyceride in atherosclerosis.

Background: Low-density lipoprotein cholesterol (LDL-C) is an established risk factor for cardiovascular disease and is usually estimated by the Friedewald formula (FF) calculated from three parameters, namely, total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C). We aimed to develop a new and simple formula (NF) for LDL-C estimation. Methods: This cross-sectional study enrolled two study populations (a testing group, n=16,749, and a validation group, n=4940). Linear regression analysis was used in the testing group to investigate the association between measured LDL-C (mLDL-C) and TC concentration, and was verified in the validation group. Results: The NF yielded an estimated LDL-C (eLDL-C) equal to 0.75×total cholesterol–0.6465 (mmol/L). For the subjects with TC between 2.58 and 7.74 mmol/L, the difference between mLDL-C and eLDL-C using the NF was less than that from the FF (testing group: –0.04 to –0.20 vs. –0.28 to –0.38 mmol/L; validation group: 0.01 to –0.12 vs. –0.23 to –0.30 mmol/L; p<0.001, respectively). The predictability of the NF was not inferior to that of the FF in subjects with different triglyceride and HDL-C concentrations, and was not affected by diabetes diagnosis and statin use. However, the NF performed similar to or worse than the FF at TC concentrations <2.58 mmol/L and >7.74 mmol/L, respectively. Conclusions: In the Chinese population, the accuracy of eLDL-C measurement with the NF was better than that with the FF, especially in subjects with TC levels between 2.58 and 7.74 mmol/L. The NF is simple and may be used for screening as well as for follow-up of patients on lipid lowering agents.