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With the increasing prevalence of cardiovascular diseases (CVD) worldwide, finding reliable and clinically relevant biomarkers to predict acute cardiovascular events has been a major aim of the scientific and medical community. Improvements of the understanding of the pathophysiological pathways of the disease highlighted the major role of lipoprotein particles, and these past decades have seen the emergence of a number of new methodologies to separate, measure and quantitate lipoproteins. Those methods, also known as advanced lipoprotein testing methods (ALT), have gained acceptance in the field of CVD risk assessment and have proven their clinical relevance. In the context of worldwide standardization and harmonization of biological assays, efforts have been initiated toward standardization of ALT methods. However, the complexity of lipoprotein particles and the multiple approaches and methodologies reported to quantify them have rendered these initiatives a critical issue. In this context and to better understand these challenges, this review presents a summary of the major methods available for ALT with the aim to point out the major differences in terms of procedures and quantities actually measured and to discuss the resulting comparability issues.

Breakpoint cluster region-Abelson ( BCR-ABL1 ) translocation is the characteristic sign of chronic myeloid leukemia (CML). The quantitation of BCR-ABL1 messenger RNA is requisite for patients with CML, and reverse-transcription real-time quantitative polymerase chain reaction (RQ-PCR) is the method used most extensively in testing laboratories worldwide. Nevertheless, substantial variation in RQ-PCR results from different laboratories makes interlaboratory comparability inconvincible owing to the lack of standardization. To facilitate interlaboratory comparative assessment and international standardization, an international scale (IS) for BCR-ABL1 was proposed. The laboratory-specific conversion factors derived from the IS can convert local different values to the IS without changing procedures. The standardization of BCR-ABL1 also includes the whole analytical process, so it is noteworthy to pay attention to the quality control before BCR-ABL1 quantitative analysis. More importantly, the World Health Organization has validated a first genetic reference panel which is limited to the manufacturers to produce and calibrate secondary reference reagents. Also, a certified reference plasmid, ERM-AD623, was internationally accepted. This article mainly focuses on BCR-ABL1 measurement and these standardization efforts in progress.

Epigenetic modifications represent an interesting landscape which can describe relevant features of human disease. Epigenetic biomarkers show several advantages as disease biomarkers because they provide information about gene function, specific endophenotypes and can even incorporate information from the environment and the natural history of disease. The improvement in genomic and epigenomic technologies has revolutionized the current comprehension of biological processes underlying health and disease. However, now is the time to adopt these new technologies to improve human health, thus converting this information into reliable biomarkers. This endeavor should be focused on improving methodologies to analyze gene methylation, histone modifications and microRNAs. Ideally, epigenetic biomarkers should be robust, routine, accurate and inexpensive in order to provide better information for patient diagnosis, prognosis, stratification and treatment monitoring. Here we describe some challenges and provide strategies to improve the adoption of epigenetic biomarkers into clinical routine. Furthermore, we summarize the recommended properties for clinical epigenetic biomarkers.

The improving quality of laboratory testing requires a deep understanding of the many vulnerable steps involved in the total examination process (TEP), along with the identification of a hierarchy of risks and challenges that need to be addressed. From this perspective, the Working Group “Laboratory Errors and Patient Safety” (WG-LEPS) of International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) is focusing its activity on implementation of an efficient tool for obtaining meaningful information on the risk of errors developing throughout the TEP, and for establishing reliable information about error frequencies and their distribution. More recently, the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has created the Task and Finish Group “Performance specifications for the extra-analytical phases” (TFG-PSEP) for defining performance specifications for extra-analytical phases. Both the IFCC and EFLM groups are working to provide laboratories with a system to evaluate their performances and recognize the critical aspects where improvement actions are needed. A Consensus Conference was organized in Padova, Italy, in 2016 in order to bring together all the experts and interested parties to achieve a consensus for effective harmonization of quality indicators (QIs). A general agreement was achieved and the main outcomes have been the release of a new version of model of quality indicators (MQI), the approval of a criterion for establishing performance specifications and the definition of the type of information that should be provided within the report to the clinical laboratories participating to the QIs project.

Background: Much attention has been focused on epidermal growth factor receptor ( EGFR ) mutation testing since the introduction of EGFR -tyrosine kinase inhibitors have improved survival in EGFR -positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used. Methods: From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis. Results: We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples. Conclusions: cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.

Background: There is increasing interest in direct patient engagement including receiving their laboratory medicine results. We previously established an appetite for Specialists in Laboratory Medicine to support patients in understanding results. The aim of this study was to establish whether patients agreed with such an approach, determined through surveying views in eight European countries. Methods: A standardized five-question survey was administered across eight European countries to a total of 1084 individuals attending medical outpatient clinics, with 100 patients each in Poland, Serbia, Netherlands, Turkey and Czech Republic, 101 in Estonia, 116 in Denmark and 367 in Norway. The responses across countries were compared using the chi-square test (p<0.05). Results: Patients wanting their results ranged from 50% to 94% (mean 65%) of those responding positively, a mean of 72% wanted additional information with their results; direct receipt was preferred over referral to a website. Specialists in Laboratory Medicine providing such information were acceptable to a mean of 62% of those respondents wishing their results; in countries where payment was possible, there was little interest in making additional payment for such a service. Conclusions: A clear proportion of patients are interested in receiving their laboratory medicine results, the majority with explanatory notes; a role for Specialists in Laboratory Medicine is acceptable and raises the potential for direct engagement by such specialists with patients offering a new paradigm for the provision of laboratory medicine activities.

Background The International Standard ISO 15189 is recognized as a valuable guide in ensuring high quality clinical laboratory services and promoting the harmonization of accreditation programmes in laboratory medicine. Examination procedures must be verified in order to guarantee that their performance characteristics are congruent with the intended scope of the test. The aim of the present study was to propose a practice model for implementing procedures employed for the verification of validated examination procedures already used for at least 2 years in our laboratory, in agreement with the ISO 15189 requirement at the Section 5.5.1.2. Methods In order to identify the operative procedure to be used, approved documents were identified, together with the definition of performance characteristics to be evaluated for the different methods; the examination procedures used in laboratory were analyzed and checked for performance specifications reported by manufacturers. Then, operative flow charts were identified to compare the laboratory performance characteristics with those declared by manufacturers. Results The choice of performance characteristics for verification was based on approved documents used as guidance, and the specific purpose tests undertaken, a consideration being made of: imprecision and trueness for quantitative methods; diagnostic accuracy for qualitative methods; imprecision together with diagnostic accuracy for semi-quantitative methods. Conclusions The described approach, balancing technological possibilities, risks and costs and assuring the compliance of the fundamental component of result accuracy, appears promising as an easily applicable and flexible procedure helping laboratories to comply with the ISO 15189 requirements.

Background: To provide its participants with an external quality assessment system (EQAS) that can be used to check trueness, the Dutch EQAS organizer, Organization for Quality Assessment of Laboratory Diagnostics (SKML), has innovated its general chemistry scheme over the last decade by introducing fresh frozen commutable samples whose values were assigned by Joint Committee for Traceability in Laboratory Medicine (JCTLM)-listed reference laboratories using reference methods where possible. Here we present some important innovations in our feedback reports that allow participants to judge whether their trueness and imprecision meet predefined analytical performance specifications. Methods: Sigma metrics are used to calculate performance indicators named ‘sigma values’. Tolerance intervals are based on both Total Error allowable (TEa) according to biological variation data and state of the art (SA) in line with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Milan consensus. Results: The existing SKML feedback reports that express trueness as the agreement between the regression line through the results of the last 12 months and the values obtained from reference laboratories and calculate imprecision from the residuals of the regression line are now enriched with sigma values calculated from the degree to which the combination of trueness and imprecision are within tolerance limits. The information and its conclusion to a simple two-point scoring system are also graphically represented in addition to the existing difference plot. Conclusions: By adding sigma metrics-based performance evaluation in relation to both TEa and SA tolerance intervals to its EQAS schemes, SKML provides its participants with a powerful and actionable check on accuracy.

Background: Potassium is an important serum ion that is frequently assayed in clinical laboratories. Quality assurance requires reference methods; thus, the establishment of a candidate reference method for serum potassium measurements is important. Methods: An inductively coupled plasma mass spectrometry (ICP-MS) method was developed. Serum samples were gravimetrically spiked with an aluminum internal standard, digested with 69% ultrapure nitric acid, and diluted to the required concentration. The 39 K/ 27 Al ratios were measured by ICP-MS in hydrogen mode. The method was calibrated using 5% nitric acid matrix calibrators, and the calibration function was established using the bracketing method. Results: The correlation coefficients between the measured 39 K/ 27 Al ratios and the analyte concentration ratios were >0.9999. The coefficients of variation were 0.40%, 0.68%, and 0.22% for the three serum samples, and the analytical recovery was 99.8%. The accuracy of the measurement was also verified by measuring certified reference materials, SRM909b and SRM956b. Comparison with the ion selective electrode routine method and international inter-laboratory comparisons gave satisfied results. Conclusions: The new ICP-MS method is specific, precise, simple, and low-cost, and it may be used as a candidate reference method for standardizing serum potassium measurements.

Background: Laboratory test ordering under time pressure may impact test-ordering behavior. Methods: To investigate the test-ordering behavior of doctors working under such pressure, we designed a questionnaire for trainees and staff in the Emergency Department (ED). This questionnaire addressed topics such as necessity of requested tests, time spent on ordering, costs and availability of tests, and the time of the day. We hypothesized that ordering behavior would be guided predominantly by the medical need of tests and aimed at identifying practical motives that also have an effect. Results: Remarkably, two-third of the respondents (67%) admitted that tests were ordered that would not influence treatment policy directly and 48% of the doctors stated that tests were ordered that do not impact treatment at all. The frequency of such orders was “sometimes” and “frequent” in a 50:50 ratio. Interestingly, tests that could prove relevant at a later stage are often ordered simultaneously to reduce burden on the patient. None of the respondents spent more than 3 min on the ordering process and very few (8%) desired more time for ordering. Most respondents (81%) declared to have limited knowledge of the costs of laboratory tests. A random survey covering four tests confirmed this. Generally, turnaround time did influence ordering behavior while time of the day did not. Conclusions: In conclusion, doctors in an ED – besides first of all medical motives – heavily exploit practical (non-medical) reasoning for laboratory test ordering, e.g. taking availability of tests into account and ordering non-immediate tests.

Background: Measurement of adequate glucose concentrations is complicated by in vitro breakdown of glucose due to glycolysis. Unlike the commonly used NaF-EDTA and NaF-oxalate phlebotomy tubes, citrated NaF-EDTA tubes are reported to directly and thereby completely inhibit glycolysis. Recently, Greiner introduced the Vacuette ® FC-Mix NaF-EDTA-citrate tube, currently the only NaF-citrate tube without volume-disturbing liquid additions available on the European market. Here we present its potential as alternative for the laborious and therefore unfeasible conditions for glucose sampling as recommended by the World Health Organization (WHO). Methods: The FC-Mix tube was tested against the WHO recommended method of optimal laboratory conditions, both in healthy volunteers and pregnant woman undergoing oral glucose tolerance test (oGTT) for screening of gestational diabetes mellitus (GDM). Glucose concentrations were measured after different incubation times (0–48 h) and temperatures (room temperature, 37 °C), both in uncentrifuged whole blood and centrifuged material. Results: Deming regression analysis shows that glucose concentrations measured in the FC-Mix tube correlate to the WHO recommended method. Stability is maintained at room temperature for 48 h and at least 24 h at 37 °C. The use of the FC-Mix tube was also validated in screening for GDM and proved comparable to the WHO recommended method in diagnostic outcome. Conclusions: The new Greiner FC-Mix tube combines the easy handling of a routine tube with dry additive with the ability to immediately inhibit glycolysis as in the WHO method for optimal pre-analytical and analytical conditions and performs equally to those conditions when screening for GDM.

Background: The pneumatic tube system (PTS) is commonly used in modern clinical laboratories to provide quick specimen delivery. However, its impact on sample integrity and laboratory testing results are still debatable. In addition, each PTS installation and configuration is unique to its institution. We sought to validate our Swisslog PTS by comparing routine chemistry, hematology, coagulation and blood gas test results and sample integrity indices between duplicate samples transported either manually or by PTS. Methods: Duplicate samples were delivered to the core laboratory manually by human courier or via the Swisslog PTS. Head-to-head comparisons of 48 routine chemistry, hematology, coagulation and blood gas laboratory tests, and three sample integrity indices were conducted on 41 healthy volunteers and 61 adult patients. Results: The PTS showed no impact on sample hemolysis, lipemia, or icterus indices (all p<0.05). Although alkaline phosphatase, total bilirubin and hemoglobin reached statistical significance (p=0.009, 0.027 and 0.012, respectively), all had very low average bias which ranged from 0.01% to 2%. Potassium, total hemoglobin and percent deoxyhemoglobin were statistically significant for the neonatal capillary tube study (p=0.011, 0.033 and 0.041, respectively) but no biases greater than ±4% were identified for these parameters. All observed differences of these 48 laboratory tests were not clinically significant. Conclusions: The modern PTS investigated in this study is acceptable for reliable sample delivery for routine chemistry, hematology, coagulation and blood gas (in syringe and capillary tube) laboratory tests.

Background: A decreased level of Aβ1-42 in cerebrospinal fluid (CSF) is characteristic of Alzheimer disease and often used to support clinical diagnosis. The measured concentration of CSF Aβ1-42, however, depends strongly on several pre-analytical and analytical “confounding” factors such as sample collection, material of testing tube, CSF handling and storage procedures (e.g. transfer to new tubes after centrifugation, freeze-thaw effects). As a consequence, substantial variations in the measured levels of this biomarker are observed even for the same sample. This study investigates whether the accuracy of quantitative analysis of CSF Aβ1-42 can be improved by pre-analytical treatment of CSF with agents that could potentially reduce a freeze-thaw and adhesion-related depletion of Aβ1-42 from CSF, including modulators of Aβ aggregation and cryoprotecting or anti-adhesion agents. Methods: The concentration of CSF Aβ1-42 was assessed with a novel Elecsys immunoassay developed for quantification of Aβ1-42 in human CSF. Results: Low-molecular weight Aβ oligomerization inhibitors, β-sheet breaker peptides, or the mid domain 4G8 antibody do not improve the stability of CSF Aβ1-42 during a repeated freeze-thaw treatment. Cryoprotecting agents reduce a freeze-thaw dependent loss of Aβ1-42 only when spiked to CSF to final concentration of 300 mM or higher. Adhesion of Aβ1-42 can be prevented by pre-treating CSF with Tween or by using tubes with a siliconized surface. Conclusions: Between-center variability in measured level of CSF Aβ1-42 can be reduced only by standardized CSF collection into one specific tube that, without centrifugation, transfer or other types of pre-analytical processing, is directly analyzed after sample collection.

Background: Cannabis has been used since ancient times to relieve neuropathic pain, to lower intraocular pressure, to increase appetite and finally to decrease nausea and vomiting. The combination of the psychoactive cannabis alkaloid Δ9-tetrahydrocannabinol (THC) with the non-psychotropic alkaloids cannabidiol (CBD) and cannabinol (CBN) demonstrated a higher activity than THC alone. The Italian National Institute of Health sought to establish conditions and indications on how to correctly use nationally produced cannabis to guarantee therapeutic continuity in individuals treated with medical cannabis. Methods: The evaluation of cannabinoids concentration and stability in standardized preparations of cannabis tea and cannabis oil was conducted using an easy and fast ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay. Results: Extraction efficiency of oil was significantly higher than that of water with respect to the different cannabinoids. This was especially observed in the case of the pharmacologically active THC, CBD and their acidic precursors. Fifteen minutes boiling was sufficient to achieve the highest concentrations of cannabinoids in the cannabis tea solutions. At ambient temperature, a significant THC and CBD decrease to 50% or less of the initial concentration was observed over 3 and 7 days, respectively. When refrigerated at 4 °C, similar decreasing profiles were observed for the two compounds. The cannabinoids profile in cannabis oil obtained after pre-heating the flowering tops at 145 °C for 30 min in a static oven resulted in a complete decarboxylation of cannabinoid acids CBDA and THCA-A. Nevertheless, it was apparent that heat not only decarboxylated acidic compounds, but also significantly increased the final concentrations of cannabinoids in oil. The stability of cannabinoids in oil samples was higher than that in tea samples since the maximum decrease (72% of initial concentration) was observed in THC coming from unheated flowering tops at ambient temperature. In the case of the other cannabinoids, at ambient and refrigerated temperatures, 80%–85% of the initial concentrations were measured up to 14 days after oil preparation. Conclusions: As the first and most important aim of the different cannabis preparations is to guarantee therapeutic continuity in treated individuals, a strictly standardized preparation protocol is necessary to assure the availability of a homogeneous product of defined stability.

Background: We evaluated the analytical performance of six different faecal calprotectin immunoassays together with their diagnostic accuracy in the discrimination between functional and organic bowel disorders. Methods: The faecal samples were obtained from inflammatory bowel disease patients (n=27) at the time of diagnosis [Crohn’s disease (n=15), colitis ulcerosa (n=12)], gastroenterologic disease control patients (n=52) and rheumatologic disease control patients (n=26). All individuals included in the study underwent a concurrent ileocolonoscopy. Analytical performance (imprecision, accuracy, carry-over, correlation and agreement) and diagnostic accuracy (sensitivity, specificity, likelihood ratios) of the different assays were evaluated. Results: All methods demonstrated good analytical performance, but within-run and total imprecision varied depending on the assay methodology used. Using Passing Bablok and Bland-Altman analyses, low quantitative agreement was observed between the assays. All assays showed excellent diagnostic accuracy, with areas under the receiver operating characteristic curves (ROC) ranging from 0.974 to 0.998. The AUCs were not significantly different between assays (p>0.05). Diagnostic sensitivity at the cut-off at a fixed specificity of 75% ranged from 95.2% to 100%. Introduction of multiple result intervals increased the clinical interpretation of all the assays. Conclusions: Analytical and diagnostic performance of the evaluated faecal calprotectin assays is good, but numerical values differ substantially between the assays necessitating the use of different clinical cut-offs. Introduction of multiple result intervals aids in clinical decision-making.

Background: Pancreatic autoantibodies (PABs) are detected in patients with inflammatory bowel disease (IBD). Their prevalence is higher in Crohn’s disease (CrD) than in ulcerative colitis (UC). Glycoprotein 2 (GP2) and, more recently, CUB and zona pellucida-like domain-containing protein 1 (CUZD1) have been identified as target autoantigens of PAB. The clinical utility of CUZD1 autoantibodies has only recently been assessed by indirect immunofluorescence (IIF) assays. In this study, we developed and validated novel immunoassays for the detection of CUZD1 autoantibodies. Methods: Recombinant CUZD1 protein was utilized as a solid-phase antigen for the development of two immunoassays for the detection of IgG and IgA CUZD1 autoantibodies. Serum samples from 100 patients with CrD, 100 patients with UC, 129 patients assessed for various autoimmune diseases (vADs) and 50 control individuals were analyzed. Results: Two immunofluorometric assays for the detection of IgG and IgA CUZD1-specific antibodies were developed. CUZD1 autoantibodies were detected in 12.5% (25/200) IBD patients, including 16% of patients with CrD and in 9% of patients with UC (CrD vs. UC, p<0.05), compared with 3.1% (4/129) patients suspected of having vADs (CrD vs. ADs, p<0.05; UC vs. ADs, p=0.08). CUZD1 autoantibody positivity was not found to be related to disease location, age of disease onset or disease phenotype. Conclusions: This is the first study to describe novel IgA and IgG CUZD1 autoantibody enzyme-linked immunosorbent assay. These immunoassays agree well with standard IIF techniques and can be utilized in multicenter studies to investigate the diagnostic and clinical utility of CUZD1 autoantibodies.

Background: Many discriminant formulas have been reported for distinguishing thalassemia trait from iron deficiency in patients with microcytic anemia. Independent verification of several discriminant formulas is deficient or even lacking. Therefore, we have retrospectively investigated discriminant formulas in a large, well-characterized patient population. Methods: The investigational population consisted of 2664 patients with microcytic anemia: 1259 had iron deficiency, 1196 ‘pure’ thalassemia trait (877 β- and 319 α-thalassemia), 150 had thalassemia trait with concomitant iron deficiency or anemia of chronic disease, and 36 had other diseases. We investigated 25 discriminant formulas that only use hematologic parameters available on all analyzers; formulas with more advanced parameters were disregarded. The diagnostic performance was investigated using ROC analysis. Results: The three best performing formulas were the Jayabose (RDW index), Janel (11T), and Green and King formulas. The differences between them were not statistically significant (p>0.333), but each of them had significantly higher area under the ROC curve than any other formula. The Jayabose and Green and King formulas had the highest sensitivities: 0.917 both. The highest specificity, 0.925, was found for the Janel formula, which is a composite score of 11 other formulas. All investigated formulas performed significantly better in distinguishing β- than α-thalassemia from iron deficiency. Conclusions: In our patient population, the Jayabose RDW index, the Green and King formula and the Janel 11T score are superior to all other formulas examined for distinguishing between thalassemia trait and iron deficiency anemia. We confirmed that all formulas perform much better in β- than in α-thalassemia carriers and also that they incorrectly classify approximately 30% of thalassemia carriers with concomitant other anemia as not having thalassemia. The diagnostic performance of even the best formulas is not high enough for making a final thalassemia diagnosis, but in countries with limited resources, they can be helpful in identifying those patients who need further examinations for genetic anemia.

Background: Rotational thromboelastometry (ROTEM) is an attractive method for rapid evaluation of hemostasis in neonates. Currently, no reference values exist for ROTEM assays in full-term and pre-term neonates. Our aim was to establish reference ranges for standard extrinsically activated ROTEM assay (EXTEM) in arterial blood samples of healthy full-term and pre-term neonates. Methods: In the present study, EXTEM assay was performed in 198 full-term (≥37 weeks’ gestation) and 84 pre-term infants (<37 weeks’ gestation) using peripheral arterial whole blood samples. Results: Median values and reference ranges (2.5th and 97.5th percentiles) for the following main parameters of EXTEM assay were determined in full-term infants: clotting time (seconds), 41 (range, 25.9–78); clot formation time (seconds), 70 (range, 40–165.2); maximum clot firmness (mm), 66 (range, 41–84.1); lysis index at 60 min (LI60, %), 97 (range, 85–100). The only parameter with a statistically significant difference between full-term and pre-term neonates was LI60 (p=0.006). Furthermore, it was inversely correlated with gestational age (p=0.002) and birth weight (p=0.016) in pre-term neonates. Conclusions: In conclusion, an enhanced fibrinolytic activity in pre-term neonates was noted. For most EXTEM assay parameters, reference ranges obtained from arterial newborn blood samples were comparable with the respective values from studies using cord blood. Modified reagents, small size samples, timing of sampling, and different kind of samples might account for any discrepancies among similar studies. Reference values hereby provided can be used in future studies.

Background: The management of IgM monoclonal gammopathies undetermined significance (IgM-MGUS) and Waldenstrom’s macroglobulinemia (WM) may be challenging. Modern immunoassays that quantify specific monoclonal heavy and light chain immunoglobulins are promising for their use in these applications. Methods: Ninety consecutive patients (39 IgM-MGUS, 32 indolent WM [iWM], and 19 WM) seen between January 2007 and March 2014 were analyzed. Heavy/light chain (HLC) and serum free light chains assays (FLC) were determined at diagnosis to study their utility as biomarkers in IgM monoclonal gammopathies. Results: The HLC involved to uninvolved IgM ratios (iHLC/uHLC) showed a progressive increase when going from IgM-MGUS, to iWM and to WM (p=0.002). Furthermore, an iHLC/uHLC>62 identified a group of iWM patients with a shorter time-to-progression (TTP) (108 vs. 133 months, p=0.033). Separate analysis of the involved and uninvolved components showed that only the suppression of the uninvolvedimmunoglobulin was predictive of shorter TTP (HR=3.04, p=0.03) suggesting that it could be the majorcontributor to the prognostic value of the Hevylite assay. Additionally, a multivariate analysis showed that immunosuppression (either classical immunoparesis or Hevylite immunosuppression) was an independent prognostic factor (p=0.016) reinforcing its relevance in the disease mechanism. Finally, monoclonal sFLC levels were highest in WM patients, with 83% presenting values>60 mg/L. Conclusions: The results suggest that the levels of immunosuppression and/or the iHLC/uHLC ratio of IgM immunoglobulins measured by Hevylite are associated with greater disease activity which significantly impacts in the outcome of WM patients and may also help in the differentiation of IgMMGUS from iWM.

Background: Risk prediction algorithms for coronary heart disease (CHD) are recommended for clinical use. However, their predictive ability remains modest and the inclusion of genetic risk may improve their performance. Methods: QRISK2 was used to assess CHD risk using conventional risk factors (CRFs). The performance of a 19 single nucleotide polymorphism (SNP) gene score (GS) for CHD including variants identified by genome-wide association study and candidate gene studies (weighted using the results from the CARDIoGRAMplusC4D meta-analysis) was assessed using the second Northwick Park Heart Study (NPHSII) of 2775 healthy UK men (284 cases). To improve the GS, five SNPs with weak evidence of an association with CHD were removed and replaced with seven robustly associated SNPs – giving a 21-SNP GS. Results: The weighted 19 SNP GS was associated with lipid traits (p<0.05) and CHD after adjustment for CRFs, (OR=1.31 per standard deviation, p=0.03). Addition of the 19 SNP GS to QRISK2 showed improved discrimination (area under the receiver operator characteristic curve 0.68 vs. 0.70 p=0.02), a positive net reclassification index (0.07, p=0.04) compared to QRISK2 alone and maintained good calibration (p=0.17). The 21-SNP GS was also associated with CHD after adjustment for CRFs (OR=1.39 per standard deviation, 1.42×10 −3 ), but the combined QRISK2 plus GS score was poorly calibrated (p=0.03) and showed no improvement in discrimination (p=0.55) or reclassification (p=0.10) compared to QRISK2 alone. Conclusions: The 19-SNP GS is robustly associated with CHD and showed potential clinical utility in the UK population.

Background: NT-proBNP level is a predictive factor in hypertrophic cardiomyopathy patients, in which left ventricular outflow tract obstruction contributes to an increased NT-proBNP level. However, studies regarding the influence of septal myectomy on NT-proBNP level in hypertrophic obstructive cardiomyopathy are lacking. Methods: One hundred and eighty-two patients who underwent septal myectomy in Fuwai Hospital from May 2011 to March 2016 and were included in the present study. Preoperative and follow-up NT-proBNP was retrospectively collected. Correlation analysis was performed to determine the factors affecting preoperative NT-proBNP and postoperative decrease. Results: The cohort had a median age of 46.2 [IQR: 36.5–53.1] years, and 106 (58.2%) patients were male. NT-proBNP decreased to 816.5 [IQR: 400.3–1661.8] pg/mL from preoperative 1732.4 [IQR: 819–3296.5] pg/mL (p<0.001). Baseline NT-proBNP was correlated with gender (p<0.001), maximum septal thickness (p<0.001), and resting pressure gradient (p=0.006). The extent of NT-proBNP decrease was positively correlated with age (p<0.001), baseline NT-proBNP (p<0.001), follow-up time (p=0.020), and enlargement of the ascending aorta (p=0.019). NT-proBNP exhibited a persistent decrease after myectomy. Conclusions: Myectomy significantly reduced NT-proBNP level in hypertrophic obstructive cardiomyopathy patients, in which a time-dependent manner of decrease indicated myocardial remodeling of the heart after myectomy.

Background: Caspase-cleaved cytokeratin (CCCK)-18 is a protein released into the blood during apoptosis. Higher circulating CCCK-18 concentrations have been found in non-survivor than in survivor septic patients at moment of sepsis diagnosis. The following questions arise now: (1) How are serum CCCK-18 levels during the first week of sepsis? (2) Is there an association between sepsis severity and mortality and serum CCCK-18 levels during the first week? The aims of this study were to answer these questions. Methods: Multicenter study with 321 severe septic patients from eight Spanish intensive care units. We determined serum concentration of CCCK-18, tumor necrosis factor (TNF)-α, and interleukin (IL)-10 during the first week. Our end-point study was 30-day mortality. Results: Non-survivor (n=108) compared to survivor patients (n=213) showed higher serum CCCK-18 levels at days 1, 4 and 8 (p<0.001). ROC curve analyses showed that serum CCCK-18 levels at days 1 (AUC=0.77; 95% CI=0.72–0.82), 4 (AUC=0.81; 95% CI=0.76–0.85) and 8 (AUC=0.83; 95% CI=0.78–0.88) could predict mortality at 30 days (p<0.001). Logistic regression analyses showed that serum CCCK-18 levels at days 1 (OR=4.367; 95% CI=2.491–7.659), 4 (OR=10.137; 95% CI=4.741–21.678) and 8 (OR=8.781; 95% CI=3.626–21.268) were associated with 30-day mortality (p<0.001). We found a positive correlation between CCCK-18, SOFA, and lactic acid at days 1, 4 and 8. Conclusions: Non-survivor septic patients showed persistently during the first week higher serum CCCK-18 levels than survivor patients, and there is an association between sepsis severity and mortality and serum CCCK-18 levels during the first week.