Volume 53, Issue 1

The profession of laboratory medicine differs between countries within the European Union (EU) in many respects. The objective of professional organizations of the promotion of mutual recognition of specialists within the EU is closely related to the free movement of people. This policy translates to equivalence of standards and harmonization of the training curriculum. The aim of the present study is the description of the organization and practice of laboratory medicine within the countries that constitute the EU. A questionnaire covering many aspects of the profession was sent to delegates of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) and Union Européenne de Médecins Spécialistes (UEMS) of the 28 EU countries. Results were sent to the delegates for confirmation. Many differences between countries were identified: predominantly medical or scientific professionals; a broad or limited professional field of interest; inclusion of patient treatment; formal or absent recognition; a regulated or absent formal training program; general or minor application of a quality system based on ISO Norms. The harmonization of the postgraduate training of both clinical chemists and of laboratory physicians has been a goal for many years. Differences in the organization of the laboratory professions still exist in the respective countries which all have a long historical development with their own rationality. It is an important challenge to harmonize our profession, and difficult choices will need to be made. Recent developments with respect to the directive on Recognition of Professional Qualifications call for new initiatives to harmonize laboratory medicine both across national borders, and across the borders of scientific and medical professions.

Reproductive function is dependent on energy resources. The role of weight, body composition, fat distribution and the effect of diet have been largely investigated in experimental female animals as well as in women. Any alteration in diet and/or weight may induce abnormalities in timing of sexual maturation and fertility. However, the cellular mechanisms involved in the fine coordination of energy balance and reproduction are largely unknown. The brain and hypothalamic structures receive endocrine and/or metabolic signals providing information on the nutritional status and the degree of fat stores. Adipose tissue acts both as a store of energy and as an active endocrine organ, secreting a large number of biologically important molecules termed adipokines. Adipokines have been shown to be involved in regulation of the reproductive functions. The first adipokine described was leptin. Extensive research over the last 10 years has shown that leptin is not only an adipose tissue-derived messenger of the amount of energy stores to the brain, but also a crucial hormone/cytokine for a number of diverse physiological processes, such as inflammation, angiogenesis, hematopoiesis, immune function, and most importantly, reproduction. Leptin plays an integral role in the normal physiology of the reproductive system with complex interactions at all levels of the hypothalamic-pituitary gonadal (HPG) axis. In addition, leptin is also produced by placenta, where it plays an important autocrine function. Observational studies have demonstrated that states of leptin excess, deficiency, or resistance can be associated with abnormal reproductive function. This review focuses on the leptin action in female reproduction.

C-reactive protein (CRP) is a marker of inflammation. Atherosclerosis is now recognized as inflammatory disease, and it seems that CRP directly contributes to atherogenesis. Oxidation of low-density lipoprotein (LDL) molecule increases the uptake of lipid products by macrophages leading to cholesterol accumulation and subsequent foam cell formation. The elevated levels of high sensitivity CRP (hsCRP) and oxidized LDL (OxLDL) in the blood were found to be associated with cardiovascular diseases (CVD). In this review, we highlighted the evidence that CRP and OxLDL are involved in interrelated (patho) physiological pathways. The findings on association between hsCRP and OxLDL in the clinical setting will be also summarized.

Background: This study investigated approaches to continuing professional development (CPD) for specialists in laboratory medicine within four European countries: Croatia, the Czech Republic, Malta and the UK. Methods: The research questions focussed on ascertaining if continued registration/licence was linked to CPD and if so, were there requirements for certain amounts and types of CPD and for CPD activities to meet specified accreditation criteria. The Professional Associations Research Network (PARN) model of CPD measurement was applied to each country’s registration/licencing body’s CPD requirements. Results: Our results indicate a spectrum of approaches to CPD within participating countries. Conclusions: It will be necessary for European employers to be familiar with these differences and to take them into account for this increasingly mobile European workforce.

Background: The Norwegian Quality Improvement of Primary Care Laboratories (Noklus) offers external quality assurance (EQA) schemes (EQASs) for urine albumin (UA) annually. This study analyzed the EQA results to determine how the analytical quality of UA analysis in general practice (GP) offices developed between 1998 (n=473) and 2012 (n=1160). Methods: Two EQA urine samples were distributed yearly to the participants by mail. The participants measured the UA of each sample and returned the results together with information about their instrument, the profession and number of employees at the office, frequency of internal quality control (IQC), and number of analyses per month. In the feedback report, they received an assessment of their analytical performance. Results: The number of years that the GP office had participated in Noklus was inversely related to the percentage of “poor” results for quantitative but not semiquantitative instruments. The analytical quality improved for participants using quantitative instruments who received an initial assessment of “poor” and who subsequently changed their instrument. Participants using reagents that had expired or were within 3 months of the expiration date performed worse than those using reagents that were expiring in more than 3 months. Conclusions: Continuous participation in the Noklus program improved the performance of quantitative UA analyses at GP offices. This is probably in part attributable to the complete Noklus quality system, whereby in addition to participating in EQAS, participants are visited by laboratory consultants who examine their procedures and provide practical advice and education regarding the use of different instruments.

Background: A triple positive antiphospholipid (aPL) antibody profile [two positive serum IgG aPL antibodies along with one positive functional plasma lupus anticoagulant (LAC) test result] is associated with an increased risk for thrombosis, whereas patients with single positive test results may have little to no increased risk. The frequency of triple positivity in outpatients with various combinations of LAC test results is unclear. Methods: We extracted from our database all LAC test results [dilute Russell viper venom times (dRVVT) and silica clotting times (SCT)] that had concomitant serum IgG aPL testing [both serum anti β 2 -glycoprotein I (anti-β2GPI) and anti-cardiolipin (aCL) antibodies]. Results: There were 3195 patients without a prolonged prothrombin time. Double antibody positivity was found in 1% (31/2955) of those with normal functional LAC test results, in 16.0% (31/81) of those with a positive dRVVT, in 12.7% (10/79) of those with a positive SCT, and in 56.3% (45/80) of those with both tests positive (p<0.001). A triple positive aPL antibody profile was found in 28.3% (68/240) of those with at least one positive LAC test result. Conclusions: We conclude that 28% of patients with elevated LAC tests have a triple positive aPL antibody profile and patients with two positive LAC tests have a higher prevalence of a triple positive profile than do those with one positive LAC test result.

Background: Despite the well-documented role of cigarette smoke in the development of chronic obstructive pulmonary disease (COPD), lung cancer and cardiovascular disease, biomarkers for screening or monitoring disease progression and outcome remain elusive, particularly for COPD and lung cancer. Inflammatory cells and mediators are likely to be involved in the disease processes, but their importance is still poorly understood. The purpose of this study was to investigate early changes in immunological markers associated with smoking in healthy monozygotic twins without a detectable disease discordant for smoking, thereby minimising data variability due to genetic background. Methods : Twenty-two monozygotic twin pairs, aged 31.5±6.3 years, entered the study. One of each twin pair was a smoker and the other a non-smoker. None of the subjects reported any diseases or clinically defined respiratory symptoms or airflow limitation. Each subject donated blood samples for determination of total leukocytes and subpopulations, lymphocyte subpopulation plus pro-inflammatory mediators (interleukin-8, tumour necrosis factor-α, soluble tumour necrosis factor-α receptors and C-reactive protein). Results : We observed a significant increase in the number of circulating leukocytes and neutrophils in smokers compared to non-smokers. Smokers also had significantly higher numbers of B cells and CD4 + T cells, plus an increased CD4/CD8 ratio. The numbers of NK cells were statistically significant lower in smokers compared to non-smokers. Conclusions: While the prognostic significance of these changes is uncertain, results suggest that smoking is associated with immune changes, independent of genetic background and environmental conditions.

Background: Calprotectin is a 36 kDa calcium and zinc binding protein. An increased level of calprotectin points towards inflammatory bowel disease. However, the biomarker calprotectin shows 14 potential cleavages sites for trypsin. Next to trypsin, also the presence of its inhibitor α 1 -antitrypsin after a gastrointestinal bleeding may affect calprotectin testing. In this study, effects of trypsin and α 1 -antitrypsin as potential confounders for faecal calprotectin testing are investigated. Methods: An in vitro model was created. As calprotectin source, leukocytes were isolated and subsequently lysed (1% Triton X-100) and diluted in faecal matrix. Trypsin digestion was carried out by adding trypsin. Incubation occurred for 24 h or 48 h (37 °C). To study the influence of α 1 -antitrypsin on trypsin, the same experiment was repeated after adding serum containing α 1 -antitrypsin. Results: In vitro experiments enabled monitoring of the faecal calprotectin digestion, leading to loss of immunoreactivity. Trypsin activity was a potential confounder in the interpretation of calprotectin, in particular for proximal lesions, where exposure of calprotectin to trypsin is prolonged. Relative calprotectin loss was proportional to the amount of trypsin. Decrease of calprotectin was more pronounced after 48 h of incubation in comparison to 24 h of incubation. Analogue experiments also showed stable calprotectin values after adding α 1 -antitrypsin. Conclusions: Transit time, trypsin activity and addition of blood as a source of α 1 -antitrypsin may be regarded as potential confounders in the interpretation of calprotectin results. Age-related cut-off values depending on the anatomical localisation of the lesions could improve the diagnostic efficiency of calprotectin testing.

Background: Tubulointerstitial damage is a final common pathway of most renal diseases. Whether urinary neutrophil gelatinase-associated lipocalin (uNGAL), a biomarker for renal tubular damage, is of prognostic value for clinical outcomes in chronic kidney disease (CKD) patients has not been well investigated. Methods: The uNGAL and proteinuria levels were measured among a cohort of 473 advanced CKD patients of various etiologies recruited during 2002–2009. Results: The estimated glomerular filtration rate (eGFR) was 32.3±22.0 mL/min/1.73 m 2 with a urine protein-to-creatinine ratio (UPCR) 680 (255–1248) mg/g and 132 (27.9%) participants had diabetes. The baseline uNGAL level was significantly associated with male gender, eGFR, UPCR, and hemoglobin. The hazard ratio (HR) of the highest uNGAL tertile for end-stage renal disease (ESRD) was 3.44 (95% CI 1.47–8.06, p=0.004). With the adjustment of urine creatinine and urine protein, HR of the highest urine NGAL-to-creatinine ratio (UNCR) tertile and the highest urine NGAL-to-protein ratio (UNPR) tertile was 3.06 (95% CI 1.19–7.90, p=0.02) and 2.10 (95% CI 1.13–3.89, p=0.02), respectively. UNPR increased the prediction of survival model for ESRD. HR of the highest UNCR tertile and UNPR tertile for cardiovascular (CV) events was 2.21 (95% CI 0.81–5.98, p=0.08) and 2.79 (95% CI 1.25–6.26, p=0.01), respectively. None of these were associated with all-cause mortality. Conclusions: Elevated uNGAL in CKD patients is associated with risks for ESRD and probably CV events. UNPR could improve the prediction for ESRD.

Background: The absolute number of adult T-cell leukemia/lymphoma (ATL) cells in peripheral blood is an essential indicator to evaluate disease status. However, microscopically counting ATL cells based on morphology requires experience and tends to be inaccurate due to the rarity of ATL. Methods: Based on our research showing that acute-type ATL cells are specifically enriched in the CD4+/CD7- (CD7N) fraction, a new analytical method to accurately quantify ATL cells was established using an internal bead standard and simple four-color flow cytometry. This method was verified by comparison with microscopic examination of 49 peripheral blood samples and used to follow up patients. Results: A strong correlation was observed between the number of CD7N cells measured by flow cytometry and the number of abnormal lymphocytes measured microscopically by experienced technicians [Pearson’s R, 0.963; Spearman’s rho, 0.921; intercorrelation coefficient, 0.962]. The linear regression coefficient was close to 1 (β=1.013). Our method could detect 1 cell/μL, and the limit of quantitation was between 2.9 and 9.8 cells/μL. The frequency of CD7N cells among CD4+ cells changed during chemotherapy, which reflected differences between chemosensitive and chemoresistant cases. Kaplan-Meier analysis with a log-rank test showed that patients with decreased CD7N proportion after chemotherapy had significantly longer disease-specific survival (p=0.003). Conclusions: Our newly established method quantified tumor cells in patients with acute-type ATL. Furthermore, this method was useful for assessing the efficacy of chemotherapy, and the change of the CD7N proportion could be more important to predict prognosis.

Background: Fucosylation is one of the most important glycosylation events involved in cancer and inflammation. We previously developed a lectin antibody ELISA kit to measure fucosylated haptoglobin (Fuc-Hpt), which we identified as a novel cancer biomarker. In this study, we investigated Fuc-Hpt as a biomarker in chronic liver diseases, especially in hepatocellular carcinoma (HCC). Methods: We measured serum Fuc-Hpt levels using our ELISA kit in 318 patients with chronic liver diseases, including 145 chronic hepatitis (CH) patients, 81 liver cirrhosis (LC) patients, and 92 HCC patients. During a long-term follow-up period of 7 years (1996–2003), Fuc-Hpt levels were measured at three different time points in 19 HCC patients. Serum Fuc-Hpt levels were also examined with a short-term follow-up period of 3 years (2009–2012) in 13 HCC patients. Results: Fuc-Hpt levels increased with liver disease progression. Patients with LC and HCC showed significantly increased Fuc-Hpt levels in comparison to CH patients or healthy volunteers. Fuc-Hpt levels tended to be higher in HCC patients than in LC patients. Fuc-Hpt was better than α-fetoprotein (AFP) and AFP-L3 for predicting HCC [diagnosed by computed tomography (CT) or ultrasound] in LC patients with long-term follow-up. More than 80% of LC patients with long-term follow-up showed increased Fuc-Hpt during hepatocarcinogenesis, and 38% of early-stage HCC patients with short-term follow-up showed a gradual increase in Fuc-Hpt before imaging diagnosis. Conclusions: These results suggest that Fuc-Hpt is a novel and potentially useful biomarker for predicting liver disease progression and HCC development.

Background: Carbamylation is a non-enzymatic post-translational modification of proteins that has been recently identified as a non-traditional risk factor for atherosclerosis. The aim of this study was to determine whether serum homocitrulline (HCit), a characteristic carbamylation-derived product, was related to the presence and the severity of coronary artery disease (CAD). Methods: Forty-five control subjects and 109 patients were included in this cross-sectional study. After coronary angiography, the patients were classified as non-CAD patients (patients with normal arteries, n=33) and CAD patients (n=76). The severity of CAD was then evaluated using the Gensini scoring system. Serum total HCit concentrations were determined by LC-MS/MS. Results: Serum HCit concentrations were significantly (p<0.001) higher in CAD patients than in control or non-CAD subjects. The receiver operating characteristic curve analysis showed an area under the curve equal to 0.908 (95% confidence interval, 0.853–0.964, p<0.001) and a threshold HCit concentration of 0.16 mmol/mol Lys for predicting the presence of CAD (78.9% sensitivity and 78.8% specificity). HCit concentrations significantly (p<0.001) increased concomitantly with the severity of CAD and were positively correlated with Gensini scores (r=0.725, p<0.001) as well as with the number of stenotic coronary arteries (p<0.001). Furthermore, in a multiple stepwise regression analysis, HCit was significantly (p<0.001) and independently associated with the presence of CAD, the Gensini score, and the number of stenotic arteries (standardized β values of 0.525, 0.722, and 0.642, respectively). Conclusions: Our results demonstrate that serum HCit concentrations are increased during CAD and are positively associated with the severity of the disease.

Background: Our aim was to test the hypothesis that metabolic and inflammatory responses of the brain perioperatively during carotid endarterectomy (CEA) might affect blood brain barrier (BBB) integrity. Methods: Twenty patients with >70% stenosis of internal carotid artery (ICA) were prospectively included. Surgery was performed under general anaesthesia. Blood was sampled from ipsilateral internal jugular vein and radial artery: just before, during, and after ICA clamping S100B protein, glucose, lactate, 20 amino acids, and key cytokines were analysed. Results: Jugular vein S100B increased during clamping and reperfusion, while a marginal systemic increase was recorded, unrelated to stump pressure during clamping. Glucose increased during clamping in jugular vein blood and even more systemically, while jugular lactate values were higher than systemic values initially. Most amino acids did not differ significantly between jugular vein and systemic levels: glutamic acid and aspartic acid decreased during surgery while asparagine increased. Jugular vein interleukin (IL)-6 showed a transient non-significant increase during clamping and decreased systemically. IL-8 and IL-10 increased over time. Conclusions: Rising jugular vein S100B concentrations indicated reduced BBB integrity, and marginal secondary increase of S100B systemically. Limited ischaemic effects on the brain during cross-clamping, unrelated to S100B concentrations, were confirmed by lower brain glucose levels and higher lactate levels than in systemic blood. The lack of increased jugular vein glutamic acid disproves any major ischaemic brain injury following CEA. The inflammatory response was limited, did not differ greatly between jugular and systemic blood, and was unrelated to S100B.

Background: Galectin-3 is an emerging biomarker of heart failure and of myocardial fibrosis risk. Monitoring of galectin-3 is essential during treatment with galectin-3 inhibitors. The aim of our study was to assess long-term biological variability in a specific group of unhealthy subjects. Methods: The biological variability of galectin-3 was measured in a group of 44 patients after heart transplantation (HTx). Six samples were taken from each patient during a 12-month period. Galectin-3 was measured with an Abbott Architect automated immunoassay. Results: Intraindividual (CV i ) and interindividual (CV g ) variabilities were calculated together with the reference change value (RCV), the log-normal RCV for increase (RCV+), and the log-normal RCV for decrease (RCV–). The CV i , CV g , RCV, RCV+, and RCV– were 28.2%, 35.6%, 78.6%, 116%, and –53.7%, respectively. The index of individuality was 0.79. Conclusions: The concentrations of galectin-3 in patients followed 12 months after HTx fluctuated around the homeostatic point, with CV i of approximately 28%. RCVs of +116% (log-normal increase) and –54% (log-normal decrease) mean that the concentration of galectin-3 would need to approximately double or decrease by half to indicate a new process.

Background: The utility of HbA 1c for the diagnosis of type 2 diabetes requires an accurate, precise and robust test measurement system. Currently, immunoassay and HPLC are the most popular methods for HbA 1c quantification, noting however the limitations associated with some platforms, such as imprecision or interference from common hemoglobin variants. Abbott Diagnostics has introduced a fully automated direct enzymatic method for the quantification of HbA 1c from whole blood on the ARCHITECT chemistry system. Methods: Here we completed a method evaluation of the ARCHITECT HbA 1c enzymatic assay for imprecision, accuracy, method comparison, interference from hemoglobin variants and specimen stability. This was completed at three independent clinical laboratories in North America and Europe. Results: The total imprecision ranged from 0.5% to 2.2% CV with low and high level control materials. Around the diagnostic cut-off of 48 mmol/mol, the total imprecision was 0.6% CV. Mean bias using reference samples from IFCC and CAP ranged from –1.1 to 1.0 mmol/mol. The enzymatic assay also showed excellent agreement with HPLC methods, with slopes of 1.01 and correlation coefficients ranging from 0.984 to 0.996 compared to Menarini Adams HA-8160, Bio-Rad Variant II and Variant II Turbo instruments. Finally, no significant effect was observed for erythrocyte sedimentation or interference from common hemoglobin variants in patient samples containing heterozygous HbS, HbC, HbD, HbE, and up to 10% HbF. Conclusions: The ARCHITECT enzymatic assay for HbA 1c is a robust and fully automated method that meets the performance requirements to support the diagnosis of type 2 diabetes.

Background: Metabolic syndrome (MS) is an important risk factor in pediatric population for the early onset of type 2 diabetes mellitus and cardiovascular disease. New non-invasive tools are required to identify MS in at risk populations; the aim of this study was to determine an optimal cut-off point for the 13 C-glucose breath test ( 13 C-GBT) for the diagnosis of MS in adolescents. Methods: A total of 136 adolescents between 10 and 16 years old were recruited. MS was defined as: waist circumference >90th percentile and at least two of the following; high density lipoprotein-cholesterol (HDL-C) <50 mg/dL, triglycerides >110 mg/dL, diastolic and/or systolic blood pressure >90th percentile adjusted by age, gender and height, and/or fasting glucose >100 mg/dL. After the ingestion of a glucose load of 1.75 g/kg of body weight (up to 75 g) and an oral dose of 1.5 mg of universally labeled 13 C-glucose/kg dissolved in water, breath samples were taken at baseline, 30, 60, 90, 120, 150 and 180 min. Exhaled 13 CO 2 in breath samples was measured by isotope ratio mass spectrometry. Results: 13 C-GBT data, expressed as adjusted cumulative percentage of oxidized dose ( A % OD) at 180 min, was significantly higher in the healthy subjects group (17.72%±4.9%) in comparison with subjects with ≥3, 2 or 1 components of the MS (9.95%±4.73%, 14.3%±4.47% and 14.62%±4.62%, respectively). The optimal cut-off point for the A % OD was 16.09, with a sensitivity of 81.5% and a specificity of 66.7%. Conclusions: Our results demonstrate that the 13 C-glucose breath test could be a valid screening method to identify MS in adolescents.

Background: Small, dense low-density lipoprotein cholesterol (sdLDL-C) has been linked to the progression of cardiovascular disease. We compared two methods for determination of sdLDL-C, a direct enzymatic (ENZ) method and a polyacrylamide tube gel electrophoresis (PGE) assay, and investigated the associations of both sdLDL-C measurements with metabolic syndrome. Methods: We analyzed 242 patient sera for sdLDL and atherosclerosis-related markers. The PGE method separates the intermediate-density lipoprotein particles into three midbands (MID-A to MID-C) and the LDL particles into seven subfractions (LDL1 to LDL7); the sdLDL-PGE result is calculated as the sum of cholesterol concentrations from LDL3 to LDL7. Results: The regression equation for sdLDL-C was [ENZ mmol/L ]=0.779[PGE]+0.67, r=0.713. ENZ showed higher sdLDL-C concentrations than PGE (0.86±0.33 vs. 0.24±0.32 mmol/L); however, the difference was not associated with sdLDL-C concentration (p=0.290). sdLDL-C, as measured with the enzymatic assay, exhibited significant positive correlations with very-low-density lipoprotein, MID-C, MID-B, and LDL2 (all p<0.001), considered atherogenic lipoproteins, but did not correlate with the less atherogenic lipoproteins MID-A and LDL1 (all p>0.600). The ENZ and PGE methods yielded similar patterns of correlation between sdLDL-C, and atherosclerosis-related markers. Using logistic regression, sdLDL-ENZ and apolipoprotein B were identified as significant predictors of metabolic syndrome (p<0.03). Conclusions: The ENZ assay for sdLDL-C correlated well with the PGE method. The ENZ method measures a broader range of atherogenic lipoprotein particles than PGE and has the potential to identify subjects with vascular risk, thus contributing in directing specific interventions for cardiovascular prevention.