Band 52, Heft 9

The analysis of intact parathyroid hormone (PTH) (PTH1-84) is useful in the diagnosis of hyper- and hypocalcaemia, hyperparathyroidism, and in the prevention of bone mineral disorders in renal patients. The analysis is complicated by the presence of PTH fragments, which may accumulate in renal failure and cross-react in immunoassays, including the most recent third-generation immunoassays. Large variability exists between different commercially available assays. This article reviews the current literature on PTH testing, with emphasis on the use of mass spectrometry-based methods, and considers the important sources of variation which still need to be addressed prior to the development of much needed candidate reference methods for PTH analysis. Recently, mass spectrometric methods have been developed for the quantitation of PTH1-84 using surrogate tryptic peptides, but even these methods are subject to significant interferences due to the presence of newly observed modified PTH species, such as oxidised and phosphorylated PTH variants, which can accumulate in patient samples. Further work, including: 1) the use of high-resolution mass spectrometry; and 2) the analysis of PTH without prior protease digestion, is required before these approaches can be considered as reference methods against which other methods should be harmonised.

Migraine is a highly prevalent and frequently disabling disorder. Since the pathogenesis of this condition has a strong inflammatory component and migraine is significantly associated with cardiovascular disease, we assess whether C-reactive protein (CRP) may be epidemiologically or casually linked with migraine. An electronic search on Medline, Scopus and Web of Science produced 17 studies reporting original data about the epidemiological association between CRP and migraine (1 retrospective, 1 interventional, 14 cross-sectional and 1 both interventional and cross-sectional). When all studies reporting sufficient data about CRP values were pooled (n=12; 6980 cases and 38,975 controls), the concentration of CRP was found to be significantly higher in patients with migraine than in controls (weighted mean difference 1.12 mg/L; 95% CI 1.01–1.25 mg/L; p<0.001). In further analysis of studies containing separate data for migraine with and without aura (n=7), CRP values remained significantly higher in both migraineur patients with aura (n=1939; weighted mean difference 0.88 mg/L; 95% CI 0.63–1.14 mg/L; p<0.001) or without aura (n=2483; weighted mean difference 1.04 mg/L; 95% CI 0.78–1.30 mg/L; p<0.001) when compared with controls (n=29,354). Despite a large inter-study heterogeneity (99.3%), our analysis provides evidence of a potential epidemiological association between increased concentration of CRP and migraine, thus paving the way for further clinical investigations about therapeutic agents that may contextually decrease the risk of cardiovascular disease and reduce the burden of migraine.

ABO blood group antigens are complex carbohydrate molecules expressed on the surface of red blood cells and a variety of human cells and tissues. It is well known that ABO blood type exerts a profound influence on hemostasis, being a major determinant of von Willebrand factor (VWF), and consequently factor VIII, plasma levels. In this review, we will focus on the molecular mechanisms underlying the interaction between ABO blood group and VWF in normal and pathological conditions.

Background: Lactase persistence is an inherited autosomal dominant trait that confers the ability to digest lactose after weaning. Lactose persistence is caused by single nucleotide variants in a regulatory element for the lactase gene ( LCT ). In Europeans, lactase persistence is associated with the nucleotide variant LCT -13910C>T. Ethnic groups from Africa and the Arabian Peninsula have other lactase persistence variants in close proximity to the -13910C>T variant. Current hybridisation probe analysis methods have been limited by the inability to detect all the known nucleotide variants. The aim of this study was to devise a method based on hybridisation probes and melting curve analysis for rapidly detecting LCT variants. Methods: We designed hybridisation probes for producing melting curve profiles that could identify seven LCT nucleotide variants: -13907C>G, -13910C>T, -13913T>C, -13914G>A, -13915T>G, -14009T>G, and -14010G>C. To validate the assay, we generated plasmid standards of all the alleles and mixed them to create artificial heterozygote samples. Results: All genotypes could be detected in a single tube assay. Rare genotypes were confirmed in a second assay with probes that had different complementary sequences. Conclusions: This assay can be used for rapidly genotyping lactase persistence in multi-ethnic populations.

Background: The measurement of serum IgE aids in the diagnosis and management of atopic allergic disease and hyper-IgE immunodeficiency syndromes. The 2nd World Health Organization (WHO) International Reference Reagent (IRR) for serum IgE (75/502; 5000 IU/ampoule), is widely used to calibrate assays for serum IgE. Exhaustion of stocks of the 2nd IRR necessitated the production of a replacement preparation and its evaluation in an international collaborative study to determine its suitability to serve as the 3rd International Standard (IS) for serum IgE. Methods: Sera and defibrinated plasma with elevated IgE levels were pooled and lyophilised in ampoules. This preparation, coded 11/234, was assayed by 18 laboratories in 11 countries using commercial assay methodology for IgE, along with the 2nd IRR, 75/502, and two lyophilised serum samples. Results: Overall, there were no consistent differences in the way that the candidate IS (11/234), the IRR (75/502), and the two serum samples behaved in the assays with respect to linearity and parallelism. The mean IgE value of the candidate IS, 11/234, relative to the IRR, 75/502, was 13,411 IU/mL based on parallel line analysis of raw assay data at NIBSC, and 13,551 IU/mL based on the laboratories’ own estimates after correcting for the values obtained for 75/502. Conclusions: The use of 11/234 will ensure that assays for serum IgE continue to be well standardised. The preparation was established by the WHO Expert Committee on Biological Standardization as the 3rd IS for serum IgE with an assigned value of 13,500 IU/mL, corresponding to 6750 IU/ampoule.

Background: Cardiovascular prevention requires a diagnostic approach for population-based screening programmes aimed at early identification of modifiable risk factors. Dyslipidaemia, which is a major risk factor for cardiovascular disease, remains largely undiagnosed and undertreated. Point-of-care testing (POCT) provides immediate results for clinical decision-making, however, quality assessment is essential to ensure system performance requirements. CardioChek PA (CCPA), a portable whole blood analyser for rapid lipid measurement, has been on the market since 2002 but with limited evidence of performance levels. The aim of the study was to assess analytical performance issues of the device and possibly contribute to their solution. Methods: Over a 3-year period, we repeatedly evaluated CCPA accuracy, precision, and discrepancies between instruments and between test strip lots by comparison with laboratory method. At our initial evaluation, the CCPA analyser underestimated total cholesterol (bias 6.5%) and gave within-assay CVs above 6% for all lipid fractions. Our results solicited sequential improvements to the CCPA system by the manufacturer up to the performance level certified by the Cholesterol Reference Method Laboratory Network in 2013: total error 1.3% for total cholesterol and 3.1% for high density lipoprotein (HDL) cholesterol. Conclusions: POCT diagnostic tests need continuous quality management, including both quality control and quality assurance, to monitor the analytical process. A 3-year external quality surveillance provided information useful in improving POCT performance. As a result, the device is now adequate for use in screening programmes aimed at early detection of lipid disorders.

Background: The assessment of proper hydration status in hemodialysis patients is difficult. None of currently available markers or measures is clinically relevant. Recently, human pre-pro-vasopressin (1-164) split product [copeptin (CPP)] – a new surrogate marker of hydration status – was introduced. The aim of the study was to analyze body weight changes in the early post-transplant period in relation to serum CPP levels before kidney transplantation. Methods: Serum CPP and NT-proBNP concentrations and osmolality were measured in 130 kidney recipients directly prior to transplantation and, additionally, in 78 of them at 14th day post-transplant. Hydration status at transplantation was calculated from the difference in the patient’s body weight before transplantation and at the discharge. Results: During the post-transplant hospitalization, the average weight change was –1.6 kg, varying from 10.5 kg loss to weight gain of 5 kg. The overall weight loss was significantly related to pretransplant serum concentration of CPP (r=0.238), but not of NT-proBNP or osmolality. Patients with the lowest initial CPP level (first tertile) had smaller post-transplant weight loss. The early kidney graft function was unrelated to pretransplant CPP. Multivariate regression model revealed that variability of post-transplant weight loss is explained by the number of antihypertensive drugs used prior to transplantation [β=0.213 (0.049–0.377)] and pretransplant CPP values [β=0.233 (0.069–0.397)]. Conclusions: Elevated serum CPP level predicts a rapid weight loss after kidney transplantation and seems to characterize the subgroup of patients with the greatest overhydration. These results suggest the dysregulation of physiological mechanisms of CPP secretion in hemodialysis patients.

Background: Thyroid disorders are common in women of reproductive age, and thyroid dysfunction during pregnancy has been associated with adverse outcomes for mother and child. Thyroid function and thyroid function tests (TFTs) can be influenced by a variety of factors, such as ethnicity, the presence of autoimmune thyroid disease (AITD), dietary iodine intake, pregnancy, and methodological differences. However, no large-scale studies have been published which examine TFTs and prevalence of AITD in Mexican pregnant women and women of reproductive age. Methods: TFTs and thyroid autoantibody testing were performed on 660 pregnant and 104 non-pregnant women from Mérida, Yucatán, Mexico. After removal of thyroid autoantibody positive individuals and women with thyroid stimulating hormone (TSH) >4.94 mIU/L, reference intervals were calculated for TFT for non-pregnant women and pregnant women by trimester. Results: Anti-thyroidperoxidase antibodies (TPO-Ab) and/or anti-thyroglobulin antibodies (Tg-Ab) were positive in 14.4% and 13.5% of non-pregnant and pregnant women, respectively. TSH values were significantly higher in women who were positive for TPO-Ab and co-positive for TPO-Ab and Tg-Ab. TSH values were also significantly higher in Tg-Ab positive pregnant women. Other TFTs were not significantly different based on antibody status. Using antibody negative women, reference intervals were determined for TFTs in pregnant (gestational age-specific) and non-pregnant women. Conclusions: Laboratory evidence of AITD is common in this population of Mexican pregnant and non-pregnant women. TFT results and reference intervals are influenced by pregnancy and thyroid autoimmunity. For optimal interpretation of TFT results, gestational age-specific reference intervals established using a local patient population should be used.

Backgrounds: This case-control study was designed to assess the possible role of fetuin-A, a multifunctional protein, in reproductive processes of women undergoing in vitro fertilization (IVF). Methods: Paired serum and follicular fluid (FF) samples were obtained from 35 unselected patients who received IVF treatment. Their main clinical chartacteristics: age 35.2±5.4 years, BMI 22.9±2.8, duration of infertility 3.2±2.0 years, gonadotropin administered 1199.4±202.3 IU, and estradiol level on day 6 of stimulation 1827±1342 pmol/L. In total 25 healthy women of similar age admitted for minor elective surgery served as controls for serum analysis. Fetuin-A and high-sensitivity C-reactive protein (hsCRP) were measured in serum and FF by using commercially available ELISA kits and turbidimetric immunoassay, respectively. Results: Serum fetuin-A levels of patients participating IVF are markedly elevated when compared to those of healthy women (1.30±0.58 g/L vs. 0.55±0.09 g/L, p<0.000). Fetuin-A in FF proved to be comparable to its serum levels (1.17±0.45 g/L) and no relationship could be detected between the respective individual values. Moreover, markers of reproductive potential (number of oocytes and embryos) appeared to be independent of serum and FF fetuin-A. hsCRP in serum and FF was not related to fetuin-A and did not influence the number of oocytes and embryos. Conclusions: Fetuin-A concentration is high in FF of patients undergoing IVF, however, it can not be used as an estimate of fertilization success.

Background: Development of non-invasive markers that can predict the stages of hepatic fibrosis without resorting to repeated liver biopsies is still an important goal to evaluate the effectiveness of antifibrotic treatment. The present work investigates the value of the assessment of peripheral circulating reelin, in which the liver represents its prime source, as a marker for monitoring hepatic fibrogenesis. Methods: Seventy-four cases with chronic hepatitis positive for serum HCV RNA and 15 healthy volunteers were enrolled in this study. Assessment of reelin in the harvested serum and in 64 corresponding liver biopsies using immunofluorescence technique was done. The results were evaluated in relation to the stages and quantitative morphometric analysis of hepatic fibrosis as well as the serum levels of the validated biomarker hyaluronic acid. Results: Significant correlation was detected between the levels of serum reelin and the semiquantitative assessment of reelin immunoreactivity in liver tissue, the stages of hepatic fibrosis, the morphometrically determined collagen and serum hyaluronic acid with a correlation coefficient of 0.675, 0.623, 0.479, 0.772, respectively with p<0.001. The sensitivity and the specificity of reelin for the determination of advanced (F2+F3) and significant fibrosis (F2–F4) were nearly comparable to the result of hyaluronic acid. In addition the area under curve (AUC) were 0.859, 0.871 for the reelin versus 0.878, and 0.891 for the hyaluronic acid. Conclusions: In conclusion serum reelin may be considered an additional useful parameter for monitoring the progression of hepatic fibrosis in HCV-infected patients specially in those with active rheumatological conditions which result in an increase in serum hyaluronic acid.

Background: Primary antiphospholipid syndrome (PAPS) is an autoimmune disease characterized by thrombosis and/or pregnancy morbidity as well as blood antiphospholipid (aPL) antibodies such as anticardiolipin (aCL), anti-β2 glycoprotein I (anti-β2GPI) antibodies of the IgG/IgM isotype and lupus anticoagulant (LA). The clinical significance of aCL and anti-β2GPI antibodies of the IgA isotype in PAPS is still a controversial issue. Methods: Sera and plasma were collected from 84 PAPS patients (54 with thrombosis and/or pregnancy morbidity and 30 with pregnancy morbidity alone), 66 seronegative patients (subjects with clinical manifestations of PAPS although with negative results on conventional antiphospholipid antibody testing), and 78 healthy blood donors. IgA aCL and IgA anti-β2GPI were determined using fluorescence enzyme immunoassay (FEIA), (EliA TM , Phadia AB, Uppsala, Sweden). For comparison purposes, the sera were also tested for IgG/IgM aCL/anti-β2GPI antibodies using the same immunoassay method. LA was assayed following internationally accepted guidelines. Results: Present respectively in 19% and 50% of the PAPS patients studied, IgA aCL and IgA anti-β2GPI antibody frequencies were both statistically significant (p=0.001 and p<0.001, respectively). The mean titers of both IgA aCL and IgA anti-β2GPI antibodies were higher in the thrombotic patients, but only the latter were significantly associated with thrombosis. Isolated IgA anti-β2GPI antibody positivity was significantly prevalent (p=0.04) in seven (10.6%) of the seronegative patients. Conclusions: Positivity to IgA anti-β2GPI antibody detected using FEIA was found to be clinically relevant in PAPS patients. Moreover the prevalence of isolated IgA anti-β2GPI antibody positivity was significant in the seronegative patients.

Background: Calculation of the cerebrospinal fluid/serum glucose (CSF/S Glu ) ratio is part of the routine CSF work-up, however, different cut-off values ranging from 0.3 to 0.5 have been suggested so far to distinguish physiological from pathological conditions. The objective of this study was to determine cut-off values for normal CSF/S Glu ratio dependent on serum glucose concentrations. Methods: We screened our database for paired CSF and serum samples, which have been collected by lumbar puncture, were processed within 1 h after withdrawal, showed cell count <15/3, erythrocyte count <1500/3 and normal CSF total protein resulting in 1036 sample pairs. Glucose concentrations in CSF and serum were measured by enzymatic spectrophotometry. Results: Median glucose concentration in CSF was approximately 60% of that in serum. CSF/S Glu ratios negatively correlated with serum glucose levels (R=–0.586, p<0.001) and cut-off values for normal CSF/S Glu ratio defined as the 5th percentile were 0.5 for patients with serum glucose concentrations <100 mg/dL, 0.4 for those with a glucose level of 100–149 mg/dL and 0.3 for serum glucose concentrations ≥150 mg/dL. Conclusions: CSF/S Glu ratio inversely correlates with serum glucose concentrations in a non-linear manner. These findings suggest that cut-off values for normal CSF/S Glu ratio must be adjusted to serum glucose levels, probably explaining the considerably varying cut-offs that have been reported so far.

Background: Chronic heart failure is a common disease with a high morbidity and mortality. In primary care, brain natriuretic peptide (BNP) is used for excluding heart failure. The Dutch and European Society of Cardiology heart failure guidelines apply two BNP cut-off levels without making distinction for gender and age. The aim of our study was to establish BNP reference ranges for use in primary care. Methods: We investigated BNP values of 9447 eligible subjects in a primary care laboratory. For establishing the reference ranges in various age and gender classes we used the Bhattacharya method. Results: Analysis of variance demonstrated that BNP data were significantly dependent on age and gender (p<0.001 and p=0.002, respectively), with the age effect being the strongest. Further, we found that the reference ranges were significantly higher than the cut-off values used in the heart failure guidelines, particularly in elderly subjects. For example, the proportion of individuals with a BNP value higher than the 29 pmol/L cut-off increased from approximately 5% in the youngest group to no less than about 50% in the oldest subjects. Conclusions: BNP reference ranges need to be age- and gender-specific. When applying a single cut-off, many healthy subjects, especially the elderly, would be falsely diagnosed as having elevated BNP, and referred for further unnecessary diagnostics.

Background: %p2PSA and prostate health index (phi) has shown valuable results in the detection of prostate cancer (PCa), improving the prediction of the aggressiveness of the tumor. The goal of the present study was to evaluate %p2PSA and phi in the detection of PCa, estimating their relationship with the aggressiveness of PCa. Methods: A total of 354 patients with positive or negative prostatic biopsy were included. Prospectively, 150 were enrolled and 204 were enrolled retrospectively proceeding from our serum bank. Results: The best performance was observed for %p2PSA and phi, obtaining an AUC of 0.723 and 0.732, respectively. The highest specificity at sensitivity around 90% was obtained for phi (27.4%). Using the cut-off of 31.94 for phi, a reduction of 19% biopsies could be obtained, while 17 PCa would have been missed, including only four patients with a Gleason score ≥7. Similarly, using a cut-off of 1.21 for %p2PSA, a reduction of 12.7% biopsies could be obtained, while 16 PCa would have been missed, including only four patients with a Gleason score ≥7. Moreover, among patients with PCa, phi (median: 69.75 vs. 48.04) and %p2PSA (median: 2.60 vs. 1.98) values are significantly higher (p<0.0001) in patients with a biopsy Gleason score ≥7. Conclusions: Our results confirm previous evaluations, showing similar AUCs and results in sensitivity and specificity to other studies.%p2PSA and phi raise the accuracy in the detection of prostate cancer, reducing the number of unnecessary biopsies and improving the prediction of the aggressiveness of the tumor.

Background: CD4+CD25+ forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) accumulate in malignant tumors and negatively regulate antitumor immunity. However, the clinical significance of Tregs in HCC remains unclear. To determine the prognostic value of Tregs, we conducted a retrospective study on 264 patients with Barcelona Clinic Liver Cancer (BCLC) stage B hepatocellular carcinoma (HCC) who underwent transcatheter arterial chemoembolization (TACE). Methods: We measured the proportion of peripheral blood Tregs in 105 healthy donors and 264 HCC patients (stage B) prior to and following TACE between 2005 and 2007. All HCC patients were followed up until December 2012. The correlations between the proportion of Tregs and clinicopathologic factors were analyzed, and long-term survival rate after TACE according to the percentage of Tregs was assessed by univariate and multivariate analyses. Results: The 1-, 2-, 3-, 4- and 5-year cumulative survival rates were 62.1%, 32.6%, 16.5%, 10.4% and 6.9%, respectively, and the median survival time was 19.0 months. The cumulative survival rate was significantly lower in patients with higher levels of peripheral blood Treg cells compared to those with lower Treg levels (p<0.001). Furthermore, we found that both pre- and post-TACE peripheral blood Treg levels showed significant negative association with overall survival time (p<0.001). Conclusions: Elevated peripheral blood CD4+CD25+FOXP3+Treg levels are an independent predictive factor of poor survival after TACE for HCC (stage B) patients. These results suggest that targeting Tregs may improve patient outcomes, and provide a strong rationale for testing these approaches in future immunotherapy-based clinical trials.

Background: Cumulative evidence has demonstrated that carbonic anhydrase IX (CAIX) is upregulated in many types of human cancers. We attempted to evaluate plasma levels of CAIX in patients with oral cancer and investigated whether plasma CAIX is correlated with the progression of this disease. Method: In total, 191 patients with oral cancer, 30 patients with oral submucous fibrosis and 100 controls were recruited in this study. The plasma samples were collected and the levels of soluble CAIX in plasma were determined by the enzyme-linked immunosorbent assay (ELISA). Furthermore, the normal buccal mucosa fibroblast was challenged by arecoline, the major areca nut alkaloid, to assess the relationship between the levels of CAIX and areca nut chewing in oral cancer patients. Results: Results showed that patients with oral cancer exhibited significantly higher levels of soluble CAIX compared to controls (p<0.001). Plasma levels of CAIX in oral cancer patients were associated with clinical stages after adjusting for age and areca nut chewing (p<0.05). In addition, patients with areca nuts chewing had higher CAIX levels than those who have not chewed areca nuts. Total carbonic anhydrase activity and CAIX mRNA levels were significantly higher in oral submucous fibrosis fibroblasts than in normal buccal mucosa fibroblasts. Moreover, arecoline elevated CAIX expression in a dose-dependent manner in normal buccal mucosa fibroblasts. Conclusions: Our results suggest that determining plasma levels of CAIX may be used as a non-invasive method for monitoring oral cancer progression and the involvement of areca quid chewing in oral carcinogenesis may be related to a higher expression of CAIX.

Background: High sensitivity C-reactive protein (hs-CRP) is an inflammatory marker associated with increased cardiovascular disease risk. Health-related behaviors and factors decrease the risk of cardiovascular disease events, but the association between ideal cardiovascular health behaviors and hs-CRP is not clear. This study evaluated the relationship between the number of ideal cardiovascular health metrics and hs-CRP levels. Methods: We conducted a cross-sectional study from 2006 to 2007 that included 93,967 healthy subjects and 7869 diabetic patients. hs-CRP was measured by high-sensitivity nephelometry. Multivariate logistic regression was performed to analyze the association between the number of ideal cardiovascular health metrics and hs-CRP levels in the general population and in non-diabetic and diabetic subjects. Results: We found that hs-CRP levels decreased as the number of ideal cardiovascular health metrics increased in the general population, different genders, non-diabetic and diabetic subgroups. After adjusting for traditional risk factors, logistic regression analyses revealed that the risk of hs-CRP >3 mg/L in the general population with 6–7 ideal cardiovascular health metrics was 0.39 times lower than in those with 0–1 ideal metrics. Similarly, the risks were 0.39 times lower than in those with 0–1 ideal metrics in non-diabetic population. The risk of hs-CRP >3 mg/L in the diabetic population with 4–6 ideal cardiovascular health metrics was 0.45 times lower than those with 0–1 ideal metrics. Conclusions: Our findings demonstrate an inverse relationship between the number of ideal health metrics and hs-CRP in the general population, and similar relationships were observed for different gender, non-diabetic and diabetic subgroups.