Volume 43, Issue 5

Traditional gene expression studies typically focus on one or a few genes of interest. An important limitation of single-gene studies is that they present a portrait of disease that is essentially static. However, disease is a dynamic process, driven by a combination of genetic, epigenetic and environmental factors. Recently, genomic technologies have permitted better characterization of the dynamic aspect of disease progression. Genome-wide expression profiles of cardiovascular diseases, heart failure in particular, using microarrays have been published and are providing new insights into this complex disease. Tissue biopsies required for traditional microarray studies, however, are often invasive and not readily available. By contrast, blood samples are relatively non-invasive and are readily available. In a number of recent studies, blood cells appear to be a viable substitute for tissue biopsy. Blood cells have the ability to mirror the body's tissues and organs in health and disease; thus, we hypothesize that blood cells can indicate at the molecular level the presence of disease. Here we review microarray gene expression profiling of blood RNA for a number of different diseases. Sieving through gene expression molecular signatures has identified groups of genes characteristic of each and has identified biomarkers associated with specific diseases.

Hyperlipidemia is the most important risk factor for atherosclerosis, which is the major cause of cardiovascular disease. The etiology of hyperlipidemia and atherosclerosis is complex and governed by multiple interacting genes. However, mutations in two genes have been shown to be directly involved, i.e., the low-density lipoprotein receptor (LDLR) and apolipoprotein E (ApoE). Genetically modified mouse models have been instrumental in elucidating the underlying molecular mechanisms in lipid metabolism. In this review, we focus on the use of two of the most widely used mouse models, ApoE- and LDLR-deficient mice. After almost a decade of applications, it is clear that each model has unique strengths and drawbacks when carrying out studies of the role of additional genes and environmental factors such as nutrition and lipid-lowering drugs. Importantly, we elaborate on mice expressing mutant forms of APOE, including the APOE3Leiden ( APOE3L ) and the APOE2 knock-in ( APOE 2k) mouse models. These models have outstanding potential, as they are highly responsive to dietary factors and pharmacological interventions.

Alcohol use produces both desirable and undesirable effects, ranging from short-term euphoria and reduction in cardiovascular risk, to violence, accidents, dependence and liver disease. Outcomes are affected by the amount of alcohol used (which is itself affected by genetic variation) and also by the drinker's genes. Genetic effects have been most clearly demonstrated for alcohol dependence, and several of the genes for which variation leads to increased dependence risk have been identified. These include genes for enzymes involved in alcohol metabolism (alcohol dehydrogenase and aldehyde dehydrogenase), and genes for receptors affected by alcohol (particularly γ-aminobutyric acid receptors). Many other gene/dependence associations have been reported but not fully substantiated. Genetic effects on phenotypes other than alcohol dependence are less well understood, and need to be clarified before a full picture of gene-alcohol interactions can be achieved.

Angiogenesis is a crucial process in tissue remodeling during growth, both in the embryo and the adult. In our study we concentrated on the direct effect of β-carotene on human umbilical cord originating from endothelial progenitor cells (EPCs). β-Carotene uptake by EPCs was measured using a HPLC method. The determination of cell surface antigens was performed by flow cytometry. The effect on cell proliferation was estimated by measuring bromo-deoxyuridine incorporation. The influence on the formation of a tubular-like structure was investigated in a 3D assay in matrigel. Quantitative gene expression was estimated using real-time PCR. We demonstrated that β-carotene in the physiological range of concentrations found in human blood is a potent activator of EPC chemotaxis, which is accompanied by a change in the expression of genes mediating cell adhesion and homing, but does not activate the final markers of endothelial differentiation. This study points to the prochemotactic and homing activity of β-carotene in undifferentiated endothelial cell progenitors for the first time, which may suggest a potential role of this carotenoid in progenitor cell therapy aimed at angiogenesis and tissue repair.

Human chitotriosidase is a chitinolytic enzyme and mainly produced by activated macrophages. Recently, we observed that prolactin, which is structurally related to several cytokines and is involved in regulating monocyte/macrophage functions, upregulates chitotriosidase gene expression in human macrophages, suggesting that chitotriosidase is not only a biochemical marker of macrophage activation in lysosomal diseases and hematological disorders, but also may reflect induction of an immunological response. To confirm this hypothesis we evaluated by quantitative real-time PCR the mRNA chitotriosidase levels in human monocytes/macrophages following treatment with pro-inflammatory stimuli such as interferon-γ, tumor necrosis factor-α, lipopolysaccharide, and interleukin-10, an anti-inflammatory cytokine. Stimulation of macrophages with interferon-γ, tumor necrosis factor-α and lipopolysaccharide resulted in increased levels of chitotriosidase mRNA, as well as chitotriosidase activity, whereas interleukin-10 decreased chitotriosidase synthesis. This finding is consistent with the hypothesis that the production of chitotriosidase by macrophages could have biological relevance in the immune response.

Advanced glycation end-products are uremic toxins that accumulate in the serum and tissues of patients with chronic renal failure. Here, we established two enzyme-linked immunosorbent assays (ELISAs) for N ε -carboxymethyllysine and imidazolone to analyze advanced glycation end-products in human serum. Both ELISAs detected advanced glycation end-products bound to human serum albumin in a dose-dependent way. Whereas the formation of imida-zolone was independent of the presence of oxygen, concentrations of N ε -carboxymethyllysine epitopes increased 20-fold under oxidative conditions. The N ε -carboxymethyllysine ELISA showed a similar response to free, peptide-bound and protein-bound N ε -carboxymethyllysine, whereas the imidazolone antibody showed slightly higher affinity toward peptide-bound compared to protein-bound imidazolone. In human serum, linear dilution ranges from 1:10 to 1:40 (N ε -carboxymethyllysine ELISA) and from 1:2 to 1:8 (imidazolone ELISA) were found. The recovery of N ε -carboxymethyllysine from serum was 101±10% and 94±12%, respectively, and 93±15% and 97±12% for imidazolone. The coefficients of variation for intra-assay variability were 0.26–2.7% (N ε -carboxymethyllysine) and 0.1–2.4% (imidazolone), and 8.3–13.4% (N ε -carboxymethyllysine) and 7.8–12.5% (imidazolone) for inter-assay variability. In serum samples from hemodialysis patients (n=20) and controls (n=20), an approximately two-fold increase was detected in the patient group (p<0.001). The combination of the N ε -carboxymethyllysine and imidazolone ELISAs is a valuable tool to measure serum concentrations of advanced glycation end-products for clinical studies.

The apolipoprotein E ( APOE ) gene in humans contains two single-base polymorphisms in exon 4, which result in three common alleles, conventionally named ε2 , ε3 and ε4 . Numerous studies have shown an important association between the ε4 variant and an increased risk of Alzheimer's disease; other data suggest a possible linkage of APOE genetic heterogeneity with lipid profile and an increased risk of atherothrombotic stroke and coronary heart disease. APOE genotyping is therefore an increasingly common assay in laboratory medicine. The most widely used technique for APOE genotyping is based upon restriction isotyping, i.e., amplification of the fragment of exon 4 containing the most common se-quence variations, followed by enzymatic digestion of the amplicon and fragments analysis by gel electrophoresis. We developed a novel, reliable and fast method that exploits the sensitivity and specificity of denaturing high-performance liquid chromatography in detecting single-nucleotide polymorphisms. We show that, in most cases, with a single chromatographic separation it is possible to correctly identify the six different APOE allelic patterns, without any manipulation after the polymerase chain reaction amplification. When compared to restriction isotyping, our method is much faster, less labor intensive and similarly inexpensive.

Thyroid-stimulating hormone (TSH) is routinely measured in blood to diagnose thyroid disorders using immunoassays. This study used recombinant TSH (recTSH) as a source of hormonal compound exhibiting a serum-type glycosylation and putatively reflecting physiopathological alterations in TSH polymorphism. Mass spectrometry revealed that in recTSH, both subunits display high-molecular-size glycoforms compared to the pituitary hormone (pitTSH), indicating more complex glycosylation. To determine how changes in TSH glycosylation may affect epitope expression, comparative epitope mapping of rec- and pitTSH was carried out using a panel of ten hormone-specific monoclonal antibodies. Three common epitopes, I, II and III, were identified as common to both preparations and allowed the design of six assays as I/II, II/I, I/III, III/I, II/III, and III/II. Highly sialylated recTSHs were produced by enzymatic remodeling to mimic the hormone circulating in blood and revealed limited expression of epitope I, but enhanced recognition of epitope II. Fractionation on a lentil lectin-Sepharose column allowed selection of non-fucosylated recTSH, thought to be associated with primary hypothyroidism. Recognition of epitope I was not modified by TSH core fucosylation, while epitope III expression was increased in non-fucosylated glycoforms. Taken together, our findings demonstrate that changes in both core and terminal glycosylation alter epitope expression in TSH and thereby induce highly variable antibody recognition, resulting in significant discordances among hormone measurements.

We observed 30% discrepancy between liquid chemistry and dry chemistry analysers for the determination of total bilirubin in human adult serum samples, which were consistent with a 20% overestimation and 10% underestimation relative to a Jendrassik-Grof reference method, respectively. In contrast, standard reference material SRM916, which was recently recommended as being the most suitable material for attaining interlaboratory agreement, shows very good agreement on both types of analysers, as well as close to 100% recovery with respect to the reference method. We show that the liquid vs. dry bilirubin discrepancies seem to originate in the presence of either conjugated or δ-bilirubin. Our observations make it clear that good interlaboratory (or inter-analyser) agreement between bilirubin reference materials does not guarantee the same for bilirubin concentrations in human serum samples.

The objective of this study was to relate multiple laboratory results of initial blood gas analysis in patients visiting the emergency department to mortality. A total of 1806 adult patients visiting the emergency department for any reason, who underwent blood gas analysis, were included in this study. The results of nine laboratory parameters (ionised calcium, potassium, sodium, glucose, lactate, pH, pCO 2 , pO 2 and saturation) were related to mortality. Comparison of the means of survivors' and non-survivors' age, pH, pO 2 , glucose and lactate showed relevant differences (p≤0.001). When dividing the group of patients into quintiles, hypo- and hyperkalaemia (<3.6 or >4.3mmol/L), hypocalcaemia (<1.12mmol/L), hyperglycaemia (>10.6mmol/L), hyperlactataemia (>2.9mmol/L) and acidosis (pH<7.34) were all related to high mortality in comparison to the quintile with the lowest mortality (p<0.0001). Interestingly, even within the reference range for lactate and ionised calcium, there was an increased risk of mortality, so these reference ranges should be interpreted with care. Glucose and lactate have additional clinical value, but are not always requested. Therefore, glucose and lactate should be routinely added to a blood gas request. Our study shows differences in mortality for laboratory parameters in patients visiting the emergency department, irrespective of the underlying pathology.

Quantitative real-time PCR techniques are increasingly being used for the measurement of nucleic acids in research applications as well as in the clinical laboratory. It is therefore important that external quality control programs (EQA) are implemented for the evaluation of the analytical aspects common to molecular tests based on quantitative PCR. The aim of this study was the development of an Italian program of external quality control for quantitative assays based on real-time PCR with Taq-Man™ probes to compare the analytical performance of 42 laboratories. Participants were provided with a set of reagents (cDNA for reference curve preparation, primers-probe mix and three unknown samples) and requested to perform a conventional assay using the master mix employed in their laboratories. The quantitative results in unknown samples were analyzed. The results of our study showed clear heterogeneity in performance. Two of the 42 laboratories provided results indicating contamination during the experiment, whereas six did not provide values for at least one of the six standard points. Only 12 laboratories gave results that were both precise and accurate for all the samples tested. Regarding imprecision, 17 laboratories appeared to deviate in at least one result, whereas inaccuracy showed an inverse dose-dependent trend. Finally, 12 laboratories were not able to measure the sample with the lowest concentration. Ten of these laboratories were equipped with the same instruments. The results of this first round of analytical EQA of real-time PCR-based methods seem to indicate high variability among laboratories carrying out the same experimental protocol. These findings could have implications for any assay based on this type of technique. This survey demonstrates the importance of experimental EQAs of methodological proficiency testing. Our approach has proved useful for comparing the analytical aspects shared by all diagnostic laboratories applying quantitative assays for the measurement of nucleic acids based on the use of Taq-Man™ probes and real-time platforms.

Standardization of aspartate aminotransferase (AST) determination is highly desirable for inter-laboratory comparison. Serum AST mean values for 20 patients suffering from viral hepatitis showed an inter-laboratory (n=13) variation of 9.4%. Part of this variation was due to two laboratories using procedures without pyridoxal-5′-phosphate. A traceable AST value was assigned to an enzyme calibrator (EC) through the appropriate International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) primary reference procedure. The EC was commutable for seven routine methods. Common calibration using the EC reduced the inter-laboratory coefficient of variation (CV=5.9% ) and allowed retention of a common reference interval for a set of routine procedures. Calibration made superfluous the expression of results in multiples of the upper reference limit, which increased inter-laboratory variation (CV=18.5%). Furthermore, for 92% of patients, calibration with the EC allowed the correction of misclassifications when taking into account the reference interval of the reference procedure. Use of this EC could be proposed to complete the AST reference system.

There are two fully automated high-throughput clinical instruments for brain natriuretic peptide (BNP) assays, the Bayer ADVIA Centaur assay, and the Abbott AxSYM assay. Although both recommend a cut-off value of 100pg/mL, we are unaware of previous studies that have compared the unadjusted results of the two methods, required for proper evaluation of patients undergoing this test on different platforms. From 43 hemodialysis patients, 80 paired samples were collected by venipuncture into plastic evacuated tubes containing EDTA. The Bayer assay yielded lower values than the Abbott assay, with linear regression of 0.53×Abbott assay (95% confidence interval, 0.50–0.56) being forced through 0, demonstrating an r 2 -value of 0.954. Regression for the Abbott assay was 1.79×Bayer assay (95% CI, 1.69–1.89). The cut-off values for abnormal BNP results analyzed on the Abbott system are not identical to those on the Bayer system, and this needs to be taken into account when comparing studies on the clinical utility of these systems.

The aim of this multicenter study was to evaluate the technical and clinical performance of the Elecsys ® S100 electrochemiluminescence immunoassay and to assess its utility as a tumor marker in patients with malignant melanoma. Imprecision studies yielded within-run coefficients of variation (CVs) of 0.7–2.0% and between-day CVs of 1.0–6.4%. Serum samples that were distributed to 11 participating laboratories for a comparability analysis resulted in excellent recoveries of 93–105% related to the median for all laboratories. The functional sensitivity of the assay was determined to be below 0.02μg/L. The lot-to-lot reproducibility of Elecsys ® S100 was tested by analyzing 110 sera with three different reagent lots on an E2010 analyzer. This lot-to-lot comparison showed excellent correlation, with a coefficient of 0.99. A 95th percentile cut-off value of 0.10μg/L was calculated from values measured in 206 healthy individuals. Using this cut-off value, sensitivity of 41% was found, with positive and negative predictive values of 0.50 and 0.91, respectively. Method comparison with the Sangtec ® 100 luminescence immunoassay, run on two different analyzers, showed correlation with coefficients ranging from 0.76 to 0.95. A comparison of S100 values obtained with both tests showed identical patterns in 68 serial samples from 15 patients with malignant melanoma during follow-up. These findings indicate that serial measurements with the Elecsys ® S100 assay are useful for the follow-up and monitoring of therapy in patients with malignant melanoma.

All analyzers with ion-selective electrodes for ionized magnesium (iMg) should yield comparable and unbiased results. The prerequisite to achieve this goal is to reach consensus on sampling, measurement and reporting. The recommended guidelines for sampling, measurement and reporting iMg in plasma (“plasma” refers to circulating plasma and the forms in which it is sampled: the plasma phase of anticoagulated whole blood, plasma separated from blood cells, or serum) or blood, referring to the substance concentration of iMg in the calibrants, will provide results for iMg that are approximately 3% greater than its true concentration, and 4% less than its true molality. Binding of magnesium to proteins and ligands in plasma and blood is pH-dependent. Therefore, pH should be simultaneously measured to allow adjustment of iMg concentration to pH7.4. The substance concentration of iMg may be physiologically and consequently clinically more relevant than the substance concentration of total magnesium.