Volume 44, Issue 2

Currently, diagnosis of type I allergy is performed using crude allergen extracts, which allow the identification of the allergen-containing source responsible for type I allergic symptoms (e.g., allergic rhino-conjunctivitis, asthma) but not the disease-eliciting molecules. With the introduction of recombinant allergens produced by molecular biology techniques, a large panel of allergenic molecules has become available. The application of these recombinant allergens for in vitro tests has led to new forms of component-resolved diagnosis (CRD) and allows the establishment of a patient's individual reactivity profile. The increasing number of recombinant allergens characterized during the last decade has allowed the development of chip-based allergy tests for simultaneous detection of up to 5000 different allergens and epitopes. The introduction of these recombinant allergen-based tests into clinical practice improves the selection of patients for traditional specific immunotherapy and allows monitoring of the immunological efficacy of specific immunotherapy by measuring allergen-specific IgG antibodies. Besides their diagnostic application, recombinant allergens and hypoallergenic derivatives thereof have also been used as vaccines in clinical trials, and recent results have shown their usefulness for the treatment of type I allergy.

Type 1 diabetes results from a specific destruction of the insulin-producing β-cells of the pancreas. The disease is characterized by the appearance of specific autoantibodies against islet cell antigens. Autoantibodies to insulin, glutamic acid decarboxylase, tyrosine phosphatase IA-2 and cytoplasmic islet cell antibodies are useful markers for the differential diagnosis of type 1 diabetes when clinical and metabolic criteria alone do not allow definite classification. Autoimmune diagnostics is of particular importance in adults to discriminate between type 1 and type 2 diabetes and to assess the diagnosis of latent autoimmune diabetes in adults.

Diagnosis of rheumatoid arthritis (RA) is mainly based on clinical criteria of symmetric polyarthritis of the hands and feet, with morning stiffness lasting usually more than 1h. Autoantibodies typical for RA, i.e., rheumatoid factors and anti-cyclic citrullinated peptide, and measurements of inflammation add more specific information, especially for early diagnosis, where clinical presentation may be oligosymptomatic involving only a few joints. These laboratory parameters are also relevant for prognosis of disease persistence, functional impairment and radiological progression.

Bullous skin diseases represent a group of organ-specific autoimmune disorders characterised by binding of circulating autoantibodies to adhesion molecules of the epidermis and the dermo-epidermal basement membrane zone. Binding of these autoantibodies to their antigenic targets results in loss of adhesion between epidermal keratinocytes and at the level of the basement membrane zone. Chronic blisters and secondary painful erosions are the clinical hallmark of autoimmune bullous disorders. Histopathology reveals the location of blister formation and helps to classify the subtype of the bullous skin disorder. Immunofluorescence is crucial for diagnosing autoimmune bullous skin disorders. Tissue-bound autoantibodies are detected by direct immunofluorescence of perilesional skin. Circulating autoantibodies can be visualised by indirect immunofluorescence using tissue substrates such as monkey oesophagus and sodium chloride-split human skin. Most of the autoantigens are available as recombinant proteins, which allows for autoantibody screening by ELISA or immunoblot analysis to confirm the primary diagnosis and, importantly, for immunoserological follow-up of patients.

Clinical laboratories have undergone major changes due to technological progress and economic pressure. While costs of laboratory testing continue to be the dominant issue within the healthcare service worldwide, quality, effectiveness and impact on outcomes are also emerging as critical value-added features. Five Italian laboratories are therefore promoting a network of excellence by investigating markers of effectiveness of laboratory services and sharing their experience of using them in clinical practice. In the present study we report preliminary data on indicators of quality in all phases of the so-called total testing process, the key to evaluating all phases of the total testing process, including the appropriateness of test requests and data interpretation. Initial findings in evaluating pre-analytical causes of specimen rejection in three different laboratories and the effects of introducing three laboratory clinical guidelines are reported. These data should stimulate debate in the scientific community and encourage more clinical laboratories to use the same indicators to improve clinical effectiveness and clinical outcomes within the healthcare service.

We have previously reported two novel mutations in the 5′-untranslated region (UTR) of the gene for folate receptor-α ( FOLR1 ). In our search for additional mutations, 92 patient samples with elevated levels of homocysteine were screened by single-strand conformation polymorphism (SSCP) between nt −425 and −782, and −712 and −1110. Between nt −425 and −782 we did not find any mutations. Between nt −712 and −1110 there were three novel mutations. One subject had two mutations very close to each other, c.−856C>T and c.−921T>C. Two subjects had a c.−1043G>A mutation. To get an idea of the prevalence of FOLR1 mutations in an unselected population, we also screened 692 healthy school children for mutations. In this cohort, between nt −188 and +272 we discovered one novel mutation, a single nucleotide substitution, c.−18C>T, in addition to five children with the 25-bp deletion mutation previously described by us. Thus, so far we have discovered six novel mutations in the 5′-UTR region of the gene for folate receptor-α. We genotyped all 17 subjects with a FOLR1 mutation for the methylenetetrahydrofolate reductase ( MTHFR ) 677C>T polymorphism, and developed new single-nucleotide polymorphism (SNP) genotyping protocols for MTHFR 1298A>C and 1793G>A utilising Pyrosequencing™ technology. None of the 17 subjects had the 677TT genotype, which ruled out this as a cause of elevated homocysteine levels, which was observed in some of the subjects. Further studies of mutations in the 5′-UTR of FOLR1 , and in particular of their interplay with folate intake status, are warranted.

Cytochrome P450 2C8 (CYP2C8) is a polymorphic phase I drug-metabolising enzyme involved in the metabolism of a wide variety of xenobiotics, as well as a proposed player in the regulation of vascular tone. Polymorphisms in this gene may have an impact on the metabolism of therapeutic drugs such as paclitaxel and verapamil. In this report we have determined the frequencies of the main non-synonymous CYP2C8 alleles, 805A>T ( CYP2C8*2 ), 416G>A/1196A>G ( CYP2C8*3 ) and 792C>G ( CYP2C8*4 ) in a sample representative of Portuguese Caucasians. The allelic frequencies determined were 1.2%, 19.8%, and 6.4% for CYP2C8*2 , CYP2C8*3 , and CYP2C*4 , respectively. The observed CYP2C8*3 prevalence is significantly different from the frequencies previously reported in North European populations.

Thyroid hormones and thyroid autoantibodies, along with serum concentrations of two phytoestrogens of the isoflavone series, daidzein and genistein, were measured in 268 children without overt thyroid diseases, screened for iodine deficiency in one region of the Czech Republic. Since both phytoestrogens have been reported to inhibit thyroid hormone biosynthesis and in high concentrations to exert goitrogenic effects, we investigated whether their presence in the circulation could influence thyroid hormone function in a population where soy consumption is not common. Correlation analysis revealed a significant positive association of genistein with thyroglobulin autoantibodies and a negative correlation with thyroid volume. Multiple regression analysis of the relationships between actual phytoestrogen levels and measured thyroid parameters revealed only a weak but significant association between genistein and thyroid variables. Higher levels of free thyroxine were found in a subgroup of 36 children who ate soy food in the previous 24h. In conclusion, only modest association was found between actual phytoestrogen levels and parameters of thyroid function. On the other hand, even small differences in soy phytoestrogen intake may influence thyroid function, which could be important when iodine intake is insufficient.

The anticarcinogenic and antioxidant properties of vitamins A, C, E and pro- or antioxidant properties of trace metals have recently attracted increased attention. We examined the levels of antioxidant vitamins (A, C and E), selenium and malondialdehyde (MDA), and trace metals (Fe, Ni, Zn, Co and Cu) in patients with prostate cancer. In total, 41 subjects (21 controls and 20 prostate cancer patients) were included in the study. The levels of trace elements and Fe in whole blood were determined by atomic absorption spectrophotometry. Serum levels of Se were determined using a fluorimetric method, while a HPLC method was used for serum levels of vitamins and MDA. The levels of vitamins A and E were significantly lower and MDA levels were significantly higher (p<0.001) in patients with prostate cancer compared to controls. Serum vitamin C was significantly lower in patients with prostate cancer when compared to controls (p<0.01). Moreover, Se and Zn levels were also significantly lower, and levels of Ni, Co, and Cu were higher (p<0.001) in patients with prostate cancer than in controls. Fe levels were not significantly different in patients compared to controls (p>0.05). Our findings, together with the results of previous animal studies, suggest that the administration of vitamins A, C, and E, and Se and Zn may be beneficial in the prevention and treatment of human prostate cancer.

The diagnostic value of Brucella IgM/IgG flow assays was evaluated in comparison with serum agglutination and 2-mercaptoethanol tests by testing a selection of serum samples submitted to the laboratory because of clinical suspicion of brucellosis. All 39 admission and 11 follow-up samples that agglutinated in the serum agglutination test tested positive in the flow assay, whereas all 20 serum agglutination negative samples with clinical suspicion of brucellosis, 23 control samples from healthy individuals and 20 control samples from cases with chronic hepatitis tested negative in the flow assay. The Brucella IgM and IgG flow assays were slightly more sensitive than the agglutination tests in discriminating between specific IgM and IgG antibodies. The Brucella IgM and IgG flow assays are easy-to-perform and quick assays that can be used for the diagnosis of brucellosis. The flow assays are very useful, especially in rural settings where brucellosis is widespread and where well-equipped laboratories to perform the laboratory tests are not readily available.

Background: pH and phosphate concentration are the major determinants of precipitation in urine of the salts of calcium and magnesium. This study aims to model the process of salt precipitation and establish whether the acidification of urine samples is necessary for the accurate measurement of calcium and magnesium in a clinical laboratory setting. Methods: Urine samples were collected from 21 patients, aliquots were taken from each patient sample and the pH was adjusted to cover the range 2.0–10.0. The analytical and biological variation for each analyte was established and used to calculate percentage changes and critical differences. The critical difference was used to assess whether there was a significant difference between acidified and un-acidified samples. The JESS (Joint Expert Speciation System) thermodynamic computer-modelling program was used to predict the distribution of salt species formed with varying pH values and phosphate levels in simulated urine. Results: The results showed that at a pH greater than 6.5, measured calcium, magnesium and phosphate significantly decreased as a result of precipitation (p<0.0001), although the critical difference was generally not exceeded. Computer modelling showed that both pH and phosphate concentration affected the distribution of salt species formed, as well as the precipitation patterns of calcium and magnesium phosphates. Overall, calcium phosphate precipitation tends to predominate at lower phosphate concentrations and at pH values below about 6.5, while both calcium and magnesium phosphate precipitation occur at higher phosphate concentrations and pH values greater than 6.5. Conclusions: For accurate analysis of these analytes in urine, the pH should be routinely measured and acidification should be undertaken prior to analysis if the pH is greater than 6.5. Based on the findings of this study, acidification or the lack of it does not result in a clinically significant change in calcium, magnesium and phosphate measured in urine. This study also predicted the likely salt species formed at varying urinary pH values and phosphate concentrations.

The intra vitam diagnosis of different dementias is still based on clinical grounds. So far, no technical investigations have been available to support these diagnoses. For tau protein and β-amyloid (1–42) in cere-brospinal fluid (CSF), promising results for the diagnosis of Alzheimer's disease (AD) have been reported; however, their differential diagnostic spectrum is limited, as was recently shown for dementia with Lewy bodies (DLB) and for AD. Therefore, further marker proteins have to be established to ameliorate, support, and differentiate these clinical diagnoses. We evaluated β-amyloid (1–40) and phosphorylated tau protein (181p), in addition to total tau protein and β-amyloid (1–42) , in 20 patients with DLB, 34 AD patients, and 20 non-demented neurological controls (NDCs). All markers could differentiate between the dementia groups (AD, DLB) and the controls. AD and DLB could be differentiated only by levels of total tau protein and by the ratio total tau protein/phosphorylated tau protein. However, values still overlapped markedly. In some cases, tau protein levels in CSF may contribute to the clinical distinction between DLB and AD, but the value of the markers is still limited, especially because of mixed pathology. We conclude that more specific markers have to be established to differentiate between these diseases.

The indocyanine green (ICG) clearance test has been used to assess the reserve of hepatic function. This method is based on the spectrometric measurement of its plasma concentration at maximum wavelength of 805nm, which requires a spectrophotometer and associated maintenance. We established an ICG clearance test using a Toshiba 200FR automatic chemistry analyzer that can be tuned to a wavelength of approximately 805nm. Five pooled sera spiked from 0 to 4.0mg/dL were analyzed for linearity test and precision was determined at five levels in the range 0.1–2.0mg/dL. The ICG retention rate at 15min (R15) was determined for 38 patients using a conventional method and our method. The ICG clearance test using the automatic chemistry analyzer showed good linearity, and precision ranged from 0.3% to 1.0% for within-run CVs and from 0.6% to 4.7% for total CVs. The degree of agreement between the two methods was also acceptable (mean difference of 1.5%). It is expected that the ICG test using the automatic chemistry analyzer can replace the conventional ICG clearance test, considering the excellent agreement, good precision and linearity over a clinically relevant range.

The role of sialic acid (SA) in the pathogenesis of atherosclerosis and as a predictor of cardiovascular events has attracted much attention in recent years. However, most studies investigating the role of total and lipid-bound sialic acids (TSA and LSA) in the pathogenesis of atherosclerosis lack information on the reason for the elevated SA concentrations in coronary heart disease and myocardial infarction. Since the inflammation-sensitive proteins are glycoproteins with SA residues, an increase in their levels due to some type of acute-phase reaction or inflammation could be responsible for the elevated TSA levels in acute myocardial infarction (AMI). Elevated serum SA levels might also be due to either shedding or secretion of free SA from the cell or cell membrane surface if neuraminidase levels are increased, or to the release of cellular SA-containing glycolipids and/or glycoproteins into plasma from myocardial cells after AMI. The aim of the present study was to investigate both the possible role of SA-rich inflammation-sensitive proteins and the cell damage due to elevated serum TSA levels in AMI. A possible role of serum LSA as an indicator of the shedding or secretion of SA from the cell or cell membrane surface in AMI was also evaluated. The study included 38 subjects with AMI and 32 healthy volunteers. Serum TSA and LSA were determined using the methods of Warren and Katopodis, respectively. The concentrations of serum SA-rich inflammation-sensitive proteins, namely α 1 -antitrypsin, α 2 -macroglobulin and ceruloplasmin were determined immunoturbidimetrically. Our data showed that: a) mean levels of serum TSA and LSA and SA-rich inflammation-sensitive proteins in patients with AMI were significantly increased; and b) there was a significant positive correlation between TSA and LSA and α 1 -antitrypsin in patients with AMI. Since the transfer of free SA to lipoproteins is required for an increase in serum LSA levels, and free SA for this transfer can be provided by the secretion of SA from the cell, it is obvious that the shedding or secretion of SA from the cell membrane surface or release of cellular SA from cells into the bloodstream due to cell damage after AMI also occur after AMI. As a result, we can report that either the shedding or secretion of SA from the cell or cell membrane surface and the increased output of SA-rich inflammation-sensitive proteins may together be responsible for the elevated TSA levels in AMI.

Cardiovascular disease is the leading cause of mortality and morbidity in Western countries. Despite its remarkable medical and social consequences, the prevalence of peripheral arterial disease (PAD) is often underestimated among atherosclerotic disorders. So far, little is known about the behavior of traditional and emerging markers of ischemic heart disease that should allow the reliable identification of PAD patients at increased risk of developing myocardial ischemia and heart failure or dysfunction. To investigate this topic, we measured cardiac troponin T (cTnT), ischemia-modified albumin (IMA) and NT-prohormone-brain natriuretic peptide (NT-proBNP)in 35 consecutive patients with clinically ascertained PAD (stage 2–4, according to Lériche-Fontaine) asymptomatic for chest pain and current heart failure, and 20 controls displaying moderate to high cardiovascular risk factors (hypertension, diabetes, hyperlipidemia), but with no clinical evidence of PAD. Although the concentrations of cTnT and IMA were not statistically increased in PAD patients, NT-pro-BNP values were substantially higher in PAD patients than in controls (62.6 vs. 7.4pmol/L, p<0.0001). Thepercentage of subjects displaying values exceeding the specific NT-proBNP diagnostic threshold (>14.8pmol/L) was also significantly different between PAD patients and controls (74% vs. 10%, p<0.001). After excluding PAD patients exceeding the 0.01ng/mL cTnT cutoff value indicative of current ischemic cardiac involvement, the median concentration of NT-proBNP remained statistically increased (28.0 vs. 5.8pmol/L, p<0.0001). Taken together, these results indicate that NT-proBNP, but not IMA, is substantially increased in PAD patients. This finding suggests that such patients, even though asymptomatic, might develop myocardial dysfunction, and thus warrant further investigation.

Despite many years of study, clinical trials of new drugs to prevent thrombosis have often been disappointing. Part of the problem lies in our incomplete understanding of the regulation of plasminogen activation and/or inhibition in vivo. We have previously shown that in vitro nitration of plasminogen in plasma by peroxynitrite resulted in decreased plasmin activity. We hypothesized that macrophages may be agents of plasminogen nitration and designed this study to prove this hypothesis. We first better characterized our previous observations using purified plasminogen instead of whole plasma, studied the time and concentration dependence of these reactions, and co-incubated plasminogen with macrophages, as well as with non-inflammatory cells as controls, to assess nitration and impaired activity. When plasminogen (10μmol/L) is incubated in the presence of SIN-1 (0.01–2mmol/L), plasmin activity (generated by streptokinase) is reduced in a time- and concentration-dependent fashion. We performed experiments incubating human plasminogen in the presence of murine RAW264.7 macrophages, allowing for free diffusion of reactive oxygen species, while preventing the action of proteases. In this way we show that incubation of plasminogen with macrophages also decreases plasmin activity, while increasing nitration of the molecule, an effect that is already apparent after 2h and reaches a plateau of 60% inhibition after 24h of incubation. This effect appears specific for macrophages, since 31EG4 murine mammary cells used in parallel and under the same conditions failed to produce any deleterious changes in plasminogen. Our data on quick functional inactivation of plasminogen by nitration, mediated by macrophages, adds a new pathophysiological dimension to our previous work showing plasminogen as a target for peroxynitrite damage. Nitrosative stress may be implicated in impaired fibrinolysis. New therapeutic approaches for nitrosative stress in atherosclerosis and diabetes should limit the formation of superoxides and peroxynitrite.

Background : In plasma, triglycerides (TG) are transported in lipoprotein particles (mainly chylomicrons, very low-density and low-density lipoprotein). Turbidimetry (bichromatically at 660 and 700nm) allows measurement of the lipemic (L) index. We explored the use of this index, in combination with a TG assay, to detect errors due to non-fasting, to assess abnormalities in TG metabolism and to detect patients with glycerol kinase deficiency (GKD). Methods : We collected 2441 patient samples. Normolipidemic (n=2347), type IV hyperlipidemic (n=80), postprandial samples (n=22) and serial dilutions of Intralipid with saline (n=6) were selected. One patient presenting with GKD was included, as well as two patients with type I and type V hyperlipoproteinemia, respectively. Results : We introduced the use of the ratio between the logarithm of serum triglycerides and that of the L-index (TG/L ratio). Conclusion : Although the proposed TG/L-index ratio cannot be regarded as an alternative for the accurate diagnosis of lipid disorders, it provides additional information about TG-containing particles.

Our study evaluates a fast and easy way to perform point-of-care testing (POCT) measurements of glycated hemoglobin HbA 1c in comparison with an immunoassay on an automated biochemistry analyzer and cation exchange chromatography, the two methods routinely used in clinical laboratories for the measurement of HbA 1c . A significant finding of our study is that although the POCT instrument insert claims that the method is not affected by the presence of HbS in the heterozygous state or in combination with β-thalassemia, discrepant results were found in some cases with such hemoglobinopathies. In these cases, the two POCT and laboratory immunoassay methods showed clinically significant positive interferences with samples containing the HbS trait. We conclude that samples with the HbS trait should be interpreted with caution when tested using the POCT instrument.

Increased C-reactive protein (CRP) concentration within the reference interval (<10.0mg/L) is a strong predictor of cardiovascular disease (CVD) in apparently healthy adults. Cutoff points for use of CRP in estimating CVD risk are <1, 1–3 and >3mg/L for low, average and high relative risk, respectively. For measuring CRP concentrations to assess cardiovascular risk, high-sensitivity CRP (hsCRP) assays have been developed. The aim of this study was to evaluate the analytical performance and clinical efficacy for cardiovascular risk estimation of the Olympus immunoturbidimetric latex CRP assay (sensitive application). The comparative method used was the CardioPhase* hsCRP assay, approved by the Food and Drug Administration for use in CVD risk assessment. The imprecision of the Olympus hsCRP assay in the concentration range 0.2–10.0mg/L was 0.38–8.16% within runs and 3.75–9.63% between runs. For method comparison studies, 194 fresh serum samples were selected to cover the interval 0.15–10.0mg/L CRP. Comparison of the Dade Behring and Olympus methods was performed using weighted Deming regression analysis (slope 0.99mg/L, intercept 0.002mg/L, S y,x =0.02mg/L, r=0.992) and a Bland-Altman relative difference plot (mean difference −0.002%, SD=0.040%). The agreement between the Dade Behring and Olympus methods for relative risk class assignments was 95.4%. Statistical analysis of the agreement between the two methods for each relative risk class showed that the differences between the methods were not statistically significant (p>0.10). Although previous reports found poor performance of the Olympus CRP tests for use in cardiovascular and peripheral vascular risk estimation, our study proved good analytical performance and clinical efficacy of the Olympus hsCRP assay for this use.