Volume 43, Issue 9

High plasma levels of triglycerides are an independent risk factor for the development of cardiovascular disease. Apolipoprotein A5 ( APOA5 ) is a new member of the apolipoprotein APOA1/C3/A4 gene cluster, found by comparative sequencing analysis. The importance of the APOA5 gene for determination of plasma triglyceride levels has been suggested by the creation of transgenic and knock-out mice (transgenic mice displayed significantly reduced triglycerides, whereas knock-out mice had a high level of triglycerides). It has now been clearly established that distinct polymorphisms in the APOA5 gene consistently influence plasma triglycerides in a wide range of human populations, although some differences between ethnic groups and males and females were described. The possible roles of APOA5 variants in determining the risk of myocardial infarction and coronary artery disease development, as well as in the determination of low-density lipoprotein-particle size or plasma concentrations of C-reactive protein and high-density lipoprotein-cholesterol, are also summarized.

The 19-bp insertion/deletion polymorphism in the 5′ flank of the dopamine β-hydroxylase ( DBH ) gene has been associated with psychiatric disorders. We have developed a simple, reliable and inexpensive closed-tube assay for genotyping of this polymorphism based upon T m determination of amplified DNA fragments. Mistyping of heterozygote samples due to preferential allele amplification was prevented by use of an optimized concentration of Mg 2+ , addition of dimethyl sulfoxide and annealing/extension at an appropriate temperature. Comparison of results achieved by the closed-tube assay and a conventional approach based upon agarose gel electrophoresis of amplified fragments revealed complete concordance between the two procedures. The insights obtained in this study may be utilized to develop assays based upon dissociation analysis of PCR products for genotyping of other insertion/deletion polymorphisms.

The objective of this work was to study the distribution of apolipoprotein E ( APOE ) genotypes in a sample of the Portuguese population, and its association with the dyslipidemias observed. Study participants were healthy users of local Public Health Laboratories in six regions of mainland Portugal (Porto, Vila Real, Viseu, Lisboa, Portalegre and Faro). A total of 779 men and 1153 women aged 15–74years agreed to participate. Fasting lipid levels and APOE genotypes were determined centrally at the National Institute of Health in Lisboa. The frequency distribution of APOE alleles was: ε2 =5.3%, ε3 =84.9% and ε4 =9.8%. Dyslipidemias were present in 66.6% of men and 60.7% of women. Comparison of APOE genotypes and relative allele frequencies showed that in dyslipidemic compared to normolipidemic subjects, the ε4 allele was more frequent in both sexes, although in a more pronounced way in men than in women due to higher frequencies of ε3 / ε4 and ε4 / ε4 genotypes. The known association of the ε4 allele with high cholesterol levels, the association of the ε2 allele with low cholesterol levels, and the association of the ε2 allele with high levels of triglycerides and low levels of high-density lipoprotein-cholesterol were confirmed in this study.

The electrophoretic pattern of serum proteins of 85 patients carrying different types of neoplasia and 85 matched healthy adults were comparatively studied by agarose gel electrophoresis, to find out if there is a specific protein pattern common to different types of cancer. Each protein fraction was analyzed quantitatively by densitometry. Multivariate logistic regression and receiver operating characteristic (ROC) analyses were performed using SPSS software. When total protein and albumin were measured by colorimetric methods, cancer patients, compared to controls, had a decreased concentration of total protein (66.0±11.5g/L vs. 76.4±6.8g/L, p≤0.0001) and of albumin (39.0±8.1g/L vs. 46.0±4.3g/L, p≤0.0001). The electrophoretic data of serum proteins showed that the ratio of albumin to globulin (0.92±0.30 vs. 1.21±0.16, p≤0.0001), percent of the fractions albumin (46.7±8.5% vs. 54.4±3.5%, p≤0.0001) and β-globulin (11.6±4.4% vs. 13.0±1.9%, p≤0.001) were decreased and α 1 - (5.3±2.5% vs. 2.9±0.8%, p≤0.0001), α 2 - (13.5±4.8% vs. 11.3±2.1%, p≤0.0001) and γ-globulins (23.0±7.7% vs. 18.3±3.1%, p≤0.0001) were significantly increased in cancer patients relative to controls. Cancer patients also had higher counts of leukocytes (7.98±3.11, ×10 9 cells/L vs. 6.33±1.68 ×10 9 cells/L, p≤0.0001) and erythrocyte sedimentation rate (35.9±23.5mm/h vs. 14.1±9.5 mm/h, p≤0.0001). On the basis of univariate analysis, a protein profile out of the normal ranges was more prevalent in cancer patients than in controls. Analysis of the data using multiple logistic regression indicated that the prevalence of cancer was strongly associated with the serum proteins' profile, and α 1 -globulin, erythrocyte sedimentation rate, total protein and the ratio of albumin to globulin were the best parameters to discriminate between malignant and healthy states. The area under the ROC curves were the same for most components of the serum proteins' profile at about 0.75±0.09, p≤0.001. We conclude that the profile of serum proteins indicates high diagnostic values for discriminating between cancer patients and healthy individuals and may be useful as an adjunct diagnosis for detection of malignancy.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of the pluripotent stem cell resulting from the somatic mutation of the X-linked PIG-A gene, involved in the synthesis of the glycosylphosphatidyl-inositol anchor of membrane proteins such as CD55, CD59 and CD14. In the past decade, flow cytometry has become a valuable diagnostic tool in the detection of deficient expression of the GPI-anchored proteins. We report the diagnosis of PNH in four patients confirmed by flow cytometry. Red blood cells, granulocytes and monocytes were classified as PNH types I, II and III according to the mean fluorescence intensities (MFI) of membrane proteins. MFI rate is a numerical data reflecting the severity of decreased antigen expression, and it is obtained by dividing the MFI of the type II or type III cells by the MFI of the respective cells obtained for a normal sample. We found that the investigation of granulocytes and monocytes was more informative than red blood cells when percent negativity was evaluated. In addition, the lowest MFI rate (mean 0.011) was obtained for CD14 on monocytes while CD59 and CD55 gave higher values on all three investigated cell types (0.021–0.34). Thus, CD14 on monocytes seems to be the most reliable marker for establishing the PNH clone size and the severity of antigen deficiency.

Disturbed metabolism of glycosaminoglycans (GAGs) has been proposed to play an important role in the pathogenesis of late diabetic complications. The effect of diabetic complications and metabolic control on both total serum GAGs content and the serum activity of lysosomal glycosidases (N-acetyl-β-D-glucosaminidase, α-L-fucosidase, β-D-galactosidase, and α-D-mannosidase) contributing to GAGs degradation, was investigated in 48 patients with type 2 diabetes mellitus. The activity of β-D-glucosidase and acid phosphatase, the lysosomal enzymes unrelated to GAGs metabolism, was determined for comparison. The elevated serum total GAG concentration in diabetic patients was strongly and positively influenced by poor metabolic compensation of diabetes and the presence of vascular complications. A similar tendency has been shown in regard to the activity of enzymes involved in GAG degradation, especially N-acetyl-β-D-glucosaminidase, α-L-fucosidase and β-D-galactosidase. Furthermore, the total serum GAG concentrations, as well as the activity of lysosomal enzymes involved in the extracellular matrix degradation, closely followed metabolic compensation, regardless of diabetic vascular complications. Thus, we suggest that increased values of the investigated parameters may indicate the degree of endothelial cell dysfunction and may be useful to predict the development of diabetic vascular pathology.

Abnormalities in thyroid hormone metabolism are common in critically ill patients. However, it is not known if these patients are truly hypothyroid at tissue level. Erythrocyte zinc has been shown to be a tissue marker of thyroid hormone status. In this study we have measured the erythrocyte zinc in critically ill patients. In this observational study we measured the zinc content of young erythrocytes in blood samples from 33 healthy subjects, 26 hypothyroid patients, four hyperthyroid patients, and 44 patients in the intensive care unit – 22 of these were admitted after a major surgical procedure (surgical group) and the other 22 patients had a variety of conditions (non-surgical group). Erythrocytes were separated according to age by centrifugation. Plasma thyroid hormone concentrations were abnormal in 70% of the critically ill group. Erythrocyte zinc was significantly lower in hyperthyroid patients and higher in hypothyroid patients. In the non-surgical patients, erythrocyte zinc of young cells (median 256μmol/L of cells) was significantly higher than (p<0.01) the corresponding cells in the healthy controls (202μmol/L of cells), whereas in the surgical group it was not different (197μmol/L of cells). We conclude that in non-surgical critically ill patients, erythrocyte zinc content is higher, suggesting that these patients may be hypothyroid at tissue level.

Multiple myeloma is characterized by accelerated production of the proteolytic enzyme matrix metalloproteinase (MMP)-9. We hypothesized that myeloma-produced MMP-9 may influence the rate of bone turnover in a paracrine manner. Thus, we examined the correlations of MMP-9 levels, disease severity, and bone turnover rate as evaluated by markers of bone formation and resorption. Thirty-seven newly diagnosed multiple myeloma patients (nine of Durie-Salmon stage I, 12 of stage II and 16 of stage III) and 12 age-matched controls were studied. Serum MMP-9 levels were significantly higher at stage II compared to stage I (188.78±91.27 vs. 59.25±33.09 ng/mL, p<0.004). Additionally, free urine pyridinolines (F-Pyd), free urine deoxy-pyridinolines (F-Dpd) and urine N-telopeptide fragment (NTx) were elevated, their level correlating with disease stage (p<0.001, p<0.03, p<0.001, respectively), as were bone marrow infiltration and serum interleukin-6 (IL-6) levels (p<0.0001, p<0.01, respectively). MMP-9 levels were lower in patients compared with controls (p<0.001), whereas IL-6 and bone resorption marker levels were higher in patients than in controls (p<0.001 in all cases). Significant correlation was found between infiltration, MMP-9, free urine pyd, free urine dpd and NTx for each stage of the disease (p<0.03, p<0.003, p<0.002, p<0.003 and p<0.001, respectively). Levels of MMP-9 and of IL-6 in multiple myeloma correlate well with bone turnover rate and may be useful in disease evaluation.

Artificial neural networks are suggested for use in predicting metal ion concentration in human blood plasma. Simulated and available experimental data are used to train the artificial neural network. Particularly, using 850 simulated samples, the network predicted the magnesium-free ion concentration with an average error smaller than 1%. Clinical data recently reported for 20 patients were considered and the artificial neural network predicted the concentration of free magnesium ion with an average error of about 6%. Overall, the approach of using artificial neural networks as an alternative or complementary strategy to deal with the analysis of human blood plasma can be useful for clinical diagnostics, if there is sufficient data to train the artificial neural network.

Plasma lactate measurements are typically performed in real time, limiting their usefulness in multicenter or longitudinal studies. To determine the stability of lactate specimens, blood was drawn in sodium fluoride/potassium oxalate tubes from 13 volunteers before and after 5min of handgrip exercise to intentionally increase lactate concentrations. Plasma was stored at −70°C. Aliquots were assayed in real time and after 1, 3, 6, 9, 12, 18, and 24months. Real-time lactate concentrations measured at baseline ranged from 0.52 to 2.23mmol/L before and from 2.91 to 11.04mmol/L after handgrip exercise. Using a linear mixed model, the estimated change from baseline at month 24 was 1.67% (95% confidence interval, −0.70% to 4.03%) for pre-exercise samples and 0.39% (95% CI, −1.13% to 1.91%) for post-exercise samples. Stored serial specimens from 232 HIV-infected subjects in a multicenter trial of antiretroviral therapy were also assayed centrally. Among those, median plasma lactate increased from baseline to 64weeks by 0.4mmol/L with zidovudine+lamivudine treatment and by 0.6mmol/L with didanosine+stavudine (each p<0.001 from baseline; p=0.04 for difference between groups over time). When performed as in this study, frozen storage with central batch lactate analysis is appropriate for prospectively collected samples in multicenter trials.

Plasma cystatin C, a new marker of glomerular filtration rate (GFR), was prospectively evaluated in surgical intensive care. Cystatin C was measured (immunonephelometry, Dade-Behring) in 10 patients selected to cover a full range of GFR (phase I) and in 28 unselected consecutive patients followed for 5days post-admission (phase II). Results were compared with 51 Cr-EDTA clearance (phase I only), plasma creatinine (kinetic Jaffe, Roche), 24-h or estimated by Cockcroft and Gault (CG) creatinine clearance (CrCl), and modified diet in renal disease (MDRD)-estimated GFR. In phase I, the highest correlation with 51 Cr-EDTA clearance (22–198mL/min) was noted for CG CrCl (r 2 : 0.883, p<0.001). During phase II follow-up, 24-h CrCl could not be calculated in 25% of daily evaluations. Cystatin C correlated with creatinine (0.856, p<0.0001) and CG CrCl with MDRD GFR (0.926, p<0.0001) in renal failure (10–78mL/min, n=60). There was a +40% (p<0.001) median difference between cystatin C and creatinine (as a % of upper normal cut-off). Sensitivity/specificity to detect a <80mL/min CG CrCl was 88/97% for cystatin C vs. 48/100% for creatinine (laboratory cut-off). In patients with normal and stable renal function (n=14), day-to-day intra-individual variation was 7.4% for cystatin C (vs. 10.6% for creatinine). In intensive care unit surgical adult patients, CG CrCl provides an easy and cost-effective estimate of GFR. Superior to creatinine, plasma cystatin C can be measured in selected patients where CG CrCl is known to be inaccurate.

Nine clinical laboratories in different regions of Spain have shared the search for reference individuals and the production of reference values for urinary component-to-creatininium concentration ratios measured in first morning urine samples using RD/Hitachi<MODULAR ANALYTICS> analysers. These urinary quantities include albumin, calcium(II), chloride, magnesium(II), phosphate, potassium ion, protein, sodium ion, urate and urea. All the logistic work was done in co-operation with the reagents' and analysers' supplier (Roche Diagnostics España, S.L., Sant Cugat del Vallès, Catalonia, Spain). From the blend of reference values obtained by each laboratory, the multicentre reference limits were estimated parametrically after mathematical transformation of original data.

Worldwide, allergic diseases are increasing in prevalence and incidence. Early assessment of the immunoglobulin E (IgE) sensitisation status has a major impact on clinical outcome and selection of therapeutic options. Recently, a number of new IgE-detecting test systems have entered the market, including screening tests allowing identification of a wide spectrum of sensitising allergens. We evaluated the analytical and diagnostic performance of the newly developed Allergy Screen test panel for atopy (Mediwiss Analytic, Moers, Germany). The evaluation was performed for four major respiratory and four major nutritional allergens in 142 patients with clinical suspicion of respiratory and/or food allergies. For all allergens, the test showed acceptable concordance to the skin-prick test and the in vitro IgE CAP system (Pharmacia, Freiburg, Germany). The analytical performance was acceptable, with CVs between 2 and 8% in the positive range and good dilution linearity (R=0.9735). Imprecision in the low IgE concentration range dramatically improved by lowering the cut-off value to 0.2IU/mL IgE. In conclusion, the Allergy Screen panel yields reliable results in the detection of allergic sensitisation to common allergens.

The IRIS IQ™ 200 automated urine analyser is a microscope associated to digital imaging software, and is able to classify urine particles into 12 categories and report quantitative results. We evaluated this analyser with respect to linearity and precision, and compared results with microscopic examination of the urine sediment, and with the measurement of urine strips in a CLINITEK 500 analyser. The assay was linear between 10 and 1030 particles/μL. The detection limit was 6 particles/μL. Intra- and inter-assay coefficients of variation were 1.9% and 2.3%. Results by the IQ™ 200 analyser showed highly significant correlations with those of the urine sediment (erythrocytes: ρ=0.68; leukocytes: ρ=0.60; epithelial cells: ρ=0.66; p<0.001), and urine strips (erythrocytes: ρ=0.67; leukocytes: ρ=0.66; p<0.001). These results indicate that the IRIS IQ™ 200 analyser may play a useful part in the automated examination and measurement of urine specimens.