Volume 44, Issue 11

Assessment and follow-up of renal dysfunction is important in the early detection and management of chronic kidney disease. The glomerular filtration rate (GFR) is the most accurate measurement of kidney disease and is reduced before the onset of clinical symptoms. Drawbacks to the measurement of GFR include the high cost and incompatibility with routine laboratory monitoring. Serum creatinine determination is a mainstay in the routine laboratory profile of renal function. The measurement of serum cystatin C has been proposed as a more sensitive marker for GFR. According to National Kidney Foundation-K/DOQ1 clinical guidelines for chronic kidney disease, serum markers should not be used alone to assess GFR. Based on prediction equations, clinical laboratories should report an estimate of GFR, in addition to reporting the serum value. In this article, information is presented on how best to estimate GFR using prediction equations for adults and for children. Using serum creatinine concentration with the Modification of Diet in Renal Disease (MDRD) study equation offers a suitable estimation of GFR in adults. The cystatin C prediction equation with the use of a prepubertal factor seems superior to creatinine-based prediction equations in children of <14years. Clin Chem Lab Med 2006;44:1295–302.

Translational research and translational medicine (referred to hereafter as translational research) are interchangeable terms that underline the pressing need to translate into practical benefits for those affected by disease the extensive investments divested by the private and public sectors in biomedical research. For people more directly involved in clinical practice (physicians, clinical laboratory professionals and patients), translational research responds to the need to accelerate the capture of benefits of research, closing the gap between what we know and what we practice. This basically means the transfer of diagnostic and therapeutic advances proven effective in large, well-conducted trials (and, therefore, evidence-based) to daily medical practice. Translational research should be regarded as a two-way road: bench to bedside, and bedside to bench. In particular, to make possible a more effective translation process, a new road map should be implemented through interaction and cooperation between basic researchers, clinicians, laboratory professionals and manufacturers. Some examples of recent developments in clinical laboratory testing, including markers of cardiovascular diseases, clinical proteomics and recombinant allergens, may explain the importance of careful evaluation of all variables that allow the introduction of such new insights into clinical practice to assure better clinical outcomes. The vital role of laboratory medicine in the delivery of safer and more effective healthcare requires more careful evaluation not only of the analytical characteristics, but also of any other variable that may affect the clinical usefulness and diagnostic performances of laboratory tests, thus allowing more accurate interpretation and utilization of laboratory information. Clin Chem Lab Med 2006;44:1303–12.

Background : Several independent population studies have reported that c.553G>T polymorphism of the apolipoprotein A5 gene ( APOA5 ) is associated with hypertriglyceridaemia. The aim of this study is to investigate the association between this genetic variation and the risk of type 2 diabetes mellitus. Methods : In this study, APOA5 c.553G>T polymorphisms in 152 healthy individuals and 71 type 2 diabetes mellitus patients were detected by PCR-restriction fragment length polymorphism and agarose electrophoresis methods, and serum levels of lipids were also estimated by biochemical methods. Results : The frequency of T alleles in the diabetes and control groups was 0.085 and 0.049, respectively. Compared with controls, there was no significant difference in the distribution of genotype and allele frequencies of c.553G>T polymorphic sites in diabetic patients (p=0.27 and p=0.15, respectively). However, the frequency of GT and TT genotypes and the T allele in the subgroup with hypertriglyceridaemia was significantly higher than that in the subgroup with normal triglyceridaemia in both the control group (p=0.034 and p=0.014, respectively) and the diabetes group (p=0.037 and p=0.007, respectively). In the diabetes and control groups, triglyceride levels in (GT+TT) genotype individuals were significantly higher than in GG genotype individuals (p=0.001 and p=0.003, respectively), and levels of low-density lipoprotein cholesterol were also significantly higher (p=0.044 and p=0.022, respectively). Conclusions : APOA5 c.553G>T polymorphism is not significantly associated with susceptibility to type 2 diabetes mellitus, but is associated with plasma triglyceride and low-density lipoprotein cholesterol levels. Clin Chem Med Lab 2006;44:1313–6.

Background : Evidence is accumulating that periconceptional folic acid supplementation may prevent congenital heart defects (CHD). The methionine synthase reductase (MTRR) enzyme restores methionine synthase (MTR) enzyme activity and therefore plays an essential role in the folate- and vitamin B 12 -dependent remethylation of homocysteine to methionine. We studied the influence of the MTRR 66A>G polymorphism on CHD risk. In addition, possible interaction between this variant and plasma methylmalonic acid (MMA) concentrations, as an indicator of intracellular vitamin B 12 status, was investigated. Methods : Case-control and case-parental studies were conducted to explore this association. In total, 169 CHD patients and 213 child controls, and 159 mothers with a CHD-affected child and 245 female controls were included. Results : The maternal MTRR 66AG and GG vs. AA genotypes revealed an odds ratio (OR) of 1.3 (95% CI 0.72–2.20) and 1.3 (0.71–2.37), respectively. Family-based transmission disequilibrium analysis did not reveal a significant association of the foetal 66G allele with the development of a heart defect in children (χ 2 =2.94, p=0.086). Maternal 66GG genotype in combination with high MMA concentration (above the 80th percentile) was associated with a three-fold (OR 3.3, 95% CI 0.86–12.50) increased risk for all types of CHD in offspring. Conclusions : These data indicate that maternal MTRR 66A>G polymorphism is not a risk factor for CHD. Maternal MTRR 66GG genotype with compromised vitamin B 12 status may possibly result in increased CHD risk. In addition to folate, vitamin B 12 supplementation may contribute to the prevention of CHD. Clin Chem Lab Med 2006;44:1317–23.

Background : Hyperhomocysteinemia may constitute a risk factor for patients with severe heart failure. This study examines the relationship between plasma homocysteine concentration and left ventricular ejection fraction with renal function in heart failure patients free of coronary artery disease. Methods : Left ventricular ejection fraction was documented in 62 patients with advanced heart failure who had no proven significant coronary artery stenosis. Glomerular filtration rate was measured using the Cockroft-Gault equation. Results : Elevated homocysteine levels (≥15μmol/L) were detected in 22 patients. Low glomerular filtration rate was observed in patients who had normal serum creatinine concentration. Homocysteine was strongly correlated with age, duration of disease, left ventricular ejection fraction, serum creatinine, and glomerular filtration rate. Statistically significant trends were observed across respective homocysteine quartiles. However, by multivariate regression, the strongest predictor of homocysteine was the glomerular filtration rate. Conclusions : Impaired renal function leads to a diminished clearance rate, which can be a prominent pathophysiological mechanism in the elevation of homocysteine concentration in heart failure. Clin Chem Lab Med 2006;44:1324–9.

Background : The UF-100 is a flow cytometer designed for automated cellular urinalysis. In this study, the usefulness of the UF-100 in laboratory investigation into the origin of hematuria was evaluated. Methods : Results from flow cytometric urinalysis were used to classify urinary red blood cells (RBCs) according to glomerular and non-glomerular origin and the classification was compared to the patient's clinical diagnosis as the gold standard. In parallel, microscopic sediment analysis was carried out. Results : A total of 206 urine samples from 129 patients were analyzed (127 from patients with glomerular hematuria, 79 from patients with non-glomerular hematuria). Of these, 136 samples (92 patients) showed overt hematuria (≥20RBC/μL). Urine flow cytometry correctly classified 61% (sediment analysis 69%) of urine samples with overt hematuria. If inconclusive results are excluded, the UF-100 correctly diagnosed 85% (sediment analysis 98%) of urine samples with overt hematuria. The UF-100 and microscopic sediment analysis both showed sensitivity of 99% for the detection of glomerular hematuria. The specificity of the UF-100 for the detection of glomerular bleeding was lower (42%) than the specificity of microscopic sediment analysis (93%). Conclusions : Owing to its low specificity, the UF-100 showed limited capacity to discriminate glomerular from non-glomerular causes of hematuria in a population with a high incidence of renal disease. Therefore, extensive microscopic urinalysis remains necessary to assess the origin of hematuria. Clin Chem Lab Med 2006;44:1330–4.

Background : Few reports of the effects of treatment with chymotrypsin on the determination of sperm parameters and seminal biochemistry markers are documented. Methods : Sperm parameters of 63 liquefied and 27 non-liquefied samples, untreated or treated with chymotrypsin, were evaluated using computer-assisted semen analysis. In addition, biochemistry markers such as γ-glutamyltranspeptidase, α-glucosidase and fructose in 50 liquefied and 39 non-liquefied samples, untreated or treated with chymotrypsin, were determined. Results : Treatment with chymotrypsin had no effect on sperm concentration, motility, motility a and b, straightness, curvilinear velocity, straight line velocity, average path velocity and beat cross frequency in both liquefied and non-liquefied semen. However, linearity (p=0.025) decreased and the amplitude of the lateral head (p=0.029) increased significantly in non-liquefied semen after treatment with chymotrypsin. The levels of γ-glutamyltranspeptidase, α-glucosidase and fructose in seminal plasma were unaffected by chymotrypsin, regardless of liquefaction status. Conclusions : Chymotrypsin had no effects on the detection of sperm parameters and biochemistry markers, and could be used to treat non-liquefied samples before semen analysis in the andrology laboratory. Clin Chem Lab Med 2006;44:1335–9.

Background : Cardiovascular morbidity is frequent after non-cardiac surgery and the early recognition of cardiac involvement is an essential tool for clinical risk stratification and management. The aim of this study was to investigate the behavior of traditional and emerging cardiac markers, including NT-prohormone-brain natriuretic peptide (NT-proBNP) and ischemia-modified albumin (IMA), in the perioperative period in patients undergoing major uncomplicated orthopedic surgery. Methods : A total of 37 patients undergoing major orthopedic surgery were longitudinally evaluated for NT-proBNP, IMA, cardiac troponin T (cTnT), creatine kinase isoenzyme MB and myoglobin 3h before surgery and 4 and 72h thereafter. Results : NT-proBNP values were significantly increased at 72h postoperative compared to both 3h preoperative and 4h postoperative (NT-proBNP: 20 vs. 4.5pmol/L, p<0.001 and 20 vs. 5.9pmol/L, p<0.001). IMA levels were significantly increased at 4 and 72h postoperative vs. 3h preoperative (132 vs. 113kU/L, p=0.02 and 151 vs. 113kU/L, p<0.001). In a stepwise regression model, the perioperative liquid amount and degree of modification in postoperative creatinine levels (delta-creatinine) were independently related to the NT-proBNP increase. Conclusions : The significant increase observed in NT-proBNP suggests that patients undergoing major uncomplicated orthopedic surgery may develop subclinical cardiac stress, presumably attributable to the considerable infusion of liquids. The clinical significance of this finding deserves further investigation, especially in patients at higher risk of heart failure. Clin Chem Med Lab 2006;44:1340–6.

Background : Positive test strip results, pathological particles in urine and the presence of proteinuria are common findings in nephropathies. A comparison between these methods and renal biopsies became available with the introduction of quantitative measurement of marker proteins (albumin, transferrin, IgG, α 1 -microglobulin, retinol binding protein, α 2 -macroglobulin, Bence Jones proteins) and standardised urine sediment analysis by flow cytometry or microscopy. Methods : A total of 400 urine samples were examined using marker protein patterns, test strips and quantitative sediment analyses. Results : Results from standardised urine sediment analyses were compared with the excretion of renal marker proteins. Increased erythrocyte and leukocyte counts in urine were observed in only 29% and 39% of the samples for which pathological protein excretion was found. The sensitivity in detecting pathological particles in urine sediment, such as casts and/or dysmorphic erythrocytes, was only 19%. Renal biopsies from 65 patients who were classified as pathological were compared with proteinuria and sediment analyses. Increased excretion of marker proteins was found in all cases, whereas only 41% of the cellular urine sediments showed pathological results. Conclusions : Quantitative measurement of marker proteins from both the glomerular and tubular sides should be used upfront as screening parameters for the early detection of renal disorders. Clin Chem Lab Med 2006;44:1347–54.

Background : Clinical chemistry reference values for elderly persons are sparse and mostly intermixed with those for younger subjects. To understand the links between metabolism and aging, it is paramount to differentiate between “normal” physiological processes in apparently healthy elderly subjects and metabolic changes due to long-lasting diseases. The Vienna Transdanube Aging (VITA) study, which began in 2000 and is continuing, will allow us to do just that, because more than 600 male and female volunteers aged exactly 75years (to exclude any influence of the “aging” factor in this cohort) are participating in this study. Methods : Extensive clinical, neurological, biochemical, psychological, genetic, and radiological analyses, with a special emphasis on consumption of medication and abuse of drugs, were performed on each of the probands. The multitude of data and questionnaires obtained made possible an a posteriori approach to select individuals fulfilling criteria for a reference sample group of apparently healthy 75-year-old subjects for our study. Specific analytes were quantified on automated clinical analyzers, while manual methods were used for hormonal analytes. All clinical chemistry analytes were evaluated using in-depth statistical analyses with SPSS for Windows. Results : In all, reference intervals for 45 analytes could be established. These include routine parameters for the assessment of organ functions, as well as hormone concentrations and hematological appraisals. Because all patients were reevaluated after exactly 30months in the course of this study, we had the opportunity to reassess their health status at the age of 77.5years. This was very useful for validation of the first round data set. Data of the second round evaluation corroborate the reference limits of the baseline analysis and further confirm our inclusion and exclusion criteria. Conclusions : In summary, we have established a reliable set of reference data for hormonal, hematological, and clinical chemistry analytes for elderly subjects. These values will be very useful for our future attempts to correlate disease states and aging processes with metabolic factors. Clin Chem Lab Med 2006;44:1355–60.

Hepcidin participates in the regulation of iron homeostasis and its precursor pro-hepcidin can be measured in serum. We evaluated pro-hepcidin serum concentrations in healthy subjects and the possible effects of iron supplementation on the results. The results suggest extensive physiological variation in serum pro-hepcidin concentrations between healthy subjects with no symptoms or signs of anaemia, infections, inflammations, chronic disease or other interpretative factors. Before pro-hepcidin measurements can be used in clinical practise, further investigations are required to identify the physiological factors affecting normal serum pro-hepcidin variations in healthy subjects. The responses of serum pro-hepcidin to a 100-mg oral dose of iron also showed considerable inter-individual variation. In male subjects, no systematic changes in serum pro-hepcidin concentrations were found and the increase in serum iron was fairly modest. In nine out of the ten female subjects who had rather low amounts of storage iron, iron supplementation was followed by an increase in both serum iron and serum pro-hepcidin concentrations. There were considerable inter-individual differences in the timing and magnitude of the response. We also evaluated the conceivable influences of sample storage and freeze-thaw cycles on the results of serum pro-hepcidin ELISA. We did not observe any changes in the results after serum samples were frozen and thawed up to four times and/or stored at room temperature for up to 6h. Clin Chem Lab Med 2006;44:1361–2.

Background : The purpose of an external quality assessment (EQA) is to evaluate the analytical capability of clinical laboratories, identify differences among the laboratories and improve analytical quality. Our EQA results show that the rates of unsatisfactory performance for thyroid hormone tests were the highest in all of our EQA programs. Therefore, the main purpose of this study was to investigate unsatisfactory results by comparing the analytical values of five routinely used analytical systems. Methods : The Kruskal-Wallis and two-sample Kolmogorov-Smirnov tests were used to identify analytical differences among and between analytical systems, respectively. Results : The rates of significantly different results compared to the total number of analytical results were 81.1%, 64.5%, 93.3%, 50.0% and 56.7% for free triiodothyronine, total triiodothyronine, free thyroxine, total thyroxine and thyroid-stimulating hormone, respectively. Conclusions : Relatively large analytical differences between analytical systems were observed, especially when the analytical systems were used to measure free thyroid hormones. Clin Chem Lab Med 2006;44:1363–6.

Background : Determination of leukocyte (WBC) counts in pleural fluid is routinely performed by microscopic examination. In this study, we evaluated the performance of automated (differential) WBC counting in comparison with manual counting. Methods : Pleural fluid samples (n=45) were obtained from patients undergoing diagnostic thoracocentesis. The manual total WBC count was determined after Samson staining in a Fuchs-Rosenthal hemocytometer; microscopic differential counts were performed on May-Grünwald Giemsa-stained cytospin slides. The Sysmex XE-2100 hematology analyzer was used for automated (differential) WBC counting. The functional detection limit was determined by serial dilution of continuous ambulatory peritoneal dialysis (CAPD) fluid and replicate measurements of each dilution. Results : The automated WBC count (×10 6 /L) was highly correlated with that of the microscopic reference method (r 2 =0.95; WBC-analyzer=0.97×WBC-reference method+16; n=45). Good agreement was also observed for the absolute lymphocyte count (r 2 =0.92; WBC-analyzer=0.99×WBC-reference method+32; n=36), neutrophil count (r 2 =0.94; WBC-analyzer=0.91×WBC-reference method+6; n=35), and monocyte count (r 2 =0.73; WBC-analyzer=0.83×WBC-reference method+6; n=38). The functional detection limit for WBCs was calculated at 50×10 6 /L (coefficient of variation 20%). Conclusions: With some limitations, total and differential WBC counts in pleural fluid can be reliably determined using the Sysmex XE-2100 instrument. Clin Chem Lab Med 2006;44:1367–71.

Background : The d-ROMs test for the evaluation of serum hydroperoxides (HP) is simple, reliable, and cheap. Furthermore, it can easily be adapted to automated analyzers. Changing from the manual to an automated procedure allows the simultaneous processing of a large number of samples in a greatly reduced time, avoiding manual handling of samples and reagents and reducing variability sources. Methods : This study was performed to adjust the manual procedure to a routine automated method in the clinical laboratory. We carried out the d-ROMs test in sera from 90 subjects of both sexes (34 men and 56 women) with age ranging from 20 to 80years (mean 51±14years). All subjects were free from acute or chronic inflammatory disease, immunological disease and history or evidence of malignancy. Subjects were not on vitamin and/or antioxidant therapies. Results : The detection limit of the assay was 40AU. Linearity was observed up to 475AU. The recovery ranged between 97% and 105%. Within- and between-run imprecision was <5%. The mean HP value was 304±8AU, with no significant difference between men (291±10AU) and women (311±11AU). A significant positive correlation was observed between age and HP in the whole population (r=0.4, p=0.0002). Conclusions : The automated test for the estimation of serum hydroperoxides represents a reliable and feasible procedure for increasing efficiency and reducing costs compared to the manual method, and is particularly suitable for evaluating oxidative stress in a variety of clinical conditions. Clin Chem Lab Med 2006;44:1372–5.

Background : The Accu-Chek Inform glucose monitor is a point-of-care system for testing blood glucose. New test strips, calibrated to deliver glucose plasma-like values, were launched on the market in May 2005. The aim of our study was to perform analytical validation of these new strips. Methods : We compared the new plasma strips with whole blood strips; results for the plasma strips with plasma values obtained using a clinical analyzer and with whole blood values given by the glucose electrode of a blood gas analyzer; and the influence of the type of blood (capillary or venous) on the results obtained by the glucose monitor with the plasma calibrated strips. Results : Plasma strips give on average 7% higher results than the previous whole blood strips. However, the results given by the plasma strips on capillary whole blood, even if well correlated, are not completely comparable with those given by an analyzer for venous plasma. Nevertheless, these plasma strips and the glucose electrode of a blood gas analyzer give comparable results. Conclusions : Accu-Chek Inform plasma strips are a good method for monitoring of blood glucose values in patients with diabetes. Clin Chem Lab Med 2006;44:1376–8.

Background : In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls. Methods : Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10g/L) and incubated for 3h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined. Results : Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (≥95%). Anti-insulin antibody interference was significant for RI and RAAs (p≤0.004) and correlated with anti-insulin antibody level in the serum (p≤0.001). Conclusions : In serum, RI and RAA cross-reactivities are slightly lower than in buffer. For RAA assessment, hemolysed samples should be discarded and anti-insulin antibodies should be removed from samples before immunoreactivity measurements. Clin Chem Lab Med 2006;44:1379–82.

Background : The accurate and rapid recognition of myocardial injury in patients presenting in the emergency department (ED) with chest pain continues to be a clinical challenge. Heart-type fatty acid-binding protein (H-FABP) appears to be one of the best candidates among the new early cardiac markers studied. Methods : We evaluated the analytical characteristics of a new quantitative and fully automated H-FABP assay (Randox Laboratories Ltd., Crumlin, UK) and compared its clinical performance with respect to the myoglobin (Myo) assay (Dade Behring, Milan, Italy). A precision study was carried out by testing three levels of quality control (QC) material and two in-house pool (P) samples. To test the accuracy of H-FABP determinations in plasma (lithium-heparin) samples, H-FABP concentrations measured in a set of matched sera and plasma samples were compared. A total of 77 non-consecutive patients (51 males and 26 females; 62±16years) who presented to the ED with chest pain suggesting myocardial ischemia were enrolled. The patients were classified into two groups (acute myocardial infarction, n=22; non-acute myocardial infarction, n=55) on the basis of the discharge diagnosis. Results : The between-day imprecision for three levels of control material and serum pool samples was 6.26%–8.04% (range 2.32–44.03μg/L) and 9.03%–12.63% (range 11.85–65.13μg/L), respectively. In the serum vs. plasma study, bias was +0.178 (95% CI −0.033 to +0.389). The best cut-off and the associated diagnostic efficacy were 95μg/L and 89.47% for Myo and 5.09μg/L and 98.70% for H-FABP, respectively. Conclusions : H-FABP determination in patients with ischemic symptoms may be a more reliable early indication of cardiac damage than myoglobin. Clin Chem Lab Med 2006;44:1383–5.