Volume 46, Issue 8

At least four premature newborn complications have been reported to be associated with low serum insulin-like growth factor-I (IGF-I) levels: bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis and brain damage. Local IGF-I concentrations have only been reported for bronchopulmonary dysplasia and these findings show that lung IGF-I levels are clearly increased (epithelial lining fluid levels), emphasizing the fact that IGF-I is differently regulated in the general circulation or at local level. The present review discusses the meaning of the association between serum IGF-I amounts and development of complications in premature newborns. Finally, some methodological indications are reported regarding the IGF-I assay procedures. It is important to establish what are the possible relationships between blood levels and those of different compartments involved in the diseases. Clin Chem Lab Med 2008;46:1061–6.

Differentiated thyroid cancer is an infrequent disease with a generally good prognosis. The initial treatment is total thyroidectomy coupled with ablation of thyroid remnants by iodine-131. Because thyroid cells are the only source of thyroglobulin in the human body, the circulating thyroglobulin measurement is the basis of differentiated thyroid cancer follow-up. Due to suboptimal sensitivity in older assays, the thyroglobulin measurements after stimulation by endogenous or exogenous thyrotropin are still recommended for unmasking occult disease. However, the development of thyroglobulin assays with improved functional sensitivity allows us to detect small amounts of thyroid tissue even when thyrotropin is suppressed. As a consequence, undetectable thyroglobulin during thyroxine treatment is sufficient evidence to forgo thyrotropin stimulation in many cases, if a highly sensitive assay is employed. On the other hand, increasing concentrations in highly sensitive assays are an early and reliable indicator of recurrent disease. This will result in a limited follow-up protocol, warranting the detection of recurrences of differentiated thyroid cancer and reducing patient burden and medical costs. Clin Chem Lab Med 2008;46:1067–73.

Background : The aim of the present study was to identify, using proteomics, changes in the expression of proteins and protein isoforms mainly associated with inflammation and vascular damage in the coronary sinus of patients undergoing off-pump coronary artery bypass (OPCAB) surgery. Methods : Plasma from the coronary sinus of 18 patients undergoing OPCAB was obtained before the ischemic procedure and after revascularization of the left anterior descending artery (LAD) with an internal thoracic artery graft. The mean LAD ischemic time was 11.1±0.7 min. For the proteomic study, two-dimensional gel electrophoresis was performed. Results : A significant decrease in the expression of three fibrinogen γ-chain (FGC) isoforms, three vitamin D-binding protein (DBP) isoforms, six haptoglobin (HPT) isoforms, two apolipoprotein (apo)-AI isoforms, one ceruloplasmin (CP) isoform and apoAIV was found after revascularization. Before anastomosis, negative correlations between blood flow and FGC isoforms 2 and 3 and positive correlations between blood flow and apoAI isoform 5 were observed. After anastomosis, we observed positive correlations between haptoglobin isoform 3 and DBP isoform 2 and blood flow. Before anastomosis, positive correlation between DBP isoform 2 and troponin I was observed. After LAD grafting, positive correlations between troponin I and HPT isoform 6, CP isoform 1 and apoAI isoforms 2 and 4 were observed. After the procedure, positive correlations between creatine kinase-MB and coronary sinus expression of FGC isoforms 1, 2 and 3 and HPT isoforms 1 and 2 were also observed. Conclusions : In blood from coronary sinus the expression of a number of proteins and protein isoforms associated with inflammation and vascular protection was modified after OPCAB. Clin Chem Lab Med 2008;46:1074–84.

Background: Genes correlated with nicotine may be important in obesity because of the role of nicotine in food intake. Methods: To determine if single nucleotide polymorphisms (SNPs) in the neuronal nicotinic acetylcholine receptor α2 subunit gene ( CHRNA2 ) are associated with overweight/obesity, 156 non-obese and 200 overweight/obese Koreans were genotyped for four CHRNA2 SNPs using an Affymetrix Array Chip. Results: Of four SNPs, only one, rs2043063, was associated with overweight/obesity in the study population (co-dominant, p=0.049; dominant, p=0.027). Associations between the significant SNP and obesity-related clinical characteristics were observed, but these were not significant. Conclusions: The CHRNA2 rs2043063 SNP might be a risk factor for overweight/obesity in Koreans. Therefore, further studies are needed to investigate whether mutations or common polymorphisms of the CHRNA2 gene represent a risk factor for obesity. Clin Chem Lab Med 2008;46:1085–9.

Background : Cytochrome P450 (CYP) and glutathione S-transferase ( GST ) gene variants have been intensively investigated for their implication in the development of different neoplasms. Methods : In the present study, we analyzed genetic polymorphisms of CYP1A1 , GSTM1 , GSTP1 , and GSTT1 in 127 head and neck cancer patients and 151 hospital controls. Results : No significant increase in risk in patients with the GSTM1 null genotype (OR=1.52, 95% CI: 0.93–2.49) or CYP1A1 462Val alleles (OR=1.60, 95% CI: 0.73–3.52) or GSTP1 105Val alleles (OR=0.97, 95% CI: 0.59–1.58) was observed. The GSTT1 null genotype was found in 30.5% of the controls and 21.3% of the head and neck cancer patients (p=0.15). The estimated head and neck cancer risk for the combination of either CYP1A1 Ile462Val or CYP1A1 Val462Val genotype with either GSTP1 Ile105Val or Val105Val genotype (OR=2.89, 95% CI: 0.71–11.71) and for the combination of either CYP1A1 Ile462Val or CYP1A1 Val462Val genotype with GSTT1 null genotype (OR=2.62, 95% CI: 0.64–10.85) suggested the absence of the modifying effect of combined variant alleles on head and neck cancer susceptibility. The joint effect of either CYP1A1 Ile462Val or CYP1A1 Val462Val genotype with GSTM1 null genotype significantly increased the risk of head and neck cancer (OR=7.15, 95% CI: 1.49–34.32). Conclusions : Our findings corroborate metabolic genes interactions, especially for CYP1A1 462Val alleles and GSTM1 homozygous deletion, in the development of head and neck cancer in the investigated population groups in Poland. Clin Chem Lab Med 2008;46:1090–6.

Background : Numerous polymorphisms of the apolipoprotein B ( APOB ) gene have been described. Particularly, the insertion/deletion (Ins/Del) polymorphism located in the coding part of the signal peptide of apoB, associated with modification of lipid concentrations and the risk of coronary artery disease and/or myocardial infarction (MI), has been reported in the general population. Moreover, conflicting results emerge from the literature and suggest that the effect is context-dependent. In the present study, the first investigation of the Ins/Del polymorphism of the APOB gene in Tunisian patients with MI, we examined a possible association between this polymorphism and MI in a subgroup of the Tunisian population. Methods : A total of 318 Tunisian patients with MI and 368 healthy controls were included in the study. Genomic DNA was extracted from white blood cells, and the Ins/Del polymorphism was determined by electrophoresis in polyacrylamide gels after PCR amplification. A binary logistic regression analysis was performed to test how the association between MI and Ins/Del polymorphism is independent from confounding factors. Results : A significant difference in genotype distribution and allele frequency was observed between patients and controls. Patients with MI had a frequency of 7.2% for the Del/Del genotype, 39.6% for the Ins/Del genotype, and 53.1% for the Ins/Ins genotype. Controls had a frequency of 3.0% for the Del/Del, 32.1% for the Ins/Del and 64.9% for the Ins/Ins genotype (χ 2 =12.93, p=0.002). The MI patient group showed a significantly higher frequency of the Del allele compared to controls (27.1% vs. 19.1%; χ 2 =12.50, p=0.0004). In comparison to the Ins/Ins homozygotes, the odds ratio (95% confidence interval) for MI was 1.51 (1.09–2.07) for Ins/Del heterozygotes and 2.95 (1.40–6.22) for Del/Del homozygotes. In multivariate analysis, age (p=0.001), smoking (p<0.001), hypertension (p=0.001), diabetes mellitus (p<0.001), and dyslipidemia (p=0.01) were independent correlates of the presence of MI, whereas the Ins/Del polymorphism (p=0.330) was not an independent predictor of MI. Conclusions : The present study shows a significant but not independent association between the Ins/Del polymorphism of the APOB gene and MI in the Tunisian population. Clin Chem Lab Med 2008;46:1097–101.

Background : A polymorphism C1019T on the connexin37 ( Cx37 ) gene has been found to associate with coronary artery disease. There are conflicting results on which allele confers risk, and the possibility of interactions between the polymorphism and risk factors has been raised. In this study, we examined interactions between the Cx37 polymorphism and common risk factors and their associations to early vascular parameters of atherosclerosis: carotid artery intima-media thickness (IMT), and carotid artery compliance (CAC) and brachial artery flow mediated dilatation (FMD). Methods : A population of 1440 healthy young adults from the Cardiovascular Risk in Young Finns Study was studied. The subjects were genotyped and their cardiovascular risk factor and ultrasound data gathered in 2001 were used for the statistical analyses. Results : In the whole population, homocysteine in subjects with the TT genotype was found to be associated with higher FMD values (p for interaction 0.038) and remained so in three different adjusted models (p for interaction 0.022–0.038). In women with the CC genotype, smoking was found to be associated with higher FMD values and the smoking-by-genotype interaction remained significant in three adjusted models (p for interaction 0.001–0.041). In women with TT genotype, the effect of smoking was opposite, i.e., FMD values for smokers were lower compared to non-smokers. In men, physical activity interacted with Cx37 on CAC in the CT and TT genotypes (p for interaction 0.011). No significant interactions were found to predict IMT. Conclusions : The effect of smoking and homocysteine levels on arterial endothelial functions and elasticity were modified by the allelic variation of the Cx37 gene. These data suggest that variation in the connexin gene may modify effects risk factors have on vascular function. Clin Chem Lab Med 2008;46:1102–8.

Background : The single nucleotide Marburg I ( MRI ) polymorphism of the factor VII-activating protease ( FSAP ) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. Methods : Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. Results : Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350–1400 mPEU/mL and 1.8–120 ng/mL, total coefficients of variation (CV): 5%–20% and 5%–14%, within-run CV: 4%–11% and 2.3%–12%, and run-to-run CV: 2%–17% and 4.3%–8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. Conclusions : The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP . Clin Chem Lab Med 2008;46:1109–16.

Background : The diagnostic potential of DNA methylation has generated a need for quality control systems in its analysis. Here, we tested whether cycle threshold (Ct) values from real-time quantitative PCR correlate better with the reliability of bisulfite-PCR methylation analyses than spectrophotometric concentrations. Methods : A total of 18 cell line DNA samples were quantified for β-actin ( ACTB ) Ct and spectrophotometric values from 1-μL volumes. A further 1 μL was analyzed in quintuplicate for bisulfite DNA ACTB by MethyLight. Correlations between Ct and spectrophotometric values, and MethyLight qualitative (detection in all replicates) and quantitative (Ct value standard deviation <1) reliability were compared. To validate the findings, the same comparisons were made in 40 formalin-fixed, paraffin-embedded tissue (FFPET) samples analyzed by methylation-specific PCR of MLH1 . Results : Using optimal thresholds, Ct values correctly identified 6/7 (86%) and 11/11 (100%) cell line samples to be qualitatively, and 7/7 (100%) and 4/4 (100%) quantitatively reliable and unreliable, respectively. The corresponding frequencies for spectrophotometry were 6/8 (75%), 10/10 (100%), 6/8 (75%) and 2/3 (67%), respectively. In FFPET DNA, the respective values for qualitative reliability for Ct values were 25/25 (100%) and 11/15 (73%), and 11/14 (79%) and 8/26 (31%) for spectrophotometry. Conclusions : Ct values could be useful indicators for gauging the suitability of samples for methylation analysis, in particular from FFPET. Clin Chem Lab Med 2008;46:1117–21.

Backgroud: The high postoperative recurrence of hepatocellular carcinoma (HCC) is a problem that would benefit from the identification of indicators of recurrence and prognosis. In the past few years, research has shown that E-cadherin and CD34 can be used as indicators of the invasion of malignant tumors. In the present study, we investigated the expression of E-cadherin and CD34 in HCC patients. Methods: Expression levels of E-cadherin and CD34 in 41 HCC samples were detected using two-step immunohistochemical methods and compared with clinical pathological parameters and survival rate. Results: The positive rates of E-cadherin and CD34 expression in 41 HCC cases were 48.78% and 100%, respectively. Expression of E-cadherin was significantly lower in patients with larger tumors, a high risk invasion and Edmondson classification III or IV (p<0.05). There was a significant relationship between CD34 expression and age and tumor invasiveness (p<0.05). There was no significant relationship between expression of CD34 and E-cadherin by Spearman statistical analysis (p>0.05). The survival rate in patients with negative expression of E-cadherin was significantly lower. Conclusions: The expression of CD34 cannot be used singly as a prognostic indicator for HCC patients. The co-expression of E-cadherin and CD34 cannot be used as a prognostic indicator for HCC patients. Clin Chem Lab Med 2008;46:1122–6.

Background : The calibration of Jaffe method for serum creatinine using one serum-based standard complemented with artificial matrix compensating standard's Jaffe-interfering substances allows two-point calibration with results well comparable with enzymatic methods. Method : Spectrophotometry was used. Results : Jaffe procedures compensating serum/plasma intereferents by subtracting a constant amount of creatinine poorly overcompensate creatinine in children. Two-point calibration with a pair of primary serum standards certified by the reference measurement procedure (isotope-dilution, mass spectrometry) or with a pair of secondary standards linked to primary materials could provide results well agreeable with enzymatic determination. Such a calibration comprises an absorbance offset corresponding to other-than-creatinine Jaffe-interfering chromogens present in standards in the calibration line, while a two-point calibration combining one standard with physiological saline/water always grossly distorts calibration line. We calculated/prepared artificial serum matrices capable of compensating Jaffe-interfering chromogens in serum standards. The combination of even one standard with its artificial matrix also enables two-point calibration with practically the same results as with a pair of primary standards. Conclusions : A two-point calibration of Jaffe method for serum creatinine combining only one serum standard with creatinine solution matching standard's allow matrix-present interferents present results well comparable with enzymatic determination, providing the standard is attested/linked to the reference measurement procedure. Clin Chem Lab Med 2008;46:1127–33.

Background : Although afflicted with stone formation, urolithiasis patients often present with normal renal excretions of lithogenic and inhibitory substances. In this study, crystal formation is not interpreted as the result of urinary excretions simply exceeding the static limits of normal ranges but rather as the consequence of relative combinations of such parameters which convert urine into becoming potentially lithogenic. Our model embraces different triplet combinations of fundamental urinary risk factors for calcium oxalate (CaOx) crystallization, to characterize different levels of urinary stone formation risk. Methods : Urinalyses and BONN-Risk-Indices (BRI) were determined for CaOx patients under home conditions, after 1 week of hospitalization, and for healthy controls. The relative urinary concentrations and interdependences of, inter alia, free ionized Ca ({Ca 2+ }), bound Ca ({Ca b }), and oxalic acid ({OA}) were compared. Results : Three levels of CaOx formation risk can be distinguished: (I): low stone formation risk with an increase in {Ca 2+ } and concomitant decrease in {Ca b }, while {OA} remains almost constant, BRI increases simultaneously; (II) moderate risk at {Ca 2+ }/{Ca b }≈1; and (III) high risk with decrease in {Ca 2+ }/{Ca b } and simultaneous increase in {OA}. Conclusions : The proposed approach of urinalysis interpretation allows complementary strategy of identification of patterns of disturbed urinary composites leading to calculus formation. Clin Chem Lab Med 2008;46:1134–9.

Background : Albumin and plasma cholinesterase are markedly reduced in critical illness. However, the plasma half-lives of these two proteins have only been determined in very limited numbers of patients mostly outside intensive care. Therefore, we determined the kinetics of decrease of albumin and cholinesterase in a large population of critically ill patients. Methods : Medical records of all patients of two university-affiliated surgical intensive care units during a time period of 8 years were retrospectively evaluated. To calculate the rate of fall, the first value in every episode of decreasing albumin or cholinesterase was arbitrarily set at 100% and the ensuing lower values were expressed as the respective fractions. Results : Records of 3591 patients were evaluated. Regression analysis exhibited an exponential evolution of albumin and cholinesterase over time with corresponding mean apparent plasma half-lives of 11.8 days [95% confidence interval (CI): 10.8–12.9] for albumin and 7.8 days (95% CI: 7.4–8.3) for cholinesterase. Conclusions : These findings suggest that the apparent plasma half-lives of albumin and cholinesterase in critically ill surgical patients are considerably shorter than previously determined. Clin Chem Lab Med 2008;46:1140–2.

Background : The aim of this study was to test the diagnostic model of combining procalcitonin (PCT) and C-reactive protein (CRP) levels in the cord blood and routinely used biochemical parameters and clinical data in the prediction of early onset neonatal infection. Methods : PCT and CRP concentrations were measured in cord blood of neonates with infection (group A, n=46) and compared with uninfected neonates (group B, n=240). Inclusion criteria for group A were based on obstetric history, clinical data and results of laboratory tests. Logistic regression was applied. The receiver operating characteristic (ROC) curves were constructed for PCT, CRP and the diagnostic model. Results : There was a highly significant (p<0.000001) difference in PCT and CRP concentrations between both groups. The cut-off point for PCT in cord blood was 1.22 ng/mL [sensitivity % (SE%) 80.43, specificity % (SP%) 71.67, positive predictive value % (PPV%) 35.24, negative predictive value % (NPV%) 95.03], and 1.0 mg/L for CRP (SE% 73.91, SP% 77.92, PPV% 39.08, NPV% 93.97). In total, seven variables were included in the model (concentrations of PCT and CRP in cord blood, tocolysis, nutritional status of the newborn, Apgar score, neutrophil ratio and red blood cell count in neonatal venous blood), which proved to offer the highest sensitivity (91.3%; 95% CI: 83–99) and specificity (90%; 95% CI: 86–94) for the detection of early onset neonatal infection. The likelihood ratio for the model was high at 9.13, with PPV% 63.64 (95% CI: 52–75), NPV% 98.18 (95% CI: 96–100) and calculated area under the curve at 0.973. Conclusions : The diagnostic model based on seven clinical and laboratory parameters, using the concentration of PCT and CRP measurements in the cord blood, could be a useful tool for the prediction of early onset neonatal infection. Clin Chem Lab Med 2008;46:1143–8.

Background : Atherosclerosis, a chronic inflammatory disease, underlies the pathogenesis of coronary artery disease. The present study assessed the diagnostic possibilities of inflammatory biomarkers, serum neopterin, nitrite/nitrate (NO 2 – /NO 3 – ), inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α), and their correlation with risk factors in patients with acute coronary syndromes and stable angina pectoris. Methods : We studied 44 patients with chronic stable angina pectoris, 46 with unstable angina, 55 with acute ST-elevation myocardial infarction and 39 age-matched healthy volunteers (control group). Serum neopterin, iNOS and TNF-α were determined with commercially available enzyme linked immunosorbent assay methods and NO 2 – /NO 3 – by the modified cadmium-reduction method. Results : Mean serum neopterin levels were significantly higher in patients with unstable and stable angina pectoris in comparison to control subjects (p<0.01 and p<0.05, respectively). Serum NO 2 – /NO 3 – values were significantly elevated (p<0.01) only in patients with unstable angina. ST-elevation myocardial infarction patients with cardiac death during follow-up showed significantly lower baseline neopterin values (p<0.001), and higher NO 2 – /NO 3 – levels (p<0.05) in comparison to those without adverse events. Significantly higher NO 2 – /NO 3 – values (p<0.05) were also found in patients who had myocardial reinfarction. Serum iNOS and TNF-α in all patient groups were within control ranges. A strong correlation was found between neopterin and both smoking (p<0.01) and triglycerides (p<0.05) in unstable angina patients. In stable angina patients, neopterin, iNOS and TNF-α significantly correlated with hypertension (p<0.01) and triglycerides (p<0.05). A significant difference in neopterin concentration was found between smokers and non-smokers (p<0.05). Conclusions : The results of this study suggest that in stable angina patients, if studied over time, serum neopterin or NO 2 – /NO 3 – levels may indicate future plaque instability. In ST-elevation myocardial infarction patients, neopterin and/or NO 2 – /NO 3 – levels may identify patients at long-term risk of death or recurrent acute coronary events after myocardial infarction. Clin Chem Lab Med 2008;46:1149–55.

Background: Serum monoclonal proteins (M-proteins) are usually quantified by electrophoresis or immunochemical measurement. A third alternative involves immunochemical measurement of the monoclonal isotype by subtraction of the polyclonal part of the isotype calculated from the other polyclonal isotypes and light chain. We experimentally calculated heavy chain/light chain equivalence factors for three immunoglobulin (Ig) isotypes and compared serum M-protein concentrations obtained using the three approaches. Methods : Equivalence factors were calculated for 1427 samples from 322 patients with a clinical diagnosis of monoclonal gammopathy of undetermined significance and validated in serum samples with and without a monoclonal component. Serum light chain recovery by nephelometry was compared to estimates using factors for serum immunoglobulin concentrations. Some 3735 samples were measured by electrophoresis and nephelometry for comparison of M-protein concentrations. Results: The experimentally calculated factors were 3.3059 (IgGκ), 3.5204 (IgGλ), 3.4567 (IgAκ), 3.5308 (IgAλ), 4.9104 (IgMκ) and 4.8020 (IgMλ). Light chains quantified by nephelometry in 984 serum samples without M-protein gave recovery of 100.97%±6.89% compared to immunoglobulin concentration estimates. Light chain recovery of <80% was observed more often for IgMκ (61.0%) and IgMλ (42.5%). Method differences for M-protein were >25% for 37.4%–52.3% of the serum samples. Electrophoresis results were higher than nephelometry results for 5% of serum samples and 23.7% of the estimates, especially for M-protein <20 g/L. Method differences for concentrations >40 g/L were <25% for most IgG and IgA isotypes, but not IgM. Conclusions : No method can accurately measure serum M-proteins over the entire concentration range. Estimation using polyclonal immunoglobulins and light chains represents a simple alternative to electrophoresis that is applicable to any serum M-protein concentration, regardless of electrophoretic migration and particularly for M-protein serum <20 g/L. Clin Chem Lab Med 2008;46:1156–62.

Background : It has been hypothesized that serum lipids measured in the early period of stroke are predictive of stroke severity and outcome. The optimal time for lipid measurement is not established. We explored whether lipid profile assessed within the first 24 h after stroke onset: (i) differs from that in stroke-free individuals; (ii) differs between stroke subtypes; and (iii) is predictive of stroke severity and outcome. Methods : We prospectively enrolled 70 acute ischemic stroke patients who presented to the Stroke Unit within 24 h of the onset of stroke symptoms, and 68 stroke-free control subjects. Results : Triglycerides (p<0.001) and high-density lipoprotein (HDL) cholesterol (p<0.001) were significantly lower in patients than in controls. HDL cholesterol was different across stroke subtypes classified according to the Trial of ORG 10172 in Acute Stroke Treatment scale (p=0.035). Patients with more severe stroke had higher serum triglycerides (odds ratio 2.755; p=0.030). Conclusions : Serum triglycerides might serve as a prognostic marker in acute stroke patients. Clin Chem Lab Med 2008;46:1163–7.

Background : It is customary to assume that pre-analytical variation is negligible if all pre-analytical sources of variation have been standardised. The aim of this study was to quantify the pre-analytical variation of some haematological quantities frequently measured in clinical laboratories. Methods : The experimental design considers different sources of pre-analytical variation that usually occur day-to-day for samples from patients. Results : The results demonstrate that for concentrations of erythrocytes, haemoglobin, leukocytes, neutrophilocytes (segmented) and lymphocytes, and for the volume fraction of erythrocytes (“packed cell volume”) in blood, the pre-analytical variance is not negligible. Conclusions : We suggest that these variances should be taken into account when estimating the uncertainty of measurement for patient results. Clin Chem Lab Med 2008;46:1168–70.

Background : B-type natriuretic peptide (BNP) is a biomarker used in the investigation of heart failure and other cardiovascular diseases. Our aim was to analyze the degradation of BNP in plasma samples kept at –20°C or at –80°C after 3 months or 1 year of storage. Methods : Using a Biosite assay-Triage BNP test, we evaluated 20 subjects with plasma BNP concentrations ranging from normal to high. Plasma aliquots were separated in five eppendorfs, one for immediate measurement after centrifugation, two for storage at –20°C and two at –80°C. We measured BNP after 3 months and 1 year. The significance level was set at p<0.05 (one-tailed test). Results : There was a significant decrease of 1% per month in BNP concentration. However, there was a significant interaction between time and group (high vs. low BNP). Low BNP values did not change over time, while high BNP decreased by 2% per month. The decrease over time did not depend on the storage temperature. Conclusions : The storage of the samples at –20°C or at –80°C did not prevent BNP degradation, especially for high BNP values. For low BNP values, we did not observe a significant decay at either 3 months or 1 year. These results are valid for the Biosite assay-Triage BNP test. Clin Chem Lab Med 2008;46:1171–4.

Background : Fibrinogen and C-reactive protein (CRP) concentrations are predictors of outcome in the atherosclerotic patient. It is important in risk stratification that these quantities are measured reproducibly in routine and research. Method : In the present study, we compare measurements of fibrinogen and high-sensitivity CRP in EDTA and citrate plasma samples (n=150) using nephelometric immunoassays. Fibrinogen was also measured in citrate plasma using a clotting method. Results : In approximately one-third of the samples, the fibrinogen concentration measured by immunoassay was higher in citrate plasma than in EDTA plasma, in spite of the dilution by citrate. The immunoassay results of fibrinogen concentration measurements in EDTA and citrate plasma differed significantly and also differed from those of functionally measured fibrinogen concentrations. A difference was found between the concentration of CRP in EDTA plasma and citrated plasma which also did not correspond to the dilution. Conclusions : Reproducibility of results is essential in risk stratification by fibrinogen or high-sensitivity CRP concentrations and small differences close to the decision limits may have a decisive impact. Immunological measurements are liable to confounding effects that may be difficult to foresee, qualitatively and quantitatively. Great care should be observed when measuring the concentration of calcium containing analytes in anticoagulated samples. Fibrinogen concentrations should preferably be measured functionally in citrate plasma. Clin Chem Lab Med 2008;46:1175–9.

Background : Clinical chemistry testing is influenced by a variety of preanalytical variables, including sample preparation. The presence of a diluted plasma layer at the top of primary tubes containing plasma citrate has recently been reported. However, no indication is available so far on the potential non-homogeneous distribution of clinical chemistry analytes during centrifugation of primary tubes containing lithium-heparin as an additive. Methods : A total of 40 lithium-heparin plasma samples were collected from volunteers and immediately centrifuged. An aliquot was obtained from the upper 0.4 mL of plasma (upper aliquot), 1.0 mL of plasma was discarded, and a second aliquot (lower aliquot) was obtained from the remaining plasma. The concentrations of alanine aminotransferase, albumin, alkaline phosphatase, amylase, amylase pancreatic, aspartate aminotransferase, direct bilirubin, total bilirubin, blood urea nitrogen, calcium, chloride, cholesterol, C-reactive protein (CRP), creatinine, creatine kinase, γ-glutamyltransferase (GGT), glucose, high-density lipoprotein-cholesterol, iron, lactate dehydrogenase (LDH), magnesium, phosphate, potassium, total protein, sodium, triglycerides and uric acid were assayed on a Roche/Hitachi Modular System P according to the manufacturer's specifications and using proprietary reagents. Sodium, chloride and potassium were measured on a Roche/Hitachi Modular System using indirect ion-selective electrode methods. Results : We observed a statistically significant difference between the upper and lower aliquots for CRP (3.88±0.67 vs. 3.94±0.68 mg/L; p=0.025), GGT (32.1±8.0 vs. 31.8±8.0 U/L; p=0.013), LDH (395±19 vs. 386±20 U/L; p=0.010) and triglycerides (1.29±0.09 vs. 1.27±0.09 mmol/L; p=0.001); results for the other analytes were not significantly different. In no case did the mean percentage bias recorded between aliquots exceed the current analytical quality specifications for desirable bias. Conclusions : The results of our investigation show that plasma layer stratification might occur in primary lithium-heparin tubes for a limited number of routine clinical chemistry tests, introducing a statistically significant bias in the measurement of GGT, LDH, triglycerides and CRP in the upper vs. the bottom section. When delayed testing is necessary for these parameters, we suggest that plasma should be separated after centrifugation and appropriately mixed before delayed/repeated analysis or aliquoting. Clin Chem Lab Med 2008;46:1180–2.

Background : To investigate the potential use of Artificial Neural Network (ANN) in the evaluation of serum protein electrophoresis, we set up a multicenter study involving six Italian laboratories. For this purpose, we developed an algorithm named CASPER (Computer Assisted Serum Protein Electrophoresis Recognizer). Methods : A total of 59,516 samples from the six centers were divided into three groups. Training and validation sets were used to develop the neural network, whereas evaluation set was used to test the performance of CASPER in recognizing abnormal electrophoretic profiles. Results : CASPER showed 93.0% sensitivity and 47.4% specificity. CASPER sensitivity and specificity ranged in the six sites from 88% (site 3) to 97% (site 5) and from 36% (site 6) to 53% (site 3), respectively. Sensitivity for γ zone was 94.6%, for β zone 89.7% and for oligoclonal patterns 92.0%. Conclusions : The sensitivity of the CASPER algorithm does not allow us to recommend its use as a replacement for the visual inspection, but it could be helpful in avoiding accidental misclassifications by the operator. Moreover, the CASPER algorithm may be a useful tool for training operators and students. This study evidenced a high inter-observer variability, which should be addressed in a dedicated study. Data set to train and validate ANNs should contain a huge range and an adequate number of different abnormalities. Clin Chem Lab Med 2008;46:1183–8.

Background : This work is the second part of a study regarding indicators and quality specifications for the non-analytical processes in laboratory medicine. Five primary care and five hospital laboratories agreed on the indicators for two strategic processes (quality planning and project development) and various support processes (client relationships, instrument and infrastructure maintenance, safety and risk prevention, purchases and storage, personnel training). Methods : In the majority of cases, the median values recorded over 1 year is considered to be the state-of-the-art in our setting and proposed as the quality specification for the indicators stated. Values have been stratified according to primary care and hospital laboratory for referred tests and group of personnel for training. In some cases, the specifications have been set equal to zero events, such as serious incidents in the infrastructure maintenance process and number of work accidents in the safety and risk prevention process. Results and conclusions : In light of this study, an effort is needed to optimize decisions regarding corrective actions and to move from a subjective individual criterion to systematic and comparative management. This preliminary study provides a comprehensive vision of a subject that could motivate further research and advances in the quality of laboratory services. Clin Chem Lab Med 2008;46:1189–94.

Background : Measurement of HbA 1c is key in monitoring diabetic patients in both laboratories and clinical units, where HbA 1c results are used as part of patient education. Methods : We have evaluated the DCA Vantage ® , a new device for immunological assay of HbA 1c . HbA 1c results obtained were evaluated in terms of precision, linearity, specificity and practicability, and were compared with results obtained by a Variant II ® HPLC method. Results : The method exhibited intra- and inter-assay coefficients of variation lower than 2.6% and 4.0%, respectively, and good correlation with the comparison HPLC method (r 2 =0.9776). No interference was noted in the presence of labile HbA 1c or carbamylated hemoglobin. The new device exhibited improved practicability characteristics and allowed better sample identification, better management of quality control routines and greater connectivity possibilities compared to the previous DCA 2000 ® analyzer. Conclusions : This new analyzer exhibited analytical and practical characteristics very suitable for HbA 1c assay for laboratory or point-of-care use according to good laboratory practice. Clin Chem Lab Med 2008;46:1195–8.