Volume 46, Issue 2

The presence of high affinity/avidity endogenous insulin antibodies in significant amounts in non-diabetic individuals could uniquely be a double whammy. Clinically it could trigger pathology, namely hypoglycaemia, and analytically it could cause erroneous and potentially misleading results (i.e., falsely high or falsely low) in the key biochemical parameters essential in the differential diagnosis of this pathology, namely serum insulin, proinsulin and even C-peptide. The purpose of this paper is to highlight the clinical and analytical sequelae of endogenous insulin antibodies in (a) delaying or even confusing the differential diagnosis of unexpected/unexplained hypoglycaemia, and (b) interfering in some immunoassays of pancreatic hormones (commercial or in-house). Clin Chem Lab Med 2008;46:153–6.

Background : Interpretation of serial data in monitoring of patients is usually performed by use of the ‘reference change value’ (RCV). While this tool for interpretation of measured differences is simple and clear, there are a number of drawbacks attached to the uncritical use of this concept. It is a dichotomised interpretation of continuous data using a fixed probability without any counter hypothesis. Therefore, a tool for better understanding and interpretation of measured differences in monitoring is needed. Theory : The concept of sensitivity, specificity, likelihood ratios and odds used for diagnostic test evaluations is applied to monitoring by substituting measured concentrations with measured differences. Thus, two frequency distributions of differences are assumed, one for a stable, steady-state, situation and one for a certain change. Values exceeding a measured difference will thus represent ‘false change’ for the stable and ‘true change’ for the change and the ratio between these will define the likelihood. By making the hypothesis of change variable and equal to the actual difference, the distribution corresponding to the true changes for the measured difference varies with this. Consequently, the likelihood ratio for change increases with growing measured difference and when used together with the pre-test odds or pre-test probability, the post-test odds and post-test probability, related to the clinical situation, can be calculated. Results : One example is acute intermittent porphyria, where increasing excretion of porphobilinogen is characteristic for an attack. The within-subject biological variation is estimated to 25%, which for two measurements gives a variation of 35% for measured differences. Three pre-test probabilities are assumed and illustrate that post-test odds and probability depends on both the pre-test probability and the measured difference. A second example is monitoring women in a follow-up after treatment of breast cancer, using the tumour marker CA 15-3. The within-subject biological variation is estimated to 14.9% and for differences 21% (2 ½ ×14.9 due to two measurements). Here, the monitoring is totally scheduled and the frequency of progression depends on the time after treatment. Thus, the pre-test probability varies with time so that a certain measured difference with a given likelihood ratio will result in varying post-test odds depending on time. Conclusions : The concept presented here expands the earlier concept of RCVs by making it possible to have an estimate of the post-test odds for a certain difference to occur based on likelihood ratios and pre-test odds. Clin Chem Lab Med 2008;46:157–64.

Background : Usually, laboratory diagnostics of hereditary hemoglobin disorders is fairly straightforward. Sometimes, however, correct diagnosis can be difficult. In this study, we describe a family with multiple mutations and sequence variations in the α- and β-globin gene clusters. Methods : Hemocytometry results were obtained using an automated cell counter. Hemoglobin variant analysis was performed by cation-exchange HPLC. PCR and DNA sequence analyses were used to identify mutations in the globin genes. Results : The proposita was referred to our laboratory for hematological evaluation [hemoglobin 145 g/L (119–155 g/L) mean corpuscular volume 72 fl (80–97 fl), mean corpuscular hemoglobin 26 pg (28–36 pg), erythrocytes 5.6×10 12 /L (3.7–5.0 10 12 /L)]. Characterization and quantification of hemoglobin variants showed 11.3% HbA 1 , 4.4% HbA 2 , 58.9% HbC and 23.0% HbF. Subsequent analysis revealed, in addition to a heterozygous HbC mutation, the presence of a β-thalassemia causing mutation (–90C>T), a heterozygous α-thalassemia (–α –3.7 /αα) and three different γ-globin sequence variations. Additional molecular analysis was performed in all family members. Conclusions : In the family presented in this study, 10 different mutations were found in the globin genes. Molecular analysis was necessary to clarify hemoglobin variant analysis, in particular the low amount of HbA 1 in the proposita. Knowledge of the molecular background facilitates in the understanding of the hematological parameters and proper counseling of the patient. Clin Chem Lab Med 2008;46:165–8.

Background : Our aim was to study genetic variability in the gene encoding cytotoxic T-lymphocyte antigen ( CTLA-4 ) and individual susceptibility to the development of preeclampsia or placental abruption. Methods : A total of 361 women (132 with preeclampsia, 117 with placental abruption and 112 healthy controls) were genotyped for 49A–G polymorphism (dbSNP: rs231775) in the CTLA-4 gene. Results : The frequency of the G alleles was significantly higher in women with preeclampsia than in controls (51.1% vs. 42.0%; OR 1.44, 95% CI 1.01–3.48, p<0.043). Women with placental abruption had decreased frequency of AA genotype (22.2% vs. 35.7%) and significantly more AG or GG genotypes compared with controls (OR 1.94, 95% CI 1.09–2.07, p<0.024). No significant differences were detected in the frequencies of genotype GG (29.5%, 21.4% and 19.6%, respectively) between the three groups. Conclusions : Our data suggest that the 49A–G polymorphism in the CTLA-4 gene is associated with the development of placental abruption and preeclampsia, with women having the G allele being at risk. Clin Chem Lab Med 2008;46:169–73.

Background : Gilbert's syndrome is a chronic or recurrent mild unconjugated hyperbilirubinemia caused by decreased activity of UDP glucuronosyltranferase ( UGT1A1 ). The most common cause of Gilbert's syndrome in Caucasians is homozygous variant of the A(TA) 7 TAA promoter polymorphism. Alleles with five or eight TA repeats have also been described, but they are very rare in Caucasian populations. Methods : Over a 6-year period (2001–2006), 1109 subjects with suspected Gilbert's syndrome were included in this study. Genotyping of (TA) 6 and (TA) 7 alleles was performed using high-resolution electrophoretic separation of amplified PCR products on Spreadex EL300 gels. In seven subjects, aberrant electrophoretic patterns were observed and additionally sequenced on an ABI Prism 310 Genetic Analyzer. Results : Genotype distributions for 1102 subjects with (TA) 6 or (TA) 7 alleles were as follows: 54.10%, 26.33% and 18.94% for the (TA) 7 /(TA) 7 , (TA) 6 /(TA) 7 and (TA) 6 /(TA) 6 , respectively. Sequencing of seven samples that could not be identified as one of these alleles identified four subjects with the (TA) 5 /(TA) 7 , two with the (TA) 7 /(TA) 8 and one with the (TA) 6 /(TA) 8 genotype. Conclusion : Genotyping of TA repeats in the promoter region of the UGT1A1 gene revealed the presence of rare alleles with five or eight TA repeats, with a very high frequency of the (TA) 7 allele in subjects suspected of having Gilbert's syndrome. Clin Chem Lab Med 2008;46:174–8.

Background : A relation between apolipoprotein E (APOE) genotypes and high sensitive C-reactive protein (hsCRP) has been observed in some studies with elderly subjects and different patient groups. We studied whether serum hsCRP levels are linked with common APOE ( ɛ2 , ɛ3 , ɛ4 ) polymorphism already in children and young adults. Methods : The study cohort included 1221 subjects participating in the Cardiovascular Risk in Young Finns Study at age 3–18 years at baseline in 1980. These subjects were reexamined at the 21-year follow-up at age 24–39 years in 2001. APOE phenotypes were examined in 1986, serum hsCRP was measured from fresh samples in 2001 and baseline hsCRP (in 1980) was measured from frozen samples in 2005. Results : Serum hsCRP was significantly associated with APOE phenotypes in children and young adults using multivariate analysis adjusted for age, body mass index, smoking, total cholesterol and low-density lipoprotein cholesterol. Male ɛ4 carriers had significantly lower hsCRP levels both in childhood (p=0.003) and in adulthood (p=0.013). hsCRP increased in both phenotype classes ( ɛ4 + and ɛ4 –) during the 21-year follow-up. Female ɛ4 carriers had lower hsCRP levels in childhood (p=0.032) but not in adulthood (p=0.995). An interaction effect between time and APOE phenotype (p=0.045) in relation to hsCRP was observed in females during the 21-year follow-up. Conclusions : Common APOE polymorphism affects the level of circulating hsCRP already in children and young adults. Male APOE ɛ4 carriers have consistently lower hsCRP levels. In females, APOE ɛ4 carriers had lower hsCRP levels in childhood but not in adulthood. Clin Chem Lab Med 2008;46:179–86.

Background : Behçet's disease (BD) is a multisystemic immuno-inflammatory disorder. Inflammatory processes may cause lipid peroxidation, alteration of lipid profile and increase the risk of atherosclerosis. The aim of this study was to evaluate the association between thiolactonase (HTLase) activity and plasma homocysteine levels (tHcy) in a BD population and to investigate their association with methylenetetrahydrofolate reductase ( MTHFR ) 677C→T genotype. Method : A total of 35 BD patients were compared to 39 healthy volunteers. Results : Significantly higher tHcy levels associated with lower HTLase activities were found in BD patients as compared to healthy controls (p<0.001). These patients also exhibited lower values of triglycerides and high-density lipoprotein cholesterol (HDL-C). Homozygosity for the T allele of the MTHFR gene was more frequent in BD patients (14.3% vs. 7.7%). It was associated with significantly higher tHcy levels (16.9 μmol/L for n=17 vs. 13.1 μmol/L for n=18; p<0.05) and markedly lower HTLase activity (362.6±156.7 U/L vs. 414.2±180.2 U/L) for the (TT+CT) and CC genotypes, respectively. Moreover, HDL-C levels were inversely correlated with tHcy (r=–0.5; p=0.004) but positively associated with HTLase activity (r=0.374; p=0.038). These correlations were also present in several clinical manifestations, such as ocular, neurological involvement or thrombosis. Conclusions : Homozygosity of the T allele of the MTHFR gene is prevalent in BD patients. High levels of tHcy associated with low HTLase activities may be one of the causes leading to thrombosis in BD patients. Clin Chem Lab Med 2008;46:187–92.

Background : Celiac disease (CD) is a complex disorder triggered by gluten affecting genetically predisposed individuals. More than 90% of patients carry human lymphocyte antigen (HLA)-DQ2 ( DQA1*05, DQB1*02 ) and/or HLA-DQ8 ( DQA1*03, DQB1*0302 ). We propose the use of the DQ-CD Typing kit that allows identification of the HLA class II alleles ( DQA1*0201,*03,*05, DQB1*02,*0302, DRB1*03,*04,*07 ) selected to be informative in the CD risk evaluation and of a second kit, namely DQ-CD Zygosis, for DQB1*02 homozygosity determination. Methods : The study was performed on a cohort of 100 individuals previously HLA typed with commercial kits. Fresh blood or previously extracted DNA was amplified in a unique PCR program using allele-specific primers and visualized on agarose gel. Results : DNA amplification yielded strong and clear products without non specific signals or ghost bands. All the samples showed the expected alleles in accordance with the previous HLA typing. Conclusions : The DQ-CD Typing and Zygosis kits are fast, simple, economical and accurate tools that can be used to determinate the HLA-DQ2/DQ8 status in laboratory practice addressed for the diagnosis of CD. Molecular HLA testing is considered a valid support in the confirmation/exclusion of CD, especially in high-risk groups, such as CD relatives, or when serological and histological data are ambiguous. Clin Chem Lab Med 2008;46:193–6.

Background : Reactive oxygen species and redox signaling play an important role in the regulation of many vital biological processes. However, they are also tightly connected with many pathological conditions. The detection and evaluation of these signaling events are very often accompanied with great difficulties. In this article, we describe the development of a novel electrochemically-based technique for monitoring the cellular redox state. Methods and results : T-cells were attached on the surface of a working electrode, which was modified with 2-palmitoylhydroquinone as a redox mediator. Using cyclic voltammetry, we were able to indirectly (via the redox mediator) monitor an electron transport from the cells towards the working electrode, which enabled us to evaluate the redox activity of the cells. Conclusions: This new technique is rather simple and sensitive and may be used in the future as a valid diagnostic procedure in various branches of biomedical science. Clin Chem Lab Med 2008;46:197–203.

Background : Midregional pro-adrenomedullin (MR-proADM) and endothelin-1 have been shown to predict mortality of patients with acute myocardial infarction. However, the prognostic value of both biomarkers in predicting long-term clinical events after acute myocardial infarction remains unclear. Methods : In a prospective study, 30 patients suffering from acute ST elevation myocardial infarction or non-ST elevation myocardial infarction were enrolled. Measurements of MR-proADM and CT-pro-endothelin-1 (CT-proET-1) were performed at initial presentation, 2 or 3 days and 4 months after acute myocardial infarction. Long-term clinical events (e.g., recurrent myocardial infarction, percutaneous transluminal coronary angioplasty, aorto-coronary venous bypass or cardiogenic shock) were documented over a period from the 4th until the 10th month. Results : Both MR-proADM and CT-proET-1 were able to differentiate patients with subsequent long-term clinical events (n=11) from those without (n=19). At the time of acute myocardial infarction, median MR-proADM level of the event group was 0.69 nmol/L as compared to 0.59 nmol/L of the no-event group (p=0.036). A difference was still observed after 3 days (event group median 0.66 nmol/L; no-event group median 0.57 nmol/L; p=0.022). Accordingly, median CT-proET-1 level was 72.9 pmol/L in the event group as compared to a median of 54.4 pmol/L in patients in the no-event group (p=0.009) 3 days after acute myocardial infarction. Within the acute phase, patients with MR-proADM levels ≥0.67 nmol/L were 3 times more likely (relative risk 2.8; 95% confidence interval 1.2–6.9; p=0.042) to suffer from a future clinical event. The area under the curve (AUC) was 0.71 (95% confidence interval 0.51–0.86; p=0.046). After 3 days, patients with CT-proET-1 levels ≥57 pmol/L were 6 times more likely (relative risk 5.9; 95% confidence interval 0.9–40.4; p=0.036) to suffer from a future clinical event. The AUC was 0.76 (95% confidence interval 0.55–0.90; p=0.015). Conclusions : Elevated levels of MR-proADM and CT-proET-1 during the acute phase of myocardial infarction may predict an adverse long-term clinical outcome. Clin Chem Lab Med 2008;46:204–11.

Background : The aim of the present study was to evaluate the effect of a single haemodialysis (HD) session on serum fetuin-A levels, considered a negative acute phase response marker; moreover, we evaluated the behaviour of fibrinogen and high sensitivity C-reactive protein (hsCRP) as acute phase response and chronic/subclinical inflammation markers, respectively, after a single HD session. Methods : Serum fetuin-A, albumin, hsCRP and fibrinogen were measured in 72 patients before and after a single HD session. Results : After a single HD session, we observed a significant increase in fibrinogen levels, while fetuin-A levels decreased (p<0.05). Also, hsCRP levels were significantly increased. Conclusions : The significant decrease of fetuin-A levels after a single HD session is consistent with the hypothesis of HD-induced inflammation; activated acute phase response and fetuin-A deficiency might account for increased cardiovascular risk and accelerated atherogenesis in dialysis patients. Clin Chem Lab Med 2008;46:212–4.

Background : The precision of the formulae used to estimate glomerular filtration rate (GFR) decreases when the serum creatinine (SCr) assay is biased compared with the assay used during the development of the formulae. Methods : For 100 children referred for 51 Cr-EDTA clearance (CL EDTA ), SCr was measured with a JAFFE (classic Jaffe colorimetric creatinine assay), a compensated Jaffe (COMP), an enzymatic (ENZ) and an HPLC assay. A population pharmacokinetics approach based on a non-linear mixed effects model (NONMEM) was used to model the relationships between the CL EDTA and physiopathological/analytical variables. Results : Unlike JAFFE values, COMP and ENZ SCr gave a high bias using the Schwartz formula for the GFR calculation (median +27.0% and +39.1%, respectively). The best equation obtained from the analysis of the curves of [ 51 Cr-EDTA] plasma vs. time was (n=67): CL EDTA (mL/min)=61.9×[SCr (μM)/Θ] Ψ × [age (years)/13.4] 0.522 ×(weight (kg)/44.2) 0.233 . The SCr assay-related coefficients and exponents were Θ=97.4, Ψ=–0.757 (–0.922; –0.592) for JAFFE; Θ=85.3, Ψ=–0.579 (–0.681; –0.477) for COMP; and Θ=82.6, Ψ=–0.560 (–0.659; –0.460) for ENZ. When applied to 33 children, this equation estimated CL EDTA without any significant bias: +3.1% (–11.8; +11.4) for COMP and +5.3% (–7.2; +16.4) for ENZ. Conclusions : As long as there is no standardization of SCr measurements, population pharmacokinetics may be a powerful tool to model inter-assay variability. Clin Chem Lab Med 2008;46:215–8.

Background : This study was undertaken to reveal how the physiological ageing process influences plasma glycosaminoglycans (GAGs) content and composition. Methods : GAGs isolated from plasma samples obtained from 105 healthy subjects were quantified using a hexuronic acid assay and subjected to electrophoretic fractionation. Results: We found that total GAGs decreased gradually with advancing age. The electrophoretic analysis allowed identifying chondroitin sulfates, dermatan sulfates and heparan sulfates and heparin in plasma of healthy individuals. Chondroitin sulfates, predominant plasma GAGs, showed declining values with age and displayed higher structural diversity in subjects from the first five decades of life compared to older subjects. An age-related increase in plasma dermatan sulfates content during the first four decades of life followed by a decrease in a later period of life was apparent. Structural heterogeneity of these glycans was found in subjects until 10 years of age. Heparan sulfates and heparin plasma levels were inversely correlated with age until 25 years of age, without age-dependent changes thereafter. These macromolecules displayed higher heterogeneity of structure in subjects over 50 years of age than in younger subjects. Conclusions : The obtained results indicate that age should be taken into account in all studies investigating blood GAGs alterations during pathological conditions. Clin Chem Lab Med 2008;46:219–24.

Background : In many countries and especially in Scandinavia, blood samples drawn in primary healthcare are sent to a hospital laboratory for analysis. The samples are exposed to various conditions regarding storage time, storage temperature and transport form. As these factors can have a severe impact on the quality of results, we wanted to study which combination of transport conditions could fulfil our pre-defined goals for maximum allowable error. Methods : Samples from 406 patients from nine general practitioners (GPs) in two Danish counties were sent to two hospitals for analyses, during two periods (winter and summer). Transport conditions (mail, courier pick-up, or brought to hospital by public coach), storage time, storage temperature and centrifugation requirements were different in the two counties. Results were tested for deviation from a “0-sample”, the blood sample taken, centrifuged and separated at the doctor's office within 45–60 min. This sample was considered as the best estimate of a comparison value. Results : The pre-set quality goals were fulfilled for all the investigated components for samples transported to hospital by courier either as whole blood or as “on gel” after centrifugation, as long as the samples were stored at 20–25°C and centrifuged/analysed within 5–6 h. A total of 4% of the samples sent by mail had mismatched identity, probably due to plasma being transferred to a new tube. Conclusions : Samples can be sent as unprocessed anticoagulated whole blood if the above mentioned conditions are met. There is no need for centrifugation in the primary sector. Neither mailing of samples with plasma “on gel” nor public transport by coach bus fulfil our analytical goals. Clin Chem Lab Med 2008;46:225–34.

Background : Several creatinine-based estimates of the glomerular filtration rate (GFR) have been advocated as reliable alternatives to the urinary clearance of exogenous filtration markers. The Cockcroft-Gault (C-G) equation requires a body weight parameter, which was not included in the Modification of Diet in Renal Disease (MDRD) Study and the Mayo Clinic Quadratic Equation (MCQE) formulas. However, there is little information on the lifestyle influences on these equations, especially the intensity and the type of physical activity. Methods : We evaluated the differences in the GFR, as estimated by the MDRD, MCQE and C-G equations, in 60 professional male cyclists at rest and 60 healthy sedentary matched controls. Results : Professional cyclists had significantly lower body weight, body mass index and serum creatinine concentration than healthy sedentary individuals. We also observed a significantly higher MDRD-estimated GFR in athletes than in controls (119 mL/min/1.73 m 2 vs. 104 mL/min/1.73 m 2 , p<0.001), whereas the GFR values estimated by both the MCQE (137 vs. 135, p=0.128) and C-G (127 vs. 127, p=0.490) formulas did not differ significantly. As compared to the MDRD values, the mean GFR calculated by the MCQE and C-G formulas was overestimated by 29% and 23% in the sedentary population, and by 17% and 7% in athletes, respectively. A lower bias was observed when comparing C-G with MCQE-estimated values in both the sedentary [mean: –5%, 95% confidence interval (CI): –32% to 22%] and athlete (mean: –6%, 95% CI: –41% to 29%) populations. The correlation among the different equations in healthy sedentary individuals was always significant, whereas the only significant association in the athletes was that between MCQE and C-G values. Conclusions : The results of this study attest that the three most widely used creatinine-based formulas produce significant variations in the estimated GFR in a population of endurance athletes at rest. Probably, the use of C-G or MCQE formulas might be more suitable in this context, as they globally appear more robust against variations of the training regimen. Clin Chem Lab Med 2008;46:235–9.

Background : The oral glucose tolerance test (oGTT) is the most common method to estimate indices of insulin sensitivity in clinical as well as in epidemiological studies. The aim of this study was to investigate the variability of insulin levels for reference intervals and of insulin sensitivity indices during oGTT by three different insulin immunoassays. Methods : Serum insulin levels were determined during oGTT of randomly selected adult subjects (total n=101, with n=68 with normal glucose tolerance) by the LIAISON (LIA), the Elecsys (EL) and the AutoDELFIA (AD) method. Results : Sensitivity and precision of all three insulin assays were comparable. Insulin levels demonstrated a close (p<0.001) interassay correlation (LIA vs. EL: r=0.987, LIA vs. AD: r=0.966, EL vs. AD: r=0.965). Insulin levels and insulin sensitivity indices (n=68) of the AD method were significantly different compared to the LIA or EL assay (p<0.001). Basal insulin levels demonstrated an assay-dependent significant body mass index (BMI) dependency (LIA: r=0.24, p=0.05; AD: r=0.44, p<0.001; EL: r=0.36, p<0.01) in the regression analysis. At 120 min post glucose, gender was the relevant influencing factor on insulin levels of the LIA (r=0.40, p<0.001), AD (r=0.37, p<0.01) and EL (r=0.40 p<0.001) method. Conclusions : We conclude that assay-dependent reference intervals should be used for the clinical interpretation of insulin levels from oGTT and of insulin sensitivity indices. Moreover, BMI and gender have to be considered as influencing factors. Clin Chem Lab Med 2008;46:240–6.

Background : The serum biomarker N-terminal proB-type natriuretic peptide (NT-proBNP), a cleaved fragment of the brain natriuretic peptide (BNP) precursor (amino acids 1–76), is accepted as a standard marker for evaluating and monitoring cardiac injury characterized by myocardial wall stress. Strenuous exercise may generate transitory ischemia, myocardial stress and diastolic left ventricular dysfunction, possibly inducing increased concentrations of NT-proBNP. A purported caveat to prolonged strenuous exercise is based on evidence for biochemical and structural signs of heart dysfunction in recreational athletes after continuous exertion. Methods : We compared NT-proBNP levels in three groups of physically fit subjects: top-level rugby players, professional soccer players and healthy controls. NT-proBNP concentrations were measured at rest and after an intensive training session followed by two different recovery strategies (passive or active). Results : A comparison of the three samples showed that NT-proBNP concentrations in the rugby players were lower than those in controls at rest and were similar to those in professional soccer players. Elevated post-training NT-proBNP levels were unaffected by the type of recovery. The relatively high NT-proBNP levels after active recovery when psychophysical stress is higher, because of cycling and cold water immersion, suggest that not only endurance exercise, but also strenuous, stressful short exercise can induce an increase in NT-proBNP concentrations. Conclusions : In this sample of professional athletes, NT-proBNP was low at rest, and the increase after physical exercise was physiological. Clin Chem Lab Med 2008;46:247–9.

Regular exercise offers protection against all-cause mortality, primarily by protection against atherosclerosis and insulin resistance. There is evidence that physical training is effective as a treatment in patients with chronic heart diseases and type 2 diabetes. With excessive repetition of the training stimulus, local inflammation can generate a systemic inflammatory response. Cytokines play an important role in these processes. Our data confirm that cytokines that are considered to induce systemic bioactivity following exercise are not alone, but also growth factors and chemokines, which are secreted in exercise without the involvement of traditional proinflammatory cytokines, could be potent angiogenic factors. Clin Chem Lab Med 2008;46:250–2.

Background : The aim of the study was to perform a comprehensive diagnostic evaluation of six popular, non-proprietary, indirect markers of liver fibrosis in a cohort of patients with chronic hepatitis C representing the full spectrum of disease severity. Methods : A total of 167 consecutive, hepatitis C virus RNA positive, untreated patients with chronic hepatitis C were studied. Liver biopsy with histological evaluation and age/platelet index, aspartate aminotransferase/alanine aminotransferase ratio, aspartate aminotransferase to platelet ratio index, Bonacini's discriminant score, Forn's fibrosis index and FibroIndex were assessed in all patients. Results : The area under the receiver operating characteristic curves of the six tests was always greater when performed to discriminate patients with METAVIR score F4 than when assessed to discriminate patients with METAVIR score ≥F2. At step-wise discriminant analysis the only indirect marker of fibrosis entered was FibroIndex, with the following correct classification of the patients: total=52.1, patients with scores F0–F1=62.2, patients with scores F2–F3=26.0 and patients with score F4=68.4. Conclusions : The ability to correctly classify patients using a panel of non-proprietary indirect markers of liver fibrosis is far from being ideal. Among them, FibroIndex appears to possess the best discriminating capacity. The simultaneous use of several indirect markers of liver fibrosis does not improve their diagnostic accuracy. Clin Chem Lab Med 2008;46:253–9.

Background : Laboratory evaluation of α 1 -antitrypsin (A1AT) deficiency is generally performed by determination of A1AT concentrations and identification of specific allelic variants by phenotyping. For this purpose, we evaluated a new Hydragel 18 A1AT Isofocusing ® kit on the semi-automatic Hydrasys ® System (Sebia) for the determination of A1AT phenotypes by isoelectrofocusing on ready-to-use agarose gels with specific immunological detection. Methods : Serum samples from 66 patients were analysed with this new kit in comparison with the conventional and manually performed isoelectrofocusing method on polyacrylamide gels with Coomassie Blue staining. Results : A1AT phenotypes showed comparable iso-electrofocusing patterns in both systems. The good within-gel reproducibility of this kit was demonstrated using two normal serum samples (M1 and M1M2 phenotypes) and six pathological serum samples with different phenotypes (MS, SS, SZ, MZ, ZZ). A sensitivity study was undertaken by performing serial dilutions on a serum with a ZZ phenotype containing 0.27 g/L A1AT. The detection limit was 0.050 g/L. Conclusions : This new method is highly specific, rapid and simple to perform. It improves identification of not only the most common but also various rare A1AT phenotypes. It appears to be suitable for routine analysis and screening applications in a clinical laboratory setting. Clin Chem Lab Med 2008;46:260–3.

Background : The compatibility of immunoassay tests in different sample matrices is extremely important during the assay validation process. In this study, we investigated the interchangeability of some Access ® (and therefore all the UniCel Family platforms) assays between serum and plasma. Methods : We tested approximately 200 samples in parallel between serum and lithium heparin plasma for seven analytes: α-fetoprotein (AFP), carcino-embryonic antigen (CEA), total prostate specific antigen (tPSA), free prostate specific antigen (fPSA), digoxin, progesterone and unconjugated estriol (uE3). We used the Access2 ® Immunoassay System (Beckman Coulter), a fully automated random access system with a chemiluminescent signal. We performed statistical comparative analysis using two commercially available programs, Analyze-it from Microsoft Excel and MedCalc Software, and a dedicated statistical program. Results : Firstly, we showed the results of the statistical tests performed on each population to verify their distribution. Analysis by several statistical tests (Passing and Bablok regression, Youden and Bland and Altman diagrams, the Mountain plot and multivariate analysis) showed that all the assays studied were valid in both serum and lithium heparin plasma matrices. Conclusions : As all Access and UniCel Family instruments use the same reagent packs, these results are transferable to all Beckman Coulter immunochemistry platforms, without a commutability problem between serum and plasma and without a need for establishment of a plasma reference interval. Clin Chem Lab Med 2008;46:264–70.

Background : GEM ® Premier™ 4000 (Instrumentation Laboratory), a new blood gas/CO-Oximeter/hematocrit/electrolyte/glucose/lactate analyzer was evaluated. The only reagents required were disposable cartridges (GEM ® Premier™ 4000 PAK comprise all components necessary for analysis, including quality controls). Methods : The evaluation was performed in two laboratories according to the Valtec protocol guidelines. Analytical performances (imprecision at three levels, linearity, inaccuracy by method comparison using patient samples, interferences) and instrument practicability were compared with three routinely used laboratory blood gas analyzers (ABL 835 and 725, Radiometer; OMNI S, Roche Diagnostics) and the centrifuged microhematocrit method. Results : Within-run and between-run imprecision yielded good coefficients of variation values for all parameters. Linearity was satisfactory and within the expected ranges provided by the manufacturer. Method comparisons demonstrated close agreement (Deming's regression correlation coefficients 0.92–1.00). No interferences of lipemia, hyperbilirubinemia and hemolysis were found on CO-Oximetry parameters. Fetal hemoglobin >35% overestimated carboxyhemoglobin measurements, but the magnitude of error was reduced with the fetal hemoglobin correction analysis mode. Conclusion : The GEM ® Premier™ 4000 analytical performance was validated for all parameters with the accuracy and the reliability of traditional systems. This blood gas analyzer fulfills most of the requirements for both point-of-care and laboratory use. Clin Chem Lab Med 2008;46:271–9.

Background : Storage and shipment conditions of clinical specimens affect the quality of nucleic acids and may interfere with molecular analysis. The aim of our study was to verify whether blood storage at room temperature affects single nucleotide polymorphisms analysis; moreover, we analysed the consequences of serum storage at +4°C on viral load determination of hepatitis B and C viruses. Methods : For single nucleotide polymorphism screening, genomic DNA was extracted from EDTA whole blood samples stored at room temperature for different times, quantified photometrically, and Factor V Leiden point mutation analysis was performed. For viral load determination, serum samples with medium or low viremias were stored at +4°C for different times and analysed by Cobas AmpliPrep/Cobas TaqMan tests for hepatitis B virus (HBV) DNA or hepatitis C virus (HCV) RNA. Results : While mutation analysis was successfully performed on all samples tested, serum storage at +4°C of HBV- and HCV-infected sera decreased viral load, in particular for low viremias. Conclusions : Storage of blood samples at room temperature up to 1 month does not affect the feasibility of genetic analysis, while serum storage at +4°C affects viral load. Clin Chem Lab Med 2008;46:280–2.

Background : Elevated circulating total homocysteine is an independent vascular risk factor. Enzymatic homocysteine measurements represent an alternative to HPLC- or immunochemistry-based assays, suitable for automation. Here, we report on analytical performance of a commercial cystathionine β-synthase-based assay, for use on Vitros ® automated analyzers. Methods : Linear range, limit of detection and analytical sensitivity were inferred from duplicate measurements of homocystine standard solutions (1–65 μmol/L). Imprecision was assessed using commercial controls according to NCCLS EP5-A2 and accuracy using NIST-SRM1955 reference material. Agreement with a clinically validated HPLC method was examined on 207 patient samples. Results : The enzymatic assay was linear from 1 to 90 μmol/L homocysteine. Total (within-day) imprecision ranged from 4.5 (3.9)% to 2.8 (1.6)% at homocysteine 9.7–43.2 μmol/L. Accuracy was acceptable at 8.9 and 17.7 μmol/L homocysteine, with +6.4% and –1.2% bias, respectively, but showed substantial negative bias (–20.1%) at 4.0 μmol/L. High triglycerides (19.8 μmol/L) negatively interfered. The enzymatic method was slightly less sensitive than the HPLC method (limit of detection 0.7 and 0.2 μmol/L, respectively) but correlated well with the latter (r 2 =0.9997, slope=1.04, intercept=–0.66 μmol/L) and was more precise (p<0.05). Conclusions : The Vitros ® homocysteine assay met the CLIA Desirable Analytical Quality Specifications at homocysteine ≥9 μmol/L. Its analytical performance and suitability for automation make the Vitros ® assay an analytically acceptable alternative to HPLC-based methods. Clin Chem Lab Med 2008;46:283–6.

Background : Standardization in collection and testing of oral fluid is lacking. Methods : A novel standardized collection and quantification system for oral fluid testing was evaluated. Sample collection volumes were determined. For determination of the saliva content in oral fluid samples, tartrazine, which is an integral part of the saliva extraction solution serving as an internal standard, was used. Results were compared with those obtained by employing glucose as a ‘supplemental internal standard’ for determination. Analytical performance of the new system for collection and quantification of oral fluid was evaluated. Calcium and magnesium analyte concentrations in oral fluid samples were measured with a routine laboratory method and compared to a reference method. Results : Volumes of oral fluid samples collected ranged from 4.9 to 10.5 mL. The mean saliva content in oral fluid samples was found to be 65.5 percent by volume (vol.-%) when determined through the tartrazine concentration and 65.0 vol.-% when determined through the glucose concentration. Evaluation of analytical performance revealed interassay imprecision coefficients of variation (CVs) ranging from 1.5% to 3.2% and intraassay imprecision CVs from 1.1% to 2.5%. When linearity was tested, a quasi-linear curve was observed (R 2 =0.99). Comparison of two different methods for determination of calcium and magnesium concentrations showed correlations of R 2 =0.96 for calcium and R 2 =0.97 for magnesium. Conclusions : The new system for collection and quantification of oral fluid helps to improve standardization of pre-analytics in oral fluid testing and to provide reliable and accurate quantification of analytes in oral fluid samples. Clin Chem Lab Med 2008;46:287–91.

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