Volume 47, Issue 1

Herein, we expound the theory of circulating melanoma cells (CMCs) and their detection with reverse transcription polymerase chain reaction as a molecular staging approach. We discuss the molecular markers that have been used for CMC detection focusing on the use of these markers for multiplex detection analysis. Finally, we comment on the contradictory data of CMC detection studies in the literature and we propose possible solutions which may contribute to the clinical significance of CMC detection in patient management. Clin Chem Lab Med 2009;47:1–11.

GB virus C (GBV-C) and hepatitis G virus (HGV) are two isolates of the same virus, independently identified in humans in the 1990s by two research laboratories, and were initially considered a potential cause of liver disease. Studies failed to associate the virus with hepatitis or any known human disease. GBV-C reappeared in the scientific scene when some research groups, in an attempt to find the interference of the virus among HIV seropositive patients, reported a lower mortality rate and slower disease progression among co-infected patients. From then on, several mechanisms have been proposed to clarify this putative benefit; however, the question whether GBV-C exerts a protective effect in HIV-infected patients remains to be resolved. Clin Chem Lab Med 2009;47:12–9.

Background : Polymorphisms of the gene for C-reactive protein (CRP) alter baseline serum CRP concentrations. The impact of polymorphisms of the CRP gene (genotype) on the normal range for CRP concentrations (phenotype) has not been determined. This study evaluated the median serum CRP concentrations in normal subjects stratified for CRP genotype, after adjustment for relevant covariates as well as polymorphisms in tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) genotypes. Methods : A total of 423 healthy adults without infectious or inflammatory conditions undergoing phlebotomy for laboratory testing were enrolled in the study. Assays for serum high sensitivity CRP (hs-CRP), IL-6, and TNF-α genotypes were measured. Results : The median hs-CRP concentration was 0.96 mg/L (range <0.1–40.15 mg/L). The CRP 1444 C/T heterozygote and homozygote genotype was associated with a significantly higher hs-CRP concentration (p<0.05), even after multivariate analysis controlling for age, gender, body mass index, hypertension, dyslipidemia and obstructive sleep apnea. The CRP 286 C/T/A heterozygote genotype was significant (p<0.05) in the multi-variable analysis, univariately was borderline significant (p=0.052). Conclusions : Selected genetic polymorphisms of the CRP gene are independently associated with higher basal hs-CRP concentrations in healthy adults. Larger studies are needed to perform haplotype analyses and to adequately evaluate the relationship between hs-CRP and genetic polymorphisms with lower allelic frequencies. Clin Chem Lab Med 2009;47:20–5.

Background : Polymorphisms in VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are considered the major genetic factors modulating warfarin dose requirement. As a result, a rapid and economic method for genotyping patients for predicting warfarin dose could be useful in clinical applications. Methods : In this study, we developed and validated a melting temperature (T m )-shift genotyping assay for detection of VKORC1 C1173T and CYP2C9 A1075C polymorphisms by using SYBR Green-based multiplex allele-specific PCR and consequent melting curve analysis. In this T m -shift genotyping assay, a GC-tail was incorporated into one of the two allele-specific primers to increase the melting temperature of PCR product, such that both alleles can be differentially detected simultaneously at their respective melting curve patterns. In total, 100 randomly selected samples of both VKORC1 and CYP2C9 genes from each genotype group determined by melting curve were validated by direct DNA sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. Results : There was 100% concordance between the results of DNA sequencing and T m -shift genotyping, and 99% concordance between the results of DHPLC assay and T m -shift genotyping. Conclusions : This allele-specific PCR assay is convenient, inexpensive and will be useful for VKORC1 and CYP2C9 genotyping in a clinic laboratory. Clin Chem Lab Med 2009;47:26–31.

Background : The activated renin-angiotensin-aldosterone system has been reported to play an important role in the pathogenesis of atrial fibrillation (AF). We hypothesized that functional genetic variations of angiotensin-converting enzyme ( ACE ) and CYP11B2 genes may influence the susceptibility to AF in patients with hypertensive heart disease. Methods : The I/D polymorphism of ACE was detected by polymerase chain reaction (PCR), and the –344 C/T polymorphism of the CYP11B2 gene was detected using PCR and subsequent cleavage by Hae III restriction endonuclease. Results : The overall distribution of ACE I/D genotypes in patients with and without AF was significantly different (p=0.001). The frequency of the DD genotype was significantly higher in patients with AF than in patients without AF (20.6% vs. 8.1%, OR 2.94, 95% CI 1.64–5.26, p<0.001). The frequency of the D allele was significantly higher in the AF group than in the non-AF group (p=0.001). After adjustment for age and left atrial dimension, multivariable analysis showed that the DD genotype of the ACE gene was an independent risk factor for AF in patients with hypertensive heart disease. No relationship between –344 C/T CYP11B2 polymorphism and AF was found in this cohort. Conclusions : Our study suggests that ACE I/D polymorphism is associated with AF and the DD genotype may be an independent predictive factor for AF in patients with hypertensive heart disease. Clin Chem Lab Med 2009;47:32–7.

Background : Considerable interindividual differences in both drug response and pharmacokinetics of telmisartan have been identified. This study was designed to investigate the influence of MDR1 C3435T polymorphism on pharmacokinetics of telmisartan after a single oral dose in healthy Chinese volunteers. Methods : A total of 61 unrelated male volunteers were genotyped for MDR1 C3435T polymorphism by using the polymerase chain reaction-restriction fragment length polymorphism method. Six 3435CC homozygotes, eight 3435CT heterozygotes, and five 3435TT homozygotes were randomly selected and received a single oral dose of 40 mg telmisartan. Plasma concentrations of telmisartan were determined by the high performance liquid chromatography-mass spectrometry method up to 48 h after telmisartan administration. Results : Interindividual variation for t max , C max , t 1/2 , AUC 0–48 , AUC 0–∞ , and clearance (CL) for telmisartan was approximately 6-, 33-, 16-, 17-, 20-, and 20-fold, respectively. C max (p=0.010), t 1/2 (p=0.029) and CL (p=0.010) of telmisartan were not normally distributed. MDR1 3435TT homozygotes seemed to show increases in C max , t max , t 1/2 , AUC 0–48 , and AUC 0–∞ . However, no significant differences in C max , t max , t 1/2 , AUC 0–48 , AUC 0–∞ , and CL among MDR1 C3435T genotypes were observed. Conclusions : MDR1 C3435T polymorphism does not affect oral pharmacokinetics of telmisartan. Clin Chem Lab Med 2009;47:38–43.

Background : Erythropoietic protoporphyria (EPP) is known to be inherited in both autosomal dominant and recessive manners. A deleterious mutation in conjunction with a polymorphic wild type allele underlies the molecular basis of the dominant type. Methods : We report a patient with EPP who was found to have a novel large deletion [c.1-9628_67+2871del12566 bp] and three polymorphisms [c.1–251A>G, c.68-23C>T and c.315-48T>C] in trans to the deletion in his ferrochelatase ( FECH ) gene. Results : The combination of the deletion and the polymorphisms reduced his FECH activity level to 20% of control. Conclusions : It is conceivable that a homozygous state for this polymorphic haplotype might be sufficient to produce clinical phenotype of EPP. The boundary between autosomal dominant and autosomal recessive inheritance may not always be distinct. Clin Chem Lab Med 2009;47:44–6.

Background : Telomere length declines with age in mature endothelial cells and is thought to contribute to endothelial dysfunction and atherogenesis. Bone marrow-derived circulating endothelial progenitor cells (EPCs) are critical to vascular health as they contribute to both reendothelialization and neovascularization. We tested the hypothesis that EPC telomere length decreases with age in healthy adult humans. Methods : Peripheral blood samples were collected from 40 healthy, non-obese, sedentary men: 12 young (age 21–34 years), 12 middle-aged (43–55 years) and 16 older (57–68 years). Putative EPCs were isolated from peripheral blood mononuclear cells and telomere length was determined using genomic DNA preparation and Southern hybridization techniques. Results : EPC telomere length (base pairs) was ∼20% (p=0.01) lower in the older (8492+523 bp) compared to the middle-aged (10,565+572 bp) and young (10,205+501 bp) men. Of note, there was no difference in EPC telomere length between the middle-aged and young men. Conclusions : These results demonstrate that EPC telomere length declines with age in healthy, sedentary men. Interestingly, telomere length was well preserved in the middle-aged compared to young men, suggesting that EPC telomere shortening occurs after the age of 55 years. Clin Chem Lab Med 2009;47:47–50.

Background : The single nucleotide polymorphism (SNP) at deoxyribonuclease I (DNase I) in exon 8 (A2317G: Gln222Arg) has been shown to be associated with several diseases. Methods : The allele frequency of the DNASE1 polymorphism in Chinese (Shenyang and Guangzhou in China), Uygurs (Urumqi), Tamils (Sri Lanka), and Tibetans (Katmandu in Nepal) was investigated, and the results were compared with those of other studies. Results : This study revealed that DNASE1*1 is more common in Africans and DNASE1*2 is more common in Caucasians. Expression vectors of DNASE1*1 and DNASE1*2 were constructed and compared to the enzyme properties secreted into a medium from transfected COS-7 cells. The activity of the type-2 enzyme was significantly higher than that of the type-1 enzyme. In addition, the type-1 enzyme was heat-labile when compared to the type-2 enzyme. Moreover, the optimum pH of the DNase I type-2 enzyme was more acidic than that of DNase I type-1. Conclusions : This study revealed that the distribution of Gln222Arg in the DNASE1 gene is different among ethnic groups and that the DNASE1 polymorphism appears to affect the specific activity, heat sensitivity, and optimum pH of the DNase I enzyme. Clin Chem Lab Med 2009;47:51–5.

Background : Sequencing based mutation screening assays of genes encompassing large numbers of exons could be substantially optimized by multiplex PCR, which enables simultaneous amplification of many targets in one reaction. In the present study, a multiplex PCR protocol originally developed for fragment analysis was evaluated for sequencing based mutation screening of the ornithine transcarbamylase ( OTC ) and the medium-chain acyl-CoA dehydrogenase ( MCAD ) genes. Methods : Single exon and multiplex PCR protocols were applied to generate PCR templates for subsequent DNA sequencing of all exons of the OTC and the MCAD genes. For each PCR protocol and using the same DNA samples, 66 OTC and 98 MCAD sequence reads were generated. The sequences derived from the two different PCR methods were compared at the level of individual signal-to-noise ratios of the four bases and the proportion of high-quality base-signals. Results : The single exon and the multiplex PCR protocol gave qualitatively comparable results for the two genes. Conclusions : Many existing sequencing based mutation analysis protocols may be easily optimized with the proposed method, since the multiplex PCR protocol was successfully applied without any re-design of the PCR primers and other optimization steps for generating sequencing templates for the OTC and MCAD genes, respectively. Clin Chem Lab Med 2009;47:56–9.

Background : Oxidative stress plays an important role in the pathogenesis of coronary artery disease. Recent work showed that high-density lipoproteins isolated from atherosclerotic lesions and blood of patients with established coronary artery disease contain elevated levels of nitrated apolipoprotein A-I. Methods to quantify nitrated apolipoprotein A-I in the plasma may facilitate in the determination of a correlation between plasma levels of nitrated apolipoprotein A-I and risk of atherosclerosis. Methods : In this report, the presence of plasma nitrated apolipoprotein A-I in subjects with cardiovascular disease was detected by Western blot analysis and quantified by a newly developed specific sandwich enzyme-linked immunosorbent assay. Results : The precision of the assay was indicated by the good correlation obtained between enzyme-linked immunosorbent assay and Western blot (r 2 =0.96). Using these two methods, we were able to detect significant elevations (3-fold increase) of plasma nitrated apolipoprotein A-I in low high-density lipoprotein cholesterol subjects as compared to high-density lipoprotein cholesterol subjects. In addition, we found that both plasma and serum samples can be used by enzyme-linked immunosorbent assay to quantify nitrated apolipoprotein A-I concentrations. More importantly, the degree of nitrated apolipoprotein A-I-containing high-density lipoprotein particles was negatively correlated with levels of circulating apolipoprotein A-I. Conclusions : Measurement of nitrated apolipoprotein A-I levels by this rapid and reproducible new method has the advantages of great sensitivity with no sample manipulations, thus we suggest that this novel method could be useful to monitor levels of circulating nitrated apolipoprotein A-I in order to investigate its value as a potential biological marker for inflammatory vascular disease. Clin Chem Lab Med 2009;47:60–9.

Background : The stable fractions of glycated hemoglobin (Hb), particularly HbA 1c , and glycated albumin (GA) were measured to monitor chronic glycemic control. Haptoglobin (Hp) is a Hb-binding protein, which plays a major role in preventing free Hb-induced tissue oxidative damage. The aim of this study was to clarify the relationships of serum Hp concentration with HbA 1c and GA concentrations. Methods : A cross-sectional study to determine the relationship of serum Hp concentration with GA and HbA 1c concentrations was conducted. The subjects were 125 Japanese type 2 diabetic patients with stable HbA 1c levels for more than 3 consecutive months. Patients with altered albumin and red blood cell turnover, which are observed in those with chronic renal failure, liver cirrhosis, and anemia among others, were excluded from the study. Results : Serum Hp concentration positively correlated with HbA 1c concentration (r=0.30, p<0.001), but not with GA concentration (r=0.15, p=0.10). There was a weak inverse correlation between serum Hp concentration and GA/HbA 1c ratio (r=−0.19, p=0.03). Moreover, GA /HbA 1c ratio inversely correlated with body mass index (BMI) (r=−0.31, p<0.001). In contrast, there was no significant correlation between Hb concentration and HbA 1c (r=0.01, p=0.88) or GA (r=0.12, p=0.21) concentrations. We also analyzed the correlation of serum Hp concentration with GA and HbA 1c concentrations in patients with the Hp 2-1 and Hp 2-2 genotypes, separately. Hp concentration positively correlated with HbA 1c concentration in patients with the Hp 2-1 (r=0.32, p=0.03) and Hp 2-2 (r=0.29, p=0.02) phenotypes. Multiple regression analysis revealed that the observed correlation between Hp and HbA 1c concentrations was significant after adjustment for age, gender, and BMI. In contrast, there was no significant correlation between GA and Hp concentrations in patients with either phenotype. Then, we analyzed how Hp concentration affects GA/HbA 1c ratio in patients with these Hp phenotypes. There was an inverse correlation between Hp concentration and GA/HbA 1c ratio in patients with Hp 2-1 (r=−0.44, p=0.003), but not in those with Hp 2-2 (r=−0.03, p=0.75). Multiple regression analysis revealed that the inverse correlation between Hp concentration and GA/HbA 1c ratio in patients with Hp 2-1 was independent of age, gender, and BMI. Conclusions : Hp phenotype and concentration should be considered in interpreting HbA 1c and GA levels as glycemic control indicators in diabetic patients. We suggest that in type 2 diabetic patients with Hp 2-1 and high Hp concentrations, HbA 1c level may be overestimated relative to GA level. Clin Chem Lab Med 2009;47:70–4.

Background : Trace element determination in laboratory medicine is widely carried out by atomic absorption or emission spectroscopy. In the last decade, there has been a rapid growth in the use of inductively coupled plasma-mass spectrometry because of its strong detection power, and the possibility of multi-elements analysis in a single run. Methods : Having the advantages of smaller sample volume and better detection limit, we developed a method for the simultaneous determinations of six trace elements by using 100 μL serum, and the assay can be accomplished within 3 min. Results : The method developed gave recovery of the six elements ranging from 97% to 117%. The method covered a wide dynamic range with manganese in the range of nmol/L, while magnesium was in the range of mmol/L. The detection limits were 0.001 mmol/L, 0.05 μmol/L, 2.0 nmol/L, 0.2 μmol/L, 0.05 μmol/L, and 0.01 μmol/L for magnesium, aluminium, manganese, copper, zinc, and selenium, respectively. All the six elements had intra-assay imprecision of less than 5%, and inter-assay imprecision of less than 8%. Conclusions : This fast and robust method is suitable for use in the clinical laboratory where short turnaround time is needed for managing patients with trace element deficiency or toxicity. Clin Chem Lab Med 2009;47:75–8.

Background : Neutrophil gelatinase-associated lipocalin (NGAL), an iron-transporting protein rapidly accumulating in the kidney tubules and urine after nephrotoxic and ischemic insults, has been put forward as an early, sensitive, non-invasive biomarker for acute kidney injury (AKI). The aim of this study was to evaluate urinary NGAL levels as a predictor of early AKI (first 5 days after injury) in multi-trauma patients. Methods : We studied multi-trauma adult patients admitted to the intensive care unit of a trauma hospital. Exclusion criteria were a) known cardiac or chronic kidney disease, and b) initial evaluation after more than 24 h had elapsed from injury. Urinary NGAL was measured using an ELISA technique upon admission and at 24 and 48 h. Presence of AKI was defined by the risk injury failure loss and end-stage kidney classification (RIFLE) criteria. Data are reported as median and interquartile range. Results : A total of 31 patients (25 male, 6 female) were studied. NGAL levels at admission were significantly higher among patients who subsequently developed AKI [155.5 (50.5–205.9) ng/mL vs. 8.0 (5.7–17.7) ng/mL, p=0.0000] and these higher levels persisted over the following 2 days. On the basis of receiver-operating characteristic analysis both NGAL and serum creatinine baseline measurements could predict AKI [area under the curve (95% confidence interval) 0.977 (0.823–0.980) and 0.789 (0.556–0.906), respectively], but the area under the curve for NGAL was significantly larger (p=0.024). A cut-off point >25 ng/mL for NGAL had a sensitivity of 0.91 and specificity of 0.95 in predicting AKI. Conclusions : Urinary NGAL can be used from the 1st day of injury as a reliable predictor of early AKI in multi-trauma patients. Clin Chem Lab Med 2009;47:79–82.

Background : The objective of this study was to develop a robust quantification method for simultaneously analyzing molecules in human plasma using the Fourier transform infrared (FT-IR) system with a partial least square (PLS) regression. Methods : Plasma spectra were analyzed from 4000 to 500 cm −1 (with 2.0 cm −1 of resolution and 32 scans), and the molecule concentrations (IgA, IgG, IgM) were measured blindly by using a cross-validation model prepared by PLS analysis of data from 135 samples. Results : There was a significant correlation between the FT-IR predicted concentration and the concentration obtained with the clinical reference method: R 2 =0.98 (IgA), R 2 =0.98 (IgG), and R 2 =0.97 (IgM). The root mean square error of prediction (RMSEP) was 0.05 g·L −1 (IgA), 0.4 g·L −1 (IgG), and 0.03 g·L −1 (IgM). Variability of inter-experimenter reproducibility was less than 2%. The interchangeability of the two methods was studied by using the Bland-Altman method. Conclusions : Together with PLS analysis, FT-IR spectrometry appears to be an easy-to-use and accurate method to determine multianalyte concentrations in dried human plasma. It could be an alternative tool for rapidly quantifying many molecules after developing a specific predictive model. Clin Chem Lab Med 2009;47:83–90.

Background : Assay of total serum protein by the biuret method calibrated with albumin standards according to the reference method provides results with a positive bias approximately 3%–5% exceeding the total error of 3.4% allowable for total protein in serum analysis made by analysers using two-part reagents and short-term procedures. Methods : We used two types of two-part biuret reagents utilised in a short-term measurement in analysers with albumin or serum calibrators, in which protein was attested by the Kjeldahl method. Results : Tests with potentially interfering substances proved that serum blanking used in a short-term biuret procedure is not capable of sufficiently eliminating effects of serum interferents. A short-term blanking is evidently capable of suppressing only an absorbance caused by serum-present coloured and turbid interferents, but its capacity to transform them (oxidise, hydrolyse, saponify, etc.) to some other not-interfering substances is very low compared with a long-term blanking. Lipids and bilirubin are responsible for significant positive bias of total protein in normal serum samples (approximately 3%) and even a greater positive offset in lipaemic and icteric sera (approximately 5%). We verified that interference tests based on a normal serum spiked with endogenous lipids and bilirubin give quite false and misleading results in the biuret reaction. A pure albumin, not depending on its bovine/human origin, gives absorbance responding only to its copper complexes with protein with a biuret regent, while its absorbance with a serum also includes the absorbance of interferents present in serum. The simplest way to improve current short-term biuret procedures is the use of a human serum calibrator with total protein attested by the Kjeldahl method. A serum calibrator, behaving analogously to serum samples, compensates for a positive bias in most normal sera. Reagents with a greater concentration of active biuret components (copper and alkali, reference method included) seem to be unnecessarily aggressive to proteins and are responsible for a lower accuracy when used in short-term measurements. Conclusions : Standard Reference Material 927c based on pure bovine albumin is still recommended and used as the primary standard for assays of total protein by colourimetric methods. The albumin calibrator is responsible for a positive bias of approximately 3%–5% in serum total protein assayed by the biuret reaction both in the reference and in current methods. Its substitution by a serum calibrator attested by the Kjeldahl method could solve this drawback. Clin Chem Lab Med 2009;47:91–101.

Background : This study aimed to assess the state-of-the-art of antinuclear antibody (ANA) testing as practiced in the Belgian and Luxembourg laboratories, using the results obtained in the Belgian National External Quality Assessment Scheme from 2000 to 2005. Methods : During this period, nine samples with different specificities were sent for analysis. Participants were surveyed for methodology used and were asked to report staining pattern and titer of ANAs. In 2002, an attempt was made to improve the comparability of quantitative ANA results by the provision of a commercial reference material and to relate observed differences to methodology. Results : With one exception, all participants employed a microscope-based indirect immunofluorescence assay with human epithelial cell line 2 cells. Most laboratories were accurate in describing the pattern. The percentage of unacceptable answers was greater for samples with borderline levels of antibody and for samples showing a cytoplasmic pattern. An improvement in the detection of anticentromere antibodies was observed. For all samples, a wide range of titers was reported. The provision of the secondary reference preparation led to improved inter-laboratory concordance. Comparison of methodology variables revealed a correlation between unstandardized titers and the power of the lamp of the microscope and the use of a dark room. Conclusions: The EQAS results presented in this work provide valuable insights into the state of the art of ANA testing as practiced in the Belgian and Luxembourg Laboratories and illustrate the important value of a national EQAS for ANA testing as a tool to improve performance and interlaboratory comparability of laboratory results. Clin Chem Lab Med 2009;47:102–8.

Background : The aim of this study was to verify the analytical range of the DIAgam XS Cardio NanoGold C-reactive protein (CRP) method proposed as a full-range assay. Methods : For this purpose, analytical specifications were verified according to the Clinical and Laboratory Standards Institute specifications and a comparison was made with the reference method for the cardiovascular risk assessment, the CardioPhase high sensitivity CRP from Siemens Healthcare Diagnostics. Results : Our results showed that the immunoturbidimetric assay of CRP using reagents from DIAgam laboratory gave accurate and precise measurements from 0.42 to 265 mg/L. The performances in limits of detection and linearity allowed a full-range determination of CRP concentrations. The comparison with the reference method for the determination of cardiovascular risk was also accurate. Conclusions : In conclusion, the DIAgam reagents might be used in a routine laboratory for the detection of acute inflammation as well as for evaluation of cardiovascular risk. Clin Chem Lab Med 2009;47:109–11.

Background : One of the most frequently requested examinations in the clinical laboratory is the differential leukocyte count. Despite many technological improvements, thousands of differential leukocyte counts are made by microscopic examination of a blood smear, counting 100 leukocytes and expressing the fraction of the specific leukocyte types as percentages (rounded to integer values) of the total leukocyte count. Although in the clinical laboratory it is not usual practice to report measurement uncertainties, currently the ISO 15189 standard considers measurement uncertainty as a very helpful element for a comprehensive interpretation of any measurement result. Methods : The estimation of the measurement uncertainty of each differential leukocyte count result was carried out according to international guidelines. The sources of standard uncertainty taken into account were: pre-metrological variation, random distribution, between-examiner reproducibility, and rounding to an integer. Results : In this example, a sample of blood with a concentration number of leukocytes of 3,5×10 9 /L 1) is taken into consideration. For each differential leukocyte count result, the standard uncertainties corresponding to each source of measurement uncertainty, as well as the combined and the expanded uncertainties, were estimated with information from the literature. Conclusions : The procedure presented here to estimate the measurement uncertainty of differential leukocyte count results is appropriate to fulfill the requirements of the ISO 15189 standard related to measurement uncertainty. Knowledge of this uncertainty is helpful in interpreting sequential results obtained in the same patient. Clin Chem Lab Med 2009;47:112–5. 1) According to ISO, IUPAC and IFCC recommendations, the comma is used as the decimal sign.