Volume 48, Issue 3

The goal of standardization for measurement of the catalytic concentration of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments, and laboratory where the assay is performed. To pursue this objective, the IFCC has established reference systems for the most important clinical enzymes. These systems are based on the following requirements: a) reference methods, well described and evaluated extensively; b) suitable reference materials; and c) reference laboratories operating in a highly controlled manner. When these reference systems are used appropriately, the diagnostic industry can assign traceable values to commercial calibrators. Clinical laboratories that use procedures with validated calibrators to measure human specimens can now obtain values that are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e., they are measuring the same catalytic quantity). Clin Chem Lab Med 2010;48:301–7.

The advent of modern instrumentation, with associated improvements in test performance and reliability, together with appropriate internal quality control (IQC) and external quality assurance (EQA) measures, has led to substantial reductions in analytical errors within hemostasis laboratories. Unfortunately, the reporting of incorrect or inappropriate test results still occurs, perhaps even as frequently as in the past. Many of these cases arise due to a variety of events largely outside the control of the laboratories performing the tests. These events are primarily preanalytical, related to sample collection and processing, but can also include post-analytical events related to the reporting and interpretation of test results. The current report provides an overview of these events, as well as guidance for prevention or minimization. In particular, we propose several strategies for the post-analytical reporting of hemostasis assays, and how this may provide the final opportunity to prevent serious clinical errors in diagnosis. This report should be of interest to both the laboratory scientists working in hemostasis and clinicians that request and attempt to interpret the test results. Laboratory scientists are ultimately responsible for these test results, and there is a duty to provide both accurate and precise results to enable clinicians to manage patients appropriately and to avoid the need to recollect and retest. Also, clinicians will not be in a position to best diagnose and manage their patient unless they gain an appreciation of these issues. Clin Chem Lab Med 2010;48:309–21.

Background: A major use of serum magnesium measurements in clinical practice is to identify patients with deficiency. However, numerous studies have shown that magnesium deficiency is common and may be present in over 10% of hospitalized patients, as well as in the general population. An important cause for under diagnosis of deficiency is that serum magnesium, the most commonly used test, can be normal despite negative body stores. This article focuses on the limitations of “normal” magnesium results and highlights the importance of lifestyle or “modus vivendi” as a pragmatic means of identifying those individuals potentially at risk for negative body magnesium stores. Methods: Researched peer reviewed articles on magnesium published between 1990 and 2008 in MEDLINE and EMBASE, using database keywords “magnesium, deficiency, diagnosis, treatment and hypomagnesaemia”. Bibliographies of retrieved articles have been searched and followed. We have also performed a manual search of each individual issue in which most of these reports have appeared. Results: In 183 peer reviewed studies published from 1990 to 2008, magnesium deficiency was associated with increased prevalence and risk in 11 major conditions. Similarly, in 68 studies performed over the same period, magnesium deficiency was found to predict adverse events and a decreased risk of pathology was noted when supplementation or treatment was instituted. Conclusions: The perception that “normal” serum magnesium excludes deficiency is common among clinicians. This perception is probably enforced by the common laboratory practice of highlighting only abnormal results. A health warning is therefore warranted regarding potential misuse of “normal” serum magnesium because restoration of magnesium stores in deficient patients is simple, tolerable, inexpensive and can be clinically beneficial. Clin Chem Lab Med 2010;48:323–7.

Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin ( HBB ) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format. Clin Chem Lab Med 2010;48:329–36.

Background: The goal of our study was to evaluate the influence of genetic polymorphisms of matrix metalloproteinases (MMP)-2, MMP-3 and MMP-7 on susceptibility to endometrial cancer. Methods: In the present study, we enrolled a total of 118 patients with endometrial cancer confirmed by histopathology, and 229 unrelated healthy individuals. Polymorphism for the MMP-2 (rs2285053), MMP-3 (rs3025058) and MMP-7 (rs11568818) genes was genotyped by polymerase chain reaction-restriction enzyme length polymorphism. Results: The frequencies of MMP-7 −181 G/G and A/G genotypes were found to be significantly higher in cancer patients compared with healthy controls (p=0.017). Stratification showed that individuals with MMP-7 −181 G allele were at increased risk for endometrial cancer when >50 years of age [odds ratios (OR)=2.03; 95% confidence interval (CI) 1.21–3.39], endometrioid (OR=1.80; 95% CI 1.11–2.92), low (stage I–II) (OR=1.73; 95% CI 1.05–2.83) or high stage (stage III–IV) (OR=2.69; 95% CI 1.16–6.24). Compared with the A/A genotype, the A/G+G/G genotype modified the risk of developing endometrial carcinoma and significance was detected in patients over 50 years old, and those with endometrioid type and high stage endometrial cancer. However, no significant difference in MMP-2 (-735 C/T) and MMP-3 (6A/5A) genotypes was observed between endometrial carcinoma cases and controls. Conclusions: This is the first report on the association of MMP-2 , MMP-3 and MMP-7 gene polymorphisms in endometrial cancer. Our results suggest that individuals with the MMP-7 −181 G/G and A/G genotype may have an increased risk of developing endometrial cancer. Clin Chem Lab Med 2010;48:337–44.

Background: Rheumatoid arthritis (RA) is currently thought to be an immune-mediated disease where the host's genes and environmental factors interact. Some of the immuno-regulatory genes that are responsible for an individual's susceptibility to RA have been identified. The co-stimulatory receptor gene cluster on chromosome 2q33 encodes for both the positive T-cell regulators CD28 molecule (CD28) and inducible T-cell co-stimulator (ICOS), and the negative regulator cytotoxic T-lymphocyte-associated protein 4 (CTLA4). The CTLA4 gene has been implicated in several immune-mediated diseases, but it is not known whether RA is associated with any of these genes. Methods: We conducted single nucleotide polymorphism (SNP) genotyping with direct sequencing and restriction fragment length polymorphism for 308 Korean patients with RA and 412 healthy control subjects. For the case-control analysis, SNPStats, SNPAnalyzer and Helixtree programs were used. Results: Although none of the polymorphisms in CTLA4 showed a significant association with RA, CD28 and ICOS showed a significant association with RA [rs2140148 in CD28, p=0.022, odds ratio (OR)=1.60, 95% confidence interval (CI)=1.07–2.40 in the dominant model, rs6726035 in ICOS, p=0.032, OR=1.28, 95% CI=1.02–1.60 in the codominant model]. Conclusions: Our results suggest that CD28 and ICOS genes may be associated with a risk of RA in Koreans. Clin Chem Lab Med 2010;48:345–53.

Background: Abnormal DNA methylation of the male germ line is proposed as a possible mechanism causing compromised spermatogenesis in some men diagnosed with idiopathic infertility. Previous studies suggested that aberrant DNA methylation of several genes is associated with disruptions in spermatogenesis. However, little information is available on DNA methylation patterns of testis-specific genes in idiopathic male infertility. Methods: To investigate the association between DAZ gene methylation patterns and spermatogenic failure, we performed an analysis of methylation patterns in 174 idiopathic infertile patients and 58 fertile controls using bisulfite-modified sequencing. Results: We found that the methylation patterns of CpG island (CGI) in the DAZ gene promoter region were different between somatic cells and spermatic cells in the control group. DAZ gene methylation patterns among groups with different spermatogenic status were the same in somatic cells, completely methylated. The results were concordant in spermatic cells, completely unmethylated, in groups with different degrees of spermatogenesis, except for the group with azoospermia (AZ). Conclusions: Our data indicate that the methylation patters of the DAZ gene are not associated with spermatogenic failure. This suggests that epigenetic modification of DAZ is unlikely to be involved in the etiology of spermatogenic failure. Clin Chem Lab Med 2010;48:355–60.

Background: In China, two common methods are used to detect serum glucose, glucose oxidase (GOD) and hexokinase (HK) methods. We evaluated five commercially available glucose reagent kits with the Center of Disease Control (CDC) HK reference method. Methods: Five routine methods were compared with the HK reference method according to Clinical and Laboratory Standards Institute (CLSI) guideline (EP9-A2). These five routine methods were performed on an “open channel” Olympus 5400 analyzer. Results: All five evaluated methods were closely correlated to the reference method (r>0.9998). Compared to the HK reference method, the Olympus-HK method and Roche-GOD method showed a very small positive bias (mean±SD: 0.08±0.09 mmol/L and 0.03±0.08 mmol/L); while the Biosino-HK method, Osborne-GOD method and Baiding-GOD method showed a very small negative bias (mean±SD: –0.07±0.06 mmol/L, –0.09±0.07 mmol/L and –0.01±0.04 mmol/L). Conclusions: All five reagents we evaluated using matching calibrators showed good correlation to the reference method and small bias. These five glucose reagents are acceptable for use in clinical laboratories. Clin Chem Lab Med 2010;48:361–4.

Background: We report a Dutch–Belgian multicentre evaluation of the Tosoh HLC-723G8 glycohaemoglobin analyser, an ion-exchange HPLC instrument for the separation and quantification of haemoglobin A1c (HbA1c) in whole blood. Methods: We evaluated the analytical performances of the Tosoh G8 analyser and compared the results for blood samples with its predecessor, the Tosoh G7, and with two other widely used analysers, the Bio-Rad Variant II and Adams Arkray HA-8160. Results: Within- and between-batch imprecision [coefficient of variation (CVs)] was <0.5% and 2%, respectively, and compared favourably with the G7. The excellent performances in terms of speed (1.6 min/analysis) did not result in increased variability of the results or carry-over between samples. The method shows no interference from carbamylated haemoglobin, and recognises the presence of haemoglobinopathies, which triggers the correction of the HbA1c result. Comparison with established methods showed good correlation, not only with the G7 but also with the Variant II and HA-8160 systems. Conclusions: With respect to reproducibility, chromatographic resolution, speed of analysis and identification of Hb variants, the Tosoh G8 analyser can be considered to be state of the art. Clin Chem Lab Med 2010;48:365–71.

Background: Acute virus infections are indicated by increased serum neopterin concentrations. Testing of blood donations for increased neopterin was introduced in the Austrian Tyrol in 1986 and throughout Austria in 1994 to improve the safety of blood transfusions. Radioimmunoassay (RIA) was the first available test, and the 98th percentile for neopterin concentrations from 76,500 donations was defined as the cut-off threshold, excluding donations with neopterin ≥10 nmol/L. When ELISAs were introduced on fully automated test systems to measure neopterin concentrations, results were generally higher and the cut-off was adjusted up to 12 nmol/L. Methods: In early 2006, new equipment (Hamilton Microlab Star ® pipettor+Dade Behring ELISA Processor III, BEP III ® ) became available which monitors sample and reagent volumes for each uptake and dispense, and takes the viscosity of the fluids into account. Using this system, the performance of neopterin determinations was evaluated over 21 months and compared to earlier data. Results: When the new apparatuses were introduced, an immediate decrease in mean neopterin concentrations and the percentage of samples with neopterin concentrations ≥10 nmol/L was observed. The 55,516 measurements performed in 2007 showed a mean value of 5.5 nmol/L. Neopterin in 680 donations (1.23%) were ≥10 nmol/L. Conclusions: The new Hamilton diluter increased the accuracy of the neopterin test procedure. Results are now identical with those of RIA and ELISA when run in a semi-automated manner. Differences in pipetting performance of standards and samples are considered as one possible explanation for the earlier discrepancies seen with the automated apparatus. Clin Chem Lab Med 2010;48:373–7.

Background: There is accumulating evidence that fibrinogen is also a biomarker of oxidative stress in human plasma. Results of in vitro studies demonstrated that fibrinogen can bind to apolipoprotein(a) [apo(a)] component of lipoprotein(a) [Lp(a)] through both lysine-sensitive and lysine-insensitive mechanisms. The goal of the present study was to investigate oxidized fibrinogen reactivity (OFR) as a biomarker of oxidative stress in human plasma in the presence and absence of lysine analogs. Methods: Citrate anticoagulated peripheral venous blood samples were collected from 65 (36 M/29 F) consecutive patients with various peripheral vascular diseases. After centrifugation, the plasma was used promptly. Plasma OFR was determined in duplicate using a recently described kinetic photometric assay (358 nm, 37°C) in the presence and in the absence of lysine analogs. Results: The inclusion of tranexemic acid (TRA) or ε-aminocaproic acid in the incubation medium resulted in a rapid increase in OFR in a dose-dependent manner. The peak effect was observed at a final concentration of 200 mmol/L TRA. OFR was significantly higher in patient plasma assayed in the presence of TRA compared with no TRA (163.1±73.5 vs. 63.4±20.7 U/L; p<0.0001). Bound OFR was also significantly higher than free OFR (99.7±56.3 vs. 63.4±20.7; p<0.001). Conclusions: On the basis of the present results it appears that oxidized fibrinogen resides in plasma in two compartments: free and bound to apo(a) of Lp(a). The relatively simple and cost-effective kinetic approach applied in this study makes routine determination of OFR available as a biomarker of oxidative stress, separately in both compartments. Clin Chem Lab Med 2010;48:379–82.

Background: Although uric acid (UA) is one of the most important antioxidants in plasma and appears to be neuroprotective in animal models, results from human studies are controversial. In this study, we investigated the kinetics of serum UA concentrations in the acute, subacute and chronic phase of ischemic stroke and its relation with initial stroke severity, stroke evolution in the subacute phase and long-term stroke outcome. Methods: Serum concentrations of UA were measured in 199 stroke patients at admission (median, 2.8 h after stroke onset), at 24 h, 72 h, day 7, month 1 and month 3 after onset of stroke. We evaluated the relationship between changes in UA concentrations and (a) stroke severity [patients with transient ischemic attack (TIA) vs. stroke patients, National Institutes of Health Stroke Scale (NIHSS) score at admission], (b) stroke evolution (stroke progression, infarct volume at 72 h), and (c) stroke outcome [modified Rankin scale (mRS) score at month 3, mortality]. Results: UA concentrations decreased significantly during the first 7 days after stroke onset before returning to baseline (p<0.001). Mean plasma UA concentrations decreased from 336.66±113.01 μmol/L at admission to 300.37±110.04 μmol/L at day 7 (p<0.001) in patients with stroke, but did not change significantly in patients with TIA. Changes in UA concentrations from admission to day 7 (ΔUA day 7 ) correlated with the NIHSS score (ρ=0.32; p<0.001), stroke progression (ρ=0.29; p=0.001), infarct volume (ρ=0.37; p<0.001), mRS score (ρ=0.28; p=0.001) and mortality (p=0.010). Conclusions: Decreases in UA during the first week after onset of stroke correlates with more severe stroke, unfavorable stroke evolution, and poor long-term stroke outcome. Clin Chem Lab Med 2010;48:383–90.

Background: Linezolid is the first synthetic compound of a new group of antimicrobials, the oxazolidinones, which inhibit protein synthesis. It shows a broad spectrum of activity against Gram positive organisms. With respect to its pharmacokinetics, linezolid shows a relatively high volume of distribution and good penetration into inflammatory fluids, bone, fat and muscle. Methods: A reversed-phase isocratic high-performance liquid chromatographic method for linezolid analysis in piglet pulmonary tissue is described. Tissue samples and controls were prepared in 1×TBE (1 M Tris, 0.9 M boric acid, 0.01 M EDTA). The mobile phase consisted of 20% ultrafiltered water and 80% of (A) 15 mM potassium monohydrogen phosphate buffer (pH=5) with (B) acetonitrile (80%/20%; v/v). Samples were homogenized and precipitated with HClO 4 3% (1/1, v/v). The injection volume was 100 μL. Ofloxacin was used as an internal standard. Results: The assay was linear over a linezolid concentration range: 1.6–100 μg/mL. The method provided good validation data (n=15): inaccuracy (3.6%), intra and inter-day variability (4.2% and 5.2%, respectively), recovery (91.8%), limit of detection (0.8 μg/mL) and quantitation (1.6 μg/mL) and acceptable stability within 24 h in the auto-sampler. Conclusions: The method offers a fast and simple approach to determine linezolid in pulmonary tissue which could be of use in pharmacokinetic studies. Clin Chem Lab Med 2010;48:391–8.

Background: The use of heparin as an alternative to EDTA in the production of plasma samples is of particular interest for B-type natriuretic peptide (BNP) measurements. Lithium heparin is now widely used for the determination of biochemical parameters, including cardiac markers. The goal of this study was to determine the feasibility of measuring BNP using heparin plasma instead of EDTA plasma with the Access 2 system (Beckman-Coulter). Methods: BNP was determined in heparin plasma and EDTA plasma from 24 patients within 1 h of blood collection. Additional measurements were performed with heparin plasma, every hour for the first 4 h, and then 8 h after the collection of blood that was stored at room temperature. Results: At H 0 , the observed BNP concentrations in heparin plasma were much higher (mean values 65% higher) than those in EDTA plasma. Using predetermined thresholds, this difference would lead to 30% discordance between samples in heparin and EDTA. BNP stability decreased over time in heparin plasma: immunoreactivity decreased approximately by 30% during the first 2 h and by 60% after 8 h. Conclusions: Heparin plasma does not seem to be a suitable alternative to EDTA plasma for measurement of BNP using the Access 2 system, even if measurements are performed immediately after blood sampling. Clin Chem Lab Med 2010;48:399–401.

Background: The objective of this study was to determine the composition of kidney stone fragments obtained after extracorporeal shock wave lithotripsy (ESWL). Methods: Kidney stone fragments from 25 patients with urolithiasis treated with ESWL were submitted for morphological analysis. The composition was determined for all the recovered fragments. Results: Thirteen patients (52%) had pure stones. The most common type of pure stone was calcium oxalate (61.6%), of which half was the monohydrate type (COM) and half was the dihydrate type (COD). The other pure stones consisted of either uric acid (30.8%) or struvite (7.6%). For mixed stones, the most frequently observed component was COM or COD (50%), followed by a mixture of COD and carbapatite (25.1%). Conclusions: Our findings indicate that the composition of kidney stone fragments recovered after ESWL can be determined. Knowledge of stone composition is fundamental to understand the etiology of lithogenesis. Clin Chem Lab Med 2010;48:403–4.

Background: Current data are conflicting about the influence of gender on cystatin C concentrations. The goal of this study was to determine the reference interval for serum cystatin C in normal Brazilian subjects, taking into account the influence of gender. Methods: Ninety-seven healthy volunteers, aged 18–70 years, 44% male, had glomerular filtration rate (GFR) measured using a 51 Cr-EDTA method and estimated with the Modification of Diet in Renal Disease (MDRD) study equation. Serum cystatin C was measured using a turbidimetric method, and creatinine by the Jaffe method. Results: Mean serum cystatin C was not significantly different between males and females, 0.62±0.12 vs. 0.65±0.12 mg/L, respectively (p=0.26). However, median serum creatinine was significantly higher in men [97 (80–115) vs. 80  (53–88) μmol/L; p=0.0001]. There were also no significant differences between genders with respect to GFR measured using 51 Cr-EDTA (103±14 for males and 106±19 mL/min/1.73 m 2 for females, p=0.47), and estimated with the MDRD equation (86±12 vs. 83±16 mL/min/1.73 m 2 , respectively, p=0.24). Conclusions: There was no effect of gender on serum cystatin C, as well as on measured and estimated GFR. Clin Chem Lab Med 2010;48:405–8.

Background: Ethnic differences necessitate a need for local reference intervals (RI), but establishing these can be challenging in some cultures that are reluctant to donate blood. Frozen sera are an alternative, but results can be questionable. Between 1998 and 2001, we collected blood samples from 343 healthy pregnant Chinese women (5–41 weeks' gestation), and in 2001 published gestational RI for thyrotropin thyroid stimulating hormone (TSH), free thyroxine (FT4) and free triiodothyronine (FT3) using the ACS180, now obsolete. As a pilot to re-establishing RI, TSH, FT4 and FT3 were re-assayed in archived specimens using contemporary assays. Methods: Thirty archived specimens (–80°C) with ACS180 TSH concentrations ranging from 0.25 to 3.7 mIU/L were measured using the Roche E170, Advia Centaur and Architect i2000 TSH, FT4 and FT3 assays, along with 10 newer contemporary samples. Results were compared to the original, and examined in context of past and present methodological performances in an external quality assurance (EQA) program. Results: All contemporary assays detected significantly lower TSH and increased FT4 and FT3 concentrations in the stored samples. Conclusions: With reference to methodological performances in EQA, the results obtained with contemporary assays suggest analyte deterioration in specimens and thus their unsuitability for re-establishing RI. Clin Chem Lab Med 2010;48:409–12.

Background: Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples has gained significance in the routine diagnostic laboratory. Methods: In this study, the analytical and clinical performance of the artus ® EBV RG PCR kit in conjunction with automated sample preparation on the QIAsymphony ® SP instrument was evaluated. Results: When the accuracy of the new test system was tested, all results were found to be within ±0.5 log 10 unit of the expected panel results. Determination of linearity showed a quasilinear curve over 4 log units. The lower limit of detection was determined to be 391 EBV DNA copies/mL in EDTA whole blood. The between day imprecision ranged from 18% to 66%, and the within run imprecision ranged from 11% to 50%. When clinical samples were tested and the results compared with those obtained with the routinely used easyMAG™ sample preparation and EBV R-gene™ test system, 60 samples tested positive and 31 samples tested negative by both assays. Nineteen samples were found to be positive using the QIAsymphony ® sample preparation and artus ® EBV RG PCR test system only, and no samples tested positive with the routinely used test system only. Conclusions: The QIAsymphony ® sample preparation and artus ® EBV RG PCR test system is suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. Clin Chem Lab Med 2010;48:413–8.