Band 47, Heft 5

Oral anticoagulant therapy (OAT) based on vitamin K antagonists (VKAs; coumarin derivatives) is the current mainstay for the prevention and long-term treatment of a variety of thromboembolic disorders. Care of patients on OAT is challenging due to considerable variability in the response to a particular dose. This has been attributed to environmental, demographic, clinical and genetic variables. Individualized responses represent a major clinical challenge because patients may experience adverse health outcomes from bleeding or thrombosis as a result of over- or under-coagulation, respectively. Growing evidence indicates that up to 60% of the individual pharmacological response to coumarins might be due to genetic variables and affected by polymorphisms in the genes encoding two enzymes, namely, vitamin K epoxide reductase (VKOR) and cytochrome P450 CYP2C9. Genetic testing has been proposed as a useful tool for allowing prediction of the dose response during initial anticoagulation therapy, to assess variability in dose maintenance and to identify warfarin ‘resistance’. However, genetic testing is not a panacea. Limitations include the optimal composition of test panels, still largely unknown, information concerning inter-individual variability, lack of analytical and quality specifications, lack of comprehensive outcome analyses to enable assessment of cost-effectiveness, lack of universal agreement related to reliable dosing algorithms and other ethical and social issues. The aim of this article is to provide a comprehensive overview of our current understanding of the pharmacogenetics of VKAs, as well as assessing potential advantages and limitations. Although it might be premature to recommend routine genetic testing, the future development and clinical validation of simple but comprehensive algorithms integrating the most informative gene polymorphisms ( VKORC1 and CYP2C9 ) with some demographic information (age, race, body mass index) and clinical variables (comorbidities, drugs interference), and standardized dietary intake of vitamin K may provide a valuable tool in the care of patients on OAT with conventional VKAs. However, the ongoing development of new anticoagulant drugs targeting thrombin and factor X will introduce a paradigm shift in long-term anticoagulation therapy, so that consideration could be given to demise pharmacogenetics testing for VKAs. Clin Chem Lab Med 2009;47:503–15.

Background : Hair is a DNA source that can be collected easily and inexpensively from participants in epidemiological studies. However, there is concern about DNA quality and quantity. Therefore, we assessed genotyping performance of whole genome amplified (WGA)-DNA extracted from hair using the GenomePlex ® method and evaluated its agreement with genotyping results of buccal cell DNA from the same individuals, using the Illumina GoldenGate platform. Methods : The Illumina DNA test panel includes 360 highly validated single nucleotide polymorphisms (SNPs) selected from the Linkage IV Panel that are distributed across the entire genome. DNA was extracted from both archived hair and buccal cell samples obtained from 44 randomly selected subjects participating in a large cohort study in Canada. Results : The genotyping success rate was 97.7% for 44 paired samples. However, WGA-DNA from hair failed more during genotyping in comparison to buccal cell DNA. Hair samples with a pre-WGA-DNA ≥1 ng/μL quantified using the PicoGreen ® assay (n=33) showed an average genotyping completion rate of 98.8% and SNP concordance of 91.2% with genotyping performance of buccal cell DNA. In contrast, samples with a pre-WGA-DNA <1 ng/μL had lower genotyping completion rate (94%) and poor SNP concordance (49%). Conclusions : Results suggest that WGA-DNA obtained from hair can produce excellent genotyping call rates and show relatively good SNP concordance with results from buccal cell DNA using high-throughput technology. DNA quantity obtained from hair samples is a crucial determinant of genotyping performance. Larger studies are needed to examine the utility of hair DNA with different genotyping platforms. Clin Chem Lab Med 2009;47:516–22.

Background: Interleukin-18 (IL-18) has been suggested to play an important role in coronary arterial disease and its sequelae. The aim of the present study was to investigate the association between IL-18 promoter functional polymorphisms ( −607C/A and −137G/C ) and acute myocardial infarction (AMI) in northern Chinese Han population. Methods: We performed a case-control study including 234 patients with AMI and 216 age- and sex-matched controls. Genotyping was performed using sequence specific primer-polymerase chain reaction (PCR-SSP). Results : There were significant differences in the genotype and allele distribution of −607C/A polymorphism of the IL-18 gene between cases and controls. Logistic regression analysis with adjustments for other well-established risk factors revealed that the −607C allele carriers had a significantly increased risk of AMI compared with the non-carriers (OR=1.890, 95% CI 1.196–2.985, p=0.006). No relationship between −137G/C polymorphism and AMI was found. Conclusions : This study shows for the first time that the IL-18 gene promoter −607C/A polymorphism may be considered a genetic risk factor for AMI in northern Chinese Han population. Clin Chem Lab Med 2009;47:523–9.

Background : Detection of tumor-associated genetic alterations in plasma of cancer patients has recently been suggested to be an accurate method for detecting early or recurrent cancer. Methods : We performed quantitative real-time PCR for MYC and GAPDH in tissue and plasma samples of 57 patients with gastric cancer and in plasma of 79 cancer-free individuals. We also performed two-color MYC fluorescence in situ hybridization (FISH) in tissue from the 57 patients with gastric cancer. Results : The tissue MYC/GAPDH ratio by real-time PCR was significantly correlated with MYC status by FISH (p<0.001). The mean ratio of plasma MYC/GAPDH was 5.226±3.578 (range: 1.25–18.35) in gastric cancer patients, and 2.436±0.881 (range: 1.00–5.00) in the healthy volunteers (p<0.001). We used receiver-operating characteristics (ROC) curve analysis to select two optimal plasma MYC/GAPDH cut-offs of 2.725 and 5.225. The sensitivity and specificity were 75.4% and 76.9% at 2.725, 38.6% and 100% at 5.225, respectively. The plasma MYC/GAPDH ratio from cancer patients was significantly correlated with the tissue MYC/GAPDH ratio (p=0.009), and tissue MYC status by FISH (p=0.024). Conclusions : These findings suggest that the plasma MYC/GAPDH ratio, as determined by real-time PCR, may be an alternative non-invasive approach for detecting gastric cancer. Clin Chem Lab Med 2009;47:530–6.

Background : The aim of this study was to assess the prognostic value of cancer antigen (CA) 125 and CA 72-4 in patients with ovarian borderline tumor (BOT). Methods : All women diagnosed and treated for BOT at our institution between 1981 and 2008 were included into this retrospective study (n=101). Preoperatively collected serum samples were analyzed for CA 125 (Architect, Abbott and Elecsys, Roche) and CA 72-4 (Elecsys, Roche) with reference to clinical data and compared to healthy women (n=109) and ovarian cancer patients (n=130). Results : With a median of 34.7 U/mL (range 18.1–385.0 U/mL) for CA 125 and 2.3 U/mL (range 0.2–277.0 U/mL) for CA 72-4, serum tumor markers in BOT patients were significantly elevated as compared to healthy women with a median CA 125 of 13.5 U/mL (range 4.0–49.7 U/mL) and median CA 72-4 of 0.8 U/mL (range 0.2–20.6 U/mL). In addition, there was a significant difference compared with ovarian cancer patients who showed a median CA 125 of 401.5 U/mL (range 12.5–35,813 U/mL), but no difference was observed for CA 72-4 (median 3.9 U/mL, range 0.3–10,068 U/mL). Patients with a pT1a tumor stage had significantly lower values of CA 125 but not of CA 72-4 compared with individuals with higher tumor stages (median CA 125 29.9 U/mL for pT1a vs. 50.9 U/mL for >pT1a; p=0.014). There was a trend for increased concentrations of CA 125 but not of CA 72-4 in the presence of ascites, endometriosis or peritoneal implants at primary diagnosis. With respect to the prognostic value of CA 125 or CA 72-4, CA 125 was significantly higher at primary diagnosis in patients who later developed recurrence (251.0 U/mL vs. 34.65 U/mL, p=0.012). Conclusions : Serum CA 125 and CA 72-4 concentrations in BOT patients differ from healthy controls and patients with ovarian cancer. CA 125, but not CA 72-4, at primary diagnosis correlates with tumor stage and tends to be increased in the presence of ascites, endometriosis or peritoneal implants. Moreover, CA 125 at primary diagnosis appears to have prognostic value for recurrence. Clin Chem Lab Med 2009;47:537–42.

Background : Antinuclear autoantibody determination relies on an initial screening step using immunofluorescence on HEp2 cells. This may be followed by anti-deoxyribonucleic acid (DNA) determination, if the fluorescence of nuclei is homogeneous. We assessed the validity of a restricted algorithm and compared this to a more comprehensive algorithm that accepted any nuclear pattern as a positive indicator. Methods : Our retrospective study analyzed routine results for antinuclear antibodies (ANA) and their anti-DNA identification [double stranded nuclear DNA (ds-DNA), membrane associated DNA (mDNA), nucleosomes] for 58 systemic lupus erythematosus (SLE) patients (690 sera). We included 158 patients with systemic or organ-specific autoimmune diseases (888 sera), 44 with viral disease (88 sera), 34 cancer patients (89 sera) and 111 patients with inflammation that served as controls (122 sera) for a total of 1187 samples. Results : 1) Anti DNA antibodies are not associated only with a homogeneous pattern, but can also be seen with a speckled or nucleolar pattern. 2) The observed pattern is typical for a particular patient rather than for a specific pathology. 3) A homogeneous pattern does not necessarily indicate SLE, nor does the presence of circulating anti DNA antibodies. 4) Determination of various specificities of anti DNA antibodies, whatever the immunofluorescent pattern, increases the sensitivity and specificity for SLE. Conclusions : If diagnosis is based exclusively on a homogenous pattern, preselection would have missed identification of SLE as high levels of anti DNA antibodies were also associated with speckled or nucleolar pattern. Clin Chem Lab Med 2009;47:543–9.

Background : Analysis of exhaled breath is a promising diagnostic method. Sampling of exhaled breath is non-invasive and can be performed as often as considered desirable. There are indications that the concentration and presence of certain of volatile compounds in exhaled breath of lung cancer patients is different from concentrations in healthy volunteers. This might lead to a future diagnostic test for lung cancer. Methods : Exhaled breath samples from 65 patients with different stages of lung cancer and undergoing different treatment regimes were analysed. Mixed expiratory and indoor air samples were collected. Solid phase microextraction (SPME) with carboxen/polydimethylsiloxane (CAR/PDMS) sorbent was applied. Compounds were analysed by means of gas chromatography (GC) and mass spectrometry (MS). Results : The method we used allowed identification with the spectral library of 103 compounds showing at least 15% higher concentration in exhaled breath than in inhaled air. Among those 103 compounds, 84 were confirmed by determination of the retention time using standards based on the respective pure compound. Approximately, one third of the compounds detected were hydrocarbons. We found aromatic hydrocarbons, alcohols, aldehydes, ketones, esters, ethers, sulfur compounds, nitrogen-containing compounds and halogenated compounds. Acetonitrile and benzene were two of 10 compounds which correlated with smoking behaviour. A comparison of results from cancer patients with those of 31 healthy volunteers revealed differences in the concentration and presence of certain compounds. The sensitivity for detection of lung cancer patients based on eight different compounds not seen in exhaled breath of healthy volunteers was 51% and the specificity was 100%. These eight potential markers for detection of lung cancer are 1-propanol, 2-butanone, 3-butyn-2-ol, benzaldehyde, 2-methyl-pentane, 3-methyl-pentane, n-pentane and n-hexane. Conclusions : SPME is a relatively insensitive method and compounds not observed in exhaled breath may be present at a concentration lower than LOD. The main achievement of the present work is the validated identification of compounds observed in exhaled breath of lung cancer patients. This identification is indispensible for future work on the biochemical sources of these compounds and their metabolic pathways. Clin Chem Lab Med 2009;47:550–60.

Background: Urolithiasis is a common disease that is increasing throughout the world. This study aimed at determining the composition of urinary stones in patients with renal lithiasis in an emerging country. Methods: A morphological analysis was performed on 340 urinary stones from 325 consecutive patients from Brazil with lithiasis. Results and conclusions: Among the 340 stones analyzed, 34.7% were pure. The most frequent stone was calcium oxalate (CaOx) (59.3%), followed by uric acid (23.7%). CaOx was more frequently seen in women (p=0.024), while uric acid was more common in men (p<0.001). Among the mixed stones, CaOx (67.1%) was the most frequent major component, followed by carbapatite (11.2%) and struvite (7.9%). CaOx (p<0.001) and uric acid (p=0.014) were more frequently the major components in men, while carbapatite (p<0.001) and struvite (p=0.011) were more frequent in women. The major component of both pure and mixed stones was CaOx (65.1%), followed by uric acid (10.9%), carbapatite (10%), struvite (6.7%), ammonium urate (5.1%), cystine (1.8%) and protein (0.4%). These findings may be related to regional factors, such as weather and nutritional habits. Clin Chem Lab Med 2009;47:561–4.

Background: The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], α-keto acids derived from BCAA [BCKA; α-ketoisovaleric acid (KIV), α-ketoisocaproic acid (KIC), α-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Methods: Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27–41 years of age and 35 males, 28–43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3–5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 μm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5±0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. Results: Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%–110%. Conclusions: We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Clin Chem Lab Med 2009;47:565–72.

Background: There is an increasing demand for easy to measure biomarkers in clinical practice. We created the relational database Utrecht Patient Oriented Database (UPOD) to develop tools for identifying new biomarkers for disease. In this study, we used UPOD to identify better biomarkers for discriminating different asthma phenotypes. Methods: We performed a prospective study at the University Medical Center (UMC) Utrecht using blood from patients with asthma and a healthy reference group. Since asthma is an inflammatory disease, absolute leukocyte counts and leukocyte differential parameters were analyzed using raw data files and a logistic regression model. Results: We compared 17 difficult-to-treat asthma (DTA) cases, 13 non-difficult-to-treat asthma cases, and 19 healthy volunteers. Absolute leukocyte counts and differential parameters for leukocytes were able to discriminate asthma patients from healthy volunteers. However, among patients with asthma, difficult-to-treat cases could be more accurately defined with a neutrophil morphology change (OR 8.0; 95% CI 1.5–42.0), compared to the absolute neutrophil count (OR 4.0; 95% CI 0.8–21.0). Conclusions: In this asthma patient population, we were able to define asthma phenotypes more precisely using neutrophil morphology parameters, compared to absolute counts. Using UPOD with differential parameters, it is possible to conduct larger scale biomarker studies, combining clinical, laboratory medicine, and epidemiological techniques. Clin Chem Lab Med 2009;47:573–8.

Background: Posaconazole is now widely used for prophylaxis of invasive fungal infections in immunocompromised patients. The pharmacokinetic properties of the drug argue for therapeutic monitoring, but so far described analytical methods have shortcomings with respect to application in a routine setting. The aim of our work was to develop an analytical method suitable for routine use. Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. For sample preparation, protein precipitation followed by on-line solid phase extraction was used. SCH 56984, a posaconazole related compound provided by the manufacturer of posaconazole, was used as internal standard. Results: The method was characterized by short hands-on time and an analytical run time of four minutes. Validation data demonstrated acceptable analytical performance. Conclusions: LC-MS/MS with on-line solid phase extraction for sample preparation allows the implementation of a convenient and reliable method for pharmacokinetic monitoring of posaconazole. Clin Chem Lab Med 2009;47:579–84.

Background: Serum β 2 -microglobulin (β2m) has been established as a marker of disease activity in malignancies, autoimmune conditions and infections. Despite its important role in prognosis assessment and disease monitoring, relatively few studies are available on its expression in healthy individuals. Furthermore, interpretation of results is hampered by the variety in reference limits due to differences in methodology, sample population and statistics. Methods: Serum β2m concentrations were measured using a microparticle-enhanced immunonephelometric method in 183 healthy blood donors aged 29–75 years. Results: The median β2m concentration was 1.67 (0.88–2.75) mg/L with no difference between men and women (1.71 mg/L vs. 1.62 mg/L, p<0.07). A linear correlation was found between β2m and age (p<0.0001), serum concentrations significantly higher in older subjects (1.55, 1.59, 1.70, and 1.87 mg/L in age groups of 29–40, 40–50, 50–60 and 60–75 years, respectively, p<0.0001). Reference intervals obtained by non-parametric estimation after partitioning by age were 1.02–2.46 mg/L vs. 1.29–2.70 mg/L in younger (29–49 years) vs. older (50–75 years) individuals. Conclusions: These data can help standardise β2m reference limits and support age-adjusted comparisons in clinical studies. Clin Chem Lab Med 2009;47:585–9.

Background: Earthquakes are major causes of morbidity and mortality. The Wenchuan region of China was devastated by a catastrophic earthquake on May 12, 2008, at 02:28 p.m. (Beijing time), registering magnitude 8.0 on the Richter scale and causing more than 69,181 deaths. As a first-line general hospital in the disaster area, Mianyang Central Hospital admitted a large number of the victims. Methods: A total of 534 victims (246 males, 288 females) were categorized as non-crush injury patients (n=239), simple crush injury patients (n=136), and crush syndrome patients (n=69) according to their traumatic conditions. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), cholinesterase (CHS), and creatine kinase (CK) levels were measured. Results: ALT, AST, LDH, CHS, and CK levels showed significant differences among the three groups by one-way analysis of variance (ANOVA). Pearson correlation analysis showed that correlative changes between any two of the following: ALT, AST, GGT, ALP, LDH, and CHS were similar among three groups, with the following exceptions. The correlation coefficients of ALT-GGT, AST-GGT, and ALP-CHS changed from positive to negative values, and ALP-LDH changed from a negative value to a positive value. Receiver-operating characteristic (ROC) curve analysis showed the highest diagnostic effectiveness of 99.4% for CK, with 100% specificity [positive predictive value (PPV)=100%] and 99.4% sensitivity [negative predictive value (NPV)=99.0%] in distinguishing crush injury (including crush syndrome) from non-crush injury. AST had the best diagnostic effectiveness in distinguishing crush syndrome from crush injury; 53.8%, with 85.5% specificity (PPV=64.4%) and 77.9% sensitivity (NPV=90.7%). Multivariate logistic analysis revealed that CK was best at distinguishing crush injury (including crush syndrome) from non-crush injury (OR 409.636, 95% CI 382.96–438.17), and AST was best for distinguishing crush syndrome from crush injury (OR 50.08, 95% CI 46.84–53.55). Conclusions: Crush injury and crush syndrome are severe in victims following accidents or natural catastrophes. Serum CK, LDH, AST, ALT, GGT, and ALP activities were all helpful biochemical parameters in estimating the severity of crush injury and/or crush syndrome and preventing the development of further complications. Clin Chem Lab Med 2009;47:590–5.

Background : Human serum from biobanks is frequently used in prospective epidemiological studies. Long-term storage may modify its composition. A better understanding of the stability of the serum components may improve the interpretation of future studies. Methods : The concentrations of selected proteins; immunoglobulins, carrier proteins and enzymes in samples stored at   –25°C for 25 years and 2 years were compared with 1-month-old samples. For each length of storage time, 130 specimens were randomly selected from apparently healthy male blood donors aged 40–49 years. We examined the distribution of values, compared dispersion and localization of central tendency, and established reference intervals for each component. Results : The study demonstrated non-significant or numerically small group differences in the concentrations of albumin, aspartate amino transferase, cystatin C, immunoglobulin E, immunoglobulin G, and sex hormone binding globulin. Mean values between fresh and 25-year-old samples suggested larger differences during storage for alanine amino transferase (–73.4%), creatinine kinase (–96.1%), insulin C-peptide (–98.7%), ferritin (–18.5%) and transferrin (+8.2%). Conclusions : The findings showed that long-term storage can introduce a considerable bias for vulnerable components. Clin Chem Lab Med 2009;47:596–603.

Background : Three adult patients presented with unexpectedly high thyrotropin (TSH) concentrations that were discordant with clinical and biochemical findings of euthyroid or hyperthyroid status. Methods : Antibody interference in the TSH immunoassay (Roche) was investigated by polyethylene glycol (PEG)-pretreatment, heterophilic blocking tube (HBT)-pretreatment, rheumatoid factor (RF) testing, immunofixation, protein A adsorption, and gel filtration chromatography (GFC). Results : PEG-precipitation yielded <20% recovery of serum TSH, whereas HBT-pretreatment did not decrease TSH test results. RF-testing and immunofixation were negative. Protein A adsorption and GFC demonstrated the presence of TSH-immunoglobulin complexes in serum. Conclusions : Interference by TSH-immunoglobulin complexes should be ruled out in euthyroid and hyperthyroid patients presenting with inappropriately increased or non-suppressed TSH values. Clin Chem Lab Med 2009;47:604–6.

Background : Samples with limited volume is a common problem in laboratories receiving samples from pediatric patients. Also, pediatric samples may contain nucleated red blood cells (NRBC) which distorts the white blood cell (WBC) count and which can only be measured by some automated cell counting systems. Differential counts are sometimes required, posing the question of validity of flagging depending on age of the patients and on predilutions. Methods : We evaluated the hematology analyzers XE-2100 and XS-800 for their suitability in measuring hematological parameters in such samples. Results : With the exception of the MCHC and partly the MCH, we observed very good agreement between complete blood counts (CBC) in diluted and undiluted samples. Dilution did not impair sensitivity in the clinically relevant range nor, accuracy of the NRBC count on XE-2100. Flagging was ineffective in undiluted samples from children <1 year of age and in all diluted samples when measuring differential counts. Conclusions : In summary, while automated measurement of CBC and NRBC is possible in diluted samples, measurement of differential counts is restricted by loss of flagging efficiency. In addition, flagging is also ineffective in children <1 year of age using the analyzers evaluated and should, for diagnostic purposes, be performed manually. Clin Chem Lab Med 2009;47:607–11.