Volume 47, Issue 12

Recent progress in the understanding of the pathophysiology of hemostasis has established that blood coagulation disorders causing bleeding or thrombosis often indicate a multi-casual nature in which the interaction between genetic and acquired factors plays a major mechanistic role. The interaction of two or more factors may attenuate clinical symptoms. However, a synergic worsening may also occur, resulting in a higher risk than expected from the combination of the separate effects. The role of genetic factors on the modulation of the phenotypic expression of coagulation disorders will be addressed in this review, with particular emphasis on the underlying pathogenic mechanisms and clinical implications. Clin Chem Lab Med 2009;47:1455–60.

Background: The use of laboratory tests has been increasing in recent years due to various factors affecting laboratories, physicians, legal aspects, or patients themselves. Methods: The efficacy of laboratory tests must be evaluated on the basis of the clinical benefits that they provide in terms of prevention, diagnosis, follow-up, or treatment; with the aim of optimizing health results in general. Results: There are techniques that can be used to determine the clinical validity of the efficacy, effectiveness, and safety of treatment or preventive measures performed on individuals with abnormal tests, as well as providing an economic evaluation of the process. Once the test is incorporated into clinical service, it must be evaluated by retrospective audits in test utilization. To improve the use of laboratory tests, many strategies have been devised that incorporate clinical practice guidelines (CPGs), the conduct of professionals and the patients, and organization of the process. We discuss the importance of the involvement of health professionals. Conclusions: Strategies in relation to CPGs, conduct of professionals, conduct of patients, or organization of health care processes improve the use of tests in relation to clinical processes. Laboratory professionals have the appropriate knowledge and can improve the quality and efficacy of health care. Clin Chem Lab Med 2009;47:1461–5.

Background: Diseases with an onset during childhood or adult life can have their origin during fetal life or at birth. Neonatal blood dried on filter paper (Guthrie cards) collected for screening purposes is routinely stored for decades. In addition to clinical use, these filters in combination with patient registers constitute an invaluable resource for epidemiological and pathophysiological research. Although RNA has been successfully recovered from such filters even after decades of storage, the potential decay of RNA over time has not previously been investigated using quantitative methods. Methods: Filter papers (n=5) with dried blood spots from the Swedish National PKU register, stored for 1, 5, 10, 15 or 20 years were randomly selected. RNA was isolated from each sample, quantitated by spectrophotometry and reverse transcribed following DNase I treatment. Amplifiable cDNA was subsequently detected by real-time PCR using primers specific for transcripts encoding β-actin. Results: Transcripts encoding β-actin were detected in all 25 samples analyzed at a mean threshold cycle (Ct) of 25 (SD 1.9). A one-way ANOVA indicated no significant effect of storage time on Ct values. Conclusions: The lack of significant decay of RNA in dried blood filters stored for up to 20 years suggests that such filters are useful for studies of RNA determinants of diseases with an onset in childhood as well as adult life. Clin Chem Lab Med 2009;47:1467–9.

Background: Rs619203 (Cys2229Ser) and rs529038 (Asp2213Asn) polymorphisms in the ROS1 gene have been studied in relation to myocardial infarction (MI) yielding inconsistent results. We investigated the role of ROS1 rs529038 polymorphism in coronary artery disease (CAD) in Greeks using a case-control study. Methods: Genotyping for rs529038 polymorphism was performed using a multiplex PCR technique in patients with CAD (n=294) and controls (n=311). Logistic regression analysis was used to calculate crude and adjusted odds ratios (ORs). Results: Logistic regression analysis did not show any statistically significant effect of ROS1 polymorphism in the occurrence of CAD (AG vs. AA, OR: 1.08, p=0.635; GG vs. AA, OR: 0.62, p=0.220). Adjustment for confounding factors gave similar results, irrespective of the type of disease (i.e., stable coronary artery disease vs. acute coronary syndrome). Conclusions: Our findings do not support the hypothesis that ROS1 rs529038 polymorphism is an important contributing factor in the etiology of CAD in the Greek population. Clin Chem Lab Med 2009;47:1471–3.

Background: Glutathione S-transferase (GST) enzymes are critical for detoxifying reactive oxygen species (ROS) and their products which have been implicated in the pathology of inflammatory diseases such as atopic dermatitis (AD). Methods: We investigated the effects of genetic polymorphisms of GST on the risk of AD in preschool age children. Biomarkers for oxidative stress were also evaluated with respect to GST genotype. Results: The GSTP1 Val105 allele was significantly associated with an increased risk of AD [odds ratio (OR)=1.62, p<0.05]. The combination of the GSTP1 Val105 allele and the GSTT1 null genotype further increased this risk by 2.3-fold (p<0.01). No association was observed for the GSTM1 null or GSTT1 null genotype alone. In children with AD, blood total antioxidant capacity was significantly less (p<0.001), while malondialdehyde was higher (p=0.12). Children with the GSTP1 Val105 allele had significantly lower concentrations of erythrocyte glutathione compared to GSTP1 Ile / Ile homozygotes (p=0.03). Conclusions: Our study suggests that the GSTP1 Val105 allele is an important determinant of susceptibility to AD in preschool age children and increased oxidative stress may play a role in the pathogenesis of AD. Clin Chem Lab Med 2009;47:1475–81.

Background: A recent genome-wide linkage study mapped blood pressure (BP)-related loci on human chromosome 1q and identified the regulator of G-protein signaling 5 ( RGS5 ) as a candidate for regulation of BP. Thus, we assessed the relationship between RGS5 genetic polymorphisms and essential hypertension (EH) in Chinese. Methods: A total of 906 patients with EH and 894 age- and gender-matched normotensive (NT) controls were enrolled. Sixteen single nucleotide polymorphisms (SNPs) in RGS5 were genotyped. Results: There were no significant differences in the overall distributions of the genotypic and allelic frequencies for each SNPs between the two groups. However, in haplotype analysis, significant differences for the overall distributions were noted for four haplotypes constructed by five SNPs (rs12041294C/T, rs10917690A/G, rs10917695T/C, rs10917696T/C and rs2662774G/A), viz. H 2 (C–A–C–T–A) (p=0.038), H 5 (C–G–T–T–G) (p=0.001), H 6 (T–G–C–T–A) (p=0.021) and H 12 (T–A–T–T–G) (p=0.023). Serum concentrations of high- and low-density lipoprotein cholesterol showed significant associations with haplotypes revealed by a global test (p=0.0001 and 0.0309). Conclusions: Multiple SNPs in combination in RGS5 may confer risk for hypertension. Our results also lend support for the effect of RGS5 SNPs on lipid metabolism. Further studies are warranted to find the causal SNPs in RGS5 for EH. Clin Chem Lab Med 2009;47:1483–8.

Background: The risk for type 1 diabetes mellitus (T1DM) and celiac disease (CD) is related to human leukocyte antigen (HLA) DQA1, DQB1 and DRB1 loci. Unfortunately, HLA typing has been too difficult and costly for frequent use. Automated genotyping focused on risk alleles could provide access to HLA typing in diagnostic evaluations, epidemiological screening and contribute to preventive strategies. Methods: A sequence specific primer amplification method requiring a total of four PCR reactions, one restriction enzyme digestion and a single electrophoretic step provides low to medium resolution typing of DQA1, DQB1 and DRB1 using Applied Biosystems 3730 DNA analyzer. The method was validated using 261 samples with genotypes determined using a reference method. Results: Specific fluorescent DQA1 , DQB1 and DRB1 amplicons were of expected size. Concordance with the reference method was 100% for DQA1 and DQB1 alleles and 99.8% for DRB1 alleles. Conclusions: We have developed a high throughput HLA typing method that accurately distinguishes risk alleles for T1DM and CD. This method allows screening of several thousand samples per week, consuming 32 ng of DNA template, low reagent volumes and minimal time for data review. Clin Chem Lab Med 2009;47:1489–95.

Background: The goal of this study was to provide reference ranges for thyrotropin (TSH) and free thyroxine (fT 4 ) based on data collected from a disease-free sample of French middle-aged adults. Methods: A total of 3218 subjects participating in the Supplémentation en Vitamines et Minéraux Antioxydants (SU.VI.MAX) study had measurements for TSH, fT 4 and urinary iodine concentrations. Thyroid volume and structure were evaluated using standardized ultrasonography. We selected a disease-free sample which included 2338 subjects (1313 females aged 35–60 years and 1025 males aged 45–60 years) with normal thyroid imaging, no previously reported thyroid disease or use of thyroid medication, and no risk factors for thyroid dysfunction. Distribution of TSH and fT 4 was estimated in males and females. Results: The median (central 95% range) TSH serum concentrations for females were 1.79 mU/L (0.29–5.21 mU/L) for ages 35–44 years and 1.98 mU/L (0.27–6.94 mU/L) for ages 45–60 years (p<0.0001, for age). The median (central 95% range) for males 45–60-year-old was 1.63 mU/L (0.28–4.54 mU/L) (p<0.0001, for sex). Sex- and age-specific mean fT 4 concentrations did not differ significantly (p=0.06) between males and females and (p=0.08) between female age groups. However, median fT 4 concentrations between 45–60-year-old males and females differed (p<0.001). Conclusions: In middle-aged adults, the TSH distribution was associated with gender and, among females, with age. Stratification according to gender and age should be considered when TSH ranges are used in the diagnosis, treatment and monitoring of thyroid disease. Clin Chem Lab Med 2009;47:1497–505.

Background: The objective of this study was to evaluate the efficiency of free light chain (FLC) analysis in comparison to serum protein electrophoresis (SPE) for detecting M-proteinemia. Methods: A total of 553 consecutive patients for whom evaluation of M-proteinemia was requested were included in this study. For all patients, serum FLC analysis and SPE followed by pentavalent immunofixation analysis was performed. Identification of monoclonal bands was performed using specific antisera. FLC analysis was performed using the Modular P analyzer in accordance with the manufacturer's recommendations. Local reference ranges for FLCs on this platform were established based on samples from patients with a normal electrophoretic pattern [no monoclonal bands, no hypo- or hypergammaglobulinemia, no acute phase pattern and normal kidney function, i.e., estimated glomerular filtration rate (eGFR) >60 mL/min]. Results: Local reference ranges (95%) were established (n=243): κ: 8.01–28.26 mg/L; λ: 8.07–23.58 mg/L and κ/λ ratio: 0.74–1.66. Negative and positive predictive values were 98.6% and 49.5%, respectively, for screening for M-proteinemia by SPE alone, 94.3% and 21.7% for FLC concentration and 95.1% and 21.4% for FLC with the κ/λ-ratio included. Combining protein electrophoresis and FLCs resulted in a negative predictive value of 99.0% and a positive predictive value of 23.4%. Conclusions: Serum FLC analysis alone is not suitable for screening for M-proteinemia. Clin Chem Lab Med 2009;47:1507–11.

Background : Neuron-specific enolase (NSE) and S100 protein are implicated in several brain injuries, including stroke. Our objective was to analyze the temporal profile and the clinical significance of NSE and S-100 in acute ischemic (IS) and intracerebral hemorrhage (ICH). Methods : We studied 224 patients with IS and 44 patients with ICH. Computerized tomography (CT) scans were performed to assess infarct volume. Stroke severity was evaluated using the National Institute of Health Stroke Scale (NIHSS), and functional outcome at 3 months with the modified Rankin Scale (mRS). Serum NSE and S100 protein were measured using an electrochemiluminescence-immunoassay. Results : Peak values were found at 72 h for NSE and at 24 h for S100 in IS. For ICH, peak values were found at 24 h for both NSE and S100. At these time intervals S100 and NSE correlated with the NIHSS score and were independently associated with poor outcome. Conclusions : High serum NSE and S100 are associated with poor outcome in IS, and high serum NSE is associated with poor outcome in ICH. These findings suggest the potential utility of NSE and S100 as prognostic markers for acute stroke. Clin Chem Lab Med 2009;47:1513–8.

Background: The blood-brain barrier is compromised in patients with stroke. The release of neuro-biochemical protein markers, such as neuron specific enolase (NSE) into the circulation may allow the pathophysiology and prognosis of patients with cerebrovascular diseases to be evaluated further. The present study was designed to measure the marker of neuronal damage, NSE, in saliva and serum of patients with acute ischemic stroke and patients with stroke related diseases as a diagnostic and/or monitoring tool for early prediction of ischemic stroke. Methods: Salivary and serum NSE concentrations were measured in 150 individuals. Fifty were patients recently diagnosed as having ischemic stroke, 75 were gender and age-matched risk-group patients (patients with hypertension, type 2 diabetes and ischemic heart disease). Another 25 were gender and age-matched healthy controls. Results: Salivary and serum NSE concentrations were significantly higher than that of healthy controls. The cut-off threshold for salivary NSE of 3.7 μg/L was optimum, showing 80% accuracy for differentiation of ischemic stroke from normal individuals. Conclusions: Salivary NSE (alone or in combination with serum) can be used as a valuable diagnostic and possibly prognostic tool for measurement of neuronal damage in patients with stroke and stroke-related diseases. Clin Chem Lab Med 2009;47:1519–24.

Background: The goal of the study was to assess the presence of antibodies to mutated citrullinated vimentin (anti-MCV) and cyclic citrullinated peptides (anti-CCP) in patients with juvenile idiopathic arthritis (JIA) compared with patients with other juvenile onset rheumatic diseases. Methods: The study included 56 patients who fulfilled the International League of Associations for Rheumatology (ILAR) classification criteria for JIA, and 17 control patients with other juvenile onset rheumatic diseases. Data on six core outcome variables and the Sharp score were collected for patients with JIA. Sera and synovial fluid, if available, were tested for anti-CCP and anti-MCV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). Results: Anti-MCV antibodies were positive in 3/56 (5.4%) and anti-CCP in 1/56 (1.8%) of patients with JIA. Two out of three anti-MCV positive patients (one of them also anti-CCP positive) were found to be rheumatoid factor (RF)-positive with polyarticular disease. Within the control group, anti-MCV was positive in 4/17 (23.5%) patients, while anti-CCP positivity was not observed. No correlation between anti-MCV with anti-CCP antibody levels was found for any of the six core outcome variables or for the adapted Sharp score. Conclusions: Our results show that antibodies targeting citrullinated proteins are not a useful diagnostic marker for JIA, but can indicate severe patterns of disease in JIA. Clin Chem Lab Med 2009;47:1525–30.

Background: The concept of developmental hemostasis has been universally accepted. Physiological reference ranges for coagulation tests are available for infants and children of different ages. However, on Oriental children they are rare. Methods: Results of preoperative activated partial thromboplastin time (APTT) in neonates, infants, children aged 1–18 years and adults with minor elective surgery in a university affiliated hospital were reviewed retrospectively. Plasma activity of factors VIII, IX, XI, XII (FVIII:C, FIX:C, FXI:C, FXII:C) and lupus anticoagulants (LAC) in 47 children with prolonged APTT and 34 adult controls were measured to investigate the causes of prolongation. Results: Compared with adults, APTT values were prolonged significantly and were age-dependent in children, especially in neonates and infants aged 1–6 months. Mean values for FXII:C and FIX:C in children with prolonged APTT values were significantly lower than those in adults (p<0.001). Prolonged APTT values correlated negatively with FXII:C and FIX:C, and weakly with the LAC Screen ratio (LAC-SR) (r 0.01 =−0.808, –0.705 and 0.372, p=0.000, 0.000 and 0.001, respectively). There was weak negative correlation between FXII:C and LAC-SR (r 0.01 =−0.277, p=0.012). No significant correlation was seen between prolonged APTT values and FVIII:C or FXI:C. Conclusions: APTT values change dynamically with age during childhood and display a distinct pattern of evolution in children. Lower values of FXII:C and FIX:C, and presence of LAC contribute to the prolongation of APTT values in Chinese children. Clin Chem Lab Med 2009;47:1531–7.

Background: The technology to recognize nucleated red blood cells (NRBC) automatically has only recently been developed. Modern hematology analyzers allow for rapid and accurate NRBC counts. The goal of our study was to evaluate NRBC counts and the concentrations of serum transferrin receptor (sTfR) in patients affected by different thalassemia syndromes and hereditary spherocytosis. We wished to gain a better understanding of the meaning of the presence of NRBC in peripheral blood and the relationship of the two parameters with effective and ineffective erythropoiesis in the different thalassemia syndromes. Methods: NRBC counts in peripheral blood were evaluated in a large group of patients with thalassemia (36 thalassemia major, 55 thalassemia intermedia and 61 Sβ-thalassemia patients) and compared with data from 29 patients with hereditary spherocytosis; in all the patients the concentration of sTfR was evaluated as an index of global erythropoiesis. Results: The NRBC count showed a good relationship with ineffective erythropoiesis: highest counts were observed in the thalassemia syndromes characterized by almost completely ineffective erythropoiesis. NRBCs were absent in patients affected by hereditary spherocitosis, a disease characterized by effective erythropoiesis. Conclusions: The NRBC count can be useful for better defining ineffective erythropoiesis in patients with thalassemia, and can help optimize transfusion therapy in severe thalassemia syndromes. Clin Chem Lab Med 2009;47:1539–42.

Background: Crimean-Congo hemorrhagic fever (CCHF) is a public health problem in many countries. Chitotriosidase (ChT) is an enzyme secreted by activated macrophages that catalyzes the hydrolysis of chitin and chitin-like substrates. The goal of this study was to assess the relationship between serum ChT activity and mortality. Methods: ChT activities on the first day of hospitalization were analyzed in serum from 46 patients with CCHF and 36 healthy controls. Serum ChT activities and other clinical and laboratory parameters for patients with non-fatal and fatal CCHF were compared. Results: The median ChT activity was increased in all patients with CCHF [189.9 (134.8–246.6) nmol/mL/h]. The median ChT activity in the non-fatal CCHF group [220.2 (180.6–290.1) nmol/mL/h] was higher compared with the fatal CCHF group [29.2 (16.5–45.7) nmol/mL/h] (p<0.001). In univariate analysis, platelet count, lactate dehydrogenase (LDH), and activated partial thromboplastin time were associated with mortality. Conclusions: This is the first study investigating the association of serum ChT enzyme activity with mortality from CCHF. This study suggested that relatively low ChT enzyme activities may be a prognostic marker in patients with CCHF. Clin Chem Lab Med 2009;47:1543–7.

Background: Preanalytical mistakes (PAMs) in samples usually led to rejection upon arrival to the clinical laboratory. However, PAMs might not always be detected and result in clinical problems. Thus, PAMs should be minimized. We detected PAMs in samples from Primary Health Care Centres (PHCC) served by our central laboratory. Thus, the goal of this study was to describe the number and types of PAMs, and to suggest some strategies for improvement. Methods: The presence of PAMs, as sample rejection criteria, in samples submitted from PHCC to our laboratory during October and November 2007 was retrospectively analysed. Results: Overall, 3885 PAMs (7.4%) were detected from 52,669 samples for blood analyses. This included missed samples (n=1763; 45.4% of all PAMs, 3.3% of all samples), haemolysed samples (n=1408; 36.2% and 2.7%, respectively), coagulated samples (n=391; 10% and 0.7%, respectively), incorrect sample volume (n=110; 2.8% and 0.2%, respectively), and others (n=213; 5.5% and 0.4%, respectively). For urine samples (n=18,852), 1567 of the samples were missing (8.3%). Conclusions: We found the proportion of PAMs in blood and urine samples to be 3-fold higher than that reported in the literature. Therefore, strategies for improvement directed towards the staff involved, as well as an exhaustive audit of preanalytical process are needed. To attain this goal, we first implemented a continued education programme, financed by our Regional Health Service and focused in Primary Care Nurses. Clin Chem Lab Med 2009;47:1549–52.

Background: Evaluation of specific urinary markers with respect to urine creatinine (uCreat) is common. However, as uCreat is a function of both glomerular filtration and tubular secretion, using uCreat for specific tubular markers, suggests that glomerular function is normal, and there is no tubular secretion. Thus, adjusting values of any tubular marker to uCreat, especially in patients with acute or even moderate chronic renal failure, can be misleading. Methods: Using urine cystatin-C (uCST3) as a model tubular marker for following 120 kidney graft recipients daily, we evaluated the utility of either uCST3 alone or the uCST3/uCreat ratio to detect tubular damage. All positive kidney biopsies were always associated with a uCST3>0.18 mg/L. Results: Using the uCST3/uCreat ratio, discrepancies regarding biopsy status were observed in nine patients (4 false positive, 5 false negative results). In two patients, variability of uCreat appeared to be the most important factor causing inconsistent uCST3/uCreat ratios. With a negative predictive value (NPV) of 85.7%, uCST3/uCreat can lead to errors in clinical interpretation. These errors can be avoided when estimates of tubular damage are based on uCST3 concentrations alone (NPV=100%). Conclusions: We recommend using the uCST3 value to evaluate the extent of renal tubular damage. Indeed, our conflicting results on uCST3/uCreat can be extended to every marker of tubular function. Evaluating a urine marker specific for renal tubular damage to a second urine marker that is itself strongly dependent upon glomerular or other renal or non-renal conditions, impairs its clinical relevance and may lead to incorrect interpretations. Correction with uCreat can be performed only in pure glomerulopathy, when specific markers of glomerular function are measured (i.e., urinary albumin). In all other cases of renal diseases, such correction is inappropriate and should be avoided. Clin Chem Lab Med 2009;47:1553–6.

Background: Pro-gastrin releasing peptide (ProGRP) concentrations in blood play an important role in the diagnosis and treatment of patients with small cell lung cancer (SCLC). The automated quantitative ARCHITECT ® ProGRP assay was developed to aid in the differential diagnosis and in the management of SCLC. The purpose of this study was to evaluate the analytical performance of this chemiluminescent microparticle immunoassay at multiple sites. Methods: ARCHITECT ProGRP measures ProGRP using a two-step sandwich using monoclonal anti-ProGRP antibodies coated on paramagnetic microparticles and labeled with acridinium. Analytical performance of the assay was evaluated at four sites: Abbott Japan, Denka Seiken, the Johns Hopkins University, and the University of Munich. Results: Total precision (%CV) for nine analyte concentrations was between 2.2 and 5.7. The analytical sensitivity of the assay was between 0.20 pg/mL and 0.88 pg/mL. The functional sensitivity at 20% CV was between 0.66 pg/mL and 1.73 pg/mL. The assay was linear up to 50,000 pg/mL using a 1:10 autodilution protocol. The calibration curve was stable for 30 days. Comparison with the Fujirebio microtiter plate enzyme-linked immunosorbent assay (EIA) ProGRP assay gave a slope of 0.93 and a correlation coefficient (r) of 0.99. Conclusions: These results demonstrate that the ARCHITECT ProGRP assay has excellent sensitivity, precision, and correlation to a reference method. This assay provides a convenient automated method for ProGRP measurement in serum and plasma in hospitals and clinical laboratories. Clin Chem Lab Med 2009;47:1557–63.

Background: Vitamin D is important to health and disease. Liquid chromatography-tandem mass spectrometry (LC-MSMS) is considered the most accurate technology for quantification of serum 25-hydroxyvitamin D (25OHD) which is the best biomarker for estimating vitamin D nutritional status. Methods: Serum was mixed with acetonitrile containing hexadeuterated 25-hydroxyvitamin D 3 (d 6 -25OHD3) and centrifuged 10 min at 15,634× g . The supernatant was injected onto a turbulent flow preparatory column then transferred to a polar endcapped C18 analytical column. The mass spectrometer was set for positive atmospheric pressure chemical ionization. Results: The analytical cycle time was 5.5 min. Inter- and intra-assay CV for both analytes across three concentrations ranged from 3.8% to 14.2%. The method was linear from 3.0 to 283.6 nmol/L for 25-hydroxyvitamin D 3 (25OHD3) and 4.6 to 277.9 nmol/L for 25-hydroxyvitamin D 2 (25OHD2), with an accuracy of 88.7%–118.7% and 90.7%–100.3%, respectively. No carryover or ion suppression was observed. Comparison with a radioimmunoassay using patient specimens (n=527) showed a mean difference of 5.2%, and diagnostic agreement of 80.9% with Deming regression of slope 0.867, intercept 12.8, standard error of estimate (SEE) 17.4, and r=0.8425. Conclusions: The LC-MSMS method coupled with turbulent flow technology for serum 25OHD quantitation is rapid, efficient, and suitable for clinical testing. Clin Chem Lab Med 2009;47:1565–72.