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October 7, 2005
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June 1, 2005
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Mitochondria produce reactive oxygen species (ROS) under physiological conditions in association with activity of the respiratory chain in aerobic ATP production. The production of ROS is essentially a function of O 2 consumption. Hence, increased mitochondrial activity per se can be an oxidative stress to cells. Furthermore, production of ROS is markedly enhanced in many pathological conditions in which the respiratory chain is impaired. Because mitochondrial DNA, which is essential for execution of normal oxidative phosphorylation, is located in proximity to the ROS-generating respiratory chain, it is more oxidatively damaged than is nuclear DNA. Cumulative damage of mitochondrial DNA is implicated in the aging process and in the progression of such common diseases as diabetes, cancer, and heart failure. Clin Chem Lab Med 2003; 41(10):12811288
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June 1, 2005
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The Directive on in vitro Diagnostic Medical Devices (IVDD 98/79/EC) was officially adopted by the European Union (EU) on December 7, 1998. The IVDD aims to supplement the legal framework of the European Community, which governs the conditions for the placing on the market of medical devices, by extending the already implemented legislation to the category of in vitro diagnostic medical devices (IVDs). They consist of those devices, including reagents and reagent products, calibrator materials or instruments, as well as specimen receptacles, intended by the manufacturer for the in vitro analysis of specimens derived from the human body. This directive has introduced at the European level common regulatory requirements across Europe for the safety, quality and performance of in vitro diagnostics (IVDs), incorporating them into medical device legislation. It harmonizes the conformity assessment procedures to be applied by manufacturers before they place IVDs on the market. For certain products expressly specified in the directive, of which the most important are used for the evaluation of the safety of blood supply and patient testing, the manufacturer will have to take into account in their performance evaluation the so-called "Common Technical Specifications" (CTS). These are needed to establish the performance characteristics of the IVDs in evaluation and have the same status as harmonized standards. In the meantime, the IVD directive has been transposed into national law in all EU countries. During a transitional period ending in December 2003, manufacturers will have the option of following pre-existing national regulatory processes or taking their IVDs through the new procedures as specified in the directive. Following this, starting from 7th December 2003, adherence to the directive regulations will become mandatory, and only IVDs bearing the "Communautés Européennes (CE) mark" can then be sold in the EU. Clin Chem Lab Med 2003; 41(10): 12891298
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June 1, 2005
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Remodelling of extracellular matrix by activated matrix metalloproteinases is considered to contribute to progression of ventricle remodelling during chronic heart failure. The aim of this study was to associate two promoter polymorphisms, -790T/G and -735C/T, in the gene for matrix metalloproteinase (MMP)-2 (gelatinase A) with chronic heart failure (CHF). For this purpose, 164 patients (124 men, 40 women, median age 56 years, range 21-91 years) with CHF (functional class NYHA II-IV, ejection fraction median 25%, cardiothoracic index more than 50%) were compared with 196 control subjects without clinical signs of cardiovascular disease (131 men and 65 women, median age 56 years, range 27-84 years) in -790T/G and -735C/T MMP-2 genotype distributions and allelic frequencies. The genotypes were determined by polymerase chain reaction (PCR) with restriction analyses. A significant increase of the T allele of the -790T/G MMP-2 polymorphism (p = 0.04), as well as of the C allele of the -735C/T MMP-2 gene polymorphism, in patients with CHF was proven (p = 0.04). The heterozygote CT of the 735C/T MMP-2 polymorphism exhibits a 7 times higher odds ratio (OR) for the CHF patients with lower levels of total cholesterol (less than 5 mmol/l), especially for nonhypertensive CHF men (OR = 7.28, 95% confidence interval 1.51-35.03, p = 0.006). Determination of MMP polymorphisms in the regulatory area of the gene could help us to comprehend individual susceptibility of patients with CHF to MMP inhibitors based on known risks of MMP genotypes. Clin Chem Lab Med 2003; 41(10):12991303
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June 1, 2005
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To investigate the relationship between the lipoprotein lipase Ser447Ter (S447X) mutation, lipid profiles and risk of atherosclerotic disease, we studied two groups of Japanese subjects. These groups consisted of a dyslipidemic group (triglyceride (TG) >1.69 mmol/l and high-density lipoprotein-cholesterol (HDL-C) = 0.91 mmol/l; n = 106) and a control group (TG = 1.69 mmol/l and HDL-C ≥1.16 mmol/l; n = 106). All subjects in the control group were confirmed to the pattern A phenotype (normal low-density lipoprotein (LDL) pattern), and 21 individuals in this group were heterozygous for the S447X mutation, but not homozygous. In the patient group, pattern A was shown by 44 of 106 patients. The rest were of the pattern B phenotype, which is associated with an increased risk of coronary heart disease. Homozygosity was concentrated in the patient group (p < 0.05) and in those with the pattern B phenotype (p < 0.05). In contrast, heterozygosity between both groups was not statistically significant. In conclusion, heterozygous and homozygous status with respect to the LPL S447X mutation appears to have different meanings with respect to biochemical and clinical phenotypes of atherosclerosis. Clin Chem Lab Med 2003; 41(10):13041308
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June 1, 2005
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To examine physical exercise-related changes in urinary excretion of protein/peptide hormones and to correlate modifications with the general increase in post-exercise proteinuria, urine C-peptide, insulin and insulin-like growth factor-I (IGF-I) and their plasma concentrations were measured. Plasma and urinary C-peptide, insulin and IGF-I before (B ex ) and at the end (E ex ) of physical exercise (a 2.5-hour competition, 102 km) were analysed in 20 young cyclists. At E ex compared with B ex , concentration of urinary C-peptide decreased slightly but significantly (21.3±2.7 vs. 13.5±1.7 nmol/l), but urinary insulin and urinary IGF-I concentrations significantly increased at E ex (92.5±4.2 vs. 131.4±15.7 pmol/l and 10.0±2.1 vs. 33.6±3.8 pmol/l, respectively). Plasma insulin and plasma C-peptide significantly decreased, whereas plasma IGF-I was unchanged. Urinary concentrations of total proteins and creatinine significantly increased. Both E ex urinary C-peptide/urinary protein and urinary C-peptide/urinary creatinine ratios were significantly reduced. The correlation between C-peptide and insulin in plasma was confirmed at B ex as well as E ex , but in urine only at B ex . An increased renal tubular reabsorption of C-peptide at the end of exercise might be suggested, but the expected values considering creatinine excretion were almost three times less. The E ex urinary insulin concentration was higher than expected, considering the circulation levels, but lower when compared with the expected concentration considering creatinine excretion. Physical exercise proteinuria, related to an increased protein filtration and a saturation of the mechanisms responsible for the reabsorption, does not appear similar for all peptide hormones. Clin Chem Lab Med 2003; 41(10):13081313
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June 1, 2005
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Neopterin, a marker of stimulated cellular immune response, is increased in unstable angina, acute myocardial infarction and possibly stable coronary artery disease. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors (statins) have antiinflammatory properties, but their effect on neopterin largely unknown. Neopterin was measured in 232 patients undergoing elective coronary angiography and compared to the degree of atherosclerosis, use of concomitant medications and demographics. Neopterin was lower in subjects using statins (n = 66) compared to those not taking statins (median (range): 6.65 (4.1-18.3) vs. 7.70 (3.6-29.1) nmol/l, p < 0.0001). This association was also found in the subgroup of patients with coronary artery disease (1-3-vessel disease, n = 164; 6.60 (4.1-18.3) vs. 7.80 (3.6-29.1) nmol/l, p = 0.0012), whereas only a slight tendency toward lower neopterin levels was found in the group without atherosclerosis (6.90 (5.1-16.0) vs. 7.60 (4.0-18.5) nmol/l, p = 0.17). In patients with coronary atherosclerosis, neopterin concentrations were lower in smokers (n = 105) compared to nonsmokers (7.20 (3.6-29.1) vs. 7.90 (4.4-18.6) nmol/l, p < 0.02), confirming previous observations. However, use of statins was associated with lower neopterin levels in both nonsmokers and smokers (6.70 (4.1-18.3) vs. 7.60 (3.6-29.1) nmol/l, p < 0.05, and 6.20 (5.2-16.0) vs. 7.80 (4.4-18.6) nmol/l, p < 0.05, respectively). Overall, similar serum neopterin concentrations were found in patients with coronary atherosclerosis and those without. In accordance with their antiinflammatory effects, the use of statins is associated with lower neopterin levels in patients undergoing elective coronary angiography. Clin Chem Lab Med 2003; 41(10):13141319
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June 1, 2005
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During carotid endarterectomy (CEA) the internal carotid artery is cross-clamped for a period of several minutes. This maneuver may cause cerebral hypoperfusion and/or impairment of the blood-brain barrier (BBB) even in cases where clinical signs are absent. The aim of the present study was to examine whether such alterations could be detected by monitoring the cerebral marker S-100B protein concentrations during and after CEA in the serum. Twentyfive consecutive patients (17 M/8 F, mean age: 64.2 years, range 47-79 years) undergoing elective CEA at our department were studied. All of these patients were without perioperative neurological deficit. Intraoperative samples were collected from internal jugular and peripheral venous blood: 1) before carotid cross-clamping; 2) immediately before declamping; 3) after clamp release. Postoperative samples were taken from peripheral blood at 6 and 24 h, respectively. S-100B was assayed in sera using an immunoluminometric technique. During carotid crossclamping, S-100B protein concentrations in the ipsilateral jugular serum significantly (p < 0.02) increased to pre-clamp values. After declamping, however, S-100B returned to the baseline level. No differences were seen between the responses of hypertensive and normotensive patients. There was no correlation between carotid occlusion time and S-100B protein concentrations. In the peripheral venous serum no significant changes in S-100B concentrations were detected during or after CEA. We presume that the elevation of S-100B protein concentration during CEA in patients with no neurological deficits indicates the transient opening of the BBB elicited by carotid cross-clamping. Clin Chem Lab Med 2003; 41(10):13201322
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June 1, 2005
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Prolidases I and II were highly purified from human erythrocytes. The effects of various amino acids, MnCl 2 and mercaptoethanol, on these two enzymes were investigated. Normal prolidase II was very labile in the absence of MnCl 2 or mercaptoethanol. The activity of prolidase II was maintained at about 76% by preincubation with MnCl 2 ; it was then activated up to 140% by treatment with mercaptoethanol for 60 minutes at 37 °C. Normal prolidases I and II showed the highest activity against glycylproline or methionylproline in the presence of MnCl 2 . The activity of prolidase I against glycylproline was enhanced strongly by glycine and MnCl 2 , but not activated in the absence of MnCl 2 . The activity of prolidase II against methionylproline was enhanced three-fold in the presence of glycine and MnCl 2 , but its activity against glycylproline was very low even in the presence of MnCl 2 . A stronger enhancement of this activity was found in normal erythrocytes, and a lower level of this activity was found in erythrocytes of patients treated with glycine, MnCl 2 and mercaptoethanol compared to those treated with glycine and MnCl2. The activity of prolidase II against methionylproline in all erythrocytes, of normal humans and of patients, was strongly activated by the addition of glycine with MnCl 2 but suppressed by the addition of mercaptoethanol. Clin Chem Lab Med 2003; 41(10):13231328
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June 1, 2005
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The measurement of insulin-like growth factor-I (IGF-I) has become an essential tool for diagnosing growth hormone deficiency and acromegaly, as well as for monitoring the efficacy of treatment in these disorders. The latter aspect gains significance in the light of epidemiological studies which indicate a relationship between IGF-I levels and the incidence of certain malignancies. We aimed to evaluate the performance of widely implemented IGF-I assays by testing four representative, commercially available immunoassays. Thus, four parallel determinations of the IGF-I levels of 427 healthy blood donors aged between 18 and 79 years were conducted. Apart from divergent performance criteria, the assays also differed systematically. These differences were, however, linear and of lower magnitude among the lower ranges. We conclude that despite the wide variance among commercially available IGF-I assays, which principally involve assayspecific normative data, each of the implemented assays was robust and thus an appropriate tool in the diagnostic workup of growth hormone deficiency in adult life, when IGF-I levels are low. Clin Chem Lab Med 2003; 41(10):13291334
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June 1, 2005
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While pathophysiology of elevated cytokines is well delineated, reference values for children are unknown, although they may vary physiologically with age and differ from those of adults. Between June and November 2001, interleukin (IL)-6, IL-10 and tumor necrosis factor-α (TNF-α) concentrations from blood samples of 79 healthy children in six different age groups (group I: 03 months; group II: 4-12 months; group III: 13-24 months; group IV: 25-36 months; group V: 37-48 months; group VI: 49-60 months) were measured with ELISA. TNF-α was within 2.2-3.5 pg/ml in all groups with a trend toward higher values in groups II and III (p = ns). IL-6 was significantly lower in group III than in groups IV (p = 0.0165) and VI (p = 0.0147). IL-10 was within 3.35.5 pg/ml in all groups (p = ns). In regression analysis no correlation between age and cytokine concentrations was found. Although not statistically significant, IL-6 was lower and TNF-α higher than the adult reference values provided by the kit manufacturer. Although reference cytokine levels seem not agerelated during early infancy, IL-6 is significantly lower during the second year of life than later. In infants aged 5 years or younger, reference levels of IL-6 should be chosen lower, and those of TNF-α higher, than the adult reference values. Clin Chem Lab Med 2003; 41(10):13351339
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June 1, 2005
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The aim of the present study was to analyze, on a double-blind basis, the relationships between the apolipoprotein(a) (apo(a)) gene and protein size polymorphisms in healthy volunteers (n = 99) and patients with premature myocardial infarction (n = 91). Apo(a) genotypes were determined by pulse-field electrophoresis and phenotypes were separated by sodium dodecyl sulfate-agarose gel electrophoresis. Results showed that phenotyping overestimated apo(a) size with respect to genotyping (mean (SD) = 3.7 (3.4) kringle units; p < 0.001) in subjects with a double-band genotype, although both measurements were highly correlated (r = 0.83; p < 0.001). We also observed that the protein band in subjects with a single-band phenotype was related more closely to the smallest allele than to the largest allele band. The correlation of plasma lipoprotein(a) (Lp(a)) concentration was stronger with the phenotype than with the genotype. We hypothesize that posttranslational modifications in the apo(a) molecule may be the most plausible explanation for the discrepancies observed. In conclusion, the present study highlights the dissimilarities between phenotyping and genotyping methods for the measurement of apo(a) size and suggests that laboratories should carefully consider these relationships and the transfer of results between such methodologies. Clin Chem Lab Med 2003; 41(10): 13401344
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June 1, 2005
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Polymorphisms in cytochrome P450 CYP3A4 and multidrug resistance (MDR)1 genes coding for the important drugmetabolising CYP3A4 and the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) are poorly documented in the Portuguese population. In this study we have determined the frequencies of CYP3A4 and MDR1 alleles in Portuguese Caucasians. Both genes were simultaneously analysed as these genes are known to be frequently co-induced and their products to show a pronounced overlap of substrates. CYP3A4 A-392G (CYP3A4*1B), T673C (CYP3A4*2) and MDR1 T-129C, G2677T and C3435T single nucleotide polymorphisms (SNPs) were analysed in 100 individuals from the southern region of the country. We observed a frequency of 4.0% for CYP3A4*1B, not significantly different from that reported on other Caucasian European populations. CYP3A4*2 was found at an allele frequency of 4.5%, constituting the first report of the presence of this allele outside the Finnish population. Significant differences were found concerning the MDR1 C3435T SNP frequency (64.5%) compared with other European populations, while no differences were found concerning G2677T (47.5%) or T-129C (5%) SNPs. Linkage between the C3435T and G2677T SNPs was observed, although not as evidently as documented in other Caucasian populations. No preferential associations were detected between CYP3A4 and MDR1 alleles. Clin Chem Lab Med 2003; 41(10):13451350
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June 1, 2005
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Lipoprotein lipase is the rate-limiting enzyme in the lipolysis of plasma triglyceride-rich lipoproteins. We studied six variants (T-93G, D9N, N291S, PvuII, HindIII and S447X) in the lipoprotein lipase (LPL) gene in 309 nondiabetic patients with angiographically assessed coronary artery disease and in 197 controls in a southern Brazilian population of European descent. The HindIII H-allele was associated with lower triglycerides (p < 0.01) and higher high-density lipoprotein cholesterol (p = 0.03) levels, and the S447X mutation was associated with lower triglyceride levels (p < 0.01) in males, but not females. No other significant lipid associations were observed. Haplotypes were derived from these two sites (HindIII/S447X), and carriers of H-S and H-X haplotypes showed lower triglycerides (p < 0.01) and increased highdensity lipoprotein cholesterol (p = 0.01) levels when compared to the H+S haplotype in males. In this gender, the H-X haplotype was associated with a protective effect (OR = 0.36, 95%CI = 0.13-0.97) for significant disease (≥60% of luminal coronary stenosis), even controlling for other classical risk factors. Clin Chem Lab Med 2003; 41(10):13511356
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June 1, 2005
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Venous thromboembolism (VTE) is a multi-factorial disease involving numerous genetic and environmental risk factors. In this study we investigated the occurrence and the risk associated with factor V Leiden, hyperhomocysteinemia and low folate and vitamin B 12 levels in young patients with thrombosis. We studied 78 patients (33 females/45 males, mean age 33 years) with a history of thrombosis in a lower limb. Additionally, 98 healthy subjects (45 females/54 males, mean age 44 years) were included. Serum levels of homocysteine (Hcy), folate and vitamin B 12 were assayed. Factor V Leiden and methylenetetrahydrofolate reductase (MTHFR) C677T mutations were investigated in all subjects. Factor V Leiden was highly prevalent in the patients (39% heterozygous, 10% homozygous vs. 6.3% heterozygous in controls). An increase in the risk of idiopathic VTE was associated with Hcy levels >15.2 µmol/l (odds ratio, OR = 2.83), folate <15.1 nmol/l (OR = 7.49) and vitamin B 12 <182 pmol/l (OR = 11.97). Low levels of folate or vitamin B 12 were independently and strongly associated with the risk of VTE in a multivariate model (OR for idiopathic thrombosis = 16.44 and 10.76, respectively). Twenty patients (53%), carriers of factor V Leiden, had low levels of vitamin B 12 , compared to 28% of patients who were noncarriers of the mutation (p = 0.03). In contrast, none of the control carriers of the mutation had a low level of vitamin B 12 . The risk of VTE associated with lower levels of vitamin B 12 and folate was stronger than that introduced by elevated Hcy levels. The increased risk of VTE, accompanied by factor V Leiden, may be related to confounding environmental factors. Clin Chem Lab Med 2003; 41(10):13571362
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June 1, 2005
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Cardiac troponin I (cTnI) is a sensitive and specific biochemical marker of myocardial damage. We assessed the analytical performance of the Vidas® Troponin I assay (Biomerieux). Controls and serum pools were used to determine the precision, analytical sensitivity and linearity; 97.5 and 99.5 percentiles concentrations were determined from the reference population. Fifty corresponding samples of serum and plasma (lithium-heparin) were tested and the results compared. The in vitro stability of serum and plasma samples was assessed at 20 °C, 4 °C and -20 °C, respectively. Samples of serum were used to assess the agreement between the Vidas® Troponin I method and the revised Dimension RxL cTnI method (Dade-Behring). The total imprecision (CVs) was 13.1-5.2% for concentrations ranging between 0.25 and 19.8 µg/l cTnI. The lower detection limit was <0.1 µg/l. The upper reference limit (97.5 and 99.5 percentiles) was 0.11 µg/l and 0.12 µg/l, respectively (CV >10%). The assay was linear up to 21 g/l. The concentrations in lithium-heparin plasma were higher compared to those of the matched serum samples. The study of the agreement between the Vidas and Dimension RxL cTnI assays showed a total concordance of 96% with a bias value of -0.042. The Vidas® Troponin I test is a fast, precise and sensitive method for the determination of cTnI. Clin Chem Lab Med 2003; 41(10): 13631368
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June 1, 2005
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The concentration of homocysteine (Hcy) rises rapidly after the collection of blood. This feature requires blood to be collected into the anticoagulants EDTA or heparin and the plasma to then be immediately separated; alternatively, the blood may be kept on ice and centrifuged within 1 hour. The use of chemical preservatives has been proposed as a means of stabilising Hcy levels in whole blood after collection. The objective of this study was to determine whether the commonly available fluoride-oxalate (Fl-Ox) and sodium citrate (Na-Cit) containers could stabilise Hcy levels in blood. Our results showed that when blood was collected into potassium EDTA (K-EDTA) tubes, Hcy levels rose from initial levels, on standing at room temperature (~25 C), by an average of 21% after 3 hours and 32% after 5 hours. The initial Hcy levels of blood collected into Fl-Ox and Na-Cit containers, however, were lower, at averages of 89% and 91%, respectively, compared to that of the same samples when collected into K-EDTA tubes. Hcy in these samples subsequently rose on standing, and after 5 hours was, on the average, 10 and 13% higher, respectively, compared with the initial levels in K-EDTA tubes. We conclude that Fl-Ox and Na-Cit do not stabilise Hcy in blood after collection and should not be used as preservatives. Clin Chem Lab Med 2003; 41(10):13691372
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June 1, 2005
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Learning curves have been described for different health technologies, mainly new surgical or diagnostic procedures, but learning curves for a new hospitals laboratory procedures have not been systematically studied. To monitor the timeliness (turnaround time) of stat tests from the Emergency Department as a marker of laboratory quality and to address the issue of a learning curve for procedures performed in a new hospital laboratory, we employed a computerized system for collecting data of turnaround time (from order entry to result verification) on stat tests from the Emergency Department of a newly opened (July 24, 2000) 471-bed general hospital. The data collection operates without user intervention. We evaluated the turnaround times of stat complete blood count and biochemistry tests from August 2000 to December 2001. Results show that it took 6 to 12 months before the turnaround times reached a plateau, we believe that this is the learning curve of a new hospital laboratory. Computer-generated turnaround times for Emergency Department stat tests appear to be a useful tool for monitoring the quality of laboratory tests and can demonstrate the learning curve of a new hospital laboratory. Clin Chem Lab Med 2003; 41(10):13731378
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