Band 43, Heft 3

Stress doses of hydrocortisone are known to have immunomodulatory effects in patients with hyperdynamic septic shock. The prognosis correlates with the presence and severity of septic encephalopathy. However, neurological evaluation is influenced by the use of analgesia sedation during artificial ventilation. The objective of this study was to demonstrate the effect of stress doses of hydrocortisone during the initial phase of human septic shock on the serum values of the neurospecific protein S-100B in comparison to the inflammation markers interleukin (IL)-8 in serum and polymorphonuclear (PMN) elastase in plasma. A total of 24 consecutive patients, who met the American College of Chest Physicians/Society of Critical Care Medicine criteria for septic shock, were enrolled in this prospective, randomized, double-blind, single-center trial. The severity of illness at recruitment was graded using the Acute Physiology and Chronic Health Evaluation II and the Simplified Acute Physiology Score II scoring systems. Multi-organ dysfunction syndrome was described by the Sepsis-related Organ Failure Assessment (SOFA) score. All patients were prospectively randomized to receive either stress doses of hydrocortisone or placebo. Hydrocortisone was started in 12 patients with a loading dose of 100mg and followed by a continuous infusion of 0.18mg/kg/h for 6days. Median S-100B serum levels of the hydrocortisone group decreased from 0.32ng/mL at study entry to 0.07ng/mL 6days later without significant differences compared to the placebo group. Initial IL-8 serum levels were significantly higher in the hydrocortisone group up to 12h after study entry, and significantly decreased from 715 to 17pg/mL at the end of the observation period. Median PMN elastase plasma levels were not affected by hydrocortisone infusion. Patients with initial S-100B serum levels >0.50ng/mL revealed significantly higher SOFA scores up to 30h, IL-8 serum levels up to 12h, and PMN elastase plasma levels up to 36h after study entry than those patients with ≤0.50ng/mL. These effects were independent of the amount of fluid correction for hemodilution. Starting S-100B, IL-8 and PMN elastase values of the hydrocortisone group were within the ranges already known in patients with out-of-hospital cardiac arrest or severe traumatic brain injury. Stress doses of hydrocortisone resulted in a significant reduction in IL-8 serum, but not in S-100B serum and PMN elastase plasma concentrations in patients with hyperdynamic septic shock. For the first time, a similar extent of S-100B increase in serum of septic patients at the time of diagnosis was shown as reported for cardiac arrest or severe traumatic brain injury.

Studies in healthy humans have shown that consumption of conjugated linoleic acid (CLA) significantly reduced very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) blood concentrations. We propose that decreased concentrations are due to the inhibition of VLDL production and secretion [measured by apolipoprotein B100 (apoB100)] from the liver. To investigate the effects of a mixture of CLA isomers on VLDL metabolism, HepG2 liver cells were incubated for 24h with 50μmol/L of the different fatty acids. Effects of CLA were compared to a saturated fatty acid (palmitic acid), an n-6 fatty acid (linoleic acid) and no treatment (control). HepG2-cell apoB100 levels were measured using Western blotting. ApoB100 secretion was significantly decreased in cells treated with CLA (44%, p<0.005) compared to control cells and those enriched with palmitic acid. Treatment of cells with CLA also decreased intracellular cholesterol levels. Collectively, these results demonstrate that CLA reduces apoB100 production and secretion compared to saturated and polyunsaturated fatty acids, possibly by limiting the availability of free cholesterol (required for apoB100 production). A reduction in apoB100 production in the body would decrease the levels of VLDL and atherogenic LDL and thus reduce the risk of developing cardiovascular disease.

The drug-metabolizing cytochrome P450 (CYP) enzyme CYP2D6 is involved in the metabolism of several clinically important drugs. So far more than 50 different CYP2D6 allelic variants have been described, and thus there is an increased need for routine high-performance liquid chromatography (HPLC) methods for the evaluation of the functional implication of CYP2D6 polymorphism. Debrisoquine is metabolized to 4-hydroxydebrisoquine by CYP2D6, and therefore it has been used widely to determine the hydroxylation capacity of the enzyme. The aim of the present study was to develop a simple, accurate HPLC method with ultraviolet detection for the measurement of debrisoquine and 4-hydroxydebrisoquine in urine for evaluation of the relationship between CYP2D6 enzyme activity and genotypes. For the HPLC determination, a C18 extraction column was used with a flow rate of 0.8mL/min and detection at 210nm. The compounds were eluted from the column in less than 10min. Coefficients of variation at all concentrations were less than 4% for both compounds. The debrisoquine/4-hydroxydebrisoquine ratio (debrisoquine metabolic ratio) was determined in a panel of 16 Caucasian healthy volunteers with zero (poor metabolizers), one, two or more than two (ultrarapid metabolizers) CYP2D6 active genes. Significant correlation (p<0.05) between the number of CYP2D6 active genes and the hydroxylation capacity of the enzyme was found. The present HPLC method was simple, fast and accurate, and thus will be useful for the evaluation of CYP2D6 hydroxylation capacity in pharmacogenetic studies.

Maternal iodine deficiency can compromise the thyroid status of the mother, fetus and newborn child. Therefore, it is important to assess the iodine excretion level of groups of pregnant women. In this study we aimed to determine iodine intake in pregnancy using a recently reported automated kinetic method for urinary iodine determination. Urinary iodine measurements of 123 pregnant women (18 first, 28 second and 77 third trimester) were carried out using a new automated kinetic assay based on the Sandell-Kolthoff reaction at 37°C and its kinetic measurement at 340nm in a random-access automated analyzer after ammonium persulfate digestion at 95°C in a water bath with ±0.1°C precision. Statistical analyses were carried out using SPSS software. Whole group, first trimester, second trimester and third trimester urinary iodine concentrations (mean±SD) in pregnant women were 1.13±0.81, 1.08±0.71, 0.86±0.58 and 1.27±0.87μmol/L, respectively. The urinary iodine concentration significantly increased with gestational age (p<0.05). We found that our study group was mildly iodine-deficient according to WHO criteria. Furthermore, the pregnant women were found to be mildly iodine-deficient in the first and third trimesters and moderately so in the second trimester. The only statistical difference was between second and third trimester values (p<0.05). Even though the increased iodine deficiency in the second trimester is not useful for early detection of iodine deficiency in pregnancy, the severity of this deficiency in the second trimester may lead to important effects on thyroid metabolism for both mother and fetus. Our study suggests that the iodine excretion of pregnant women living in iodine-deficient areas could be assessed using this fast and automated method.

Total butyrylcholinesterase activity (EC 3.1.1.8) was previously suggested as a marker for metabolic syndrome. The present study examined total butyrylcholinesterase activity and the relative and absolute activities of two butyrylcholinesterase electrophoretic bands (C 4/5 and C OF ) in 99 obese individuals (body mass index ≥30kg/m 2 ) presenting the CHE2 C5 − phenotype of the CHE2 gene. Anthropometric, hormonal and biochemical variables already associated with metabolic syndrome were also examined. The data from these obese individuals of the CHE2 C5− phenotype show that total butyrylcholinesterase activity and the absolute activities of the C 4/5 and C OF electrophoretic bands are associated with metabolic syndrome and with variables related to it. These butyrylcholinesterase activities do not behave as independent risk factors for metabolic syndrome, but can be considered as secondary markers for this syndrome in obese individuals with the CHE2 C5− phenotype.

The non-thyroidal illness syndrome (NTIS) is considered a transient and completely reversible phenomenon, but it has been shown that it may last for several days postoperatively after coronary artery bypass grafting (CABG) surgery. This study was undertaken to assess thyroid function 6 months after uncomplicated CABG. The thyroid profile was evaluated in 40 consecutive patients undergoing CABG preoperatively, at 0, 12, 48, and 120h postoperatively, and at 6-month follow-up. Triiodothyronine (T 3 ), free T 3 (FT 3 ), free thyroxine (FT 4 ) and thyroid stimulating hormone (TSH) were assayed using a microparticle enzyme immunoassay. T 4 and total serum thyroid hormone-binding capacity (T-uptake) were measured on the same samples using a fluorescence polarization immunoassay. Patients with severe systemic illness and patients treated with amiodarone were excluded. All patients were euthyroid at admission. Mean age was 67.4±9.0years. There were 31 (77.5%) men. Typical NTIS was observed in all patients, and the FT 3 concentration was still reduced by postoperative day 5 (p<0.0001). At 6-month follow-up, all patients were free from cardiac symptoms, and no new cardiac events were recorded. The thyroid profile was normal in 35 patients (87.5%). One patient (4.5%) had developed overt hypothyroidism. Two patients had isolated low T 3 and FT 3 levels with normal TSH. Two patients had moderately increased FT 3 levels with suppressed TSH. In most uncomplicated patients, thyroid function returns to normal 6months after CABG. However, we observed significant alterations of the thyroid profile in 5 out of 40 patients. Further studies are needed to define the long-term consequences of postoperative NTIS.

Oxidative stress is known to be involved in many human pathological processes. Although there are numerous methods available for the assessment of oxidative stress, most of them are still not easily applicable in a routine clinical laboratory due to the complex methodology and/or lack of automation. In research into human oxidative stress, the simplification and automation of techniques represent a key issue from a laboratory point of view at present. In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP), was detected in the plasma of chronic uremic patients. Here we describe in detail an automated version of the originally published microplate-based technique that we adapted for a Cobas Mira Plus clinical chemistry analyzer. AOPP reference values were measured in plasma samples from 266 apparently healthy volunteers (university students; 81 male and 185 female subjects) with a mean age of 21.3years (range 18–33). Over a period of 18months we determined AOPP concentrations in more than 300 patients in our department. Our experiences appear to demonstrate that this technique is especially suitable for monitoring oxidative stress in critically ill patients (sepsis, reperfusion injury, heart failure) even at daily intervals, since AOPP exhibited rapid responses in both directions. We believe that the well-established relationship between AOPP response and induced damage makes this simple, fast and inexpensive automated technique applicable in daily routine laboratory practice for assessing and monitoring oxidative stress in critically ill or other patients.

Urinary stone analysis is the most important diagnostic step after stone removal from the body. The methods employed for these analyses are based on diverse analytical principles. Chemical methods are used for detecting individual ions. Infrared spectroscopy is used for examining molecular structures, and X-ray diffraction for determination of the crystalline structure of a substance. Since 1980, a twice-yearly ring trials quality control survey has been on offer to examine the quality of urinary stone analyses. A summary of the results of 44 ring trials (1980–2001) has been compiled for individual pure substances and binary (two-component) mixtures. On average, 100 laboratories have participated in these ring trials. Initially, over 80% of the participants carried out their analyses using chemical methods. In 2001, this figure decreased to a mere 13%. In contrast, a progressive increase in the use of infrared spectroscopy was observed, up to 79% of all participants employed this method. X-Ray diffraction was only employed in a small number of specialised laboratories (5–9%). The chemical methods produced a very high proportion of errors (6.5–94%) with both the pure substances and binary mixtures, whereas high error rates for infrared spectroscopy and X-ray diffraction were confined to individual substances only. Due to the poor results in the ring trials, the majority of laboratories stopped using chemical analysis, which is now considered to be obsolete. Regarding mixtures, error rates of over 10% also occurred with infrared spectroscopy and X-ray diffraction. Ring trials are indispensable for the quality management of urinary stone analysis.

The performance of suitable secondary reference material for the use of trueness control of six routinely measured clinical enzymes in the Dutch External Quality Assessment (EQA) scheme is described. The reference material of choice was selected using the split-patient-sample between-field method (twin study) design as described in an earlier study of the Calibration 2000 project in The Netherlands. This material, which was proven to be commutable for all wet chemistry systems, was implemented as the national enzyme calibrator. It consisted of a cryo-protected lyophilised serum with additions of recombinant human enzymes. Various batches of the frozen version of this material without cryo-protection additive, called native EQA samples, were used in the general EQA scheme for performance evaluation. The results of Calibration 2000 calibrated and non-Calibration 2000 calibrated laboratories were compared for both the regular (spiked with non-human enzymes) and native EQA samples in terms of precision and bias with established reference method values for the native samples. The regular samples showed mean between-laboratory CV ranges for all six enzymes involved (low–high) of 5.5–10.3% for the non-calibrated users vs. 4.6–10.8% for the calibrated users. For the native samples these respective ranges were 5.2–9.9% vs. 2.2–4.9%. Without exception, the group of Calibration 2000 calibrated users showed the lowest bias against the reference method values. Regular EQA samples (spiked with non-human enzymes) showed poorer performance than native samples and are not suitable for accuracy assessment purposes, the main aim of EQA schemes. Native samples that are commutable should be used for trueness control in current EQA schemes.

C-Reactive protein has been associated with several complications of pregnancy. The aims of the present study were: (1) to evaluate a turbidimetric immunoassay for the measurement of C-reactive protein; and (2) to investigate the chronological changes of the levels of this protein from preconception throughout normal pregnancy and its relationship with variables associated with preconception and pregnancy outcome. Inter-assay imprecision was <5% for C-reactive protein >1mg/L and 18% at a mean value of 0.33mg/L. The limit of detection was 0.10mg/L. The method was linear between 0.10 and 30mg/L. There were no observed interferences from jaundice, hemolysis, lipemia or paraproteinemia at the levels studied. There was good agreement with the nephelometric method. A total of 39 women were studied at preconception, at 8, 20 and 32weeks of pregnancy, and in labor. Preconception C-reactive protein concentration was 1.17±0.18mg/L and increased (p<0.001) throughout pregnancy up to 5.69±0.82mg/L. Body mass index at preconception and weight gain during pregnancy were the main factors associated with this increase in C-reactive protein.

An important number of patients dependent on renal dialysis prefer peritoneal dialysis to hemodialysis. In the case of peritoneal dialysis, the glucose polymer icodextrin is frequently added to the dialysis fluid as an osmotic agent, since this polymer is able to maintain an osmotic gradient across the peritoneal membrane longer than monomeric glucose, leading to a prolonged effective ultrafiltration time. It was previously shown that icodextrin is partly able to enter the blood via the lymphatic system, where hydrolysis to glucose oligomers such as maltose and maltotriose occurs. The presence of these oligomers in the blood appears to cause significant overestimations of the glucose values in several point-of-care (POC) glucose analyzers, with potentially dramatic consequences. This effect has been investigated for a series of POC glucose analyzers, both by analyzing the blood of peritoneal dialysis patients and by an in vitro investigation of the quantitative effects of maltose and maltotriose. In particular, POC analyzers utilizing the bacterially produced enzyme glucose dehydrogenase seem to lack glucose specificity.

The predominant technique used to draw blood for laboratory testing is a conventional straight needle attached to an evacuated tube system. However, alternative tools might be advantageous in exceptional circumstances. The use of butterfly devices has been traditionally discouraged for reasons of costs and due to the high risk of obtaining unsuitable samples, but there is no convincing evidence to support the latter indication. The purpose of this study was to compare results of hematological and clinical chemistry testing, after drawing blood into evacuated tubes, employing either a traditional 21-gauge straight needle or a 21-gauge butterfly device with 300-mm-grade polyvinyl chloride tubing. Blood samples and complete sets of data were successfully obtained for 30 consecutive outpatients. Of the 43 hematological and clinical chemistry parameters measured, means for paired samples collected by the two alternative drawing techniques did not differ significantly, except for serum sodium, white blood cells and platelets counts. Bland-Altman plots and limits-of-agreement analysis showed mean bias of between −7.2% and 1.7% and relative coefficients of variation ranging from 0.2% to 21.2%. The 95% agreement interval in the set of differences was acceptable and was mostly within the current analytical quality specifications for desirable bias. The rate of hemolysis in plasma was not statistically different between the two collection techniques. Taken together, the results of the present investigation suggest that, when a proper technique is used and within certain limitations, the butterfly device may be a reliable alternative to the conventional straight needle to draw blood for laboratory testing.

The aim of the study was to evaluate the clinical efficiency of soluble transferrin receptor and transferrin receptor-ferritin index (sTfR/logF) in the diagnosis of iron deficiency anemia, as well as the differential diagnosis of iron deficiency anemia and anemia in rheumatoid arthritis. The study included 96 patients with anemia and 61 healthy volunteers as a control group. In healthy subjects there were no significant sex and age differences in the parameters tested. The study results showed these parameters to be reliable in the diagnosis of iron deficiency anemia, as well as in the differential diagnosis of iron deficiency anemia and anemia of chronic disease. The results indicate that sTfR/logF could be used to help differentiate coexisting iron deficiency in patients with anemia of chronic disease. Receiver operating characteristic analysis showed a higher discriminating power of transferrin receptor-ferritin index vs. soluble transferrin receptor in the diagnosis of iron deficiency anemia, as well as in the differential diagnosis between iron deficiency anemia and anemia of chronic disease. In patients with anemia in rheumatoid arthritis, the parameters tested showed no significant differences with respect to C-reactive protein concentration. These results suggested that the parameters tested are not affected by acute or chronic inflammatory disease.

Imipenem (thienamycin formamidine), a broad-spectrum β-lactam antibiotic, is always used in combination with cilastatin in order to avoid the premature breakdown of imipenem by renal tubular dipeptidase. As this dipeptidase also hydrolyzes the glutathione metabolite cysteinylglycine, the therapeutic asso-ciation of imipenem and cilastatin might cause an accumulation of the aminothiol cysteinylglycine. We demonstrate here that when patients are treated with imipenem-cilastatin, their plasma levels of cysteinylglycine are significantly and specifically increased, while cysteine levels are decreased and homocysteine levels are unaffected. We conclude that antibiotic treatment using imipenem-cilastatin induces important metabolic changes that should not remain unrecognized.

The number of consultant clinical chemists (NCCC) in the 15-nation European Union (EU) (Austria, Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, The Netherlands, Portugal, Spain, Sweden, United Kingdom) has been ascertained. These data were analysed in relation to several established national parameters, including demographics, gross domestic product (GDP), cost of healthcare, cost of in vitro diagnostic (IVD) testing and the number of physicians and pharmacists. Large differences in the population-corrected costs of IVD testing (range ∼2.4-fold) and NCCC (range ∼30-fold) were observed between the countries, which could not be satisfactorily explained by any of the parameters assessed. The differences in IVD testing and NCCC might reflect different practices in laboratory medicine across the EU, but could not be estimated independently. In recognition of the different scope of laboratory medicine practised under the title of clinical chemistry, a simple staffing model was derived in an attempt to give a better estimate of the appropriate number of consultant laboratory medicine specialists. This model allocated a fixed number of laboratory specialists per million inhabitants for the five disciplines: clinical chemistry, 10; haematology, 10; serology, 7.5; microbiology, 12.5; and blood banking, 2. The staffing model also allowed for the contribution of the primary care sector by including one full-time consultant laboratory medicine specialist for each small private laboratory and two extra consultant laboratory specialists per million inhabitants where there are not large numbers of private laboratories. Application of the model to the available data helped to reduce the variation observed in the primary analysis of NCCC (range ∼9-fold) but still revealed important differences between countries. These differences could arise from the poor quality of published data as much as from true differences in laboratory medicine practice. We conclude that a more sophisticated analysis of laboratory practice and of all professionals working in laboratory medicine disciplines would be required before any conclusions could be drawn about relative staffing, efficiency or cost effectiveness. The staffing model derived is a first step towards objective estimation of the number of consultant laboratory specialists in the EU.