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June 1, 2005
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June 1, 2005
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In humans the iron status is influenced by environmental and genetic factors. Among them, the genetic polymorphism of the hemoglobin (Hb)-binding plasma protein haptoglobin (Hp) has been shown to affect iron turnover. The best known biological function of Hp is capture of free Hb in plasma to allow hepatic recycling of heme iron and to prevent kidney damage during hemolysis. In healthy males, but not in females, the Hp 2-2 phenotype is associated with higher serum iron, higher transferrin saturation, and higher ferritin than Hp 1-1 and 2-1. Moreover, serum ferritin correlates with monocyte L-ferritin content, which is also highest in Hp 2-2 subjects due to endocytosis of multimeric Hb-Hp 2-2 complexes by the recently identified Hb scavenger receptor CD163 in macrophages. This iron delocalization pathway, occurring selectively in Hp 2-2 subjects, has important biological and clinical consequences. The Hp polymorphism is related to the prevalence and the outcome of various pathological conditions with altered iron metabolism such as hemochromatosis, infections, and atherosclerotic vascular disease.
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June 1, 2005
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An increasing incidence of male infertility has been noted over the past few decades. This adds urgency to the need to develop immunohistochemical markers for better evaluation of testicular biopsies. We provide evidence that a histopathological evaluation performed according to morphological criteria and assisted by immunohistochemical staining on consecutive sections enhances the sensitivity and accuracy of the diagnosis based on testicular biopsies from infertile men.
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June 1, 2005
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Different types of cancer are naturally resistant to many anticancer drugs. Additionally, these tumours develop acquired drug resistance, which includes the classical multidrug resistance (MDR) accompanied by the synthesis of P-glycoprotein, a member of the superfamily of ATP-binding cassette (ABC) transporters. Furthermore, atypical MDR is mediated by several different, some unkown, mechanisms. To overcome chemoresistance problems, antineoplastic drugs are often combined with other modes of therapy, e.g. hyperthermia, where good response has been reported in several experimental tumour models and in advanced cancer patients. The success of this combined anticancer treatment may be limited by an increase in chemoresistance and thermoresistance. A model system to study resistance phenomena is the use of chemoresistant and thermoresistant cancer cell lines. We have established chemoresistant cancer cell lines (gastric and pancreatic carcinoma, fibrosarcoma, melanoma) and now thermoresistant cell lines derived from gastric and pancreatic carcinoma cells and their counterparts that were resistant towards daunorubicin (classical MDR) and mitoxantrone (atypical MDR). Using proteomics, in this paper we evaluate the drug resistance of chemoresistant melanoma cells (parental cell line MeWo and sublines exhibiting drug resistance towards etoposide, cisplatin, fotemustine and vindesine) as a paradigm for analysis of drug resistance phenomena. Additionally, we investigate heat resistance and the interaction of chemoresistance and thermoresistance to identify common pathways using the parental and drug resistant stomach cancer cell lines EPG85-257, EPG85-257RNOV, EPG85-257RDB and their thermoresistant counterparts. Possible implications of differential protein expression will be discussed.
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June 1, 2005
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We present an indicator of aging based on eight quantitative variables, measured during the periodical health examination offered by French social security to the general population in France. The used sample derived from the population examined each year. We selected a reference group of 24,510 adults older than 25 years (non-smokers, drinking less than 44 g alcohol per day, with body mass index less than 35, and not using medication). A multiple regression analysis by gender was used to select significant variables to predict age from all quantitative variables measured during the health examination. The resulting biological age could be calculated from the measured variables and the aging indicator was defined as the difference between the calculated age and the chronological age. By definition, this indicator is independent of age in the reference population and gives an indication of the difference between an individual's status in the process of aging and the mean status of people of the same chronological age. Associations between this indicator and life style habits such as tobacco and alcohol use, and occupational status were observed. A geographical analysis also exhibited significant variation over France. Such an indicator can be used at the individual level as a pedagogical tool to explain the observed individual health status. It is also of interest for the epidemiological studies, where it could contribute to a better understanding of the aging process and associated factors.
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June 1, 2005
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Cardiac troponins (cTnT and cTnI) are useful tools for risk stratification in patients with unstable angina. However, their value in patients with renal failure has been questioned. In this study, we determined cTnT and cTnI at 3-month intervals during 9 months in 97 chronic renal failure (CRF) patients treated with hemodialysis. cTnT was measured using a third generation immunoassay and cTnI by fluorimetric immunoassay with a detection limit similar to that of cTnT (0.01 μg/l). In the renal patients without coronary heart disease (CHD(−) group), cTnT was more frequently elevated above cut-off for acute myocardial infarction (AMI) (up to 21.6%) than cTnI (no patient). In the absence of CHD, cTnT levels were positively correlated to age, and more than half of the CHD(−) patients aged over 60 years had cTnT levels above the upper reference limit (URL) of 0.04 μg/l (0.059±0.042 μg/l). cTnI increased with age in parallel to cTnT but mean levels did not exceed the URL of 0.08 μg/l in the CHD(−) patients aged over 60 years (0.036±0.031 μg/l). In the patients with documented cardiac events (CHD(+)) we found higher troponin levels than in the CHD(−) patients of the corresponding age, but for cTnI the differences between CHD(+) and CHD(−) patients were significant in the patients aged ≤60 years only (0.049±0.054 vs. 0.019±0.018 μg/l, p<0.05). For cTnT, the differences between patients with and without coronary events also tended to be less important in the eldest patients. There was a significant correlation between cTnI and cTnT levels in the CHD(−) and in the CHD(+) groups. Changes in the plasma levels of cardiac troponins are common in hemodialysis patients in the absence of CHD, and advanced age appears to amplify these changes. The reason could be that most hemodialysis patients with advanced age have subclinical lesions and demonstrate release characteristics of troponins that compare to those in patients with symptomatic coronary events. Therefore, it will be important to analyze troponin elevations above the URL or above the cut-off concentration for AMI in asymptomatic renal patients in relation to prognosis.
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June 1, 2005
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Point-of-care testing is concerned with the immediacy of response, primarily because of the need to act in a life-threatening crisis or to provide counsel in the ongoing management of a chronic disease. There are both clinical, operational and economic benefits that can accrue from this testing modality which may be observed from several perspectives – the patient, the clinician, the healthcare provider, the healthcare purchaser and society. Thus point-of-care testing can improve the management of chronic diseases such as diabetes, compromised coagulation status and epilepsy – both in terms of optimisation of, and compliance with, therapy. There are also life-threatening crises that can be averted by rapid provision of test results. Each of these scenarios can lead to more efficient use of healthcare resources.
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June 1, 2005
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It has been suggested that evidence-based laboratory medicine (EBLM) could help to improve the pertinence and accuracy of medical guidelines. In order to demonstrate this, we have used an EBLM approach ( i.e. a systematic review) to examine three recently published guidelines that gave quite conflicting recommendations regarding the use of laboratory variables in the management of primary non-small cell lung cancer patients. In recommending the routine measurement of serum albumin, and, to a lesser extent, that of serum calcium in the pre-therapeutic prognostic evaluation of the advanced disease, the American Thoracic Society and the European Respiratory Society were probably correct with regard to calcium but perhaps mistaken regarding albumin. Some of the recommendations of the European Group on Tumour Markers regarding the usefulness of routine measurements of tumour markers (carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), tissue-polypeptide antigen (TPA)) in the pre- and/or post-therapeutic prognostic evaluation can also be criticised. In addition, the latter society as well as the Société de Pneumologie de Langue Française did not even try to list laboratory variables, others than tumour markers, that would be useful to stratify patients participating in clinical trials ( i.e. lactate dehydrogenase (LDH), albumin, calcium, blood cell count, etc. ), and the laboratory variables listed by the two former societies were probably not the right ones in this context: in particular LDH and tumour markers (fragments of cytokeratin 19 (Cyfra 21–1), tissue-polypeptide-specific antigen (TPS), neuron-specific enolase (NSE)) were not mentioned. Most, if not all of these discrepancies in the current medical practice guidelines might have been avoided had an EBLM approach been used by the authors.
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June 1, 2005
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Angiotensin-converting enzyme (ACE) is a well known zinc-metallopeptidase that converts angiotensin I to the potent vasoconstrictor angiotensin II and that degrades bradykinin, a powerful vasodilator, both for regulation of vascular tone and cardiac functions. Other natural substrates of ACE were identified broadening the functions of this enzyme within different physiological contexts such as neuronal metabolism, hematopoiesis, digestion and reproduction. Synthetic substrates were developed for the determination of ACE activity in various biological fluids, mostly human plasma, for the diagnosis of sarcoidosis and other granulomatous diseases. After the successful use of captopril, the first ACE inhibitor in the treatment of hypertension, a number of molecules were synthesized and used in the treatment of congestive heart failure and for preventing cardiac impairment after myocardial infarction. This class of antihypertensive drugs benefited from structural data on carboxypeptidases active site, as ACE molecule has not yet been crystallized. In the last two decades ACE gene has been cloned that allowed the identification (i) of two isoenzymes, one called somatic ACE resulting from gene duplication and primarily expressed in endothelial cells, and the other, called germinative or testicular ACE, resulting from the transcription in the male reproductive system of a more simple gene, (ii) of an hydrophobic C-terminal peptide for membrane-anchoring and specifically cleaved by a metalloprotease to release soluble forms of both isoenzymes, and (iii) of several allelic polymorphisms, one of them consisting of an insertion/deletion (I/D) polymorphism in a short intronic Alu sequence that could account for half the variance in plasma ACE level and resulting in a large inter- individual variability; moreover this I/D polymorphism was proposed as a genetic marker for identifying individuals at high risk of ischemic heart disease and of anticipating in one individual the efficacy of the antihypertensive therapy, although conflicting data arose from the past decade literature. Moreover, ACE gene cloning has confirmed the expression of the enzyme in endothelial cell, in particular as an ecto-enzyme facing the vascular lumen, but not to the same extent with regard to the vascular origin of the cells. Plasma ACE in healthy subjects arises essentially from the endothelium. On the other hand, in granulomatous diseases where a local stimulation of macrophages leads to an abnormal ACE secretion, it can also be found in other biological fluids such as cerebrospinal and broncho-alveolar fluids. Low plasma ACE levels result from endothelium impairment such as in deep vein thrombosis or in endothelio-toxic anticancer therapies. Another cause of low, sometimes undetectable, plasma ACE levels is the use of an ACE inhibitor, but this is without any significance with regard to its clinical benefits. Albeit molecular cloning has provided a number of new details on ACE structure and function, many questions still remain, in particular about its tertiary structure including glycosylations, about its tissue-specific expression and regulation, and also about the exact significance of the I/D polymorphism in cardiovascular pathology including the pharmacogenomic field.
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June 1, 2005
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In idiopathic calcium urolithiasis the relationships between oxypurines, accompanying proteins and glucose in urine and plasma, and the associated metabolic activity (MA) are unknown. To establish whether MA is related to these parameters and to calcium oxalate crystallization, or whether it reflects a reaction of metabolism to systemic insults was the major goal of the work. One hundred fifty one males were studied in three trials: trial 1 (n=130 patients) and trial 2 (n=24 patients) were cross-sectional; trial 3 included 11 patients and 14 controls). Mean age was 46 years (trials 1 and 2) and 29 years (trial 3). In trial 1 the stratification was based on the median urinary oxypurine excretion, in trial 2 on the median plasma oxypurine concentration (below or above: Low and High subgroups). No dietary restrictions were imposed, but standardized ambulatory laboratory testing was carried out. MA was quantitated by a score. Established analytical methods were used, except for oxypurine measurement which was done by high performance liquid chromatography. Patients with kidney stones tended to be overweight (body mass index >25 kg/(m) 2 ) and to have fasting hyperglycemia. In trial 1 severe oxypurinuria, and especially severe xanthinuria, was associated with an increase in urinary pH, creatinine clearance, proteins, uric acid, malonedialdehyde (indicator of lipid peroxidation), systolic blood pressure, and with a decrease in plasma uric acid (synonymous with a decrease of antioxidant capacity). Tubular reabsorption of proteins and stone-forming substances was diminished but MA remained unchanged despite slightly increased calcium oxalate crystal growth. In trial 2 high adenosine and xanthine coincided with elevated systolic and diastolic blood pressure, high uric acid with high urinary malonedialdehyde, high summed oxypurines minus uric acid with an increase of diastolic blood pressure, glycemia and MA; urinary nitrate (indicator of systemic vasodilation) was unchanged. In trial 3 patients' oxypurinemia and proteinuria were normal, but body mass index, glycemia and insulinemia were increased. Urinary total protein, albumin and non-albumin proteins were positively predicted (multivariate regression analysis) by urinary xanthine, glucose and pH (trial 1); MA was positively (trial 3) or negatively (trial 2) predicted by urinary total protein. In calcium urolithiasis, a disorder of affluence, 1) oxypurinuria and proteinuria and oxypurinemia and MA appear causally linked, presumably via oxidant/antioxidant imbalance-induced renal tissue damage; 2) urinary proteins may act as inhibitors or promoters of stone-forming processes; 3) a stone-initiating role of impaired vasodilatation is conjectural; 4) overweight, obesity, mild glucosuria and hyperdynamic blood circulation are regular signs.
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June 1, 2005
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Fatty acid status in humans is usually related to plasma or red blood cell fatty acid profiles. The aim of the study was to explore whether a maternal deficiency in dietary essential fatty acids would differentially affect lipid fractions in several tissues of the offspring, including brain. Female Wistar rats were fed an essential fatty acid-deficient diet during 3 months before mating. The fatty acid composition of different lipid fractions was examined in maternal milk, and in plasma, red blood cells, liver, adipose tissue, cerebral cortex and hippocampus of the offspring using thin layer and capillary column gas chromatography. Lipid fractions from most tissues of deprived offspring showed a common fatty acid profile characterized by elevated 20:3 ω9/20:4 ω6 ratio, and decreased docosahexaenoic acid and arachidonic acid. However, arachidonic acid was not affected in brain, even though 22:5 ω6 was increased in phospholipids of cerebral cortex and hippocampus. The present results demonstrate different degrees of resistance to essential fatty acid deficiency in lipid fractions and tissues. This suggests a priority distribution of arachidonic acid to preferential areas and shows that blood phospholipid fatty acids do not exactly reflect brain phospholipid status.
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June 1, 2005
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We developed a universal liquid chromatographymass spectrometry (LC-MS) assay with automated online extraction to quantify simultaneously the immunosuppressants sirolimus, everolimus, tacrolimus, and cyclosporine. Whole blood (300 μl) plus 6 μl 32-desmethoxyrapamycin (1 ng/μl) as internal standard was treated with 600 μl methanol/0.2 M ZnSO 4 (80/20 v/v). After vortexing (30 s) and centrifugation (20000 g , 5 min) 50 μl of the supernatant were loaded on an extraction column, were washed by 0.35 ml/min water for 3 min and, after activation of a column-switching valve, were back-flushed by 0.25 ml/min methanol/ water (90/10 v/v) onto a C 18 analytical column. After 22 min the extraction column was washed for 2 min with methanol and for 3 min with water before starting the next run. Column temperatures were kept at 33 °C. Sodium adduct ions [M+Na] + ions were detected in the selected ion mode. For sirolimus, everolimus and tacrolimus the assay was linear from 0.3 to 200 μg/l and for cyclosporine from 5 to 1000 μg/l (all r 2 >0.999). Recovery of all immunosuppressants and the internal standard was >90% and in general, inter-day and intra-day precision was <10%. The simultaneous quantification of blood levels by LC-MS seems to be the method of choice in transplanted patients receiving multiple immunosuppressants.
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June 1, 2005
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Remarkably elevated levels of phospholipase A 2 (PLA 2 ) are measurable in human blood samples in cases of acute pancreatitis. The source of the enzyme was first thought to be exclusively the pancreas, but now it is generally accepted that two isoenzymes – the pancreatic PLA 2 , group I, and the extrapancreatic PLA 2 , group II – contribute to the raised activity. In contrast to the group II-PLA 2 , the pancreatic PLA 2 is heat-resistant for 1 hour at 60 °C. The catalytically inactive proenzyme of the pancreatic PLA 2 can be activated by trypsin. The aim of our study was to evaluate the diagnostic value of PLA 2 isoenzyme activity measurements to identify patients with severe complications in acute pancreatitis. Blood samples from patients suffering from acute pancreatitis were analyzed for catalytically active pancreatic PLA 2 on day 1 and 2 of hospitalization with a modified radiometric Escherichia coli -based PLA 2 assay. In 10 of 41 patients clearly elevated values of catalytically active, heat-resistant pancreatic PLA 2 (7.2 to 81.2 U/l) were observed. This group of patients was characterized by severe complications (necrotizing pancreatitis, shock, sepsis, respiratory problems) of which two patients subsequently died. Patients with low or undetectable activity (<7 U/l) of pancreatic PLA 2 recovered rapidly. According to these results the presence of catalytically active pancreatic PLA 2 in serum is associated with severe complications of acute pancreatitis. In contrast to total serum-PLA 2 , the catalytic concentration of pancreatic PLA 2 can serve as a prognostic marker in acute pancreatitis.
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June 1, 2005
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Serum carbohydrate antigen 15.3 (CA 15.3) and carcinoembryonic antigen (CEA) are currently employed in clinical practice as markers for breast cancer, particularly in the follow-up and therapy monitoring. However, the American Society for Clinical Oncology (ASCO) stated in its clinical practice guidelines for the use of tumour markers in breast carcinoma that neither CA 15.3 nor CEA are recommended for routine use in screening, diagnosis and surveillance after primary treatment, or in monitoring response to treatment, because current literature data are insufficient. Cytokeratin fragment 21.1 (CYFRA 21.1) assay detects a serum fragment of cytokeratin 19 (CK19) and is employed in the diagnosis and management of lung cancer, particularly of squamous cell histotype. Breast carcinoma has been demonstrated to express CK19 fragments in the primary and metastatic lesions and CK19 mRNA is detectable in peripheral blood from patients affected by breast cancer. We measured serum markers CYFRA 21.1, CEA and CA 15.3 in the sera from 212 females affected by histologically proven breast carcinoma. Patients comprised 96 individuals with untreated primary disease (54 stage I-II, 18 stage III and 24 stage IV), 30 regional (chest-wall and/or lymph-nodes) relapsing disease and 68 metastatic (haematogenous metastases) relapsing disease. Forty-eight patients previously treated by surgery and without any evidence of disease were enrolled to evaluate the role of serum markers in the monitoring for recurrence of the disease. One hundred healthy age-matched females and 65 patients affected by benign mammary gland disease (including 38 patients with mastopathy and 27 with fibroadenoma) were enrolled as controls. Serum levels of all markers increased from controls to patients affected by breast cancer, from stage I-II to stage IV of the breast cancer and from local to advanced recurrence. The comparison of diagnostic accuracy in the detection of primary and relapsing breast cancer showed no significant differences between markers. Univariate and multivariate survival analysis showed a significant statistically prognostic value for CA 15.3 and CYFRA 21.1 but not for CEA. However, the factors N and M were confirmed to be very strong predictors of the patients' survival. Finally, CEA and CYFRA 21.1 detected less recurrences than CA 15.3. In conclusion, our data show no significant improvement in the diagnosis, prognostic evaluation and follow-up of breast cancer by CYFRA 21.1 and CEA assays compared to CA 15.3 assay. Considering the ASCO statement on tumour markers in breast cancer, the CYFRA 21.1 assay should not be employed in clinical practice.
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June 1, 2005
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Hypocholesterolemia, which often accompanies infectious diseases has been suggested to serve as a prognostic marker in hospitalized patients. Even though patients with chemotherapy-induced leukopenia are at high risk of infection and mortality, only limited information is available on serum cholesterol levels in these patients. We therefore measured serum cholesterol levels in 17 patients with hematological malignancies during chemotherapy-induced neutropenia and correlated it with clinical outcome. Patients with fever (>38.5 °C) showed a significant decrease in serum cholesterol levels within 24 hours. Eight days after onset of the fever non-survivors had significantly lower serum cholesterol levels (median 2.09 mmol/l, range 0.49–2.79, n=6) compared to survivors (median 3.23 mmol/l, range 1.68–4.86, n=11). Cholesterol levels in survivors returned to baseline levels at the time of discharge from the hospital. At the onset of fever, serum levels of inflammatory cytokines interleukin-6, tumor necrosis factor (TNF) and soluble TNF receptors p55 and p75 were elevated in all patients, but only TNF and TNF receptor p75 levels were significantly different in survivors and non-survivors. Our data suggest that a decrease in serum cholesterol levels is a prognostic marker in neutropenic patients with fever. Release of inflammatory cytokines may in part be responsible for hypocholesterolemia in these patients.
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June 1, 2005
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Monitoring the ionized magnesium (Mg 2+ ) concentration in critically ill patients can prevent development of serious and potentially fatal complications. The analyzers KONE Microlyte 6 (KONE Instruments, Espoo, Finland) and NOVA CRT (NOVA Biomedical, Waltham, MA, USA) provide the discontinuous measurement of Mg 2+ and were evaluated in several studies. It was our objective to integrate the Mg 2+ -selective electrodes into a device for continuous on-line measurements. This device is suitable not only for research but also for a specific evaluation of electrode characteristics. It allowed us to compare the genuine electrodes and reference systems independently of their specific analyzers. Precision, accuracy, response time, limit of detection, drift and interferences were assessed by continuous flow-through measurements and discussed in comparison to the results of previous studies. The NOVA electrode proved to be superior regarding accuracy, sensitivity and selectivity, especially with respect to calcium. It was demonstrated that current commercial serum-like control materials were not appropriate for quality control of the assessed Mg 2+ -electrodes. However, despite the fact that the electrodes are commercially used for discontinuous measurements, both sensor types can be used for continuous on-line measurements in an extracorporeal circulation in a rat model. The NOVA electrode showed superior characteristics with this application as well. This study should also be understood as a contribution to the development of devices for on-line analyzers used in point-of-care-testing.
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June 1, 2005
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The professional duties of the specialists in clinical chemistry differ from country to country in Europe. One of the main goals of the Strategic Plan of the Forum of the European Societies of Clinical Chemistry and Laboratory Medicine (FESCC; IFCC-Europe) is to promote a high scientific and professional standard in the field of clinical chemistry and laboratory medicine in Europe. This can be stimulated by the knowledge of the local conditions in each country and by striving towards a strong and harmonised position in all the European countries. In order to enhance the knowledge of the managerial situation of the specialists in clinical chemistry in Europe, FESCC launched a survey in September 2000. This survey provides information about the position of the specialists in clinical chemistry in the various disciplines in the medical laboratories and in hospitals, and about the advisory tasks and the managerial education during the post-graduate training in clinical chemistry. Of the 35 FESCC member countries 33 have participated in the survey (94%). The results show a rather heterogeneous situation in Europe caused by the local historical developments, the differences in academic background and the relative numbers of private and physicians' office laboratories. Large differences exist between the European countries in the disciplines of laboratory medicine that are headed by a specialist in clinical chemistry. In the different countries the clinical chemistry laboratories are headed by specialists in clinicazl chemistry in between 20% and 100% of the laboratories. The haematology, immunology, microbiology, therapeutic drug monitoring, molecular biology and haemostasis laboratories and departments of blood banking are headed by specialists in clinical chemistry in between 0% and 100% of the laboratories. The responsibilities for the various managerial tasks of the specialists in clinical chemistry show no uniformity in Europe. In the majority of the countries the general management, the purchase of equipment and reagents and the education of technicians are in >90% the responsibility of the specialists in clinical chemistry. In most countries the majority of the specialists in clinical chemistry are members of the medical staff of the hospitals and have a position equivalent to the position of specialists in other medical disciplines. In some countries, however, it only holds true for the specialists with a medical background. In 79% of the countries the law regulates the profession of the specialists in clinical chemistry and in 60% of the countries the law regulates their position in the medical staff of the hospital. The advisory tasks to physicians, general practitioners and other users of laboratory tests are practised by >90% of the laboratories in 64% of the countries. Information is given directly to the patients by >90% of the laboratories in 30% of the countries. Only in a few countries laboratories give information to the public. The post-graduate training in clinical chemistry includes a managerial training in 58% of the countries, the study of information technology in 61% of the countries and an economy and/or a business administration study in 15% of the countries. In 27% of the countries no managerial education forms part of the post-graduate study in clinical chemistry. Harmonisation of the managerial aspects of the profession is one of the challenges for the European specialists in clinical chemistry. A European syllabus for post-graduate training could be helpful.
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