Volume 44, Issue 1

In human breast cancer, estrogen receptor-α (ERα), progesterone receptor (PR) and human epidermal growth factor receptor (ERBB2) status are currently determined using different techniques. We propose to assess the mRNA expression of these three clinically relevant markers using a unique technique, real-time nucleic acid sequence-based amplification (NASBA). Gene expression of hormone receptors was analyzed and compared to the cytosolic functional protein content as determined with a ligand binding assay (LBA), while ERBB2 mRNA expression was compared to quantitative PCR and ELISA. We observed that the three markers are significantly overexpressed at the mRNA level in positive tumors, as measured by DNA- or protein-based techniques. Biostatistical analysis of the receiver operating characteristic (ROC) curve demonstrated high concordance between NASBA and LBA [area under the curve (AUC) for ROC of 0.899] and showed that ERα status could be predicted using the molecular assay with a sensitivity of 72.7% and a specificity of 93.5%. Similar results were obtained for PR (AUC ROC 0.938, sensitivity 75.3%, specificity 100%). Moreover, excellent concordance was observed between NASBA, quantitative PCR and ELISA with respect to ERBB2 (AUC ROC 0.92, sensitivity 90%, specificity 89.7%; and AUC ROC 0.98, sensitivity 100%, specificity 91.5%, respectively). These results suggest that NASBA is well suited for assessing ER, PR and ERBB2 status in breast tumor samples. This approach is rapid, highly sensitive and a standardized method that could be complementary to the existing techniques, especially for small tumors.

Background : Raised levels of plasma cell-free DNA have been detected in various patient groups, including trauma patients. We hypothesized that plasma DNA is increased in burn patients and may represent an objective indicator of burn severity and have predictive as well as prognostic significance. Methods : This was a prospective clinical study with full ethical approval. With informed consent, blood samples were collected from 28 burn patients within 24h of injury and from 12 control subjects. Plasma cell-free DNA was measured by real-time quantitative polymerase chain reaction (PCR) assay for the β-globin gene. Descriptive analysis, non-parametric data comparison tests (Mann-Whitney) and correlation tests (Spearman rank) were performed on the data. Results : Samples were taken at a mean time of 5.7h after injury from 13 patients with flame/flash burns and 15 patients with scalds. Median plasma DNA levels in the control, scald and flame/flash burn patient groups were 287, 648 and 2685 kilogenome-equivalents/L, respectively. Plasma DNA levels correlated with the length of hospital stay, but not with admission to the intensive care unit (ICU) nor the length of ICU stay. DNA levels correlated with the burn surface area (Spearman rank r=0.54, p=0.04) and the number of operations needed (Spearman rank r=0.55, p=0.03) for scalds, but not for flame/flash burns. Conclusions : Plasma DNA is increased after burn injury and is significantly correlated with some outcome measures, including the length of hospital stay. DNA levels are higher in flame/flash patients than in scald patients; the difference may provide an objective indication of burn depth and inhalation injury.

Recently a revolutionary technique for quantitative PCR determination was introduced in diagnostic laboratories. To determine the influence of technical variability on the reliability of the quantitative assay, it is crucial to use External Quality Assurance (EQA) programs. An EQA program was developed in Italy to check the analytical performance of real-time PCR procedures based on Taq-Man™ probes. This article suggests a new statistical approach to discriminate, using a bivariate technique, laboratory performance that appears to be questionable, by separately considering the two main features of the standard curve: analytical sensitivity and efficiency. Furthermore, specific indexes to evaluate the impact of these two features on the determination of the initial number of molecules are given to help to improve the assay procedure.

Background : Increased homocysteine (Hcy) blood levels are correlated with vascular and neurological problems. Aim : The aim of the present study was to investigate erythrocyte membrane acetylcholinesterase (AChE) activity in subjects with the MTHFR C677T genotype in relation to Hcy. Methods : Blood was obtained from 22 individuals with the MTHFR C677T genotype before and after folic acid supplementation and once from controls (n=30). Plasma folate, vitamin B 12 and total antioxidant status (TAS) were measured with commercial kits, Hcy by a HPLC method and membrane enzyme activity spectrophotometrically. Results : In MTHFR C677T carriers, AChE activity was significantly higher (4.20±0.12 × mg protein) and decreased to normal levels (3.14±0.10 × mg protein; p<0.001) after therapy. TAS differed slightly before and after treatment. Hcy levels were significantly higher before (22.4±2.8μM) compared to after (12.1±2.0μM; p<0.001) therapy and compared to controls (10.5±2.5μmol/L; p<0.001). In an in vitro study, incubation of Hcy-activated membrane AChE from controls with phenylalanine resulted in restoration of activity, but failed to reverse the stimulated enzyme from hyperhomocysteinaemic MTHFR C677T subjects before therapy. Alanine incubation protected the enzyme from Hcy activation in controls. Conclusions : Increased membrane AChE activity may be due to high Hcy levels. In vitro, phenylalanine reversed the Hcy activation of the membrane enzyme from controls and alanine protected it from Hcy action.

It has been reported that the dimeric isoform of the enzyme pyruvate kinase M2 was overexpressed in various solid tumor cells. Hence, it was suggested that circulating levels of the so-called tumor M2-pyruvate kinase (Tu M2-PK) could be used as a tumor marker for monitoring systemic therapies of various solid tumors. We analyzed its validity as a tumor marker by comparing plasma levels of Tu M2-PK in patients with different non-malignant diseases to levels in healthy individuals and in patients with hematological diseases. Plasma levels of Tu M2-PK were measured using an ELISA assay in a total of 284 patients. The mean Tu M2-PK concentration of 32 U/mL was significantly higher in the group of patients with hematological malignancies (n=121) (p<0.001). However, 37% of healthy individuals (n=63) and 44% of patients with non-malignant diseases (n=100), especially patients with an acute inflammatory reaction (67%), were found to have elevated levels of Tu M2-PK using a cutoff level of 15U/mL. The specificity was 59% and the sensitivity was 51%. There was no significant correlation between the prevalence of a hematological malignancy and positive Tu M2-PK result. Thus, our data imply that Tu M2-PK is not a useful tumor marker for hematological malignancies and solid tumors, as a significant number of false positive results were detected in healthy individuals and patients with non-malignant diseases.

Identification of genetic polymorphisms as risk factors for complex diseases affecting older people can be relevant for their prevention, diagnosis and management. The –1131T→C polymorphism of the apolipoprotein A-V gene ( APO A-V ) is tightly linked to lipid metabolism and has been associated with increased triglyceride levels and familial dyslipidemia. The aims of this study were to analyze the allele and genotype frequencies of this polymorphism in a Brazilian elderly population and to investigate any association between the polymorphism and major morbidities affecting elderly people. This polymorphism was investigated in 371 individuals, aged 66–97years, in a Brazilian Elderly Longitudinal Population Study. Major morbidities investigated were: cerebrovascular diseases (CVD); myocardial infarction (MI); type 2 diabetes; hypertension; obesity; dementia; depression; and neoplasia. DNA was isolated and amplified by PCR and its products were digested with restriction enzyme Tru1 I. T and C allele frequencies were 0.842 and 0.158, respectively. Our population showed allele frequencies that were similar to European and Afro-American and different from Asiatic populations. Genotype distributions were not within Hardy-Weinberg equilibrium only for the obesity subject sample. On the other hand, a significant association between the C allele and obesity in the presence of CVD×depression interaction was observed. Logistic analysis showed no association of the polymorphism with each morbidity group. Therefore, the C allele in elderly Brazilian subjects may represent a risk factor for these morbidity interactions, which may lead to better comprehension of their pathophysiology.

Fibronectin is a known chemoattractant for several cell types that play a role in the wound healing process, including fibroblasts, endothelial cells and macrophages. It also generates a scaffold that allows attachment of other extracellular matrix components. Large amounts of fibronectin have been detected in atherosclerotic plaques, suggesting that it may play a role in the pathogenesis of atherosclerosis. To examine the possible involvement of fibronectin in the etiology of atherosclerotic coronary heart disease, we analyzed four polymorphisms in the human fibronectin gene and determined the plasma fibronectin levels in patients with coronary heart disease (n=109) and age- and gender-matched controls (n=123) in Chinese Han people. No significant positive association was observed between these polymorphisms and coronary heart disease. The levels of circulating plasma fibronectin, however, were significantly lower in patients with coronary heart disease (mean±SD 245±87mg/L) compared with controls (354±88 mg/L) (p<0.001). The odds ratio (OR) for plasma fibronectin was 0.94 in a multivariate unconditional logistic regression model (OR=0.94, 95% CI 0.91–0.96, p<0.001). We conclude that, in our population, the four fibronectin gene polymorphisms detected are not associated with clinical coronary heart disease. Our data suggest that low circulating fibronectin levels might be a new marker of coronary heart disease.

The associations of serum calcium and phosphorus concentrations as well as other cardiovascular risk factors were investigated in relation to the existence and severity of coronary heart disease (CHD) in 260 clinically stable, angiographically defined CHD patients aged 40–70years. The subjects were classified as CHD + cases if one or more coronary arteries had a significant stenosis (≥70%) and CHD − controls if there was no stenosis (≤10%) in any artery. The severity of coronary occlusion was scored on the basis of the number and extent of lesions, as normal, mild, moderate or severe. Fasting serum concentrations of electrolytes, lipids and (apo)lipoproteins were determined. The concentrations of serum total calcium (2.41±0.14 vs. 2.33±0.22mmol/L, p≤0.05), albumin-corrected calcium (2.33±0.25 vs. 2.23±0.25 mmol/L, p≤0.01), phosphorus (1.32±0.21 vs. 1.25±0.17mmol/L, p≤0.007) and the ion product of calcium and phosphorus (3.16±0.58 vs. 2.91±0.50, p≤0.0001) were significantly higher in the CHD + compared to the CHD − group. Patients with CHD compared with controls had increased serum levels of triglyceride, total cholesterol, low-density lipoprotein-cholesterol (LDL-C), apolipoprotein B (apoB), lipoprotein(a) [Lp(a)] and decreased serum levels of high-density lipoprotein (HDL)-C and apoAI. Multiple logistic regression analysis showed strong and significant association between diabetes mellitus (odds ratio, OR=5.24, p≤0.0001), male gender (OR=8.84, p≤0.0001), Lp(a) (OR=1.014, p≤0.006), hypertension (OR=2.61, p≤0.02), apoB (OR=1.031, p≤0.001), age (OR=1.055, p≤0.003), phosphorus (OR=2.438, p≤0.01), albumin-adjusted calcium (OR=1.532, p≤0.05), cholesterol (OR=1.009, p≤0.05) and the occurrence of CHD. On the basis of bivariate correlation analysis, serum-adjusted calcium was positively correlated with the levels of cholesterol (r=0.285, p≤0.0001), LDL-C (r=0.320, p≤0.0001), Lp(a) (r=0.173, p≤0.005), apoB (r=0.237, p≤0.0001), LDL-C/apoB ratio (r=0.180, p≤0.007), apoAI (r=0.181, p≤0.003) and inversely to HDL-C (r=−0.146, p≤0.02) and HDL-C/apoAI ratio (r=−0.263, p≤0.0001). Serum phosphorus concentration was a significant correlate of triglyceride (r=0.199, p≤0.001) and Lp(a) (r=0.129, p≤0.04). The results demonstrated that serum calcium and phosphorus are associated with the prevalence and severity of CHD, probably through correlation with atherogenic lipids and (apo)lipoproteins. Serum calcium and phosphorus and their ion product were also independent risk factors for CHD.

Urinary calcium levels in women with mild preeclampsia, severe preeclampsia and eclampsia were evaluated in this study. We collected 24-h urine samples from 35 mild preeclamptic (Group 1), 30 severe preeclamptic (Group 2), and 17 eclamptic patients (Group 3). The control group (Group 4) consisted of 35 healthy pregnant women. Serum levels of total calcium and creatinine, and urinary calcium were measured. These values were compared in the four groups. The mean maternal age and parity were similar in all groups. There were no statistically significant differences in the serum levels of total calcium and creatinine (p>0.05). Urinary calcium excretion in patients with preeclampsia and eclampsia was significantly lower than in controls (p<0.0001). Urinary calcium levels between mild preeclampsia and severe pre-eclampsia, and severe preeclampsia and eclampsia were similar (p>0.05), but were lower in eclampsia than in mild preeclampsia (p<0.05). In conclusion, urinary calcium excretion is reduced in patients with severe preeclampsia or eclampsia. However, the decrease in urinary calcium excretion cannot be used to identify the severity of preeclampsia, or to predict impending eclampsia.

The aim of this study was to develop an in vitro re-mineralization model in human serum. For this purpose a commercially produced demineralized human bone matrix (DBM) was incubated in samples of human serum pools obtained from two physiologically different groups. The first group consisted of young males and the second of older females. After incubation periods of 4 and 7days at 37°C, changes in the levels of calcium and inorganic phosphate content of the serum and DBM samples were measured. The results of the study showed that the change in mineral content of serum and DBM samples in both study groups was statistically significant. The decrease in serum calcium content and increase in DBM inorganic phosphate content were significant in the young group for longer incubation times. In the older group, both serum calcium and inorganic phosphate decreased and DBM mineral content increased for the same incubation time. When the two physiological groups were compared, statistically significant differences were identified for changes in mineral levels in both serum and the DBM samples. These data indicate that the mineral content of human serum decreases and that of DBM increases when these two materials are incubated together. These changes provide evidence for the re-mineralization of DBM. The model described here could also detect a difference in re-mineralization capability between two different groups of human sera.

Serum amyloid A (SAA), a plasma precursor of reactive amyloid deposits, is a multigene product. SAA1 and SAA2, with primary structures that are 93% identical (98 of 104 amino acids), behave as acute phase proteins, as demonstrated by their increasing levels in plasma. Heretofore, it has been understood that SAA1 predominates and functions as an isotype in plasma. However, accurate measurements differentiating the two isotypes have not been reported. In this study, using monoclonal antibodies specific for SAA1, we developed an enzyme-linked immunosorbent assay (ELISA) for SAA1. The levels and ratios of SAA1 in total SAA (TSAA) were investigated in healthy subjects and patients with rheumatoid arthritis (RA). The SAA1/TSAA ratio was 74±12% and 77±12% in healthy subjects and RA patients, respectively. In RA patients, the ratios were not influenced by SAA1 genotype, which has been proposed to affect plasma SAA values. The kinetics of SAA1 in inflamed patients undergoing hemodialysis was found to be parallel with total SAA and C-reactive protein. Finally, this study confirmed that SAA1 is a major isotype of acute phase SAA and may determine total SAA values. This specific assay could be used in the evaluation of SAA behavior in several clinical conditions.

Studies were carried out in 183 non-dialyzed, 123 hemodialysis, 81 continuous ambulatory peritoneal dialysis and 35 post-transplant patients and in 103 healthy subjects as a reference group. Lipids and apolipoprotein (apo)AI and apoB were determined using Roche kits. An anti-apoB antibody was used to separate apoB-containing apoCIII and apoE-triglyceride-rich lipoprotein (TRL) in the non-high-density lipoprotein (non-HDL) fraction from apoCIIInonB and apoEnonB in the HDL fraction in four groups of patients with chronic renal failure (CRF) and healthy subjects. Multivariate linear regression analysis was used to investigate the relationship between triglyceride (TG) or HDL-cholesterol (HDL-C) concentrations and lipoproteins. Dyslipidemia varied according to the degree of renal insufficiency, the type of dialysis and therapy regime in CRF patients. Lipoprotein disturbances were manifested by increased TG, non-HDL-C and TRL concentrations, and decreased HDL-C and apoAI concentrations, whereas post-renal transplant patients showed normalization of lipid and lipoprotein profiles, except for TG levels and total apoCIII and apoCIIInonB. The present study indicates that CRF patients have disturbed lipoprotein composition, and that hypertriglyceridemia and low HDL-C concentrations in these patients are multifactorial, being secondary to disturbed lipoproteins. The method using anti-apoB antibodies to separate apoB-containing lipoproteins in the non-HDL fraction from non-apoB-containing lipoproteins in HDL can be used in the diagnosis and treatment of patients with progression of renal failure or atherosclerosis. The variability of TG and HDL-C concentrations depends on the variability of TRL and cholesterol-rich lipoprotein concentrations, but the decreases in TG and increases in HDL-C concentrations are caused by apoAI concentration variability. These relationships, however, need to be confirmed in further studies.

Low levels of serum testosterone typically found in women and children cannot be reliably measured by immunoassay. We developed a simple and sensitive method using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Sample preparation involved protein precipitation of serum (1.0mL) with acetonitrile containing the internal standard (testosterone- d3 ) followed by liquid extraction with methylene chloride. The chromatographic cycle per specimen was 10min. The performance was evaluated according to the CLSI EP10-A2 protocol. Within- and between-run imprecision was 20.9%, 2.29% and 1.80%, and 1.81%, 3.58% and 2.97% at mean concentrations of 0.17, 14.1 and 28.8nM, respectively, with no apparent carryover. The method was linear from 0.21 to 53.1nM and the analytical recovery was 100.5–106.2% across the concentrations tested. There was no interference observed from other steroids that were tested. Correlation using de-identified patient specimens with a commercial HPLC-MS/MS method showed a slope of 0.991, an intercept of −0.017 and a correlation coefficient (R 2 ) of 0.998 by linear regression over concentrations ranging from 0.21 to 16.7nM. In conclusion, we report here an HPLC-MS/MS method suitable for clinical measurement of serum testosterone.

Phenylketonuria is an inherited metabolic disease, which is characterized by an increased level of serum phenylalanine. Quantitative measurement of phenylalanine in the serum of phenylketonuria patients is necessary to confirm the disease, and to distinguish phenylketonuria from other forms of hyperphenylalaninemia. In this study, we report a rapid and inexpensive micro-assay for simultaneous detection and quantitative measurement of serum phenylalanine on dry blood-spots. Analysis of the standard curve showed a broad linear range for phenylalanine from 120 to 1800μmol/L. Application of this method, the standard Guthrie bacterial inhibition assay and a high-performance liquid chromatography (HPLC) method for analysis of 34 samples from phenylketonuria patients and control samples produced comparable results, with the regression equation Y=0.994X+0.996. The advantage of this method over the Guthrie bacterial inhibition assay is its ability to measure serum phenylalanine quantitatively without false positive results. The method was successfully applied to dried blood-spots, serum and whole blood. The cost per sample is approximately 20–50 US cents, which is much less than for HPLC and commercial enzyme kits. The method can be automated, and is thus suitable for neonatal and mass screening for phenylketonuria, especially in developing countries where funding is a limiting factor.

The aim of the present study was the investigation of N-terminal pro-brain natriuretic peptide (NT-proBNP) in the pediatric population. This is essential for adequate monitoring and classification of pediatric patients with heart disease, but no consistent data are available yet. In addition, the comparability of two commercially available NT-proBNP assays and the inter-laboratory variability for the most suitable one were assessed. For this purpose, 408 subjects (1–29years) were included. NT-proBNP was determined with a non-competitive electrochemiluminescent immunoassay (Roche NT-proBNP; n=402) and a competitive enzyme-immunoassay (Biomedica NT-proBNP; n=402). Inter-laboratory variability was evaluated for the Roche assay by stepwise inclusion of four and 11 centers throughout Germany, respectively. Roche NT-proBNP ranged from 5.0 to 391.5ng/L, with higher values for younger children. The 97.5th (75th) percentile curve ranged from 319.9ng/L (231.2 ng/L, 1–3years) to 114.9ng/L (53.3 ng/L, 18years). In contrast, Biomedica NT-proBNP ranged from 253.7 to 7602.8 ng/L, with no significant age dependency. The mean difference between the assays was 1649.7ng/L (95% confidence interval 1546.3–1753.1ng/L). Inter-laboratory variability ranged from 6.5% to 3.8%, covering a range from 51.3 to 6618.1ng/L. The assay seems to influence the interpretation of resulting NT-proBNP values and therefore has to be chosen carefully. For the monitoring and classification of pediatric patients with congenital heart disease, age-based NT-proBNP values should be used.

A new quality control model using performance goals based on biological variation in External Quality Assurance Schemes (EQAS) is described. The proposed model aims to use assay (analytical) CV A , bias and total error available from participation in EQAS to describe assay performance using minimum, desirable and optimum quality specifications based on biological variation. The model provides further analysis of EQAS data and should be useful in better management of laboratory quality control, as it provides further information that can facilitate trouble-shooting. Additionally, it can help in evaluating the performance of current and proposed new laboratory methods by applying a unifying system if different EQAS are used to cover a range of analytes.

Background: Hemoglobin A 1c (HbA 1c ) has been used in controlled trials for the last 10years but has never been evaluated in clinical practice as an effective parameter for clinical outcome. We investigated the trend for glycemic control over 11years in one county of 350,000 citizens. Methods: We studied 226,382 HbA 1c-DCCT-aligned from 39,455 patients in whom routine monitoring for diabetes was initiated in 1993, 1996, or 2001. The trend in glycemic control was investigated in groups by probit plots, and in individual patients by target plots. Results: From 1993 to 2001, the number of HbA 1c measurements increased three-fold. The number of new monitoring series increased from 0.22% to 0.27% of the county population, and the number of patients monitored using HbA 1c as a proxy for diabetes increased from 0.5% to 1.5%. A proportional reduction in high HbA 1c concentrations of 5% was identified in the 1993 group, compared to 15% in the 1996 group, and 20% in the 2001 group. The percentage of patients with diabetic first HbA 1c experiencing normalization increased from 8% to 30% for males and from 9% to 24% for females (1993–2001). The percentage of HbA 1c concentrations that were not normalized decreased from 78% to 53% for males and from 83% to 59% for females (1993–2001). The median HbA 1c at initiation of monitoring decreased from 8.7% in 1993 to 7.5% in 2001 (p<10 −5 ). The number of normal first HbA 1c results in monitoring series increased from 7% to 17% for males and from 8% to 22% for females. Up to 10% of subjects developed diabetic concentrations during monitoring. Conclusion: On average, patients with diabetic first HbA 1c concentrations (≥6.62%) showed an improvement in glycemic control from 5% in 1993 to 20% in 2001. High concentrations were easiest to reduce. In patients with originally diabetic HbA 1c levels, 66% on average showed improved glycemic control in the 2001 series compared to 50% in the 1993 series. An average of 6% (1993) vs. 9% (2001) with originally normal HbA 1c levels showed an upward trend inHbA 1c levels. Median HbA 1c at initiation of monitoring decreased from 8.7% in 1993 to 7.5% in 2001 (p<10 −5 ). The incidence of new cases was constant.

We examined the clinical usefulness of antepartum clinical characteristics, along with measures of glucose tolerance, in Dutch multiethnic women with gestational diabetes mellitus (GDM) for their ability to predict type 2 diabetes within 6months of delivery (early postpartum diabetes). The present study comprised a cross-sectional 5-year investigation (1998–2003) of a consecutive series of 168 women with GDM identified by a two-stage protocol at 16–33weeks of gestation. The following data were collected for all women: age and gestational age at entry into the study; prepregnancy body mass index (BMI); ethnicity; obstetric and clinical history, including the onset of early postpartum diabetes; pregnancy outcome; level of fasting C-peptide; and glycemic parameters of 50-g 1-h glucose challenge test and 100-g 3-h oral glucose tolerance test (diagnostic OGTT). We used receiver operating characteristic (ROC) curve analysis to test the clinical usefulness of the glycemic parameters. A total of 11 women (6.5%) developed early postpartum diabetes. Apart from family history of diabetes (p=0.052), anthropometric, maternal, and neonatal clinical parameters showed no association with early postpartum diabetes in univariate analyses. The level of fasting glucose, and both the glucose challenge test and diagnostic OGTT post-load glucose levels and glucose areas were associated with early postpartum diabetes. ROC curve analysis identifiedall three glucose challenge-test parameters, including fasting glucose concentration, as poor diagnostic tests, with a positive predictive value of ∼22%, whereas the positive predictive value associated with the area under the diagnostic OGTT curve increased progressively over monitoring time from 20.6% to 100%. Using a 3-h OGTT glucose area threshold of 35.7mmol·h/L resulted in 100% sensitivity and 100% specificity, identifying the 11 women who developed early postpartum diabetes. In summary, we can conclude from the present analysis that early postpartum diabetes is rare in GDM women (6.5%), and that the clinical usefulness of the total area under the diagnostic 3-h OGTT is superior to all other glycemic parameters for detecting early postpartum diabetes.

Measurement of estradiol is useful in women undergoing ovarian stimulation for in vitro fertilization and embryo transfer (IVF-ET). The analytical performance of a new two-step estradiol assay (ADVIA Centaur ® estradiol-6 III from Bayer Diagnostics) was evaluated in 41 sera from 11 women undergoing ovarian stimulation. The results were compared to those obtained with two radioimmunoassays (RIAs; RIA Estradiol Immunotech IM 1663 from Beckmann Coulter and Coat-A-Count Estradiol from Diagnostic Products Corporation) and with one chemiluminescent immunoassay (CLIA; ADVIA Centaur ® estradiol-6). The ADVIA Centaur ® estradiol-6 III assay was the most sensitive assay, with a functional sensitivity of 55pmol/L. Within- and between-run coefficients of variation calculated for the new ADVIA Centaur ® assay ranged from 3.3% to 9%, which was better than the precision obtained for the other assays. A dilution test showed serum interferences when estradiol was measured in non-diluted samples. No statistical difference was observed between the estradiol results obtained in diluted sera with the new two-step ADVIA Centaur ® assay and those measured with the Immunotech RIA and the other CLIA. In conclusion, this new, two-step estradiol assay performed on the ADVIA Centaur ® system displays suitable sensitivity, precision and intermethod agreement with the Immunotech RIA for the measurement of serum estradiol concentrations in women undergoing ovarian stimulation and IVF-ET. For correct linearity, estradiol measurement should be performed on diluted samples.

The EC4 Syllabus for Postgraduate Training is the basis for the European Register of Specialists in Clinical Chemistry and Laboratory Medicine. The syllabus: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. The syllabus is not primarily meant to be a training guide, but on the basis of the overview given (common minimal programme), national societies should formulate programmes that indicate where knowledge and experience is needed. The main points of this programme are: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. Knowledge in biochemistry, haematology, immunology, etc.; Pre-analytical conditions; Evaluation of results; Interpretations (post-analytical phase); Laboratory management; and Quality insurance management. The aim of this version of the syllabus is to be in accordance with the Directive of Professional Qualifications published on 30 September 2005. To prepare the common platforms planned in this directive, the disciplines are divided into four categories: Indicates the level of requirements in postgraduate training to harmonise the postgraduate education in the European Union (EU); Indicates the level of content of national training programmes to obtain adequate knowledge and experience; Is approved by all EU societies for clinical chemistry and laboratory medicine. Knowledge in biochemistry, haematology, immunology, etc.; Pre-analytical conditions; Evaluation of results; Interpretations (post-analytical phase); Laboratory management; and Quality insurance management. General chemistry, encompassing biochemistry, endocrinology, chemical (humoral), immunology, toxicology, and therapeutic drug monitoring; Haematology, covering cells, transfusion serology, coagulation, and cellular immunology; Microbiology, involving bacteriology, virology, parasitology, and mycology; Genetics and IVF.