Volume 39, Issue 10

Urinary tract infection (UTI) represents a significant burden for the National Health Service. Extensive research has been directed towards rapid detection of UTI in the last thirty years. A wide range of microbiological and chemical techniques are now available to identify and quantify bacteria in urine. However, there is a clear and present need for near, rapid, sensitive, reliable analytical methods, preferably with low-running costs, that could allow early detection of UTI and other diseases in urine. Here we review the “state of the art” of current practice for the detection of bacteria in urine and describe the advantages of the recent “e-nose” technology as a potential tool for rapid, near-patient diagnosis of UTI, by sensing volatile organic compounds (VOCs).

Risk stratification is a key element of clinical management not only in the primary and secondary prevention, but also during the acute stages of cardiovascular disease. The current risk assessment algorithms in primary prevention are based on established risk factors: gender and age, cigarette smoking, the presence of hypertension and diabetes mellitus, and serum concentrations of total cholesterol, low-density lipoprotein (LDL)-cholesterol and high-density lipoproteincholesterol. However, many individuals who are assessed as “low risk” on the basis of traditional risk factors, still develop cardiac events. This article addresses current issues relevant to the assessment of cardiovascular risk. It emphasizes the potential importance of disturbed energy supply for atherogenesis, by introducing the concept of fuel transport (chylomicron, VLDL, and remnants) and overflow (LDL) pathways of lipid metabolism. It highlights the present lack of routine methods to monitor the fuel transport pathway. It considers the measurements of serum C-reactive protein and plasma fibrinogen as new additions to the cardiovascular risk factor profiles. Finally, risk stratification based on the traditional and the new risk factors is linked to that based on the markers of acute myocardial damage such as cardiac troponin I or troponin T. It is concluded that the combined use of the markers of myocardial damage and the “new” cardiovascular risk factors is the way ahead for the assessment of cardiovascular risk.

There is a growing pressure on clinical chemistry laboratories to conform to quality standards that require the evaluation and expression of the uncertainty of results of measurement. Nevertheless, there is some reluctance to accept the uncertainty concept in the analytical community due to difficulty in evaluating uncertainty in practice. For example, often the uncertainty of some uncertainty components is not known very well in clinical chemistry measurements, such as those associated with matrix effects or with the values of the calibrators. Moreover, it is not clear how to interpret uncertainty in relation to diagnostic criteria, reference ranges and other decision limits in clinical chemistry practice. Hence, the value of reporting the uncertainty of the measurement result is not obvious. In this paper it is suggested a relatively simple, logical procedure for evaluating measurement uncertainty based on the principles in the Guide for the Expression of Uncertainty of Measurement (GUM). The measurement process is partitioned into elements that are well known to the analyst, namely sampling, calibration, and analysis. The corresponding model function expresses the result of a measurement as the value obtained by the analytical procedure multiplied by the correction factors for sampling bias, for bias caused by the calibrators, and for other types of bias. Under normal conditions, when the measurement procedure is validated and corrected for all known bias, the expected value of each correction factor is one. The uncertainty that remains with regard to sampling, manufacturing of calibrators and other types of bias is combined with the analytical imprecision to yield a combined uncertainty of a result of measurement. The advantages of this approach are: (i) Data from the method validation, internal quality control and from participation in external quality control schemes can be used as input in the uncertainty evaluation process. (ii) The partition of the measurement into well-defined tasks highlights the different responsibilities of the clinical chemistry laboratory and of the manufacturer of reagents and calibrators. (iii) The approach can be used to harmonize the uncertainty evaluation process, which is particularly relevant for laboratories seeking accreditation under ISO 17025. The application of the proposed model is demonstrated by evaluating the uncertainty of a result of a measurement of prolactin in human serum. In the example it is shown how to treat the uncertainty associated with a calibrator supplied with a commercial analytic kit, and how to evaluate the uncertainty associated with matrix effects.

Liver transplantation is the only therapeutic option for patients with end-stage liver disease. Nitric oxide, a free radical produced from L-arginine, a potent vasodilator, also inhibits platelet adhesion and aggregation, reduces adhesion of leukocytes to the endothelium and suppresses proliferation of vascular smooth muscle cells. The inducible form of the nitric oxide synthase may generate large quantities of nitric oxide, and may be induced by the action of cytokines and lipopolysaccharides. Nitric oxide can be released from the hepatic vascular endothelium, platelets and Kupffer cells as a response to ischemia-reperfusion injury and circulatory shock. We analyzed the relationships between the levels of nitric oxide, hepatic enzymes and other clinical parameters (glucose, total proteins, total bilirubin, creatinine, albumin) obtained in serum samples before liver transplantation and every 48 h till day 15 in 15 patients aged 40 ± 13 years. Aspartate aminotransferase and alanine aminotransferase levels changed from high at the beginning, to almost normal at the end of the study, cholinesterase levels remained decreased throughout the study and nitric oxide remained high, never reaching normal values.

Carbohydrate-deficient transferrin (CDT) is widely accepted as screening test for excessive alcohol consumption. However, results from subjects with transferrin variants must be interpreted with caution since chromatography-based methods may give false-positive results. Furthermore, due to the co-elution in HPLC or the co-migration in capillary zone elctrophoresis (CZE) of the di- and trisialylated C transferrins with the tetrasialylated D peak, exact measurement of CDT is impossible in CD-variants. Therefore, in this study, we tried to offer a different solution, including only the asialo-D, asialo-C, monosialo-D, monosialo-C, disialo-D and trisialo-D transferrins in the CDT calculation and referring to a different cut-off value for CDT in transferrin CD-variants. Comparison of alcohol consumers with teetotalers demonstrated area under the receiver operating characteristic curve of 0.79 and 0.76 for carbohydrate-deficient transferrin, 0.71 and 0.71 for mean corpuscular volume and 0.51 and 0.68 for γ-glutamyltransferase in 43 subjects with transferrin CD-variants and 225 subjects with CC-phenotypes, respectively. Since false-positive carbohydrate-deficient transferrin results due to a transferrin CD-variant have major social implications, capillary electrophoresis-based or similar methods (HPLC, FPLC) should be preferred in populations carrying a high D-allele frequency.

A liquid chromatographic-mass spectrometric method for the determination of cortisol in serum using atmospheric pressure electrospray ionization and tandem mass spectrometry is described. During sample preparation, 150 μl of serum were deproteinized with methanol/zinc sulfate followed by on-line solid phase extraction employing column switching. Tri-deuterated cortisol was used as the internal standard. The following transitions were monitored: cortisol, 363>309 m/z; d3-cortisol, 366>312 m/z. The total run-time was 5 minutes. The method proved linear (0–500 μg/l; r=0.999), precise (total coefficient of variation between 5.0% and 3.2% at a mean cortisol concentration of 15.1 μg/l and 269 μg/l, respectively; n=16) and specific with regard to relevant endogenous and exogenous steroids.

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR in hepatitis C virus (HCV) genotyping were examined for informativeness and reliability. The analysis of HCV isolates included seven type 1 isolates, two type 2 isolates, and two type 3 isolates. We also analyzed five isolates that presented as mixed infections determined by type-specific PCR. Among mixed isolates, one isolate was 1a/1b and four isolates were 1b/3a. SSCP and LSSP-PCR were applied to the analysis of 5′ non-coding region of HCV (−289 to −5) that contains genotype-specific sequences. Direct cycle sequencing of this region determined sequence divergences that define genotype and sequence alterations within the same genotype. Optimized conditions for the SSCP analysis clearly distinguished between genotypes 1, 2 and 3. In addition, the SSCP analysis detected sequence variants within the same genotype. However, the SSCP analysis and DNA sequencing did not confirm the presence of mixed infections. LSSP analysis, not previously employed in HCV genotyping, enabled clear distinction between genotypes 1, 2 and 3, however, this method did not differentiate between sequence variants within a genotype. Importantly, the LSSP profile demonstrated distinction between mixed infection isolates.

Wilson's disease is an inherited autosomal recessive disorder of copper transport characterized by progressive copper accumulation in the liver and the central nervous system. The disease is caused by mutations in the ATP7B gene. Although many different mutations in this gene were described, a substitution of a histidine by a glutamine residue at codon 1069 (H1069Q) accounts for approximately 30–60% of all mutations in Caucasian patients. We describe a DNA-based method using fluorescence resonance energy transfer probes on the LightCycler for rapid determination of the common H1069Q mutation in the ATP7B gene. We screened 53 patients with Wilson's disease for the H1069Q mutation by the melting curve analysis. The reliability and discriminating power of this technique were documented by comparing results of the LightCycler assay with direct DNA sequencing. The protocol allows genotyping of 30 samples in less than 1 hour without a need for restriction enzyme digestion or gel electrophoresis.

Three groups of patients have been studied longitudinally for 24 months to analyze the role of hepatitis G virus (HGV) in hepatic disease. Group 1 consisted of 50 patients with non-B, non-C chronic hepatitis, group 2 consisted of 44 hemodialyzed patients, and group 3 consisted of 50 healthy blood donors. The presence of HGV RNA was detected by both reverse transcription-polymerase chain reaction (RT-PCR) and capillary zone electrophoresis (CZE). At the baseline visit the HGV RNA was detected in seven out of 50 patients with non-B, non-C chronic hepatitis, in two out of 44 hemodialyzed patients, and in three out of 50 healthy blood donors. HGV-infected hemodialyzed patients and HGV viremic blood donors had serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels within normal limits. During the follow-up period the two HGV-positive hemodialyzed patients and the three infected healthy blood donors did not show any sign of hepatic disease. There were no significant differences between HGV-positive patients in the three groups at the beginning and at the end of the follow-up. No considerable deterioration of general health conditions was observed on the basis of clinical and laboratory data in HGV-positive chronic hepatitis patients. Finally, HGV does not seem to be responsible for hepatic disease.

Glycoprotein 90K/MAC-2BP is a member of the scavenger receptor cystein-rich protein superfamily, which is thought to be involved in immune surveillance, defending the body against pathogens and cancer. 90K serum levels are elevated in patients with cancer of various origins and in viral infections, such as human immunodeficiency virus and hepatitis C virus (HCV). Because in patients with HCV-related cirrhosis the incidence of hepatocellular carcinoma (HCC) is high, in the present paper we examined, by means of an enzyme-linked immunosorbent assay, the 90K serum levels in 103 patients with liver cirrhosis, and in 69 with HCC, and compared them to α-fetoprotein, the reference tumor marker for this neoplasm. Serum levels of 90K (cut-off 14 μg/ml) were elevated both in cirrhosis (39%) and HCC (46%) compared to controls (14.1 μg/ml vs . 10.6 μg/ml in cirrhosis, and 14.8 μg/ml vs . 9.1 μg/ml in HCC, p≤0.001). There was a significant association with the presence of anti-HCV antibodies. 90K was found to be a non-specific tumor marker which is complementary to α-fetoprotein on the basis of its probable different biological significance. In fact, 74% of HCC patients had at least one positive marker. Combined use of 90K and α-fetoprotein could improve the sensitivity of a single test in the diagnosis of HCC.

When introducing a new parameter, it is necessary to compare the power of the new measure with already established ones. For a new method it is quite difficult to compete with established methods which have already ascertained sets of data over many years. A formal comparison of the new parameter with the actual “gold-standard” method can be a useful approach to reduce that problem. It cannot be expected that a new measure would reflect the “gold-standard” method in a simple proportionality. Therefore, it is important to find out the accuracy of the prediction of one parameter from the other, based on simple, e.g. linear, functions. A number of methods exist to determine the crystallization risk of calcium oxalate salts from urine. The most established method is the calculation of the relative urinary supersaturations with respect to these salts using the EQUIL -program, a program computing the equilibrium concentrations of complexes of primary cations and anions commonly found in urine. The Bonn-Risk-Index (BRI) is a new strategy for the evaluation of the risk of calcium oxalate formation, by performing crystallization experiments on native unprepared urine samples. Although the analytical and computational efforts of both approaches are quite different (relative supersaturation = high, BRI = low), the measurements revealed a considerable and significant linear relationship between the relative urinary calcium oxalate supersaturation, and BRI. We were, therefore, interested in predicting the relative supersaturation from the BRI and in the accuracy of this prediction.

We report age-related reference intervals for capillary zone electrophoresis for children between 1 and 14 years of age.

Disorders in thyroid function can impair normal development in children. Therefore it was our aim to establish reference intervals for serum triiodothyronine (T 3 ), free T 3 (fT 3 ), thyroxine (T 4 ), free T 4 (fT 4 ), thyroxine binding globulin (TBG) and thyrotropin (TSH) which are applicable from birth to adulthood by using the non-isotopic automated chemiluminescence immunoassay system, Immulite ® (DPC Los Angeles, USA). Serum samples from 762 euthyroid newborns, children and adolescents (369 female, 393 male; age 1 day to 19 years) were examined; of these, 381 were classified as pubertal. Due to non-normal distribution, the 2.5th, 50th and 97.5th percentiles (the central 95% interval) were calculated for each group. The median concentrations of T 4 , fT 4 and TSH were up to 3.2-fold higher during the first 2 weeks, while T 4 increased during the first month of life. The concentrations in all age groups showed no sex differences. From 1 year onwards, the concentration of all parameters tended to decrease until adult age, with the exception of TBG which increased by >60% (p<0.02) and reached a maximum at approximately 5 years of age. The findings underscore the fact that thyroid hormones are not associated with sexual development, except for TBG, which decreased slightly (p<0.04) between Tanner stages 1 and 5. However, the reference intervals established here demonstrate that marked changes occur in concentrations of thyroid hormones after the neonatal period. Our findings complement these of earlier studies. The developed reference intervals can be used to assess the thyroid status of patients, particularly if the measurements are done on the Immulite/Immulite 2000 system.

Pediatric reference ranges for osteocalcin measured by a new, fully automated, chemiluminescent immunometric assay on the Immulite ® immunoanalyzer are presented. Samples from 627 children, ranging from newborns to 18 years of age, were measured. Osteocalcin values are generally higher in children than in adults, highest levels being reached during the puberty growth spurt at about 12 years in girls and 14 years in boys, thereafter rapidly declining towards adult levels.

Interference caused by human anti-animal antibodies has been described in many laboratory assays. Usually the antigen giving rise to these antibodies is not known. When present in sufficiently high titer, these antibodies may have a significant effect on laboratory assays, causing falsely increased or decreased values, to the detriment of patient care and laboratory diagnosis. We report a case of an 86-year old man who had greatly increased C-reactive protein (CRP) levels in serum (>500 mg/l), suggestive of an unusually strong inflammatory response such as seen in septicemia. Because this diagnosis was not consistent with his symptoms, CRP measurements were repeated using alternative methods, none of which gave CRP results higher than 12 mg/l. Protein electrophoresis followed by immunofixation showed a monoclonal IgM-λ paraprotein (7 g/l), which bound the goat anti-CRP antibodies used in the first CRP assay. Precipitation of the IgM-λ paraprotein eliminated the interference. Subsequent bone marrow tests did not reveal any blood malignancy, and the monoclonal IgM component was attributed to monoclonal gammopathy of unknown significance. In conclusion, an IgM-λ paraprotein was shown to cause falsely elevated CRP values in an automated immunometric assay using goat anti-CRP antibodies.

The aim of the study was to discover how an implemented quality system succeeded in fulfilling the personnel and management expectations and to identify the factors that facilitate or hinder quality management implementation in clinical laboratories. The concepts assessed include leadership (commitment and change management), clear and common goals, human recourses focus, client focus, management by fact and process improvement. The quality process in the laboratories had not, even after 3–4 years, reached a level of acceptance allowing its use as a daily development tool. The factors that predict a success of the quality system include willingness to improve the laboratory services and to keep the process going and good atmosphere at work. However, the study showed that the senior managers of the laboratory should take a more visible role in leading the change, and emphasize more explicitly the long-term goals. The middle managers (physicians, biochemists and head technologists) should arrange opportunities for the staff to participate in the system and disseminate the information on, and practical applications of, the quality principles and tools. The staff should be more active in finding new information and in participating in the system.

There are multiple interrelationships between science, medicine and visual arts. This article discusses aspects of architecture associated with the Greek healing cult of Asklepios, the case in point being the Asklepios temple on the island of Kos in the Aegean. Further, the cult is contrasted with the beginnings of the observation-based medicine practised by Hippocrates (460–c.370 BC). Finally, it is suggested that including elements of visual arts in medical education is consistent with the aims of the new medical curricula.