Volume 38, Issue 10

Cholesteryl ester transfer protein (CETP) is a plasma glycoprotein that mediates the transfer of cholesteryl ester from high density lipoproteins (HDL) to triglyceride-rich lipoproteins in exchange for triglycerides. Several approaches are currently being used in research laboratories to measure its activity and/or mass. However, these assays are not standardized and it is not possible to compare data from different laboratories. Also, we lack enough information to assess the value of this variable as a coronary heart disease (CHD) predictor. Several genetic variants at CETP locus have been identified and they have been generally associated with increased HDL-cholesterol concentrations. However, there is no consensus about the association of this CETP-related increase in HDL-cholesterol and protection against CHD. Nevertheless, the most recent evidence from the common CETP-TaqI-B polymorphism shows that the lower CETP activity associated with the presence of this polymorphism decreases CHD risk in men. Based on this and previous evidence, there has been an interest in the development of CETP inhibitors as a tool to increase HDL-cholesterol, thus reducing CHD risk. However, it should be noted that the evidence about the cardioprotective role of these drugs is not yet available.

The aim is to determine if a single measurement of blood 2,3-diphosphoglycerate combined with gas analysis (pH, PCO 2 , PO 2 and saturation) can identify the cause of an altered blood-oxygen affinity: the presence of an abnormal haemoglobin or a red cell disorder. The population (n=94) was divided into healthy controls (A, n=14), carriers of red cell disorders (B, n=72) and carriers of high oxygen affinity haemoglobins (C, n=8). Those variables were measured both in samples equilibrated at selected PCO 2 and PO 2 and in venous blood. In the univariable approach applied to equilibrated samples, we correctly identified C subjects in 93.6% or 96.8% of the cases depending on the selected variable, the standard P 50 (PO 2 at which 50% of haemoglobin is oxygenated) or a composite variable calculated from the above measurements. After introducing the haemoglobin concentration as a further discriminating variable, the A and B subjects were correctly identified in 91.9% or 94.2% of the cases, respectively. These figures become 93.0% or 86.1%, and 93.7% or 94.9% of the cases when using direct readings from venous blood, thereby avoiding the blood equilibration step. This test is feasible also in blood samples stored at 4°C for 48 h, or at room temperature for 8 h.

We studied a possible effect of the extent of the acute phase response after acute myocardial infarction on the cumulative release of troponin T. The height of the acute phase response might influence the cumulative release of troponin T, bound to the myofibrillar structures of the heart, in a different way compared to the free cytoplasmic cardiac marker hydroxybutyrate dehydrogenase (EC 1.1.1.27). To investigate this, the cumulative amount of C-reactive protein in plasma, i.e. the quantified acute phase response, was related to the cumulative plasma release of hydroxybutyrate dehydrogenase (an established method for infarct sizing) on the one hand and to that of troponin T on the other hand. The study was performed in patients receiving (n=16) and in patients not receiving (n=6) thrombolytic therapy. Cumulative protein release was calculated using a two-compartment model for circulating proteins. Conclusions: The cumulative amount of plasma C-reactive protein is significantly higher in the patients not receiving thrombolytic therapy, as is in accordance with earlier studies. The cumulative amount of troponin T released is significantly related to the cumulated concentration of C-reactive protein, especially in patients not receiving thrombolytic therapy. The intensity of the acute phase response, estimated from cumulative plasma C-reactive protein response, has no effect on the relative proportions of troponin T and hydroxybutyrate dehydrogenase released into plasma.

Water metabolism may be non-invasively investigated using deuterium elimination from the body. Evaluation of elimination constants (k D ) and pool sizes (N) becomes complicated when turnover rates vary periodically - a realistic scenario in free-living subjects. Unfortunately, phase and frequency of periodic variations are not a priori known and can therefore not be used for a sampling protocol. This study investigates the impact of periodical flux variations on the measurement of water turnover. For two models of periodic variation with identical apparent k D , data sets of tracer concentrations in body water were generated for two-hour intervals using a monoexponential decay equation. Data were analyzed using the two- and the multi-point method increasing observation periods stepwise. Apparent values for k D , N and water turnover (R H₂O ) were compared with values originally used in the model. Periodically varying turnover rates introduce considerable errors for k D and R H₂O when the two-point method is used. The multi-point method gives a more robust estimate of R H₂O already after short observation periods, however, due to slope-intercept correlations, still tends to overestimate R H₂O . Errors are more pronounced the more uneven flux rates are distributed. Stepwise analysis of tracer enrichments using the multi-point approach identifies periodical variation of flux rates.

Congenital disorders of glycosylation (CDG) are genetic multisystemic diseases due to various defects in the biosynthesis or processing of glycoproteins. Our aim is to present our experience in the selective screening of CDG syndrome in a paediatric population (421 patients) with clinical suspicion of the disease, analysing serum carbohydrate-deficient transferrin (CDT) by radioimmunoassay and/or immunoturbidimetry. We established the normal values for our paediatric population. The abnormal results were confirmed and classified by isoelectric focusing of serum sialotransferrins, and by enzymatic and molecular studies. We found 14 patients (3.3%) with abnormal serum CDT; 11 of them were classified as CDG type Ia (CDG-Ia) and the other three showed altered isoelectrofocusing patterns but remain untyped and are under investigation. In conclusion, both CDT assays proved to be useful tools for CDG screening. Isoelectric focusing is a simple procedure but it requires specific instruments that are not always available. Since the immunoturbidimetric procedure is commonly used to monitor for recent excessive alcohol consumption in clinical laboratories and does not require special equipment, it may also be reliably used to screen for CDG in children under clinical suspicion.

The differential diagnosis of menopause and amenorrhea is currently based on the assay of circulating follicle stimulating hormone, luteinising hormone, and estradiol. The diagnostic performance of the three hormone assays, both as single and combined tests, was evaluated considering as reference data the results from 300 subjects for either condition, and assuming menopause – amenorrhea prevalence ratios corresponding to 1 and 10. In the calculation an “allocation” scheme was adopted, and the uncertainty associated with the diagnostic performance parameters was accounted for. The results obtained clearly demonstrate that the addition of a test for luteinising hormone or estradiol (or both) to the testing for follicle stimulating hormone is not justified in terms of improvement of the diagnostic information.

By governmental mandate, Japanese school children are screened annually for proteinuria, hematuria, and glucosuria to identify children with possible renal disorders. We added urine dipstick tests for albumin and creatinine to the Japanese screening protocol, and used their dipstick results for blood, glucose and protein. The sulfosalicylic acid precipitation test was used to confirm “trace” positive protein dipsticks. The Japanese and our screening protocol have in common the same data for glucosuria and proteinuria. Their scheme has an algorithm for repeat testing of children with abnormal results, and further testing and medical evaluation for those showing persistently abnormal values. Out of the 23,121 students, we found seven with likely nephritis, one with confirmed nephritis, one with nephrotic syndrome, 170 with persistent unexplained hematuria, 19 with persistent unexplained proteinuria, 14 cases of urinary tract infection, and 20 cases of likely diabetes mellitus. We conclude that dipstick testing for albumin, protein, creatinine, glucose and occult blood has significant value in a multilevel testing scheme for identifying children with urinary tract abnormalities or diabetes. The assay of albumin increases the sensitivity of the screening, and dividing the albumin by the creatinine concentration reduces the potential errors arising from concentrated or dilute urines.

The measurement of single parameters of oxidative stress in biological fluids can often give results difficult to interpret as to the real involvement of oxidative processes in a given disease condition. In the present study we propose a novel integrated parameter, called “redox compensation index”, obtained by combining the results of two established and convenient procedures, i.e. the Fox-2 assay for plasma lipid hydroperoxides and the ferric reducing/antioxidant power (FRAP) assay for total antioxidant potential of plasma. These procedures were employed for the evaluation of oxidative stress in a group of patients with type 2 diabetes mellitus, a condition in which oxidative processes are implicated in the development of complications. In type 2 diabetic patients, plasma lipid hydroperoxides were directly correlated with levels of glycated hemoglobin. On the other hand, a significant inverse correlation was o b s e rved between levels of glycated hemoglobin and redox compensation values. The data reported suggest that the redox compensation index could represent a convenient parameter for the direct appraisal of oxidative status in clinical subjects, and are in support of the proposed role of protein glycation in production of oxidative alterations during type 2 diabetes mellitus.

A self-developing solid-phase immunoassay (B.R.A.H.M.S. PCT-Q, B.R.A.H.M.S.-Diagnostica GmbH, Hennigsdorf, Germany) has recently become available for the semi-quantitative and rapid measurement of procalcitonin (PCT). In this study we examined the validity of this assay at daily clinical routine conditions at five different hospitals in a prospective study. After development of the assay (200 μl plasma, 30 minutes incubation), PCT levels were categorized into four groups (< 0.5 μg/l; ≥ 0.5−< 2 μg/l; ≥ 2−< 10 μg/l; ≥ 10 μg/l) according to the provided reference scale. Samples from patients with suspected elevation of PCT of different etiology (n=237) were read by various analyzers and compared with the results of the Lumitest ® PCT (B.R.A.H.M.S.-Diagnostica GmbH, Hennigsdorf, Germany). A total of 74.7% of measurements were categorized according to the results of the Lumitest ® PCT, 24.5% were read within the next lower or higher category. Using a ± 10% range at the reference concentrations (20% at 0.5 μg/l), 82.7% of samples were correctly categorized and 16.4% within the next categories. Using a cut-off value of 2.0 μg/l, 92.0% (94.1% for ± 10%) of the results were correctly categorized. The semi-quantitative solid phase immunoassay allows a rapid, simple and semi-quantitative measurement of plasma PCT. The validity of the test results and its ease of use are sufficient to support acute diagnostic decisions. However, for the follow-up of PCT concentrations and routine daily measurements, the quantitative luminometric assay should be preferred, when available.

The intermethod variability of control materials and patient blood samples for the measurement of hemoglobin A 2 (HbA 2 ) were compared. A set of 54 blood samples and 10 control materials were analyzed in duplicate by HPLC and microcolumn methods. For each set of methods the distances of the materials from the regression line of patient blood results (expressed as normalized residuals) were calculated. Four out of ten controls had normalized residuals exceeding three standard deviations from the regression line. Moreover, total Hb and Hb derivatives analysis proved that only a minority of the controls could be considered similar to patients' blood samples. Intermethod calibration performed “ a posteriori ” by the two best performing control materials improved intermethod variability among all the five tested methods. We conclude that the use of high resolution HPLC methods together with appropriate commutable control materials allows for better harmonization of results in the field of diagnosis of hemoglobin disorders in research and clinical practice.

Serum C-reactive protein (CRP) concentration was determined for 3605 subjects using an immunonephelometric assay improved to provide greater sensitivity. Subjects were from 5 to 75 years old and belonging to 1003 nuclear families recruited from the Stanislas Coh o rt Study between January 1994 and August 1995. Sample values for CRP ranged from 0.17 mg/l to 100 mg/l. Geometric means (mean − SD; mean + SD) were in the 5–14 years old group 0.37 (0.17–1.07) mg/l, in the 15–28 years old group 0.47 (0.17–1.38) mg/l and in the 29–75 years old group 0.98 (0.34–2.85) mg/l. For women, the geometric means were 0.38 (0.17–1.10) mg/l, 0.62 (0.20–1.90) mg/l and 0.98 mg/l (0.31–3.13) mg/l respectively. The interindividual variability ranged from 138% to 759% among different age classes. Biological factors associated with CRP concentration variations were examined and accounted for 25% of the CRP variability in men and 40% in women. The main biological factors statistically associated with CRP concentration variations in men were: drugs, leukocyte count, body mass index, tobacco consumption, age, and in women: drugs, leukocyte count, age, body mass index and hemoglobin concentration. These factors were used to define the exclusion and partition criteria when obtaining the reference samples. Medians for reference values ranged from 0.20 to 0.68 mg/l in males and from 0.20 to 0.78 mg/l in women.

Ideally, every laboratory should derive their own reference intervals for all analytes, but this is difficult in practice. A survey, by questionnaire, of UK laboratories using the Chiron Diagnostics ACS:180 (Chiron Diagnostics Limited, Halstead, Essex, UK), for thyroid function tests, showed that 10% of laboratories derived their own reference intervals, 60% quoted values “adapted” from intervals for previous methods, whilst the remaining 40% quoted (often incorrectly) reference intervals supplied by the manufacturer. In addition only 13% of respondent laboratories derived their own reference intervals for testosterone. As a result of this survey, a study was devised to enable the users of the Chiron Diagnostics ACS:180 immunoassay system to develop and use within-method, between-laboratory reference intervals for thyroid hormones and testosterone. Laboratory collaboration provided the recommended minimum number of data points by establishing a reference sample group. This sample group was used for the calculation of appropriate reference intervals for each hormone according to the guidelines published by the IFCC. We propose this approach as a model for laboratories using identical instrumentation to produce, through collaboration, within-method, between laboratory reference intervals.

A test and a reference analytical method are usually compared for agreement based on paired data obtained from several independent subjects. Bias between two methods can be classified as constant and proportional. Bias is modeled as an average bias between two methods (constant bias) and proportional bias related to individual measurements. A weighted least square approach is followed for estimating the parameters. Estimates of constant and proportional biases are tested individually and their significance can be used to explain the sources of disagreement between two methods and help deciding a remedial strategy. The proposed model can also be used to determine an optimum combination of subjects and runs per subject and facilitate the allocation of the resources.

Two methods for the determination of iodine in urine or serum based on the Sandell-Kolthoff reaction were compared. The first was an autoanalyser method (AAII, Technicon) where the mineralisation takes place in a continuous flow manner. This procedure was used at the Department of Clinical Chemistry of the University Hospital of Berne (Switzerland) from 1968 until 1993. The second method was evaluated and adapted in our own laboratory. Each sample and the iodate-standards are mineralised in a Pyrex glass tube and the decolorisation reaction takes place in a 96-well-microtiter plate which was read by a PC-controlled photometer at 405 nm. This method, with a detection limit of 0.1 μmol/l showed good analytical recovery (90 to 110%) and a low imprecision (intra-assay coefficient of variation (CV) of 5% and an inter-assay CV of 7.5%). In contrast to the autoanalyser method, the microtiter plate-method is suitable both for series up to 24 samples (3-fold) and for single samples. A comparison of 87 samples in the range of 0.1 to 60 μmol/l which were measured with both Sandell-Kolthoff based methods showed no obvious discrepancy. These two methods showed a good agreement for the determination of urinary iodine. This guarantees that the results of earlier epidemiological studies can be compared with recent studies performed in our and many other laboratories.

We evaluated two enzyme immunoassays (EIA) for detection of antinuclear antibodies (ANA) which became recently available and which were designed for application on a fully automated system; (i) the EIA-ANA screen kit from Sigma Diagnostics (St. Louis, MO, USA) applied on APTUS™, an automated EIA analyser from Sigma Diagnostics and (ii) the Cobas ® Core Hep-2 EIA-ANA assay applied on the fully automated Cobas Core immunochemistry analyzer from Roche Diagnostics (Basel, Switzerland). The evaluation was done by an analytic comparison of the automated systems to an established indirect immunofluorescence (IF) method performed on SSA transfected human epitheloid cell substrate slides. Three hundred and thirty six samples were tested with the Sigma EIA-ANA assay and 603 samples with the Cobas ® Core Hep-2 EIA-ANA assay. For both EIA systems, there was a trend of generally increasing signal from the assay system with increasing IF-ANA titers. With an IF-ANA reference range of < 1:160, concordance between Sigma ® EIA-ANA and IFANA was 86% and concordance between Roche ® EIA-ANA and IF-ANA was 85%. When compared to IF-ANA with a reference range of < 1:160, sensitivity and specificity of the EIA-ANA screen was 0.65 and 0.92, respectively, for Sigma ® EIA-ANA and 0.61 and 0.91, respectively, for the Cobas ® Core assay. The low sensitivity observed with both methods is a major concern. Sigma EIA-ANA screen revealed the presence of autoantibodies in 23 of the 29 samples containing antibodies to extractable nuclear antigens (ENA) and/or double stranded DNA and the Cobas ® Core Hep-2 EIA-ANA assay revealed the presence of autoantibodies in 40 of the 43 samples containing antibodies to ENA and/or double stranded DNA.

We evaluated the analytical performance of the Immulite 2000 ® immunoassay analyzer (Diagnostic Products Corporation, Los Angeles, USA) based on a new detection technology, electrochemical luminescence. The evaluated analytes were thyrotropin, triiodothyronine, free thyroxine, follitropin, lutropin, prolactin, cortisol, estradiol and progesterone. We tested the assay precision, linearity, recovery, and correlation with comparison methods for these analytes. For most assays, within-run and between-day imprecisions were less than 8% and 10%, respectively. The linearity and recovery were acceptable for all assays. The correlation between the Immulite 2000 ® assays and comparison methods showed satisfactory results.

The Olympus AU400 analyser (Olympus, Tokyo, Japan) is an automated chemistry instrument for turbidimetric, spectrophotometric and ion selective electrode measurements. Overall analytical performances of the AU400 and the reagents provided by Olympus were evaluated according to the French Society of Clinical Biology guidelines. Twenty parameters including specific proteins, substrates, enzyme activities and electrolytes were tested. The linearity exceeded the specifications given by the manufacturer. Within- and between-run imprecision (CV%), evaluated at two levels, was below 1.5% for ion selective electrode parameters and 3% for other analytes, except for CO 2 , alkaline phosphatase at low levels and magnesium. Results compared well with those obtained with the analysers routinely used in our laboratory (Behring BNII, Olympus AU800 and Beckman CX3 Delta). The usual positive interferences from lipaemia and haemoglobin on total protein measurement were observed. Creatine kinase and alkaline phosphatase assays were the subject of positive and negative interference by haemoglobin, respectively. There was a negative interference by bilirubin in the uric acid, aspartate-amino transferase, creatine kinase and lactate dehydrogenase assays and a positive interference in the calcium assay. The system was found to be very easy to use and the workstation is user-friendly.

Many medical laboratories have made a start with the introduction of quality management systems. However, it is still not clear against which standards such systems should be measured. The existing ISO and CEN standards do not cover essential aspects of medical laboratories. The publication of the EC4 Essential Criteria has stimulated the development of the ISO/Draft International Standard 15189. This standard seems adequate for our type of laboratories. However, it is not easy to read. The EC4 Essential Criteria could well serve as a guide, covering additional aspects, e.g. on total quality management and budget management as required in the EFQM model, that are not (yet) included in the ISO standard. In the present article the EC4 Essential Criteria are cross-referenced with two new international ISO standards, ISO/FDIS 15189 and ISO/FDIS 17025, the latter being the successor of ISO guide 25 and EN 45000. Both new ISO documents are in compliance with the new ISO 9000:2000 standard.

Ion-selective electrodes (ISEs) respond to ion-activity and therefore do not sense substance concentration directly. However, it is recognized that sodium and potassium in plasma will continue to be expressed for clinical purposes in terms of substance concentration (mmol/l). A convention is proposed whereby for routine clinical purposes results of ISE measurements of sodium and potassium in undiluted plasma should be reported in terms of substance concentration (mmol/l). In specimens with normal concentrations of plasma water, total CO 2 , lipids, protein and pH, the values will concur with the total substance concentration as determined for example by flame atomic emission spectrometry (FAES) or ISE measurements on diluted samples. In specimens with abnormal concentrations of plasma water, the results will differ. However, under these circumstances, measurements of sodium and potassium by ISE in the undiluted sample will more appropriately reflect the activity of sodium and potassium and are therefore clinically more relevant than the determination in diluted samples. Detailed recommendations are made about practical procedures to achieve this. The recommended name for this quantity is the substance concentration of ionized sodium or ionized potassium in plasma, as opposed to total sodium or total potassium determined by, e.g. FAES, or ISE measurements on diluted samples.