Volume 42, Issue 1

Cardiac markers are measured by a number of different immunoassays using specific antibodies directed to the respective antigens. Lacking assay standardization, different results from different assays measuring the same marker may be obtained and this problem may cloud interpretations of reported data. Presently, there are no reference procedures for cardiac markers; certified reference materials should still be established and, at least for cardiac troponins, the analyte in the patients' blood is significantly different from newly synthesized protein. It is therefore clear that the problems of cardiac marker standardization will not be quickly solved. A number of projects are, however, underway under the auspices of the IFCC and other organizations. The aim of this opinion is to reflect on some concepts related to the implementation of a metrologically correct measurement system, giving practical examples on how these concepts can be applied to immunoassays measuring cardiac markers.

The relationship between ageing and nutrition is considered with collagen as the intermediate target. Some data showed that diet restriction resulted in decreased collagen accumulation and collagen ageing. Conversely, being overweight reduced the lifespan and increased collagen ageing.Collagen ageing, which includes low turnover and glycoxidation, involves an increase in both stiffness and weakness. Their consequences concern all tissues including those with vital importance such as cartilage, heart ventricle or arterial wall.

Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is a new efficient method for single-nucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMS-PCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allele-specific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was non-specific amplification of the shorter products. To reduce the amplification of these nonspecific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 5–10 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetra-primer ARMS-PCR to SNP genotyping.

Hereditary haemochromatosis is an autosomal recessive disorder of iron regulation that results in abnormal intestinal iron absorption with progressive iron overloading of parenchymal cells. Two specific, single point mutations of the HFE gene (C282Y and H63D) have been described in haemochromatosis patients. Epidemiological studies have revealed a strict association between hereditary haemochromatosis and C282Y homozygosis or C282Y/H63D compound heterozygosis, suggesting that these mutations may provide a useful tool for diagnosis. However, recent investigations from southern Europe have reported lower allelic frequencies of the C282Y mutation among haemochromatosis patients, apparently depending on the geographical area of the population analysed. To assess the predictive value of the detection of the C282Y and H63D HFE mutations in our geographical area, we have evaluated their occurrence in 46 haemochromatosis patients from southern Italy. We found that only 19.6% of our patients were homozygous for the C282Y mutation and 21.7% were compound C282Y/H63D heterozygotes. Among the remaining 59%, approximately 40% did not display any of the known HFE mutations. We conclude that, in southern Italy, another genetic determinant/s must be responsible for many haemochromatosis cases and that a genetic screening for the C282Y and H63D HFE mutations is not sufficient for hereditary haemochromatosis diagnosis.

We have evaluated the usefulness of the PFA-100™ system (collagen/ADP and collagen/epinephrine cartridges) to assess the in vitro effects of a few platelet function inhibitors: Aspisol ® (60 μg/ml), 4-[4-[4-(aminoiminomethyl]-1-piperazinyl]-1-piperidineactetic acid, hydrochloride trihydrate (GR144053F, fibrinogen receptor antagonist, 100 nM), adenosine-3′,5′-diphosphate (A3P5P, P2Y 1 ADP receptor antagonist, 500 μM) and Bis[(adenosine-5′- O -phosphorodithioyl) methylene]-phosphinic acid (APTMPA, P2Y 12 ADP receptor antagonist, 500 μM) on platelet function, as compared with the other commonly used diagnostic technique, a whole blood electrical aggregometry (20 μM ADP-or 0.5 mM arachidonic acid). The in vitro studies were carried out on a group of 38 subjects. Whereas all the examined platelet antagonists and inhibitors almost completely blocked the 20 mM ADP-or 0.5 mM arachidonic acid-induced (in the case of acetylsalicylic acid) whole blood aggregation, only two inhibitors (Aspisol ® and GR144053F) remained effective in a significant prolongation of the PFA-100™ occlusion time. Otherwise, using the PFA-100™ system we were not able to detect the inhibitory actions of ADP receptor antagonists–P2Y 1 and P2Y 12 . Our findings point to a limited usefulness of the PFA-100™ system for the monitoring of the effectiveness of ADP receptor antagonists. The outcomes of this study show that platelet aggregometry in whole blood is characterised by the highest sensitivity in the monitoring of the investigated blood platelet inhibitors.

Small-dense low-density lipoprotein (SD-LDL) is associated with coronary heart disease risk. Current methods for its quantification are expensive, complex and time-consuming. Plasma was adjusted to a density (D) of 1.044 g/ml in a volume of 0.18 ml and centrifuged in a Beckman Airfuge at 160 000× g for 3 h 7 min and apolipoprotein B (apoB) then determined in the infranatant. Results were compared with centrifugation of 5 ml of plasma at D=1.044 g/ml at 144 000× g for 18 h in a preparative ultracentrifuge (UC). We obtained blood samples from healthy subjects (n=73) and from dyslipidaemic patients (n=112). SD-LDL apoB levels as determined using the UC method ranged from 0.01–0.43 g/l and there was a good correlation with Airfuge results (r=0.925; p<0.0001; n=185). There was a mean difference of 0.0125 g/l between methods. SD-LDL apoB levels as determined using the Airfuge showed a reasonable agreement with results obtained using density gradient ultracentrifugation (r=0.773; p<0.01; n=12). Airfuge results correlated directly with triglyceride concentration, in both healthy men (r=0.296; p<0.05) and dyslipidaemic men (r=0.520; p<0.001) and also in dyslipidaemic women (r=0.463; p<0.005). Airfuge results correlated inversely with high-density lipoprotein-cholesterol (HDL-C) concentration, in both healthy men (r=−0.237; p<0.05) and dyslipidaemic men (r=−0.293; p<0.005). Using a micro-method, we obtained results which establish that the Airfuge provides a more rapid and less expensive method for quantification of SD-LDL.

We evaluated the analytical performance of a fully-automated electrochemiluminescence “sandwich” immunoassay method for the N-terminal fragment of the pro-peptide of brain natriuretic peptide (BNP). We then compared the diagnostic accuracy of this method in discriminating between normal subjects and patients with cardiomyopathy with that found with two previously described immunoradiometric assay methods for the assay of atrial natriuretic peptide (ANP) and BNP. We studied 193 consecutive patients (mean age 64.4±12.3 years, range 20–89 years, including 56 women and 137 men) with chronic cardiomyopathy and a group of 85 healthy subjects (mean age 52.3±12.0 years, 42 women and 43 men, range 20–79 years). N-terminal fragment of proBNP 1–76 (NT-proBNP) was measured with a fully-automated “sandwich” electrochemiluminescence method using an Elecsys ® 2010 analyzer, while ANP and BNP were measured with immunoradiometric assay methods. The low detection limit of the NTproBNP assay was 4.2 pg/ml (0.50 pmol/l), while the functional sensitivity was 22 pg/ml (2.60 pmol/l) with a working range (imprecision profile ≤ 10% coefficient of variation) extended up to about 30 000 pg/ml (3540 pmol/l). Healthy women (64.3±41.6 pg/ml, 7.59±4.91 pmol/l) showed significantly higher values than men (46.9±30.9 pg/ml, 5.53±3.64 pmol/l, p=0.0118). Moreover, age and sex were significantly and independently related to the NT-proBNP values in healthy subjects, as assessed by a multiple linear regression analysis (R=0.389, F-value=7.316, P-value=0.0012). As expected, the NT-proBNP values of patients with cardiomyopathy were significantly higher than those of normal subjects and progressively increased with the severity of heart failure. The respective diagnostic accuracy of the ANP, BNP and NT-proBNP assays in discriminating between the group of normal subjects and that of patients with cardiomyopathy was tested by the response operating characteristic curve analysis. Our data indicated that the NT-proBNP assay is significantly better than either of the ANP or BNP immunoradiometric assays in discriminating affected patients from healthy subjects, especially when only patients with mild disease severity (New York Heart Association class I and II) are considered.

Centrifuged and filtered urine is often used to evaluate in vitro the crystallization processes of calcium oxalate (CaOx), but even such simple manipulations can alter the composition of the urine, as regards its protein and lipid concentrations. In urine samples taken from 17 normal male adults, we evaluated CaOx crystallization by simultaneously using three different types of urine: untreated (U), centrifuged at 2000 rpm (800 g )and filtered at 0.22 μm (CF), and centrifuged-filtered and ultrafiltered at 10 000 Da (CFU). The addition of 1.2 mmol/l of oxalate to each type of urine produced notably different results. The total amount of CaOx crystals (expressed as calcium oxalate dihydrate crystals (COD)+oxalate monohydrate crystals (COM)area/total area × 100)was on average 13.2% in U urine, 70.7% in CF urine and 11.1% in CFU urine (CF>U and CFU, U=CFU); the relative prevalence of COD and COM (expressed as COD area/COM area) was on average 71.4 in U urine, 0.0026 in CF urine and 5.5 in CFU urine (U>CF and CFU, CFU>CF); the diameter of COD (expressed in microns)was on average 15.2 in U urine, 3.7 in CF urine and 24.3 in CFU urine (CFU>U and CF, U>CF); the diameter of COM (expressed in microns)was on average 5.2 in U urine, 2.6 in CF urine and 8.9 in CFU urine (CFU>U and CF, U>CF); the total amount of CaOx aggregates (expressed as CaOxAgg area/total area×100)was on average 8.5% in U urine, 22.1% in CF urine and 2.9% in CFU urine (CF>U and CFU, U>CF). We conclude that CaOx crystallization processes in manipulated urine are extremely different, probably due to changes in macromolecular compounds.

Immunological reactions are influenced by various factors including antigens, antibodies and other variables. We focused on two items: i) matrix effects, especially of detergents and ii) temperature effects: preheating sera, especially effects on rheumatoid factor (RF) measurement and false-positive reactions in ELISAs, and cold storage of sera, especially effects on complement. Among various additives, detergents affected the agglutination reaction for fecal hemoglobin and hepatitis B surface (HBs) antigen. Some of the detergents examined abolished these antigenicities, however, polyethylenglycols enhanced the reactions. Heat-inactivation of sera at 56°C for 30 min was employed in serological testing. However, in RF measurement, 10 min of preheating was sufficient to abolish C1q (subcomponent of C1), which could participate in the agglutination reaction. In ELISA for antibodies, false-positive reactions were caused by preheating sera. By the analyses of assays for antibodies to hepatitis C virus (HCV) and cardiolipin, it was found that they were induced by immunoglobulin G (IgG) modified by preheating. Cold storage induced activation of complement (cold activation) in anti-HCV antibody positive sera. CH50 titers in the sera were lowered by one cycle of freezing at −20°C and thawing, and the decrease was affected by the containers.

This study describes the performance of the different methods used for blood alcohol analysis by the Belgian laboratories participating in the external quality assessment scheme. The best performance was achieved by the gas chromatography (GC) and the enzymatic methods. The enzymatic methods are easier to use but more apparatus-dependent, whereas GC methods display higher precision, especially at concentrations near the critical legal limit of 0.5 g/l. The study highlights the need to introduce a methodology other than the chemical oxidation-based method of Casier, the sole legal method for forensic analyses in drinking-driving cases in Belgium. This method with poor intra-laboratory, inter-laboratory and linearity performances has definitely become obsolete. Even though GC clearly appears as the method of choice, mainly due to its higher specificity, the present study also shows the need for practical guidelines to improve the quality of GC alcohol analyses used in Belgium.

Because routine assays for pancreatic lipase catalytic activity are not yet standardized, between-method comparability is very poor. This is mainly due to the lack of reference materials (RMs). The aim of this study was to assign values of catalytic concentration to two human pancreatic lipase RMs, one prepared from human pancreatic juice (BCR 693), the other obtained by recombinant technology (BCR 694). Lipase catalytic activity was assayed in five experienced laboratories, using aliquots from the same lot of triolein emulsion and a standardized titrimetric procedure, optimized with regard to substrate, cofactors and pH. The accepted sets of data (n=4) gave a mean ± the corresponding uncertainty expressed as the 0.95 confidence interval of 1732±72 U/l and 1043±60 U/l for BCR 693 and 694, respectively. Transferability of the whole operating procedure proved to be quite satisfactory. The authors conclude that both RMs can be used to verify the correct implementation of the standardized measurement procedure and to assign values to secondary lipase materials (commercial calibrators, control products) which, in turn, ensures traceability to the standardized procedure in this study, and contributes to the harmonization of laboratory results according to the Directive for in vitro Diagnostic Medical Devices.

Polyethylene terephthalate (PET) tubes have several advantages over glass tubes: they are unbreakable, lighter and more easily disposed of. Despite a steady increase in their use and an expansion of the range of available tubes, few studies validating their use have been published in the literature. This paper describes the various studies that have been performed to compare VENOJECT glass, VENOSAFE PET and VENOSAFE PET/heparin tubes for the assay of a panel of analytes in routine clinical chemistry, immunochemistry, hormone and tumor marker analysis and trace metal determination. These studies demonstrate that VENOSAFE PET tubes are a suitable alternative to glass tubes.

CYP2C9, an isoform of the cytochrome P450 enzyme, is involved in the metabolism of most of the drugs of choice for the treatment of thromboembolic disorders. Functional polymorphism is associated with two variant alleles (alleles *2 and *3 ) encoding CYP2C9 enzymes with a potentially different catalytic activity. The aim of the study was to determine the frequency of the CYP2C9 polymorphism in a representative sample of the Croatian population (n=177) and to assess the association between the CYP2C9 polymorphism and the warfarin dose in patients with thromboembolism (n=181). The CYP2C9 genotype was determined by polymerase chain reaction-restriction fragment length poymorphism (PCR-RFLP). According to the CYP2C9 genotype distribution, 31.2% of the healthy subjects were identified with a heterozygous or homozygous CYP2C9 variant genotype. The frequency of 2C9*2 and 2C9*3 alleles was 12.4% and 3.7%, respectively. There was no gender-related genotype or allele difference. In thromboembolism patients, the frequency of CYP2C9 alleles *2 and *3 was 17.4% and 6.6%, respectively, and did not differ significantly from the control group. Almost half (42.5%) of the patients carried at least one variant CYP2C9 genotype. The allele difference between patient subgroups receiving warfarin doses lower and higher than the optimal warfarin dose (4.1 mg/day) was significant (p=0.027), especially for allele 2C9*3 (p=0.019; OR 3.250, 95%, CI 1.263–8.413). Comparison of the warfarin dose between patients with different genotypes yielded a significant dose difference between the patients with wild-type genotype and those with variant genotypes (Kruskall-Wallis, χ 2 =9.745, p=0.008). The results of the association of each of five genotype combinations with the warfarin maintenance dose revealed it to be significantly related to the genotype (Kruskall-Wallis, χ 2 =12.854, p=0.025). Expressed as percentage of the mean dose in patients with wild-type alleles, the mean warfarin maintenance dose was 92% in 2C9*2 heterozygotes, 74% in 2C*3 heterozygotes, 61% in 2C9*2 homozygotes, 34% in 2C9*3 homozygotes and 63% in compound heterozygotes for 2C9*2 and 2C9*3 . Although the mean maintenance dose in homozygous *2/*2 and compound *2/*3 genotype patients was markedly lower (mean 2.66 mg and 2.75 mg, respectively, vs. 4.37 mg), statistical analysis yielded no significance because of the small number of patients carrying these genotypes. A significantly lower maintenance dose was observed in the subgroup of heterozygous *1/*3 genotype patients (p=0.022). These preliminary results suggest a significant association of the CYP2C9 polymorphism with the warfarin dose and underline the importance of pre-therapeutic genotyping to identify the subjects likely to develop undesirable drug effects.

Allowable imprecision and bias reference limits for laboratory data can be calculated based on measurements of biological variation. Although biological variation of clinical chemical data has been reported from many laboratories, there have been few reports of biological variation in coagulation tests. In this study, we calculated the biological variation of 13 coagulation tests in the clinical laboratory of Kyushu University Hospital and determined allowable imprecision and bias limits of variation. The participating subjects were 17 healthy individuals: three males and two females in their 20s, two males and two females in their 30s, one male and four females in their 40s, and two males and one female in their 50s. Monthly measurements were performed before breakfast 12 times from June 2001 to May 2002 and allowable imprecision and bias limits were calculated. Taken together with coefficient of variation of control plasma used in daily laboratory work at the hospital, the allowable imprecision limits of intra-laboratory variation determined in this study appear to be in attainable ranges.

Several methods to assess the total antioxidant capacity (TAC) are available. However, the final value of measured TAC in the sample depends on the procedure used in every specific assay. This makes crucial the comparison of different analytical methods. The aim of our study was to evaluate analytical characteristics and laboratory reliability of two different assays: the ferric-reducing ability (FRAP) assay and a new spectrophotometric test (OXY-adsorbent test, Diacron, Italy). Unselected outpatients referred to the Institute of Clinical Physiology were studied (n=187, 58 females, 129 males, mean age: 65±13 years). All blood samples were maintained on ice, centrifuged within 15 minutes after blood collection and then stored at −80°C until performance of assay procedures. OXY assay: The lower limit of sensitivity was 6 μmol HClO/ml. The assay was found to be linear up to 440 μmol HClO/ml (r=−0.99, p<0.001). Absorbance was linear over a wide concentration range with solutions containing uric acid in purified form (0–1000 μmol/l, r=−0.996, p<0.001), serum (r=−0.99, p<0.01) or plasma serially diluted (r=−0.99, p<0.01). Mean value in plasma samples accounted for 366.2±7.2 μmol HClO/ml. Mean OXY value in females (353.4±13.2 mmol HClO/ml) was not different from that detected in males (372±8.6 mmol HClO/ml). A significant difference was observed between subjects without and with hypertension in serum OXY levels (344.8±9.9 and 383.2±10 μmol HClO/ml, p<0.01, respectively). FRAP assay: The lower limit of sensitivity was 15 μmol/l. Linearity was observed up to 1000 μmol/l (r=−0.998, p<0.001). Absorbance was linear over a wide concentration range with solutions containing uric acid in purified form (0–1000 μmol/l, r=0.997, p<0.001), serum (r=0.99, p<0.01) or plasma serially diluted (r=0.99, p<0.01). FRAP mean value in plasma samples, evaluated in 102 patients, accounted for 514.1±19.1 μmol/l. Mean FRAP in females (469±22.5 μmol/l) was not different from that detected in males (535±25.6 mmol/l). FRAP vs. OXY: A significant direct relationship was observed when comparing FRAP with OXY levels in the whole population (r=0.22, p<0.05). Neither of the methods are expensive and they are speedy and simple to perform. Values are reproducible and linearly correlated to the concentration of antioxidants present in the samples. For this reason, these methods may be considered practicable indicators of total antioxidant capacity, for routinely potential use in every laboratory and useful in all the studies concerning the evaluation of oxidative stress.

Introduction: Biochemical markers of bone metabolism have been mainly determined manually until now and the precision and accuracy of these methods have not always been satisfactory. This has been shown in several external quality assessment schemes (EQAS). Objective and study design: A study named BIOROSE was undertaken to evaluate new automated assays for serum markers of bone metabolism. The main focus was to evaluate the assay performance in a multicenter setting with 20 laboratories participating in Germany. The evaluation consists of a familiarization phase to determine precision and accuracy and an EQAS to evaluate the comparability between laboratories. Materials: The parameters β-CrossLaps (CTX), N-MID-Osteocalcin (OC) and intact parathyroid hormone (PTH) were measured with reagents including calibrators and control sera obtained from Roche Diagnostics, Mannheim, Germany, with electrochemiluminescence immunoassays (ECLIA) on the automated analyzer Elecsys 2010. Results: We calculated for the control samples, PCB 1-3, the mean and median values from the measured values of all participating laboratories and used these as target values. From these target values, a recovery range for the participating laboratories was calculated for β-CrossLaps, OC and intact PTH of better than 80–126% for PCB 2 and PCB 3, and for PCB 1 (low concentration range) for β-CrossLaps 79–129%, OC 90–120% and intact PTH 78–126%. The between-day imprecision was 2.4–7.2% for β-CrossLaps, 1.1–5.9% for OC and 1.7–5.5% for intact PTH in the elevated range (sample PCB 2). In the EQAS, the inter-laboratory imprecision for β-CrossLaps in the sample with a value of 0.8 ng/ml (above the upper limit of normal, which is 0.6 ng/ml) was 9.8% on day 1 and 9.7% on day 2. Conclusion: The performance evaluation of automated assays for β-CrossLaps, N-MID-Osteocalcin and intact parathyroid hormone in the BIOROSE multicenter study showed that the participating laboratories had no problems in setting up these methods and they yielded results for precision and accuracy that are superior to results achieved in external quality assessment schemes for manually performed methods. In addition, at the clinically important decision level of the upper limit of the normal range, all three tested analytes gave precise results that improved medical decisions.

During the past few decades, new technologies have allowed the fabrication of miniaturized sensors and the development of analyzers well designed for point-of-care testing (POCT). They combined the ease-of-use and portability required for POC with the accuracy and the reliability of traditional systems. Instrumentation Laboratory introduced the GEM ® Premier ™3000, a compact and portable system to rapidly analyze pH, pCO 2 , pO 2 , Ca 2+ , Na 2+ , K + , lactate, glucose and hematocrit, from 135 μl of whole blood with a simple component: a cartridge, the GEM ® Premier Pak. The analytical performance was compared with the Radiometer ® ABL 725 instrument for blood gas/electrolytes/metabolites, and for hematocrit with the centrifuged microhematocrit method. Aqueous quality control materials (Instrumentation Laboratory) were used for precision and recovery studies. The evaluation was performed according to the Valtec protocol, designed by the French Society of Clinical Biology, for imprecision, linearity and accuracy. Within-run and between-run imprecisions for all the parameters gave good CV values. Linearity was good and results comparison showed close agreement with correlation coefficients between 0.91 and 0.99. The analyzer is very easy to use; the simplicity and the convenience of only one consumable make the GEM ® Premier ™ 3000 very suitable for a STAT laboratory or for POCT.