Volume 46, Issue 7

Direct detection technologies for pathogenic microorganisms are emerging to be applied in the diagnosis of serious bloodstream infections and infections at sterile body sites, as well as for quality control measures prior to the release of sterile blood products and to ascertain microbial safety of food. Standard blood cultures as the current gold standard for detection of bacteraemia/sepsis and other culture-based microbiological identification procedures are comparatively slow and have limited sensitivity for fastidious or slow-growing microorganisms. Rapid nucleic acid-based technologies with PCR amplification or hybridisation probes for specific pathogens, broad-range bacterial or fungal assays, flow cytometry, as well as protein-based characterisation by mass spectrometry, aim at identification of pathogenic microorganisms within minutes to hours. Interpretation of direct detection of panbacterial or panfungal nucleic acids instead of living microorganisms in blood is complex, given the risk of contamination, the ubiquitous presence of bacterial and fungal DNA, and the lack of a gold standard. Since many of the infections at sterile sites, particularly sepsis, are medical emergencies requiring immediate therapeutic responses, rapid technologies could contribute to reduction of morbidity, mortality, and of the economic burden. This review summarises the currently available data on rapid non-culture-based technologies and outlines the potential clinical usefulness in infectious disease diagnosis. Clin Chem Lab Med 2008;46:888–908.

Nucleic acid techniques (NATs), such as species-specific and universal polymerase chain reactions (PCRs), are finding ever wider use in the diagnosis of bacterial infection. However, although universal PCR assays, in particular, approach a type of modern Petri dish, they have a number of limitations which restrict their applicability. The sensitivity of universal PCR is lower than that of many species-specific PCRs, and the contamination of samples and PCR reagents with irrelevant DNA from various sources remains a problem. Thus, NATs in general and universal PCR assays in particular require careful validation to be of value for the diagnosis of infection. Validation includes sampling, DNA extraction/isolation, template amplification and visualisation of the results. Furthermore, it implies the establishment of measures of asepsis, the inclusion of positive and negative controls, techniques to optimise the release of DNA from bacterial cells, adequate repetition of the amplification reaction, and routine testing of reagent negative and inhibition controls. Finally, it entails the comparison of results obtained by NATs with those obtained by conventional microbiological methods and matching with clinical evidence of infection. Validation of NATs in clinical diagnosis remains an ongoing challenge. Because of these limitations, NATs can only serve as adjunct tools for the diagnosis of infection in selected cases; they cannot replace conventional culturing techniques. Clin Chem Lab Med 2008;46:909–18.

Successful reduction of the risk of viral transmission via blood components has focused attention on the risk of transfusion-associated bacterial sepsis. The incidence of transfusion reactions due to bacteria is currently estimated at between 1 per 100,000 and 1 per 1,000,000 units in the case of packed red blood cells and between 1 per 900 and 1 per 100,000 units in the case of platelet concentrates. For autologous transfusion, only isolated case reports are available which do not permit a quantitative risk assessment. No reliable data on the risk of morbidity and mortality due to transfusion-associated bacterial infection are available for Germany at present. As platelet concentrates provide more favourable conditions for bacterial growth by virtue of being stored at room temperature, these blood components are thought to bear a considerably greater risk of bacterial contamination than red blood cells. The rate of contamination of platelet concentrates is cited at between 0.02% and 1.2%, depending on the production and bacterial culture techniques used, whereas the rate for packed red blood cells is between 0.1% and 0.2%. Risk reduction strategies include careful selection of blood donors, optimisation of donation and production techniques, and detection of bacteria in samples of blood components prior to transfusion. Also of importance for patient safety are quality assurance measures in the preparation for transfusion, in measures of patient monitoring, and investigation of transfusion reactions. Clin Chem Lab Med 2008;46:919–25.

Since the impressive reduction of transfusion-transmitted virus infections, bacterial infections by blood transfusion represent the most important infection risk. Platelet concentrates are the current focus of attention, as they are stored under temperature conditions which allow growth of contaminating bacteria up to 10 10 and more microbes per platelet bag. This paper does not consider the pathogen reduction methods but will assess suitable screening methods. Beside conventional microbiological approaches or surrogate markers, several efficient methods able to detect bacterial contamination in platelets are available on the market. They need to be divided into two different methodological principles: the cultivation methods and rapid methods. Cultivation or incubation methods require some time for signal production as they depend on growth of microbes. Thus, they have to be combined with early sampling, i.e., the sample to be examined has to be drawn from the blood component 1 day after donation. Their advantage is the relatively uncomplicated implementation into the logistics of blood banks. Because of the initially very low count of bacteria after donation, a certain small sampling error in application of that strategy remains. Rapid methods are able to produce the diagnosis within a short time. Therefore, they allow postponing of sample drawing, ideally up to the time immediately before transfusion. However, this procedure causes logistic complications. On the other hand, late sampling combined with a rapid method will prevent the transfusion of highly contaminated platelet concentrates leading to acute septic shock up to the death of the patient. Considering the sum of different aspects including the supply of patients, the potential improvement of microbial safety of platelet concentrates is comparable in both strategies. Clin Chem Lab Med 2008;46:926–32.

Culture-based and molecular assays have been developed for the screening of platelet concentrates and other blood components for bacterial contaminations. In this review, the principles of the assays are outlined. The focus of this review is the assessment of the analytical qualities of the methods. Spiking studies by adding defined levels of a wide range of bacteria to the complex biological matrix provide the first basis to evaluate and compare the qualities of methods for bacterial detection. The sensitivity acceptable for reliable screening for bacteria critically depends on the timing of either early sampling (within a period of up to 24 h after preparation of the blood component) or late sampling (a few hours before issuing the blood component). Large screening studies are essential to confirm both adequate sensitivity and specificity of the testing. In the ideal setting, these studies are prospectively planned and include systematic surveillance of adverse events in response to the administration of the screened products. The findings from sterility testing (predominantly with automated systems for detection of bacteria based on CO 2 generation) of more than 550,000 platelet concentrates in 13 studies are summarised. The limitations of the early sampling and the “negative-to-date” strategy to issue platelet concentrates are addressed. A few reported cases of probable transmission of bacteria by platelet transfusion despite negative screening tests emphasise the need to further develop optimised methods for testing of bacteria blood components. Clin Chem Lab Med 2008;46:933–46.

Since bacterial infection of the recipient has become the most frequent infection risk in transfusion medicine, suitable methods for bacteria detection in blood components are of great interest. Platelet concentrates are currently the focus of attention, as they are stored under temperature conditions, which enable the multiplication of most bacteria species contaminating blood donations. Rapid methods for bacteria detection allow testing immediately before transfusion in a bed-side like manner. This approach would overcome the sampling error observed in early sampling combined with culturing of bacteria and would, at least, prevent the transfusion of highly contaminated blood components leading to acute septic shock or even death of the patient. Flow cytometry has been demonstrated to be a rapid and feasible approach for detection of bacteria in platelet concentrates. The general aim of the current study was to develop protocols for the application of this technique under routine conditions. The effect of improved test reagents on practicability and sensitivity of the method is evaluated. Furthermore, the implementation of fluorescent absolute count beads as an internal standard is demonstrated. A simplified pre-incubation procedure has been undertaken to diminish the detection limit in a pragmatic manner. Additionally, the application of bacteria detection by flow cytometry as a culture method is shown, i.e., transfer of samples from platelet concentrates into a satellite bag, incubation of the latter at 37°C, and measuring the contaminating bacteria in a flow cytometer. Clin Chem Lab Med 2008;46:947–53.

Although blood component transfusion is currently regarded as safe, adverse events may occur in recipients of these products. Among those, blood borne viral, bacterial and parasitic infections are best known. For detection of bacterial contamination in platelet concentrates various methods are available or under investigation. One of these methods, real-time polymerase chain reaction (PCR) with particular focus on real-time 16S rDNA detection, will be discussed in this review. Clin Chem Lab Med 2008;46:954–62.

Today, sterility of established parenteral drugs including biologicals, such as plasma derived products, is practically guaranteed. Bacterially contaminated products are extremely rare exceptions owing to the efficiency of the manufacturing processes in the pharmaceutical industry. In contrast, the manufacturing processes of cell based medicinal products or tissue preparations show much less defined conditions. The sterility of source materials cannot be guaranteed in many cases. As a rule, these source materials cannot be sterilised, as it holds true for the final products. Furthermore, the established methods for sterility testing are not applicable for cell preparations. Sterility of a restricted sample does not guarantee sterility of the whole preparation. Thus, small amounts of residual bacteria in the product can be overlooked and can grow up to enormous numbers during storage and shipping of cell based medicinal products. Considering these problems, there are some parallels in the warranty of microbial safety of cellular blood components. Therefore, the experiences collected in transfusion medicine in the past decade can be successfully used in the production of cell based medicinal products. Comparable to the situation regarding cellular blood components, there is a need for new principles in rapid bacteria detection. Clin Chem Lab Med 2008;46:963–5.

Background : Although mutations in the interferon (IFN) sensitivity determining region (ISDR) of hepatitis C virus (HCV) have been reported to be useful as a predictive viral factor for IFN therapy in patients infected with HCV-1b, such laboratory research has not been favorably translated into the clinic. To promote such translation, we attempted the establishment of a rapid and simple polymerase chain reaction (PCR) combined with melting curve analysis (MCA) to screen for mutations in the ISDR and for the monitoring of HCV quasispecies. Methods : A PCR-MCA protocol was established using in-house primers and hybridization probes designed according to the results of direct sequencing of 34 HCV-1b samples. Then, the performance of PCR-MCA was verified by comparing with mutation profiles obtained by direct sequencing and sequencing after cloning. Results : The MCA assay revealed that melting temperature (Tm) was inversely correlated with the number of nucleotide (nt) and amino acid substitutions in the ISDR deduced on the basis of the results of direct sequencing. A boundary Tm of 58.0°C allowed us to discriminate HCV genomes into two groups: one with a Tm >58.0°C had no or a low number of nt substitutions, while the other genomes with a Tm <58.0°C had a high number of nt substitutions, corresponding to wild-type in the former and mutant-type in the latter in respect of a clinical setting for IFN therapy. Moreover, this MCA assay provided precise discrimination of Tm between clones, reflecting the degree of the genetic complexity of HCV genomes. Conclusions : This study indicates that the MCA assay is useful to rapidly and simply screen the mutational status of the ISDR of HCV, as well as in using the ISDR as one of the targets for discriminating the genetic complexity of HCV genomes. The MCA assay could also be applicable as a convenient and useful screen of the genetic heterogeneity of clones relating to HCV quasispecies. Clin Chem Lab Med 2008;46:966–73.

Background : Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme involved in the metabolism of thiopurine drugs. To date, at least 25 single nucleotide polymorphisms have been reported in the TPMT gene, 23 of these are associated with reduced enzyme activity. Methods : The aim of the present study was to sequence the whole coding region of TPMT (exons 3–10) to identify known and novel TPMT sequence variants amongst healthy Estonians. Erythrocyte TPMT activity was also measured to carry out a genotype-phenotype comparison. Results : A total of 21 subjects were heterozygous for known TPMT alleles (* 2 , *3A , *3C , *9 , *12 ). Several other previously described intronic and exon polymorphisms were identified. Three novel mutations were detected −30T>A in exon 3, 10A>G in intron 3, and 145A>G in intron 10. Association analysis revealed four markers (114T>A, 94T>A, 460G>A, 719A>G) whose frequencies were significantly different in intermediate (enzyme activity ≤60 ng/mL/h) methylators compared to normal (enzyme activity 61–139 ng/mL/h) and high (enzyme activity ≥140 ng/mL/h) methylators (p<0.001). Haplotype analysis found one haplotype to be associated with intermediate TPMT activity. Conclusions : Our results point to several markers that predict reduced enzyme activity. None of the identified markers were associated with high enzyme activity. Clin Chem Lab Med 2008;46:974–9.

Background : Adult-type hypolactasia is characterized by the inability to digest lactose during adulthood, due to lactase (LCT) deficiency. It is usually diagnosed by the measurement of breath hydrogen increase after a lactose load (breath hydrogen test, BHT). A substitution of C to T at position –13910 bp upstream the LCT gene (rs4988235), in a regulatory region, was found to be strongly associated with the lactase persistence phenotype in North-European populations. Methods : We investigated the –13910 C/T polymorphism to determine LCT genotype distribution and to validate genetic testing for adult-type hypolactasia in a Southern European population. A total of 43 children referred for suspected lactose malabsorption were enrolled in the study, their parents and siblings (whole sample=112 individuals) also took the breath test, and all were enrolled for clinical monitoring and genotype determination. In addition, 125 unrelated blood donors from the same geographic area were genotyped for the calculation of allelic frequencies. The frequency of C/C genotypes was 70%. Results : The correlation between the C/C genotype (which should correspond to lactose non-digesters) and positive BHT in unrelated family founders was significant (χ 2 =16.7, p<0.002). The genetic test compared to the BHT had a sensitivity of 95% and 91% and a specificity of 48% and 55% in adults and children, respectively. Conclusions : Low specificity might be due to intrinsic limitations of the standard BHT or to other possible mutations, although no sequence variation was found upon sequencing a 253 bp fragment of the LCT regulatory region in asymptomatic individuals. Clin Chem Lab Med 2008;46:980–4.

Background : Genetic predisposition to hemochromatosis involves several different point mutations in the HFE gene. Routine testing for two such mutations (H63D and S65C) using real-time genotyping deserves special care, as these mutations are in close proximity (6 bp) of each other. Methods : A novel assay was designed for these two mutations based on a SimpleProbe assay format that allows for more flexibility in assay design, as it requires only one detection probe instead of the two probes that are commonly used with a hybridization probe based assay. Results : The SimpleProbe assay format yielded data that were easily interpreted without significant optimizing efforts. Conclusions : The SimpleProbe assay format offers some unique advantages compared to hybridization probes and offers a robust and interesting alternative to hybridization probes in the detection of genetic variations. This is the first time that the use of this assay format is described in the clinical literature. Clin Chem Lab Med 2008;46:985–6.

Background : 5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism. Previous studies have suggested an association between the MTHFR 1298A/C polymorphism and several diseases, such as cardiovascular and psychiatric diseases, neural tube defects, diabetes, and cancer. Currently, either PCR-restriction fragment length polymorphism (RFLP) technique or real-time PCR using Taqman assay are used to determine the MTHFR 1298 genotype. Methods : We developed a simple and efficient approach that employs mutagenically separated PCR to genotype MTHFR 1298A/C polymorphism. Two forward mutagenic allele-specific primers of different lengths for MTHFR 1298A/C were paired with the same reverse primer in a one-tube assay to genotype 20 genomic DNA samples. Results : Electrophoresis on 2.5% agarose gel showed two allele-specific fragments, a 113-bp A allele-specific and a 93-bp C allele-specific PCR product. The results were confirmed by the conventional PCR-RFLP method. Conclusions : We conclude that mutagenically separated PCR could be used as an alternative simple, reliable, and cost-effective method for the genotyping of MTHFR 1298A/C polymorphism. Clin Chem Lab Med 2008;46:987–9.

Background : Aging is associated with low-grade elevation of circulating inflammatory markers, leading to increased risk of morbidity and mortality. The Mediterranean diet has been suggested as a determinant of longevity. In the current study, we investigated the impact of the Mediterranean diet on inflammatory status in old subjects. Methods : Within the ZINCAGE study, 957 healthy old subjects (≥60 years old) from five European countries were recruited. Plasma interleukin (IL)-6, IL-8, monocyte chemoattractant protein, tumor necrosis factor-α, high-density lipoprotein cholesterol (HDL-C) and erythrocyte sedimentation rate (ESR) were measured. Dietary data were collected applying a food frequency questionnaire and were used to estimate adherence to the Mediterranean diet. Results : The Italians presented the greatest adherence to the Mediterranean diet, while the Polish the poorest. In females, higher diet score was significantly associated with lower body mass index and ESR and higher HDL-C levels (β=−0.127, p=0.003; β=−0.144, p=0.001; β=0.144, p=0.029, respectively). In males, diet score was negatively associated with IL-8 levels (β=−0.101, p=0.044). The Mediterranean diet was associated with reduced IL-8 concentrations in Greeks (β=−0.213, p=0.007). Conclusions : There were significant effects of the components of the Mediterranean diet on inflammation markers. The Mediterranean diet score is useful in assessing nutritional influence on immune status. Clin Chem Lab Med 2008;46:990–6.

Background : The role of proinflammatory cytokines in pelvic inflammatory disease (PID) is unclear. We therefore determined whether plasma proinflammatory cytokines, interleukin-1β (IL-1β), IL-6, IL-8 and tumor necrosis factor-α (TNF-α) were useful plasma markers in PID patients. Methods : Multiplex bead array analysis was used to measure the plasma levels of proinflammatory cytokines in 50 healthy controls as well as in 41 PID patients before and after routine protocol treatments. Results : IL-1β, IL-6, IL-8 and TNF-α were significantly elevated in PID patients before antibiotic treatment than after treatment. However, IL-8 was not significantly different between healthy controls and PID patients. The relative increase in ratio of IL-6 was significantly correlated with white blood cell count (r=0.448, p=0.003), neutrophil count (r=0.472, p=0.002) and C-reactive protein level (r=0.412, p=0.008). Conclusions : IL-1β, IL-6, IL-8 and TNF-α may play an important role in the pathogenesis of PID. These biomarkers, particularly IL-6, could be useful adjuncts for the clinical diagnosis of PID. Clin Chem Lab Med 2008;46:997–1003.

Background : Free radical-mediated oxidative stress has been implicated in the etiopathogenesis of several autoimmune disorders. Also, there is growing evidence supporting the role of reactive oxygen species in the pathogenesis of thyroid disorders. The aim of this study was to investigate the influence of hypothyroidism, hyperthyroidism, and their treatments on the metabolic state of oxidative stress, and antioxidant status markers. Methods : A total of 20 newly diagnosed patients with overt hypothyroidism due to Hashimoto's thyroiditis, 20 patients with overt hyperthyroidism due to Graves' disease, and 20 healthy subjects as the control group were enrolled in the study. Fasting blood samples (12 h), taken at the initiation, after the 30th and 60th day of therapy were analyzed for malondialdehyde, nitrite, vitamin E, vitamin A, β-carotene, ascorbate, and myeloperoxidase and superoxide dismutase activity. No patient presented additional risk factors for increased reactive oxygen species levels. Results : Malondialdehyde, nitrite, vitamin E, and myeloperoxidase activity increased in patients with hypothyroidism. After 2 months, the levels of nitrite and vitamin E were reduced to control levels by treatment. The patients with hyperthyroidism had increased levels of malondialdehyde and myeloperoxidase activity in comparison with the controls. Treatment with propylthiouracil attenuated these increments after 1 month. Conclusions : Our results reveal an increased generation of reactive oxygen species and impairment of the antioxidant system in patients with hyperthyroidism, and particularly in patients with hypothyroidism. These findings indicate that thyroid hormones have a strong impact on oxidative stress and the antioxidant system. Clin Chem Lab Med 2008;46:1004–10.

Background : This study was performed to clarify variations in breath isoprene concentrations with age, gender, body mass index (BMI) and total serum cholesterol. Our cohort consisted of 205 adult volunteers of different smoking background without health complaints. Total cholesterol in blood serum was measured in 79 of these volunteers. Methods : Mixed expiratory exhaled breath was sampled using Tedlar bags. Concentrations of isoprene were then determined using proton transfer reaction-mass spectrometry. Results : Isoprene concentrations ranged from 5.8 to 274.9 ppb, with an overall geometric mean (GM) of 99.3 ppb. There was no statistically significant difference in mean isoprene in breath between males and females (GM 105.4 and 95.5 ppb, respectively). Ageing led to a decrease in concentration in men, with an estimated slope of the regression line for log-transformed isoprene concentrations of –0.0049, but did not influence isoprene levels in women. We did not observe any significant correlation between isoprene breath content and cholesterol level in blood, even after adjusting for the possible influence of age. Similarly, no correlation was found between isoprene levels and BMI. Conclusions : Isoprene concentrations in exhaled breath showed gender-specific correlations with respect to age. Further investigations are necessary to clarify the relation between isoprene concentrations in exhaled breath and cholesterol levels and synthesis rates in blood. Clin Chem Lab Med 2008;46:1011–8.

Background : Brain natriuretic peptide (BNP), N-terminal proBNP (NT-proBNP) and to a lesser extent prohormone proBNP are recognized as biochemical markers of left ventricular dysfunction. In renal failure, interpretation of natriuretic peptide remains unclear, as natriuretic peptide levels may be not only be dependent on cardiac function and dimensions but also on renal function, fluid volume and removal by dialysis procedure including hemodiafiltration (HDF). The purpose of this study was (i) to assess BNP, NT-proBNP and proBNP levels and their correlation with clinical and echocardiographic data in chronic hemodialysis patients, and (ii) to investigate basal level alteration following HDF. Methods : Baseline clinical and echocardiographic parameters were collected in 31 dialysis patients without evidence of cardiac failure. Pre- and post-HDF BNP, NT-proBNP and proBNP concentrations were measured. Correlations between echocardiographic measurements and basal circulating peptides, between changes in peptide values and changes in fluid volume after HDF were investigated. Results : Baseline plasmatic levels were elevated (BNP=517±840 pg/mL, NT-proBNP=5340±6132 pg/mL and proBNP=3569±4683 pg/mL) and correlated with left auricular diameter and left ventricular mass index. HDF session induced a significant decrease of 39%, 59% and 36% for BNP, NT-proBNP and proBNP levels, respectively. This decrease was not correlated to post-HDF fluid removal or weight decrease. Correlation between BNP and proBNP was stronger (r 2 =0.88) than between NT-proBNP and proBNP (r 2 =0.54). Conclusions : Despite their elimination, BNP, NT-proBNP and proBNP could be potential markers of left ventricular remodeling in chronic renal failure patients on hemodialysis. According to these results, their cut-off values, however, need to be re-evaluated. Clin Chem Lab Med 2008;46:1019–24.

Background : Brain injury after head trauma can be detected by S-100B measurements in serum. Recent preliminary studies indicate that urinary levels of S-100B are also increased after head injury, a finding that is of possible clinical value. The aims of the present study are two-fold: to compare serum measurements of two assays, the Liaison Sangtec ® 100 system and the Elecsys ® S100 test, and to investigate to what extent they can detect and measure S-100B in urine. Methods : A total of 191 serum and 174 urine samples from 107 patients (children aged between 1 and 18 years following head trauma) were measured with both assays. The results were compared using correlation analysis and Bland-Altman difference plots. Results : Serum values of the Sangtec ® system ranged from 0.02 to 2.28 μg/L, and from 0.005 to 2.13 μg/L for the Elecsys ® test. Comparisons showed a clear correlation (correlation coefficient 0.80) but not an agreement between the methods. The Sangtec ® system could only detect S-100B in 20 out of 174 urine samples (range 0.02–0.06 μg/L), whereas the Elecsys ® test could detect S-100B in 171 samples (range 0.005–0.14 μg/L). No clear relation was observed between the two methods in urine analysis (correlation coefficient 0.60). Conclusion : The Sangtec ® and Elecsys ® assays are not interchangeable methods when analyzing S-100B in serum or urine samples after head injury. Clin Chem Lab Med 2008;46:1025–9.

Background : At present, measurements of troponins play a key role in the diagnosis of myocardial infarction. There is a consensus that a turnaround time (TAT) of 1 h or less should be achieved for cardiac marker assays. However, little is known about the real delays between the patient's arrival at the emergency department (ED) and the reporting of the test. Methods : In the present study, the TATs of a cardiac marker, troponin T (TnT), were analyzed at a central laboratory at Kuopio University Hospital in a population consisting of 215 patients who were admitted to the hospital ED. Four specific time points were recorded: 1) the time of the patient's arrival to the ED, 2) the time of the ordering of the test, 3) the time when blood samples arrived at the laboratory, and 4) the time of completion and reporting of the test. Results : The median diagnostic TAT from patient's arrival to the reporting of the TnT result was 122 min. The clinical median TAT was 46 min and the laboratory median TAT was 69 min. Laboratory TAT consisted of the preanalytical TAT at the ED (median 14 min), pre- and postanalytical TAT in the laboratory (median 40 min), and the analytical TAT (on average 10 min). The laboratory TAT was approximately half of the complete diagnostic TAT. Conclusions : On the basis of this analysis, we propose new concepts for the TATs in the diagnostic process: diagnostic TAT which consists of clinical and laboratory TAT. The focus should be shifted from the analytical TAT to the diagnostic process as a whole. Clin Chem Lab Med 2008;46:1030–2.

Background : Immunoassays allow the specific detection and quantitation of biological molecules in complex samples at physiologically relevant concentrations. However, there are concerns over the comparability of such techniques when the same assay is performed by different operators or laboratories. An international intercomparison study was performed to assess the uncertainty involved in the estimation of a protein cytokine concentration using a fluorescent ELISA. Methods : The intercomparison study method was based on a non-competitive sandwich immunoassay with an enhancement step to generate a fluorescent readout. The intercomparison was performed in two phases, with the uncertainty of the instrument determined separately from that of the assay. The 11 laboratories participating in the study represented national metrology institutes or nominated expert laboratories. Results : Participants were asked to determine an undisclosed concentration of interferon using a supplied standard. The mean participant estimate and experimental standard deviation of the mean was 3.54±0.22 mg/L, with the spread of data ranging around ±35% of the mean. The quantitation range of the ELISA and of participants' instruments displayed large variation that contributed to the overall uncertainty. Conclusions : Identified sources of uncertainty within the ELISA methodology included pipetting, data fitting, model selection and instrument/plate variation. Clin Chem Lab Med 2008;46:1033–45.

Background : A scatter plot combined with correlation and regression analysis to assess the agreement between analytical methods that measure the same quantity has a long tradition in clinical chemistry. However, only when linearity is absolute this approach may be useful to estimate whether the methods under comparison produce the same errors in measurement. The aim of this study is to provide a new original regression-based tool which does not suffer from the problems of the classical regression/correlation method and that is also easy to use and to interpret. Methods : To evaluate the performance of the new method, the results of some analytical determinations were compared by the new procedure, the Bland-Altman test, Passing-Bablok regression and Mountain plot. Results : All the tests used to compare the data allowed superimposable interpretations, both when methods under comparison were in agreement and in disagreement. Conclusions : The understanding of the new test is truly intuitive and it is reduced to the appraisal of how much the equation of the best-fit line (Y=X) is able to predict the value of the Y. The goodness of this prediction can be evaluated by the coefficient of determination, whose value is closely connected to the differences between the methods under comparison. The result of our simulation is that quite high agreement between the methods occurs for determination coefficient equal or greater than 0.98. Clin Chem Lab Med 2008;46:1046–9.