Volume 49, Issue 2

Background: The objectives of this article were 1) to identify current trends in critical values practices in North America, Europe, and other regions; 2) to describe progress toward harmonization of critical limits; and 3) to synthesize strategies that will encourage global consensus. Critical limits are described in national surveys. Critical value practices are guided by federal statutes, The Joint Commission regulations, and accreditation requirements in the US; by provincial healthcare agencies in Canada; by thought leaders and ISO EN 15189:2007 in Europe; and in SE Asia, mostly by ad hoc policies lacking statutory grip. Methods: Review of databases, literature, websites, federal statutes, litigation, official policies, current affairs, and accreditation agency requirements. Results and conclusions: Practical strategies will accelerate harmonization of critical values practices, as follows: a) continue national and international survey comparisons; b) clarify age, ethnic, and subject dependencies; c) standardize qualitative and quantitative decision levels for urgent clinician notification; d) monitor compliance and timeliness for safety; and e) alert high frequencies of critical values related to adverse events. New expectations and communication technologies present opportunities for enhanced performance using wireless closed-loop reporting with recipient acknowledgment to reduce phone calls and improve efficiency. Hospitals worldwide can benefit from developing consensus for critical values practices.

Ischemia modified albumin (IMA), as measured using the albumin cobalt binding test, is currently the most promising biomarker for early detection of ischemia before the onset of irreversible cardiac injury. This paper reviews the information available on IMA, including its pathophysiology, analysis, clinical applications and future perspectives. The data provided was identified by a search of MEDLINE using the terms IMA, biomarkers and ischemia. IMA may be useful to cover the complete diagnostic window of patients presenting with acute coronary syndromes (ACS) in the Emergency Department, along with the electrocardiogram and cardiac troponins. Preliminary data regarding the significance of IMA in the prognosis of either ACS or following revascularization need further study.

Background: Decreased brain-derived neurotrophic factor (BDNF) has been demonstrated in animal models and patients with depression. However, little is known about changes in BDNF in post-stroke depression (PSD). This study investigated the changes in serum BDNF in patients with PSD, and evaluated whether serum concentrations of BDNF were associated with BDNF gene Val66Met polymorphism. Methods: PSD patients were diagnosed in accordance with DSM-IV criteria, and the severity of depression was evaluated with the Hamilton Rating Scale for depression. Serum BDNF was measured twice, first at 7 days after the onset of stroke and then at 3–6 months after stroke. Val66Met polymorphisms of the BDNF gene were determined using the polymerase chain reaction-restriction fragment length polymorphism method. BDNF concentrations and Val66Met polymorphisms were also measured in 30 healthly controls. Results: A total of 93 patients admitted as a result of first time acute ischemic stroke were included. During the 6-month follow-up, 35 patients (37.6%) were diagnosed with PSD. Serum BDNF concentrations were decreased in PSD patients at 3–6 months after stroke (p<0.05). The serum BDNF concentrations were not associated with BDNF gene Val66Met polymorphisms in either patients or healthy controls. Conclusions: Serum concentrations of BDNF decrease in PSD patients and BDNF may play an important role in the pathogenesis of PSD. However, Val66Met polymorphisms are not associated with serum concentrations of BDNF. The mechanism of decreased serum BDNF requires further study.

Background: DNA somatic mutations of EGFR, KRAS, BRAF and PIK3CA in the epidermal growth factor receptor (EGFR) signaling pathway play critical roles in the response or resistance of tumors to targeted therapy with tyrosine kinase inhibitors (EGFR-TKIs). To provide a high-throughput (HTP) clinical testing service for detecting these mutations, we developed a novel platform, SurPlex ® -xTAG70plex-EGFR liquidchip. Methods: This platform was developed based on a universal 100-tag system. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were optimized and standardized. Results: A total of 70 alleles of somatic mutations of EGFR , KRAS , BRAF and PIK3CA can be detected simultaneously in one reaction from one formalin-fixed and paraffin-embedded (FFPE) slide within one day. Cross-reaction was <8% between individual amplimers and 70 different ASPE primers. The sensitivity for detecting mutants in the wild-type DNA was 1%–5%. Seventy-three FFPE samples with somatic mutations were used to validate the 70plex. Seventy-one showed a complete match, while two were not detected. Conclusions: A simple, accurate, sensitive HTP technology was developed and standardized for detecting simultaneously 70 different alleles of EGFR , KRAS , BRAF and PIK3CA gene mutations from FFPE tumor slides.

Background: Hepcidin has emerged as the primary regulator of iron homeostasis. Previous studies on assessing urinary hepcidin are limited. We developed a method for quantifying hepcidin-25 (Hep-25) in plasma using surface-enhanced laser-desorption-ionization time-of-flight mass spectrometry (SELDI-TOF/MS) and a 25-AA peptide as reference standard. The aims of the study were 1) to assess the performance of this method in different conditions of iron metabolism disorders; 2) to assess the diagnostic validity of non-invasive serum biomarkers in the identification of iron overload. Methods: Validation of the method was performed in 10 patients with type I hemochromatosis (HE) and in 177 subjects previously enrolled in a general population epidemiological study. Among the latter group, 17 had non-alcoholic fatty liver disease, 10 had chronic hepatitis C, and 150 subjects had normal ultrasound, normal liver function tests (LFTs), an alcohol intake <20 g ethanol/day and were negative for the C282Y mutation. The following biomarkers were assayed in each case: plasma Hep-25, C282Y and H63D mutations of the HFE gene; serum iron, ferritin (SF), transferrin saturation, transaminases, γ-glutamyltransferase (GGT), glucose, insulin, total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglycerides. Results: Plasma Hep-25 concentrations were higher in HCV+ patients (26.3±7.2 nmol/L) than in controls, and correlated positively with SF (p<0.001). H63D heterozygous subjects revealed a pattern of iron overload that was significantly higher than H63D wild type subjects. Analyzing the data with the Biomarker Pattern 5.0.2. software to identify the most significant biomarkers for discriminating between HE cases and controls allowed us to produce an algorithm with four terminal nodes, which included glucose >4.8 mmol/L and Hep-25/SF ratio ≤6.6 as the main splitters. These variables enabled the correct diagnosis of HE with 100% sensitivity, 93% specificity and an area under the receiver operating characteristic (ROC) curve of 0.993. Conclusions: Our plasma Hep-25 mass spectrometry method yields measurements that reflect pathological and genetic influences; simple non-invasive biomarkers (Hep-25/SF ratio and glucose) can predict the presence of HE.

Background: Biochemical markers have problems in distinguishing iron deficiency anemia (IDA) from anemia of chronic disease (ACD), and the combined state of iron-restricted erythropoiesis (IRE) with ACD (ACD/IRE). We investigated the extent to which hepcidin-25, a potential marker for the evaluation of iron metabolism, enables the differentiation of the states above, and to assess its correlation with convential markers of iron deficiency. Methods: One hundred and fifty-five patients with anemia were classified as having IDA, ACD or ACD/IRE using clinical findings, biochemical markers and hematological indices. The diagnostic performance of hepcidin-25 alone or in combination with the reticulocyte hemoglobin content (CHr) was evaluated using receiver-operating characteristic curve analysis and multivariate analysis. Hepcidin-25 was determined using an isotope-dilution micro-HPLC-tandem mass spectrometry method. Results: Hepcidin-25 correlated with biochemical markers of iron deficiency but not with hematological indices. Use of a hepcidin-25 cut-off of ≤4 nmol/L allowed the differentiation of IDA from ACD and ACD/IRE, but not the discrimination of ACD from ACD/IRE in patients with severe inflammation. Furthermore, the discrimination of ACD/IRE from ACD required the combination with CHr. Conclusions: Hepcidin-25 is primarily an indicator for decreased body iron levels, but not for IRE. The combination of hepcidin-25 with CHr in a diagnostic plot (hepcidin-25 plot) might be useful for the differentiation of ACD from ACD/IRE and IDA.

Background: This cross-sectional multicentric survey study aimed to assess the quality of the extra-analytical phase of laboratory activities in some developing European countries and Mexico. We assessed the quality of the extra-analytical practices in participating laboratories regarding the: a) sample acceptance criteria; b) phlebotomy procedures; c) test results reporting and d) recording non-conformities. Methods: A survey was performed during the April–May 2009. A total of 15 clinical laboratories from the following countries were included: Bosnia, Croatia, Czech Republic, Hungary, Mexico, Poland, Portugal, Romania, Serbia and Ukraine. Questions were scored (scores from 1–4) and average scores was calculated for each category. Results: The overall score for all respondents (n=443) was 3.10±0.33. The average score was 3.11±0.56 for sample acceptance criteria, 2.76±0.58 for phlebotomy and 3.34± 0.53, for test results reporting (F=116.49; p<0.001). Laboratory accreditation was associated with better practices and higher overall quality of the extra-analytical procedures (F=16.62; p<0.001). Moreover, the highest scores for sample acceptance criteria (F=8.32; p<0.001), phlebotomy procedures (F=13.28; p<0.001) and for reporting non-conformities (F=33.62; p<0.001) were observed for accredited laboratories or laboratories under preparation for accreditation. Conclusions: The overall quality of the extra-analytical practices in countries in this survey is not satisfactory. Phlebotomy practices are the most critical extra-analytical activity. Since laboratory accreditation was associated with better practices and higher overall quality of the extra-analytical procedures, we believe that the most significant improvement could be made by implementing the total quality management system and standardizing laboratory procedures.

Background: Blood biomarkers are subject to pre-analytical variability. In many cases, the stability of important new tissue biomarkers during freeze cycles and storage has not been studied sufficiently. Methods: To test the stability of matrix metalloproteinases-7 (MMP-7) and their tissue inhibitors (TIMP-1), vascular growth factors (VEGF) and VEGF-receptor, serum samples were frozen and then thawed up to six times. The impact of storage temperature was investigated using an accelerated stability testing protocol. Stability at –20°C and –75°C was calculated using the Arrhenius equation. Results: The average concentration of TIMP-1 was stable, even after six freeze/thaw cycles. One thawing did not change the concentration of MMP-7 and VEGF-receptor. However, repeated freeze/thaw cycles increased the measured values significantly. Decreases in VEGF concentrations were dramatic, even after the first freeze/thaw cycle. According to the Arrhenius calculation, MMP-7 showed excellent stability, at least 5 years at –20°C and several 100 years at –75°C. The VEGF-receptor maintains 90% of its initial concentration at –20°C over 3 months, and decades at –75°C. TIMP-1 and VEGF showed poor stability with cryopreservation, even at –75°C. Conclusions: The stability of MMP-7, TIMP-1, VEGF or VEGF-receptor in biobanking is highly variable, and this should be taken into account in the interpretation of results. A temperature –20°C is unsuitable for prolonged storage of the biomarkers investigated, and repeated thawing of sera is not recommended. VEGF is especially unstable and should be quantitated using serum that has never been frozen.

Background: The relationship between asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) plasma concentrations and acute heart failure is unknown. We evaluated ADMA and SDMA in patients with acute dyspnea. Methods: We studied 57 dyspneic subjects (50–95 years), with estimated glomerular filtration rate (eGFR) ≥30 mL/min/1.73 m 2 , presenting to the emergency department. Troponin I, N terminal-proBNP (NT-proBNP), ADMA, and SDMA were measured. Electrocardiogram, chest X-ray and lung ultrasound were performed. Patients were classified into cardiogenic dyspnea and non-cardiogenic dyspnea, and were also classified on the basis of renal function according to their eGFR. Results: Two-way analysis of variance demonstrated that ADMA and SDMA did not differ for type of dyspnea, but increased in renal dysfunction. NT-proBNP significantly increased both in cardiogenic dyspnea and renal dysfunction. Multiple regression analysis demonstrated that after adjustment for troponin and dyspnea, the only variables which significantly correlated with SDMA plasma concentrations were renal function (β=–0.47, p<0.001) and NT-proBNP (β=0.28, p=0.02). Conclusions: Neither type of dimethylarginine showed cardiogenic dyspnea to be a determinant for plasma concentrations. Renal dysfunction was a confounder for both ADMA and SDMA.

Background: Stone composition has changed substantially over the past decades in developed countries. We studied urinary stone composition in Tunisian children. Methods: We studied 205 children ranging from 3 months to 16 years old age and who were diagnosed as having urinary stones (122 boys and 83 girls). Stone analysis was performed respectively using a stereomicroscope and by infrared spectroscopy to determine, respectively, the morphological type and molecular composition of each. Results: Of 205 stones analysed, 77 (37.5%) were composed of a single constituent. Calcium oxalate (CaOx) stone were predominant, being a constituent of 54.7% of stones, followed by calcium phosphate and purines; 14.6% of cases. Calcium oxalate monohydrate was more frequently seen in girls (p<0.01), while struvite was more common in boys (p<0.001). Teenagers were more affected by whewellite stones compared with infants (p<0.05). Purine stones were predominant in 20% of cases, but its prevalence decreased with age. Conclusions: The increase in calcium oxalate stones in school age children and the decrease of struvite and purines stones confirm the change in the etiology of urolithiasis with respect to age.

Background: Heparin binds positively charged electrolytes. In blood gas syringes, electrolyte-balanced heparin is used to prevent a negative bias in electrolyte concentrations. The potential pre-analytical errors introduced by blood gas syringes are largely unknown. Here, we evaluate electrolyte concentrations in non-anticoagulated blood compared with concentrations measured in electrolyte-balanced blood gas syringes. Methods: Venous blood was collected into plain tubes. Ioni-zed calcium, potassium, sodium and hydrogen ions were analyzed directly using a blood gas analyzer and the remaining blood was collected into different blood gas syringes in random order: Preset (Becton Dickinson), Monovette (Sar-stedt) and Pico 50-2 (Radiometer). Results: Ionized calcium and sodium concentrations were significantly lower in blood collected in Becton Dickinson and Sarstedt syringes compared to non-heparinized (NH) blood. The mean bias exceeded biological variation-based total allowable error, which in most cases leads to clinically misleading individual results. In contrast, ionized calcium concentrations in blood collected in Pico 50-2 syringes were identical to values obtained from NH blood. Sodium showed a minor, yet statistically significant, bias. Conclusions: Despite the fact that blood gas syringes now contain electrolyte-balanced heparin, one should be aware of the fact that these syringes can introduce pre-analytical bias in electrolyte concentrations. The extent of the bias differs between syringes.

Background: Since the introduction of liquid chromatography-mass spectrometry (LC/MS) for assessing vitamin D status, it has been shown that the C-3 epimer can account for a significant proportion of circulating 25-hydroxyvitamin D 3 (25OHD 3 ) concentrations in infants. However, some question whether monitoring a single MS transition at a chromatographic retention time typical for 3-epi-25OHD 3 sufficiently warrants conclusions about the identity of the substance generating the signal. Therefore, we aimed to substantiate the evidence for 3-epi-25OHD 3 in infants by collision induced dissociation (CID)-MS/MS product ion scans. A second objective was mass spectrometric investigation of the presence and prevalence of the 3-epi metabolite in serum from adults. Methods: Serum samples from six infants and 32 adults were studied using an ultra performance LC/tandem MS (UPLC/MS/MS) method designed to separate the 3-epi-25OHD 3 from 25OHD 3 . Samples were submitted to liquid/liquid extraction and Sephadex LH-20 fractionation, prior to column-switching UPLC with MS/MS recording of CID product ion spectra of the [M+H] + precursor ion. The respective standards were analyzed under identical UPLC/MS/MS conditions for comparison. Results: In the chromatograms of all samples, two peaks eluted with retention characteristics and spectra closely matching those observed for the 25OHD 3 and the 3-epi standards. The percentage of the 3-epi metabolite relative to 25OHD 3 in infants ranged from 15% to 41%, and in adults from 2.5% to 17%. Conclusions: This preliminary finding suggests that the prevalence of 3-epi-25OHD 3 in serum of infants is considerable, and that even in adults the concentrations of this form should not be neglected.

Background: Several studies suggest that the detection of nucleated red blood cells (NRBCs) in hospitalized patients indicates an increased risk of mortality. This study evaluates the impact of low arterial oxygen partial tension (pO 2 ) on NRBC appearance and prognosis in NRBC positive patients. Methods: NRBCs in blood, arterial blood gases, and other laboratory parameters were monitored daily in 234 surgical intensive care patients. pO 2 was assessed in relation to mortality and the detection of NRBCs. Results: NRBCs were found in 67 patients (28.6%). Mortality was significantly higher in NRBC positive patients (41.8%, 28/67) than in those that were NRBC negative (3.0%, 5/167). Multivariate logistic regression showed an increased mortality in NRBC positive patients (odds ratio 5.79; 95% confidence interval (CI) 1.07–31.33, p<0.05). NRBC positive patients showed significantly lower pO 2 levels during intensive care treatment than NRBC negative patients. Prior to the initial detection of NRBCs in the peripheral blood, pO 2 levels were significantly lower in patients who died than in surviving patients. After the first appearance of NRBCs, no significant difference in pO 2 between these groups was found. Conclusions: The detection of NRBCs is of prognostic significance concerning patient mortality. In NRBC positive patients, hypoxemia occurs more frequently. Low-levels of pO 2 seem to precede the appearance of NRBCs, especially in those patients with high risk of mortality.

Background: The potential use of cystatin C was recently assessed in kidney transplantation. A new particle-enhanced turbidimetric immunoassay (PETIA) that uses sheep antibodies (Binding Site human cystatin C immunoassay) has been developed. Analytical performance of this new assay was evaluated. Clinical relevance was determined by comparison with a reference method in a cohort of kidney transplant patients. Methods: First, the analytical performance of the Binding Site cystatin C kit was tested on SPA PLUS ® and Hitachi ® analyzers. Second, a comparison study was performed using SPA PLUS ® analyzer against two other cystatin C methods (the Siemens-PENIA method on BNII ® and the Dako-PETIA application on Olympus AU640 ® ). Third, the glomerular filtration rate (GFR) was estimated using several predictive cystatin C- and creatinine-based equations and compared to GFR measured by an isotopic method ( 99m Tc-DTPA). These predictive algorithms were analyzed with respect to bias, precision and accuracy. Results: Total intra-assay and inter-assay coefficients of variation were below 5%. Values obtained with the SPA PLUS ® correlated with the Siemens-PENIA and the Dako-PETIA methods. The creatinine and cystatin C-based equation allowed reliable assessment of GFR in our population of renal transplantation. Conclusions : The use of algorithms based on cystatin C and creatinine could provide a reliable estimate of GFR in kidney transplantation.

Background: We validated the DiaSorin Liaison Calcitonin_II-Gen, an improved method for calcitonin (CT) measurements, compared this method with the Cisbio_h-CT kit and established the reference range of CT in a normal adult population. Methods: We determined the precision, functional sensitivity, traceability to the 2nd IS 89/620, linearity and measurement uncertainty, accuracy profile and β-expectation limits. We evaluated the specificity, the susceptibility to human anti-animal antibodies (HAMA), hook-effect and carry over. To establish a reference range, we selected 267 adults without renal insufficiency presenting with normal thyroid stimulating hormone (TSH), free thyroxin (T4) and calcium concentrations and without anti-thyroglobulin antibodies as our “reference” healthy population. We compared the method with Cisbio on 250 consecutive and 45 samples from a post-pentagastrin stimulation test. Results: Precision (expressed as CV) was <10% for the measurement range, functional sensitivity: 5.3 ng/L and the method was found linear until to a 1/10 dilution. Uncertainty ranged from 25% to 7.2%, and the risk that one result falls out of the ±20% acceptance limits was <5% between 2.9 and 1513 ng/L. The Bland and Altman plot showed no systematic bias between the two methods. The test is still prone to HAMA influence, does not present any hook-effect, although carry over was observed. Ninety-five percent of our adult reference population showed CT concentrations <7.4 ng/L, with an important gender difference: 95% of the men showed CT values <9.8 ng/L, whereas 95% of women were <4.0 ng/L. Conclusions: The Liaison Calcitonin_II-Gen is an analytically robust method. The important difference in gender observed in our population might lead to re-evaluation of the generally used “10 ng/L” cut-off in a multicentre prospective study.

Background: The ImmunoCAP © ISAC allows for the determination of specific immunoglobulin E (IgE) against 103 recombinant or purified allergen components in a single analytical step. The aim of our study was to perform a comparison of the specific IgE results measured with the microarray method to those obtained using the traditional method of ImmunoCAP © . Methods: We selected 86 clinically relevant patients on the basis of their specific IgE for recombinant allergens (ImmunoCAP © 250, Phadia). Also, we selected two patients with a high total IgE to evaluate the non-specific binding of IgE. All samples were screened with the ImmunoCAP © ISAC. Then, we compared the 555 specific IgE antibodies results provided by ImmunoCAP © ISAC with the specific IgE levels obtained with ImmunoCAP © 250. Results: We observed that 82 of the 384 results found to be positive with ImmunoCAP © were negative with ISAC © (concordance 78.65%). Of 171 negative results obtained with ImmunoCAP © , 11 were positive with ISAC © (concordance 93.57%). No non-specific binding was observed. Conclusions: Our results show that the ImmunoCAP © ISAC has good analytical performance when compared with the ImmunoCAP © 250 method. We did not observe any non-specific binding. However, better sensitivity for some clinically relevant allergen components, such as rPru p 3 is needed.

Background: High temperature and long heating time can be considered the main drawbacks of the conventional digestion methods of urinary iodine determination. The aim of this study was to assess the usefulness of a microwave for digestion and decomposition of urine interfering substances. It may help for shortening the time and increasing the safety of the method. Methods: In this study, two digestion methods were tested on urine samples. Random urine samples were processed by conventional electrical heat as well as new microwave digestion methods. The urine samples were digested using the two methods mentioned, and then urinary iodine was determined using a colorimetric reaction according to the Sandell-Kolthoff reaction. In order to increase speed and precision of the test, a microplate ELISA reader was used. Sensitivity, precision, and comparison of the results were assessed in both of the methods. Results: In the case of microwave digestion, only a 10-min period was needed for complete digestion of urine samples. The precision and recovery of the new digestion step was acceptable. Furthermore, comparison of final results of iodine content obtained using the microwave and conventional method showed good correlation. Conclusions: These results show that a microwave method for urine digestion is advantageous due to its safety and ease of use, and quick preparation of urine samples to eliminate interfering substances and release iodine. Thus, it can replace the conventional electrical heat digestion method.

Background: Creatinine concentrations in athletes could be higher than in sedentary people due to higher muscle mass; a correlation between body mass index (BMI) and creatinine concentrations has been described only in male athletes participating in different sports. Methods: Elite swimmers (28 males and 26 females) belonging to the National Teams of Slovenia were recruited during the period 1999–2009. The evaluation of possible modifications to serum creatinine concentrations during a single season were performed in 10 males and seven females who were evaluated four times during a season. Serum creatinine was analyzed using the Jaffe method. Pearson's simple correlation analysis was used to test the association between serum creatinine and BMI. Creatinine values and BMI were compared using the unpaired Student t-test. Creatinine values during the different phases of the season were compared using a one-way analysis of variance test. Results: The values for creatinine are related to BMI in male and female swimmers. It should be noted that there is a significant difference (p<0.001) for estimated glomerular filtration rate (eGFR) values obtained by Cockcroft and Gault equation, but not for the Modification of Diet in Renal Disease (MDRD) equation. The values of creatinine were stable in each athlete during various training cycle periods in the observed competition year. Conclusions: The study confirms that in a large and representative group of elite swimmers, creatinine concentration is strictly correlated to BMI. The use of creatinine-based eGFR formulas should be used with caution in athletes.

Background: Trisomy 21 risk estimation in first trimester pregnancies can be performed by a combined test based on ultrasound measurement of fetal nuchal translucency thickness and maternal plasma concentrations of free β human chorionic gonadotropin (hCGβ) and pregnancy-associated plasma protein A (PAPP-A). However, little knowledge exists regarding the biological variation of hCGβ and PAPP-A when the time interval between sampling increases. Methods: We estimated these variations from double mea-surements of hCGβ and PAPP-A in first trimester pregnancies in 167 women. Data were divided into three groups based on the number of days between sampling. The correlation coefficients and biological variation were estimated for each group. Results: The correlation coefficient between the first and second samples was 0.841 for hCGβ, and 0.706 for PAPP-A. The ranges for biological variation were 11.9%–48.5% for hCGβ and 31.6%–63.3% for PAPP-A, increasing with time between sampling. The average overall biological variation for hCGβ was 29%, and 49.7% for PAPP-A. Conclusions: We found high biological variation for plasma concentrations of hCGβ and PAPP-A, increasing with longer time intervals between sampling. From our data that showed high correlation of hCGβ and PAPP-A in the first and second sample, we found no reason to recommend retesting. However, new studies should clarify whether PAPP-A should be collected early, and hCGβ late, in the first trimester of pregnancy.

Background : Our aim was to analyze C-reactive protein (CRP) values in term and preterm infants and correlate non-infection-associated increases with various neonatal disorders. Methods : Retrospective cohort study that included all newborns hospitalized at a tertiary care center between 2004 and 2007 with documented CRP values in the first 3 days of life. Analysis of differences in CRP values between term and preterm newborns and cases with CRP increases in sepsis negative newborns. Results : For diagnosis of blood culture proven sepsis (19 and 14 cases, respectively) in 353 preterm and 179 term newborns, CRP at a cut-off of 8 mg/L had sensitivities of 53% and 86% and specificities of 91% and 88%, respectively. The area under the receiver operating characteristics curves were 0.799 and 0.890, respectively. Preterm newborns had lower median values compared to term newborns in sepsis positive (9 vs. 18.5 mg/L, p<0.001) and negative newborns (0.5 vs. 2 mg/L, p<0.001). Increases in individuals without infection were correlated significantly with meconium aspiration syndrome and surfactant application in term newborns (p=0.009 and 0.025, respectively) and with surfactant application and higher birth weight in preterm newborns (p<0.001 and 0.031, respectively). Conclusions : CRP values were significantly lower in preterm compared to term newborns, and its application in the diagnosis of sepsis in preterm newborns was not as reliable as in term newborns. Meconium aspiration syndrome, surfactant application, and high birth weight were associated significantly with increased CRP values.

Background: In the management of breast cancer, several algorithms for interpretation of measured concentrations during monitoring have been introduced, without objective evaluation of their performance regarding the distance of the starting concentration from the cut-off concentration. Methods: A computer simulation model has developed using parameters for the tumour biomarker CA 15-3 regarding biological variation and different rates of increase during progressive disease. Seven different algorithms, which include the cut-off point in the calculations, are applied to the simulated data corresponding to 1000 surrogate patients. Steady-state variation (CV-within-subject=14.9%) is based on Gaussian random numbers and an exponential increase in tumour growth (λ=0.009, 0.021, and 0.090 kU/day). Results: The main outcome of the simulations was that low starting concentrations (baseline concentrations) delay the detection of progressive disease (true-positive), whereas, baseline concentrations just below the cut-off value results in false-positive results of progression during steady-state situations. The algorithms investigated show varying susceptibility for baseline concentrations approaching the cut-off. Thus, three of the algorithms show >90% false-positives and three algorithms <5% false-positives when baseline concentrations were just below the cut-off point. Conclusions: Based on the simulations, it is possible to select the optimal algorithms for early detection of progressive disease and a low percentage of false-positives.

Background: Cystoscopy with urinary cytology is the gold standard for the diagnosis and follow-up of patients with tumors of the urinary bladder. The aim of the study was to evaluate the performance of the nuclear matrix protein 22 (NMP22) tumor marker test, BladderChek ® point-of-care test and voided urinary cytology for the detection and follow-up of bladder tumors. Methods: NMP22 was measured using an ELISA assay in stabilized voided urine and using the BladderChek ® test. Voided urinary cytology was performed on urine samples. Results were compared to cystoscopic findings and histopathological examination results after transurethral resection of the bladder lesion. Results: For the prediction of malignant histopathological result, sensitivity and specificity were 45.2% and 75.0%, respectively, for NMP22 at a cut-off of 7.5 kU/L, 17.7% and 100% for the BladderChek ® test and 37.0% and 100% for voided urine cytology. For the prediction of suspicious or positive cystoscopic finding, sensitivity and specificity were 40.4% and 72.1%, respectively, for NMP22 at a cut-off of 7.5 kU/L, 14.8% and 93.8% for the BladderChek ® test and 26.8% and 98.1% for voided urine cytology. Conclusions: The NMP22 quantitative test showed higher sensitivity and lower specificity compared with voided urine cytology, whereas the sensitivity of the BladderChek ® test was low. We could not recommend any of the three non-invasive tests as a replacement for cystoscopy for the diagnosis or follow-up of urinary bladder tumors.

Background: The identification of reliable markers for diagnosis of breast cancer has been thoroughly addressed by metabolic profiling using nuclear magnetic resonance (NMR) spectroscopy or imaging. Several clear diagnostic indicators have emerged using either in vitro analysis of tissue extracts, ex vivo analysis of biopsies or in vivo direct spectral observations. Most of the breast cancer characteristic metabolites could be assayed by mass spectrometry (MS) to exploit the superior sensitivity of this technique and therefore reduce the traumatic impact of current biopsy procedures. Methods: Following extraction, aqueous metabolite mixtures were obtained that were submitted to liquid-chromatography, electrospray-ionization, mass spectrometry (LC/ESI-MS) analysis to estimate the content of choline (Cho) and its phosphorylated derivatives, phosphocholine (PCho) and glycerophosphocholine (GPCho). The determinations were performed using 10 samples from breast tissue biopsies, surgical specimens and one single sample of a hepatic metastasis. In addition, some measurements were also repeated using high-resolution 1 H NMR spectroscopy to complement the mass spectrometry results. Results: The contents of Cho, PCho and GPCho in breast tissue extracts were estimated by LC/ESI-MS based on standard compound calibration curves. Sharply increased ratios of phosphorylated-to-unphosphorylated metabolites, PCho/ Cho and (PCho+GPCho)/Cho, were observed in all tumor samples, although without discrimination between benign and malignant lesions, contrary to samples from healthy individuals and from those with fibrocystic disease. Conclusions: The assessment of breast cancer markers by LC/ESI-MS is feasible and diagnostically valuable. In addition to high sensitivity, the approach also shows a resolution advantage for assaying choline derivatives compared to NMR, and could complement the latter.

Background: The current preoperative diagnosis of a thyroid mass relies on microscopic evaluation of thyroid cells obtained by fine needle aspiration biopsy (FNAB). More recently, FNAB has been combined with molecular analysis to increase the accuracy of the cytological evaluation. In this mono-institutional prospective study, we evaluated whether the routine introduction of BRAF testing in thyroid FNAB could help ameliorate the preoperative recognition of papillary thyroid carcinoma (PTC) in “suspended” or malignant cytological categories. Moreover, we investigated the prognostic role of the BRAFV600E mutation in PTC. Methods: BRAFV600E analysis was performed in thyroid FNAB from 270 patients classified into one of five cytological categories THY1, THY2, THY3, THY4, THY5. All subsequently underwent thyroidectomy±node dissection, from October 2008 to September 2009 in our Department. For each cytological category, we considered the definitive histological diagnosis of PTC and the presence of the BRAFV600E mutation. In 141 patients with a final tissue diagnosis of PTC, we correlated the presence of BRAFV600E with gender, age, histotype, TNM, size of the lesion, extracapsular extension, node metastases and multifocality. Results: The prevalence of the BRAFV600E mutation, among PTCs at final tissue diagnosis, was 69%. It improved the FNAB diagnostic accuracy from 88% to 91%. The BRAFV600E mutation was correlated with older age, classical variant of PTC, advanced stages in patients >45 years. Conclusions: BRAFV600E testing could play a role in improving the diagnostic accuracy of FNAB for PTC, representing a useful adjuvant tool in presurgical characterization of thyroid nodes in particular cases. There is an association between the BRAFV600E mutation and some clinico-pathological characteristics of PTC.