Volume 55, Issue 4

Inflammatory bowel diseases (IBDs) are chronic diseases that result from the inflammation of the intestinal wall, suspected in any patient presenting with intestinal symptoms. Until recently, the diagnosis was mainly based on both clinical and endoscopic arguments. The use of an easy, fast, reliable, non-invasive, and inexpensive biological assay is mandatory not only in diagnosis but also in evolutionary and therapeutic monitoring. To date, the fecal calprotectin is the most documented in this perspective. This marker allows the discrimination between functional and organic bowel processes with good performance. The determination of the fecal calprotectin level contributes to the evaluation of the degree of disease activity and to monitoring of therapeutic response.

The activation of the extrinsic coagulation pathway occurs after endothelial injury when the tissue factor (TF), a transmembrane protein located outside the vasculature, binds factor VII (FVII) or activated FVII (FVIIa). Once formed, the TF-VIIa complex activates both factor IX and X and initiates the coagulation process. The TF-VIIa complex is inhibited by both TF pathway inhibitor (TFPI) and antithrombin (AT). The interaction between TF-VIIa and AT induces FVIIa-AT complex formation, which is released into the plasma. Because AT reacts with FVIIa only when it is bound to TF, the circulating levels of FVIIa-AT reflect the degree of exposure of TF to blood. Preliminary clinical studies have shown higher plasma levels of FVIIa-AT complex both in patients with a prior arterial or venous thrombotic event. Increased plasma levels of FVIIa-AT have also been reported in a number of other prothrombotic conditions – antiphospholipid antibodies, solid and hematological malignancies, pre-eclampsia (PE), obesity and cardiac surgery. However, most of the studies published so far are retrospective and with a limited sample size. Larger prospective clinical studies are needed to confirm these findings and to assess the prognostic role of this possible new biomarker for activated coagulation.

It is now undeniable that laboratory testing is vital for the diagnosis, prognostication and therapeutic monitoring of human disease. Despite the many advances made for achieving a high degree of quality and safety in the analytical part of diagnostic testing, many hurdles in the total testing process remain, especially in the preanalytical phase ranging from test ordering to obtaining and managing the biological specimens. The Working Group for the Preanalytical Phase (WG-PRE) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has planned many activities aimed at mitigating the vulnerability of the preanalytical phase, including the organization of three European meetings in the past 7 years. Hence, this collective article follows the previous three opinion papers that were published by the EFLM WGPRE on the same topic, and brings together the summaries of the presentations that will be given at the 4th EFLM-BD meeting “Improving quality in the preanalytical phase through innovation” in Amsterdam, 24–25 March, 2017.

Comorbidities associated with obesity have become a worldwide public health concern. Obesity-associated hepatic steatosis is not benign, and the risk of developing severe liver disease is high. Currently, biopsy is the only clinical tool available for the diagnosis of pathological alterations in the liver. However, the procedure is painful and not without risk. As such, there is a need to identify non-invasive biomarkers of steatosis. There has been considerable progress in this area, but research appears to be limited to measurements of levels of certain parameters in patients with liver impairment relative to those of healthy controls. The clinically relevant aim should be to distinguish, at an early stage, those obese individuals with liver steatosis from those obese individuals without it. Plasma constituents that act as surrogates of altered hepatic energy metabolism in response to food intake are likely candidates. Targeted metabolomics, combined with quantitation of the metabolites involved, has been shown to be an efficient measurement tool. Indeed, the evaluation of exhaled volatile compounds might be sufficient, while other rapid, sensitive, and reproducible methods have been validated in preliminary studies in various clinical settings. Metabolomics methods are promising but require considerable expertise and sophisticated (and expensive) equipment not readily available in all centers. The challenge is to adapt this newly acquired, expanding knowledge to current, reasonably equipped clinical laboratories, while substantially reducing costs. Good outcomes are urgently required if effective prevention programs are to be developed to decrease the prevalence of liver disease.

Background: Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5. Methods: Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (T m ). A standardized software algorithm was used to assign amplicons as ‘reference’ or ‘variant’ as compared to duplicate reference sequence DNA controls for each SNP. Results: Intra-assay (n=5) precision of T m s for all SNPs was ≤0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/μL. Conclusions: The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of ‘reference’ or ‘variant’ available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

Background: Determination of the hepatitis C virus (HCV) genotype and discrimination between HCV subtypes 1a and 1b is still mandatory prior to anti-HCV treatment initiation. The aim of this study was to evaluate the performance of the recently introduced cobas ® HCV GT assay (Roche) and to compare it to two comparator assays. Methods: The cobas ® HCV GT assay is based on primer-specific real-time polymerase chain reaction (PCR). For comparison, the TRUGENE ® HCV 5′NC Genotyping Kit (Siemens) and the VERSANT ® HCV Genotype 2.0 Assay (Siemens) were employed. Accuracy of the new assay was determined using proficiency panels. For clinical evaluation, 183 residual clinical samples obtained from patients with chronic hepatitis C infection were included. Results: When accuracy was tested, panel members containing HCV subtypes 1a, 1b, and 3a were identified as expected; however, the new assay failed to identify low titer panel members containing HCV subtype 5a correctly. Of 183 clinical samples, 160 gave concordant results. For seven samples, an indeterminate result was reported with the cobas ® HCV GT assay and the remaining 16 samples were found discordant with one of the comparator assays. When time-to-results of the assays were compared, the new assay showed shorter total time and similar hands-on time per sample. Conclusions: The cobas ® HCV GT assay showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Due to the high level of automation, fast and reliable results are obtained with short hands-on time.

Background: Serum dihydrotestosterone (DHT) is an important analyte for the clinical assessment of disorders of sex development. It is also reportedly a difficult analyte to measure. Currently, there are significant gaps in the standardisation of this analyte, including no external quality assurance (EQA) program available worldwide to allow for peer review performance of DHT. We therefore proposed to establish a pilot EQA program for serum DHT. Methods: DHT was assessed in the 2015 Royal College of Pathologists of Australasia Quality Assurance Programs’ Endocrine program material. The material’s target (i.e. “true”) values were established using a measurement procedure based on isotope dilution gas chromatography (GC) tandem mass spectrometry (MS/MS). DHT calibrator values were based on weighed values of pure DHT material (>97.5% purity) from Sigma. The allowable limits of performance (ALP) were established as ±0.1 up to 0.5 nmol/L and ±15% for targets >0.5 nmol/L. Results: Target values for the six levels of RCPAQAP material for DHT ranged from 0.02 to 0.43 nmol/L (0.01–0.12 ng/mL). The material demonstrated linearity across the six levels. There were seven participating laboratories for this pilot study. Results of the liquid chromatography (LC) MS/MS methods were within the ALP; whereas the results from the immunoassay methods were consistently higher than the target values and outside the ALP. Conclusions: This report provides the first peer comparison of serum DHT measured by mass spectrometry (MS) and immunoassay laboratories. Establishment of this program provides one of the pillars to achieve method harmonisation. This supports accurate clinical decisions where DHT measurement is required.

Background: Preanalytical variables, such as sample collection, handling and transport, may affect patient results. Preanalytical phase quality monitoring should be established in order to minimize laboratory errors and improve patient safety. Methods: A retrospective study (2001–2013) of the results obtained through the Spanish Society of Clinical Biochemistry and Molecular Pathology (SEQC) External quality assessment (preanalytical phase) was performed to summarize data regarding the main factors affecting preanalytical phase quality. Our aim was to compare data from 2006 to 2013 with a previously published manuscript assessing the 2001–2005 period. Results: A significant decrease in rejection rates was observed both for blood and urine samples. For serum samples, the most frequent rejection causes in the first period were non-received samples (37.5%), hemolysis (29.3%) and clotted samples (14.4%). Conversely, in the second period, hemolysis was the main rejection cause (36.2%), followed by non-received samples (34.5%) and clotted samples (11.1%). For urine samples, the main rejection cause overall was a non-received sample (up to 86.1% of cases in the second period, and 81.6% in the first). For blood samples with anticoagulant, the number of rejections also decreased. While plasma-citrate-ESR still showed the highest percentages of rejections (0.980% vs. 1.473%, p<0.001), the lowest corresponded to whole-blood EDTA (0.296% vs. 0.381%, p<0.001). Conclusions: For the majority of sample types, a decrease in preanalytical errors was confirmed. Improvements in organization, implementation of standardized procedures in the preanalytical phase, and participation in a Spanish external quality assessment scheme may have notably contributed to error reduction in this phase.

Background: Laboratory evaluation through external quality assessment (EQA) schemes is often performed as ‘peer group’ comparison under the assumption that matrix effects influence the comparisons between results of different methods, for analytes where no commutable materials with reference value assignment are available. With EQA schemes that are not large but have many available instruments and reagent options for same analyte, homogenous peer groups must be created with adequate number of results to enable satisfactory statistical evaluation. We proposed a multivariate analysis of variance (MANOVA)-based test to evaluate heterogeneity of peer groups within the Croatian EQA biochemistry scheme and identify groups where further splitting might improve laboratory evaluation. Methods: EQA biochemistry results were divided according to instruments used per analyte and the MANOVA test was used to verify statistically significant differences between subgroups. The number of samples was determined by sample size calculation ensuring a power of 90% and allowing the false flagging rate to increase not more than 5%. When statistically significant differences between subgroups were found, clear improvement of laboratory evaluation was assessed before splitting groups. Results: After evaluating 29 peer groups, we found strong evidence for further splitting of six groups. Overall improvement of 6% reported results were observed, with the percentage being as high as 27.4% for one particular method. Conclusions: Defining maximal allowable differences between subgroups based on flagging rate change, followed by sample size planning and MANOVA, identifies heterogeneous peer groups where further splitting improves laboratory evaluation and enables continuous monitoring for peer group heterogeneity within EQA schemes.

Background: Sodium concentration is a frequently used marker to discriminate between differential diagnoses or for clinical follow-up. Pseudonatremia, as a result of indirect ion-selective electrode (ISE) measurements in automated chemistry analyzers, can lead to incorrect diagnosis and treatment. We investigated whether the estimated water content, based on total protein and lipid concentrations, can be used to reduce diagnoses of pseudonatremia. Methods: Indirect and direct ISE measurements of sodium were compared in blood samples from intensive care unit (ICU) (n = 98) and random non-ICU patients (n = 100). Differences between direct measurements using whole blood and lithium-heparin plasma were also determined. Water content, estimated by a linear combination of total protein and lipid concentrations, was used to correct indirectly measured sodium concentrations. The prevalence of pseudonatremia was evaluated in the ICU patient group. Results: An absolute difference of 3 mmol/L was observed between direct measurements using lithium-heparin plasma and whole blood, with higher concentrations in plasma. Additionally, we observed that differences between indirect and direct measurements displayed a linear relationship with the estimated water content. The prevalence of pseudohypernatremia after indirect measurements (32%) was reduced when measurements were corrected for water content (19%). Conclusions: In critically ill patients , sodium concentrations should be preferably measured by direct measurements. Whole blood is the preferred material for these measurements. For routine sodium analyses in other patients, correction using the estimated water content appears promising in reducing the prevalence of pseudohypernatremia by indirect measurements.

Background: Cerebrospinal fluid (CSF) leakage is a rare condition that can potentially lead to the development of serious complications. In the last decade, β-trace protein (β-TP) has been shown to be a valuable immunological biomarker that allows prompt and non-invasive identification of CSF leakage. At our institution, the measurement of β-TP has been included in the diagnostic work-up of CSF leakage for more than 10 years. According to our diagnostic algorithm, the presence of CSF in secretion is excluded when β-TP values are <0.7 mg/L, whereas β-TP values ≥1.3 mg/L indicate the presence of CSF in secretion. β-TP values between 0.7 and 1.29 mg/L indicate the presence of CSF if the β-TP ratio (β-TP secretion/β-TP serum) is ≥2. This study aimed to validate this diagnostic algorithm using clinically defined nasal/ear secretions. Methods: We performed a retrospective statistical analysis of three β-TP interpretation strategies using data of 236 samples originating from 121 patients with suspect CSF leakage received at our laboratory between 2004 and 2012. Results: The highest odds ratio was obtained when the proposed algorithm has been used for the interpretation of β-TP results, showing a sensitivity of 98.3% and a specificity of 96%. Positive and negative predictive values were 89.2% and 99.4%, respectively. Conclusions: Our data suggest that the proposed β-TP interpretation algorithm is a valuable tool for the diagnosis of CSF leakage in the clinical practice.

Background: Cost-impact models have indicated that in the USA, the use of antibiotic stewardship protocols based on procalcitonin (PCT) levels for patients with suspected acute respiratory tract infection results in cost savings. Our objective was to assess the cost impact of adopting PCT testing among patients with acute respiratory infections (ARI) from the perspective of a typical hospital system in urban China. Methods: To conduct an economic evaluation of PCT testing versus usual care we built a cost-impact model based on a previously published patient-level meta-analysis data of randomized trials including Chinese sites. The data were adapted to the China setting by applying the results to mean lengths of stay, costs, and practice patterns typically found in China. We estimated the annual ARI visit rate for the typical hospital system (assumed to be 1650 beds) and ARI diagnosis. Results: In the inpatient setting, the costs of PCT-guided care compared to usual care for a cohort of 16,405 confirmed ARI patients was almost 1.1 million Chinese yuan (CNY), compared to almost 1.8 million CNY for usual care, resulting in net savings of 721,563 CNY to a typical urban Chinese hospital system for 2015. In the ICU and outpatient settings, savings were 250,699 CNY and 2.4 million CNY, respectively. The overall annual net savings of PCT-guided care was nearly 3.4 million CNY. Conclusions: Substantial savings are associated with PCT protocols of ARI across common China hospital treatment settings mainly by direct reduction in unnecessary antibiotic utilization.

Background: After the re-introduction of ImmunoCAP ® ISAC sIgE 112 on the market, we undertook a study to evaluate the performance of this multiplex-based immunoassay for IgE measurements to allergen components. Methods: The study was carried out at 22 European and one South African site. Microarrays from different batches, eight specific IgE (sIgE) positive, three sIgE negative serum samples and a calibration sample were sent to participating laboratories where assays were performed according to the manufacturer’s instructions. Results: For both the negative and positive samples results were consistent between sites, with a very low frequency of false positive results (0.014%). A similar pattern of results for each of the samples was observed across the 23 sites. Homogeneity analysis of all measurements for each sample were well clustered, indicating good reproducibility; unsupervised hierarchical clustering and classification via random forests, showed clustering of identical samples independent of the assay site. Analysis of raw continuous data confirmed the good accuracy across the study sites; averaged standardized, site-specific ISU-E values fell close to the center of the distribution of measurements from all sites. After outlier filtering, variability across the whole study was estimated at 25.5%, with values of 22%, 27.1% and 22.4% for the ‘Low’, ‘Moderate to High’ and ‘Very High’ concentration categories, respectively. Conclusions: The study shows a robust performance of the ImmunoCAP ® ISAC 112 immunoassay at different sites. Essentially the same results were obtained irrespective of assay site, laboratory-specific conditions and instruments, operator, or the use of microarrays from different batches.

Background: AMiDot is a microdot array-based immunoassay that allows simultaneous detection of multiple autoantibodies on a single patient. We evaluated the AMiDot “Systemic Autoimmune Disease” (SAD) panel, which detects antibodies to 17 different antigens. Methods: AMiDot was performed on 184 samples from blood donors and on 280 randomly selected clinical samples containing antibodies to extractable nuclear antigens or to dsDNA. The results obtained by AMiDot on the clinical samples were compared to results obtained by EliA (Thermo Fisher) for anti-Ro60, anti-La, anti-RNP, anti-Scl-70, anti-CENPB, anti-Sm, and anti-Jo-1 and by Farr assay for anti-dsDNA. Discordant results were further analyzed by immunodot (D-tek). Results: Concordance between AMiDot and EliA was ≥87% and κ agreement ≥0.44. When compared to EliA and immunodot (in case of discordance between AMiDot and EliA), concordance improved to ≥91% and κ agreement to ≥0.77. The sensitivity of AMiDot (compared to EliA and immunodot, in case of discordance between AMiDot and EliA) was ≥93%, except for anti-Ro60 (84%). The concordance and κ agreement of AMiDot with the Farr assay (for dsDNA antibodies) was, respectively, 84% and 0.33. The sensitivity of AMiDot for dsDNA (compared to Farr assay) was 25%. The specificity was ≥97% (in blood donors as well as in clinical samples). The within-run imprecision was 9%–27% and the between-run imprecision 29%–39%. Conclusions: AMiDot offers an alternative to line immunodot assay. Individual antibody assays might suffer from low sensitivity.

Background: Myocardial injury after noncardiac surgery, as measured by troponin elevation, is strongly associated with mortality. However, it is unknown in which patients prognosis can be improved. The presence of kinetic changes of troponin may be associated with a worse prognosis and warrant more aggressive management. Therefore, we aimed to study the kinetics of troponin in patients with postoperative myocardial injury, and to determine the added predictive value of kinetic changes of troponin on mortality. Methods: This cohort study included patients with myocardial injury after noncardiac surgery. Troponin I (TnI) was measured on the first three postoperative days. The primary outcome was all-cause 1-year mortality. We studied both absolute and relative TnI changes, and determined the delta TnI that was associated with mortality to distinguish a rise-and-fall TnI pattern from a stable TnI pattern. Next, we determined the added predictive value of a rise-and-fall TnI pattern for mortality. Results: In total, 634 patients were included. The risk ratio (RR) for mortality increased significantly with an absolute delta TnI of ≥200 ng/L (RR 1.5, 99.4% CI 1.0–2.2, p=0.003). Using this delta TnI to define a rise-and-fall pattern, 459 patients (72%) had a stable TnI pattern and 175 patients (28%) had a rise-and-fall pattern. When added to a model including the highest TnI value and variables from the revised cardiac risk index (RCRI), the TnI pattern did not increase the predictive value for mortality. Conclusions: A postoperative TnI rise-and-fall pattern was associated with 1-year mortality, but had no added value in addition to the highest TnI level to predict 1-year mortality. Therefore, postoperative TnI kinetics are not useful for further mortality risk stratification in patients with myocardial injury after noncardiac surgery.

Background: P35 and P22 Toxoplasma gondii proteins are recognized by specific IgG at the early infection stage, making them ideal for acute toxoplasmosis pregnancy control. Both proteins have been studied to discriminate between acute and chronic toxoplasmosis. However, results were hardly comparable because different protein obtainment procedures led to different antigens, the reference panels used were not optimally typified, and avidity tests were either not performed or narrowly examined. Methods: We bioinformatically predicted P35 and P22 regions with the highest density of epitopes, and expressed them in pET32/BL21DE3 alternative expression system, obtaining the soluble proteins rP35a and rP22a. We assessed their diagnostic performance using pregnant woman serum samples typified as: not infected, NI (IgG−, IgM−), typical-chronic, TC (IgM−, IgG+), presumably acute, A (IgG+, IgM+, low-avidity IgG), and recently chronic, RC (IgG+, IgM+, high-avidity IgG). Results: rP35a performed better than rP22a to differentiate A from RC, the areas under the curve (AUC) being 0.911 and 0.818, respectively. They, however, performed similarly to differentiate A from TC+RC (AUC: 0.915 and 0.907, respectively). rP35a and rP22a evaluation by avidity ELISA to discriminate A from RC rendered AUC values of 0.974 and 0.921, respectively. The indirect ELISA and avidity ELISA results analyzed in tandem were consistent with those obtained using commercial kits. Conclusions: rP35a and rP22a features suggest that, with complementary use, they could replace parasite lysate for toxoplasmosis infection screening and for acute toxoplasmosis diagnosis. Our proposal should be validated by a longitudinal study and may lead to a reliable toxoplasmosis pregnancy control, performing tests in only one serum sample.