Volume 52, Issue 4

Inflammatory bowel diseases (IBDs), which comprise the two major clinical subtypes, Crohn’s disease and ulcerative colitis, incur high morbidity and potential mortality. The present study reviews data on the pathogenesis and diagnosis of IBDs. The pathogenesis depends on complex interactions between susceptibility genes, environmental factors, and innate and adaptive immunity, the understanding of which is crucial to discovering novel laboratory biomarkers. Traditional laboratory tests for the diagnosis, prognosis and assessment of disease activity of IBDs are reported on, and the biochemical properties, pre-analytical and analytical aspects and clinical utility of the fecal markers lactoferrin and calprotectin are described. DNA testing and established (ASCA and pANCA) and emerging (ACCA, ALCA, AMCA, OmpC) serum markers are described; a further aspect to be addressed is the clinical use of pharmacogenetics for the treatment of IBDs.

Crohn’s disease (CrD) and ulcerative colitis (UC) are the main inflammatory bowel diseases (IBD). IBD-specific humoral markers of autoimmunity in the form of autoantibodies have been reported first in the late 1950s by demonstrating the occurrence of autoimmunity in UC, while humoral autoimmunity in CrD can be traced back to the 1970s. Ever since, the pathophysiological role of autoimmune responses in IBDs has remained poorly understood. Notwithstanding, autoreactive responses play a major role in inflammation leading to overt IBD. In CrD, approximately 40% of patients and <20% of patients with UC demonstrate loss of tolerance to antigens of the exocrine pancreas. Glycoprotein 2 (GP2) has been identified as a major autoantigenic target of the so-called pancreatic antibodies. The previously unsolved contradiction of pancreatic autoreactivity and intestinal inflammation in IBD was elucidated by demonstrating the expression of GP2 at the site thereof. Intriguingly, GP2 has been reported to be a receptor on microfold cells of intestinal Peyer’s patches, which are believed to represent the origin of CrD inflammation. The development of immunoassays for the detection of antibodies to GP2 has paved the way to investigate the association of such antibodies with the clinical phenotype in CrD. Given the recently discovered immunomodulating role of GP2 in innate and adaptive intestinal immunity, this association can shed further light on the pathophysiology of IBD. In this context, the association of anti-GP2 autoantibodies as novel CrD-specific markers with the clinical phenotype in CrD will be discussed in this review.

In the past decades, the recognition of several inherited thrombophilic traits has greatly improved our knowledge of the pathogenesis of venous thromboembolism, explaining about half of all idiopathic cases. As a consequence, thrombophilia testing has enormously increased in the past years for various clinical conditions. In this paper, the current indications of the most commonly tested thrombophilic abnormalities (i.e., Factor V Leiden, prothrombin G20210A mutation, protein C, S and antithrombin deficiencies) are analysed. When used appropriately thrombophilia testing has a positive impact on the health care of the people tested and their relatives.

There has been increasing recognition of various laboratory markers of thrombophilia that are associated with increased risk of thrombosis either through hereditary (especially Factor V Leiden, prothrombin G20210A mutation, and protein C, S and antithrombin deficiencies) and/or acquired means (e.g., antiphospholipid antibodies) over past decades. This has led to an explosion of clinical requests for these markers, that has now become virtually uncontrolled, and seemingly inclusive of everyone who has had a thrombotic event. Although these haemostasis-related defects should be assessed in selective cases, the overuse (or misuse) of testing causes serious adverse outcomes and leads to the conclusion that, in general, testing for thrombophilia is futile.

Background: Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. Methods: We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. Results: The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. Conclusions: COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.

Background: Several recent studies have shown some discrepancies between 25-hydroxyvitamin D [25(OH)D] assay methods, despite some improvement in the past few years. The accuracy of 25(OH)D assay methods is still a real challenge for clinical laboratories. The aim of this study was to assess the agreement between a large panel of routine assays and a two-dimensional liquid chromatography/tandem mass spectrometry (2D LC-MS/MS) method, selected as the reference method. Methods: Forty-nine human plasma samples with only endogenous 25(OH)D 3 were analyzed with 11 different methods, especially with three LC-UV methods that differed in the extraction step. Seven routine immunoassays were also tested: two manual (RIA and EIA from IDS) and five fully-automated methods. The results of the 25(OH)D 3 assays were compared with those of the 2D LC-MS/MS method using weighted Deming regression analysis, Bland-Altman plots and concordance correlation coefficient (CCC). The ability of these methods to properly classify patients was evaluated by sorting results depending on vitamin D status. Results: The CCC was >0.90 for the three LC-UV methods and for most of the automated IA, meaning substantial agreement with 2D LC-MS/MS results. The ability to properly classify patients according to their vitamin D status was overall satisfactory for most of the methods tested (concordance >90%). Conclusions: The immunoassays available on Liaison, Isys, Architect and Elecsys, together with our in-house LC-UV method preceded by an SLE step met the minimum requirements for the assessment of vitamin D status in clinical laboratories.

Background: The aim of this study was to compare pregnancy-associated plasma protein A (PAPP-A) and the β-subunit of human chorionic gonadotropin (hCGβ) measured in maternal plasma at the first trimester screening, in women who later developed early or late pre-eclampsia (PE) to women with normal pregnancies. Methods: In the study were included 161 cases with PE and 88 controls with uncomplicated pregnancies. Plasma PAPP-A and hCGβ were measured between gestational age (GA) 8+2 and 14+0, and cases with early and late onset PE were compared with controls. Results: Median of hCGβ (MoM) was lower in women with early (p<0.05) and late PE (p<0.05) compared to healthy pregnant women. Median (range) of hCGβ (MoM) for women with early onset PE, late onset PE, and women with healthy pregnancies, were 0.75 (0.30–1.21), 0.93 (0.26–3.18), and 0.97 (0.33–4.12), respectively. No significant difference in the median of PAPP-A (MoM) was found between the groups. Conclusions: hCGβ was significantly lower in pregnancies that subsequently developed PE and might be of use in the effort of trying to find ways to predict PE. No significant difference was found for PAPP-A in our study.

Background: S100B protein measurement in blood is proposed to exclude the presence of computed tomography (CT) lesions after minor head injury (MHI). We aimed to validate S100B as an accurate and valuable screening tool for MHI diagnosis in a large multicenter study, as well as: 1) to evaluate whether a second S100B blood level determination 3 h after the first one would be informative; 2) to compare the bioclinical performances of the two commercially available automated methods of measurement of S100B for the screening of patients. Methods: Four thousand and thirty MHI subjects were enrolled in a prospective observational multicenter study; results for serum S100B measurement determined within 3 h after the clinical event (H0) then at H3 were compared to that of cranial CT scans performed with 6 h following the presentation to emergency department. Both the Diasorin and the Roche Diagnostics assays were systematically performed. Results: Cerebral lesions on CT scan were identified with sensitivity and negative-predictive value (NPV) of 96.3% and 99.4% (Diasorin, 1 dissonant case), and of 100% and 100% (Roche Diagnostics, no dissonant case). Sensitivity and NPV at H3 appeared lower than those at H0, due to the rapid decrease in S100B levels. Conclusions: Serum S100B level on admission of patients with MHI is an accurate and useful screening tool to exclude intracranial lesions. Performing a second late S100B level determination is not informative. The two automated immunoassays appear usable in a similar manner, although the two methods are not interchangeable.

Background: New urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid binding protein (L-FABP), and kidney injury molecule-1 (KIM-1) open the opportunity to detect kidney injuries in early stages. Our study aimed at evaluating NGAL, L-FABP, and KIM-1 in comparison to established markers of urine protein differentiation for detection of renal dysfunction. Methods: On the basis of the PROTIS expert system (for differentiation of glomerulo-/tubulopathy) urine and plasma samples of 263 randomly selected patients were routinely examined (urine: total protein, albumin, IgG, α 1 -microglobulin, creatinine, and dip stick results for leukocytes, blood, protein, glucose, pH, and nitrite; plasma: creatinine and cystatin C) followed by the analysis of the new urine biomarkers NGAL (CMIA), L-FABP (ECLIA), and KIM-1 (ELISA). Results: Of the three new markers L-FABP showed the highest correlation with α 1 -microglobulin (r=0.76, p<0.01) and was closest associated with the degree of tubular proteinuria assessed by the PROTIS system. NGAL distinguished the PROTIS proteinuria groups with distinctive tubular proteinurias from the controls as well, but revealed a marked diagnostic influence by leukocyturia. Urinary KIM-1 revealed only a weak diagnostic value for the detection of renal injury. Conclusions: Urinary NGAL and L-FABP proved to be promising candidates for detecting injuries of the renal tubular system over a broad range of clinical conditions. L-FABP showed a better diagnostic performance and a lower interference by leukocyturia and hematuria than NGAL. Both markers may serve as sensitive tissue injury markers in addition to the established markers of renal functional impairment.

Background: Automated systems for antinuclear antibody analysis are being introduced. The aim was to evaluate whether automated quantitative reading of fluorescence intensity is clinically relevant and allows for value-added reporting of test results. Methods: Consecutive samples (n=260) were used to correlate fluorescence intensity with end-point titer. Moreover, 434 samples from controls (150 healthy blood donors, 150 chronic fatigue syndrome, and 134 diseased controls) and 252 samples (obtained at diagnosis) from patients with systemic rheumatic diseases were screened for antinuclear antibodies (1:80) on HEp-2 cells using NOVA View ® , and likelihood ratios were calculated for fluorescence intensity result intervals. Results: There was a significant correlation between end-point titer and fluorescence intensity. Likelihood ratios for a systemic rheumatic disease increased with increasing fluorescence intensity. The likelihood ratio for a systemic rheumatic disease was 0.06, 0.18, 0.51, 5.3, and 37.5 for a fluorescence intensity of ≤66, 67–150, 151–300, 301–1000, >1000, respectively. A range of 31%–37% of the patients with Sjögren’s syndrome, systemic sclerosis or systemic lupus erythematosus had fluorescence intensities >1000. Conclusions: Estimation of fluorescence intensity by automated antinuclear antibody analysis offers clinically useful information. Likelihood ratios based on fluorescence intensity test result intervals aid with the interpretation of automated antinuclear antibody analysis and allow value-added reporting.

Background: Highly-sensitive cardiac troponin (cTn) assays are being introduced into the market. In this study we argue that the classification of cTn assays into sensitive and highly-sensitive is flawed and recommend a more appropriate way to characterize analytical sensitivity of cTn assays. Study: The raw data of 2252 cardiac troponin I (cTnI) tests done in duplicate with a ‘sensitive’ assay was extracted and used to calculate the cTnI levels in all, including those below the ‘limit of detection’ (LoD) that were censored. Duplicate results were used to determine analytical imprecision. Results: We show that cTnI can be quantified in all samples including those with levels below the LoD and that the actual margins of error decrease as concentrations approach zero. Conclusions: The dichotomous classification of cTn assays into sensitive and highly-sensitive is theoretically flawed and characterizing analytical sensitivity as a continuous variable based on imprecision at 0 and the 99th percentile cut-off would be more appropriate.

Background: The relationship between fractional cholesterol esterification rate in plasma or serum high-density lipoprotein (HDL) (FER HDL ) and lipoprotein subfractions and other cardiovascular disease (CVD) risk factors has been demonstrated. However, the current method for measuring FER HDL requires fresh serum samples and radioactive labeling of the samples, making it impractical for use in clinical laboratories. In this study, we developed a simple and precise HPLC method for the measurement of FER HDL . Correlations between FER HDL and CVD risk factors were evaluated in 119 healthy volunteers. Methods: Fasting blood samples were collected and serum was isolated within 2 h. Serum HDL was prepared by precipitation of apolipoprotein B (apoB)-containing lipoproteins with dextran sulfate and magnesium chloride. HDL fractions were divided into two aliquots and incubated at 0°C and 37°C, respectively, for 1 h. Free cholesterol in the HDL fractions was analyzed by HPLC. FER HDL was calculated as the percent decrease of free cholesterol during incubation. Results: The esterification reaction of HDL free cholesterol was not linear, but the measured FER HDL was stable when serum samples were stored at room temperature for <4 h, or at 4°C for <24 h. The intra-assay and total CVs for FER HDL measurements were 1.0%–2.1% and 1.6%–3.8%, respectively. Results of 119 healthy volunteers showed that FER HDL was positively correlated with age, BMI, blood pressure, total cholesterol (TC), triglyceride (TG) and small dense low-density lipoprotein-cholesterol (LDLb-C), and negatively correlated with HDL-C and HDL2-C. FER HDL has shown to be a predictor of HDL and LDL subfraction distributions. Conclusions: This method is simple, non-radioactive and precise and will be useful in prediction of lipoprotein subfraction distributions and in clinical assessment of CVD risks.

Background: The unique properties of metallic nanoparticles have enabled their utilization in biosensing applications. A novel assay for the detection of hepatitis C virus (HCV) RNA in serum specimens has been developed using magnetic nanoparticles and unmodified cationic gold nanoparticles (AuNPs). Methods: HCV RNA was extracted using magnetic nanoparticles functionalized with an oligonucleotide specific to HCV RNA. Extracted RNA is reacted with oligonucleotide sequence specific for HCV RNA in presence of unmodified cationic AuNPs. In positive samples, AuNPs are aligned onto the phosphate backbone of the RNA and their aggregation changes the solution color from red to blue. In the absence of target, solution color remains red. The assay has been tested on 50 serum clinical samples (25 HCV positive and 25 controls). Results: The dual nanoparticles assay detected HCV RNA in serum and generated comparable results to real-time PCR. The assay had specificity and a sensitivity of 96% and 96.5%, respectively, and a detection limit of 15 IU/mL. Conclusions: The developed colorimetric dual nanoparticles HCV RNA assay is simple and inexpensive and can be used for rapid detection of unamplified HCV RNA in serum. Similar sensing platforms can be developed to detect other nucleic acid targets.

Background: The pathophysiology of sepsis is incompletely understood. Impaired bioavailability of L-arginine, the substrate for NO synthesis, is linked to sepsis severity, and plasma arginase has been linked to hypoargininemia in other disease states. Circulating neutrophils are increased in sepsis and constitutively express arginase. We investigated whether plasma arginase activity is increased in human sepsis and whether this is associated with neutrophil numbers and activation. Methods: We used HPLC and a radiometric assay to evaluate plasma amino acid concentrations and plasma arginase activity. The relationships between plasma arginase activity, neutrophil count, neutrophil activity and plasma L-arginine and arginine metabolites were evaluated in 44 sepsis patients and 25 controls. Results: Plasma arginase activity was increased in sepsis patients, correlated with neutrophil count (r=0.44; p=0.003), but was independent of sepsis severity (SOFA or APACHE II score). Plasma HNP1-3 correlated with neutrophil count (r=0.31; p=0.04), was elevated in shock (median 180 ng/mL vs. 83 ng/mL sepsis without shock, p=0.0006) and correlated with SOFA score. Sepsis patients with high neutrophil counts had significantly higher plasma HNP1-3 and arginase activity and lower plasma L-arginine concentrations than those with lower neutrophil counts and controls. Conclusions: Plasma arginase activity, potentially derived in part from neutrophil activation, is elevated in sepsis, and may contribute to impaired bioavailability of L-arginine in sepsis.