Diagnostic value of immunoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis.

Immunoassays are essential in the workup of patients with suspected heparin-induced thrombocytopenia. However, the diagnostic accuracy is uncertain with regard to different classes of assays, antibody specificities, thresholds, test variations, and manufacturers. We aimed to assess diagnostic accuracy measures of available immunoassays and to explore sources of heterogeneity. We performed comprehensive literature searches and applied strict inclusion criteria. Finally, 49 publications comprising 128 test evaluations in 15 199 patients were included in the analysis. Methodological quality according to the revised tool for quality assessment of diagnostic accuracy studies was moderate. Diagnostic accuracy measures were calculated with the unified model (comprising a bivariate random-effects model and a hierarchical summary receiver operating characteristics model). Important differences were observed between classes of immunoassays, type of antibody specificity, thresholds, application of confirmation step, and manufacturers. Combination of high sensitivity (>95%) and high specificity (>90%) was found in 5 tests only: polyspecific enzyme-linked immunosorbent assay (ELISA) with intermediate threshold (Genetic Testing Institute, Asserachrom), particle gel immunoassay, lateral flow immunoassay, polyspecific chemiluminescent immunoassay (CLIA) with a high threshold, and immunoglobulin G (IgG)-specific CLIA with low threshold. Borderline results (sensitivity, 99.6%; specificity, 89.9%) were observed for IgG-specific Genetic Testing Institute-ELISA with low threshold. Diagnostic accuracy appears to be inadequate in tests with high thresholds (ELISA; IgG-specific CLIA), combination of IgG specificity and intermediate thresholds (ELISA, CLIA), high-dose heparin confirmation step (ELISA), and particle immunofiltration assay. When making treatment decisions, clinicians should be a aware of diagnostic characteristics of the tests used and it is recommended they estimate posttest probabilities according to likelihood ratios as well as pretest probabilities using clinical scoring tools.

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

Multiplex assays for biomarker research and clinical application: translational science coming of age

Over the last decade, translational science has come into the focus of academic medicine, and significant intellectual and financial efforts have been made to initiate a multitude of bench-to-bedside projects. The quest for suitable biomarkers that will significantly change clinical practice has become one of the biggest challenges in translational medicine. Quantitative measurement of proteins is a critical step in biomarker discovery. Assessing a large number of potential protein biomarkers in a statistically significant number of samples and controls still constitutes a major technical hurdle. Multiplexed analysis offers significant advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. The two contemporary approaches in multiplexed and quantitative biomarker validation, antibody-based immunoassays and MS-based multiple (or selected) reaction monitoring, are based on different assay principles and instrument requirements. Both approaches have their own advantages and disadvantages and therefore have complementary roles in the multi-staged biomarker verification and validation process. In this review, we discuss quantitative immunoassay and multiple reaction monitoring/selected reaction monitoring assay principles and development. We also discuss choosing an appropriate platform, judging the performance of assays, obtaining reliable, quantitative results for translational research and clinical applications in the biomarker field.

Rapid exclusion or confirmation of heparin-induced thrombocytopenia: a single-center experience with 1,291 patients

The current gold-standard for diagnosing heparin-induced thrombocytopenia is the detection of platelet-activating antibodies by means of functional assays which, since they are time consuming and not widely available, are not suited to guiding acute treatment decisions. The objective of our study was to assess the ability of more rapid immunoassays to predict the presence of functionally relevant anti-platelet factor 4/heparin-antibodies.

Effect of repetitiveness on the immunogenicity and antigenicity of Trypanosoma cruzi FRA protein

Repetitive proteins (RP) of Trypanosoma cruzi are highly present in the parasite and are strongly recognized by sera from Chagas' disease patients. Flagelar Repetitive Antigen (FRA), which is expressed in all steps of the parasite life cycle, is the RP that displays the greatest number of aminoacids per repeat and has been indicated as one of the most suitable candidate for diagnostic test because of its high performance in immunoassays. Here we analyzed the influence of the number of repeats on the immunogenic and antigenic properties of the antigen. Recombinant proteins containing one, two, and four tandem repeats of FRA (FRA1, FRA2, and FRA4, respectively) were obtained and the immune response induced by an equal amount of repeats was evaluated in a mouse model. The reactivity of specific antibodies present in sera from patients naturally infected with T. cruzi was also assessed against FRA1, FRA2, and FRA4 proteins, and the relative avidity was analyzed. We determined that the number of repeats did not increase the humoral response against the antigen and this result was reproduced when the repeated motifs were alone or fused to a non-repetitive protein. By contrast, the binding affinity of specific human antibodies increases with the number of repeated motifs in FRA antigen. We then concluded that the high ability of FRA to be recognized by specific antibodies from infected individuals is mainly due to a favorable polyvalent interaction between the antigen and the antibodies. In accordance with experimental results, a 3D model was proposed and B epitope in FRA1, FRA2, and FRA4 were predicted.

Adjunctive dexamethasone affects the expression of genes related to inflammation, neurogenesis and apoptosis in infant rat pneumococcal meningitis

Streptococcus pneumoniae is the most common pathogen causing non-epidemic bacterial meningitis worldwide. The immune response and inflammatory processes contribute to the pathophysiology. Hence, the anti-inflammatory dexamethasone is advocated as adjuvant treatment although its clinical efficacy remains a question at issue. In experimental models of pneumococcal meningitis, dexamethasone increased neuronal damage in the dentate gyrus. Here, we investigated expressional changes in the hippocampus and cortex at 72 h after infection when dexamethasone was given to infant rats with pneumococcal meningitis. Nursing Wistar rats were intracisternally infected with Streptococcus pneumoniae to induce experimental meningitis or were sham-infected with pyrogen-free saline. Besides antibiotics, animals were either treated with dexamethasone or saline. Expressional changes were assessed by the use of GeneChip® Rat Exon 1.0 ST Arrays and quantitative real-time PCR. Protein levels of brain-derived neurotrophic factor, cytokines and chemokines were evaluated in immunoassays using Luminex xMAP® technology. In infected animals, 213 and 264 genes were significantly regulated by dexamethasone in the hippocampus and cortex respectively. Separately for the cortex and the hippocampus, Gene Ontology analysis identified clusters of biological processes which were assigned to the predefined categories "inflammation", "growth", "apoptosis" and others. Dexamethasone affected the expression of genes and protein levels of chemokines reflecting diminished activation of microglia. Dexamethasone-induced changes of genes related to apoptosis suggest the downregulation of the Akt-survival pathway and the induction of caspase-independent apoptosis. Signalling of pro-neurogenic pathways such as transforming growth factor pathway was reduced by dexamethasone resulting in a lack of pro-survival triggers. The anti-inflammatory properties of dexamethasone were observed on gene and protein level in experimental pneumococcal meningitis. Further dexamethasone-induced expressional changes reflect an increase of pro-apoptotic signals and a decrease of pro-neurogenic processes. The findings may help to identify potential mechanisms leading to apoptosis by dexamethasone in experimental pneumococcal meningitis.

Prolyl hydroxylase inhibitors increase the production of vascular endothelial growth factor in dental pulp-derived cells

Introduction: Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp–derived cells. Methods: To test the response of dental pulp–derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, 3[H]thymidine, and 3[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF). Results: We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp–derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels. Conclusions: These results show that PHD inhibitors can stimulate VEGF production of dental pulp–derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Steroid hormone related male biased parasitism in chamois, Rupicapra rupicapra rupicapra

Parasites are linked with their host in a trophic interaction with implications for both hosts and parasites. Interaction stretches from the host's immune response to the structuring of communities and the evolution of biodiversity. As in many species sex determines life history strategy, response to parasites may be sex-specific. Males of vertebrate species tend to exhibit higher rates of parasites than females. Sex-associated hormones may influence immunocompetence and are hypothesised to lead to this bias. In a field study, we tested the prediction of male biased parasitism (MBP) in free ranging chamois (Rupicapra rupicapra rupicapra), which are infested intensely by gastrointestinal and lung helminths. We further investigated sex differences in faecal androgen (testosterone and epiandrosterone), cortisol and oestrogen metabolites using enzyme immunoassays (EIA) to evaluate the impact of these hormones on sex dependent parasite susceptibility. Non-invasive methods were used and the study was conducted throughout a year to detect seasonal patterns. Hormone levels and parasite counts varied significantly throughout the year. Male chamois had a higher output of gastrointestinal eggs and lungworm larvae when compared to females. The hypothesis of MBP originating in sex related hormone levels was confirmed for the elevated output of lungworm larvae, but not for the gastrointestinal nematodes. The faecal output of lungworm larvae was significantly correlated with androgen and cortisol metabolite levels. Our study shows that sex differences in steroid levels play an important role to explain MBP, although they alone cannot fully explain the phenomenon.

Vulvovaginal-swab or first-catch urine specimen to detect Chlamydia trachomatis in women in a community setting?

Screening for chlamydia in women is widely recommended. We evaluated the performance of two nucleic acid amplification tests for detecting Chlamydia trachomatis in self-collected vulvovaginal-swab and first-catch urine specimens from women in a community setting and a strategy for optimizing the sensitivity of an amplified enzyme immunoassay on vulvovaginal-swab specimens. We tested 2,745 paired vulvovaginal-swab and urine specimens by PCR (Roche Cobas) or strand displacement amplification (SDA; Becton Dickinson). There were 146 women infected with chlamydia. The assays detected 97.3% (95% confidence interval [CI], 93.1 to 99.2%) of infected patients with vulvovaginal-swab specimens and 91.8% (86.1 to 95.7%) with urine specimens. We tested 2,749 vulvovaginal-swab specimens with both a nucleic acid amplification test and a polymer conjugate-enhanced enzyme immunoassay with negative-gray-zone testing. The relative sensitivities obtained after retesting specimens in the negative gray zone were 74.3% (95% CI, 62.8 to 83.8%) with PCR and 58.3% (95% CI, 46.1 to 69.8%) with SDA. In community settings, both vulvovaginal-swab and first-catch urine specimens from women are suitable substrates for nucleic acid amplification tests, but enzyme immunoassays, even after negative-gray-zone testing, should not be used in screening programs.

Increased lipopolysaccharide-induced tumour necrosis factor-alpha, interferon-gamma and interleukin-10 production in atopic dermatitis

BACKGROUND: Atopic dermatitis (AD) is based on a genetic predisposition, but environmental factors may trigger skin inflammation. According to the hygiene hypothesis, decreased exposure to microbial products in early childhood does not allow sufficient maturation of the immune system that is associated with an increased risk of atopic sensitization. OBJECTIVES: The effect of lipopolysaccharide (LPS) on the cytokine production of peripheral blood mononuclear cells (PBMC) of AD patients and nonatopic controls was studied. PATIENTS AND METHODS: PBMC were isolated from heparinized blood of 10 patients with AD and 10 nonatopic individuals, suspended in culture medium and stimulated with LPS. Cytokine levels in the supernatants were measured by immunoassays. Results Upon stimulation with LPS, PBMC from AD patients produced significantly higher amounts of tumour necrosis factor-alpha, interferon-gamma and interleukin (IL)-10 compared with control PBMC. LPS stimulation blocked the increased spontaneous production of IL-4 and IL-5 by PBMC from AD patients, but had no effect on IL-13 production. CONCLUSIONS: These results demonstrate that the effects of LPS stimulation depend on both the type of cytokine and the origin of PBMC. Endotoxin exposure is suggested to modulate the disease course of AD.

[What you always wanted to know about HbA1c]

Irreversible, nonenzymatic glycation of the haemoglobin A beta chain leads to the formation of haemoglobin A1c (HbA1c), a stable minor haemoglobin component with enhanced electrophoretic mobility. The rate of formation of HbA1c is directly proportional to the ambient glucose concentration. HbA1c is commonly used to assess long-term blood glucose control in patients with diabetes mellitus, because the HbA1c value has been shown to predict the risk for the development of many of the chronic complications in diabetes. There are currently four principal glycohaemoglobin assay techniques (ion-exchange chromatography, electrophoresis, affinity chromatography and immunoassays) and over 20 methods that measure different glycated products. The ranges indicating good and poor glycaemic control can vary markedly between different assays. At the moment values differ between methodologies and even between different laboratories using the same methodology. Optimal use of HbA1c testing requires standardisation. There is progress towards international standardisation and improved precision of HbA1c which will lead to all assays reporting results in a standardised way. Clinicians ordering HbA1c testing for their patients should be aware of the type of assay method used, the reference interval, potential assay interferences (e.g. haemoglobinopathies, chronic alcohol ingestion, carbamylation products in uraemia) and assay performance. And they should know that a variety of factors have been shown to directly influence HbA1c values, e.g. iron deficiency anaemia, chronic renal failure and shortened red blood cell life span.

Soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 are produced at sites of inflammation and are markers of arthritis activity in Behçet's disease

OBJECTIVE: We analysed the production of soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 at sites of inflammation and measured their plasma concentrations to evaluate them as biological markers of disease activity. METHODS: Plasma samples of 35 patients with Behçet's disease (BD) were collected prospectively at monthly intervals and grouped for inactive disease, active BD without arthritis, and active BD with arthritis. sTNFR1 and sTNFR2 concentrations were measured using immunoassays and compared with other biological disease activity parameters. Plasma sTNFR levels were compared to synovial fluid (SF) levels in seven patients. Sixteen tissue samples of mucocutaneous lesions were stained for TNFR2 expression by immunohistochemistry. RESULTS: sTNFR1 and sTNFR2 were found at increased plasma concentrations in active BD, with the highest concentration in active BD with arthritis (p<0.001). Concentrations of both sTNFRs were at least three times higher in SF of arthritic joints than in the corresponding plasma samples (p = 0.025). A change of more than 1 ng/mL of sTNFR2 plasma concentrations correlated with a concordant change in arthritic activity (96% confidence interval). Sensitivity to change was superior to that of sTNFR1, and other biological disease activity parameters such as erythrocyte sedimentation rate (ESR), immunoglobulin (Ig)G, IgA, and interleukin (IL)-10 plasma concentrations. A strong staining for TNFR2 was found in mucocutaneous lesions, where mast cells were identified as the major source for this receptor. CONCLUSIONS: This longitudinal study demonstrates that sTNFR2 plasma concentrations are closely linked with active BD, and especially with arthritis. Taken together with the expression of TNFR molecules in mast cells of mucocutaneous lesions, our results indicate a fundamental role for the TNF/TNFR pathway in BD.

[Diagnostics of autoimmune diseases]

Autoantibodies play a key role in diagnostic laboratories as markers of autoimmune diseases. In addition to their role as markers they mediate diverse effects in vivo. Autoantibodies with protective effect have been described. Natural protective IgM autoantibodies against tumour-antigens of malignant cells or their precursors may contribute to increased survival rates of carcinoma patients. In a mouse model of systemic lupus erythematosus it has been shown that anti-dsDNA IgM autoantibodies protect from glomerular damage. In contrast, a direct pathogenic role of autoantibodies has been well established e.g. in myasthenia gravis or in Goodpasture syndrome. Similarly autoantibodies against SSA Ro52 are detrimental in neonatal lupus erythematosus with congenital heart block. Moreover, putatively protective autoantibodies may become pathogenic during the course of the disease such as the onconeuronal autoantibodies whose pathogenicity depends on their compartmentalisation. In patients with paraneoplastic syndromes tumour cells express proteins that are also naturally present in the brain. Anti-tumour autoantibodies which temporarily suppress tumour growth can provoke an autoimmune attack on neurons once having crossed the blood-brain barrier and cause specific neurological symptoms. Only a restricted number of autoantibodies are useful follow-up markers for the effectiveness of treatment in autoimmune diseases. Certain autoantibodies hold prognostic value and appear years or even decades before the diagnosis of disease such as the antimitochondrial antibodies in primary biliary cirrhosis or anti-citrullinated protein (CCP)-antibodies in rheumatoid arthritis. It is crucial to know whether the autoantibodies in question recognise linear or conformational epitopes in order to choose the appropriate detection methods. Indirect immunofluorescence microscopy remains a very useful tool for confirmation of results of commercially available immunoassays and for detection of special and rare autoantibodies that otherwise often remain undetected. Standardisation of autoimmune diagnostics is still underway and requires joint efforts by laboratories, clinicians and industry.

Effects of ursodeoxycholic acid in combination with vitamin E on adipokines and apoptosis in patients with nonalcoholic steatohepatitis

BACKGROUND/AIMS: Adipokines and hepatocellular apoptosis participate in the pathogenesis of nonalcoholic steatohepatitis (NASH). In a randomized trial ursodeoxycholic acid (UDCA) with vitamin E (VitE) improved serum aminotransferases and hepatic histology. The present work evaluates the effect of this combination on adipokines and hepatocellular apoptosis. METHODS: Circulating levels of adiponectin, resistin, leptin, interleukin (IL)-6, IL-8, retinol binding protein-4, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha were measured by enzyme-linked immunoassays at the beginning and after 2 years of treatment with either UDCA+VitE, UDCA+placebo (P) or P+P. Apoptosis was assessed by immunohistochemistry for activated caspase-3 and circulating levels of apoptosis-associated cytokeratin 18 fragments (M30). RESULTS: Levels of adiponectin increased in patients treated with UDCA+VitE, whereas they decreased in the two other groups (P<0.04) and correlated with the improvement of liver steatosis (P<0.04). M30 levels worsened in the P/P group and improved in the other two groups. They correlated with hepatocellular apoptosis (P<0.02) and steatosis (P<0.02) as well as negatively with adiponectin levels (P<0.04). CONCLUSIONS: UDCA+VitE improves not only aminotransferase levels and liver histology of patients with NASH, but also decreases hepatocellular apoptosis and restores circulating levels of adiponectin. These results suggest that the UDCA+VitE combination has metabolic effects in addition to its beneficial cytoprotective properties.