Laboratory testing of anticoagulants: the present and the future

This review provides an update on laboratory testing and monitoring for existing and emerging anticoagulants, starting with an overview of haemostasis and the routine coagulation tests currently employed within most haemostasis laboratories, including the prothrombin time (PT)/international normalised ratio (INR) and the activated partial thromboplastin time (APTT). Current anticoagulant therapy and laboratory monitoring is then discussed in terms of benefits and limitations, followed by a similar brief discussion of the new and emerging anticoagulants. The main focus, however, is laboratory testing related to vitamin K antagonists, heparin, lepirudin and the new agents dabigatran etexilate and rivaroxaban. Although the newer agents do not require laboratory monitoring, laboratory testing will occasionally be required, and pathology laboratories should become proactive in developing appropriate strategies. The tests most likely to fulfill this role are the ecarin clotting time (or chromogenic alternatives), and the chromogenic anti-Xa assay. Nevertheless, the dilute Russell viper venom time (dRVVT) assay may provide another alternative, and existing routine tests are also likely to be utilised for the foreseeable future, potentially also for laboratory testing of the new anticoagulants, albeit perhaps in modified form.     

Laboratory identification of factor inhibitors: an update

Coagulation factor inhibitors comprise antibodies that bind to and then neutralise specific pro-coagulant plasma proteins. Coagulation factor inhibitors can develop against any coagulation factor, although the most common are against factor VIII (FVIII). These can develop in individuals with inherited haemophilia A (HA) as an immune response to factor replacement therapy, or as auto-antibodies leading to the condition of acquired HA. Clinical suspicion for inhibitors may arise when individuals present with bleeding symptoms without any prior bleeding diathesis, or when a patient with known mild haemophilia presents with a bleeding diathesis more extreme to their usual presentation, or when there is failure of factor replacement therapy to arrest bleeding in a known haemophiliac. The laboratory identification of factor inhibitors requires a careful and systematic approach that excludes other possible causes of prolonged screening tests, most commonly the activated partial thromboplastin time (APTT), and sometimes prothrombin time (PT). Coagulation factor inhibitor studies, including the Bethesda assay, are then undertaken to measure inhibitor titre, which guides treatment. This paper overviews the laboratory investigation of factor inhibitors, and also briefly reviews recent cross-laboratory inhibitor studies and the most recent evidence related to differential inhibitor formation according to type of therapy.     

The centrally projecting Edinger–Westphal nucleus—I: Efferents in the rat brain

The oculomotor accessory nucleus, often referred to as the Edinger–Westphal nucleus [EW], was first identified in the 17th century. Although its most well known function is the control of pupil diameter, some controversy has arisen regarding the exact location of these preganglionic neurons. Currently, the EW is thought to consist of two different parts. The first part [termed the preganglionic EW—EWpg], which controls lens accommodation, choroidal blood flow and pupillary constriction, primarily consists of cholinergic cells that project to the ciliary ganglion. The second part [termed the centrally projecting EW—EWcp], which is involved in non-ocular functions such as feeding behavior, stress responses, addiction and pain, consists of peptidergic neurons that project to the brainstem, the spinal cord and prosencephalic regions. However, in the literature, we found few reports related to either ascending or descending projections from the EWcp that are compatible with its currently described functions. Therefore, the objective of the present study was to systematically investigate the ascending and descending projections of the EW in the rat brain. We injected the anterograde tracer biotinylated dextran amine into the EW or the retrograde tracer cholera toxin subunit B into multiple EW targets as controls. Additionally, we investigated the potential EW-mediated innervation of neuronal populations with known neurochemical signatures, such as melanin-concentrating hormone in the lateral hypothalamic area [LHA] and corticotropin-releasing factor in the central nucleus of the amygdala [CeM]. We observed anterogradely labeled fibers in the LHA, the reuniens thalamic nucleus, the oval part of the bed nucleus of the stria terminalis, the medial part of the central nucleus of the amygdala, and the zona incerta. We confirmed our EW–LHA and EW–CeM connections using retrograde tracers. We also observed moderate EW-mediated innervation of the paraventricular nucleus of the hypothalamus and the posterior hypothalamus. Our findings provide anatomical bases for previously unrecognized roles of the EW in the modulation of several physiologic systems.     

Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na⁺–K⁺-ATPase α/β, Na⁺–K⁺–2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na⁺–K⁺-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na⁺–K⁺-ATPase β in the secretory coils was the same as Na⁺–K⁺-ATPase α, but Na⁺–K⁺-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.     

Orexinergic innervation of urocortin1 and cocaine and amphetamine regulated transcript neurons in the midbrain centrally projecting Edinger–Westphal nucleus

Orexin is a neuropeptide that has been implicated in several processes, such as induction of appetite, arousal and alertness and sleep/wake regulation. Multiple lines of evidence also suggest that orexin is involved in the stress response. When orexin is administered intracerebroventricular it activates the hypothalamic pituitary adrenal (HPA)-axis, which is the main regulator of the stress response. The HPA-axis is not the only player in the stress response evidence suggests that urocortin 1 (Ucn1), a member of the corticotropin releasing factor (CRF) neuropeptide family, also plays an important role in the stress response adaptation. Ucn1 is primarily synthetized in the centrally projecting Edinger–Westphal nucleus (EWcp), which also receives dense innervation by orexin terminals. In this study we tested the hypothesis that orexin would directly shape the response of EWcp-Ucn1 neurons to acute cold stress. To test this hypothesis, we first assessed whether orexinergic axon terminals would innervate EWcp-Ucn1/CART neurons, and next we exposed orexin deficient (orexin-KO) male mice and their male wild-type (WT) littermates to acute cold stress for 2 h. We also assessed stress-associated changes in plasma corticosterone (CORT), as well as the activation of Ucn1/CART neurons in the EWcp nucleus. We found that orexin immunoreactive axon terminals were juxtaposed to EWcp-Ucn1/CART neurons, which also expressed orexin receptor 1 mRNA. Furthermore, acute stress strongly activated the EWcp-Ucn1/CART neurons and increased plasma CORT in both WT littermates and orexin-KO mice, however no genotype effect was found on these indices. Taken together our data show that orexin in general is not involved in the animal's acute stress response (plasma CORT) and it does not play a direct role in shaping the response of EWcp-Ucn1 neurons to acute stress either.     

Subcellular localization and activation of ADAM proteases in the context of FasL shedding in T lymphocytes

The “A Disintegrin And Metalloproteinases” (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.     

Chicken TBK1 interacts with STING and is involved in IFN-β signaling regulation

TANK-binding kinase 1 (TBK1) is an essential serine/threonine-protein kinase required for the Toll-like receptor (TLR)- and retinoic acid-inducible gene I (RIG-I) -mediated induction of type I IFN. Through endogenous Co-IP and LC-MS/MS, we identified chicken TBK1 (chTBK1) as a chSTING-interactive protein. Through exogenous Co-IP assay in transfected cells, we confirmed the interaction between chSTING and chTBK1. To better understand the biological role of chTBK1 in the chSTING-mediated IFN pathway, we cloned the chTBK1 and investigated its biological functions. Quantitative RT-PCR showed that chTBK1 mRNA was widely expressed in different tissues. The overexpression of chTBK1 in DF-1 cells induced the expression of IFN-β and ISGs and inhibited AIV viral replication. We identified indispensable domains of chTBK1 on IFN-β production via the generation of various chTBK1 mutant forms. Together, we identified the chTBK1 as a chSTING interactive protein and concluded that chTBK1 is involved in chSTING-triggered IFN-β signaling in chicken cells.     

The use of coroner's autopsy reports to validate the use of targeted swabbing rather than tissue collection for rapid confirmation of virological causes of sudden death in the community

Background and objectivesIn this study, coroner's autopsy reports were used to validate results obtained from respiratory virus screening of swabs rather than tissue collected during autopsy in cases of adult death of unknown cause.Study designCoroner's autopsy samples collected for respiratory virus screening between October 2010 and February 2011, were identified. Autopsy reports were requested from cases positive for a virus. Each report was reviewed to correlate findings at autopsy with the virology result and to determine whether the virus found was listed as a contributing factor in the death.ResultsSixty-four coroner's autopsy cases were identified and a respiratory virus was found in 25 cases. Influenza A(H1N1)pdm09 virus was found most frequently, then RSV and influenza B with a dual influenza A and B infection and a parainfluenza type 1. Where multiple sites were swabbed, the virus was detected in all sites. Autopsy reports for 12 cases were obtained each reporting findings consistent with respiratory infection. Influenza A was always listed as a contributing factor in the death whereas RSV was listed once and influenza B was omitted in one case. The quality of the reports was variable and full histology was less likely to be performed in the elderly.ConclusionsWhile coroner's reports supported the use of swabbing rather than tissue collection, the lack of consistency and omission of the virology findings as contributing factors to death means that the burden of viruses on mortality statistics will remain under-estimated particularly in the elderly.     

A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures

A significant challenge when investigating autonomic neuroanatomy is being able to reliably obtain tissue that contains neuronal structures of interest. Currently, histochemical staining for acetylcholinesterase (AChE) remains the most feasible and reliable method to visualize intrinsic nerves and ganglia in whole organs. In order to precisely visualize and sample intrinsic cardiac nerves and ganglia for subsequent immunofluorescent labeling, we developed a modified histochemical AChE method using material from pig and sheep hearts. The method involves: (1) chemical prefixation of the whole heart, (2) short-term and weak histochemical staining for AChE in situ, (3) visual examination and extirpation of the stained neural structures from the whole heart, (4) freezing, embedding and cryostat sectioning of the tissue of interest, and (5) immunofluorescent labeling and microscopic analysis of neural structures. Firstly, our data demonstrate that this modified AChE protocol labeled intrinsic cardiac nerves as convincingly as our previously published data. Secondly, there was the added advantage that adrenergic, cholinergic and peptidergic neuropeptides, namely protein gene product 9.5 (PGP 9.5), neurofilament (NF), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT2), neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene related peptide (CGRP), and substance P may be identified. Our method allows the precise sampling of neural structures including autonomic ganglia, intrinsic nerves and bundles of nerve fibers and even single neurons from the whole heart. This method saves time, effort and a substantial amount of antisera. Nonetheless, the proof of specific staining for many other autonomic neuronal markers has to be provided in subsequent studies.     

Impact of an autologous oxygenating matrix culture system on rat islet transplantation outcome

Disruption of the pancreatic islet environment combined with the decrease in oxygen supply that occurs during isolation leads to poor islet survival. The aim of this study was to validate the benefit of using a plasma-based scaffold supplemented with perfluorodecalin to improve islet transplantation outcome.Rat islets were cultured in three conditions: i) control group, ii) plasma based-matrix (P-matrix), and iii) P-matrix supplemented with emulsified perfluorodecalin. After 24 h culture, matrix/cell contacts (Integrinβ1, p-FAK/FAK, p-Akt/Akt), survival (caspase 3, TUNEL, FDA/PI), function, and HIF-1α translocation were assessed. Afterwards, P-matrices were dissolved and the islets were intraportally transplanted. Graft function was monitored for 31 days with glycaemia and C-peptide follow up. Inflammation was assessed by histology (macrophage and granulocyte staining) and thrombin/anti-thrombin complex measurement.Islet survival correlated with an increase in integrin, FAK, and Akt activation in P-matrices and function was maintained. Perfluorodecalin supplementation decreased translocation of HIF-1α in the nucleus and post-transplantation islet structure was better preserved in P-matrices, but a quicker activation of IBMIR resulted in early loss of graft function.“Oxygenating” P-matrices provided a real benefit to islet survival and resistance in vivo. However, intraportal transplantation is not suitable for this kind of culture due to IBMIR; thus, alternative sites must be explored.     

Time to think outside the box? Prothrombin time,international normalised ratio,international sensitivity index,mean normal prothrombin time and measurement of uncertainty a novel approach to standardisation

BACKGROUND: The prothrombin time (PT) assay is the most clinically ordered coagulation test, and most often used for monitoring of vitamin K antagonist therapy (e.g., warfarin), where results are expressed as an international normalised ratio (INR). The INR is in essence the patient's PT 'mathematically adjusted' to a standardised value taking into account the peculiarities of the test system as defined by an ISI (international sensitivity index) and MNPT (mean normal prothrombin time). Although some manufacturers provide assigned ISI values for specific PT reagents and instrumentation, it is still recommended practice that laboratories check or validate these ISIs, as well as estimate the MNPT. Where an ISI is not provided by a manufacturer, the laboratory needs to estimate its own value. Current recommendations suggest the use of commercial reference-plasma calibration sets, but there is limited information on the performance of these in the field. RESULTS: We report a comparative study that assessed the utility of three such commercial calibration plasma sets, used as recommended, as well as alternate or supplementary procedures for estimation of ISI and MNPT. The latter included one novel approach using comparative data of 'existing' versus 'replacement' reagent, as well as assessment of external quality assurance data. Although MNPT value estimates were not grossly disparate, a wide variety of ISI values (e.g., 1.12-1.30 for our primary instrument) was obtained with the different plasma sets. CONCLUSION: Because of the above, further verification checks are required prior to acceptance of ISI and MNPT estimates generated from commercial plasma calibration sets. We also provide some recommendations regarding the process of standardisation of INR testing.     

Cytotoxicity evaluation and antioxidant enzyme expression related to heavy metals found in tuna by-products meal: An in vitro study in human and rat liver cell lines

Heavy metals can accumulate in organisms via various pathways, including respiration, adsorption and ingestion. They are known to generate free radicals and induce oxidative and/or nitrosative stress with depletion of anti-oxidants. Tuna by-product meal (TBM) is rich in proteins and can, therefore, offer an attractive protein source for animals. This study was undertaken to assess the effects of metals present in TBM, namely cadmium (Cd), lead (Pb), and mercury (Hg), separately or in combination with oxidative stress, on cell viability. Three cell models: rat liver FTO2B, human hepatoma HepG2, and human hepatic WRL-68, were used. Cell viability was determined following exposure to various concentrations of the metals. Two antioxidant genes, catalase (CAT) and superoxide dismutase (SOD), were measured to obtain a better understanding of oxidative stress-associated gene expression. Among the metals present in TBM, only Cd at a concentration of 30 μM was noted to exhibit cytotoxic effects. This cytotoxicity was even more pronounced after co-stimulation with H₂O₂, used to mimic systemic oxidative stress. At non-toxic concentrations, Hg and Pb were noted to aggravate oxidative stress toxicity. The results further revealed that exposure to Cd, Pb, and a co-stimulation of H₂O₂ with Hg resulted in the increased expression of antioxidant gene SOD. A risk assessment of toxic contaminants in TBM indicated that food safety objectives should consider the human health impacts of foods derived from animals fed on contaminated meal and that much care should be taken when TBM is used in animal diet.     

Biosynthesis of collagen I,II, RUNX2 and lubricin at different time points of chondrogenic differentiation in a 3D in vitro model of human mesenchymal stem cells derived from adipose tissue

The first aim of the study was to identify the most appropriate time for differentiation of adipose tissue derived mesenchymal stem cells (MSCs) to chondrocytes, through the self-assembly process. For this purpose, the expression of some chondrocyte markers, such as collagen type I, collagen type II, RUNX2 and lubricin was investigated at different times (7, 14, 21 and 28 days) of chondrogenic differentiation of MSCs, by using immunohistochemistry and Western blot analysis. The second aim of the study was to demonstrate that the expression of lubricin, such as the expression of collagen type II, could be a possible biomarker for the detection of chondrocytes well-being and viability in the natural self-assembling constructs, called ‘cell pellets’. Histology (hematoxylin and eosin) and histochemistry (alcian blue staining) methods were used to assess the chondrogenic differentiation of MSCs. The results showed that after 21 days the differentiated chondrocytes, when compared with MSCs cultured without chondrogenic medium (CD44, CD90 and CD105 positive; CD45, CD14 and CD34 negative), were able to produce significant quantities of collagen type I, collagen type II, and lubricin, suggesting hyaline cartilage formation. During the differentiation phase, the cells showed a reduced expression of RUNX2, a protein expressed by osteoblasts. Our studies demonstrated that 21 days is the optimum time for the implantation of chondrocytes differentiated from adipose tissue-derived MSCs. This information could be useful for the future development of cell-based repair therapies for degenerative diseases of articular cartilage.     

Long-term persistence of immunity induced by OVA-coupled gas-filled microbubble vaccination partially protects mice against infection by OVA-expressing Listeria

Vaccination aims at generating memory immune responses able to protect individuals against pathogenic challenges over long periods of time. Subunit vaccine formulations based on safe, but poorly immunogenic, antigenic entities must be combined with adjuvant molecules to make them efficient against infections. We have previously shown that gas-filled microbubbles (MB) are potent antigen-delivery systems. This study compares the ability of various ovalbumin-associated MB (OVA-MB) formulations to induce antigen-specific memory immune responses and evaluates long-term protection toward bacterial infections. When initially testing dendritic cells reactivity to MB constituents, palmitic acid exhibited the highest degree of activation. Subcutaneous immunization of naïve wild-type mice with the OVA-MB formulation comprising the highest palmitic acid content and devoid of PEG2000 was found to trigger the more pronounced Th1-type response, as reflected by robust IFN-γ and IL-2 production. Both T cell and antibody responses persisted for at least 6 months after immunization. At that time, systemic infection with OVA-expressing Listeria monocytgenes was performed. Partial protection of vaccinated mice was demonstrated by reduction of the bacterial load in both the spleen and liver. We conclude that antigen-bound MB exhibit promising properties as a vaccine candidate ensuring prolonged maintenance of protective immunity.     

Characterization,expression, and evolutionary analysis of new TLR3 and TLR5M genes cloned from the spiny eel Mastacembelus armatus

Toll-like receptors (TLRs) play an important role in innate and adaptive immunity. Here, we identify two new TLRs from the spiny eel Mastacembelus armatus (TLR3 and membrane TLR5M). Both MaTLR3 and MaTLR5M were expressed in all tested tissues; expression was highest in liver and spleen, respectively. After infection with Vibrio parahaemolyticus, expression of both TLRs fluctuated and differed significantly from controls at several time points. The predicted three-dimensional model of the MaTLR3 and MaTLR5M proteins indicates that most sites under positive selection were located in the extracellular domains of TLRs. Evolutionary analysis detected positively selected sites in the ancestral lineages of vertebrates, amphibians and reptiles. Multiple ML methods recovered 10 positively selected sites in teleost TLR3 and 24 in TLR5M, and most sites were located in leucine-rich repeat domain, possibly related to an “arms-race” co-evolution with pathogens.     

Electromagnetic field and brain development

Rapid advances in technology involve increased exposures to radio-frequency/microwave radiation from mobile phones and other wireless transmitting devices. As cell phones are held close to the head during talking and often stored next to the reproductive organs, studies are mostly focused on the brain. In fact, more research is especially needed to investigate electromagnetic field (EMF)’s effects on the central nervous system (CNS). Several studies clearly demonstrate that EMF emitted by cell phones could affect a range of body systems and functions. Recent work has demonstrated that EMF inhibit the formation and differentiation of neural stem cells during embryonic development and also affect reproductive and neurological health of adults that have undergone prenatal exposure. The aim of this review is to discuss the developing CNS and explain potential impacts of EMF on this system.     

The unique myelopoiesis strategy of the amphibian Xenopus laevis

Myeloid progenitors reside within specific hematopoietic organs and commit to progenitor lineages bearing megakaryocyte/erythrocyte (MEP) or granulocyte/macrophage potentials (GMP) within these sites. Unlike other vertebrates, the amphibian Xenopus laevis committed macrophage precursors are absent from the hematopoietic subcapsular liver and instead reside within their bone marrow. Presently, we demonstrate that while these frogs’ liver-derived cells are unresponsive to recombinant forms of principal X. laevis macrophage (colony-stimulating factor-1; CSF-1) and granulocyte (CSF-3) growth factors, bone marrow cells cultured with CSF-1 and CSF-3 exhibit respectively archetypal macrophage and granulocyte morphology, gene expression and functionalities. Moreover, we demonstrate that liver, but not bone marrow cells possess erythropoietic capacities when stimulated with a X. laevis erythropoietin. Together, our findings indicate that X. laevis retain their MEP within the hematopoietic liver while sequestering their GMP to the bone marrow, thus marking a very novel myelopoietic strategy as compared to those seen in other jawed vertebrate species.