Calprotectin blockade inhibits long-term vascular pathology following peritoneal dialysis-associated bacterial infection.

Bacterial infections and the concurrent inflammation have been associated with increased long-term cardiovascular (CV) risk. In patients receiving peritoneal dialysis (PD), bacterial peritonitis is a common occurrence, and each episode further increases late CV mortality risk. However, the underlying mechanism(s) remains to be elucidated before safe and efficient anti-inflammatory interventions can be developed. Damage-Associated Molecular Patterns (DAMPs) have been shown to contribute to the acute inflammatory response to infections, but a potential role for DAMPs in mediating long-term vascular inflammation and CV risk following infection resolution in PD, has not been investigated. We found that bacterial peritonitis in mice that resolved within 24h led to CV disease-promoting systemic and vascular immune-mediated inflammatory responses that were maintained up to 28 days. These included higher blood proportions of inflammatory leukocytes displaying increased adhesion molecule expression, higher plasma cytokines levels, and increased aortic inflammatory and atherosclerosis-associated gene expression. These effects were also observed in infected nephropathic mice and amplified in mice routinely exposed to PD fluids. A peritonitis episode resulted in elevated plasma levels of the DAMP Calprotectin, both in PD patients and mice, here the increase was maintained up to 28 days. In vitro, the ability of culture supernatants from infected cells to promote key inflammatory and atherosclerosis-associated cellular responses, such as monocyte chemotaxis, and foam cell formation, was Calprotectin-dependent. In vivo, Calprotectin blockade robustly inhibited the short and long-term peripheral and vascular consequences of peritonitis, thereby demonstrating that targeting of the DAMP Calprotectin is a promising therapeutic strategy to reduce the long-lasting vascular inflammatory aftermath of an infection, notably PD-associated peritonitis, ultimately lowering CV risk.

Therapeutic targeting of chronic kidney disease-associated DAMPs differentially contributing to vascular pathology.

Chronic Kidney Disease (CKD) is associated with markedly increased cardiovascular (CV) morbidity and mortality. Chronic inflammation, a hallmark of both CKD and CV diseases (CVD), is believed to drive this association. Pro-inflammatory endogenous TLR agonists, Damage-Associated Molecular Patterns (DAMPs), have been found elevated in CKD patients' plasma and suggested to promote CVD, however, confirmation of their involvement, the underlying mechanism(s), the extent to which individual DAMPs contribute to vascular pathology in CKD and the evaluation of potential therapeutic strategies, have remained largely undescribed. A multi-TLR inhibitor, soluble TLR2, abrogated chronic vascular inflammatory responses and the increased aortic atherosclerosis-associated gene expression observed in nephropathic mice, without compromising infection clearance. Mechanistically, we confirmed elevation of 4 TLR DAMPs in CKD patients' plasma, namely Hsp70, Hyaluronic acid, HMGB-1 and Calprotectin, which displayed different abilities to promote key cellular responses associated with vascular inflammation and progression of atherosclerosis in a TLR-dependent manner. These included loss of trans-endothelial resistance, enhanced monocyte migration, increased cytokine production, and foam cell formation by macrophages, the latter via cholesterol efflux inhibition. Calprotectin and Hsp70 most consistently affected these functions. Calprotectin was further elevated in CVD-diagnosed CKD patients and strongly correlated with the predictor of CV events CRP. In nephropathic mice, Calprotectin blockade robustly reduced vascular chronic inflammatory responses and pro-atherosclerotic gene expression in the blood and aorta. Taken together, these findings demonstrated the critical extent to which the DAMP-TLR pathway contributes to vascular inflammatory and atherogenic responses in CKD, revealed the mechanistic contribution of specific DAMPs and described two alternatives therapeutic approaches to reduce chronic vascular inflammation and lower CV pathology in CKD.

Neutrophil-derived miR-223 as local biomarker of bacterial peritonitis.

Infection remains a major cause of morbidity, mortality and technique failure in patients with end stage kidney failure who receive peritoneal dialysis (PD). Recent research suggests that the early inflammatory response at the site of infection carries diagnostically relevant information, suggesting that organ and pathogen-specific "immune fingerprints" may guide targeted treatment decisions and allow patient stratification and risk prediction at the point of care. Here, we recorded microRNA profiles in the PD effluent of patients presenting with symptoms of acute peritonitis and show that elevated peritoneal miR-223 and reduced miR-31 levels were useful predictors of bacterial infection. Cell culture experiments indicated that miR-223 was predominantly produced by infiltrating immune cells (neutrophils, monocytes), while miR-31 was mainly derived from the local tissue (mesothelial cells, fibroblasts). miR-223 was found to be functionally stabilised in PD effluent from peritonitis patients, with a proportion likely to be incorporated into neutrophil-derived exosomes. Our study demonstrates that microRNAs are useful biomarkers of bacterial infection in PD-related peritonitis and have the potential to contribute to disease-specific immune fingerprints. Exosome-encapsulated microRNAs may have a functional role in intercellular communication between immune cells responding to the infection and the local tissue, to help clear the infection, resolve the inflammation and restore homeostasis.

Guidance for quality control practices and precision goals for CBCs based on IQMH patterns-of-practice survey.

INTRODUCTION: Effective medical laboratory quality management systems ensure confidence in analyzing and reporting accurate and reliable patient results. To guarantee quality assurance, each laboratory needs appropriate internal quality control (IQC) procedures to monitor their test systems. The Institute for Quality Management in Healthcare (IQMH) Centre for Proficiency Testing conducted a survey on quality control (QC) practices in routine hematology. METHODS: An online survey was sent to 184 Ontario laboratories performing complete blood counts (CBC) and leukocyte differentials. RESULTS: All participants used three levels of commercial QC for test system monitoring. Eighty percent of laboratories supplement with in-house patient QC. The frequency of QC analysis was variable based on: Manufacturer recommendations (80%) Parameter stability (25%) Clinical impact of incorrect results (21%) Number of samples potentially requiring retesting if there is a QC failure (11%). All laboratories used established QC rules and limits to monitor results. They utilized various methods in establishing limits including: Standard deviation of QC results (60%) Manufacturer precision goals (55%) Published precision goals (24%) IQMH allowable performance limits (APLs) (37%). CONCLUSION: Considerable variation in QC practices of Ontario laboratories was identified, and consensus practice recommendations and precision goals were developed to guide and standardize QC practice.

Preventing Peritoneal Dialysis-Associated Fibrosis by Therapeutic Blunting of Peritoneal Toll-Like Receptor Activity.

Peritoneal dialysis (PD) is an essential daily life-saving treatment for end-stage renal failure. PD therapy is limited by peritoneal inflammation, which leads to peritoneal membrane failure as a result of progressive fibrosis. Peritoneal infections, with the concomitant acute inflammatory response and membrane fibrosis development, worsen PD patient outcomes. Patients who remain infection-free, however, also show evidence of inflammation-induced membrane damage and fibrosis, leading to PD cessation. In this case, uraemia, prolonged exposure to bio-incompatible PD solutions and surgical catheter insertion have been reported to induce sterile peritoneal inflammation and fibrosis as a result of cellular stress or tissue injury. Attempts to reduce inflammation (either infection-induced or sterile) and, thus, minimize fibrosis development in PD have been hampered because the immunological mechanisms underlying this PD-associated pathology remain to be fully defined. Toll-like receptors (TLRs) are central to mediating inflammatory responses by recognizing a wide variety of microorganisms and endogenous components released following cellular stress or generated as a consequence of extracellular matrix degradation during tissue injury. Given the close link between inflammation and fibrosis, recent investigations have evaluated the role that TLRs play in infection-induced and sterile peritoneal fibrosis development during PD. Here, we review the findings and discuss the potential of reducing peritoneal TLR activity by using a TLR inhibitor, soluble TLR2, as a therapeutic strategy to prevent PD-associated peritoneal fibrosis.

Targeting Toll-like receptors with soluble Toll-like receptor 2 prevents peritoneal dialysis solution-induced fibrosis.

Peritoneal membrane failure due to fibrosis limits the use of peritoneal dialysis (PD). Peritoneal fibrosis may potentially be induced by sterile inflammation caused by ongoing cellular stress due to prolonged exposure to PD solutions (PDS). Effective therapies to prevent this process remain to be developed. Toll-like receptors (TLRs) mediate sterile inflammation by recognizing damage-associated molecular patterns (DAMPs) released by cellular stress. We evaluated the involvement of TLRs and DAMPs in PDS-induced fibrosis models and the therapeutic potential of TLR-DAMP targeting for preventing fibrosis. A range of PDS elicited pro-inflammatory and fibrotic responses from PD patient peritoneal leukocytes, mesothelial cells and mouse peritoneal leukocytes. TLR2/4 blockade of human peritoneal cells or TLR2/4 knockouts inhibited these effects. PDS did not induce rapid ERK phosphorylation or IκB-α degradation, suggesting that they do not contain components capable of direct TLR activation. However, PDS increased the release of Hsp70 and hyaluronan, both TLR2/4 DAMP ligands, by human and mouse peritoneal cells, and their blockade decreased PDS-driven inflammation. Soluble TLR2, a TLR inhibitor, reduced PDS-induced pro-inflammatory and fibrotic cytokine release ex vivo. Daily catheter infusion of PDS in mice caused peritoneal fibrosis, but co-administration of soluble TLR2 prevented fibrosis, suppressed pro-fibrotic gene expression and pro-inflammatory cytokine production, reduced leukocyte/neutrophil recruitment, recovered Treg cell levels and increased the Treg:Th17 ratio. Thus, TLR2/4, Hsp70 and hyaluronan showed major roles in PDS-induced peritoneal inflammation and fibrosis. The study demonstrates the therapeutic potential of a TLR-DAMP targeting strategy to prevent PDS-induced fibrosis.

Lack of grading agreement among international hemostasis external quality assessment programs.

: Laboratory quality programs rely on internal quality control and external quality assessment (EQA). EQA programs provide unknown specimens for the laboratory to test. The laboratory's result is compared with other (peer) laboratories performing the same test. EQA programs assign target values using a variety of methods statistical tools and performance assessment of 'pass' or 'fail' is made. EQA provider members of the international organization, external quality assurance in thrombosis and hemostasis, took part in a study to compare outcome of performance analysis using the same data set of laboratory results. Eleven EQA organizations using eight different analytical approaches participated. Data for a normal and prolonged activated partial thromboplastin time (aPTT) and a normal and reduced factor VIII (FVIII) from 218 laboratories were sent to the EQA providers who analyzed the data set using their method of evaluation for aPTT and FVIII, determining the performance for each laboratory record in the data set. Providers also summarized their statistical approach to assignment of target values and laboratory performance. Each laboratory record in the data set was graded pass/fail by all EQA providers for each of the four analytes. There was a lack of agreement of pass/fail grading among EQA programs. Discordance in the grading was 17.9 and 11% of normal and prolonged aPTT results, respectively, and 20.2 and 17.4% of normal and reduced FVIII results, respectively. All EQA programs in this study employed statistical methods compliant with the International Standardization Organization (ISO), ISO 13528, yet the evaluation of laboratory results for all four analytes showed remarkable grading discordance.

Serum Sclerostin Increases After Acute Physical Activity.

Physical activity has a major impact on bone density and on osteoporosis prevention. Sclerostin is produced by osteocytes and inhibits bone formation. The impact of exercise on sclerostin secretion has not been studied so far. This pilot study aimed to explore circulating sclerostin levels immediately after acute exercise. Healthy young women practicing physical activity less than 120 min per week were enrolled. The exercise was a 45-min, low-speed, treadmill running test. Blood samples were taken at rest before exercise and within 5 min after the end of exercise. We assessed serum creatinine, 25-OH vitamin D, alkaline phosphatase, C-telopeptide of type I collagen, bone-specific alkaline phosphatase, and sclerostin. Sclerostin stability at rest was also validated over the same period of time among women fulfilling the same inclusion criteria. The study included 23 participants (mean ± SD age: 22.9 ± 1.5 years) for the exercise test and 9 participants for the resting test (26.1 ± 3.1 years). There was no difference in body mass index between the two groups. Sclerostin increased after exercise in comparison to baseline (mean ± SEM: 410 ± 27 vs. 290 ± 19 pg/mL; p < 0.001) corresponding to an increase of +44.3 ±5.5%. In the resting test, sclerostin remained stable (303 ± 20 vs. 294 ± 20 pg/mL, p = 0.76). There was a substantial increase in serum sclerostin in untrained healthy young women immediately after physical activity. These results suggest the existence of an acute release of systemic sclerostin in response to physical activity.

Soluble Toll-like receptor 2 is a biomarker for sepsis in critically ill patients with multi-organ failure within 12 h of ICU admission.

Soluble TLR2 levels are elevated in infective and inflammatory conditions, but its diagnostic value with sepsis-induced multi-organ failure has not been evaluated. 37 patients with a diagnosis of severe sepsis/septic shock (sepsis) and 27 patients with organ failure without infection (SIRS) were studied. Median (IQR) plasma sTLR2 levels were 2.7 ng/ml (1.4-6.1) in sepsis and 0.6 ng/ml (0.4-0.9) in SIRS p < 0.001. sTLR2 showed good diagnostic value for sepsis at cut-off of 1.0 ng/ml, AUC:0.959. We report the ability of sTLR2 levels to discriminate between sepsis and SIRS within 12 h of ICU admission in patients with multi-organ failure.

Toll-Like Receptors 2 and 4 Are Potential Therapeutic Targets in Peritoneal Dialysis-Associated Fibrosis.

Peritoneal dialysis (PD) remains limited by dialysis failure due to peritoneal membrane fibrosis driven by inflammation caused by infections or sterile cellular stress. Given the fundamental role of Toll-like receptors (TLRs) and complement in inflammation, we assessed the potential of peritoneal TLR2, TLR4 and C5a receptors, C5aR and C5L2, as therapeutic targets in PD-associated fibrosis. We detected TLR2-, TLR4-, and C5aR-mediated proinflammatory and fibrotic responses to bacteria that were consistent with the expression of these receptors in peritoneal macrophages (TLR2/4, C5aR) and mesothelial cells (TLR2, C5aR). Experiments in knockout mice revealed a major role for TLR2, a lesser role for TLR4, a supplementary role for C5aR, and no apparent activity of C5L2 in infection-induced peritoneal fibrosis. Similarly, antibody blockade of TLR2, TLR4, or C5aR differentially inhibited bacteria-induced profibrotic and inflammatory mediator production by peritoneal leukocytes isolated from the peritoneal dialysis effluent (PDE) of noninfected uremic patients. Additionally, antibodies against TLR2, TLR4, or the coreceptor CD14 reduced the profibrotic responses of uremic leukocytes to endogenous components present in the PDE of noninfected patients. Enhancing TLR2-mediated inflammation increased fibrosis in vivo Furthermore, soluble TLR2 (sTLR2), a negative modulator of TLRs that we detected in PDE, inhibited PDE-induced, TLR2- or TLR4-mediated profibrotic responses. Notably, sTLR2 treatment markedly reduced Gram-positive and -negative bacteria-induced fibrosis in vivo, inhibiting proinflammatory and fibrotic genes without affecting infection clearance. These findings reveal the influence of peritoneal TLR2 and TLR4 on PD-associated fibrosis and describe a therapeutic strategy against fibrosis.

Therapeutic Boosting of the Immune Response: Turning to CD14 for Help.

The Toll-like family of immune receptors (TLRs) are critical for an efficient immune response to a variety of microorganisms and other antigens that may cause pathology. Modulating immune responses by targeting TLRs therefore has substantial therapeutic potential, and a number of TLR-based therapeutic strategies have been developed. Minimizing the adverse effects that may result from the therapeutic manipulation of these signalling receptors nevertheless remains a major challenge. Efficient responses via TLRs require the activity of the co-receptor CD14, which enhances TLR responses. In an attempt to boost the immune response for therapeutic purposes, we have sought to target CD14 to achieve TLR modulation. Here we discuss the design, activity and therapeutic development options of TLR-derived peptides that interact with CD14 and enhance its co-receptor activity, thus amplifying TLR-mediated responses. This strategy represents a promising alternative to current TLR-based therapies, as it has the potential to amplify responses to different pathogens mediated by different TLRs by targeting the common TLR co-receptor, CD14.

Selective antibody intervention of Toll-like receptor 4 activation through Fc γ receptor tethering.

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

Targeting the TLR co-receptor CD14 with TLR2-derived peptides modulates immune responses to pathogens.

Dysregulation of Toll-like receptor (TLR) responses to pathogens can lead to pathological inflammation or to immune hyporesponsiveness and susceptibility to infections, and may affect adaptive immune responses. TLRs are therefore attractive therapeutic targets. We assessed the potential of the TLR co-receptor CD14 as a target for therapeutics by investigating the magnitude of its influence on TLR responses. We studied the interaction of CD14 with TLR2 by conducting peptide screening and site-directed mutagenesis analysis and found TLR2 leucine-rich repeats 5, 9, 15, and 20 involved in interaction with CD14. Peptides representing these regions interacted with CD14 and enhanced TLR2- and TLR4-mediated proinflammatory responses to bacterial pathogens in vitro. Notably, the peptides' immune boosting capacity helped to rescue proinflammatory responses of immunosuppressed sepsis patients ex vivo. In vivo, peptide treatment increased phagocyte recruitment and accelerated bacterial clearance in murine models of Gram-negative and Gram-positive bacterial peritonitis. Up-modulating CD14's co-receptor activity with TLR2-derived peptides also enhanced antigen-induced dendritic cell (DC) maturation and interleukin-2 production and, most notably, differentially affected DC cytokine profile upon antigen stimulation, promoting a T helper 1-skewed adaptive immune response. Biochemical, cell imaging, and molecular docking studies showed that peptide binding to CD14 accelerates microbial ligand transfer from CD14 to TLR2, resulting in increased and sustained ligand occupancy of TLR2 and receptor clustering for signaling. These findings reveal the influence that CD14 exerts on TLR activities and describe a potential therapeutic strategy to amplify responses to different pathogens mediated by different TLRs by targeting the common TLR co-receptor, CD14.

Lupus anticoagulant testing.

Antiphospholipid antibodies are a heterogenous group of autoantibodies directed against glycoproteins in concert with anionic phospholipids. In clinical laboratory practice, antiphospholipid antibody evaluations usually consist of a combination of the following: anticardiolipin antibody assay, anti-beta 2 glycoprotein I assay, and at least two lupus anticoagulant assays with an appropriate confirmatory test. Lupus anticoagulants produce their laboratory effect by prolonging recalcification times in assays within which phospholipid content is limited. Although many assays are available, all are based on the fundamental principle of demonstrating normalization of prolonged recalcification times with the addition of exogenous phospholipid. The antibody specificity of an individual lupus anticoagulant is difficult or impossible to determine; however a small proportion do demonstrate avidity for selected proteins such as prothrombin or beta 2 glycoprotein I. The mechanism by which these antibodies cause their clinical manifestations remains unknown; however their relationship to increased risk of thrombosis, pregnancy loss, and autoimmune thrombocytopenia is undoubted. There is no correlation between the "strength" of lupus anticoagulants and the level of thrombotic risk; thus it is important to identify both "weak" and "strong" lupus anticoagulants.

Anticardiolipin antibody and anti-beta 2 glycoprotein I antibody assays.

Antiphospholipid syndrome (APS) is an autoimmune disease and is a risk factor for a number of clinical manifestations; the classic presentations include fetal death or thrombosis (arterial or venous thromboembolism), in the presence of persistently increased titers of antiphospholipid (aPL) antibodies. The actual cause of APS is unknown but thought to be multifactorial. The disease is characterized by the presence of a heterogenous population of autoantibodies against phospholipid-binding proteins. APS presents either in isolation with no evidence of an underlying disease or in concert with an autoimmune disease such as systemic lupus erythematosus or rheumatoid arthritis. The wide diversity in clinical presentation often causes difficulty in identifying and treating patients and therefore a concise laboratory report containing interpretative comments is required to provide needed guidance to the clinician. For a diagnosis of APS to be made both clinical and laboratory classification criteria must be met. Laboratory testing to identify aPL antibodies includes lupus anticoagulant (liquid-based clotting assays) and immunological solid-phase assays (usually enzyme-linked immunosorbent assay formats) for IgG and/or IgM anticardiolipin (aCL) antibodies and anti-beta 2 glycoprotein I (ß2-GPI) antibodies. Other autoantibodies, such as those directed against anionic phospholipids, can also be assayed; however they are not of clinical significance. Participation in a quality assurance program and an in-depth technical and clinical understanding of testing for aPL antibodies are required, as methods are limited by poor robustness, reproducibility, specificity, and standardization. Testing is further complicated by the lack of a "gold standard" laboratory test to diagnose or classify a patient as having APS. This chapter discusses the clinical and laboratory theoretical and technical aspects of aCL and anti-ß2GPI antibody assays.

TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2.

TLR and complement activation ensures efficient clearance of infection. Previous studies documented synergism between TLRs and the receptor for the pro-inflammatory complement peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. We demonstrate a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identify an underlying mechanistic target. Pre-exposure of PBMCs and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4 signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR upregulation or modulation of C5a-induced Ca(2+) mobilization. Rather, TLRs targeted another C5a receptor, C5L2 (acting as a negative modulator of C5aR), by reducing C5L2 activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defense at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation.

Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors.

BACKGROUND: Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC). METHODS: Primary HPMC were isolated from omental biopsies and TLR expression detected by real-time polymerase chain reaction (PCR), reverse transcription (RT)-PCR and flow cytometry. The responsiveness of HPMC to specific bacterial TLR agonists was determined using chemokine production as a biological readout. The requirement for CD14 in HPMC responses to a clinically relevant Staphylococcus epidermidis cell-free supernatant (SES) was investigated using soluble CD14 or anti-CD14-blocking antibodies. RESULTS: Real-time PCR detected TLR1-6 messenger RNA expression in HPMC and responses to TLR2/1 and TLR2/6 ligands and SES. No cell surface TLR4 expression or responses to lipopolysaccharide were detectable in HPMC, but they did respond to flagellin, a TLR5 ligand. SES-mediated responses were dependent on TLR2 but did not require CD14 in HPMC for optimal efficiency, unlike peripheral blood mononuclear cells. HPMC expression of TLR2 was also modulated by TLR2 ligands and inflammatory cytokines. CONCLUSIONS: These data suggest that mesothelial cell activation by TLR2/1, TLR2/6 and TLR5 contributes to bacterial recognition influencing the course of the infective process and has implications for improving treatment of infection in PD patients.

Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry.

Platelet function testing is important for the diagnostic evaluation of common and rare bleeding disorders. Our study goals were to promote best practices and reduce unnecessary testing variances by developing North American guidelines on platelet function testing. Guidelines were developed by consensus for expert recommendations (minimum level for approval, 70%) that included recommendations on the evaluation and interpretation of light transmission platelet aggregometry (LTA). To assess consensus, medical opinions on recommendations were gathered from diagnostic laboratories that perform LTA, in collaboration with the Quality Management Program-Laboratory Services (QMP-LS) in Ontario, Canada (10 laboratories), and the North American Specialized Coagulation Laboratory Association (NASCOLA; 47 laboratories, 5 overlapping the QMP-LS group). Adequate consensus was achieved for all and 89% of recommendations for the QMP-LS and NASCOLA groups, respectively. The recommendations adopted provide North American laboratories with additional guidance on platelet function testing, including how to interpret LTA abnormalities.

Interlaboratory variation in heparin monitoring: Lessons from the Quality Management Program of Ontario coagulation surveys.

Unfractionated heparin (UFH) monitoring is subject to substantial interlaboratory variation. We analysed results of annual coagulation surveys administered by the Quality Management Program - Laboratory Services (Toronto, ON, Canada) from 2003 to 2007 to evaluate variation in UFH monitoring across Ontario. Participating laboratories performed an activated partial thromboplastin time (APTT) utilising their local methodology on lyophilised human plasma spiked with UFH. In the 2006 and 2007 surveys, laboratories licensed to perform anti-Xa assays also reported anti-Xa activity results. The APTT differed significantly between heparin-sensitive and heparin-insensitive methods (p<0.0005). Within-method variation was observed and increased with increasing heparin concentration. Among laboratories performing an APTT and anti-Xa, the coefficient of variation was greater in the anti-Xa than in the APTT for both the 2006 (64.0% vs. 10.5%) and 2007 (15.0% vs. 11.6%) surveys. Substantial interlaboratory variation in UFH monitoring, both between and within APTT methods, was observed and was not reduced by use of an anti-Xa assay.