Long-term stability of clinically relevant chemistry analytes in pleural and peritoneal fluid.

INTRODUCTION: Our aim was to investigate the stability of clinically relevant analytes in pleural and peritoneal fluids stored in variable time periods and variable storage temperatures prior to analysis. MATERIALS AND METHODS: Baseline total proteins (TP), albumin (ALB), lactate dehydrogenase (LD), cholesterol (CHOL), triglycerides (TRIG), creatinine (CREA), urea, glucose and amylase (AMY) were measured using standard methods in residual samples from 29 pleural and 12 peritoneal fluids referred to our laboratory. Aliquots were stored for 6 hours at room temperature (RT); 3, 7, 14 and 30 days at - 20°C. At the end of each storage period, all analytes were re-measured. Deviations were calculated and compared to stability limits (SL). RESULTS: Pleural fluid TP and CHOL did not differ in the observed storage periods (P = 0.265 and P = 0.170, respectively). Statistically significant differences were found for ALB, LD, TRIG, CREA, urea, glucose and AMY. Peritoneal fluid TP, ALB, TRIG, urea and AMY were not statistically different after storage, contrary to LD, CHOL, CREA and glucose. Deviations for TP, ALB, CHOL, TRIG, CREA, urea and AMY in all storage periods tested for both serous fluids were within the SL. Deviations exceeding SL were observed for LD and glucose when stored for 3 and 7 days at - 20°C, respectively. CONCLUSIONS: TP, ALB, CHOL, TRIG, CREA, urea and AMY are stable in serous samples stored up to 6 hours at RT and/or 30 days at - 20°C. Glucose is stable up to 6 hours at RT and 3 days at - 20°C. The stability of LD in is limited to 6 hours at RT.

Analytical verification and comparison of chromogenic assays for dabigatran, rivaroxaban and apixaban determination on BCSXP and STA Compact Max analysers.

INTRODUCTION: The aim of the study was to perform analytical verification and comparison of chromogenic assays for determination of dabigatran, rivaroxaban and apixaban concentration on BCSXP and STA Compact Max analysers. MATERIALS AND METHODS: Precision, linearity, measurement uncertainty estimation and determination of limit of blank, limit of determination and limit of quantification were calculated. Analytical performance specifications were set according to manufacturer specifications and literature data on between laboratory variability. Comparison of the methods was done using Bland-Altman and Passing-Bablok regression analysis. RESULTS: Obtained results have shown acceptable precision on STA Compact Max only for dabigatran (CV = 3.5%) at lower concentration level comparing to manufacturer declaration (CV = 3.6%). On BCSXP, the highest coefficient of variation has been shown for apixaban (6.1%) at lower concentration level. Within laboratory precision was not met on STA Compact Max for all assays. Bland-Altman analysis has shown statistically significant bias for dabigatran (23.2%, 95%CI 11.2 - 35.3; P < 0.001) and apixaban (8.4%, 95%CI 1.2 - 15.6; P = 0.023). Passing-Bablok regression analysis has shown systematic and proportional deviation between methods for rivaroxaban (y = 6.52 (2.94 to 11.83) + 0.84 (0.80 to 0.89) x. CONCLUSION: Chromogenic assays for dabigatran, rivaroxaban and apixaban on BCSXP and STA Compact Max analysers are shown as methods with satisfactory long-term analytical performance specifications for determination of direct oral anticoagulants in clinical laboratories. However, we cannot recommend interchangeable use because of the significant bias between assays.

The response of HEK293 cells transfected with bovine TLR2 to established pathogen-associated molecular patterns and to bacteria causing mastitis in cattle.

Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.

poly(I:C) and LPS induce distinct IRF3 and NF-kappaB signaling during type-I IFN and TNF responses in human macrophages.

Macrophages play major roles in the onset of immune responses and inflammation by inducing a variety of cytokines such as TNF and IFN-beta. The pathogen-associated molecular pattern, polyinosinic-polycytidylic acid [poly(I:C)], and LPS were used to study type-I IFN and TNF responses in human macrophages. Additionally, activation of the key signaling pathways, IFN-regulatory factor 3 (IRF3) and NF-kappaB, were studied. We found that TNF production occurred rapidly after LPS stimulation. LPS induced a strong IFN-beta mRNA response within a short time-frame, which subsided at 8 h. The IFN-stimulated genes (ISGs), ISG56 and IFN-inducible protein 10, were strongly induced by LPS. These responses were associated with NF-kappaB and IRF3 activation, as shown by IRF3 dimerization and by nuclear translocation assays. poly(I:C), on the other hand, induced a strong and long-lasting (>12 h) IFN-beta mRNA and protein response, particularly when transfected, whereas only a protracted TNF response was observed when poly(I:C) was transfected. However, these responses were induced in the absence of detectable IRF3 and NF-kappaB signaling. Thus, in human macrophages, poly(I:C) treatment induces a distinct cytokine response when compared with murine macrophages. Additionally, a robust IFN-beta response can be induced in the absence of detectable IRF3 activation.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS.

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Human monocytoid cells as a model to study Toll-like receptor-mediated activation.

THP-1 2A9, a subclone of the monocytoid cell line THP-1 and known to be exquisitely sensitive to LPS, was tested for TNF production following triggering by excess doses of TLR ligands. TLR2, TLR4 and TLR5 agonists, but neither TLR3 nor TLR9 agonists, induced TNF production. When used at lower concentrations, priming by calcitriol strongly influenced the sensitivity of cells to LPS and different TLR2 triggers (lipoteichoic acid (LTA), trispalmitoyl-cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam3Cys) and peptidoglycan (PGN)). Priming by calcitriol failed to modulate TLR2 and TLR4 mRNA and cell surface expression of these receptors. TNF signals elicited by TLR2 agonists were blocked by the TLR-specific antibody 2392. CD14-specific antibodies showed variable effects. CD14-specific antibodies inhibited TNF induction by LTA. High concentrations partially inhibited TNF induction by Pam3Cys. The same antibodies failed to inhibit TNF induction by PGN. Thus, THP-1 2A9 cells respond by TNF production to some, but not all TLR agonists, and the wide variety of putative TLR2 agonists interact to variable degrees also with other cell-surface-expressed binding sites such as CD14. THP-1 2A9 cells might provide a model by which to investigate in more detail the interaction of pathogen-associated molecular patterns and monocytoid cell-surface-expressed pattern recognition receptors.

International validation of novel pyrogen tests based on human monocytoid cells.

It is a requirement that parenteral medicines be tested for pyrogens (fever causing agents) using one of two animal-based tests: the rabbit pyrogen test and the bacterial endotoxin test. Understanding the human fever reaction has led to novel non-animal alternative tests based on in vitro activation of human monocytoid cells in response to pyrogens. Using 13 prototypic drugs, clean or contaminated with pyrogens, we have validated blindly six novel pyrogen tests in ten laboratories. Compared with the rabbit test, the new tests have a lower limit of detection and are more accurate as well as cost and time efficient. In contrast to the bacterial endotoxin test, all tests are able to detect Gram-positive pyrogens. The validation process showed that at least four of the tests meet quality criteria for pyrogen detection. These validated in vitro pyrogen tests overcome several shortcomings of animal-based pyrogen tests. Our data suggest that animal testing could be completely replaced by these evidence-based pyrogen tests and highlight their potential to further improve drug safety.

Toll-like receptor-4 is involved in eliciting an LPS-induced oxidative burst in neutrophils.

The lipopolysaccharide (LPS) receptor complex of mononuclear phagocytes is composed of Toll-like receptor-4 (TLR4), MD-2 and CD14. Other phagocyte populations may express similar LPS receptors. The transmembrane glycoprotein TLR4 was shown to induce or upregulate a variety of gene products, which collectively are the mediators of an LPS effect. In this study, an involvement of TLR4 in mediation of an oxidative burst was determined using murine peritoneal exsudate neutrophils and lucigenin-enhanced chemiluminescence (CL). The CL response was dependent on the LPS dose and the presence of serum, putatively a source of lipopolysaccharide-binding protein (LBP). In the absence of serum, a CL signal was elicited by 4 microg/ml LPS in peritoneal exsudate cells (PEC) from TLR4-sufficient (C3H/HeN) but not TLR4 deficient (C3H/HeJ) mice. The signal obtained in PEC from TLR4-sufficient mice was completely abrogated by superoxide dismutase (SOD), which indicated that the response depended on the formation of superoxide anion, and was also seen in purified neutrophils but not purified macrophages (Mphi). In the presence of serum, lower LPS concentrations (e.g. 40 ng/ml) elicited a strong CL response in PEC from TLR4-sufficient, and a weak signal in cells from TLR-4-deficient mice. This suggests that TLR4 engagement is involved in promoting an oxidative burst in murine neutrophils.