When results matter: reliable creatinine concentrations in hyperbilirubinemia patients.

Background Failure to report a creatinine concentration, especially in icteric patients who are eligible for a liver transplant, can result in a life-threatening situation. We assessed the influence of bilirubin interference on several creatinine assays and investigated ways to circumvent icteric interference without interfering with our normal automated sample logistics. Methods Using icteric patient sera (total bilirubin >255 µmol/L) we determined creatinine concentrations using an enzymatic and Jaffé assay (Roche Diagnostics) in both normal (i.e. undiluted) and decreased mode (i.e. diluted) as well as an enzyme-coupled amperometric assay on a Radiometer ABL837 FLEX analyzer. Creatinine concentrations from the five methods were compared with an in-house developed LC-MS/MS method. Passing and Bablok (proportional and constant bias) as well as difference plot parameters (bias and 95% limits of agreement [LoA]) were calculated. Interferograph-based regression analysis of the enzymatic and Jaffé results was used to investigate if such an approach could be used to report corrected creatinine concentrations in icteric patient sera. Results In icteric patient sera the enzyme-coupled amperometric assay was hardly influenced by icteric interference as shown by a difference plot bias of -1.5% (95% LoA -11.6 to +8.5%). The undiluted Jaffé method had a bias of -1.4% with a very broad 95% LoA (-35.1 to +32.2%) emphasizing the poor specificity of this method. The undiluted enzymatic method had the largest bias (-13.4%, 95% LoA -35.8 to +9.0%). Diluting sera in the enzymatic method did not improve the bias (-10.5%, 95% LoA -25.4 to 4.4%), while diluting the Jaffé method resulted in a bias increase (+11.4%, 95% LoA -14.7 to 37.5%). Using interferograph-based regression analysis we were able to reliably correct enzymatic creatinine concentrations in 97 out of 100 icteric patient sera. Conclusions Analytically, quantifying creatinine in icteric sera using the Radiometer ABL837 FLEX analyzer is the method of choice within our laboratory. However, not all laboratories are equipped with this method and even if available, the limited number of highly icteric patient sera makes this method costly. For those laboratories using the Roche enzymatic method, mathematically correcting an icteric creatinine concentration using an interferograph based on an LC-MS/MS reference method is a suitable alternative to report reliable creatinine results in icteric patients.

The production of secretory leukocyte protease inhibitor by dendritic cells.

Secretory leukocyte protease inhibitor (SLPI) is produced at mucosal sites where it plays an important role in the homeostatic control of local inflammation. In addition to its anti-protease activity SLPI is able to reduce LPS activity by interfering with the transfer of LPS to CD14. In addition SLPI can be taken up by cells where it can prevent signaling via the NF-κB route. It is preferentially expressed in dendritic cells from mucosal sites, suggesting a role in the maintenance of a tolerogenic environment, but it is unclear how this differential expression is regulated. Here we analyzed the regulation of SLPI expression in dendritic cells and found that activation by TLR ligands but not via antiCD40 leads to its expression, which is predominantly dependent on p38 activation. This induced expression is late compared to the induction of cytokines and co-stimulatory molecules, is not dependent on factors that are secreted by the cell itself and may be related to cellular feedback mechanisms involved in inflammation and immunity. In correlation with the differential expression by TLR ligands and antiCD40, SLPI deficient mice show enhanced specific immunity when antigen is co-injected with LPS, but not with antiCD40. The results underscore the importance of SLPI as a modulator of specific immunity that can also function at peripheral sites under pathogenic pressure.

Primary nasal epithelium exposed to house dust mite extract shows activated expression in allergic individuals.

Nasal epithelial cells form the outermost protective layer against environmental factors. However, this defense is not just physical; it has been shown that epithelial cells respond by the production of inflammatory mediators that may affect local immune responses. In this research we set out to characterize potential differences between the responses of nasal epithelium from healthy and allergic individuals to house dust mite (HDM) allergen. These differences will help us to define local mechanisms that could contribute to allergic disease expression. Epithelial cells were cultured from nasal biopsies taken from five healthy and five allergic individuals. These cultures were exposed for 24 hours to culture medium containing HDM allergen, or to culture medium alone. Isolated RNA was used for microarray analysis. Gene-ontology of the response in healthy epithelium revealed mainly up-regulation of chemokines, growth factors, and structural proteins. Moreover, we saw increased expression of two transcription factors (NF-kappaB and AP-1) and their regulatory members. The expression pattern of epithelium from allergic individuals in the absence of the HDM stimulus suggests that it is already in an activated state. Most striking is that, while the already activated NF-kappaB regulatory pathway remained unchanged in allergic epithelium, the AP-1 pathway is down-regulated upon exposure to HDM allergen; this is contrary to what we see in healthy epithelium. Clear differences in the expression pattern exist between epithelial cells isolated from healthy and allergic individuals at baseline and between their responses to allergen exposure; these differences may contribute to the inflammatory response.