The color interference assay indicated possible color interference in more than 50% of the root canal samples analyzed by the endpoint QCL, even after considering serial dilution method to 10−4, a strategy usually attempted to minimize possible sample color interference. In fact, because the endotoxin samples were suspended in a noncolored medium (LAL water), it can be speculated that

the use of 25% acetic acid as a stop reagent might interfere with the assay because of its capacity to turn yellow by increasing the intensity of the yellow color and consequently overestimating the levels of endotoxin. Regarding the endotoxin detection, the sample ZD1839 datasheet GSK-3 phosphorylation by itself presents critical points that must be considered for an optimal LAL

reaction. First of them is the microbiota profile (primary vs secondary infection), particularly in secondary endodontic infection in which gram-positive bacteria (32) are predominantly involved. An unusual reactivity with peptidoglycan from the cell wall of gram-positive bacteria (≈0.00025%) (33) might account for a positive LAL assay at concentrations 1.000 to 400.000 times higher than the required one because of the alternative glucan pathway (19), requiring specifically blockage with laminarin (34). The pH variation in the root canals after the use of chemical substances during the treatment also plays an important role in the LAL reaction. In order to get an ideal pH (6.0-8.0) 30 and 31 for LAL enzyme activation, an adjustment of the pH of the root

canal samples might be required, particularly after the use of chemical substances (eg, sodium hypochlorite, chlorhexidine, and ethylenediaminetetraacetic acid). Moreover, a prior cleaning of the root canal samples by centrifugation or filtration might be necessary, particularly in the analysis Baf-A1 cost of the endotoxin samples after the use of an intracanal medicament (eg, calcium hydroxide), because the turbidity of the samples might interfere in the endotoxin measurement. In view of the results, the present study indicated that it is not possible to reconcile the levels of endotoxin determined by the endpoint QCL with the kinetic LAL methodology. Foremost, future endotoxin comparison studies must take into consideration the method used for the quantification of bacterial LPS before establishing any comparisons of the levels of endotoxin, always comparing endpoint with endpoint-QCL LAL studies, as well as kinetic to kinetic LAL investigations.