Murine Nasal Lavage Fluid Collection without Blood Contamination

Upper airway samples, including nasal lavage fluid (NLF) and nasal swabs, are useful in detecting pathogens, including viruses and bacteria, which can cause respiratory diseases. NLF has been used to examine cellular and humoral components in the respiratory system, for example, in evaluating the induction of immunoglobulin (Ig) A following mucosal vaccinations. In experimental rodents, the NLF samples can be collected by either the trans-pharyngeal or trans-tracheal route. Although the trans-pharyngeal route has been reported to be more efficient in collecting the NLF samples than the trans-tracheal route, the NLF samples collected by the trans-pharyngeal route were often contaminated with blood, affecting the levels of cellular and humoral contents in the original NLF samples. Thus, this study aimed to establish a novel NLF collection method in experimental mice to minimize blood contamination. Briefly, before the lower jaw of the mouse was separated, cotton balls were placed in the mouth to absorb blood. When the bleeding was stopped, the NLF samples were collected by inserting a micropipette into the choana and flushing with 200 µL of phosphate-buffered saline twice (final volume: 400 µL/mouse). To detect blood contamination, a simple and sensitive forensic luminol test that detects hemoglobin was used. The luminol test demonstrated that blood contamination was detectable in the NLF samples harvested by the conventional method (without cotton balls), but not those harvested by the novel method (with cotton balls). Due to the contamination of blood, the total IgA and IgG concentrations in the NLF samples were higher in the conventional method than in the novel method; blood has been known to contain much higher levels of IgA and IgG than NLF. Therefore, this unique method can be used as a simple and sensitive method to collect NLF samples from experimental mice to prevent blood contamination.