Novel applications for a noninvasive sampling method of the nasal mucosa

Abstract

Reliable methods for sampling the nasal mucosa provide clinical researchers with key information regarding respiratory biomarkers of exposure and disease. For quick and noninvasive sampling of the nasal mucosa, nasal lavage (NL) collection has been widely used as a clinical tool; however, limitations including volume variability, sample dilution, and storage prevent NL collection from being used in nonlaboratory settings and analysis of low abundance biomarkers. In this study, we optimize and validate a novel methodology using absorbent Leukosorb paper cut to fit the nasal passage to extract epithelial lining fluid (ELF) from the nasal mucosa. The ELF sampling method limits the dilution of soluble mediators, allowing quantification of both high- and low-abundance soluble biomarkers such as IL-1β, IL-8, IL-6, interferon gamma-induced protein 10 (IP-10), and neutrophil elastase. Additionally, we demonstrate that this method can successfully detect the presence of respiratory pathogens such as influenza virus and markers of antibiotic-resistant bacteria in the nasal mucosa. Efficacy of ELF collection by this method is not diminished in consecutive-day sampling, and percent recovery of both recombinant IL-8 and soluble mediators are not changed despite freezing or room temperature storage for 24 h. Our results indicate that ELF collection using Leukosorb paper sampling of ELF provides a sensitive, easy-to-use, and reproducible methodology to collect concentrated amounts of soluble biomarkers from the nasal mucosa. Moreover, the methodology described herein improves upon the standard NL collection method and provides researchers with a novel tool to assess changes in nasal mucosal host defense status.

Keywords: biomarkers; epithelial lining fluid; innate immune status; nasal mucosa; storage conditions.

Fig. 1.

Percent recovery of recombinant IL-8 from Leukosorb strips using various storage conditions. A: flowchart describing methods for detecting the percent recovery of IL-8 recombinant protein from Leukosorb strips. B: image of a Leukosorb strip and its relative size. C: diagram and measurements of cut Leukosorb strips. D: percent recovery of recombinant IL-8 from Leukosorb strips at varying storage conditions (n = 5). A known amount of recombinant IL-8 with no carrier was spiked onto Leukosorb strips. The strips were then stored in triplicate in four storage conditions (immediate elution, −20°C, room temperature, or 37°C). Protein was then eluted from strips and the eluant was stored at −20°C until analysis (at least 24 h). The strips were then batch-analyzed by ELISA and compared with protein of a known concentration. Compared with the strips that were immediately eluted, there was a significant difference in percent recovery only in the 37°C group. Values are presented as means ± SE. *P ≤ 0.05.

Fig. 2.

Intranare, day-to-day, and interindividual variability of IL-8 production in epithelial lining fluid (ELF). A: flowchart describing methods for determining variability in ELF samples within nares, from day to day, and between individuals using Leukosorb paper and IL-8 ELISA. B: intranare variability in IL-8 production using the average of the right nare over 3 days of sampling and left nare over 3 days of sampling (biological replicates = 3). There was no significant difference in IL-8 concentration intranare. Means are graphed for each individual. C: day-to-day variability in IL-8 production. The left and right nare were averaged for each day for a biological replicate of two. There was no significant variability in IL-8 concentration over the 3 days. Means are graphed for each individual at each day. D: interindividual variability in IL-8 production. Right and left nare measurements for each of the 3 days is graphed for each individual. There is significant interindividual variability. There is also a significant sex difference, IL-8 production is greater in men than it is in women. Values are presented as means ± SE. In B–D, n = 5; open squares indicate men, closed circles indicate women. ***P ≤ 0.001.

Fig. 3.

Effectiveness of a variety of storage conditions for collection and analysis of ELF for biomarkers of immune status from Leukosorb strips. A: flowchart describing methods for determining the effectiveness of varying storage conditions on analysis of ELF from Leukosorb strips for biomarkers of immune status. B: IL-1β collected per strip. Levels were lower in the 37°C group than in the immediate-elution group. C: IL-6 collected per strip. D: IL-8 collected per strip. Levels were lower in the 37°C group than the immediate-elution group. E: IP-10 collected per strip. F: neutrophil elastase collected per strip. Groups of strips stored at room temperature (RT) and 37°C had lower values of neutrophil elastase than the group that underwent immediate elution. G: IL-8 concentration resultant from elution of Leukosorb strips. The groups stored at RT and 37°C were dried out during storage. They were thus eluted in a lower volume of liquid, making the resultant protein more concentrated. The concentration of the eluant per milliliter is shown. The eluent at room temperature was more concentrated than the group that underwent immediate elution (Immed. Elution) and thus would be better for detecting low-abundant protein than the more dilute eluant of the immediate-elution group. Values are presented as means ± SE. In B–G, n = 10. Statistical analysis included one-way ANOVA with a Dunnett’s post hoc test, comparing all groups with the immediate-elution group. *P ≤ 0.05, ***P ≤ 0.001.

Fig. 4.

Comparison of ELF from Leukosorb strips and traditional nasal lavage fluid (NLF) for detection of biomarkers of immune status. The immediate-elution group from the ELF storage condition study was compared with visit 1 (v1) NLF from the same subjects. A: IL-1β levels in ELF vs. NLF. Significantly more IL-1β was detected in the ELF than the NLF. B: IL-6 levels in ELF vs. NLF. C: IL-8 levels in ELF vs. NLF. Significantly more IL-8 was detected in ELF than in NLF. In addition, there was a significant sex difference in the immediate-elution group, in which IL-8 levels were higher in men than in women. D: IP-10 levels in ELF vs. NLF. Significantly more IP-10 was detected in ELF than in NLF. E: neutrophil elastase (NE) levels in ELF vs. NLF. Significantly more NE was detected in ELF than NLF. In addition, there was a significant sex difference in the immediate elution group, in which NE levels were higher in men than in women. F: day-to-day variability in IL-8 NLF. NLF from the 2 days of collection were compared, and there was no variability detected between the 2 days; n = 10; v2 indicates visit 2. Statistical analysis included a paired t-test comparing ELF with NLF. Sex difference was detected using a two-way ANOVA with sex and storage condition as factors with a Dunnett’s post hoc test comparing all groups with the immediate elution group. *P ≤ 0.05, ***P ≤ 0.001.

Fig. 5.

Ability of ELF to measure viral markers of disease in subjects given the live attenuated influenza virus (LAIV) vaccine and the presence of bacteria. A: flowchart describing methods for administration of LAIV to healthy volunteers. B: measurement of influenza FluB gene expression before LAIV administration (day 0) and 1 day after exposure (day 1) from samples collected through both ELF and NLF sampling methods. C: measurement of gene expression of the antibiotic-resistant bacteria marker mecA before and after LAIV in ELF and NLF samples; n = 6.