Validation of synthetic absorptive matrices for sampling airway mucosal lining fluid

Sampling airway mucosal lining fluid (MLF) using a synthetic absorptive matrix (SAM) aims to overcome the problems of mediator dilution and variable return of lavage fluid inherent to nasal and bronchoalveolar lavage sampling. Accuwick Ultra (catalog no. SPR0730; Pall Life Sciences, Ann Arbor, Mich), a synthetic, fibrous, hydroxylated polyester medium is one such SAM designed for sample collection, storage, and conjugate release.

At the start of this study in April 2009 Accuwick was commercially available in sheets 8 inches x 10 inches which was then cut into small strips by Parafix Tapes & Conversions Ltd (UK) for the purposes of this study. However, despite the apparent success of Accuwick in previously published studies it had not yet been properly validated. The experiments described below aimed to answer the following questions:

1. What were the cytokine recovery capabilities of Accuwick Ultra?

2. Did the recovery differ from one cytokine to another?

3. Was recovery reproducible?

4. Was there a more suitable SAM to Accuwick?

2.1.1 Assessment of Accuwick Ultra

Strips of Accuwick approximately 3cm x 0.5cm were spiked with 70µL of known concentrations of Meso Scale Device (MSD) protein standards (see section 2.18 for full details of the MSD platform). A pro-inflammatory 4-plex MSD plate was used measuring the following cytokines: IFNγ, IL-1β, IL-6, TNFα.

49 In total, 38 strips of Accuwick Ultra were spiked with the following concentrations: 2.4pg/mL (n=6), 9.8pg/mL (n=6), 39pg/mL (n=6), 156pg/mL (n=6), 625pg/mL (n=6), and 2500pg/mL (n=8).

These were then spun using a microcentrifuge filter system to isolate the spiked matrix. A volume of 70µL was chosen to ensure that a minimum of 50 µL (enough for 2 replicates on the MSD plate per strip of Accuwick) was recovered. The standard curve ran from 2500pg to 0.15pg using an eight point dilution series.

2.1.2 Comparison of absorptive matrices

A sputum supernatant from an individual with COPD (courtesy of Dr Joseph Footitt’s study, ethics approval 07/H0712/138) acted as the test sample for comparison of Accuwick Ultra with a second matrix: Whatman’s filter paper (GE Healthcare, UK). The Whatman’s paper was cut into similar sized strips to the Accuwick and spiked in an identical fashion to the Accuwick. In this instance a 7-plex pro-inflammatory plate was utilised allowing measurement of a wider range of cytokines namely:

IFN-γ, IL-10, IL-12 p70, IL-6, IL-8 and TNFα.

To ensure that the cytokines were not being lost in transit through the spin filter itself, 40µl of the sputum supernatant was also spiked onto the spin filter directly without first being spiked onto the SAMs. The recoveries were then compared with the concentrations obtained following direct application of the sample to the well of the MSD plate (i.e. a neat sample).

Following discussion with Pall, a third SAM: ‘Leukosorb’ (Pall) was tested against Accuwick Ultra and Whatman’s filter paper. In addition, in order to assess the potential impact of differing biological sample matrices, both a sputum sample and a nasal sample were used to test the SAMs.

However, unfortunately there was insufficient sample volume to complete a full 3-way comparison and so the Leukosorb was tested against Whatman’s using the nasal sample and against Accuwick using the sputum sample.

50 2.1.3 Optimising protein recovery

I next investigated the potential for improving cytokine recovery by addition of a buffer to the SAM prior to spinning off the fluid. This was done in 2 parts, either by using a mix of Dulbecco’s PBS (H15-002, PAA) with 1% Bovine Serum Albumin (BSA) (A7906-500G, Sigma-Aldrich) or by using this mix with the addition of 1% Triton X-100 (9002-93-1, Sigma-Aldrich).

Finally, the possibility of improving recovery by allowing the sample to air dry on the SAM prior to eluting with a buffer was also a condition that was tested.

As in the previous experiment, a sputum supernatant from a subject with COPD was used in this series of experiments.

40µL of the sputum supernatant sample was spiked onto each of 24 strips of Leukosorb:

 8 strips were placed in the spin filter whilst still wet and spun immediately with no addition of buffer

 8 strips were eluted with buffer whilst still wet:

Of these:

o 4 had 60µL of PBS/ 1%BSA added prior to being placed in the spin filter and centrifuged

o 4 had 60µL of PBS/ 1%BSA with 1% Triton X added prior to being placed in the spin filter and centrifuged

 8 strips were left to air dry for 1 hour:

o 4 then had 60µL of PBS/ 1%BSA added prior to being placed in the spin filter and centrifuged

o 4 had 60µL of PBS/ 1%BSA with 1% Triton added prior to being placed in the spin filter and centrifuged.

51 Two MSD plates were used, each with a set of standards: Triton X was included in the standards of the plate in which Triton containing samples were analysed. The second plate did not include Triton in the standards or the samples.

25µL of sample was pipetted directly into 4 wells on each plate to act as a baseline level for calculation of % recoveries.

2.2 2

The bronchosorption device that was eventually used to sample bronchial MLF from the majority of subjects in this study was the end result of a series of stepwise revisions to earlier prototypes made by a medical device manufacturer called Hunt Development Ltd, (Midhurst, UK). Dr Onn Min Kon, (St Mary’s Hospital, London) and myself suggested a handle device similar to those found in other bronchoscopic sampling devices (such as bronchial biopsy forceps and bronchial brushes) to increase ease of use. The material of the catheter sheath had to be modified following initial testing to allow a more fluid advancement of the sampling probe, whilst the medical adhesive and site of attachment of the introducer to the handle had to be strengthened following a detachments in one of the first attempts. The design of the final device can be seen in figure 2.1.

52 Figure 2.1 Bronchosorption device, Hunt Developments Ltd.