A Validation Study on a Sputum Home Collection Method with Immediate Freezing and Delayed Processing: Impact on Proteomic, Mucin and RNA/DNA Endpoints

Abstract

Introduction: Due to Covid-19 restrictions on collecting and processing sputum samples in real time in clinic, we designed a novel sputum home collection method with immediate freezing and delayed processing (“home”). A validation study was carried out to compare key sputum endpoints using the “home” vs “real time (RT)” collection and processing methods. Sputum soluble phase proteomics, mucins and RNA/DNA endpoints were measured and compared between the 2 methods to assess the validity of the “home” method. Methods: Spontaneous sputum samples were collected from N=10 healthy adult volunteers. Each sample was split evenly by weight and processed, half by the “home” method and half by the RT method. Home method samples were first aliquoted into 3 collection tubes (T) as follows: T1: 100-250mg for mucin analysis (refractive index, gel chromatography, and CsCl gradients);T2 and T3: equal weights each, T2 for proteomic analysis (MesoScale Discovery) and T3 for RNA/DNA analysis (Isohelix collection kit). Each was immediately frozen at -20 deg C (24-48hr), then at -80 deg C (2-4 weeks) without any processing. Thawed home T1 and T2 samples were processed by treating with 8M Urea (1:1) to deactivate SARS-CoV-2 if present. T1 was then stored at 2-4 deg C, and T2 was processed with 7x DPBS, centrifuged and recovered supernatants stored at -80 deg C. In contrast, the RT sputum was first treated with 8M Urea (1:1) soon after collection, and then processed for mucins and proteomics per the “home” method above. The remaining cell pellet from the RT processed sample was stored in Zymo research RNA/DNA shield (0.5ml) and, along with home T3 samples, extracted and analyzed for qualitative and quantitative yield, as well as for genes of interest. Paired T-Test analysis compared all sputum endpoints between the home and RT method. Results: There were no statistically significant differences (p<0.05) between the home and RT method for any mucin (MUC5B, MUC5AC, MUC5AC:MUC5B ratio, total mucin) or proteomic endpoint (IL-1a, IL-6, IL-8, TNFalpha, TIMP1, TIMP2, MMP-9, CRP, MPO). In addition, except for CRP and MUC5AC, correlation between sample pairings was strong (correlation coefficient R, range = 0.5-0.9) and statistically significant (p<0.05) for all sputum endpoints. RNA/DNA results are still pending. Conclusion: The sputum “home collection method with immediate freezing and delayed processing” does not result in significantly different proteomic and mucin measurements when compared to the same samples being processed in real time in an identical manner.