Objectives: CFTR function was measured in vivo by JJ Wine and coworkers (Wine JJ, et al. PLoS One. 2013;8:e77114; Char JE, et al. PLoS One. 2014;9:e88564) by computing a ratio between CFTR-independent (M-sweat, stimulated by methacholine) and CFTR-dependent (C-sweat, induced by a beta-adrenergic cocktail) sweat secretion rates by multiple individual glands in a series of subjects. We have been focusing on develop- ing a simplified method suitable for implementation in multicentre studies and possibly for supporting controversial diagnosis, and on setting up the method in Brussels. Methods: We adapted the published procedure in order to obtain com- mercial availability of instruments, reproducibility of image analysis and cost reduction. Secretion rates were given by changes of volume of sweat drops secreted in an oil layer, including the presence of a water-soluble blue dye for the C-sweat. We utilized this dye for both M and C phases in order to facilitate image analysis and simplify the equipment. Considering the results of the above cited study we estimated an appropriate sample size for detecting significant differences among CF, non-CF and carriers of 11 subjects per group. Results: We first reproduced the method and the results of the above study at the CF Center of Verona, then we simplified procedure and equip- ment and finally demonstrated operator dependent variability of image analysis. Then we set suitable experimental conditions for image analysis software developed by researchers at Politecnico di Milano. We are now aiming at automating the analysis procedure originally performed using the ImageJ software (NIH, USA). We developed a standardized operating procedure (SOP) and then confirmed previous results as we obtained aver- age ratios providing an approximately linear readout of CFTR function: the carriers’ mean ratio was 0.52 the value of non-CF controls while for all CF subjects the ratio was zero. All groups were discriminated clearly since following the standardization of this bioassay we found among these groups significant differences of C/M ratios (p<0.0001 for three-groups compari- son). In two patients with CFTR-related disease we obtained a C/M ratio of about 0.2 the non-CF control. Conclusions: We optimized and standardized a protocol and found statistically significant differences among CF, carriers and non-CF sub- jects. This bioassay is complementary to sweat tests already available and applicable to multicentre studies and is useful as well to aid in solving controversial diagnosis of CF. Support: Lega Italiana Fibrosi Cistica Associazione Veneta Onlus.
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