Figure 4

Diagnostic algorithm. (A) Monocyte cell membrane depolarization assay. Single cell fluorescence assay was performed as described¹⁰. Measurements of the cell membrane depolarization were obtained in a extracellular environment lacking chloride-ion and were measured at two different conditions: under basal conditions (empty boxes), or with a stimulus-cocktail consisting of 8-Br-cAMP, Forkolin and IBMX (filled boxes) added at the 5th minute. Percentage of DiSBAC2(3) fluorescence variation (∆F) is shown over time according to the equation: ∆Ft = [(Ft-F0)/F0]*100, where Ft and F0 are the fluorescence values at time t and at the time t0 (5th minute). The difference between the maximum fluorescence values obtained on stimulation (filled boxes) and the matching fluorescence under basal conditions (empty boxes) represents the CF index. We defined the CF index outcome for this assay being a positive value in healthy subjects and a negative value in CF patients. A typical non-CF pattern, where a higher signal can be observed after stimulation, is represented at the top-left panel, with a CF index of +116. On the other hand, when monocytes from a subject carrying a non-sense mutation on both alleles were used, the opposite trend was observed (bottom-left panel), with a negative CF index of −25,2. With the monocytes from the patient (186-8T/C) a positive CF index was obtained (43 ± 12; n = 2; right-bottom panel), with a response to the cAMP-analogue similar to those obtained from the other HTZ subject (CF = +22; right-up panel). (B) CF index. Plotting of the CF index values obtained from the subject in comparison with recording from patients and controls performed in our laboratory using the same experimental setting. Number of Samples: CF subjects n = 40, HTZ subjects n = 28 and non CF subjects n = 31. Analysis of the 186–8 T/C subject was repeated twice (technical duplicate).