Background: A major problem of venom-specific immunotherapy (VIT) is the absence of reliable parameters for deciding treatment discontinuation. Aim of the Study: Intracutaneous tests (ICTs), the basophil activation test (BAT), specific IgEs (sIgEs) and blocking factor (BF) activity were measured during VIT. We made an evaluation by means of a protective index (PI) including ICT, BAT and BF values. Material and Methods: A population of 45 patients who had experienced a systemic reaction after an insect sting were tested before VIT (T0), at 1 week (T1w), at 10 weeks (T10w) and at 21 weeks (T21w), and, for a subgroup of 17 patients, at 3-5 years (T3-5y). Basophil activation (expressed in % CD63 and in the area under the curve) and BF activity were measured by flow cytometry using the CCR3/CD63 protocol. Results: The first 21 weeks of follow-up showed no significant variation in the ICT, sIgE and BAT measurements, except for BAT, by eliminating weak negative anti-IgE responses. In these conditions, the decrease in basophil activation was significant at T10w (p = 0.009) and T21w (p = 0.009). Increased BF activity was also significant at T10w (p = 0.008) and T21w (p = 0.002). The PI threshold calculated from the mean ± 3 standard errors (SE) was 64.8 (14.7 ± 16.7, n = 25) at T0. PI increase was significant at T3-5y (3,430 ± 6,282; p < 0.001). Conclusion: VIT induced a significant decrease in ICT values and basophil activation, along with an increase in serum BF activity, significant after 10 weeks of VIT. Evaluated in a larger population, the PI could represent a new tool for the clinico-biological follow-up of VIT efficacy.

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