Immunocap tryptase

Human ROSA KITWT MCs 18, 19 were cultured exactly as previously described: they were cultured in Iscove's Modified Dulbecco's Medium (ref 21980-032, Gibco, Thermo Fisher Scientific, Waltham, Mass) including the following supplements (penicillin 1%, streptomycin 1%, sodium pyruvate 1%; Minimum Essential Medium Vitamin solution 1%; Minimum Essential Medium Non Essential Amino Acids Solution 2%; and L-Glutamine 1%, Insulin-Transferrin-Selenium 1%, Gibco; Bovine Serum Albumin 0.3% CHoKL 10%, Sigma-Aldrich, St Louis, Mo), in the presence or the absence of recombinant IL-33 (10 ng/mL) or of neutralizing anti-IL33 (10 mg/mL), IgE (2 mg/mL; ref 401152, Merck Millipore, Burlington, Mass). MCs were also cultured with quercetin (Q4951, Sigma-Aldrich), cromolyn (C0399, Sigma-Aldrich), and neutralizing IL-6 antibody (MAB2061, R&D Systems, Minneapolis, Minn). Levels of histamine (ELISA Kit, IBL International GmbH, Tecan Group, M€ annedorf, Switzerland) and total tryptase (a and b) (ImmunoCAP Tryptase, Thermo Fisher Scientific) were measured in culture supernatants 1 hour after stimulation. Levels of platelet-derived growth factor (PDGF) and of TGF-b (Milliplex, Merck Millipore) were measured 5 days after stimulation.

Serum IL-31 levels were measured using an enzyme-linked immunoabsorbent assay (ELISA) standard kit (Bender MedSystems GmbH, Vienna, Austria). All procedures were performed according to the manufacturer's protocol. Total serum tryptase levels were measured using a fluoroimmunoenzyme assay (ImmunoCAP Tryptase; Thermo Fisher Scientific, Uppsala, Sweden) in all children and adults with mastocytosis enrolled in the study. The assessment of pruritus severity was performed using a visual analogue scale (VAS). Spontaneous itch severity in the last 24 h was measured at the beginning of the visit at the Department of Dermatology before physical examination and provoking Darier's sign.

We performed serial ID with Apis mellifera (Apis) venom (ALK Abelló SA, Madrid, Spain) at increasing concentrations from 0.00001 to 0.1 µg/mL. Degree of sensitization was established by the lowest concentration that produced a positive reaction (wheal with a mean diameter of 5 mm).
Serum sIgE and sIgG4 to Apis were determined using ImmunoCAP (Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative results were expressed in kU/L (sIgE) and μg/mL (sIgG4).
Baseline serum tryptase levels were measured using ImmunoCAP Tryptase (Thermo Fisher Scientific).

Patients of any age diagnosed with hymenoptera venom allergy who agreed to start VIT and continue receiving it for at least 6 months were included. Patients who dropped out before month 6 of VIT without having experienced a SAR were excluded.
Hymenoptera venom allergy was diagnosed according to the recommendations of the European Academy of Allergy and Clinical Immunology [15] in their recent guidelines [16] and was based on a detailed clinical history and a complete allergy work-up performed at least 4 weeks after the sting reaction.
The allergy work-up comprised skin prick and intradermal tests for the main hymenoptera venoms (Apis mellifera, Vespula species, and Polistes dominula [ALK-Abelló]), baseline ST (ImmunoCAP tryptase, Thermo Fisher), total IgE (Immulite 2000, Siemens Diagnostics), and specific IgE to Apis species, Vespula species, and Polistes dominula (ImmunoCAP, Thermo Fisher). Patients' clinical and demographic characteristics were collected.
In the case of suspected mast cell disease, a complete bone marrow study was performed to diagnose SM according to the current World Health Organization (WHO) criteria [17] and cMCAS according to published proposals [2, 18, 19] . The REMA score was used as a screening tool for cMCAS [19] .