Flow cytometry for basophil activation markers: the measurement of CD203c up-regulation is as reliable as CD63 expression in the diagnosis of cat allergy.

The flow cytometric basophil activation test (BAT), based on the detection of allergen-induced CD63 expression, has been proved effective in the diagnosis of various IgE-mediated allergies. However, there is not yet consensus about the suitability of CD203c expression as a specific basophil activation marker and its diagnostic reliability. The goal of the present study was to compare measurement of CD63 and CD203c expression using BAT in a model of cat allergy and to determine optimal experimental conditions for both markers. Heparinized whole blood samples from 20 cat allergic patients and 19 controls were incubated with Fel d1 (relevant allergen) or anti-FcepsilonRI (positive control) either in IL-3 or IL-3-free conditions. An optimal gating of basophils was achieved in triple staining protocols: anti-IgE PE/anti-CD45 PerCP/anti-CD63 FITC or anti-IgE FITC/anti-CD45 PerCP/anti-CD203c PE. We demonstrated that IL-3 significantly enhanced CD63-induced expression by basophils obtained from cat allergic patients in response to Fel d1. Sensitivity was found to be 100%. The CD203c protocol, when performed under IL-3-free conditions, also demonstrated 100% sensitivity. Only one of the control subjects was positive in both tests. In conclusion, using well-defined experimental conditions, the measurement of CD203c up-regulation on basophils in response to specific allergens is as reliable as CD63-BAT for the in vitro diagnosis of patients with IgE-mediated allergy.

IL-4 and IL-13 mRNA real-time PCR quantification on whole blood to assess allergic response.

Although routinely used in clinical practice, skin prick tests and serum specific IgE often fail to distinguish between IgE-sensitization and symptomatic IgE-mediated allergy. There is therefore a need for new laboratory tests relating allergic symptoms to the offending agent. In this way, we evaluated the diagnostic reliability of a new whole blood quantitative real-time PCR assay for IL-4 and IL-13 mRNAs. We compared the response of cat allergic patients and non-cat allergic controls upon anti-IgE and cat allergen (Fel d1) stimulation of whole blood. Allergen addition led to a significant increase of IL-4 and IL-13 mRNAs in allergic patients compared to non-allergic controls (p<0.0001). Both cytokine mRNA levels were strongly correlated and peaked within 2 h after Fel d1 or anti-IgE addition. This rapid increase as well as purification experiments led us to the conclusion that basophils represent an important if not the main source of both transcripts in this setting. The effect was allergen-specific since not observed when stimulating blood from cat allergic patients with birch pollen. Interestingly, we found that IFN-gamma mRNA, contrary to IL-4 mRNA, reached higher levels in response to Fel d1 with control individuals than with patients allergic to cat. This study shows that whole blood real-time PCR is a valuable method for IL-4 and IL-13 mRNA measurement after in vitro allergen challenge. We suggest that it might be useful, complementing conventional markers, for the diagnosis and the follow-up of allergic diseases. Further assessments are required to evaluate the clinical potential of this technique.