Background
The identification of the primary sensitizing pollen is difficult in Southern European patients with Seasonal Allergic Rhinitis (SAR) if sensitized to various pollen sources with overlapping seasonality.

A more precise diagnosis is obtained by IgE assays to allergen molecules, currently available as singleplex or microarrays.

 

Objectives
To test the analytical performance of a multi‐parameter immunoblot molecular “Pollen Test” specifically designed to test IgE antibodies to pollen extracts and molecules clinically relevant in Southern Europe.

 

Methods
Sera were obtained from 101 children and 98 adults with SAR and tested with a customized multiplex immunoblot assay (EUROLINE Southern European Pollen Profile [ESEP]; EUROIMMUN AG, Luebeck, Germany) containing a comprehensive panel of allergen extracts and molecules. ESEP's outcomes were then compared in selected sera (ESEP positive to negative = 2:1) with those of singleplex IgE assays (ImmunoCAP; ThermoFisher Scientific, Uppsala, Sweden). For each of the examined reagents, qualitative (sensitivity, specificity, accuracy), semi‐quantitative (classes) and quantitative (Spearman's rank correlation, Bland‐Altmann plots) comparisons were performed.

 

Results
Compared to ImmunoCAP, cumulative ESEP's sensitivity and specificity were 87% (95% CI 84%‐90%) and 88% (83%‐93%) for extracts and 99% (98%‐100%) and 87% (83%‐91%) for molecules. Cohen's kappa coefficients (κC) ranged for extracts from 0.18 (Pellitory) to 0.50 (Cypress) and for molecules from 0.21 (Ole e 1) to 0.68 (Phl p 7). The quantitative outcomes of the two diagnostic tests were highly correlated, with Spearman's rank correlation coefficients always exceeding 0.80. Bland‐Altmann plots showed a tendency of ESEP to overestimate serum specific IgE levels, when compared to ImmunoCAP.

 

Conclusions and clinical relevance
Sensitivity and specificity of ESEP in testing serum IgE antibodies against pollen allergen extracts and molecules, in Italian patients with SAR, both exceeded 85%. The advantages and limitations of a multiplex customized immunoblot assay, in the routine clinical use of molecular diagnostics in Southern European pollen allergic patients, deserve to be tested.