Basophil activation test compared to skin prick test and fluorescence enzyme immunoassay for aeroallergen-specific Immunoglobulin-E

Abstract

Background: Skin prick test (SPT) and fluorescence enzyme immunoassay (FEIA) are widely used for the diagnosis of Immunoglobulin-E (IgE)-mediated allergic disease. Basophil activation test (BAT) could obviate disadvantages of SPT and FEIA. However, it is not known whether BAT gives similar results as SPT or FEIA for aeroallergens.

Objectives: In this study, we compared the results of SPT, BAT and FEIA for different aeroallergens.

Methods: We performed BAT, SPT and FEIA in 41 atopic subjects (symptomatic and with positive SPT for at least 1 of 9 common aeroallergens) and 31 non-atopic subjects (asymptomatic and with negative SPT).

Results: Correlations between SPT and BAT, SPT and FEIA, and BAT and FEIA results were statistically significant but imperfect. Using SPT as the "gold standard", BAT and FEIA were similar in sensitivity. However, BAT had lower specificity than FEIA. False positive (BATposSPTneg) results were frequent in those atopic subjects who were allergic by SPT to a different allergen and rare in non-atopic subjects. The false positivity in atopic subjects was due in part to high levels of serum Total-IgE (T-IgE) levels in atopic individuals that lead to basophil activation upon staining with fluorochrome-labeled anti-IgE.

Conclusion: As an alternative to SPT in persons allergic to aeroallergens, BAT in its present form is useful for distinguishing atopic from non-atopic persons. However, BAT in its present form is less specific than FEIA when determining the allergen which a patient is allergic to. This is due to IgE staining-induced activation of atopic person's basophils and/or nonspecific hyperreactivity of atopic person's basophils.

Figure 1

Example of basophil activation assay. Buffy coat cells suspended in an interleukin-3 (IL-3)-containing buffer were stimulated with (a) negative control (Glycerol Saline); (b) positive control (anti-FcεRI) and (c) Timothy allergen. Basophills were defined as cells not expressing CD3, CD8, CD14, CD19 or HLA-DR (P2) and expressing CD123 and IgE (P3). Activated basophils were defined as CD63+ basophils (P4).

Figure 2

Basophil activation assay performed (1) with or (2) without IgE staining. Buffy coat cells suspended in an IL-3-containing buffer were (A) stimulated with birch allergen or (B) not stimulated (negative control - Glycerol Saline). Subsequently, the cells were stained with (1) IgE-FITC, CD63-PE, CD123-PC5, and HLA-DR/CD3/CD19-APC or (2) IgG1 Mouse Isotype Control-FITC, CD63-PE, CD123-PC5, and HLA-DR/CD3/CD19-APC. Basophills were defined as cells that do not express CD3, CD8, CD14, CD19 or HLA-DR (P2), but express CD123 (P3). Activated basophils were defined as CD63+ basophils (P4). Density plot displaying only P4 is shown for the negative control for both versions of staining (with or without IgE). Percentages of activated basophils are shown in the respective P4 gates. Corrected percentage of activated basophils was calculated by subtracting saline control percentage from allergen stimulated percentage of activated (CD63+) basophils. In this example, the corrected percentage of activated basophils on stimulation with Birch is 37.4% (38.4-1.0%) when anti-IgE was used for basophil staining and is 13.7% (14.2-0.5%) when anti-IgE was not used for basophil staining.

Figure 3

Receiver operating characteristics (ROC) curves used for the determining the cutoffs for positive BAT results. Sensitivity and Specificity of BAT was calculated using different cutoffs (10%, 15%, 20%, 25% and 30% above background) for positivity. The cutoffs at which sensitivity of BAT was similar to FEIA were selected. Selected cutoff value included 15% above background for Cat and Timothy, 25% for DP and 30% for Dog and Birch.

Figure 4

Results of BAT (left) and FEIA (right) in atopic patients (n = 39, squares and asterisks) and non-atopic persons (n = 28, diamonds). The atopic patients are divided into those allergic to the allergen of interest per SPT result ("Atopic SPT^pos", squares) and those allergic to a different allergen(s) ("Atopic SPT^neg", asterisks). The numbers of Atopic SPT+ patients were 19 for cat, 14 for dog, 11 for D.pteronyssimus, 26 for Timothy, and 21 for birch. The numbers of Atopic SPT^neg patients can be calculated for each allergen as 39 minus the number of Atopic SPT^pos patients (eg, 39-19 = 20 for cat). Significance of the difference between the Atopic SPT^pos and Atopic SPT^neg groups and between Atopic SPT^neg and Non-atopic groups is given in the upper section of each plot. BAT results are displayed as corrected percentage of activated (CD63+) basophils (saline control percentage subtracted). Undetectable IgE levels by FEIA are displayed as 0.05 kU/L. Dashed lines denote cutoffs for positivity. Cutoff for FEIA positivity was 0.1 kU/L, while that for BAT varied for different allergens - 15% above background for Cat and Timothy, 25% for DP and 30% for Dog and Birch.

Figure 5

Comparison of BAT performed with vs without IgE in healthy (non-atopic) persons and atopic patients. The atopic patients are divided into those atopic to the allergen of interest per SPT result (middle) and those allergic to a different allergen(s) (right). Significance of the difference between groups stained with and without IgE is given in the upper section of each plot. BAT results are displayed as corrected percentage of activated (CD63+) basophils (saline control percentage subtracted).

Figure 6

Comparison of T-IgE levels in atopic patients and non-atopic individuals. T-IgE levels were measured in 36 atopic patients and 28 non-atopic individuals. Significance of the difference between the two groups is given in the upper section of the plot.

Figure 7

Comparison of T-IgE levels in BAT^posSPT^neg and BAT^negSPT^neg atopic patients. The numbers of SPT^neg atopic patients analyzed for T-IgE levels were 19 for cat, 24 for dog, 27 for D.pteronyssimus, 11 for Timothy, and 16 for birch. Significance of the difference between the two groups is given in the upper section of the plot.