**6. Conclusions**

Reports about sensitivity and specificity for BAT indicate that determination of both, CD63 and CD203c, considerably increases the sensitivity up to 92% and specificity in a range of 86–90% [60, 61]. Today, BAT is also used to determine sensitization to several allergens such as diverse types of pollen and house dust mites. It has been reported that BAT has the same sensitivity but lower specificity when compared with FEIA. BAT could be used as an alternative to SPT in some patients with allergy to aeroallergens [62] and as a useful test preventing

**Figure 14.** Basophils activation test. (a) After activation basophils upregulate CD63 and CD203c on membrane surface. Both CD63 and CD203c are detected by antibodies conjugated to fluorochromes; (b) the histogram shows increased

BAT assay is performed with 100 µL of peripheral blood; the drug is incubated with the

internal control, the same volume of blood is incubated with negative or positive controls. N-formyl-methionyl-leucyl-phenylalanine (f-MLP) is used as positive control. f-MLP is an N-formylated tripeptide that functions as a chemotactic peptide for polymorphonuclear (PMN) cells but is a potent activator of basophils too. After incubation, cells are labeled with monoclonal antibodies for 30 min, and then erythrocytes are lysed and results are analyzed by flow cytometry. To ensure that CD63 expressing cells are basophils, analyzed cells are also labeled against CD123 and Human leukocyte antigen-DR (HLA-DR). CD123 is the IL-3Rα, granulocytes including basophils, that constitutively express this cluster of differentiation [64]; whereas HLA-DR is expressed on B lymphocytes, monocytes, macrophages, activated T lymphocytes, activated natural killer (NK) lymphocytes but is absent in basophils. First, we analyzed cells by their complexity (SSC) and expression of CD123 and HLA-DR, basophils would be CD123+HLA-DR−, and only if activated by allergen or drug-medication, basophils

during 1 h; as an

blood at 1 mg/mL, in 36.5°C of temperature and atmosphere of 5% CO<sup>2</sup>

would be CD63+ CD203c+ (SSC/CD123+HLA-DR−CD63+CD203c+) (**Figure 15**).

preoperative anaphylaxis [63].

expression of CD203c on gated basophils.

92 Allergen

The analytical and functional methods described in this chapter are evolved significantly since the first clinical report related to the identification of a triggering allergen in an asthmatic patient. All the allergen-based diagnostic methodologies revised in this chapter are grounded in the antigen-antibody reaction; recognizing the advantages and disadvantages of each analytic method is essential to make adequate choices. Although the apparent simplicity of methods is described here, some technical considerations have to be considered to avoid human errors when performing and interpreting sIgE tests.

It is important to note that the understanding of these techniques could be easy, but to apply them to make therapeutical decisions is not as easy. Allergic diseases are the best example of precision medicine. In this context, the therapeutical interventions through allergen-specific desensitization and addition of biologicals to block the function of certain molecules must be argued not only with evidence-based medicine but also with a personalized analysis of every single patient. Today technology is under service of science, and we have to be aware of that. The concept of"molecular allergy" is not only to request the laboratory technician for determinations of sIgE by sophisticated methods, but also to understand these techniques and apply all this knowledge to benefit our patients. The usage of allergen-based diagnostic methodologies must reach the patient and not only remain for investigation.
