Detection of basophils and their activation

A rapid increase of patients with allergic hypersensitivity in last two or three generations has become a serious social and economical burden. According to WHO, e.g. the current numbers of asthmatic patients and of patients with food allergy are around 300 million and 250 million, respectively. Not only the worldwide increase of allergic patients, but also the increase of particular allergens occurs. To face this problem the reliable diagnostics is crucial. The commonly used method of skin prick tests (SPT) is not optimal due to its low specificity and the risk of further aggravating the condition. More suitable are in vitro approaches, such as the determination of specific IgE in the serum (sIgE) or the analysis of basophil activation markers. In most cases, the specificity of sIgE detection is sufficient, but the sensitivity is usually only about 75%, and in cases of some allergens (mainly food and drug) both sensitivity and specificity of sIgE detection are substantially lower. Basophil Activation Test (BAT), however, is known not only for very high specificity, but also for high sensitivity, even in cases of many problematic antigens. This test is based on the detection of basophil activation markers using flow cytometry.

The optimal marker for basophil detection is CD203c, as it is in the blood expressed only on them (in the tissues it is also on functionally similar mast cells)[1]. Markers of basophil activation are related to exposition of luminal membrane leaflet of intracellular granules on the cell surface during the process of activation-dependent degranulation. Among them the optimal one is CD63 (LAMP-3), which is on basophil surface exposed only in response to their activation, it is exposed quickly, and this process well correlates with histamine release. These two antigens, CD203c for basophil identification, and CD63 for detection of their activation, were used by BasoFlowEx kit, which was designed for common flow cytometry devices equipped with blue (488 nm) excitation laser. Anti-CD203c antibody is there in PE format and anti-CD63 in FITC format.

CD203c, however, can be used also for other purposes than for simple identification of both resting and activated basophils. In response to basophil activation, it is upregulated and it can thus serve as a supplemental activation marker in thorough analysis of causal allergen effects [2]. Besides it, increased basal expression of CD203c is a marker of chronical urticatia.

Fig.1: Activated basophil with visualized CD203c (PE, red) and CD63 (FITC, green) distribution. Colocalization of these two markers appears as yellow colour. DNA indicated by DAPI (blue). Heterogeneity of  CD63 distribution is a result of incorporation of intracellular vesicles membrane into the plasma membrane upon basophil activation-mediated degranulation.

Basophil activation test utilizing CD203c and CD63 can also be used for determination of sensitivity to a particular allergen. Whereas detection of CD63 externalized on basophils upon in vitro stimulation of patients blood sample with several allergens gives an answer about reactivity to particular allergens, in case that a dilution series of causal allergen is used, a specific individual dose-dependent activation curve is obtained for this patient, revealing further information about sensitivity to that allergen. This information is important for monitoring time-dependent development of patients allergic hypersensitivity, and can help to adjust the medication strategy.
Fig.2: Dose-dependent activation curves of six individuals upon in-vitro challenge of their basophils with allergen extract of Dermatophagoides pteronyssinus (European house dust mite). Evaluated using BasoFlowEx kit and one of valuated allergens from EXBIO collection. Three individuals were not responding to Dermatophagoides pteronyssinus allergens. Three responding (allergic) patients differed substantially in their dose-dependent activation curves, revealing that two of them reacted only to very high allergen doses, whereas one of them reacted even to three orders lower allergen doses.
 

Basophil activation kit:

Basophil antigen markers in EXBIO portfolio:

Allergens in EXBIO protfolio:

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[1] : Another basophil markers are less specific, e.g. CD193/CCR3 is expressed also on eosinophils, and is also detected in Th1 and Th2 cells (not counting e.g. airway epithelial cells); IgE is also attached to other cell types, e.g. to monocytes, and antibody binding to IgE leads to basophil activation, which is a serious complication for its use as a basophil marker.
[2] : For this application, however, one must keep in mind that CD203c expression in basophils is enhanced by IL-3, which is often a part of basophil activation buffers (such as in e.g. BasoFlowEx kit). Although induction of CD203c expression by IL-3 is quite slow (maximum of IL-3 induced CD203c upregulation is after 180 minutes of exposition to IL-3, whereas e.g. IgE-mediated upregulation of CD203c has its maximum after 15 minutes) this effect must be taken in account, or an alternative stimulation buffer without IL-3 must be used.