Immunotherapy in allergy and cellular tests

Figures & data

Table 1. Recent allergy immunotherapy reports and associated basophil activation tests (BATs)

Type of bat	Phenotyping strategy	Activation	Pro	Con	Cost effectiveness		References
Commercial					(A) comm/ (B) home
FlowCAST® Bühlmann	SSC/CCR3-PE	CD63-FITC	SSC allows to well separate eosinophils from basophils	CCR3 co-expressed by CD3+ T-cells		Prep: 1 hour	Sanz et al., J Invest Allerg Clin Immunol, 2002
FlowCAST® 2 Bühlmann		CD63-FITC, CD203c-PE-DY647	Activation measured as CD63+CD203c up-regulation	CCR3 downregulated by basophil activation	A	Perf: 2 hrs	Chim and Cho, All Asthma Immunol Res, 2012
FlowCAST® Higsense Bühlmann			Performance features reported in the sheet.	Inhability in evaluating CD63 or CD203c by alone and or discriminate their up-regulation in Highsense		Prep: 2.5 hrs
		CD63+CD203c-PE-DY647			B	Perf: 1 hour
					A ³100-200% more expensive
Basotest®	Anti-IgE-PE	CD63-FITC	Easy to handle	Activation based only on CD63	A = B		Rodriguez-Trabado et al., All Asthma Proc, 2008
Orpegen Pharma			Low expenditure, easy interpretation and low time-consuming	Low sensitivity respect to CD203c
			Performance features reported in the sheet.	Dependence on CD63 response
Basotest™	Anti-IgE-PE	CD63-FITC	Easy to handle	Activation based only on CD63	Less qualified than Orpegen		None reported
Glycotope			Low expenditure, easy interpretation and low time-consuming	Low sensitivity respect to CD203c
				Dependence on CD63 response
Flow cytometry protocol (1)	CD45-PC5/CRTH2/DP2-FITC	CD203c-PE or CD63-PE	Increase in sensitivity due to CD203c	Low diffusion of CRTH2 marker (high cost)	More expensive and time consuming than a common commercial kit		Boumiza et al., Clin Mol All, 2005; Boumiza et al., Clin exp all, 2003
			Capturing marker less dependent on basophil activation status	CRTH2 co-expressed on eosinophils and Th2 lymphocytes
				Need of a CD3 dissecting marker
Flow cytometry protocol (2)	SSC/CD203c-PE	CD63-FITC	CD203c is a basophil specific marker	CD203c shift basophil gate following activation = loss in CD63 expressing cells	Cost likewise a test equipped 2-colors FC		Mikkelsen et al., Clin Mol All, 2010
Flow cytometry protocols (3)	CD45/CD123/	CD63 or/and CD203c	Better discrimination of basophil capture	Elevated cost	Feasibility and handling to address		Chirumbolo et al., 2008
-4	HLA-DR		Electronic purificaton (>95%)	Time consuming			Ford et al, JACI, 2013
	CD45/CD123/		CD123 does not change the gate with activation
	HLA-DR/CD41a

Flow2CAST. 2. Home-made; 3. Basotest® (Orpegen Pharma); OAS, Oral allergy syndrome; AIT, allergy immunotherapy; SLIT, sublingual immunotherapy.

Table 2. Basophil activation tests (BATs): potential, handling, cost-effectiveness, and reliability

Type of bat	Phenotyping strategy	Activation	Pro	Con	Cost effectiveness		References
Commercial					(A) comm/ (B) home
FlowCAST® Bühlmann	SSC/CCR3-PE	CD63-FITC	SSC allows to well separate eosinophils from basophils	CCR3 co-expressed by CD3+ T-cells		Prep: 1 hour	Sanz et al., J Invest Allerg Clin Immunol, 2002
FlowCAST® 2 Bühlmann		CD63-FITC, CD203c-PE-DY647	Activation measured as CD63+CD203c up-regulation	CCR3 downregulated by basophil activation	A	Perf: 2 hrs	Chim and Cho, All Asthma Immunol Res, 2012
FlowCAST® Higsense Bühlmann			Performance features reported in the sheet.	Inhability in evaluating CD63 or CD203c by alone and or discriminate their up-regulation in Highsense		Prep: 2.5 hrs
		CD63+CD203c-PE-DY647			B	Perf: 1 hour
					A ³100-200% more expensive
Basotest®	Anti-IgE-PE	CD63-FITC	Easy to handle	Activation based only on CD63	A = B		Rodriguez-Trabado et al., All Asthma Proc, 2008
Orpegen Pharma			Low expenditure, easy interpretation and low time-consuming	Low sensitivity respect to CD203c
			Performance features reported in the sheet.	Dependence on CD63 response
Basotest™	Anti-IgE-PE	CD63-FITC	Easy to handle	Activation based only on CD63	Less qualified than Orpegen		None reported
Glycotope			Low expenditure, easy interpretation and low time-consuming	Low sensitivity respect to CD203c
				Dependence on CD63 response
Flow cytometry protocol (1)	CD45-PC5/CRTH2/DP2-FITC	CD203c-PE or CD63-PE	Increase in sensitivity due to CD203c	Low diffusion of CRTH2 marker (high cost)	More expensive and time consuming than a common commercial kit		Boumiza et al., Clin Mol All, 2005; Boumiza et al., Clin exp all, 2003
			Capturing marker less dependent on basophil activation status	CRTH2 co-expressed on eosinophils and Th2 lymphocytes
				Need of a CD3 dissecting marker
Flow cytometry protocol (2)	SSC/CD203c-PE	CD63-FITC	CD203c is a basophil specific marker	CD203c shift basophil gate following activation = loss in CD63 expressing cells	Cost likewise a test equipped 2-colors FC		Mikkelsen et al., Clin Mol All, 2010
Flow cytometry protocols (3)	CD45/CD123/	CD63 or/and CD203c	Better discrimination of basophil capture	Elevated cost	Feasibility and handling to address		Chirumbolo et al., 2008
-4	HLA-DR		Electronic purificaton (>95%)	Time consuming			Ford et al, JACI, 2013
	CD45/CD123/		CD123 does not change the gate with activation
	HLA-DR/CD41a

Figure 1. Cartoon showing a possible panel of decision strategy for the optimization of BAT use during AIT. Each step, indicated by a capital letter, represent an “analytical barrier,” which the operator must address in order to reach the best quality performance of the assay. Starting from a dual choice (commercial or home made) a BAT must address “analytical barriers” represented by the proper choice of gatig markers (A) and activation/intracellular markers (B). Usually, due to limited needs regarding cost/effectiveness and feasibility/handling, practitioners select commercial one-step, dose-related 2-colors FC protocols (C). Once chosen the methodology, further barriers are represented by the allergen sensitization protocol (D), desensitization process (E), and follow up during or post maintanance (F). Home made protocols with a dose-dependent method of allergen response calibration (D) allow a better interpretation of the cellular response during AIT.