Development of a microfluidic protocol for the measurement of basophil activation

Abstract

The prevalence of allergy is increasing worldwide, creating substantial demands on health services in establishing diagnoses and managing patients with allergic sensitivity. The basophil activation test represents a valuable tool for investigation of allergic sensitivity and even for prediction of symptom severity, though practical considerations and the high capital, training and running costs associated with flow cytometry have limited its clinical uptake. In seeking to develop a more rapid and cost effective technology for the diagnosis of allergic disease, we have investigated microfluidic approaches for the efficient enrichment of basophil populations while retaining their functional characteristics to enable basophil activation on a chip.
Acoustofluidic based basophil separation was developed by taking advantage of both the active-force movement of cells and specific surface binding of basophils to anti-CD203c under microfluidic conditions. At flow rates of 30 ml/h, up to 94 % of basophils from whole blood adhered within the acoustofluidic device, as determined by measurements of cell-associated histamine, CD63 expression by flow cytometry and basophil counts based on flow cytometric analysis and confocal microscopy imaging studies. Erythrocyte depletion agents saponin, ammonium chloride and distilled water were evaluated based on their noted affect on basophil viability. The measurement of net histamine release achieved 100% net histamine release upon allergen response and thus was applied to deplete whole blood samples prior to entry into the device. The acoustofluidic platform developed represents a novel way to isolate basophils significantly faster and more efficiently than established methods such as density gradient filtration and negative immunomagnetic separation.
Assays for basophil activation in response to house dust and grass pollen allergens, and to stimuli including anti-IgE antibody and formyl-methionyl-leucyl-phenylalanine (FMLP) peptide, were performed that involved measurement of the release of the granule components histamine (using an enzyme immunoassay) and basogranulin (by dot blotting with monoclonal antibody BB1), or measurement of upregulation of the cell surface marker CD63 (by fluorescence-activated cell sorting: FACS). Concentration response curves for IgE-dependent activation of basophils determined by each of these methods generally followed a bell-shaped curve, though there was considerable inter-subject variation. When the area under the curve was compared between experimental methods, it was found that CD63 expression was strongly correlated with histamine release (p = <0.0001, r = 0.9143, n=5) and basogranulin (p = 0.0048, r = 0.6794, n=6), and fluorescent detection of CD63 was employed as a means for investigating activation status of basophils bound within the device. Promising data has been obtained from the initial imaging studies performed to identify basophils bound within the device. Analysis performed confirmed approximately 23,450 basophils were adhered when using dip pen nanolithography slides and 11,380 using in-house printed slides when compared to controls (445 basophils in the absence of antibody and 56 when no ultrasound was present).
The findings have created a platform for a basophil activation protocol through the implementation of an acoustofluidic device, that could later be further developed as a diagnostic aid to determine allergic sensitivity in clinic.

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ORCID for Xunli Zhang: orcid.org/0000-0002-4375-1571

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