Studies on cell-on-chip technology and basophil regulation for improved allergy diagnostics
Time: Fri 2020-09-11 09.00
Subject area: Medical Technology
Doctoral student: Frida Kalm , Nanobioteknologi, Science for Life Laboratory, SciLifeLab, Department of Clinical Science and Education, Karolinska Institutet, Södersjukhuset, Stockholm, Sweden.
Opponent: Associate professor Stephanie Descroix,
Supervisor: Docent Anna Nopp Scherman, Karolinska institutet
Allergic diseases affect approximately 30% of adults and has an impact on both the individual’s quality of life as well as an economic impact on society. Two effector cells involved in allergic disease are mast cells and basophils, where basophils are more readily available in blood and therefore of great interest when studying allergy. Basophils can be recruited into the tissue during inflammation originating from for example allergic reactions or parasite infections. Allergy diagnostics starts with evaluation of the patient’s medical history followed by in vivo and/or in vitro testing. All diagnostic tests have different advantages and disadvantages are chosen depending on the patient and the circumstances. In vivo tests include the gold standard of allergy diagnostics, which is the challenge tests, but also the commonly used skin prick test (SPT). Allergy diagnostics can also be done in vitro using allergen-specific IgE antibody assays and the basophil activation test (BAT). BAT is useful to study the cellular response to an allergen but is only available at university hospitals and therefore often require long transportations of blood samples. A research field that is growing fast is microfluidics which can miniaturize and improve existing methods and diagnostic tests. The aims of this thesis were to improve the existing BAT using microfluidic techniques to enable fast and cheap point-ofcare (POC) diagnostics as well as to further study basophil adhesion and activation for a better understanding of basophil behavior and regulation both in vivo and in vitro in a microfluidic chip. In paper I, we developed a novel microfluidic immunoaffinity-based basophil activation test (miBAT) assay to investigate whether it was possible to capture and activate basophils from whole blood in a microfluidic chip. The yield of captured basophils from whole blood was 64% at a capture flow rate of 3 µl/min. The captured basophils were activated using an anti-FceRI antibody and the basophil identification marker CD203c and the activation marker CD63 were detected using fluorescence microscopy. This was done using blood from both healthy donors and allergic patients and showed comparable results between BAT and miBAT. In paper II, we further investigated whether it was possible to detect a dose-dependent allergen activation for basophils captured in a microfluidic chip. We detected a significant difference in CD63-expression between the negative control and allergen-activated basophils from allergic patients but no difference between the negative control and the non-relevant allergen (an allergen to which the patient had no IgE antibodies). The healthy donors showed no significant difference in activation between the negative control and the allergens. The miBAT results were comparable to BAT. In paper III, we studied basophil adhesion and activation to better understand both basophil function as well as the effect that basophil capture and stimulation in a microfluidic chip has on the cell. The basophil capture in a microfluidic chip could potentially mimic basophil adhesion to the endothelium and was therefore of interest due to the elevated background activation seen in unstimulated basophils captured in a microfluidic chip, reported in paper I and II. Basophils did not upregulate CD63 after passage through a microfluidic chip, but there was a slight but significant activation after crosslinking of CD203c, which is the surface marker used for basophil-specific capture in miBAT, giving one potential factor for the background activation. IgE-dependent (anti-IgE) basophil degranulation after crosslinking of adhesion molecules, to mimic adhesion before transmigration into tissue, showed a significant decrease in CD63-expression compared to anti-IgE activation, which indicate a regulatory function. Cytokine stimulation followed by IgE-independent (fMLP) basophil degranulation on the other hand showed a significantly increased CD63-expression compared to non-primed fMLP activation. In conclusion, we have developed a novel microfluidic-based technique (miBAT) able to detect basophil activation (CD63-expression) using allergens in allergic patients. miBAT was also able to discriminate between relevant and non-relevant allergen activation as well as between allergic patients and healthy controls. miBAT has the potential to be used at POC for allergy diagnostics. We have also shown that crosslinking of CD203c is a potential contributor to the basophil activation seen in the negative controls in miBAT but also that IgE-dependent activation is downregulated from crosslinking of some adhesion molecules, which is of interest both in the microfluidic chip but also in vivo to better understand basophil functions