Background: The basophil activation test (BAT) is a flow-based functional assay that rely on ex vivo basophil activation through exposure to allergenic substances.
Objective: This work aims at assessing our capabilities to simplify, standardize and miniaturize the experimental procedure of BAT.
Method: BATs were performed on whole blood and all tests were carried out within 24h of blood collection. To enable workflow simplification and standardization, we leveraged a dry and room temperature stable reagent technology. Not only staining reagents but also allergenic substances and anti-IgE for positive controls were dried. CD203c and C63 expression on basophils were monitored to characterize basophil activation upon exposure to different allergenic extracts. Water soluble protein extracts of milk and peanut were prepared in house and used throughout the study. A robotic platform was also used to fully automate sample preparation when plate-based assays were considered.
Results: After having optimized the staining reagent panels, flow cytometry performances as well as intermediate precision were characterized. Utility and relevance of the developed strategy were demonstrated through the analysis of blood samples from non-allergic and allergic donors. We further demonstrated that the developed procedure can be realized on plates with a fully automated sample preparation, extending further the standardization capabilities of the method while enabling its miniaturization.
Conclusion: A no-wash, whole blood based procedure for BAT, relying on the use of dry, room temperature stable and ready to use reagents was developed. This could be a major step toward fully leveraging the capability of BAT in clinical research.
Jean-Marc Busnel– Beckman Coulter Life Sciences