A hand-operated microfluidic sample preparation-to-analysis workflow for simplifying the basophil activation test

Abstract

Access to the basophil activation test (BAT) has been hindered by the requirement for fresh blood analysis, specialized laboratory equipment, and advanced technical expertise. To address these issues, we have developed a hand-operated microfluidic sample preparation “µF-prep” device to perform the most time sensitive steps of the assay and stabilize the sample, effectively extending the time window before flow cytometry analysis. The µF-prep device performs concurrent basophil stimulation and staining for eight conditions in parallel. Barcoded staining of basophils allows the pooling of all conditions into one lyse/fix buffer tube for sample stabilization. After flow cytometry analysis, an XGBoost-enabled analysis pipeline unpools the eight conditions and generates basophil counts and activation levels directly from raw flow cytometry data. To characterize the µF-prep device, we stimulate whole blood samples from peanut-allergic and non-allergic donors. We compare µF-prep with a conventional BAT sample preparation protocol (“conv-prep”), and assess the stability of stimulated samples stored in the lyse/fix buffer. The µF-prep device performs sample preparation with <2 minutes of active user engagement. Our analysis pipeline shows excellent agreement with manual gating analysis. Compared with conv-prep, µF-prep exhibits similar activation levels at peanut doses of 1 – 100 ng/mL, maximum activation levels, area under the dose response curve, and EC50 values. Activation levels of basophils from anonymous and presumed non-allergic donors in samples stored in the lyse/fix buffer for up to 7 days at 4°C are similar to those analyzed on day 0. In summary, we demonstrate the potential of µF-prep to facilitate access to the BAT by simplifying sample preparation, stabilizing samples to remove the need for overnight blood shipping for flow cytometry analysis, and automating the data analysis pipeline.