Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis

Background Basophil activationtests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies. Objective We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample. Methods Blood from 46 healthy donors and 120 patients with peanut allergywas collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs. Results Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophilCD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63hi population of basophilswas not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophilCD203c and identification of a population of CD63hi basophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C. Conclusion BATs to measure upregulation of basophilCD203c and induction of a CD63hi basophilpopulation can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.