Validation of inducible basophil biomarkers: Time, temperature and transportation

Abstract

Background: The short stability window of several hours from blood collection to measuring basophil activation has limited the use of flow cytometry-based basophil activation assays in clinical settings. We examine if it is possible to extend this window to 1 day allowing for shipment of samples between laboratories. Several options exist for reporting the results including reporting all the measured values directly, calculating ratios and reporting a single value covering all measured results. Each of these options have different stability and value to the physician.

Methods: Whole blood samples from peanut allergic patients were stimulated with four different peanut concentrations at Day 0, Day 1, and Day 2. Samples were stored under temperature-controlled conditions. Flow cytometry was used to analyze the samples. The basophil activation and degranulation were measured as percentage of positive CD63 basophils and CD203c MFI fold change. Shipped samples were transported under ambient conditions.

Results: The results show that CD63 is a stable marker at Day 1. The CD203c ratio decreases significantly at Day 1. Calculating the CD63/IgE ratio proves to be more stable than CD63 alone. The most stable readouts are the semi-quantitative results and the trajectory of the dose response curve. Finally, we confirmed that the stability can be extended to samples shipped overnight to the laboratory.

Conclusions: It is possible to extend the stability of the basophil activation assay to 1 day for samples stored at 18-25°C as well as samples shipped under ambient conditions as long as the temperature is within the 2-37°C range.

Keywords: CD63; allergy; basophil activation test; basophils; peanut allergy.

FIGURE 1

Design of the basophil assay. (a) Gating strategy. Initially an FCS/SSC, singlet (FCS‐A/ FCS‐H) and CD45/SSC are applied. This is followed by gating on basophils using both a CD123+/CD193+ and an IgE+/SSClow gate. Basophil activation is determined by measuring percentages of CD63 positive basophils as well as the fold change in CD203c MFI compared to the negative control. (b),(c) Whole blood was stimulated with peanut allergen concentrations as indicated in the figure or anti‐IgE as a positive control or unstimulated negative control (PBS). After processing, the samples were analyzed by flow cytometry. (b) Assay precision in triplicate assays. The CD63 and CD203c activation markers were measured in samples from three donors processed in triplicate. (c) Whole blood from healthy controls were stimulated with PBS, Anti‐IgE and peanut allergen concentrations as indicated in the figure at Day 0, Day 1, and Day 2 after blood collection [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Representative dose response from four patients. The individual % positive CD63 and CD203c MFI fold change dose response curves for each day were created by connecting the data points for the various peanut concentrations, PBS and anti‐IgE at Day 0 (red circles), Day 1 (blue squares) and Day 2 (green triangles). Four representative patients have been shown. The dose response curves for all 30 patients are included in Supplementary Figure S1. (a) % positive CD63 basophils. (b) CD203c MFI fold change [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Temperature stability. Blood collected from six peanut allergic patients was stored under temperature‐controlled conditions for 22–26 h at 2–8, 18–25, 30, and 37°C before stimulated with PBS, Anti‐IgE and peanut allergen concentrations as indicated in the Figure (a) sample from the same patient that was stimulated within 4 hours of collection is included as Day 0. The expression of (a) CD63, measured as percentage of positive basophils, and (b) CD203c, measured as fold change in median fluorescent intensity (MFI) of the basophil population, was determined by flow cytometry [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Stability of shipped samples. Blood collected from six peanut allergic patients was stimulated with PBS, Anti‐IgE and peanut allergen concentrations as indicated in the figure. The expression of (a) CD63, measured as percentage of positive basophils, and (b) CD203c, measured as fold change in median fluorescent intensity (MFI) of the basophil population, was determined by flow cytometry at an out of state collection site validated to run the basophil test (Day 0). The samples were then shipped overnight to our central laboratory at ambient conditions where the basophil responses were measured on the day the samples arrived (Day 1) and the following day (Day 2) [Color figure can be viewed at wileyonlinelibrary.com]