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

Abstract

Background: Basophil activation tests (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 allergy was 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 basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63^hi population of basophils was 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 basophil CD203c and identification of a population of CD63^hi 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 basophil CD203c and induction of a CD63^hi basophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.

Keywords: Basophils; CD203c; CD63; EDTA; anti-coagulants; cytometry by time-of-flight mass spectrometry; heparin; peanut allergy; platelets.

FIG 1. Overview of basophil activation tests

Blood from each subject was collected separately into EDTA and heparin tubes, and stored at 4°C for 24 hours, then incubated with RPMI, anti-IgE, or IL-3. CD123⁺HLA-DR^- cells are gated as basophils (left panels) and histograms show their expression of CD203c (middle panels) and CD63 (right panels). Gray shaded histograms are of RPMI (unstimulated) cells, red lines show anti-IgE stimulation and green lines show IL-3 stimulation.

FIG 2. Comparison of anti-coagulants

Blood of the same 10 healthy donors anti-coagulated with EDTA or heparin was stored at 4°C for 24 h before cells were stimulated with IL-3 or anti-IgE. (A) (Left) ΔCD203c MFI, (Right) % of CD63^hi basophils. (B) (Left) CD203c MFI, (right) % of CD63^hi basophils after incubation with RPMI alone. Data shown (individual values [dots] and means ± SD) are the combined results of all the measurements performed in 3 independent experiments. ***P < .0005; **P < .005; *P < .05; no asterisks, P > .05. P values are stated when they are between 0.05–0.1.

FIG 3. Effect of calcium/magnesium on CD203c and CD63

Blood of healthy donors was treated with IL-3, anti-IgE with or without Ca/Mg, or Ca/Mg only. (A) Representative basophil plots of CD63 for one donor, and (B) CD63 results from 5 donors. (C) Representative basophil histograms of CD203c for one donor, and (D) CD203c MFI of 5 donors. (B, D [that share the X axis labels in D]) Data shown (individual values [dots] and means ± SD) are from 1 of 3 independent experiments, each of which gave similar results. ***P < .0005; **P < .005; *P < .05; no asterisks: P > .05 between EDTA and heparin for each condition of stimulation. None of the P values for any other comparisons are between 0.05–0.1.

FIG 4. Assessment of platelet attachment to basophils

Blood of healthy donors anti-coagulated with EDTA or heparin was incubated with RPMI or CaCl₂/MgCl₂. (A) Representative photographs of the individual blood cell types identified. (Left) DIC (Differential interference contrast) with labels indicating one basophil, several platelets, and three dendritic cells (DCs). Three panels on far right: FcεRIα, CD41, and DAPI staining alone. Second and third panels from left: Merged staining indicated as Merge1 (DIC plus antibody staining) and Merge2 (antibody staining alone). (B) Representative pictures of basophils and platelets after incubating blood with RPMI (Heparin, EDTA, left and middle panel) or RPMI with Ca/Mg (EDTA + Ca/Mg, right panel). Pictures highlighted by yellow borders are higher magnifications of the corresponding picture. Scale bars in A and B = 10 μm. Data shown in A and B are from 1 of 3 independent experiments, each of which gave similar results. (C, D) Blood was treated as in Fig 1 and CD41 positive basophils were assessed by flow cytometry of the cells of one subject (C) or by assessing the cells of 11 subjects (D). ***P < .0005; **P < .005; *P < .05; no asterisks: P > .05. (E) Blood anti-coagulated with heparin was incubated with anti-IgE and stained for CD63 and CD41. Representative dots plot are displayed. Blue dots: CD63^hi basophils, red dots: CD63^neg/low basophils, and black dots: all other cells. (F) Blood anti-coagulated with EDTA or heparin was incubated with RPMI or CaCl₂/MgCl₂ and stimulated with RPMI, anti-IgE, or IL-3 for 30 minutes. Representative images of (Upper panel) DIC and (Lower panel) merged IgE, CD63, and DAPI staining. Scale bars = 10 μm.

FIG 5. Comparison of anti-coagulants for performing BATs in peanut allergic patients

Blood from peanut allergic patients was treated with IL-3 or anti-IgE or peanut extract. (A) ΔCD203c MFI, (B) Absolute CD203 MFI values, (C) % CD63^hi basophils. (D) ΔCD203c MFI (left) and % CD63^hi basophils (right) of non-releasers identified among peanut allergic patients. Lower/higher 5 % of all values are plotted as individual values [dots]. Boxes extend from the 25^th to 75^th percentiles and whiskers represent 5^th and 95^th percentiles. Bars in the boxes indicate medians, and crosses indicate means. (A–C) n=98. (D) n=9. ***P < .0005; **P < .005; *P < .005; no asterisks: P > .05. P values are stated when they are between 0.05–0.1. Red asterisks are comparisons between EDTA and heparin at each condition of stimulation. Black asterisks are for comparisons of (B) CD203c MFI or (C) % of CD63 high basophils between RPMI and each condition of stimulation for cells analyzed in the same anti-coagulant.

FIG 6. BAT using the CyTOF platform

(A) Blood from healthy donors was collected separately into EDTA and heparin tubes, stored at 4°C for 24 hours, and then incubated with RPMI, anti-IgE, or IL-3. DNA⁺ CD235^-CD61⁻CD45⁺CD123⁺HLA-DR^- cells were gated as basophils (left panels) and histograms show their expression of CD63 (right panels) after mock stimulation with RPMI (gray shaded histograms) or after stimulation with anti-IgE (red lines) or IL-3 (green lines). (B) % of CD63^hi basophils upon stimulation with anti-IgE (left panel) or IL-3 (right panel). n = 5 for EDTA, n = 7 for Heparin. Individual values are plotted [dots] with means ± SD. **P < .005. (C) (Left) Blood anticoagulated with heparin was incubated with anti-IgE. Representative CD61 staining after basophils were gated as (DNA⁺CD123⁺HLA-DR^-). (Right) Comparison of CD63 expression between platelet^- basophils (CD61^-) and platelet⁺ basophils (CD61⁺). (D) Blood from peanut allergic patients was collected into heparin tubes, stored at 4°C for 24 hours, and then stimulated with peanut extract (100 ng/mL) for 30 min (Flow: fluorescence-based flow cytometry; upper panel) or 20 min (CyTOF; lower panel). The histograms show basophil CD63 from one representative patient. (E) Data from 5 representative patients showing % of CD63^hi basophils with conventional flow cytometry vs. CyTOF.