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Published in final edited form as: J Allergy Clin Immunol. 2016 Jul 15;139(3):889–899.e11. doi: 10.1016/j.jaci.2016.04.060

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

Kaori Mukai a,b, Nicolas Gaudenzio a,b, Sheena Gupta c, Nora Vivanco d, Sean C Bendall d, Holden T Maecker c,e, Rebecca S Chinthrajah b,f, Mindy Tsai a,b, Kari C Nadeau b,f, Stephen J Galli a,b,e
PMCID: PMC5237629  NIHMSID: NIHMS803372  PMID: 27527263

Abstract

Background

Basophil activation tests (BATs) have promise for research and for clinical monitoring of subjects 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

To attempt 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 peanut allergic patients was collected into ethylenediaminetetraacetic acid (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-Immunoglobulin E (anti-IgE) or interleukin-3 (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 CD63hi 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 for identification of a population of CD63hi basophils, whether the specimens were analyzed by conventional flow cytometry or by Cytometry by Time-of-Flight mass spectrometry (CyTOF), 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 CD63hi basophil population can be conducted using blood obtained in heparin tubes and stored at 4°C for 24 hours.

Keywords: Basophils, CD63, CD203c, anti-coagulants, heparin, EDTA, peanut allergy, Cytometry by Time-of-Flight mass spectrometry, CyTOF, platelets

INTRODUCTION

Basophils and mast cells are major effector cells of IgE-dependent immune and allergic responses.1–3 These cells express large numbers of the high affinity IgE receptor, FcεRI, on their surface and crosslinking of their FcεRI-bound IgE by bi- or multi-valent allergens induces the secretion of multiple stored and newly synthesized mediators, cytokines, and chemokines.4–7 Although basophils typically represent <1% of peripheral blood leukocytes, analysis of basophil function has become increasing popular, both because basophils may have certain unique roles in immunity and allergic diseases8–10 and because blood basophils are much more readily available for analysis than tissue-resident mast cells.

Studies of basophil activation ex vivo, i.e., basophil activation tests (BATs), are flow cytometry-based assays to assess basophil activation by various stimuli. The BAT was first developed as a diagnostic test in 1991 and the usage of such tests has subsequently increased.11–13 However, several different BATs are now used, including commercial kits and tests developed and employed by research groups. Various BATs differ in the choice of anticoagulant, temperature and duration of blood storage, activation markers measured, consideration of the effects of possible platelet attachment to basophils, reproducibility, whether basophils are studied in whole blood or after various purification steps, and stimulants used to activate the cells.14–22

However, to compare most meaningfully results of BATs obtained with different patient populations analyzed at various sites, it would be optimal to employ standardized methods at all sites. Moreover, such protocols ideally could be used at reference laboratory sites after overnight shipment of samples from widespread clinical sites. We developed a simple protocol which permitted us to perform BATs on whole blood stored for up to 24 h before analysis, and showed that this protocol could be used to analyze basophils both by conventional flow cytometry and by the newly introduced method, Cytometry by Time-of-Flight mass spectrometry (CyTOF).23

METHODS

Outline of experiments

We used blood specimens from both healthy donors and patients with peanut allergy to compare BAT results obtained in blood anticoagulated with EDTA vs. heparin and stored before BAT analysis at room (or ambient, for shipped specimens) temperature or at 4°C for 4 or 24 h. See Table E1 for a summary of the design of the experiments depicted in the various regular and supplemental figures.

Blood specimens

Blood from randomly selected anonymous donors (allergy status unknown) was obtained from the Stanford Blood Center (Palo Alto, CA, USA) and blood from peanut allergic patients (see Tables E2 and E3 in the Online Repository) was obtained as part of enrollment into an IRB-approved clinical trial (ClinicalTrials.gov Identifier: CT02103270). Peanut allergy was defined as having a reaction to a double-blind, placebo-controlled food challenge to peanut (up to 500 mg total of peanut protein) and a positive skin prick test to peanut (>= to 5 mm).

Basophil Activation Tests

Blood specimens were gently rotated at room temperature or at 4°C for 4 or 24 h after blood collection. Immediately before starting BAT assays, samples were put into a water bath at 37°C for 30 seconds. 100 μL of whole blood were mixed with 100 μL of medium only or each stimulant. More details about the BAT protocols, and the reagents employed, can be found in this article's Online Repository at www.jacionline.org.

Cytometry by Time-of-Flight mass spectrometry (CyTOF)

Metal labelled antibodies used for CyTOF analysis are shown in Table E4. Other details can be found in this article's Online Repository at www.jacionline.org.

Basophil quantification using fluorescence beads, basophil and platelet analysis by confocal microscopy, and preparation of peanut extract

Details can be found in this article's Online Repository at www.jacionline.org and 24.

Statistical Analysis

Mann-Whitney U tests were performed (the groups analyzed are described in the figure legends) and the results reported in figures as ***P < .0005; ** P < .005; *P < .05. We considered P < .05 as statistically significant.

RESULTS

BATs can be performed 24 hours after collection of heparin anti- coagulated blood stored at 4°C

We sought to identify conditions of blood collection and storage that would permit conducting basophil activation tests (BATs) using specimens stored as long as 24 h before analysis. This interval would permit shipping specimens obtained at one location to another for analysis. We performed anti-IgE or IL-3 stimulation of basophils in whole blood and used changes in CD203c25–27 and CD6328–30 as basophil activation markers. Basophils were gated as CD123 positive and HLA-DR negative cells31 and expression of CD203c and CD63 in gated basophils was shown as histograms (Fig 1).

FIG 1. Overview of basophil activation tests.

FIG 1

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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.

Basophils exhibited upregulation of both CD203c and CD63 upon anti-IgE or IL-3 stimulation in samples from normal blood donors that were collected in either EDTA or heparin, although the CD63 upregulation in EDTA was minimal (Fig 1). CD203c was uniformly upregulated in both EDTA and heparin samples. In heparin, but not EDTA, specimens, anti-IgE stimulation induced a strongly bimodal upregulation of CD63, yielding a basophil population with high levels of fluorescence intensity (in Fig 1, the “CD63hi” basophil population represented 0.02% in EDTA and 22% in heparin samples, respectively). In subsequent experiments we compared the intensity of responses under different protocols of testing using mean fluorescence intensity (MFI) to quantify CD203c and the % of CD63hi basophils to quantify CD63.

We first compared results obtained 4 or 24 h after blood storage at 4°C or at room temperature. When blood samples in EDTA were stimulated with anti-IgE or IL-3, the most significant and largest differences in basophil CD203c expression (ΔCD203c) were in specimens stored for 24 h at 4°C (see Fig E1, A in the Online Repository). In heparin specimens, ΔCD203c was similar under all 4 conditions after anti-IgE stimulation, but, as with EDTA specimens, the most significant and substantial ΔCD203c after IL-3 stimulation was in specimens stored for 24 h at 4°C (see Fig E1, A in the Online Repository). CD63hi basophils were observed only in specimens collected with heparin and stimulated with anti-IgE, but the results obtained in the 4 conditions of storage were very similar (Fig 1 and Fig E1, B in the Online Repository). Absolute MFI values for CD203c without stimulation (RPMI media) were low under all conditions, with values in EDTA specimens being higher in samples stored 24 h at either temperature whereas the opposite was the case for specimens collected in heparin (see Fig E1, C in the Online Repository). No obvious CD63hi populations were observed without anti-IgE or IL-3 stimulation in any of the conditions (see Fig E1, D in the Online Repository). However, in tests of heparin specimens, 3 of 11 donors barely responded to anti-IgE stimulation (both ΔCD203c and % of CD63hi basophils were nearly zero), but the cells responded to IL-3 stimulation. We therefore considered these 3 subjects to be “non-releasers”.32–36

Importance of exogenous or added calcium/magnesium for BAT measurements

In tests of blood collected into either EDTA or heparin tubes from the same healthy donors, no significant difference was observed in ΔCD203c between EDTA and heparin (P = 0.0627) upon anti-IgE stimulation, but anti-IgE significantly induced CD63hi basophils only in the heparin samples (P < 0.0001, Fig 2, A). Absolute levels of CD203c MFI were higher in basophils in heparin than in EDTA (Fig 2, B) both without stimulation and after anti-IgE stimulation (data not shown), which resulted in similar values for ΔCD203c in the EDTA and heparin samples.

FIG 2. Comparison of anti-coagulants.

FIG 2

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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 CD63hi basophils. (B) (Left) CD203c MFI, (right) % of CD63hi 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.

The addition of calcium/magnesium to blood collected in EDTA tubes just before anti-IgE stimulation resulted in induction of a CD63hi population in response to anti-IgE that was similar to that induced in heparin samples (Fig 3, A and B) (EDTA vs heparin; P = 0.0079 without Ca/Mg, P > 0.05 with Ca/Mg). By contrast, addition of calcium/magnesium did not significantly influence the % of CD63hi basophils in heparin (P > 0.05 % CD63hi in anti-IgE vs anti-IgE + Ca/Mg conditions in heparin). We next examined whether calcium/magnesium also affected CD203c expression in EDTA and/or heparin, because it has been reported that calcium ionophore can induce CD203c upregulation.31 Without stimulation, CD203c levels were higher in heparin vs. EDTA specimens (Fig 2, B, Fig 3, C and D). Adding only calcium/magnesium significantly upregulated CD203c in basophils in EDTA (P = 0.008 RPMI vs Ca/Mg only) but not in heparin (P = 0.222 RPMI vs Ca/Mg only). Also, while anti-IgE stimulation elicited higher absolute CD203c expression in heparin compared to standard EDTA samples, adding calcium/magnesium to anti-IgE resulted in induction of similar absolute levels of basophil CD203c MFI in the two anti-coagulants. These results indicate that calcium/magnesium contributes to both baseline levels and upregulation of basophil CD203c surface expression.

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

FIG 3

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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.

Because RPMI contains calcium nitrate and magnesium sulfate, we compared RPMI and calcium/magnesium-free PBS (CMF-PBS) as vehicles. We found no differences between RPMI and CMF-PBS for either CD203c upregulation or induction of CD63hi basophils (data not shown). This provided additional evidence that chelation of extracellular calcium/magnesium by EDTA was the reason for the failure of anti-IgE stimulation to induce CD63hi basophils in EDTA specimens. Our data thus indicate that addition of calcium/magnesium is essential to obtain CD63hi basophils in EDTA specimens.

Finally, we found no significant differences in the % of basophils among the 4 conditions of storage in EDTA or heparin (see Fig E2, A in the Online Repository). Nor were there significant differences in the % of basophils in EDTA and heparin samples after storage of blood at 4°C for 24 h (see Fig E2, C in the Online Repository). However, there were slightly lower numbers of basophils in heparin specimens after storage at 4°C for 24 h (see Fig E2, B in the Online Repository).

CD63 expression is predominantly due to basophils rather than platelets

CD63 can be expressed on platelets in addition to basophils and mast cells,29,37,38 and activated platelets can bind to various leukocytes, raising concerns that platelet binding to basophils can falsely elevate levels of “basophil” CD63.15,17,39–41 We used the platelet/megakaryocyte specific marker CD41 (gpIIB) to search for CD41 staining associated with basophils. Immunocytochemical analysis revealed that platelets were attached to some basophils in both EDTA and heparin specimens (Fig 4, A and B). Adding calcium/magnesium to EDTA specimens caused extensive aggregation of platelets, and some of these large aggregates attached to basophils (Fig 4, B). By flow cytometry, basophils in heparin samples exhibited more platelet attachment than did those in standard EDTA specimens (Fig 4, C and D). This is consistent with a report that the majority of CD63+ basophils were CD41- when EDTA, which is known to inhibit platelet activation,42,43 was used as an anti-coagulant. The mean % of basophils that were CD41+ after anti-IgE stimulation was somewhat higher in the CD63hi than in the CD63-/lo population (Fig 4, D). However, basophils exhibited similar levels of high or low CD63 expression regardless of the expression level of CD41 (Fig 4, E). Furthermore, we confirmed morphologically that CD63 was expressed on basophils after anti-IgE but not IL-3 stimulation in heparin but not at all in EDTA unless exogenous calcium/magnesium was added (Fig 4, F). Our observations are consistent with those of others, who concluded that CD63 is expressed on basophil itself when the cells are activated via the FcεRI.44,45 Moreover, in heparin specimens, CD41 was positive on some basophils after IL-3 stimulation even though IL-3 did not increase basophil CD63 expression. Finally, we found that leukocytes other than basophils were also CD41+ (between 21–73% of cells, Fig 4, E), but CD63 was expressed only on basophils by flow cytometry or direct microscopy. Together, these results indicate that the appearance of many CD63hi basophils is predominantly or entirely due to basophil-derived rather than platelet-derived CD63.

FIG 4. Assessment of platelet attachment to basophils.

FIG 4

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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: CD63hi basophils, red dots: CD63neg/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.

BATs by flow cytometry in peanut allergic patients

We next tested blood collected in heparin tubes from 21 peanut allergic participants enrolled in a trial of oral immunotherapy (OIT) for peanut allergy (Table E2 in the Online Repository), after storage of the blood for 24 h at 4°C or room temperature. Values for anti-IgE-induced ΔCD203c were not significantly different among the 4 conditions of blood storage (see Fig E3, A in the Online Repository), like with blood specimens from healthy donors (see Fig E1, A in the Online Repository). In cells stimulated with the 3 highest concentrations of peanut extract, those stored at RT for 24 h had lower ΔCD203c values vs. at 4 h, but there were no significant differences between the corresponding 4 and 24 h values at 4°C. Values for CD63hi basophils after treatment with anti-IgE or the 3 highest concentrations of peanut extract were not significantly different between 4 and 24 h at either 4°C or RT (see Fig E1, B in the Online Repository). Slightly higher values for CD63hi basophils were observed after incubation with RPMI, IL-3 or a low (1 ng/mL) concentration of peanut extract at 24 h vs. 4 h at 4°C, but these changes were more marked in RT specimens, even in non-releasers (see Fig E3, B in the Online Repository), a finding we did not observe in blood from healthy donors (see Fig E1, D in the Online Repository). Finally, higher mean “basal” levels of CD63hi basophils (in specimens treated only with RPMI), and higher variation in the individual values, was especially notable in RT specimens in comparisons between 4°C and RT at 24 h (see Fig E3, C in the Online Repository).

Given those results and in light of other possible pitfalls of performing BATs after blood stored at RT for 24 h18,46, and considering that there were little or no differences in results obtained with blood stored at 4°C for 4 or 24 h, we decided to store blood routinely at 4°C for 24 h before performing BATs. We tested heparin vs. EDTA anticoagulated blood obtained in the same venipuncture from each of 98 peanut allergic patients (see Table E3 for their demographic characteristics in the Online Repository) at their screening for entry into the OIT trial. Upon stimulation with anti-IgE, IL-3, or 5 concentrations of peanut extract, CD203c upregulation occurred in basophils using either anti-coagulant (Fig 5, A). Values for ΔCD203 upon IL-3 stimulation were slightly lower in heparin vs. EDTA but all doses of peanut extract resulted in significantly higher values in heparin vs. EDTA. As in specimens from healthy blood donors (Fig 2, B), levels of CD203c surface expression without stimulation were slightly higher in heparin vs. EDTA (P < 0.0001, Fig 5, B) and induction of many CD63hi basophils after stimulation with anti-IgE or peanut extract was observed only in heparin (Fig 5, C). However, we observed a small amount of CD63 upregulation at lower fluorescence intensity both in EDTA and heparin, like with specimens from healthy blood donors (Fig 1, data not shown). As with healthy blood donors, 9 of the 98 patients were non-releasers, with basophils responding neither to anti-IgE nor peanut antigen (Fig 5, D). We found that very similar results were obtained in BATs done in duplicate at the same time using blood from the same patients (see Fig E4, A in the Online Repository) or when we tested blood from the same subjects at screening or weeks later at the beginning (week 0) of the OIT trial (see Fig E4, B in the Online Repository). To examine the feasibility of performing BATs in specimens after shipment at 4°C, we conducted BATs 24 h after blood collection in aliquots of blood from the same subjects with/without overnight shipment (over a distance of 130 km) at 4°C or at room or ambient temperature. ΔCD203c upon anti-IgE stimulation didn’t differ significantly between specimens kept at different temperatures with or without shipment but IL-3-induced ΔCD203c was lower in specimens which had been kept at room/ambient temperature (regardless of shipment) as in Figure E1 (Figure E5, A). CD63hi basophil values were more variable at RT with/without shipment, also as in Figure E1 (Figure E5, B).

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

FIG 5

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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) % CD63hi basophils. (D) ΔCD203c MFI (left) and % CD63hi 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 25th to 75th percentiles and whiskers represent 5th and 95th 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.

BATs can be performed by CyTOF mass spectrometry in blood stored 24 h before analysis

Finally, we investigated whether our protocol for preparing whole blood for BATs could also be used for the recently established CyTOF mass spectrometry approach, which employs metal-labeled antibody probes instead of antibodies labeled with fluorescent compounds.23 The appearance of a CD63hi basophil population upon stimulation with anti-IgE was observed in blood from healthy donors that was obtained with heparin but not with EDTA (Fig 6, A and B), which is consistent with the results we obtained using conventional flow cytometry (Fig 1, Fig E1 in the Online Repository, Fig 2). Notably, it appeared that few platelets were attached to basophils analyzed by CyTOF (only 0.6–6% of basophils were positive for the platelet marker CD61) (Fig 6, C, left). Moreover, the expression pattern of CD63 was equivalent between CD61+ and CD61− basophils (Fig 6, C, right). These findings support the conclusion that CD63 expression is predominantly due to basophils rather than platelets. In blood from peanut allergic donors obtained in heparin tubes, CyTOF also can be used to assess upregulation of CD63 and the appearance of a CD63hi population of basophils in specimens stimulated with peanut extract (Fig 6, D), and the results obtained with CyTOF correlate very well with those obtained by flow cytometry (Fig 6, E).

FIG 6. BAT using the CyTOF platform.

FIG 6

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(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 CD63hi 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 CD63hi basophils with conventional flow cytometry vs. CyTOF.

DISCUSSION

We wished to define a practical protocol for performing BATs in blood stored for up to 24 h before analysis. We used whole blood because of its convenience, compared to first having to purify basophils (which also might result in some changes in basophil function and activation parameters), and also because tests conducted in whole blood may be more physiologically relevant. We decided against using IgE or FcεRIα as gating markers both because their expression could markedly change depending on the concentration of serum IgE and because other cells can express these markers, particularly in allergic patients.47–51 Also, FcεRI crosslinking by allergen causes their internalization and this could interfere with basophil gating.52

Our findings indicate that, for conducting BATs 24 h after sample collection, using heparin as the anti-coagulant and storage at 4°C are the optimal conditions among those tested. Using heparin (vs. EDTA) permitted a much more robust induction of CD63hi basophils after anti-IgE. Moreover, performing BATs after storage of heparinized blood for 24 h at 4°C also permitted strong anti-IgE- or IL-3-induced upregulation of CD203c surface expression. Notably, there appears to be no consensus about the optimal time at which to conduct BATs. Sturm et al.19,20 reported a time-dependent drop of basophil reactivity in EDTA specimens stored at 4°C. However, Sousa et al.53 observed that the basophil response was intact at 24 h after blood collection using the same conditions as Sturm et al.19,20

We confirmed that upregulation of CD63 requires physiological extracellular concentrations of calcium/magnesium (Fig 3), consistent with current understanding that degranulation of basophils and mast cells is dependent on extracellular calcium/magnesium.54–57 The lack of induction of CD63hi basophils in EDTA samples can be compensated by addition of exogenous calcium/magnesium. Indeed, some groups and commercial kits use EDTA as an anti-coagulant and add calcium/magnesium to enable the stimulus-dependent induction of a CD63hi basophil population.58,59 However, adding exogenous calcium/magnesium lengthens the procedure and could cause variable results. Moreover, we found that calcium/magnesium affected not only CD63 but also CD203c expression levels, including baseline levels (Fig 3, C and D). Finally, addition of calcium/magnesium caused extensive platelet aggregation, resulting in large aggregates of platelets that might affect BAT results (Fig 4, B).

Some BATs are conducted with IL-3 priming.60–62 Because stimulation with IL-3 alone induced upregulation of surface CD203 (Fig 1, Fig E1, A in the Online Repository, Fig 2), this has the potential to partially mask CD203c upregulation in response to allergens.63 Therefore, we recommend that not adding IL-3 routinely during BATs, particularly when assessing CD203 responses.

Importantly, we examined whether our protocol (4°C/24 hours) could be used to perform BATs in allergic patients, including assessing stimulation with peanut antigen. When we assessed BATs in blood samples from 98 peanut allergic patients collected separately into EDTA or heparin, we obtained results with anti-IgE or IL-3 stimulation that were essentially the same as those from blood of healthy donors. Because some subjects are “non-releasers”,32–36 it is essential to include a non-FcεRI mediated stimulant, such as IL-3, as a positive control. Consistent with prior reports, we found that approximately 10–20% of healthy donors and allergic patients are non-releasers who do not respond to FcεRI-mediated stimulation (data from such “non-releasers” are included in the data shown in our figures).

In conclusion, we report that BATs can be performed by flow cytometry in blood obtained with heparin as an anti-coagulant and after storage for 24 h at 4°C. Such specimens can be analyzed by both conventional flow cytometry and CyTOF mass spectrometry. While the two methods gave very similar results for stimulus-induced changes in basophil CD63 surface expression, CyTOF can permit multiple additional basophil surface structures or intracellular proteins to be analyzed simultaneously. Given the cost of the equipment, and the expertise, needed for performing CyTOF analyses, it is useful that these tests, as well as conventional flow cytometric BATs, can be performed on blood stored for 24 h prior to analysis.

Supplementary Material

Key Messages.

  • Heparin is superior to EDTA as an anticoagulant for BATs since heparin permits assessment of both CD203c upregulation and CD63hi basophils.

  • BATs can be performed using heparinized blood stored for 24 hours at 4°C.

  • Using heparinized blood, BATs can be performed either by conventional flow cytometry or by Cytometry by Time-of-Flight mass spectrometry.

Acknowledgments

We thank Dr. Chen Liu for excellent technical assistance, and Drs. Alexandra F. Santos and Gideon Lack for critical comments on the manuscript.

Declaration of funding sources: Supported by National Institutes of Health (NIH)/National Institute of Allergy and Infectious Disease (NIAID) grant 5U19AI104209.

Abbreviations used

APC

Allophycocyanin

BAT

Basophil activation test

CMF-PBS

Calcium/magnesium free phosphate buffered saline

CyTOF

Cytometry by Time-of-Flight mass spectrometry

DC

Dendritic cell

DIC

Differential Interference Contrast

EDTA

Ethylenediaminetetraacetic acid

FITC

Fluorescein isothiocyanate

HLA-DR

Human leukocyte antigen-D related

IgE

Immunoglobulin E

IL-3

Interleukin-3

MFI

Mean fluorescence intensity

OIT

Oral immunotherapy

PE

Phycoerythrin

PerCP

Peridinin chlorophyll protein

RPMI medium

Roswell Park Memorial Institute medium

RT

Room temperature

Footnotes

Conflicts of interest: none

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