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Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis  Kaori Mukai, PhD, Nicolas Gaudenzio,

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Presentation on theme: "Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis  Kaori Mukai, PhD, Nicolas Gaudenzio,"— Presentation transcript:

1 Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis  Kaori Mukai, PhD, Nicolas Gaudenzio, PhD, Sheena Gupta, PhD, Nora Vivanco, BSc, Sean C. Bendall, PhD, Holden T. Maecker, PhD, Rebecca S. Chinthrajah, MD, Mindy Tsai, DMSc, Kari C. Nadeau, MD, PhD, Stephen J. Galli, MD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 3, Pages e11 (March 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Overview of BATs. Blood from each subject 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. CD123+HLA-DR− cells are gated as basophils (left panels), and histograms show their expression of CD203c (middle panels) and CD63 (right panels). Gray histograms show RPMI (unstimulated) cells, red lines show anti-IgE stimulation, and green lines show IL-3 stimulation. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Comparison of anticoagulants. Blood of the same 10 healthy donors anticoagulated with EDTA or heparin was stored at 4°C for 24 hours before cells were stimulated with IL-3 or anti-IgE. A, Left, ΔCD203c MFI; right, percentage of CD63hi basophils. B, Left, CD203c MFI; right, percentage of CD63hi basophils after incubation with RPMI alone. Data shown (individual values [dots] and means ± SDs) are the combined results of all the measurements performed in 3 independent experiments. *P < .05, **P < .005, and ***P <  No asterisks, P > .05. P values are stated when they are between .05 and .1. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Effect of calcium/magnesium on CD203c and CD63. Blood of healthy donors was treated with IL-3, anti-IgE with or without calcium/magnesium, or calcium/magnesium only. A, Representative basophil plots of CD63 for 1 donor. B, CD63 results from 5 donors. C, Representative basophil histograms of CD203c for 1 donor. D, CD203c MFI of 5 donors. Fig 3, B and D, share the x-axis labels in Fig 3, D. Data shown (individual values [dots] and means ± SDs) are from 1 of 3 independent experiments, each of which produced similar results. *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 .05 and .1. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Assessment of platelet attachment to basophils. Blood of healthy donors anticoagulated with EDTA or heparin was incubated with RPMI or CaCl2/MgCl2. A, Representative photographs of the individual blood cell types identified. Left, Differential interference contrast (DIC) with labels indicating 1 basophil, several platelets, and 3 dendritic cells. The 3 panels on far right indicate FcεRIα, CD41, and DAPI staining alone. The second and third panels from the left indicate 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 calcium/magnesium (EDTA plus calcium/magnesium, right panel). Pictures highlighted by yellow borders are higher magnifications of the corresponding picture. Scale bars in Fig 4, A and B = 10 μm. Data shown in Fig 4, A and B, are from 1 of 3 independent experiments, each of which produced similar results. C and D, Blood was treated as in Fig 1, and CD41+ basophils were assessed by means of flow cytometry of the cells of 1 subject (Fig 4, C) or by assessing the cells of 11 subjects (Fig 4, D). *P < .05, **P < .005, and ***P <  No asterisks, P > .05. E, Blood anticoagulated with heparin was incubated with anti-IgE and stained for CD63 and CD41. Representative dots plots are displayed. Blue dots, CD63hi basophils; red dots, CD63−/low basophils; and black dots, all other cells. F, Blood anticoagulated 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 differential interference contrast (upper panel) and merged IgE, CD63, and DAPI staining (lower panel) are shown. Scale bars = 10 μm. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Comparison of anticoagulants for performing BATs in patients with peanut allergy. Blood from patients with peanut allergy was treated with IL-3 or anti-IgE or peanut extract. A, ΔCD203c MFI. B, Absolute CD203 MFI values. C, Percentage of CD63hi basophils. D, ΔCD203c MFI (left) and percentage of CD63hi basophils (right) of nonreleasers identified among patients with peanut allergy. 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 boxes indicate medians, and crosses indicate means. Fig 5, A-C, n = 98. Fig 5, D, n = 9. *P < .05, **P < .005, and ***P <  No asterisks, P > .05. P values are stated when they are between .05 and .1. Red asterisks are comparisons between EDTA and heparin at each condition of stimulation. Black asterisks are comparisons of CD203c MFI (Fig 5, B) or percentage of CD63 high basophils (Fig 5, C) between RPMI and each condition of stimulation for cells analyzed in the same anticoagulant. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 BAT with 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, Percentage of CD63hi basophils on 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 ± SDs. **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-negative (CD61−) and platelet-positive (CD61+) basophils. D, Blood from patients with peanut allergy was collected into heparin tubes, stored at 4°C for 24 hours, and then stimulated with peanut extract (100 ng/mL) for 30 minutes (Flow, fluorescence-based flow cytometry; upper panel) or 20 minutes (CyTOF; lower panel). Histograms show basophil CD63 from 1 representative patient. E, Data from 5 representative patients showing percentage of CD63hi basophils with conventional flow cytometry versus CyTOF. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 Comparison of time and temperature of blood storage. Blood from the same healthy donors was stored at 4°C or RT and then analyzed at 4 and 24 hours. Blood (from separate donors) that was anticoagulated with EDTA (n = 10 donors) or heparin (n = 11 donors) was incubated with RPMI (medium only control), anti-IgE, or IL-3. A, ΔCD203c MFI. B, Percentage of CD63hi basophils. C, CD203c MFI. D, Percentage of CD63hi basophils after incubation with RPMI medium only. Data shown (individual values [dots] and means ± SDs) are the combined results from 3 independent experiments. *P < .05, **P < .005, and ***P <  No asterisks, P > .05. P values are stated when they are between .05 and .1. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 Comparison of basophil percentages and numbers in different conditions of specimen preparation. Percentage of basophils (A) and basophil numbers (B) in the 4 conditions of temperature and time of blood storage tested and comparison of percentage of basophils in EDTA versus heparin specimens stored at 4°C for 24 hours (C) are shown. Blood from the same healthy donors was stored at 4°C or RT and then analyzed at 4 and 24 hours (Fig E2, A and B). Fig E2, A, EDTA, n = 15; heparin, n = 16. Fig E2, B, n = 10. Fig E2, C, n = 15. Data shown (individual values [dots] and means ± SDs) are combined results from 3 independent experiments. *P < .05, **P < .005, and ***P <  No asterisks, P > .05. P values are stated when they are between .05 and .1. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 Comparison of time and temperature of specimen storage in patients with peanut allergy. Heparin-anti-coagulated blood from patients with peanut allergy was stored at 4°C or RT and then analyzed at 4 and 24 hours after treatment with IL-3, anti-IgE, or peanut extract. A and B, ΔCD203c MFI (Fig E3, A) and percentage of CD63hi basophils (Fig E3, B). 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 boxes indicate medians, and crosses indicate means. Fig E3, A, n = 19 for 4 hours (4°C/RT) and n = 21 (including the 19 subjects studied at 4 hours) for 24 hours (4°C/RT). Fig E3, B, n = 16 for 4 hours (4°C/RT), n = 18 (including the 16 subjects studied at 4 hours) for 24 hours (4°C/RT) for releasers, and n = 3 for nonreleasers. C, Comparison of ΔCD203c MFI and CD63hi percentage basophils between 4°C and RT at 24 hours from Fig E3, A and B. *P < .05, **P < .005, and ***P <  No asterisks, P > .05. P values are stated when they are between .05 and .1. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 Assessment of reproducibility of BATs in the same subjects. Blood from patients with peanut allergy anticoagulated with heparin was stored at 4°C for 24 hours before treating the cells as in Fig 5. A, Duplicate samples from the same specimens were analyzed at the same time. Values shown with open circles or open squares are from the 2 duplicate tubes. B, Specimens from the same patients tested at 2 different times (as indicated in the figure). Solid black circles are values obtained at screening, and solid black squares are values obtained at week 0 of the OIT trial. Both panels show absolute CD203c MFI values (upper panel) and percentages of CD63hi basophils (lower panel). Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E5 Assessment of BATs in blood shipped at 4°C or ambient temperature. Blood from the same healthy donors was stored at 4°C or RT without shipment in the laboratory or during overnight shipment for 130 km at 4°C or ambient temperature (Ambient Temp) and then analyzed at 24 hours. Blood that was anticoagulated with heparin (n = 10) was incubated with RPMI (medium only control), anti-IgE, or IL-3. A, ΔCD203c MFI after incubation with anti-IgE (left) or IL-3 (right). B, Percentage of CD63hi basophils after incubation with anti-IgE. Data shown (upper panel, individual values [dots] and means ± SDs; lower panel, individual values [dots] from aliquots of blood from the same subjects are connected by lines) are the combined results from 2 independent experiments (ie, in 2 experiments in each of which blood from 5 different subjects was aliquoted and then shipped or maintained in our laboratory before performing BATs). *P < .05, No asterisks, P > .05. P values are stated when they are between .05 and .1. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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