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Novel method for assessing basophil activation by measuring altered expression of membrane-bound and intracellular basogranulin stores

Novel method for assessing basophil activation by measuring altered expression of membrane-bound and intracellular basogranulin stores
Novel method for assessing basophil activation by measuring altered expression of membrane-bound and intracellular basogranulin stores
Background
Basophil activation tests can provide valuable information on allergic sensitivity to a range of different allergens. The most widely employed methods involve flow cytometric detection of increased expression of membrane-bound CD63 or CD203c following addition of allergen to basophils in vitro.
The identification of basogranulin, a unique basic protein stored in basophil granules, has opened the way for new basophil activation methods to be explored.

Methods
Blood was collected from healthy subjects and patients with a history of allergy to food, drugs, house dust and grass pollen. Basophils were stimulated with specific allergen, anti-IgE antibody, or the peptide f-met-leu-phe (FMLP). Flow cytometry was performed with non-permeabilised and permeabilised cells with antibody specific for basogranulin (BB1) or CD63, and data analysed with CellQuest software.

Results
Flow cytometry with permeablised cells indicated depletion of intracellular stores of basogranulin following basophil activation. Associated with basogranulin release was the presence of increased quantities of this marker on the basophil membrane following cellular activation. Increased membrane expression of basogranulin mirrored that for CD63; and with allergens and other stimuli tested the measurement of cell surface basogranulin represented a more sensitive means for assessing basophil activation in vitro. The flow cytometric assays for basogranulin were optimised for use with samples of whole blood so as to avoid the need for basophil purification.

Conclusions
The rapidity, simplicity and sensitivity of basogranulin-based methods for measuring basophil activation will facilitate their application to clinical samples and allow better assessment for allergic sensitivity.
1939-4551
Alzahrani, Mohammad Omar J
81268c53-ba88-4742-bb75-ced5f3e16717
Walls, Andrew
aaa7e455-0562-4b4c-94f5-ec29c74b1bfe
Kyyaly, Mohammed Aref
7bd69b33-fec8-405c-9f40-b7157f0242f0
Arshad, Syed
917e246d-2e60-472f-8d30-94b01ef28958
Alzahrani, Mohammad Omar J
81268c53-ba88-4742-bb75-ced5f3e16717
Walls, Andrew
aaa7e455-0562-4b4c-94f5-ec29c74b1bfe
Kyyaly, Mohammed Aref
7bd69b33-fec8-405c-9f40-b7157f0242f0
Arshad, Syed
917e246d-2e60-472f-8d30-94b01ef28958

Alzahrani, Mohammad Omar J, Walls, Andrew, Kyyaly, Mohammed Aref and Arshad, Syed (2020) Novel method for assessing basophil activation by measuring altered expression of membrane-bound and intracellular basogranulin stores. World Allergy Organization Journal, 13 (8), [100351]. (doi:10.1016/j.waojou.2020.100351).

Record type: Article

Abstract

Background
Basophil activation tests can provide valuable information on allergic sensitivity to a range of different allergens. The most widely employed methods involve flow cytometric detection of increased expression of membrane-bound CD63 or CD203c following addition of allergen to basophils in vitro.
The identification of basogranulin, a unique basic protein stored in basophil granules, has opened the way for new basophil activation methods to be explored.

Methods
Blood was collected from healthy subjects and patients with a history of allergy to food, drugs, house dust and grass pollen. Basophils were stimulated with specific allergen, anti-IgE antibody, or the peptide f-met-leu-phe (FMLP). Flow cytometry was performed with non-permeabilised and permeabilised cells with antibody specific for basogranulin (BB1) or CD63, and data analysed with CellQuest software.

Results
Flow cytometry with permeablised cells indicated depletion of intracellular stores of basogranulin following basophil activation. Associated with basogranulin release was the presence of increased quantities of this marker on the basophil membrane following cellular activation. Increased membrane expression of basogranulin mirrored that for CD63; and with allergens and other stimuli tested the measurement of cell surface basogranulin represented a more sensitive means for assessing basophil activation in vitro. The flow cytometric assays for basogranulin were optimised for use with samples of whole blood so as to avoid the need for basophil purification.

Conclusions
The rapidity, simplicity and sensitivity of basogranulin-based methods for measuring basophil activation will facilitate their application to clinical samples and allow better assessment for allergic sensitivity.

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More information

e-pub ahead of print date: 23 September 2020

Identifiers

Local EPrints ID: 444593
URI: http://eprints.soton.ac.uk/id/eprint/444593
ISSN: 1939-4551
PURE UUID: 88d8f971-af14-4db2-832b-a24dc029d7d4
ORCID for Andrew Walls: ORCID iD orcid.org/0000-0003-4803-4595
ORCID for Mohammed Aref Kyyaly: ORCID iD orcid.org/0000-0002-1684-9207

Catalogue record

Date deposited: 26 Oct 2020 17:33
Last modified: 13 Dec 2021 03:16

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Contributors

Author: Mohammad Omar J Alzahrani
Author: Andrew Walls ORCID iD
Author: Syed Arshad

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