Introduction
Sensitisation to inhalant allergens such as house dust mites, cats and
dogs, is commonly associated with asthma, but is neither necessary nor
sufficient for disease expression. Similarly, a positive skin prick test
(SPT) or IgE test to a food does not equate to clinical food allergy,
and false positive results are common. Challenge testing (either oral
food challenge or inhaled allergen challenge) can be offered to
patients, but is time consuming, carries the risk of severe reaction and
is not suitable for all patients. Therefore, tests with superior
diagnostic accuracy than IgE that are safe to conduct in all patients
would be of value in clinical practice, especially amongst patients
sensitised to many allergens.
Basophils and mast cells (MC) are the two primary effector cells in
allergic responses (1). Cellular
degranulation triggers the release of preformed and newly synthesized
mediators inducing a potent biological response in a sensitized person
following allergen exposure (2). While
basophils are found in the circulation, MC are localized in peripheral
tissues. The two cell types may have different roles in an allergic
response but this is currently poorly understood
(2, 3).
The more accessible circulating basophils have been used as cell models
for studying allergy (4). Basophils
account for <1% of blood leukocytes however, making
purification a challenge. To obviate the need for purification, the
basophil activation test (BAT) was developed using immediately analysed
fresh whole blood (4,
5). Following stimulation of whole blood
with allergen (or control), the responsiveness of the basophils can be
quantified using fluorochrome-coupled antibody markers of basophil
activation (e.g. CD63 and CD203c) by flow cytometry. The advantage of
the BAT is that it takes account of many factors which influence
basophil responsiveness to an allergen such as IgG4/IgE ratio
(6, 7),
heterogeneity of sIgE to allergen components
(8), medication
(9) and innate responsiveness of the cells
(10). The disadvantages are that blood
needs to be analysed immediately after being drawn
(5), requiring the allergy clinic to have
instant access to a staffed flow cytometry facility. In addition,
10-20% of people carry “non-releaser” basophils, which are
non-responsive in the BAT, despite having clinical allergy
(11). Consequently, this test is not
generally available for clinical diagnostics, but used only in
specialist laboratories for hymenoptera venom and drug allergy testing.
The passive BAT, which uses basophils from a donor that are passively
sensitized with the serum from the patient, was developed as an
alternative method that circumvents some of these problems
(12). Stored serum samples from subjects
can be analysed in batches, providing greater flexibility and allowing
humoral factors to be investigated separately from cellular factors
(7). However, the donors’ basophils must
be stripped of endogenous IgE with a mild acid treatment before the
cells can be passively sensitized with patient serum samples, which can
damage the donor basophils and lead to auto-basophil activation
(13) and reduced sensitivity
(14). Due to these limitations, passive
BAT has only been used in a few studies. Although the passively
sensitized approach has also been used on basophilic cell lines such as
RBL-2H3, there are a number of disadvantages, including the gradual loss
of cell responsiveness within weeks of cultures
(15).
Recently, Bahri et al developed a robust and reproducible effector cell
assay based on human progenitor cell derived MC, the mast cell
activation test (MAT). This assay appeared to confer superior diagnostic
accuracy in distinguishing peanut allergic from peanut tolerant subjects
compared with existing diagnostics such as sIgE (to whole peanut or Ara
h 2), SPT and BAT (16). In this study we
use a similar approach to generate functional progenitor cell-derived
basophils (PCB) and provide detailed characterization of basophil
differentiation, and demonstrate the functionality and reproducibility
of this technique. We then explore the potential clinical application of
progenitor cell basophil activation test (PCBAT) by passively
sensitizing the cells with sera/plasma from five groups of patients with
allergic asthma and food allergy and testing degranulation to two
allergens (cat and peanut).