Discussion
We have developed a high-throughput progenitor cell derived basophil
activation test (PCBAT), which was a better predictor of clinical
reactivity to cat and peanut allergen (as measured on challenge testing)
than conventional markers of allergy such as SPT or allergen specific
IgE. PCBAT can be used with stored serum, removing the need for
immediate access to expensive flow cytometry facilities not generally
available in the clinic. By passively sensitising basophils with sera
from our well characterised patient populations then culturing with the
relevant allergen, we demonstrated dose dependent and allergen specific
basophil activation with wide variability in trajectories. Importantly,
PCBAT was negative amongst those reporting oral tolerance to peanut but
with detectable specific IgE to peanut components.
Technical aspects of the
PCBAT
The combined use of flow cytometry and immunostaining suggest the
optimal window for maturation for this culture protocol was between day
16 and 21, similar to previous reports.
(23), and reflecting the 5 days basophil
lifespan in vivo (24). Our culture
was highly enriched for mature basophils (25-50%), enabling
high-throughput barcoding, improving efficiency.
PCB responsiveness and sIgE levels
The responsiveness of PCBAT was allergen specific showing a
dose-dependent response with good association with levels of
corresponding sIgE. In addition, patients receiving omalizumab treatment
showed completely muted responsiveness in the PCBAT, in accordance with
a previous study (25).
However, one subject, with low but positive sIgE to cat (0.5KU/L) did
not show responsiveness in PCBAT; this patient was not on omalizumab
treatment. As this subject had not undergone allergen challenge, it
remains unclear whether this subject was sensitized but tolerant to cat.
One non-sensitized control subject showed minor degranulation at the
highest concentration of cat allergen used. We have confirmed that this
subject had a positive dog sIgE (5KU/L); we speculate that this weak
response to cat allergen might reflect cross-reactivity between cat
allergen and dog sIgE, which has been previously reported
(26-28). We also found quantifiable
traces of Can f 1 in the cat allergen extracts used which may provide an
alternative explanation (Figure E10).
The clinical relevance of
PCBAT
We investigated the clinical relevance of PCBAT using two cohorts of
patients who had undergone challenge testing—to inhaled cat allergen
or to peanuts.
Although a significant association was observed between PCBAT AUC and
inhalant challenge results, two subjects showed negative results in the
PCBAT but positive in the inhalant challenge. Interestingly, these two
subjects exhibited the smallest reaction in the skin test to cat at the
time of challenge (2x2mm) and had significantly greater reactivity to
other allergen including dust mite and grass (Table E4). In addition,
for some of these subjects, the blood samples were collected up to 2
years after the inhaled allergen challenge, so a change in clinical
reactivity during this time remains a possible explanation
We explored PCBAT in peanut allergy, as an exemplar of an allergic
disease where clinical reactivity is not reliably predicted by serum
sIgE, but where oral food challenge tests are used to confirm reactivity
and identify thresholds of responsiveness. All 30 patients with
physician diagnosed peanut allergy showed a positive PCBAT, and this
correlated significantly with serum sIgE to whole peanut extract and to
Ara h 1, 2 and 3. As half of the subjects had previously undergone oral
food challenge to peanut, we were able to compare the dose of peanut at
which the subject showed an objective reaction with the reactivity on
the PCBAT (as AUC). We identified that AUC on PCBAT was a better
predictor of clinical reactivity on oral food challenge than serum sIgE
to whole peanut extract or to Ara h 2 and 3, and was similar to Ara h 1.
Importantly we identified a small population with positive sIgE to 1 or
more peanut allergen component that reported regularly eating peanuts,
and found that none of them showed a positive PCBAT. This suggests that
in principle, PCBAT may be a useful test in predicting clinical
reactivity to peanut, but further testing on more subjects would be
required.