3.6 Component-resolved differential diagnosis approach with a
single flow cytometry assay
To enhance the component-resolved diagnostic performance of our CytoBas
technique for RGP allergy, multiplex flow cytometry was conducted with
[Lol p 1]4-APC and [Lol p
5]4-BV711 tetramers in a single tube. Basophils in
thawed PBMC from an allergic patient were successfully discriminated
from those of a control by a higher MFI for one or both allergen
tetramers (Figure 5A-C ). Multiplex flow cytometry on thawed
PBMC with both RGP allergen tetramers in a single tube exhibited clear
resolution between a series of RGP-allergic patients and controls
(Figure 5D ).
To test for discrimination between multiple allergic sensitizations, Api
m 1, Lol p 1 and Lol p 5 tetramers, each labelled with a different
fluorochrome, were included simultaneously in a single multiplex flow
cytometric test. Since the MFI of the allergen tetramers on pDCs was
shown to be similar to the MFI of streptavidin control conjugates on
basophils, pDCs provided a suitable background control in blood samples
for multiplex flow cytometry (Suppl. Fig 5 ). Basophils from
thawed PBMC of RGP-allergic patients demonstrated selective binding to
only [Lol p 1]4 or [Lol p 5]4tetramers and not [Api m 1]4, whilst basophils from
a BV-allergic patient only bound [Api m 1]4(Figure 6A ). The MFI of staining on basophils for each of the
three allergens correlated strongly with serum allergen-specific IgE
(each p<0.0001; Figure 6B-D ). This is in contrast to
the frequency of activated (CD63+) basophils stimulated with Api m 1 and
Lol p 1 tetramers, which showed a poorer correlation with Api m
1-specific IgE (r2 = 0.03561, p = 0.2436) or Lol p
1-specific IgE (r2 = 0.1313, p = 0.0114),
respectively. The CytoBas technique therefore reflects allergen-specific
IgE titers in serum with greater accuracy than basophil activation.
Taken together, these data demonstrate that a single flow cytometric
assay with multiple allergen tetramers can provide a rapid
component-resolved and differential diagnostic test for allergy.
DISCUSSION
We describe here a novel methodology for a component-resolved diagnostic
test for allergy using multiplex flow cytometry involving direct
staining of basophils with recombinant allergen tetramers – CytoBas.
This flow cytometric approach enabled rapid detection with high
sensitivity and specificity of BV allergy with Api m 1 and of RGP
allergy with Lol p 1. Combined staining of Api m 1 and Lol p 1 reliably
enabled differential detection of allergen sensitization, and inclusion
of both Lol p 1 and Lol p 5 facilitated quantification of the relative
sensitization to distinct components of RGP in a single-tube test. The
combination of multiple allergens from a single species provided
complete discrimination between RGP-allergic patients and controls,
highlighting the utility of multiplex CytoBas in diagnosing allergies
that exhibit sensitization to multiple allergen components.
The allergens in our study were recombinantly-produced proteins. This
approach facilitates the introduction of mutations that disrupted
enzymatic function and the addition of protein tags for biotinylation
and purification. Api m 1, Lol p 1 and Lol p 5 contain glycan
structures.39,40 Because the glycosylation patterns of
proteins in invertebrates and plants are more similar to each other and
differ strongly from mammals,41-43 we here produced
their recombinant forms in the Sf21 insect cell line. Similar to a
previous report utilizing Sf9 cells,44 we obtained two
main glycosylated products of the recombinant Api m 1, fitting with the
native paucimannosidic type of glycosylation.43 Both
recombinant RGP allergens had greater sizes than predicted for
non-glycosylated variants, and showed high sensitivity and specificity
for detection of RGP allergy. The high sensitivity and specificity of
our recombinant allergen components for confirmation of BV and RGP
allergies indicates that our approach preserves the major epitopes for
IgE binding. The fact that invertebrate- and plant-derived proteins can
be generated as such in an insect cell line, as confirmed here,
demonstrates the potential to apply our CytoBas assay for detection of
sensitization to a huge diversity of food-, aero- and stinging
insect-derived allergens.
Our CytoBas study exploits the concept that basophils present high
levels of IgE on their surface through FcεRI binding, and that this IgE
is polyclonal and reflective of the specificities produced in an
individual. This is the same concept that is utilized for BAT, even
though there is typically a fraction of cells that does not degranulate
and express CD63 after allergen stimulation. Our direct staining
approach showed that in the case of allergen-sensitization, all
basophils become positive with the population showing a Gaussian
distribution of staining intensity. The median staining intensities
correlated strongly with allergen-specific serum IgE levels, and so
could be used in a similar manner for quantitating the response.
Furthermore, the polyclonal nature of IgE on basophils enabled detection
of multiple specificities on the total population in a single test.
RGP-allergic patients with Lol p 1 and Lol p 5 co-sensitization showed
double-positive basophil populations. This is in contrast to a recent
study where distinct basophil subsets were positive for either
recombinant Api m 1 or recombinant Api m 2 conjugated to fluorescent
quantum dots (Qdots).45 The reason for this difference
is unclear, as it is unlikely that within an individual, distinct
basophil populations will present unique allergen specificities.
Therefore, it will be important for applications of multicolor flow
cytometry to incorporate appropriate controls and establish standardized
instrument settings for reproducible and robust
measurements.37,38
Another novel observation from this study is that degranulated basophils
(CD63+) bind allergen tetramers with similar intensity
as basophils that have not degranulated (CD63–) in
the same assay. Typically, the proportion of basophils that degranulate
in a BAT forms a bell-shaped curve with increasing concentrations of
allergen. The maximum frequency of CD63+ cells will
differ between individuals, and the dose-response curve is thought to be
affected by the affinity of IgE to the antigen, the density of the
epitope-specific IgE on the cell surface, and the functional capacity of
the basophil itself.46-48 Our observation argues
against differences in basophils from the same individual with regards
to IgE affinity or density of epitope-specific IgE. Rather, intrinsic
characteristics of the basophil, e.g. signaling threshold, degranulation
capacity and maturity, could underlie the differences between those
cells that do degranulate and the fraction of cells that does not.
Our allergens were tetramerized using streptavidin-fluorophore
conjugates. A previous study has reported that activated basophils may
have the capacity to bind streptavidin via positively-charged molecules
that are expressed on the basophil surface upon
degranulation.49 This could potentially lead to
non-specific binding of allergen tetramers to degranulated basophils. In
our experiments, we did not observe such an effect and the fluorescence
intensities of our allergen tetramers were similar between basophils
that had degranulated and those that had not. Still, it is recommended
for multiparameter analysis of allergen binding to perform incubations
for staining at room temperature or even at 4ºC to minimize the
potential for activation and degranulation of basophils that might
mediate non-specific binding of streptavidin.
Using a multiparameter staining approach with three allergen components,
we demonstrated that CytoBas enabled detection of sensitization to
multiple components of the same allergen (RGP; Lol p 1 and Lol p 5) as
well as differential sensitization to distinct allergens (BV; Api m 1 vs
RGP; Lol p 1 and/or Lol p 5). This differential and component-resolved
analysis through staining of basophils has several advantages over BAT:
1) The CytoBas assay only requires direct staining with allergen
tetramers and mAbs without the need for in vitro stimulation,
making it less dependent on maintaining basophil function ex
vivo , less labor intensive and easier to standardize; 2) CytoBas has
the potential for a single staining with multiplex detection of distinct
allergen components, rather than the need for multiple (7-10)
independent conditions for titration of each allergen in the BAT; 3) For
BV and RGP allergy, two distinct allergic diseases, CytoBas with Api m 1
and Lol p 1 distinguished between allergic patients and controls with
greater sensitivity and specificity than the BAT; 4) Basophil staining
with recombinant allergen tetramers is possible on fresh whole blood,
fresh PBMC and cryopreserved PBMC enabling more flexibility for
transport or batch analysis of clinical samples than is currently
possible with the BAT, which requires processing of fresh blood within
24 hrs.50 A current limitation is the availability of
recombinant allergens with the correct modifications for tetramerization
with fluorescently-labeled streptavidin. However, with the potential for
use of insect cells to generate plant and invertebrate proteins, and the
vast experience with recombinant proteins in serology-based component
resolved diagnostics, this will only be a temporary hurdle.
CytoBas with Lol p 1 tetramers exhibited greater sensitivity and
specificity than previously reported for clinical history, SPT and
allergen-specific IgE in allergic rhinitis patients sensitized to pollen
areoallergens.51 With the potential for multiplex
analysis in a single assay, CytoBas has the potential to compete with
serology-based IgE tests. Theoretically, a combination of 2-4 major
allergen components could be applied to a) detect allergen reactivity in
a highly sensitive manner; and b) detect specific sensitization and risk
of systemic response, without the need for repeat tests that are
currently recommended for e.g. peanut allergy.17 In
practice, the composition of a CytoBas assay and its performance will
need to be tested and compared to the standard diagnostic workflow for
each diagnostic process. The availability of high-end clinical
cytometers that allow 10 or more components to be multiplexed in one
assay provides the potential for CytoBas to provide a rapid
component-resolved diagnostic test for allergy whilst avoiding allergen
challenge.