38th Meeting of the Danish Society for
Flow Cytometry
Joint meeting of the Danish Society for Flow Cytometry
and the Danish Society for Allergology
The
basophil activation test in clinical and basic research
24 October 2006, 17:30–20:30.
Det Blå Auditorium, Victor Albæk
Bygning, Vennelyst Boulevard, 8000 Aarhus C.
All are welcome. Registration is not necessary.
Organizers: Hans Jürgen Hoffmann
(DSFCM) and Lars Peter Nielsen (DSA).
Program
Session 1, chair: Per Stahl Skov (Reflab,
Introduction of the
Basophil Activation Test.
CD-sens: basophil allergen sensitivity.
Session 2, chair:
Characteriation of activated basophil granulocytes.
BAT in clinical assessment of penicillin allergy.
Flowcytometric
measurement of in vitro activation and desensitization of human basophils.
Travel information
DSB train IC 149 departs from
DSB train IC 168 departs from
Abstracts
CD-sens: basophil
allergen sensitivity
Anna
Nopp, Department of
Medicine, Division of Clinical Immunology and Allergy,
Background: Monitoring of
the allergen sensitivity of a patient is most important for optimal patient care
and a basic prerequisite for immune modulating treatment. The objective of these
studies was to investigate how basophil allergen sensitivity can be measured
and be applied in monitoring the efficacy of different treatments e.g. anti-IgE
treatment (ESIT) and allergen immunotherapy (ASIT).
Methods: Basophils from non-treated or ESIT/ASIT-treated allergic patients were,
with flow cytometry, analysed for allergen threshold sensitivity (CD-sens) by
measuring CD63 up-regulation on CD203c identified basophils. The results were
compared to maximal percentage CD63 up-regulation at one allergen dose
(CD-max), skin prick test end-point allergen titration (SPT-sens), nasal and
bronchial provocation titration tests and serum IgE and IgE antibody
concentrations.
Results: There was a significant correlation between CD-sens and the following
parameters: SPT-sens, IgE antibody
concentration in percentage of “total-IgE” (relative IgE antibody
concentration) and nasal and bronchial provocation. In contrast, CD-max did not
correlate with any of the sensitization parameters.
Conclusions: CD-sens seems
to be very useful for determination of a patient’s allergen sensitivity and
should be evaluated for measurement and monitoring the efficacy of different
treatments e.g. ESIT and ASIT. CD-max, the conventional approach to basophil
allergen challenge, which mirrors cell reactivity, gives incorrect information.
BAT: Evaluation of
lysing procedure & testing of wasp- and penicillin-allergics
Background: Flow
cytometric activation tests detecting either the expression of CD63 or CD203c
on activated basophils have been investigated as alternatives to specific IgE
determination or skin prick tests.
Objectives: In the evaluation of BAT as a rapid, ex vivo tool to support
diagnosis of penicillin allergy in a clinical setting, we aimed to determine
any influence of the lysing procedure on the expression of basophil activation
markers (CD63 & CD203c) and to establish the saponin-lysing-solution with
the highest gain of basophils. Verified wasp allergic patients were used as
positive control.
Methods: We exposed heparinized
donor-blood to anti-FceRI (CRA1) to activate basophils, labelled with CD63-FITC
(Caltag) and CD203c-PE (Immunotech) and lysed with either a saponin-based
solution (WBL) or one based on formic acid (Immunoprep); both from Coulter. For
optimizing the yield of basophils, we carried out titrations with
saponin-solutions for lysing.
Testing of our BAT-setting was performed employing donor-blood from
verified wasp-allergics and also with verified pencillin-allergics. The blood
was exposed to either Vespula-allergen (Alkabello) or penicillin-allergens (the
MDM/PPL penicillin-allergens in the Allergopen-kit, Allergopharma, or
PenG-/PenV-allergens, Alkabello)
Results: More basophils are detected with WBL than with Immunoprep. The
recommendable saponin-concentration is 0,15 mg
saponin/mL PBS for the BAT. The Vespula-BAT showed a marked difference between
the allergics and controls. The preliminary data with penicillin-allergics
showed no significance when testing with MDM/PPL or PenV/PenG.
Conclusion: The BAT is a promising diagnostic tool for
IgE-mediated allergies. The current diagnostic procedure (anamnesis &
provocation tests) is time-consuming, expensive and at times even hazardous for
the patient. The BAT, as used here,, is no alternative
to clinical provocation and determination of specific IgE. More studies must be
accomplished to establish the proper conditions for the test.
Flowcytometric
measurement of in vitro activation and desensitization of human basophils
Gitte Lund, ALK-Abelló A/S,
In vitro activation and measurement of basophile
activation markers:
Measurement of basophile activation markers are widely used either in
addition to or as a replacement for histamine release assays. In our laboratory
basophiles are activated in vitro by
culturing heparinised whole blood, diluted in RPMI with allergen for 1 h, 37◦c.
The extents of basophile cell surface marker expression of CD63, CD203c,
CD164 and CD107a are subsequently measure by FACS analysis. Histamine
released to the supernatants is measured by ELISA.
Comparable endpoint results are obtained by histamine release and
CD63/CD203c measurement.
Kinetics of different basophile activation markers:
In order to investigate the kinetics of the different basophile
activation markers, in vitro, whole
blood was cultured 1-2 h with allergen extract and activation of CD63, CD203c,
CD164 and CD107a were measured at different time points upon allergen-mediated
activation. A clear difference in kinetics were show as the activation of
surface markers CD203c and CD164 were much faster compared to the slower
kinetics characterising activation of markers CD63, CD107a and the release of
histamine.
Desensitization of basophiles by allergen culturing:
Heparinised blood
from allergic individuals was cultured with different concentrations
(0,001-1SQ) of grass pollen extract and the extent of basophile activation
was subsequently measured at different time points. The effect on surface
marker expression/ histamine release following allergen challenge (0,05-5 SQ) of basophiles pre-cultured
in the presence of activating (optimal) or non-activating (suboptimal)
concentrations of allergen was subsequently measured.
Allergen induced initial surface marker activation decreases by
culturing for 1-2 days with optimal concentration of allergen, but no second
activation by adding higher allergen concentrations could be
obtained. Interestingly, we were able to show the same
non-responsiveness by culturing with suboptimal allergen concentrations. This
desensitization of basophiles was obtained despite very low initial activations
of surface marker expression and histamine release.
Activation of basophiles sensitized with monoclonal
IgE antibodies:
Basophiles was
sensitized with different combinations of recombinant human monoclonal IgE
antibodies. Activation of surface markers CD63 and CD203c was subsequently
measured after allergen challenge. Allergen cross-linking of two antibodies
were shown to be sufficient for basophile activation and degree of activation
depends on antibody affinity.
Key references
1) Lysis with
Saponin improves detection of the response through CD203c and CD63 in the
basophil activation test after crosslinking of the high affinity IgE receptor
FcepsilonRI.
Clin Mol Allergy. 2005 Jul 4;3:10.
Hoffmann HJ, Bogebjerg M, Nielsen LP, Dahl R.
Department of Pulmonary Medicine,
BACKGROUND: The basophil activation test (BAT), in which translocation of
markers to the surface of blood basophils is measured in response to allergen
by flow cytometry, is a rapid assay that is gaining popularity. Two markers are
currently being evaluated for the BAT; CD63 and the lineage-specific CD203c. In
a recent report, detection of CD203c after lysis with Saponin was shown to be
superior to detection of CD63 after lysis with formic acid. We wanted to
compare a) lysis with formic acid and lysis with Saponin, b) the response through
CD203c and CD63, and c) the definition 10% activated cells above background
with the probability binning metric T(chi) > 4, on
sets of data generated with blood basophils stimulated with varying
concentrations of anti-FcepsilonRI antibody. METHODS: Blood from volunteers was
incubated with serial logarithmic dilutions of anti-FcepsilonRI and
subsequently with antibodies to CD203c PE and CD63 FITC. Sets of samples set up
in parallel were lysed with either Saponin based Whole Blood Lysing reagent or
with formic acid based Immunoprep/Q-prep. Samples were acquired on a FACS
Calibur, but were compensated and analysed offline. Responders were defined as
persons who had 10% or more activated basophils above background, or a T(chi) > 4, for two consecutive dilutions of
anti-FcepsilonRI antibody. RESULTS: More basophils (median 1164 vs. median 397)
and better discrimination of upregulated CD203c and CD63 amongst responders
were obtained after lysis with Saponin than after lysis with formic acid. We
suggest that CD203c may be a more sensitive marker for the BAT than CD63, as
6/11 responders were found with CD203c, compared with 3/11 with CD63. Most
responders (7/11) were identified with probability binning. CONCLUSION: A
combination of lysis with Saponin and the markers CD203c and CD63 computed by
probability binning may be the most sensitive method of detecting activation of
basophils after stimulation through FcepsilonRI.
2)
Basophil allergen threshold sensitivity: a useful approach to anti-IgE
treatment efficacy evaluation.
Allergy.
2006 Mar;61(3):298-302.
Nopp
A, Johansson
SG, Ankerst
J, Bylin
G, Cardell
LO, Gronneberg
R, Irander
K, Palmqvist
M, Oman
H.
Clinical Immunology and Allergy Unit, Department of Medicine,
Karolinska Institute,
BACKGROUND: Monitoring of the allergen sensitivity of a patient is most
important for optimal patient care and a basic prerequisite for
immunomodulating treatment. The objective of this study was to investigate how basophil
allergen sensitivity can be applied in the monitoring of anti-immunoglobulin E
(IgE) treatment. METHODS: Basophils from timothy grass pollen allergic patients
were, by flow cytometry, analysed for allergen threshold sensitivity (CD-sens)
by measuring CD63 up-regulation on CD203c-identified basophils. The results
were compared with maximal percentage CD63 up-regulation at one allergen dose
(CD-max), skin prick test end-point allergen titration, (SPT-sens), nasal
provocation titration tests (nasal provocation titre) and serum IgE and IgE
antibody concentrations. RESULTS: There was a significant correlation (r =
0.50, P = 0.01) between CD-sens and SPT-sens, CD-sens and the IgE antibody
concentration in percentage of 'total IgE' (relative IgE antibody concentration)
(r = 0.72, P < 0.001) as well as between CD-sens and nasal provocation titre
(r = 0.54, P < 0.05) but, in contrast, CD-max did not correlate with any of
the sensitization parameters, i.e. SPT-sens, nasal provocation titre, absolute
and relative IgE antibody concentration or CD-sens. CD-sens could be used to
monitor omalizumab treatment efficacy while, based on CD-max, four of seven
symptom-free patients on omalizumab would have been classified as having
ongoing allergy. CONCLUSIONS: CD-sens seems to be very useful for the
determination of a patient's allergen sensitivity and should be evaluated for
the measurement and monitoring of anti-IgE treatment efficacy. CD-max, the
conventional approach to basophil allergen challenge, which mirrors cell reactivity,
gives incorrect information.
Revised, 25 October
2006 /JKL