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).
Session 1, chair: Per Stahl Skov (Reflab,
Introduction of the Basophil Activation Test.
. Anna Nopp, Karolinska Institute, Stockholm:
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.
DSB train IC 149 departs from
DSB train IC 168 departs from
CD-sens: basophil allergen sensitivity
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
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.
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
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.
Revised, 25 October 2006 /JKL