Development of the basophil activation test.

There are multiple tests available to diagnose an allergy. In Type-4 reactions
epicutaneous patch-tests are used as well as the lymphocyte transformation test
(LTT). In Type-1 reactions (immediate type reactions) mostly proteins function
as allergens. Prick tests and in vitro determination of specific IgE are the
tests of choice in these cases. There are however patients that have an
immediate type allergic reaction to small molecular substances, which indicates
that mast cells and basophils are involved. IgE involvement in these cases is
not always clear. The basophils will degranulate through activation and
histamine and other mediators are released. Because of this mechanism symptoms
of an immediate (pseudo) allergic reaction occur. Identifying specific IgE is
difficult in this situation, because the suspected substance has to be bound to
a carrier. In prick tests and provocation challenges there is the risk of
patient having an anaphylactic reaction. Therefore more diagnostic tests are
desired.

The basophil activation test by flow cytometry is a recently developed test to
mimic in vitro the contact between allergens and the cells responsile for
symptoms (basophils and mastcells). By flow cytometry (FACS) it is possible to
identify basophil in blood, even if a small number is available. With
monoclonal antibodies to degranulation markers on the surface of the basophils,
activation of the basophils can be detected. Multiple studies have demonstrated
that the basophil activation test by flow cytometry is a reliable tool for
identifying degranulation markers on basophils by different allergens. By
identifying the activation of basophils through the basophil activation test by
flow cytometry, diagnosis and treatment of some patients will substantially
improve.

The aim of this study is to develop and validate the basophil activation test
by flow cytometry in our hospital as an extra diagnostic procedure for
patients, who can not be diagnosed with existing tests or who have the risk of
developing anaphylaxis by performing diagnostic tests in vivo.

To validate the basophil activation test in our laboratory, birch pollen will
be used as an allergen, because the basophil activation for this allergen is
well documented. Subject non-allergic to birch pollen will serve as controls.
As a second step the basophil activation test for diclofenac will be developed
with patients with a positive oral provocation test to diclofenac in the last 3
years and subjects who tolerate diclofenac. Finally the basophil activation
test for diclofenac will be developed for allergens from cosmetic substances,
in particular benzophenone-3.
For this purpose 5 ml of blood will be needed from all subjects. The blood will
be incubated with the allergen and with anti IgE as positive contol and
activation buffer as negative control. Then antihuman IgE and CD63 and CD203c
will be added. By flowcytometric analysis activated basophils will be isolated.
The optimum concentration of the allergens will be searched to separate the
allergic subjects from the non-allergic subjects.

The effort of patients will be kept to a minimum. Blood will be taken by
experienced and qualified employees from the laboratory of the UMCG. The
investment in time will be about 5 to 10 minutes per patient and will only
include giving a blood sample. It will not be necessary to fill in
questionnaires or any other forms.