A new basophil activation test using CD63 and CCR3 in allergy to antibiotics

A wide range of reported evidence in the literature has shown that the quantification of basophil activation by flow cytometry (basophil activation test, BAT) has proven to be a useful tool for the assessment of immediate-type responses to allergens mediated by IgE or other mechanisms in allergic patients. The usefulness of BAT in anaphylactic adverse reactions, late-onset allergy and immunotherapy follow-up has also been demonstrated. To date, most BAT studies reported in the literature involved the capture of basophils only with a fluorochrome-labeled anti-IgE antibody and application of CD63 upregulation in order to evaluate the basophil response to allergens. Many issues need to be addressed, such as optimizing the analytical performance of the test, checking preanalytical conditions, the selection of the flow cytometry best gating protocol, the introduction of new algorithms and parameters, the search for new activation markers and the introduction of anti-IgE controls. BAT is certainly a useful technique, also for isolated cases of hypersensitivity to various other compounds and drugs, and an update of its application is certainly an interesting topic to expand the debate on allergy diagnosis.

Section: Introductionsupporting

confidence: 52%

Section: The Use Of Basophil Flow Cytometry In Allergy Diagnosis: Stamentioning

confidence: 99%

Basophil Activation Test in Allergy: Time for an Update?

Chirumbolo

2012

“…The positive basophil activation tests with cells from patient 2 corroborated the diagnosis of hypersensitivity to succinylated corticosteroids, particularly as challenge tests with the suspected agents could not be performed. The discrepant results of the basophil activation test obtained with cells from patient 1 or patient 2 may be related to the different methodology used, but cells from patients with definite IgE-mediated allergy do not always react to the respective allergen in this test system [27,28,29]. …”

Section: Discussionmentioning

confidence: 99%

Immediate-Type Hypersensitivity to Succinylated Corticosteroids

Walker

Räwer

Sieber³

et al. 2010

Background: Despite their frequent use, systemic corticosteroids have rarely elicited immediate-type reactions. Objective: We report two male patients, aged 26 and 70 years, respectively, with severe immediate-type hypersensitivity secondary to the administration of corticosteroids esterified with succinate. Methods: Skin tests, basophil activation tests and challenge tests were performed for diagnostic evaluation. Results: In both patients, immediate-type skin test reactions were found to methylprednisolone sodium hemisuccinate (MSH) and prednisolone sodium hemisuccinate (PSH). In contrast, nonsuccinylated corticosteroids (including methylprednisolone and prednisolone in one patient) yielded no test reactions. Basophils from one patient exhibited a stimulated expression of the activation marker CD63 upon in vitro incubation with PSH or hydrocortisone sodium succinate, but not with hydrocortisone. Skin tests and basophil activation tests were negative in controls. One patient was challenged with the incriminated drugs. He developed flush, conjunctivitis, tachycardia and dyspnea 2 min after injection of MSH, and dyspnea shortly after intravenous administration of PSH. Oral and intravenous challenge tests with nonsuccinylated corticosteroids were tolerated well by both patients. Conclusions: These case reports should alert clinicians to rare, but severe immediate-type reactions to corticosteroids, related to the succinate moiety in our patients. In case of allergic reactions to corticosteroids, it is mandatory to identify the causative agent and find safe alternatives.

“…The Flow2 CAST was performed according to the manufacturer's instructions (Bühlmann Laboratories, Allschwil, Switzerland) and has been fully described elsewhere [29]. Flow cytometric analysis was performed using an Accuri C6 flow cytometer (Accuri Cytometers Ltd., UK) with CFlow Plus software.…”

Section: Methodsmentioning

confidence: 99%

Mapping of the Immunodominant Regions of Shrimp Tropomyosin Pan b 1 by Human IgE-Binding and IgE Receptor Crosslinking Studies

Myrset

Fæste

Kristiansen³

et al. 2013

Background: Epitope mapping of an allergen is generally done by IgE-binding assays with short synthetic peptides, but this provides little information about which domains are responsible for IgE receptor crosslinking on effector cells. Our aim was to map the immunodominant regions of shrimp tropomyosin by both IgE-binding and IgE-receptor crosslinking studies. Methods: Five overlapping fragments covering Pandalus borealis tropomyosin were cloned, expressed in Escherichia coli and characterized by circular dichroism spectroscopy, native PAGE and bis(sulfosuccinimidyl) suberate-crosslinking. IgE binding was detected by Western blot, indirect ELISA and inhibition ELISA, and IgE receptor crosslinking was investigated by basophil activation test and skin prick test with Norwegian shrimp allergic adults. Results: The N- and C-terminal fragments of tropomyosin showed the highest amount of secondary structure. Western blot studies showed preferential binding to the terminal fragments, while indirect and inhibition ELISA studies showed binding to all fragments, but with individual variations. Basophil CD63 expression was upregulated by all fragments at high concentrations (1 µg/ml) and showed individual variations comparable to ELISA results. A mixture of the fragments with equal molar ratios induced comparably strong CD63 activation as for tropomyosin. Skin prick test studies showed positive responses to the terminal and middle fragments and increased responses to the fragment mixture compared to whole tropomyosin. Conclusions: The terminal and middle fragments of tropomyosin had the highest IgE reactivity, but overall no clear immunodominant region was observed in this study. These results correlated well with previous studies with short peptides. Dividing shrimp tropomyosin into five fragments did not reduce the allergenicity of the protein.