Basophil Markers for Identification and Activation in the Indirect Basophil Activation Test by Flow Cytometry for Diagnosis of Autoimmune Urticaria

Abstract

BackgroundThe indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c).MethodsDonor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20).ResultsFor basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity.ConclusionsFor basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

The EAACI/GA²LEN/EDF/WAO guideline for the definition, classification, diagnosis and management of urticaria

“…BATs assess histamine release or upregulation of activation markers of donor basophils in response to stimulation with the serum of CSU patients. BATs can help to co‐assess disease activity in patients with urticaria as well as to diagnose autoimmune urticaria . Furthermore, BAT can be used as a marker for responsiveness to ciclosporin A or omalizumab …”

Using a CD45dim/CD123bright/HLA-DRneg Phenotyping Protocol to Gate Basophils in FC for Airway Allergy. CD123 Does Not Decrease

“…If CD123 is really down-regulated by activation, it would be very interesting to assess if a gating strategy using CCR3 (CD193), the eotaxin receptor, might bear the same concern reported by others elsewhere [3,4], as CCR3 appears to be down-regulated upon basophil activation [2,14,15]. Interestingly, some authors reported a paper where a phenotyping protocol using a combination of CD123 and CCR3 markers, improved significantly the performance of BAT, respect to the only CD123 [16].…”

How I investigate basophilia in daily practice

“…shows the best performance to identify basophils. 24 After gating on CD45 moderately positive and low SSC cells, CD123 associated with HLA-DR seems to be a robust combination to identify basophils that are strongly positive for the first marker and negative for the second one. Moreover, this combination permits to separate basophils from plasmacytoid dendritic cells, which express bright HLA-DR. Basophils can be segregated from myeloblasts by the negativity of immaturity markers (CD34-HLA-DR-CD117-); however, CD38 is strongly positive in basophils.…”