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

BACKGROUND: The 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). METHODS: Donor 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). RESULTS: For 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. CONCLUSIONS: For 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.

Chemokine receptor 3 and eosinophilir cationic protein: as novel laboratoyt markers for assessment of airway inflammation in bronchial asthma

The aim of this work was to trace and follow up the profile of the chemokine receptor3 [CXCR3], eosinophilic cationic protein [ECP] and immunoglobulin E [IgE] in atopic asthmatic patients during and between the attacks and to outline their relations to other parameters denoting disease activity and severity. Moreover, this study also aimed at testing the ability of the three markers to monitor the patients response to treatment in order to tailor relevant therapeutic modalities to alleviate the patients' allergic condition. Thirty seven atopic asthmatic patients [Group 1] were enrolled in this study. They were sub classified according to their peak expiratory flow [PEF] into mild, moderate and severe asthmatics [subgroups IA, IB and IC, respectively]. Their results were compared to those of 13 healthy subjects [Group II]. For all subjects; number of eosinophils in peripheral blood, serum total lgE, serum ECP and plasma CXCRJ% expression by flow cytometry were estimated. For group I only laboratory parameters and PEF were done twice; during acute asthmatic attack and between the attacks [controlled condition after one week of efficient treatment of corticosteroids and supportive therapy]. Highly significant results were found in asthmatic patients' group [Group I] during the attack regarding the number of eosinophils, ECP values and CXCR3% [decrease], while only a significant difference was found regarding IgE levels when compared to healthy controls [Group II]. Comparative study between patients' subgroups was done revealing highly significant differences for patients in subgroups IB and IC regarding CXCR3%, ECP and IgE [in subgroup IC only], when compared to control group. While, in subgroup IA highly significant difference was found regarding ECP values and significant differences were found regarding.. CXCR3% [decrease] and IgE values when compared to controls. Similar results were found when patients subgroups results were compared to each others. Paired t test was used to compare patients' results during acute attack and between attacks [after treatment] to monitor the inflammatory events in both situations, where highly significant differences were found regarding CXCR3% [increase] and ECP levels [decrease], while only a significant difference was found for IgE levels [decrease]. Correlation matrix was performed for patients' results during acute attack revealing strong negative correlations between CXCR3% expression and ECP, IgE and number of eosinophils in peripheral blood [r= -0.8, -0.7 and -0.5, respectively]. While, ECP values had strong positive correlation with IgE [r=0.7] and a weak positive correlation with number of eosinophils in peripheral blood [r= 0.4]. Stepwise multi regression analysis was done to choose the best parameter [s] which can be used for monitoring patients' good response to treatment, where both CXCR3 and ECP were found to be the best for that purpose [F=7.8, p<0.01]. One way analysis of variance [ANOVA] testing and positive likelihood ratio were done to choose the best parameter [s] which can discriminate patients with severe asthma among other asthmatics. ANOVA test revealed that CXCR3% was the best for this purpose [F=47.2, p<0.01] followed by ECP, lgE and number of eosinophuls in peripheral blood [p<0.01, <0.01 and <0.05, respectively]. Moreover, positive likelihood ratio revealed that both CXCR3% and ECP had comparable excellent ratios [ratio =10, respectively] followed by IgE [ratio=7]. This study revealed an integrated explanation of the immunoinflammatory events in acute atopic asthma. Where, a drop of CXCR3 expression paves the way for the immediate hypersensitivity reaction of allergy including T helper2 [Th2] cells with their chemokine receptors leading to eosinophilic recruitment and degranulation releasing ECP with the help of IgE bound on their cell surface resulting in airway inflammatory response and typical allergic reaction of asthma. Hence, new therapeutic modalities for asthmatic patients should include agonists for CXCR3 or Th2 antagonists to alleviate the patient's condition. Moreover, this study demonstrated that short term oral corticosteroids modulate the balance of chemokine receptors' expression in favor of CXCR3 in asthmatic patients. In addition, this work provides evidence that CXCR3 and ECP assays can be used efficiently for monitoring of treatment efficacy in such patients. Lastly, CXCR3, ECP and to a lesser extent IgE assays were found to be good prognostic markers to distinguish patients with severe asthma among other asthmatic patients