Increased CD123 + HLA-DR- Granulocytes in Allergic Rhinitis and Influence of Allergens on Expression of Cell Membrane Markers.

BACKGROUND: It is reported that CD123 + HLA-DR- cells in PBMC are basophils, and CD203c, CD63, and FcεRI molecules are activation markers of basophils. However, little is known of CD123 + HLA-DR-cells in blood granulocytes. OBJECTIVE: To investigate the presence of CD123 + HLA-DR- cells in the blood granulocytes and peripheral PBMC of patients with allergic rhinitis (AR), as well as the impact of allergens on the cell membrane markers of basophils. METHODS: Flow cytometry was used to detect the expression of the membrane molecules. RESULTS: While CD123 + HLA-DR- PBMCs are representative of basophils, their presence did not significantly change in patients with AR. In contrast, both the percentage and number of CD123 + HLA-DR- granulocytes, which make up only up to 50% of basophils, were significantly increased in patients with seasonal (sAR) and perennial AR (pAR). CD63+, CD203c+, and FcεRIα+ cells within CD123 + HLA-DR- granulocytes also showed enhanced activity in patients with AR. Allergen extracts from house dust mite allergen extract (HDME) and Artemisia sieversiana wild extract further increased the number of CD123 + HLA-DR- cells in granulocytes of sAR and pAR patients, as well as in PBMCs of pAR patients. CONCLUSIONS: The use of CD123 + HLA-DR- granulocytes and PBMC may not be sufficient for diagnosing AR. Allergens could potentially contribute to the development of AR by influencing the number of CD123 + HLA-DR- cells, as well as the expression of CD63, CD203c, and FcεRIαin these cells.

CD203c is expressed by human fetal hepatoblasts and distinguishes subsets of hepatoblastoma.

Background & Aims: Hepatocytic cells found during prenatal development have unique features compared to their adult counterparts, and are believed to be the precursors of pediatric hepatoblastoma. The cell-surface phenotype of hepatoblasts and hepatoblastoma cell lines was evaluated to discover new markers of these cells and gain insight into the development of hepatocytic cells and the phenotypes and origins of hepatoblastoma. Methods: Human midgestation livers and four pediatric hepatoblastoma cell lines were screened using flow cytometry. Expression of over 300 antigens was evaluated on hepatoblasts defined by their expression of CD326 (EpCAM) and CD14. Also analyzed were hematopoietic cells, expressing CD45, and liver sinusoidal-endothelial cells (LSECs), expressing CD14 but lacking CD45 expression. Select antigens were further examined by fluorescence immunomicroscopy of fetal liver sections. Antigen expression was also confirmed on cultured cells by both methods. Gene expression analysis by liver cells, 6 hepatoblastoma cell lines, and hepatoblastoma cells was performed. Immunohistochemistry was used to evaluate CD203c, CD326, and cytokeratin-19 expression on three hepatoblastoma tumors. Results: Antibody screening identified many cell surface markers commonly or divergently expressed by hematopoietic cells, LSECs, and hepatoblasts. Thirteen novel markers expressed on fetal hepatoblasts were identified including ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP-3/CD203c), which was found to be expressed by hepatoblasts with widespread expression in the parenchyma of the fetal liver. In culture CD203c+CD326++ cells resembled hepatocytic cells with coexpression of albumin and cytokeratin-19 confirming a hepatoblast phenotype. CD203c expression declined rapidly in culture whereas the loss of CD326 was not as pronounced. CD203c and CD326 were co-expressed on a subset of hepatoblastoma cell lines and hepatoblastomas with an embryonal pattern. Conclusions: CD203c is expressed on hepatoblasts and may play a role in purinergic signaling in the developing liver. Hepatoblastoma cell lines were found to consist of two broad phenotypes consisting of a cholangiocyte-like phenotype that expressed CD203c and CD326 and a hepatocyte-like phenotype with diminished expression of these markers. CD203c was expressed by some hepatoblastoma tumors and may represent a marker of a less differentiated embryonal component.

High Infiltration of CD203c+ Mast Cells Reflects Immunosuppression and Hinders Prognostic Benefit in Stage II-III Colorectal Cancer.

Background: Activated mast cells (AMCs) have been fully researched in inflammation and allergic reactions. However, the protumoral role of AMCs and their biomarker CD203c has not yet been investigated in colorectal cancer (CRC). Methods: We retrospectively collected 449 postoperative patients with stage II-III CRC at two different hospitals as the training (n=310) and validation (n=139) cohorts. These findings were further validated in the independent cohort (Integration of GSE39582 and GSE17536, n=489). The AMC density was assessed using CD203c staining or the CIBERSORT method. The main analysis was recurrence-free survival (RFS) and overall survival (OS). Results: As an independent factor, high AMC infiltration was associated with worse RFS/OS in the training (hazard ratio [HR]=3.437/3.014, all p<0.001) and validation (HR=3.537/2.382, all p<0.001) cohorts. We developed and validated an AMC-based nomogram for better stratification for postoperative recurrence in these two cohorts. The role of AMC density was further validated in the independent cohort. High AMC infiltration was associated with decreased RFS/OS after adjuvant chemotherapy (all p<0.05). Approximately 74.2% of intramural CD203c+ AMCs expressed a high level of PD-L1. Multiple immunosuppressive pathways were enriched in high AMC infiltration tumors, including upregulation of the TNF-α/NF-κB and angiogenesis pathways and downregulation of the IFN-γ and IFN-α responses. AMC infiltration was reversely associated with CD8+ T-cell infiltration (all p<0.05). Conclusion: High AMC infiltration is associated with worse survival outcomes in stages II-III CRC. AMC density may serve as a potential biomarker for survival benefit in patients receiving adjuvant chemotherapy. This AMC-based nomogram could provide better recurrence stratification. Immunosuppression in tumors with high AMC infiltration might contribute to promoting tumor progression.

Basophil Activation Test Shows Poor Sensitivity in Immediate Amoxicillin Allergy.

BACKGROUND: In light of the pandemic of spurious penicillin allergy, correct diagnosis of amoxicillin (AX) allergy is of great importance. The diagnosis of immediate hypersensitivity reactions relies on skin tests and specific IgE, and although reliable, these are not absolutely predictive. Therefore, drug challenges are needed in some cases, which contain the risk of severe reactions. Safe in vitro diagnostics as an alternative for the drug challenge in the diagnostic workup of AX allergy would be more than welcome to fill this gap. In this respect, the basophil activation test (BAT) has shown potential, but its clinical reliability is doubtful. OBJECTIVE: To investigate the reliability of the BAT to AX and determining its exact place in the diagnostic algorithm of AX allergy. METHODS: BAT for AX was performed in 70 exposed control individuals and 66 patients diagnosed according to the European Academy of Allergy and Clinical Immunology guidelines for AX allergy. Upregulation of both CD63 and CD203c was flow-cytometrically assessed. RESULTS: Analyses revealed that 1370 µmol/L and 685 µmol/L were the most discriminative stimulation concentrations for CD63 and CD203c upregulation, respectively, and a diagnostic threshold of 9% for positivity for both markers was identified. At these concentrations, sensitivity and specificity for CD63 upregulation were 13% and 100%, respectively, and for CD203c upregulation, 23% and 98%. CONCLUSIONS: BAT with dual analysis of CD63 and CD203c is of poor performance to document AX allergy. The sensitivity is too low to let it occupy a prominent role in the diagnostic algorithm.

What happens to basophils and tryptase, LXA4 and CysLTs during aspirin desensitization?

INTRODUCTION: Aspirin desensitization (AD) is an effective treatment in patients with non-steroidal anti-inflammatory drugs (NSAID)-exacerbated respiratory disease (NERD) by providing inhibitory effect on symptoms and polyp recurrence. However, limited data is available on how AD works. We aimed to study comprehensively the mechanisms underlying AD by examining basophil activation (CD203c upregulation), mediator-releases of tryptase, CysLT, and LXA4, and LTB4 receptor expression for the first 3 months of AD. METHODS: The study was conducted in patients with NERD who underwent AD (group 1: n = 23), patients with NERD who received no desensitization (group 2: n = 22), and healthy volunteers (group 3, n = 13). All participants provided blood samples for flow cytometry studies (CD203c and LTB4 receptor), and mediator releases (CysLT, LXA4, and tryptase) for the relevant time points determined. RESULTS: All baseline parameters of CD203c and LTB4 receptor expressions, tryptase, CysLT, and LXA4 releases were similar in each group (p > 0.05). In group 1, CD203c started to be upregulated at the time of reactions during AD, and continued to be high for 3 months when compared to controls. All other study parameters were comparable with baseline and at the other time points in each group (p > 0.05). CONCLUSION: Although basophils are active during the first 3 months of AD, no releases of CysLT, tryptase or LXA4 exist. Therefore, our results suggest that despite active basophils, inhibition of mediators can at least partly explain underlying the mechanism in the first three months of AD.

Evaluation of peripheral basophil activation during exercise provocation test for desensitized patients.

Some food allergic patients who have undergone oral immunotherapy develop exercise-induced allergic reactions on desensitization (EIARDs). This study investigated basophil activation status during the exercise provocation test (EPT) performed to diagnose EIARD. EPT was performed on 20 participants, and in vivo basophil activation status was analyzed using activation markers CD203c and CD63. The results showed that there was no significant difference between EPT-positive and negative subjects for basophil activation status throughout EPT. Consequently, in vivo basophil activation after ingestion of the causative food may not be associated with EIARDs. New tests are desired for predicting EIARDs.

Basophil activation test has high reproducibility and is feasible in the clinical setting.

BACKGROUND: The basophil activation test (BAT) has high accuracy to diagnose peanut allergy and can reduce the need for oral food challenges (OFC); however, so far it has not been incorporated in clinical practice. METHODS: We assessed the reproducibility of BAT within the same laboratory and between two different laboratories and the feasibility of using BAT in the clinical setting. RESULTS: One hundred and two children being assessed for peanut allergy were tested on BAT (72 allergic, 30 sensitized tolerant). There was little internal variation (coefficient of variation <15%) in the BAT and a very strong correlation (Rs > .95) between BAT performed across laboratories. The 2 BAT methods were strongly correlated but not interchangeable. In the cases of discrepancy, our in house BAT method was 100% accurate. BAT was feasible and well-accepted by clinicians: no patient with positive BAT was referred for OFC, leading to reduction in the number of OFC required. Twenty one percent of patients who underwent OFC reacted to peanut. A negative BAT also encouraged the performance of OFC in sensitized children who would otherwise be considered allergic, 50% of whom did not react and incorporated peanut in the diet. CONCLUSIONS: The BAT is a robust test that can reliably be transferred between laboratories; however, different BAT methods are not interchangeable. BAT was well integrated in the clinical decision-making process in a specialized center.

Increased expressions of CD123, CD63, CD203c, and Fc epsilon receptor I on blood leukocytes of allergic asthma.

Background : Altered basophil identification markers have been discovered to associate with allergic asthma (AA) in recent years. However, little is known about the expression of basophil markers in blood granulocytes. Aim: To parallel test blood basophils in peripheral blood mononuclear cell (PBMC) and granulocyte populations of patients with AA and AA combined with allergic rhinitis (ARA) Methods: The expressions of surface molecules were determined via flow cytometry. CD123 expressing cells in blood were isolated using a cell sorting technique, and mouse AA models were employed for in vivo study. Results: The numbers of CD123+HLA-DR- cells in the granulocytes of AA and ARA patients markedly increased. However, only 49.7% of CD123+HLA-DR- cells in granulocytes and 99.0% of CD123+HLA-DR- cells in PBMCs were basophils. Almost all CD123+HLA-DR- cells expressed CD63 regardless in granulocytes or PBMC. The numbers of CD63, Fc epsilon receptor I (FcεRI), and CD203c expressing cells markedly enhanced in CD123+HLA-DR- granulocytes of AA and ARA patients. Mean fluorescence intensity (MFI) of CD63 and CD203c expressions on CD123+HLA-DR- PBMC and granulocytes of AA and ARA patients dramatically elevated. House dust mite extract (HDME) and Artemisia sieversiana wild allergen extract (ASWE) enhanced the numbers of CD63+CD123+HLA-DR- granulocytes and PBMC and the MFI of CD203c expression on CD123+HLA-DR- granulocyte of AA and ARA patients. Histamine, tryptase, and PGD2 enhanced proportions of CD123+ KU812 cells. ASWE- and HDME-induced AA mice showed upregulated CD63 expression on basophils. In conclusion, upregulated expressions of CD123, CD203c, CD63, and FcεRIα in PBMC and granulocytes of patients with AA and ARA suggest that CD123+HLA-DR- cells may contribute to the development of AA and ARA.

Mas-related G protein-coupled receptor MRGPRX2 in human basophils: Expression and functional studies.

Background: Occupancy of MRGPRX2 heralds a new era in our understandings of immediate drug hypersensitivity reactions (IDHRs), but a constitutive expression of this receptor by basophils is debated. Objective: To explore the expression and functionality of MRGPRX2 in and on basophils. Methods: Basophils from patients with birch pollen allergy, IDHRs to moxifloxacin, and healthy controls were studied in different conditions, that is, in rest, after stimulation with anti-IgE, recombinant major birch pollen allergen (rBet v 1), moxifloxacin, fMLP, substance P (SP), or other potential basophil secretagogues. In a separate set of experiments, basophils were studied after purification and resuspension in different media. Results: Resting whole blood basophils barely express MRGPRX2 on their surface and are unresponsive to SP or moxifloxacin. However, surface MRGPRX2 is quickly upregulated upon incubation with anti-IgE or fMLP. Pre-stimulation with anti-IgE can induce a synergic effect on basophil degranulation in IgE-responsive subjects after incubation with SP or moxifloxacin, provided that basophils have been obtained from patients who experienced an IDHR to moxifloxacin. Cell purification can trigger a "spontaneous" and functional upregulation of MRGPRX2 on basophils, not seen in whole blood cells, and its surface density can be influenced by distinct culture media. Conclusion: Basophils barely express MRGPRX2 in resting conditions. However, the receptor can be quickly upregulated after stimulation with anti-IgE, fMLP, or after purification, making cells responsive to MRGPRX2 occupation. We anticipate that such "conditioned" basophils constitute a model to explore MRGPRX2 agonism or antagonism, including IDHRs originating from the occupation of this receptor.

Identification of CD203c as a New Basophil-Specific Flow-Marker in Ph+ Chronic Myeloid Leukemia.

Basophilia is a crucial prognostic variable in Ph-chromosome-positive chronic myeloid leukemia (CML). The ectoenzyme CD203c is an activation-linked surface antigen that is expressed specifically on basophil-committed progenitor cells and mature basophils. We examined the expression of CD203c on progenitors and/or basophils in 21 healthy donors and 44 patients with CML. As expected, the numbers of CD203c+ blood leukocytes were significantly higher in CML patients compared to controls (percentage of CD203c+ cells among viable cells in CML at diagnosis: 4.19 ± 3.68% vs. controls: 0.53 ± 0.23%, p < 0.05). Moreover, CML basophils expressed higher levels of CD203c compared to normal basophils (median staining-index in CML at diagnosis: 29.41 ± 19.14 versus controls: 20.44 ± 13.45). We also found that the numbers and percentage of circulating CD203c+ cells at diagnosis correlate with the disease-related risk-profile. Incubation of CML basophils with an anti-IgE-antibody resulted in further upregulation of CD203c. After successful treatment with imatinib and/or other BCR::ABL1 inhibitors leading to major or complete molecular responses, the numbers of CD203c+ basophils decreased substantially in our CML patients compared to pre-treatment values. Together, CD203c is overexpressed on CML basophils, is further upregulated by IgE receptor cross-linking, and may serve as a biomarker to quantify basophilia in patients with CML at diagnosis and during therapy.

Basophil activation test: Assay precision and BI 1002494 SYK inhibition in healthy and mild asthmatics.

BACKGROUND: Application of basophil activation test (BAT) in clinical trials requires assay validity. Whether assay variability differs between healthy and asthmatic subjects is mostly unknown. This study compares basophil stimulation using blood from healthy and asthmatic subjects with or without inhibition of spleen tyrosine kinase (SYK). METHODS: Whole blood of healthy and mild asthmatic subjects was stimulated with anti-dinitrophenyl (DNP) IgE/DNP bovine serum albumin and anti-IgE. Basophil activation was detected by CD63 and CD203c expression. CD63 expression levels were compared with serum IgE levels. Three operators repeated experiments with three subjects each from both groups at 3 days to observe assay precision. The effect of the SYK inhibitor BI 1002494 was assessed in BAT for both healthy and asthmatic subjects. RESULTS: BAT was reproducible in both groups. Acceptance criteria of <25% CV were mostly fulfilled. Stimulation with anti-DNP (p < 0.001, r = -0.80) but not anti-IgE (p = 0.74, r = 0.05) was related to serum IgE with levels > 200 IU/ml limiting anti-DNP stimulation. BI 1002494 IC50 values were 497 nM and 1080 nM in healthy and 287 nM and 683 nM in asthmatics for anti-DNP and anti-IgE stimulation, respectively. CONCLUSION: BAT, performed with blood from healthy or asthmatic subjects, is a robust test for the measurement of a physiological response in clinical trials. Blood from asthmatic donors with serum IgE > 200 IU/ml is less feasible when using anti-DNP stimulation. SYK inhibition was not affected by disease status.

Basophils priming in patients with chronic spontaneous urticaria.

INTRODUCTION: Basophils are one of the main target cells in chronic spontaneous urticaria (CSU). If cells present higher susceptibility to production and degranulation of pro-inflammatory factors, priming may be associated with severity of symptoms and therapy ineffectiveness. AIM: To evaluate the spontaneous state of increase in basophil activity and their priming profile in patients with CSU. MATERIAL AND METHODS: The study sample included 22 patients diagnosed with CSU and 20 healthy volunteers without either allergy symptoms or CSU. In this study, we evaluate the presence of CD63 and CD63+CD203c at basophils surface by flow cytometry test (basophil activation test - BAT). RESULTS: We found that the percentage of activated basophils was higher in patients with CSU than in the control group and this difference was statistically significant (p < 0.05). CONCLUSIONS: Our results indicate a greater degree of basophils activation in patients with CSU in remission than in the control group; it might be useful for identification of patients with predominance of the autoimmune variant of CSU and typing patients responding (responders) and refractory (non-responders) to treatment with antihistamines.

Adhesion molecule cross-linking and cytokine exposure modulate IgE- and non-IgE-dependent basophil activation.

Basophils are known for their role in allergic inflammation, which makes them suitable targets in allergy diagnostics such as the basophil activation test (BAT) and the microfluidic immunoaffinity basophil activation test (miBAT). Beside their role in allergy, basophils have an immune modulatory role in both innate immunity and adaptive immunity. To accomplish this mission, basophils depend on the capability to migrate from blood to extravascular tissues, which includes interactions with endothelial cells, extracellular matrix and soluble mediators. Their receptor repertoire is well known, but less is known how these receptor-ligand interactions impact the degranulation process and the responsiveness to subsequent activation. As the consequences of these interactions are crucial to fully appreciate the role of basophils in immune modulation and to enable optimization of the miBAT, we explored how basophil activation status is regulated by cytokines and cross-linking of adhesion molecules. The expression of adhesion molecules and activation markers on basophils from healthy blood donors was analysed by flow cytometry. Cross-linking of CD203c, CD62L, CD11b and CD49d induced a significant upregulation of CD63 and CD203c. To mimic in vivo conditions, valid also for miBAT, CD62L and CD49d were cross-linked followed by IgE-dependent activation (anti-IgE), which caused a reduced CD63 expression compared with anti-IgE activation only. IL-3 and IL-33 priming caused increased CD63 expression after IgE-independent activation (fMLP). Together, our data suggest that mechanisms operational both in the microfluidic chip and in vivo during basophil adhesion may impact basophil anaphylactic and piecemeal degranulation procedures and hence their immune regulatory function.

Improved FcεRI-Mediated CD203c Basophil Responsiveness Reflects Rapid Responses to Omalizumab in Chronic Spontaneous Urticaria.

BACKGROUND: Omalizumab is effective in patients with chronic spontaneous urticaria (CSU) although its mechanism of action is poorly understood. Several studies reported that decreased high-affinity IgE receptor (FcεRI)-mediated histamine release and/or responsiveness was characteristic of basophils in patients with CSU. However, few studies have focused on the relationship between changes in basophil responsiveness via FcεRI after omalizumab treatment and the therapeutic effect in patients with CSU. OBJECTIVE: To assess basophil responsiveness via FcεRI stimulation, as well as FcεRI expression and IgE binding on blood basophils from patients with CSU before and after omalizumab treatment and its possible association with the clinical response. METHODS: We analyzed 34 patients with CSU treated with omalizumab who were categorized as fast responders (FRs) (n = 20) and non or slow responders (N/SRs) (n = 14). CD203c expression induced by FcεRI stimulation, and IgE and FcεRI expressions on blood basophils from patients with CSU before and after omalizumab treatment were analyzed. Basophil responsiveness via FcεRI stimulation was observed in vitro using basophils pretreated with omalizumab. RESULTS: FRs had increased CD203c responsiveness after treatment with omalizumab compared with N/SRs. This improvement of basophil responsiveness via FcεRI stimulation in FRs was not observed in peripheral blood basophils preincubated with omalizumab in vitro, suggesting that omalizumab does not directly affect circulating pre-existing abnormal basophils. CONCLUSION: Increased basophil responsiveness via FcεRI after omalizumab treatment is associated with the therapeutic effect and mechanism of action of omalizumab.

A new standardized phytoextract from red orange and lemon wastes (red orange and lemon extract) reduces basophil degranulation and activation.

Citrus fruits are rich sources of bioactive compounds and their consumption is associated to health-promoting effects. Citrus processing wastes contain bioflavonoids and other high added value compounds. The aim of this study was to evaluate the antiallergic properties of a new phytoextract obtained by citrus wastes and peels. Blood orange and lemon processing wastes were used to produce a Red orange and Lemon Extract (RLE). Blood samples from 30 allergic donors were collected and used to evaluate the basophil activation (CD203c) and degranulation (CD63) by stimulation trough allergen with and without the RLE. Reduced basophil expression of CD203c and CD63 were observed in RLE + Allergen treated samples, with -20.21% of CD203c expression (p < 0.0001) and -54.11% of CD63 expression (p < 0.0001), compared to Allergen treated samples. The RLE evidenced a good antiallergic activity, mainly acting on basophils degranulation, and therefore reducing the key event of pro-inflammatory mediators release after allergic stimuli.

Principles, potential, and limitations of ex vivo basophil activation by flow cytometry in allergology: A narrative review.

The major challenge of allergy diagnosis lies in the development of accessible and reliable diagnostics allowing correct prediction of the clinical outcome following exposure to the offending allergen(s) and cross-reactive structures. Since the late nineties, evidence has accumulated that flow-assisted analysis and quantification of ex vivo-activated basophils (according to the basophil activation test [BAT]) might meet this requirement for different IgE-dependent allergies and particular forms of autoimmune urticaria. Other so-called nondiagnostic applications of the BAT involve therapeutic monitoring, follow-up of natural histories, and identification of allergenic recognition sites. However, it has also become clear that appropriate use of the BAT necessitates knowledge about degranulation metrics and guidance to guarantee correct execution and interpretation of the results. Here, we have reviewed the most relevant applications and limitations of the BAT. Some personal statements and views about its perspectives are made.

Vascular endothelial growth factor-activated basophils in asthmatics.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is an angiogenic cytokine and a potential stimulator of permeability and lung neovascularization in asthmatics. It also plays an important role in the development of airway remodelling and in activation of many cells, including basophils. AIM: To reveal the possible role of VEGF in the activation of basophils in asthmatics - subgroups with and without irreversible bronchoconstriction. Protein CD203c on the basophils surface was used as the activation marker. To define the possible pathway of basophils VEGF-activation, the influence of a genetic factor (polymorphism del18/ins -2549 -2567 in the VEGF-promoter region) was also considered. MATERIAL AND METHODS: Our study involved 82 patients with asthma (40 patients without and 42 patients with irreversible bronchoconstriction) and a group of 40 controls. The flow cytometric methods with anti-CD203c in the samples of basophils with increasing concentrations of VEGF was used for analysis of their activity. Genotyping for VEGF-promoter region was performed by PCR-based methods. RESULTS: Patients with asthma and del/del genotype showed more significant differences in the basophils activation after stimulation with increasing concentrations of VEGF than asthmatics with ins/ins and ins/del genotype (p = 0.023) and controls with del/del genotype (p = 0.0006). CONCLUSIONS: Raised basophils VEGF-activation is characteristic for examined patients with asthma and might be associated with presence of polymorphism del18/ins -2549 -2567 in the VEGF-promoter region. Furthermore, it may contribute to the development of airways remodelling - this pathway has not been considered yet.

Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques.

The basis of flow cytometric allergy diagnosis is the quantification of changes in the expression of basophilic surface membrane markers (Ebo et al., Clin Exp Allergy 34: 332-339, 2004). Upon encountering specific allergens recognized by surface receptor FcεRI-bound IgE, basophils not only secrete and generate quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies (Ebo et al., Cytometry B Clin Cytom 74: 201-210, 2008). Here, we describe two flow cytometry-based protocols which allow the detection of surface marker activation (Method 1) and changes in intragranular histamine (Method 2), both reflecting different facets of basophil activation.

Basophil Activation Techniques: Staining of Exteriorized Granule Matrix.

The basis of traditional flow cytometry allergy diagnosis is measurement of the expression of basophilic surface activation and/or degranulation markers. Basophils, upon encounter with a specific allergen that cross-links surface FcRI-bound IgE antibodies, not only secrete and release quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies. Here, we describe a novel technique that relies upon the staining of exteriorized anionic proteoglycans from a basophil granule matrix by cationic fluorescent avidin probes.

Basophil Activation Experiments in Immediate Drug Hypersensitivity: More Than a Diagnostic Aid.

BACKGROUND: Correct diagnosis of immediate drug hypersensitivity reactions (IDHRs) can pose a significant challenge, mainly because of the absence of reliable in vitro tests, uncertainties associated with skin testing, and incomplete understanding of the underlying mechanisms. AIM: To summarize and hypothesize on the potential of basophil activation test (BAT) as a safe aid to explore the mechanistic endotypes of IDHR, to identify antibody recognition sites, and to monitor drug desensitization. METHODS: A literature search was conducted using the keywords "allergy, basophil activation, CD63, CD203c, diagnosis, drugs, hypersensitivity, flow cytometry"; this was complemented by the authors' own expertise. RESULTS: At present BAT has mainly been employed as a diagnostic aid. However, evidence is emerging that the technique might also deepen our insights in immune (allergic) and nonimmune (nonallergic) mechanistic processes of IDHR. It is anticipated that BAT might also benefit the identification of antibody recognition sites and benefit our understandings of desensitization strategies. CONCLUSION: Although the nondiagnostic application of BAT in IDHR is still in its infancy, with increasing employment, we can expect the technique to become a valuable asset to study many domains of IDHR that remain poorly understood.