Flow Cytometric Basophil Activation Test Research Articles

Diagnostic value of the lymphocyte transformation test for non‐IgE mediated food allergy

The aim of our study was to verify the diagnostic value of lymphocyte transformation test (LTT) for non-IgE mediated food allergy. We investigated 3 groups of patients for identifying the food hypersensitivity. 1. Group - 60 patients with symptoms of irritable bowel syndrome (IBS) without a diagnosis of organic gastrointestinal disease. 2. Group - 19 patients with atopic dermatitis, polysenzitization to inhalant, not to food allergens. 3. Group - 6 infants (3-12 months old) with intestinal symptoms (bloody stool and abdominal distention) and positive elimination diet with cow's milk. Control group -10 healthy non-atopic patients. We performed skin prick test (SPT) and the flow-cytometric basophil activation test(BAT) with commercial extracts of wheat, rye flour, egg, milk and soy allergens Alyostal (Stallergenes) in all patients. Serum levels of milk, casein, wheat, rye flour, egg and soy specific IgE were measured by Immulite. The lymphocyte transformation test was performed with the same food allergens. Open food challenge test (OFC) was completed after 14 days of elimination diet. All patients had negative SPT, sIgE and BAT with milk, casein, wheat, rye, egg and soy allergens. They had negative celiac disease serology. In 1.group 42 patients (70%) had positive LTT for wheat flour, none of them for other allergens. Open food challenge test by ingestion of daily serving cooked wheat pasta were positive in all 42 positive patients. 18 patients (30%) had negative LTT for all allergens. In 2.group 15 patients had positive LTT for wheat flour, 2 for milk, 1 for soy allergens Elimination diet of wheat or milk or soy improved eczematic manifestations. In 3.group all 6 infants had positive LTT to cow/s milk and casein allergens. They had positive OFC test with cow/s milk after elimination diet. Patients of control group were negative in all vitro tests. In our study the lymphocyte transformation test were useful diagnostic tools for non-IgE mediated food allergy.

Use of in vitro tests to assess the causative drugs for NSAIDs‐induced type I hypersensitivity

Non-steroidal anti-inflammatory drugs (NSAIDs) are associated with type I hypersensitivity, including cutaneous manifestations (e.g., urticaria and angioedema), respiratory manifestations (rhinitis, nasal polyposis, asthma), and anaphylaxis. To assess the sensitivity/specificity of in vitro tests for verifying the causative/tolerant drugs and cross-reactivity of different NSAIDs, we used three assays, including two ELISA tests for histamine/leukotriene C4 (LTC4) release, and a flow cytometric basophil activation test (BAT). We recruited 82 patients with NSAIDs-induced type I hypersensitivity (angioedema of the most cases), and isolated the peripheral leukocytes to perform histamine/LTC4 release tests in 38 patients, and BAT in 44 patients. The cell response to NSAIDs was examined in the incubation containing suspected or tolerant drugs with concentrations equal to 1-fold (physical level) or 10-fold Cmax. Comparing with the data of solve controls of the same subject, positive response was considered if the histamine/LTC4 release showed 1.2-fold increase, and BAT detected more than 5% increase of CD63+/CCR3+ cells. We found that histamine release test had a sensitivity of 41.9% (26/62), and specificity of 100% (10/10).LTC4 release test had a sensitivity of 32.3% (20/62), and specificity of 90% (9/10). BAT had a sensitivity of 46.4% (39/84) and specificity of 90% (18/20) for NSAIDs-induced type I hypersensitivity. Regarding drug classification, the four most common clinically suspected agents were aspirin, ibuprofen, diclofenac, and acetaminophen. The three assays have consistently >50% sensitivity for verifying aspirin, but various sensitivity and diverse individual response for the other NSAIDs. Heterogeneous mechanisms including both immunology and pharmacology may be involved in NSAIDs-induced type I hypersensitivity.

Identification of Autoclave-Resistant Anisakis simplex Allergens

Anisakis simplex is a fish parasite able to induce allergic reactions in humans infected when eating raw or undercooked fish parasitized with viable third-stage larvae. Some authors claim that exposure to nonviable Anisakis material can result in allergic symptoms in previously sensitized patients, indicating that parasite allergens are resistant to the thermal treatments of usual cooking procedures. Furthermore, some patients report symptoms after eating canned fish. The aim of this work was the analysis of parasite allergen stability in heating to 121 °C in an autoclave to simulate the thermal process applied to canned fish. Third-stage larvae were subjected to autoclaving for 20, 40, and 80 min, and parasite crude extracts were analyzed by electrophoresis, immunoblotting, and a flow-cytometric basophil activation test. Allergens resistant to autoclaving were separated by reversed-phase high-performance liquid chromatography and identified by ion trap mass spectrometry. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that autoclaving considerably reduced the number and intensity of identifiable protein bands in a time-dependent manner. Several allergens were detected by immunoblotting with a pool of A. simplex allergic patients' sera after autoclaving. Allergens of 9 and 14 kDa resistant to autoclaving were identified as Ani s 4 and Ani s 1 allergens, respectively. Functional analysis showed that allergens retain their capacity to activate basophils even after autoclaving for 80 min. In conclusion, some relevant A. simplex allergens retain their capacity to bind immunoglobulin E and activate basophils after being subjected to autoclaving, which is a method equivalent to that used in industrial canning processes.

Non-Celiac Gluten Sensitivity: Literature Review

Background: A significant percentage of the general population report problems caused by wheat and/or gluten ingestion, even though they do not have celiac disease (CD) or wheat allergy (WA), because they test negative both for CD-specific serology and histopathology and for immunoglobulin E (IgE)-mediated assays. Most patients report both gastrointestinal and nongastrointestinal symptoms, and all report improvement of symptoms on a gluten-free diet. This clinical condition has been named non-celiac gluten sensitivity (NCGS). Aim: We attempt to define the current pathogenic, clinical, and diagnostic criteria of this “new” disease, to provide a practical view that might be useful to evaluate, diagnose, and manage NCGS patients. Methods: We reviewed the international literature through PubMed and Medline, using the search terms “wheat (hyper)sensitivity,” “wheat allergy,” “wheat intolerance,” “gluten (hyper)sensitivity,” and “gluten intolerance,” and we discuss current knowledge about NCGS. Results: It has been demonstrated that patients suffering from NCGS are a heterogeneous group, composed of several subgroups, each characterized by different pathogenesis, clinical history, and, probably, clinical course. NCGS diagnosis can be reached only by excluding CD and WA. Recent evidence shows that a personal history of food allergy in infancy, coexistent atopy, positive for immunoglobulin G (IgG) antigliadin antibodies and flow cytometric basophil activation test, with wheat and duodenal and/or ileum–colon intraepithelial and lamina propria eosinophil counts, could be useful to identify NCGS patients. Conclusions: Future research should aim to identify reliable biomarkers for NCGS diagnosis and to better define the different NCGS subgroups. Key teaching points: • Most patients report both gastrointestinal and nongastrointestinal symptoms, and all agree that there is an improvement of symptoms on a gluten-free diet. • NCGS diagnosis can be reached only by excluding celiac disease and wheat allergy. • Patients suffering from NCGS are a heterogeneous group, composed of several subgroups, each characterized by different pathogenesis, clinical history, and, probably, clinical course. • A personal history of food allergy in infancy, coexistent atopy, positive IgG antigliadin antibodies (AGA) and flow cytometric basophil activation test, with wheat and duodenal and/or ileum–colon intraepithelial and lamina propria eosinophil counts, could be useful to identify NCGS patients. • Future research should aim to identify reliable biomarkers for NCGS diagnosis and to better define the different NCGS subgroup.

Assessing basophil function

Basophils play an important and non-redundant role as inductor and effector cells of the Th2 response, e.g., helminth infection. In addition, they are involved in a broadening range of pathological conditions including systemic lupus erythematosus, eosinophilic oesophagitis and IgG-mediated systemic anaphylaxis. There is a real need to develop robust <i>in vitro</i> techniques to enable study their role in human disease. Basophil histamine release, sulfidoleukotriene synthesis and the flow cytometric basophil activation test are three methods for diagnosing IgE-mediated allergy <i>in vitro</i>. This talk will describe the potential application of these tests in both allergic and non-allergic contexts, as well as outlining challenges in the performance and interpretation of these functional assays.

Diagnosis of stinging insect allergy: utility of cellular in-vitro tests

Diagnosis of stinging insect allergy is based on a detailed history, venom skin tests, and detection of venom-specific IgE. As an additional diagnostic tool, basophil responsiveness to venom allergens has been shown to be helpful in selected patients. This review summarizes the current diagnostic procedures for stinging insect allergy and discusses the latest developments in cellular in-vitro tests. Cellular assays have been evaluated in patients with Hymenoptera venom allergy. The diagnostic performance of the cellular mediator release test is similar to that of the flow cytometric basophil activation test (BAT), but the BAT has been the most intensively studied. BAT offers the possibility to assess basophil reactivity to allergens in their natural environment and to simultaneously analyze surface marker expression and intracellular signaling. It has been demonstrated that BAT represents a valuable additional diagnostic tool in selected patients when used in combination with other well established tests. A major limitation is the current lack of unified, standardized protocols. Flow cytometry offers huge possibilities to enhance knowledge of basophil functions. The BAT may be used as an additional test to confirm the diagnosis of stinging insect allergy in selected patients, provided that it is performed by an experienced laboratory using a validated assay. Test results have to be interpreted by clinicians familiar with the methodological aspects. The utility of the BAT to confirm allergy diagnosis and to predict the risk of subsequent systemic reactions may be improved by combined analysis of multiple surface markers and intracellular signaling pathways.

The Usefulness of CD203c Expression Measurement on Basophils after Activation with Grass Pollen and Dermatophagoides pteronyssinus Antigens. Preliminary Study

Introduction: 'Gold standard' in the diagnosis of atopic disease are skin prick tests and specific IgE evaluation. Well-established in vitro tests, such as the histamine release test, the leukotriens release test and the flow cytometric basophil activation test can be very helpful in diagnostics, especially when the skin prick test is contraindicated. The aim of this study was to evaluate the usefulness of antigen CD203c expression, as a marker of basophil activation by grass pollen or D. pteronyssinus antigens. Material and Methods: Peripheral blood from 13 allergic patients and nine healthy volunteers was analysed. Basophils activation was measured by the breakdown of antigen CD203c expression with Allergenicity Kit (Beckman Coulter), using Cytomics FC 500 flow cytometer (Beckman Coulter). Results: The sensitivity was 92,3% and specificity of test was 100%. 50.95 ± 15.7% of basophils (median 49.7%, 1.91–72.42%) were activated after grass pollen stimulation in atopic patients sensitised to this allergen, in comparison to 1.91% (0.00–7.96%) in control patients (p = 0.002). The percentage of activated basophils after D. pteronyssinus antigens stimulation was 40.6 ± 25.2% in allergic patients, compared to only 2.51 ± 1 96% of basophils from non-atopic controls (p = 0.0003). Basophils from 21 patients responded after anti-IgE stimulation (44.1 ± 18.9%), and none of the analysed samples was activated after PBS stimulation (2.03 ± 1.19%, p &lt; 0.0001). Conclusions: These results demonstrate that basophil activation test based on antigen CD203c expression is very accurate in the diagnosis of atopic diseases.

Is this time for CRTH2/DP2 in a flow cytometric basophil activation test?

Is this time for CRTH2/DP2 in a flow cytometric basophil activation test?

Flow cytometry for basophil activation markers: The measurement of CD203c up-regulation is as reliable as CD63 expression in the diagnosis of cat allergy

The flow cytometric basophil activation test (BAT), based on the detection of allergen-induced CD63 expression, has been proved effective in the diagnosis of various IgE-mediated allergies. However, there is not yet consensus about the suitability of CD203c expression as a specific basophil activation marker and its diagnostic reliability. The goal of the present study was to compare measurement of CD63 and CD203c expression using BAT in a model of cat allergy and to determine optimal experimental conditions for both markers. Heparinized whole blood samples from 20 cat allergic patients and 19 controls were incubated with Fel d1 (relevant allergen) or anti-FcεRI (positive control) either in IL-3 or IL-3-free conditions. An optimal gating of basophils was achieved in triple staining protocols: anti-IgE PE/anti-CD45 PerCP/anti-CD63 FITC or anti-IgE FITC/anti-CD45 PerCP/anti-CD203c PE. We demonstrated that IL-3 significantly enhanced CD63-induced expression by basophils obtained from cat allergic patients in response to Fel d1. Sensitivity was found to be 100%. The CD203c protocol, when performed under IL-3-free conditions, also demonstrated 100% sensitivity. Only one of the control subjects was positive in both tests. In conclusion, using well-defined experimental conditions, the measurement of CD203c up-regulation on basophils in response to specific allergens is as reliable as CD63-BAT for the in vitro diagnosis of patients with IgE-mediated allergy.

A New Combined Test with Flowcytometric Basophil Activation and Determination of Sulfidoleukotrienes Is Useful for in vitro Diagnosis of Hypersensitivity to Aspirin and other Nonsteroidal Anti-Inflammatory Drugs

Background: We assessed whether nonsteroidal anti-inflammatory drugs (NSAIDs) may provoke blood basophil activation in vitro in aspirin- and NSAID-hypersensitive patients, as detected by a flowcytometric technique using the CD63 marker – flowcytometric basophil activation test (FAST) assay – in addition to the sulfidoleukotriene (sLT) release – the cellular allergen stimulation test (CAST). Methods: Sixty aspirin- and/or NSAID-hypersensitive patients were studied. Thirty control patients without history and negative provocation challenge were also included. The percentage of activated basophils after in vitro stimulation with NSAIDs at 3 different concentrations was evaluated by an anti-CD63 phycoerythrin conjugate (FAST assay) and the amount of sLTs released in the cell supernatant by ELISA (CAST assay). Results: For aspirin, the FAST indicated a sensitivity of 41.7%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 99.4%; for paracetamol 11.7 and 100%, for metamizol 15 and 100%, for diclofenac 43.3 and 93.3%, and for naproxen 54.8 and 74.1%. Many patients showed positive tests to more than 1 NSAID. When considering the first 4 NSAIDs, the global sensitivity increased to 66.7%, while the specificity remained at 93.3%. The addition of the CAST results still increased the sensitivity up to 73.3%, but with a decrease of the specificity to 71.4%. Conclusions: The FAST shows a high percentage of positive reactions, which may reach 60–70% when 4 NSAIDs are tested and even 88% when the test is performed within 1 month of the last clinical drug exposure and reaction. The test has a high specificity above 90%. The addition of sLT determinations yields additional information in a few isolated cases. It is suggested that this test, when properly used, may help avoid some cumbersome and dangerous provocation challenges.

Anaphylaxis to viscotoxins of mistletoe ( Viscum album) extracts

Extracts of mistletoe (Viscum album) are used in many countries for adjuvant cancer therapy. These extracts contain mistletoe lectins and viscotoxins that are supposed to have immunostimulating and cytotoxic effects, respectively. The treatment is usually well tolerated. To report a case of severe anaphylaxis secondary to mistletoe extract administration with demonstrable anti-IgE antibodies to mistletoe. Skin prick tests, basophil histamine release, basophil activation test, and immunoblotting were performed to characterize the pathophysiology of this reaction. The patient had immediate-type skin prick test reactions to the whole commercial preparation and to its mistletoe extract component. A histamine release test and a flow cytometric basophil activation test performed with the patient's peripheral blood leukocytes by incubation with the mistletoe extract yielded a concentration-dependent histamine release and expression of the activation marker gp53 in up to 98% of anti-IgE-positive cells. Immunoblotting revealed IgE binding to 5-kDa proteins of mistletoe in the patient's serum, which corresponds to the molecular weight of viscotoxins. The results of all these tests were negative in controls. Until now, anaphylaxis to mistletoe extracts has been only rarely reported. In our patient, viscotoxin specific IgE evidently had induced an anaphylactic reaction.

IgE-mediated anaphylaxis to viscotoxins of mistletoe (Viscum album) extracts

Rationale Extracts of mistletoe (Viscum album) are used in many countries for adjuvant cancer therapy. The extracts contain mistletoe lectins and viscotoxins supposed to have immunostimulating and cytotoxic effects, respectively. The treatment usually is well tolerated. We here report on a 59-year-old female who had received for adjuvant treatment of colon cancer injections of a mistletoe extract (Iscador® M c. Hg, Weleda, Germany) every two weeks for more than two years without any side effects. Two months prior to presentation she suddenly had developed generalized urticaria, swelling of lips and tongue, nausea, vomiting, and shortness of breath within 10 minutes after the injection. Methods Allergologic tests were performed to characterize the pathomechanism of this reaction. Results In the patient there were immediate type skin prick test reactions to the whole commercial preparation and to its mistletoe extract component (kindly supplied by the manufacturer). A histamine release test and a flowcytometric basophil activation test performed with the patient's peripheral blood leucocytes by incubation with the mistletoe extract yielded a concentration-dependent histamine release up to 100% of total histamine and expression of the activation marker gp53 in 98% of anti-IgE positive cells. Immunoblotting revealed in the patient's serum IgE binding to 5-kD-proteins of mistletoe which corresponds to the molecular weight of viscotoxins. All these tests were negative in controls. Conclusions Anaphylaxis to mistletoe extracts has been rarely reported. In our patient evidently viscotoxin-specific IgE had induced an anaphylactic reaction.

Flow cytometric basophil activation test by detection of CD63 expression in patients with immediate‐type reactions to betalactam antibiotics

In this study, we used flow cytometry to determine the percentage of activated basophils that expressed the CD63 marker after in vitro stimulation by different betalactam antibiotics. The diagnostic reliability of the technique was assessed, as well as its correlation with specific IgE. Fifty-eight patients with clinical allergy to betalactam antibiotics and presenting positive skin tests to at least one of the allergens (minor determinant mixture (MDM), benzylpenicilloyl-polylysine (PPL), penicillin, ampicillin, amoxicillin, cephalosporins) were tested. Thirty subjects non-allergic to betalactams were also studied as controls. The flow assay stimulation test (FAST) uses flow cytometry to determine the percentage of basophils that express CD63 as an activation marker after in vitro stimulation with allergen. Double labelling with monoclonal antibodies anti-CD63-PE and anti-IgE FITC was used. The allergic patients show a statistically greater number of activated basophils than the control subjects, after the incubation of cells with all the betalactams at various concentrations. The sensitivity of the technique is 50%, the specificity 93.3%, the likelihood ratio for a positive value 7.46 and the likelihood ratio for a negative value 0.54. In spite of having a greater sensitivity (37.9%) and specificity (86.7%) than CAP, differences between sensitivity and specificities of both techniques (CAP and FAST) do not reach statistical significance. The basophil activation test is a particularly useful technique in the diagnosis of patients with IgE-mediated allergy to betalactams and allows the identification of 50% of patients. Used in conjunction with CAP, it allows the identification of 65.5% of such patients.

The use of a flowcytometric basophil activation test (FAST) for the in vitro diagnosis of latex allergy

The use of a flowcytometric basophil activation test (FAST) for the in vitro diagnosis of latex allergy