Name: basophil activation test for allergy diagnosis
Uploaded: 2021-05-31T14:00:00
Description: Watch this Scientific Journal Video about Basophil Activation Test for Allergy Diagnosis at JoVE.com

We thank Claudia Corazza for her invaluable English language support. This work was supported by Institute of Health &#39;&#39;Carlos III&#39;&#39; (ISCIII) of MINECO (grant cofunded by ERDF: &#34;Una manera de hacer Europa&#34;; Grants Nos. PI20/01715; PI18/00095; PI17/01410; PI17/01318; PI17/01237 and RETIC ARADYAL RD16/0006/0001; Andalusian Regional Ministry of Health (Grant Nos. PI-0127-2020, PIO-0176-2018; PE-0172-2018; PE-0039-2018; PC-0098-2017; PI-0075-2017; PI-0241-2016). ID is a Clinical Investigator (B-0001-2017) and AA holds a Senior Postdoctoral Contract (RH-0099-2020), both supported by Andalusian Regional Ministry of Health (cofunded by ESF: &#34;Andaluc&#237;a se mueve con Europa&#34;).

The protocol performance was conducted according to the Declaration of Helsinki principles and approved by the local Ethics Committee (Comit&#233; de &#201;tica para la Investigaci&#243;n Provincial de M&#225;laga, Spain). All subjects were informed orally about the research study and they signed the corresponding informed consent form.
NOTE: The present protocol details the BAT procedure that the authors use daily. However, this is not a standardized method and differences with proce...

BAT is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions that has shown to be useful in the diagnosis of reactions induced by different triggers such as drugs, food, or inhalants, as well as in some forms of chronic urticaria. In general, BAT performance should be considered if i) the allergen/drug produces false positive results in ST; ii) the allergen/drug is not available to be used for ST or sIgE quantification; iii) discordance between clinical history and ST or ...

IgE-mediated allergy diagnosis is based on clinical history, skin tests (STs), quantification of serum specific IgE (sIgE), and, if it is required and indicated, controlled challenge tests (CCTs)1,2,3,4,5,6. However, clinical history can be unreliable since there may be a lack of accurate information, and STs and CCTs are not risk-free procedures that can be contraindicated in subjects experiencing severe life-threatening reactions1,2,3,4,5,6. These issues, together with the fact that the determination of sIgE by validated and commercial fluoro-enzyme immunoassays is only available for few allergens and drugs, have highlighted the important role of other in vitro functional assays such as basophil activation test (BAT).
Basophils are key effector cells involved in IgE-mediated allergic reactions that are activated upon cross-linking of adjacent sIgE bound on high-affinity receptors (Fc&#949;RI) on cell surface after allergen/drug exposure. Basophil activation triggers cell degranulation and release of pre-formed and new synthesized inflammatory mediators contained in intracytoplasmatic secretion granules7,8,9. BAT is an in vitro method that tries to mimic the basophil activation in the presence of a stimulus (allergen or drug) and determines changes in the expression of basophil activation markers by flow cytometry7,10. There are different strategies to identify basophils (IgE+, CCR3+, CRTH2+, CD203c+) and to measure cell activation (mainly upregulation of CD63 and CD203c) using combinations of fluorochrome-labelled antibodies7,10. CD63, the best clinically validated activation marker11,12,13,14, is a membrane protein anchored at the secretory granules containing histamine that, after cell activation and fusion of the granules with the membrane, is expressed on basophil surface15,16,17,18,19,20,21. CD203c is a surface marker that is constitutively expressed on basophils and upregulated after Fc&#949;RI stimulation, which has also shown reliable results in BAT15,22,23,24,25. Besides, it appears to co-express with CD6326.
In the last decades, BAT has shown to be useful in the diagnosis of IgE-mediated allergic reactions induced by different triggers like drugs, food, or inhalants, as well as in some forms of chronic urticaria, as described below. However, the position of this technique in the diagnostic algorithms is highly variable and not well determined.
Drug hypersensitivity 
BAT has shown to be useful as a complementary test for selected drugs and patients, particularly for those who experience severe reactions due to the fact that diagnostic value of ST is not well established for most drugs, as they are validated and standardized for a limited number of drugs27,28,29,30. In addition, quantification of sIgE is available only for a limited number of drugs, with lower sensitivity than ST27,28,29,30,31,32. Therefore, the diagnosis of drug hypersensitivity usually relies upon drug provocation test, which can be contraindicated in subjects experiencing severe life-threatening reactions33.
Promising results have been reported for the use of BAT in selected patients reporting immediate hypersensitivity reactions to different drugs like betalactams (BLs)20,34,35,36,37,38,39, neuromuscular blocking agents (NMBAs)19,22,40,41,42,43,44,45, fluoroquinolones (FQs)46,47,48,49, pyrazolones50,51,52, radiocontrast media (RCM)53,54,55,56, and platinum compounds57,58,59. BAT has been reported to have a sensitivity and specificity between 51.7-66.9% and 89.2-97.8%, respectively; and positive and negative predictive values are described to range between 93.4% and 66.3%, respectively27,31. Moreover, BAT has been proposed as a predictive biomarker for breakthrough reactions during desensitization with platinum compounds, as CD203c expression is increased compared to CD63 in patients with high risk of adverse reactions during drug desensitization57.
It is of note that BAT is only useful in drug hypersensitivity when the reaction involves basophil degranulation; therefore, it is not useful in reactions resulting from enzymatic inhibition of cyclooxygenase 142.
Food allergy 
BAT has emerged as a potential diagnostic tool for food allergy because determination of serum sIgE to the whole allergen extract or single allergens is often ambiguous, requiring oral food challenge to confirm the diagnosis, which, similarly to drug hypersensitivity, is a costly and not risk-free procedure60. Several studies have shown relevant results with cow&#39;s milk61,62, egg61,63, wheat64,65,66,67,68, peanut63,69,70,71,72, hazelnut73,74,75,76,77, shellfish78, peach79,80,81, apple21, celery, and carrot82,83.
The main added value of BAT in the diagnosis of food allergy compared to STs and sIgE in serum is that it shows a higher specificity and similar sensitivity. Thus, BAT is a useful tool to differentiate clinically allergic patients from sensitized, but tolerant, subjects that have both high specificity (75-100%) and sensitivity (77-98%)63,69,84. Sensitivity and specificity values depend on the allergen and other factors as phenotypes (e.g., oral allergy syndrome versus anaphylaxis), age, and geography-related sensitization patterns63,85.
BAT using single allergen components can potentially improve diagnostic accuracy for some food allergens61,80. There are studies using seed storage proteins (e.g., Ara h 1, Ara h 2, Ara h 3 and Ara h 6 from peanut)86; lipid transfer proteins (e.g., Pru p 3 from peach and Ara h 9 from peanut)80,86; and Bet v 1 homologues (e.g., Ara h 8 from peanut)87. Other potential utilities are related to the identification of the culprit allergen in cases of pollen-food allergy syndrome21,87,88, allergy to red meat89, or food-dependent exercise-induced anaphylaxis66.
Interestingly, BAT can provide information about the severity and the threshold of allergic reactions since patients with more severe reactions show a greater proportion of activated basophils, as observed in studies of peanut and cow&#39;s milk-allergic patients84,90,91; and patients reacting to trace amounts of the allergen show a greater basophil sensitivity84,90,92. These data suggest that BAT may be useful to identify high-risk allergic patients who require closer follow-up and more intensified education93. Moreover, it has been reported that BAT can predict food challenge responses70,91,92,94 and thresholds of reactivity90,95 to help determine when food can be safely (re)introduced84. However, these findings are controversial in some studies63,96 and more research are required.
On the other hand, BAT has been used to monitor resolution of food allergy, either naturally or under immunomodulatory treatments, over time, which until now has been only assessed by oral food challenge, with the associated risks and costs84,97,98,99,100,101,102,103,104,105,106,107,108. Moreover, it has also been used to monitor the effect of omalizumab in food allergy as basophil activation decreases during treatment with omalizumab, but it increases after cessation of treatment109.
Inhalant allergy 
BAT is rarely beneficial in inhalant allergy as diagnosis can be routinely established by sIgE quantification and ST. However, in cases of local allergic rhinitis (undetectable levels of sIgE and negative STs with positive nasal provocation tests), BAT has allowed diagnosis in 50% of cases110. It has been further reported a correlation between basophil sensitivity and response to nasal/bronchial provocation tests, as well as between asthma severity and efficacy of treatment with omalizumab111,112.
BAT has also been used to monitor allergen immunotherapy for house dust mite and pollens, as basophil sensitivity decreases during immunotherapy, likely due to interference of blocking IgG antibodies113,114,115,116,117.
Hymenopteravenom allergy 
Diagnosis of hymenoptera venom allergy is routinely based on ST and serum sIgE. BAT has shown a high sensitivity (85-100%) and specificity (83-100%) and it has been reported to be useful in cases that yield equivocal results or in patients with a suggestive clinical history of venom allergy but undetectable sIgE and negative ST118,119. However, BAT appears not to be predictive of severity for these reactions120,121.
Up to 60% of patients exhibit sIgE to both wasp and bee venom, and the identification of dominant allergen is crucial for adequate immunotherapy treatment. In these cases, BAT has been reported to be useful in the identification of the dominant allergen119,122,123,124. Although sIgE to the major allergens of bee and wasp venoms may reduce the utility of BAT in patients with double positivity to both venoms, it provides useful information mainly in subjects with negative results in sIgE determinations123.
Some studies suggest that BAT may be useful as a predictive biomarker for side effects during the build-up phase of venom immunotherapy as this treatment option has been reported to decrease basophil sensitivity. However, reactivity does not decrease and this BAT utility is nowadays controversial13,120,125,126,127,128,129,130.
Urticaria and angioedema 
A subset of chronic urticaria patients have an autoinmune pathophysiology, due to IgE autoantibodies to autoallergens and IgG autoantibodies that target Fc&#949;RI or IgE-Fc&#949;RI complexes present on the mast cell surface131,132. In clinical practice, diagnosis of this type of chronic urticaria has relied on positive autologous serum ST, which has risk of accidental infection. BAT has been proposed as an in vitro test for diagnosing and monitoring patients with suspected chronic urticaria. Both CD63 and CD203c expression on the surface of basophils has been reported to be increased following stimulation with sera from chronic urticaria patients, showing the detection of active autoantibodies133,134,135,136,137. Recently, it has been reported that patients with positive BAT often experience the most active disease state, assessed by urticaria activity score, and require higher doses of antihistamines together with third-line treatments (cyclosporine A or omalizumab), compared to those with negative BAT138.

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			<td>5 mL Round Bottom Polystyrene Test Tube, without Cap, Nonsterile</td>
			<td>Corning</td>
			<td>352008</td>
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			<td>APC anti-human CD193 (CCR3) Antibody</td>
			<td>BioLegend</td>
			<td>310708</td>
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			<td>BD FACSCalibur Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
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			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
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			<td>FITC anti-human CD63 Antibody</td>
			<td>BioLegend</td>
			<td>353006</td>
			<td></td>
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			<td>HEPES (1 M)</td>
			<td>Thermo-Fisher</td>
			<td>15630106</td>
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			<td>Lysing Solution 10x concentrated</td>
			<td>BD Biosciences</td>
			<td>349202</td>
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			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
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			<td>N-Formyl-Met-Leu-Phe</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td></td>
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			<td>PE anti-human CD203c (E-NPP3) Antibody</td>
			<td>BioLegend</td>
			<td>324606</td>
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			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
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			<td>Purified Mouse Anti-Human IgE</td>
			<td>BD Biosciences</td>
			<td>555857</td>
			<td></td>
		</tr>
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			<td>Recombinant Human IL-3</td>
			<td>R&#38;D Systems</td>
			<td>203-IL</td>
			<td></td>
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			<td>Sheath Fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td></td>
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			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
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			<td>TUBE 9 mL LH Lithium Heparin</td>
			<td>Greiner Bio-One</td>
			<td>455084</td>
			<td></td>
		</tr>
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			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
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basophil activation test for allergy diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

BAT performed with allergens or drugs allows investigation of IgE-dependent hypersensitivity reactions. Basophil reactivity should be measured in at least two optimal concentrations in order to obtain the best results34 and activation is visualized by the upregulation of CD63 on the cell surface. In the case of allergens, moreover, to confirm the basophil reactivity, the basophil sensitivity should be analyzed by measuring the reactivity at multiple decreasing allergen concentrations<sup class="xr...

Watch this Scientific Journal Video about Basophil Activation Test for Allergy Diagnosis at JoVE.com

Basophil Activation Test for Allergy Diagnosis

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

The basophil activation test is a complimentary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions that have shown to be useful for reactions induced by drugs, food, or inhalants, as well as in some forms of chronic urticaria. This test is based on determining basophil response to an allergen or drug cross-linking IgE activation through the measurement of activation markers by flow cytometry. The basophil activation test can be a useful and complimentary tool to avoid controlled challenge tests to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions.Moreover, this test has the advantage of being able to analyze the response against any drug or allergen compared to other in vitro methods that are only available for a few drugs and allergens. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy. The need to perform the test no longer than 24 hours after sample extraction and the lack of standardization between laboratories and diagnostic algorithms.Begin by collecting peripheral blood in nine milliliter heparinized tubes, and place the samples in a rotor to maintain them at room temperature until they are required for the experimental protocol. Label two five milliliter cytometer tubes each for negative control and positive control. Also label one tube each for the different allergen or drug concentrations being tested.Then place the tubes in a rack so that the tubes fit perfectly without slipping. For the first positive control, prepare a four micromolar solution of N-Formylmethionyl-leucyl-phenylalanine or fMLP in 0.05%PBS-T To confirm the quality of basophils. For the second positive control, prepare a 0.05 milligrams per milliliter anti-IgE solution using PBS-T.Prepare the staining mix by adding one microliter each of CCR3-APC, CD203c-PE, and CD63-FITC antibodies per 20 microliters of the stimulation buffer. Then add 23 microliters of this staining mix to each tube. Add 100 microliters of PBS-T to the negative control tubes.Add 100 microliters of fMLP to one of the positive control tubes and add 100 microliters of anti-IgE E to the other to the rest of the tubes. Add 100 microliters of the different allergen or drug concentrations and incubate at 37 degrees Celsius for 10 minutes. After incubation, add 100 microliters of blood to each tube gently to avoid hemolysis.Then gently vortex the tubes and incubate them for 25 minutes at 37 degrees Celsius in the thermostatic bath with medium agitation. Keep the tubes at four degrees Celsius for at least five minutes to stop the degranulation. To each tube, add two milliliters of lysing buffer to lyse the erythrocytes.Vortex each tube and then incubate the tubes for five minutes at room temperature. Centrifuge the tubes at 300 g at four degrees Celsius for five minutes. Then overturn the rack into the sink to decant the supernatant while the cells remain at the bottom of the tubes.Add three milliliters of PBS-T to each tube to wash the cells and vortex the tubes. Perform a second round of centrifugation using the same set of parameters, and decant the supernatant by overturning the rack into the sink. Then keep the samples at four degrees Celsius protected from light until flow cytometry.Connect the flow cytometer to the computer software and wait until the cytometer is ready. Load the template and instrument settings as described in the text manuscript. Start the process of sample acquisition.To select activated basophils, first, gate the lymphocytes from the Side Scatter-Forward Scatter plot. Then gate the basophils from the lymphocyte population as CCR3+CD203c+cells and acquire at least 500 basophils per tube. Set the CD63-threshold to approximately 2.5%using the negative control tubes and analyze the samples.IgE-dependent hypersensitivity reactions were investigated by performing a basophil activation test, or BAT, using allergens and drugs. Basophil sensitivity was analyzed by measuring the reactivity at multiple decreasing allergen concentrations that help determine the allergic concentration that induces the response of 50%basophils or EC50. First, lymphocytes were gated from the side scatter-forward scatter plot.Then basophils were gated from the lymphocyte population as CCR3+CD203c+cells. Then a CCR3-CD63 plot was used to analyze basophil activation using CD63 as an activation marker for two drugs and different concentrations of an allergen. The test is performed with fresh whole blood and must be performed no longer than 24 hours after blood extraction.The stimulus used in the test should not include any excipients and the chemical characteristics of the drugs need to be considered. Other additional in vitro methods for the diagnosis of IgE-mediated allergic reactions, or the determination of sIgE, or the histamine release test.

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