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.
