Basophil Activation Tests (BAT): Degranulation, Cytometry and Chemotaxis in Drug Allergy

Maria de Lourdes Irigoyen Coria1*, María Isabel Rojo Gutierrez2, David Solís Hernández3, Ruben Humberto Meyer Gómez4, Isabel Leyva Carmona5, Jaime Mellado Abrego6, Gloria Castillo Narváez7, Isabel Guerrero Vargas8 1Laboratorio Servicios, Alergia, Reumatología y Trasplante Hospital Especialidades IMSS Cd.de México, México (1982-2000), Laboratorios Clínicos Especializados Integrales Lindavista. Cd de México, México


Background
Von Pirquet in 1905 described "the serum sickness" in patients using treatment with (diphtheria and tetanus horse serum) and in 1906, introduces the term "allergy" as a special type of defensive or immunological response to foreign substances that normally would not induce a response. In 1930, sulfonamides were also associated with more frequent reactions, which later was denominated as "drug fever" [1][2][3][4]. Since 1940, penicillin has been the most used antibiotic and currently remains as the most frequent cause of allergies [5].
The pharmaceutical industry has created new penicillins keeping the β-lactam ring, but changing the side branch without losing any antibiotic capacity; which produce cross-reactivity in allergic reactions and anaphylaxis [6][7][8][9][10]. During the 20th century and the beginning of the 21st century, the increase in the synthesis of drugs (Nonsteroidal anti-inflammatory drug [NSAIDs], analgesics, anesthetics, antihypertensives, steroid contraceptives, chemotherapy, antiretrovirals, etc.) caused a growth in the use of these drugs and increased allergies; some authors consider drug allergy as the epidemic of the 21st century.

Classification of Adverse Drugs Reactions (ADRs)
Drugs may produce predictable ADRs (type A, 80%) associated with pharmacological activity, dose dependency, toxicity and slow metabolizers; they can also produce unpredictable adverse reactions (type B, 20%) [13][14][15] which are dose independent (low concentration) and associated with aggravated intolerance, pseudo-allergies and more severe and lethal allergies. Hypersensitivity is an exacerbated immune response that produces a clinical pattern with systemic and dermal disorders (skin as the most affected organ), anaphylaxis and sudden death [8]. Gell and Coombs created the classification of hypersensitivity which was modified in 1996 [2,7,13,16] to distinguish 4 types of hypersensitivity: I, II, III, IV; later on, Pichler [2,17] subdivided type IV into a, b, c and d [17]. The International Consensus on Drug Allergy (ICON, 2013) defines the drugs hypersensitivity reactions (DHRs) [17] and says that they are caused by activation of the immune adaptive system considering immune mechanisms and activation responses supporting the diagnosis, treatment, follow-up and prevention. This classification is based on the laps of time it takes for the signs and symptoms to occur after the drug administration: 1) immediate (0-1 hour), 2) accelerated (1-72 hours), 3) delayed (more than 72 hours) (Figures 1 and 2, Table 1) [17]. to any predictable noxious reaction that appears at therapeutic doses, depends on the doses and is related to pharmacological actions. Within this group are other [14] unpredictable reactions [18], independent of the dose recognized as: hypersensitivity or allergies (DHRs) associated with immunological mechanisms, susceptibility (atopy) and polymorphism (pharmacogenetic, MHC-HLA). Adapted from Gibaldi [19], Lares-Asseff & Trujillo-Jimenez [14] and Giner-Munoz [6,7]. Drugs (generally non-immunogenic haptens) that are chemical substances of low molecular weight (less than 1000 Da) in the form of aromatic, heterocyclic components, -p-NH2Cl, sulfonamides, sulfide, OH components, halogens, with high resonance and instability (β-lactam with low polarity and hydrophily which do not facilitate covalent bonds with autologous proteins). Drugs are eliminated through metabolism with bioactivated detoxifying enzymes (hepatic N-acetylation, oxidation of cytochrome P450-CYP); this occurs mainly in the liver (microsomal), and also in the kidneys, lungs, intestine, plasma and nervous tissue. There are variabilities or ethnic polymorphisms, especially in slow acetylators, in which drugs remain more time in circulation, haptens bind to the protein, become a Hapten-Protein carrier complex (H-P), are englobed and introduced into the Antigen Presenting Cells (APC) for processing and presentation on the membrane with the major Histocompatibility Complex APC-MHC complex to induce a response cellular or humoral with IgE, IgG and IgM [7,13,22].

Active-reactive
Drugs with aromatic, polar groups and nitrogen, facilitate the direct binding or nucleophilic attacks to membranes in order to create a covalent bond with autologous proteins and induce an APC-MHC immune response [7,13,22]

p-I concept (pharmacological interaction with immune receptors)
Drugs without that lack hapten characteristics can bind (non covalently) to TCR and sending signals to create a hypersensitivity response. This explains a fast occurrence of Clinical symptoms without previous sensitizations and sometimes chaotic immune reaction, some crossreactions to the drug or its metabolites [7,14,17,[22][23][24].

HLA restriction in hypersensitivity
HLA class I , mainly HLA-B, described for several sever reactions in DHRs; for example, it has been found that abacavir is strongly associated with the HLA-B * 5701 allele in white population; carbamazepine like an inductor of Steven Johnson Syndrome (SJS) has been associated with the HLA-B * 1502 and HLA-B * 5801 alleles in Chinese patients, while allopurinol has been associated with adverse reactions in SJS and necrolysis epidemical toxic (NET) with HLA-B 5701 (Figures 3 and  4 [2,17,20] and ICON [16]. Giner-Munoz [6,7], Rojas-Espinosa [2], Demoly et al. [16], Irigoyen-Coria et al. [13] and Mayorga et al. [21]. : Immunological response to drugs and polarization at TH1, TH2 or both. The immunological mechanism of the hypersensitivity response to drug, hapten or its metabolites can be processed and presented as antigens in the APC in the context MHC (I ó II)-TCR, to activate TH0 and polarize TH2 (Humoral immunity-hypersensitivity type I), TH1 (cellular immunity-hypersensitivity type IV). Adapted from Montes-Montes [3], Giner-Munoz [6] and Irigoyen-Coria et al. [13]. J Cell Immunol. 2020 Volume 2, Issue 3

Atopy
It refers to an individual's genetic predisposition or susceptibility to develop sensitivity towards an allergen; it requires previous contact (intermittent or continuous) and depends on factors such as environment, temperature, sex (steroidal hormones) and age. This exaggerated immune response is mediated primarily by IgE antibodies, this antibody binds to FCeR1, increasing the half-life of IgE (2 -60 days). We can find IgE synthesis from fetal stage to end of life. Higher concentrations of high and low affinity receptors have been reported in mast cells and basophils associated with severity, IL-4 inhibits the development of TH1 cells and activating TH2 cells, IL-3 favors the change of the isotype of B lymphocytes for the production of IgE, and the induction of the inflammatory process in DHR ( Figure 3) [2,7,[31][32][33][34][35].

Medical history
It is the cornerstone of the diagnosis and has a better predictive value. It is based on clinical criteria, anamnesis, diagnostic algorithm, records of clinical history (signs and symptoms), followed by provocation and skin tests, as well as laboratory tests suggested by physicians in some healthcare systems ( Figure 5) [7,16,36].

Natural history
IgE antibodies may persist for years. Memory T lymphocytes (TLm) are more intense during nonimmediate DHRs: consequently, it is recommended to avoid the use of drug for long time [16,23].

In vivo tests (provocation tests)
They are considered the gold standard to confirm or rule out a drug as the main inductor of hypersensitivity reactions.
Skin tests is the most frequently used, for better results is used with different dilutions and with controls negative and positive (histamine). Medical monitoring is necessary to avoid risks of anaphylaxis [7,16,18,31,37].  J Cell Immunol. 2020 Volume 2, Issue 3
• Count the number of cells can degranulate in exposition to the antigen (drug) ( Figure 6).

Secretion methods:
In the last five decades of the twentieth century, it was demonstrated that activated leukocytes released histamine and other vasoactive amine molecules as a response to in vivo and in vitro allergens; the fast or slow release by cytokines was also demonstrated, as well as the disappearance of basophils, all evaluated through electronic microscopy [18,31,38].
There are 2 types of participating cells: mast cells and basophils; the latter are more accessible for functional studies, since they are found circulating in peripheral blood (mononuclear cells of 7-9 μm that constitute 0.5%-1% of the leukocyte total) [7,13,18].
• Histamine: It is a premade mediator immediately released as a response to allergens or drugs. It is among the firstly used elements for in vitro and in vivo tests (erythema evaluation), for fluorometry, spectrophotometry and ELISA. Nevertheless, not all donors or patients release the total amount of histamine, and it may vary between 80% and 95%; therefore, basophil population needs to be spiked. There is also spontaneous release without stimuli that might be associated with higher sensitivity, reactivity and lack of specificity [10,13,18].

Modified basophil degranulation (MBD):
It refers to the disappearance of basophils (count of cells) after activating and releasing their granulations; therefore, losing their morphology. It is determined by

Dose-response time curves of basophils in murines and humans with different allergens
The most common stimulus for basophil studies depends on the activation through antigen-specific IgE (allergen or drug) of the cell surface connected to FcεRI (high affinity) which consists of an α chain and β and γ chains associated with the α chain as αγ2 and αβγ2. The role of β (chromosome 11q and 5q) is to increase the expression of the receptor by promoting the maturation and intracellular transit of the α chain, as well as the survival of IgE. The FcεRI-mediated signaling requires the binding of this receptor to the antigen (simple bivalent dimers inducing signaling); these binding depends on the concentration and affinity, which increases up to an optimal level and later returning to the basal state (bell curve as an ideal form); however, in human and murine models, more complex antigens (drugs) with higher affinity presented non-ideal dose-response curves (several forms). The basophil-IgE-epitope-antigen (drug) reaction can be considered as an antigen-antibody precipitation reaction; this results in the reduction of the receptor's mobility and RcεI immobilization. The β and γ chains contain ITAM sequences that initiate the activation signaling of MAP kinase. The other receptor for IgE is low affinity Fcε II (CD63), which controls the growth and differentiation of B cells, increases the synthesis of IgE and also blocks the binding between IgE and eosinophils (Figures 3, 6-9  There are 4 metrics in common (1) 50% maximum response CD sens (2) maximum Histamine release response concentration (bivalent hapten that reflects affinity in the Ag-Ab binding, (3) maximum amount of secretion (4) Area under the dose response complete curve is not ideal, it is more difficult to obtain and it is due to the complexity of the allergens, source of preparation and the individual distribution of the specific antibodies of the epitope, affinity for the IgE. Taken from MacGlashan Jr. [18].

Figure 8:
Curve dose response of allergens-drugs in the human (allergic) basophils. We observed a typical curve at 30 min (A. ideal in bell) and B. not ideal curve in 60 min that implies complexity, different affinities according to the IgE of allergic individuals with atopy, difference in age and sex. Therefore, it is suggested to perform the MDB test at 2 different concentrations ranging from 0.1 to 1 mg/mL. Own elaboration and only mentioned in Irigoyen-Coria et al. [13].
In essence, the binding of the paratope (2 IgE molecules) to the epitope induces intermembrane chain crosslinking and signaling transduction through kinase proteins (PKC), which provokes degranulation and release of histamine, heparin, proteoglycans and vasoactive amines that amplify the allergic reactions (Figures 9,10 and Table  3) [11].   J Cell Immunol. 2020 Volume 2, Issue 3

Methods of activation markers expression
Since the last decade of the twentieth century and especially at the beginning of the twenty-first, a wide variety of techniques have been developed at research and commercial kits levels to identify the expression of basophil activation markers that use flow cytometry and monoclonal antibodies. The main clinical experience is with CD63, CD203c and CD69. Pharmacology bases BAT specificity on sign transductions through PKC, which stimulates the expression of gp53 receptor (CD63) (transmembrane lysosomal protein tetraspanin LAMP-31) on the basophil surface, as CD63 may or may not require IL-3 and its specificity depends more on the epitope (drug)-paratope-IgE complementarity, affinity, avidity, atopy. The other three reported mechanisms are: a) proteins constituting the membrane and that express themselves with fast release of CD11c vesicles, b) recently synthetized proteins which express in higher time frames of hours and require transcription, translation and transportation to the plasma membrane, and c) expression on the basophil surface through the C5a receptor, releasing histamine when activated by a positive formyl-methyl-leucyl-phenylalanine (fMLP) which can be used as positive control for the determination of CD63. Other markers include CD69, CD203c, CD13, CD11b, etc. ( Figure 6 and Table 4) [7,9,13,18,38,46,47].  J Cell Immunol. 2020 Volume 2, Issue 3 J Cell Immunol. 2020 Volume 2, Issue 3 CD63-Flow cytometry: The determination is possible by staining of monoclonal antibodies marked with fluorochromes CD63-FITC, CD123-PE and PerCP HLA-DR with reading by flow cytometry, a negative control of PBS/Ca-albumin, pH 7.2 and a positive control of fMLP (joined to the C5a receptor). Reliability is determined by specificity and sensitivity (≥ 85%-99.4%); it is reported as CD63 %, as well as the stimulation index (SI); the RV vary depending on the author with CD63 % = 0-5, 0-30, SI or activation >2%-4%. Factors can also vary depending on the ethnicity, C5a receptors, IgE-drug affinity, IgE concentration, LAMP-31 distribution and titration of monoclonal antibodies marked with two-color fluorochrome: green (fluorescein isothiocyanate-FITC) and red (phycoerythrin-PE) (Tables 4,5, and Figure 11) [18,44].  Table 5: Results of BAT-CD63. Report of allergic patient to 2 drugs (our group patient), the most sensitive was the anesthetic lidocaine at lower concentration and greater, parameters: %CD63 (Expression of activation markers), MFI (Medium Immunoflourescence), %SI (Stimulation Index), %IA (Activation Index) the negative control (PBS+Ca+Albumine pH 7.2) was 0 and the positive control (fMLP) was 68.4 percent correspond to 100% of SI. Own elaboration (The Histogram Cytometry corresponds to the Figure 11).

A)
Modified Leukocyte Migration Inhibition Factor (MLIF) Type IV a, b and c, associated with anaphylactic degranulation: It has been reported that leukocytes, including basophils (BAT-Chemotaxis) also play a directional chemotaxis role; therefore, when microhematocrits are incubated in Bloom chambers with drugs in two dilutions (1 mg/mL and 0.1 mg/mL) in an RPMI medium, with negative and positive controls, at 37°C, the early (20 min to 2 hrs) and delayed (4, 6 and 18 hrs) migration can be measured; the % of MLIF can also be calculated vs the negative control, as well as the reference values (RV) for MLIF (0%-25% of leukocytes migration inhibition) ( Figure 12) [13,37,48,49].  [48][49][50] and Lymphocyte transformation test (LTT) : It is done with lymphocyte incubation with drugs and DNA concentration with dyes to avoid radioactivity [2,13].

Selection of BAT Diagnostic Methodology
The decision is made based on simple, inexpensive and analysis (manual and automated complexity, equipment, cost and time) [9,18,31] Advantages and disadvantages of BAT [18,31] Advantages: • In vitro methods reduce the risk in hypersensitive patients or patients with severe pharmacodermy, which may lead to anaphylactic events or sudden death.
• Basophils and mast cells have a similar response to crosslinkings for FcεRI and FcεRII activation when increasing the half-life and FcεRI receptors.
• CD63 has a positive regulation in basophils and mast cells, which corresponds to an anaphylactic response.
• The activation of basophils is a fast functional test as a response to an allergen or drug.
• It is possible to separate the intrinsic factors (chemical structure of the drug) from the extrinsic factors (additives, stain, etc.).
• The study may suggest therapeutic alternatives to avoid cross-reactions.
• They have a high predictive value for drugs, allergens (food and inhaled), anesthetics and occupational toxic elements.
• They can determine the degree of sensitivity, reactivity and reading of a thousand events in the flow cytometer in allergic patients.
• There is a follow-up and post-treatment monitoring.

Disadvantages:
• It requires an implementation and standardization process for reliability.
• The concentration of basophils must be increased by separation methods with gradients of Percoll, sedimentation with dextran and only centrifugation.
• There are specific conditions, such as type of anticoagulant, preservation, transportation (5-8°C) in horizontal position, neutral pH of the solvent (pH of 3.7 allows the IgE to dissociate from the FcεRI in 10 to 30 seconds), drug stability.
• Reaction kinetics must be performed (concentration and response time), which include viability and functionality response time.
• The requirement of: sampling, anticoagulant, viability (preservation and transport) to perform the BAT should not be greater than 48 hours.
• There are non-responding or secretagogue individuals.
• There is a need for standardization, precision, coefficient of variation (CV) and reference values (RV) obtained in normal population (without infection), as well as for age groups and preventive culture.
• The skin provocation test and BAT may not correspond to the diagnosis (some cases of chronic spontaneous urticaria and neurodermatitis).

Discussion
Currently the synthesis and consummation of drugs has increased; in a collateral manner also an increase prevalence DHRs (urticaria, anaphylaxis, SJS, TEN, AGEP) [4,7,13,51], as well as to autoimmunity and malignancy processes [25] since postnatal stage to the old age (7% in elderly adults, 18% in children with an interval of 15%-24%, 5%-15% in hospitalized patients); more frequent in women between 40 and 60 years age. Trigger drugs include, in first place: antibiotics like penicillin, beta-lactams and cephalosporins (50%), followed by NSAIDs and analgesics (40%), and finally anesthetics, antihypertensive, hormones, antiretrovirals, among J Cell Immunol. 2020 Volume 2, Issue 3 others (10%). Therefore, it is necessary to select a reliable methodology (BAT and complementary alternatives test with dilution, controls negative and positive) for better improvement in the diagnosis and public health [7,12,13,18,16,52,53]. The selection of studies according to the type of DHRs, associated with the activation of the adaptative immune system; facilitated by Gell and Coombs classification using a diagnostic algorithm [16].
Due to all of the above and since discovery in 1906 of "Allergy "and their association with mast cells and basophils: during several decades of the 20 th century and the beginning of the 21 st century, have been an important focus of study and research models of human species, murine and other; for the development and implementation of in vivo and in vitro tests. Based on mechanisms of adaptive immune response that provoke the release of histamine, heparin, vasoactive amines and activation markers (CD63, CD203c), Mac Glashan Jr. [18] has focused on the BAT studies in murine models; these models represent a good strategy to acquire knowledge about the functions of the effects, mechanisms of molecular and immunological activation, the development of techniques that they can be applied in humans. Several researchers, including Hoffman et al. and others [7,13,18,31,38,46,47] propose that, at present time, the most developed and widely used tests at clinical and research level are the IgE-dependent BAT; there are two principal types: 1) secretion of granules (degranulation), histamine, heparin, vasoactive amines, and 2) activation markers expression (CD63, CD69, CD203c, etc.) [18]; when comparing the essays in murine and human models, similar responses to the stimulus with allergens and drugs, two more were found during the development and standardization of BAT methods for anaphylactic degranulation (3.-MDB-Microscopy and 4.-MLIF-chemotaxis) in our laboratory (Figures 6 -10) [18].
Mac Glashan Jr reflects: What do we know? 1) If flow cytometry methods and Kits provide us with a reasonable alternative to traditional tests, especially CD63 (is more closely associated with degranulation) [18] over CD203c. We think there may be a problem with target because the basophil has a c5a receptor which can be activated via the complement (infectious or autoimmune inflammation); consequently, It loses some specificity? Although our article does not consider it. 2) Is intrinsic sensitivity of basophils a useful measure to know the sensitivity, we probably think that it is in accordance with the sensitivity and reactivity of the basophils since some patients show greater degranulation, a higher %CD63 and a %SI (responders, atopy) of the allergic patient, in addition to the reactivity and severity increase; 3) How is BAT diagnostic methodology selected? This decision is based on the clinical study requested by the physician (DHRs situation), these tests are simple, inexpensive and give great support in the diagnosis of drug reactions with coverage of several types of hypersensitivity, time, as well as basophil viability and report urgency) [13,18].
Giner-Munoz is especially concerned about children and other age group; because the first worldwide cause of allergy is infections and this also with antibiotics (50%), mainly penicillin and β-lactams, prescribed by physicians due to their wide spectrum and low toxicity, therefore it is considered that BAT and alternative tests are important to avoid cross reactions and give future therapeutic options [6,7,54].
At present there are several groups of patients that require special attention in order to promote the implementation and development of BAT and other alternatives, like chemotaxis (MLIF-Type IV) [13] that may help to reduce the prevalence, risk factors, severity, lethality, and assist in solving a public health issue: 1) The prevention of allergies to penicillin and β-lactams: improve during the diagnostic algorithm, the epidemiology, history of previous anaphylactic allergic reactions, atopic family, as well as microbiological cultures with antibiograms as a preventive measure before prescribing antibiotics in attention primary services and hospitals (public and private); in this manner, 65% of allergic patients would be identified and avoid the use of lidocaine as a solvent in lyophilized antibiotic [13,55]; 2) Patients with surgeries, accidents, autoimmune diseases or traumatological events require concomitant prescription of antibiotics and NSAIDs, which may cause DHRs in many cases; for example, diclofenac (TH1 and TH2) (39%) [13,56], and anesthetics [57] causing perioperative and operative anaphylaxis (60%) [13] hypnotic drugs (2 -10%); 3) Patients with HIV [58] make frequent use of antiretroviral treatment in the form of abacavir dose (nucleoside analogue that inhibits retrotranscription) and sulfonamide antibiotics which, in severe DHRs, activate TH1 and TH2 (80%), especially in slow acetylators [53,57,[59][60][61][62]; 4) For symptoms associated with SJS (such as erythema, general discomfort, fever, nausea and vomiting), 78% of the patients had the HLA-B*57:01 allele [25]; hypersensitivity to sulfonamides and carbamazepine was also found [31,63,64]; 5) Neurological patients with epilepsy and bipolar disorder treated with carbamazepine developed SJS and had the HLA-B*15:02 allele [17,55,65]. 6) Patients with gout, persistent renal lithiasis, even leishmaniasis, as well as patients with chemotherapy and control therapy for high uric acid levels, treated with alopurinol, had the HLA-B*58:01 allele [17,24,54,63,64,66].