Basophil activation test in the diagnosis of allergic reactions to local anesthetics

Introduction

During the last two decades, the appearance of a large number of low-toxic local anesthetics (LAs) has led to a significant expansion of their use in various fields of clinical dentistry in order to relieve pain in patients suffering from a variety of therapeutic and surgical manipulations. Although the occurrence of allergic manifestations with local anesthesia is not so common, the mass use of LAs in dentistry has led to an increase in the absolute number of patients who have clinical manifestations of allergic reactions while using certain Las [1–3].

It is obvious that while using LAs, in which allergic reactions have not been registered before, there is an increase in cases of allergy intolerance. The percentage of people with polyallergic reactions to LAs, that is, increased sensitivity of the patient's body to three or more LAs of different groups, increases. These data are fully consistent with numerous studies indicating that in the modern world, an allergy “epidemic” is developing in highly developed countries. Symptoms of allergic reactions to LAs belong to the group of “unpredictable complications” [1][3].

Most reactions to LAs are psychogenic (vasovagal) [2–4]; they often refer to as pseudoallergic, that is, associated with hyperreactivity of Toll-like receptors on the surface of basophils and mast cells, with which various medications are directly connected.

According to the academic literature, the true IgE-mediated reaction to LAs is extremely rare and amounts to 0.1–1% [5]. However, two types of hypersensitivity reactions to LAs of both groups have been described (according to the classification of Jell and Coombs): IgE-mediated (type I) – urticaria, anaphylaxis and type IV – allergic contact dermatitis, delayed edema at the site of administration of the medicine [2][4].

Thus, the majority of clinical manifestations of allergy to LAs are accompanied by mixed allergic reactions of different types; therefore, one or another nosological form cannot be attributed to only one type of allergic reaction. Some authors attribute allergy to LAs to pseudoallergic reactions, others to allergic reactions, but at the same time point to various mechanisms of allergy to LAs (T-cell, IgE-dependent), and antibodies can be both free and associated with leukocytes. The exact mechanisms of non-immune reactions to LAs are still difficult to be understood. It is generally believed that they are the result of indirect pharmacological stimulation of mast cells and basophils, causing the release of inflammatory mediators. In addition, non-immune activation of the complement system by an alternative pathway is possible. Pseudoallergic reactions may have other mechanisms [4][5].

The diagnosis of allergy to local anesthesia medicines is complicated by the fact that in some cases, it is not possible to determine the nature of the reaction (allergic, pseudoallergic, toxic) only on the anamnesis basis. Skin tests are contraindicated in patients with a history of life-threatening anaphylaxis, and they can also often give false positive results due to the direct irritating effect of medicines. In some cases, specific IgE antibodies to certain LAs are determined as well. However, these tests are informative only for allergic reactions of the immediate type, IgE-mediated one. The presence of specific Ig belonging to other classes weakly correlates with clinical manifestations and is not considered a reliable sign of allergy [2][5][6].

Currently, a promising laboratory method for the diagnosis of hypersensitivity to medicines is the basophil activation test (BAT). BAT is a provocative test carried out in vitro using a specific allergen that activates basophils that carry antibodies to it on the surface. This test, conducted by flow cytometry, allows evaluating a large number of medicines at a time without any risk to the patients’ life and health; this test also allows evaluating IgE and non-IgE-dependent reactions, it is complementary to the results of skin tests and has high sensitivity and specificity [6–8].

The use of the BAT in order to detect drug allergies in the practice of the long-term work of the Federal State Budgetary Institution of the Russian Ministry of Emergency Situations named after A.M. Nikiforov (Ph.D., Senior Scientific Researcher N.V. Bychkov) showed the high diagnostic significance of this test in determining sensitization to radiopaque substances and LAs [6].

The aim of this particular study was to evaluate the detection of hypersensitivity in vitro to local anesthesia medicines by flow cytometry.

Materials and methods

Therefore, 106 patients (154 medicines) aged from 1 to 79 years (median age – 36.8 years) were examined retrospectively. All the examined patients were assessed for the activation of basophil granulocytes in vitro (BAT) for LAs of groups I, II (dilution 1:25) and T-lymphocytes of type 2 immune response (CD3+CD294+ cells) by flow cytofluorometry using the Allergenicity kit test system. The examined patients had a history of certain food and/or medicine intolerance.

The criterion for inclusion in the study was the informed consent of the patient to participate it.

The formulation of the BAT (Allergenicity kit) method included incubation of basophils with specific (allergen) and nonspecific (positive control) substances, the addition of CD294-FITC/CD203c-PE/ CD3-PC7 monoclonal antibodies in order to identify basophils and evaluate their activation process, and lysis of erythrocytes with subsequent washing and accounting on a flow cytometer (at least 500 basophils) in a multiparametric protocol with multi-stage gating. The calculation unit was the stimulation (activation) index – the ratio of the number of activated basophils in the allergen sample to the number of activated basophils in the buffer solution sample. With an index of more than 1.05, the reaction in the allergen sample was considered a positive one.

The analysis of flow cytometry data was performed by means of the BD FACSDiva Software. The Statistica 7.0 software package was used for the statistical processing of the results.

Results

The results of the analysis showed that an in vitro hypersensitivity reaction was observed to 40.9% (63 out of 154) of the examined local anesthesia medicines. The highest level of hypersensitivity detectability in the BAT was observed for the anesthetic of group I of ethers (novocaine) 10 out of 12, which was 83.3% (see Table).

Among LAs of the second amide group of the articaine series, hypersensitivity to articaine containing vasoconstrictors, stabilizers, and preservatives was detected with the highest frequency – the medicines such as “Ultracaine D-C” (64.0%), “Ultracaine D-C forte” (58.6%) compared with articaine, in which these excipients are absent – “Ultracaine D”, 20.0%, respectively.

The least sensitization in vitro was observed to LAs of the mepivacaine group (Skandonest, 5.6%) and Lidocaine (12.5%), which do not contain vasoconstrictors, stabilizers, and preservatives.

The comparative analysis of the values of the basophil activation index for LAs showed the highest frequency of detection of moderately high indicators (from 1.11 to 2.00) – 71.4%.

The quantitative analysis of CD3+CD294+ cells in various age groups in patients with in vitro sensitization to the examined medicines showed an increase in the level of T-lymphocytes of type 2 immune response with age (Figure 1).

In addition, patients with medicine hypersensitivity showed a significant increase in the level of T-lymphocytes of type 2 of the immune response (1.23± 0.11, P<0.05) compared with patients without sensitization to the examined LAs (0.82±0.16).

Discussion

The results obtained are consistent with the academic literature data [1–6] and the studies conducted in the Laboratory of Clinical Immunology of the A.M. Nikiforov of the Federal State Budgetary Institution of the Ministry of Emergency Situations of Russia (N.V. Bychkov). Indeed, it is believed that LAs of the ether group (Novocaine) are more allergenic than anesthetics of the amide group. It is associated with paraaminobenzoic acid (PABA) or methylparaben. PABA is a metabolite of Novocaine and a derivative of many medicines, it has a similar structure with parabens (preservatives), which can cause cross-allergic reactions [4]. LAs of the II amide group of the articaine series do not release a metabolite of the PABA type, but contain vasoconstrictors, stabilizers, preservatives [3-5]. According to the academic literature data, the most common cause of adverse reactions to LAs is not the active substance, but the presence of auxiliary components-fillers (vasoconstrictors (adrenaline), stabilizers (sodium or potassium disulfite), preservatives (parabens, EDTA)), which are part of these medicines. Russo et al. have also proved that pronounced allergic reactions are more often caused by preservatives, in particular, disodium salt EDTA, which is part of some LAs [4][5].

An increase in the level of T-lymphocytes of type 2 immune response in patients with hypersensitivity to LAs may be associated with the ability of these cells to support the synthesis of immunoglobulin E by plasma cells. An increase in the level of CD3+CD294+ cells in patients with in vitro sensitization to the examined medicines with age may indicate the duration of development and severity of pathological processes associated with hypersensitivity reactions.

Conclusion

Thus, the greatest hypersensitivity was revealed for the LA of group I of ethers (Novocaine) and the least for the medicines of the Mepivacaine and Lidocaine groups. Among the anesthetics of the articaine series of amide group II, the safest one was Ultracaine D. A significant increase in the number of T-lymphocytes of type 2 of the immune response was found in patients with hypersensitivity to local anesthesia medicines.