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Allergy testing


Abstract

Background

Immunoglobulin E (IgE)-associated allergen conditions are increasing worldwide, affecting the quality of life of millions of individuals and are a burden on the health care system. The increasing availability of clinically relevant allergenic molecules has begun to change the way allergen-specific IgE antibody diagnostics are performed. Allergic diseases, respiratory infections, and autoimmune conditions have similar clinical presentations, and self-reported symptoms have low positive predictive value. Thus laboratory allergy and immunologic testing are useful in clarifying diagnosis and guiding treatment. They are also useful in identifying causative allergen in atopic dermatitis (eczema), contact dermatitis, urticaria, angioedema, and food or drug allergies. Testing helps provide clinically relevant information for avoidance and treatment.

Content

This chapter describes the basic concepts of allergy and hypersensitivity reactions of the immune system, cells involved in the allergy, and their functions. Fetal origins of allergy, including the route of exposure, and maternal and dietary influences, are discussed. Technological innovations are also changing the way allergies are diagnosed. Starting from skin testing to specific IgE measurements that are clinically relevant component tests, functional tests like basophil activation tests, microarrays, and nanosensor- and biosensor-based assays are evaluated. Details of various types of allergy including food, contact, inhalants, pollen, pets, fungal, trees, latex, animal epithelia, insect sting, ocular, occupational, and red meat allergy are outlined. Non-IgE and mixed IgE and non–IgE-mediated food allergies are discussed as they can be challenging due to the overall lack of noninvasive confirmatory tests. Various allergy-related disorders like asthma, COPD, hypersensitivity pneumonitis, rhinitis, urticaria, and angioedema are reviewed.

Atopy, sensitization, and allergy

Each year, millions of Americans experience the annoying and familiar symptoms of allergy. Suspected and diagnosed allergies account for more than 17 million physician appointments, 30,000 emergency department visits, and several hundred deaths each year in the United States. Allergic diseases represent a significant public health problem in homes, schools, and the workplace, especially as the incidence of allergic diseases is increasing.

The terms “ atopy ,” “ sensitization ,” and “ allergy ” are often used interchangeably. “ Atopy ” is the genetic predilection to produce specific Immunoglobulin E (sIgE) following exposure to allergens and refers to immediate hypersensitivity mediated by IgE antibodies. “ Sensitization ” refers to the production of allergen sIgE. Sensitization to an allergen is not equal to being allergic to that allergen, because individuals may produce IgE to allergens in each instance, but not develop symptoms upon exposure to that substance.

On the other hand, “ allergy ” refers to a hypersensitivity reaction initiated by immunologic mechanisms. It is an abnormal immunologic response to an otherwise harmless environmental stimulus (e.g., food, pollen, animal dander). Individuals are considered to have clinically significant allergy or allergic disease when they have both allergen sIgE and develop symptoms upon exposure to substances containing that allergen. Therefore higher numbers of people are sensitized to an allergen than are clinically allergic to it. Allergens are antigens that may trigger allergic reactions. They are low molecular weight compounds that can enter the body by inhalation, food consumption, or drug administration.

Hypersensitivity reactions and classification

Hypersensitivity reactions are abnormal immunologic responses of the immune system to an otherwise ordinarily harmless substance that can harm the body. It is a condition in which the normally protective immune system harms the body.

Hypersensitivity reactions can occur in response to different types of antigens, which could be either environmental, infectious, or self-antigens. Environmental antigens are small molecules like dust that can enter through the respiratory tract and trigger an antibody response. An immediate hypersensitivity reaction associated with IgE, such as “rhinitis” or “asthma,” can occur. If the same molecules enter through the skin, a delayed hypersensitivity reaction (dermatitis) can occur. Drugs administered either orally or by injection can also provoke a hypersensitivity reaction mediated by IgE, IgG, or T lymphocytes. Some elemental metals like nickel can also cause a hypersensitivity reaction. Infectious agents like the influenza virus can generate a hypersensitivity reaction and can cause damage in epithelial cells in the respiratory tract. In general, some tolerance to self-antigens exist in most people, and when hypersensitivity reaction becomes an exaggerated response, tolerance is lost, and hypersensitivity reactions can happen. ,

Types of hypersensitivity reactions

There are four types of hypersensitivity reactions named type I–IV, and they are defined by the mechanism responsible for a specific cell or tissue injury that happens during an immune response. Type I, II, and III are antibody dependent, and type IV is a cell-mediated response. Even though there is an overlap between different types, the way they are diagnosed and treated is different.

Type I—immediate (atopic or anaphylactic)

An allergic reaction provoked by a re-exposure to a specific type of antigen. They are naturally occurring, and source of exposure is not always known. The reaction can be either local or systemic and can range from milder to life-threatening anaphylactic reactions. The most common agents are drugs and insect stings.

These reactions are the result of an IgE antibody response and based on the acute allergic reactions caused by molecules released by the mast cells. The difference between a normal immune response and a type I response is that the plasma cells secrete IgE. These IgE antibodies bind to Fc receptors on the surface of mast cells, basophils, and are sensitized. If the exposure to the same allergen happens again, these sensitized cells degranulate and release active mediators like histamine, leukotriene, and prostaglandins that act on surrounding tissues leading to vasodilation and smooth muscle contraction. Some of the examples of type 1 hypersensitivity include food allergies, allergic asthma, allergic conjunctivitis, allergic rhinitis, anaphylaxis, angioedema, atopic dermatitis (eczema), urticaria (hives), and eosinophilia.

Anaphylactic reactions are the clinical response to immunologic formation and fixation between a specific antigen and a tissue fixing antibody, are mediated by IgE, and occur in three stages. First, the antigen attaches to the IgE antibody fixed to the surface membrane of mast cells and basophils. Cross-linking of two IgE molecules is necessary for the release of mediators from mast cells. Second, these activated mast cells release various mediators, and third, these mediators produce various vascular changes, including activation of platelets, eosinophils, and neutrophils, and activation of the coagulation cascade.

Type 1 reaction involves the initiation of T helper-2 cytokines, such as interleukin (IL)-4 and IL-13, leading to IgE production by B cells. Once IgE is produced and secreted, it binds to mast cells and basophils. Upon activation, these cells degranulate and release soluble allergic mediators, such as histamine and leukotrienes, which act on smooth muscles, sensory nerves, mucous glands, arteries, and eosinophils. Common clinical outcomes of an IgE-mediated reaction are increased vascular permeability, smooth muscle cell contraction, and vasodilation. IgE-mediated reactions manifest within minutes to hours of exposure. Depending on the site(s) and frequency of allergen exposure, these reactions may occur in one or more organs resulting in diseases such as asthma, allergic rhinitis, urticaria, and anaphylaxis. ,

Physical allergies (heat, cold, UV light) cause a physicochemical derangement of proteins and polysaccharides of the skin and transform them into autoantigens responsible for allergic reactions.

Type II—antibody dependent (cytotoxic)

Type II hypersensitivity reactions are mediated by IgG or IgM antibodies. They are directed against cell surface antigens, which elicit complement-mediated cytotoxicity. There are three different ways this injury can happen.

Antibody-dependent, complement-mediated cytotoxic reaction.

This reaction is characterized by the interaction of IgG or IgM antibody to cell-bound antigen. This binding results in the activation of complement and destruction of the antigen bound cell (erythrocytes, leukocytes, and platelets). Examples of this type of reaction include acute transfusion reactions and immune hemolytic anemias.

Antibody-dependent, cell-mediated toxicity.

Dependent on the initial binding of specific antibodies to target cell surface antigens. Cells exhibiting the foreign antigen are tagged with IgG or IgM antibodies. These tagged cells are recognized by NK cells and macrophages, which in turn kill the tagged cells. These reactions include those seen in Goodpasture syndrome, where IgG antibodies bind a glycoprotein in the basement membrane of the kidney’s glomeruli and the lungs. Anti-basement membrane antibodies activate the complement that triggers an inflammatory response.

Anti-receptor antibodies.

These antibodies disturb the normal function of receptors. These reactions include autoimmune hypersensitivity against solid tissues like thyroid disease and hyperacute graft rejection.

Type III—immune complex

They are caused by deposition of insoluble immune complexes (aggregations of antigens and IgG, IgM antibodies) in blood vessels and tissues (skin, kidney, and joints). This triggers activation of the complement system, macrophages and leukocytes are attracted, and immune-mediated damage occurs. Some examples include common skin conditions, allergic vasculitis, and erythema nodosum. Pulmonary reactions include hypersensitivity pneumonitis, an Arthus type of reaction.

Arthus reactions manifest as local vasculitis due to the deposition of immune complexes in dermal blood vessels. These reactions are infrequently reported after vaccination of diphtheria and tetanus by CDC, characterized by severe pain, swelling, induration, edema, hemorrhage, and occasionally by necrosis.

Type IV—cell mediated (delayed type hypersensitivity, DTH)

These are T-cell-mediated responses and not antibody-mediated and typically take 2 to 3 days to develop identified by excessive inflammation (e.g., allergic contact dermatitis [ACD]). They are characterized by the secretion of proinflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interferon-γ IL-3, IL-12, and tumor necrosis factor-β) that activate and recruit macrophages and other immune cells. Due to the time it takes for these cytokines to attract and activate macrophages at sites of exposure, the effector phase typically occurs 24 hours following exposure, and it generally peaks at 48 to 72 hours after exposure. It involves antigen sensitized T cells or particle remains phagocytized in a macrophage and encountered previously activated T-cells for a second or subsequent time. T cells (CD4) respond directly or by the release of lymphokines (IL-2 and interferon-gamma) to exhibit contact dermatitis and allergies of infection. Activated CD8 cells destroy target cells on contact, while activated macrophages produce hydrolytic enzymes and, on presentation with specific intracellular pathogens, transform into multinucleated giant cells. Examples include TB, leprosy, celiac disease, and contact dermatitis (poison ivy rash).

Cells involved with allergy

There are three types of cells involved in the pathogenesis of the allergic disease: basophil, eosinophil, and mast cells. They all express many of the same receptors and cytokines and have different effector functions. The pathologic roles of these cells in allergy are directly or indirectly related to the presence of allergen specific IgE in allergic individuals.

Basophils

Basophils are the least abundant granulocytes and play a pivotal role in the development of IgE mediated chronic allergic inflammation as an initiator rather than as an effector. They are involved in the pathogenesis of several disorders, including allergic contact dermatitis, atopic dermatitis, allergic drug reactions, immediate hypersensitivity reactions, asthma, bullous pemphigoid, lupus nephritis, Crohn disease, skin and kidney allograft responses, acute and chronic myelogenous leukemia. They share some characteristics with mast cells like the presence of basophil granules in the cytoplasm, surface expression of high-affinity IgE receptor, and release of histamine when activated. Basophils contribute to the development of allergic inflammation in the skin, respiratory, and gastrointestinal tracts, as well as systemic anaphylaxis.

Studies have shown that developmental and functional heterogeneity exists within basophil populations. These cells can be differentiated into two different categories: IL-3 and thymic stromal lymphoprotein (TSLP) elicited basophils. IL-3 elicited basophils operate in an IgE-dependent manner, and TSLP elicited basophils work in an IgE-independent manner.

Eosinophils

Eosinophils are of the granulocytic lineage, and their exact physiologic function remains largely unknown. They are likely to be involved in host immune response to infection, allergy, asthma, eosinophilic gastrointestinal disorders, and maintenance of other immune cells. Eosinophils develop and differentiate in the bone marrow under the influence of IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). They have a bi-lobed nucleus with highly condensed chromatin and two major types of granules, specific and primary. Specific granules have a distinctive ultrastructural appearance and contain cationic proteins like major basic protein (MBP), eosinophil peroxide (EPO), eosinophil cationic protein (ECP), and eosinophil derived neurotoxin (EDN). Primary granules are like other granulocyte lineages, enriched in Charcot-Leyden crystal protein. Eosinophils express an array of cell surface molecules, including receptors of IgG, IgA, complement and cytokine receptors, etc. They can be activated by cross-linking of IgG or IgA Fc receptors and can be primed for activation by several mediators, including IL-3, IL-5, GM-CSF, and chemokines. Eosinophils respond to the invasion of the body by an allergen by moving to the area and producing a variety of toxins that can cause damage to the offending allergen and to the host tissues. Many eosinophils are found in the nasal mucosa in patients with allergic rhino-conjunctivitis.

Mast cells

Mast cells are derived from the bone marrow and play an essential role in the body’s defense against parasites and allergic reactions. They are found in large numbers at mucosal surfaces of the respiratory tract and gut and in skin and around blood vessels. They can be activated by allergens, toxins, and drugs to produce a variety of inflammatory mediators. This activation can be IgE dependent or independent.

Mast cells are coated with IgE antibodies, which are specific to antigens to which the host has become sensitized. The base of the Fc region of the antibody attaches to the surface of the mast cell via a high-affinity Fc-epsilon-R1 IgE receptor. The Fab segment is free to bind to the antigen. Activation of mast cells occurs when multivalent antigens cross-link membrane bound IgE antibodies, leading to many events. Cell degranulation leads to the release of the contents of secretory granules into the surrounding tissues or bloodstream. These granules contain histamine, cytokines, leukotrienes, prostaglandins, and other inflammatory mediators. Histamines cause increased vascular permeability and smooth muscle contraction. Cytokines IL-4 and IL-13 promote Th2 differentiation and the production of IgE by plasma cells. TNF-a promotes tissue inflammation. Lipid mediators such as leukotrienes and prostaglandins increase vascular permeability, cause smooth muscle contraction, stimulate mucus secretion, and chemically attract T cells, eosinophils, mast cells, and basophils. They result in inflammation leading to clinical outcomes typically seen in asthma, urticaria, angioedema, food allergies, or anaphylaxis.

Mast cells, basophils, and eosinophils have different effector functions but express many of the same receptors and cytokines. Mast cells are tissue cells required for delayed hypersensitivity reactions. Basophils are large circulating leukocytes active in areas of allergic inflammation during a late-phase allergic response. Eosinophils not only present in the GI tract but also allergic inflammatory sites. Basophils express plenty of IL-4 and IL-13 but very little IL-5; on the other hand, mast cells produce IL-5 and IL-13 and low IL-4.

Th1 versus Th2 immune response

Cytokines are the hormonal messengers responsible for most of the biological effects in the immune system, such as cell-mediated immunity and allergic-type responses. They can be functionally divided into two groups: those that are pro-inflammatory and those that are primarily anti-inflammatory but that promote allergic reactions. T lymphocytes are a significant source of cytokines. These cells bear antigen-specific receptors on their cell surface to allow recognition of foreign pathogens.

T-cells may be broadly classified as either helper T cells (Th cells, CD4+) or cytotoxic T cells (Tc cells, CD8+). The individual clones of helper T cells can be separated into two classes (Th1 and Th2) depending upon the specific cytokines the cells secrete in response to antigenic stimulation. Th1 cells primarily produce interferon (IFN)-g and IL-2, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13. Fig. 99.1 shows an overview of cytokines needed for the differentiation of the Th subsets, transcription factors, cytokines, and primary function for each Th subset.

FIGURE 99.1, Overview of cytokines needed for the differentiation of the Th subsets, transcription factors, cytokines, and primary function for each Th subset. IFN-γ , Interferon-γ; IgE, immunoglobulin E; IL, interleukin; TGF-β , transforming growth factor-β; TNF-β , tumor necrosis factor-β.

The two helper T cell classes also differ by the type of immune response they produce. Th1-type cytokines tend to provide the pro-inflammatory responses responsible for killing intracellular parasites and for perpetuating autoimmune responses. Interferon-gamma is the main Th1 cytokine. Excessive pro-inflammatory responses can lead to uncontrolled tissue damage, so there needs to be a mechanism to counteract this. The Th2-type cytokines include interleukins 4, 5, and 13, which are associated with the promotion of IgE and eosinophilic responses in atopy, and IL-10, which has more of an anti-inflammatory response. In excess, Th2 responses will counteract the Th1-mediated microbicidal action. The optimal scenario would, therefore seem to be that humans should produce a well-balanced Th1 and Th2 response, suited to the immune challenge.

Fetal origins of allergy

Dominant maternal and placental influence on the immune responses of the developing fetus exists. There is an interaction between inherited genetic characteristics of both parents and the in-utero environment during pregnancy. The presence of maternal IgG and IgE antibodies may influence the type of response elicited in the fetus to antigens present in the amniotic fluid and the maternal plasma.

Allergy and pregnancy

Pregnancy is a unique event in which a genetically and immunologically foreign fetus usually survives to full term without apparent rejection by the mother’s immune system. Pregnancy and allergic diseases are postulated as T-helper 2 (Th2) phenomena. A normal pregnancy is traditionally described as a Th2 derived condition. An imbalance between Th1 and Th2 immunity in pregnancy, leading to increased Th1-like immune response, is associated with spontaneous abortion, , preeclampsia, and preterm labor.

Under normal circumstances, the maternal-fetal interface creates a TH2 and T regulatory environment by the generation of IL-4, -10, -13, and TGF-b. They downregulate the maternal Th1 response against the fetal-maternal antigen. It is an essential event to sustain pregnancy and enhance fetal growth, and failure to achieve balance results in early miscarriage or intrauterine growth restriction. The Th2-promoting cytokine milieu impacts the fetus, and there is evidence that Th2-biased immune responses, in some cases, may be irreversibly committed due to factors like maternal and fetal genes, maternal allergy, nutrition, allergen, and antibiotic exposure, and the maternal gut microbiome. The rapid development of the diversity of the gut microbiome in the infant is likely to be a key factor switching the neonatal Th2-biased response to a more balanced pattern.

Maternal antigen exposure during pregnancy is likely to lead to the development of tolerance mechanisms in the infant, except when the exposure occurs against a background of heightened Th2-like activity when it could lead to the establishment of an allergic-type response. These responses can be reinforced by the exposure of the infant to the same antigens in the first month of life, leading to persistent IgE response and allergen-induced IL-5 and IL-13 levels during pregnancy, possibly exposing the fetus to an active Th2 environment during gestation.

There is a growing interest in how modern environmental changes influence fetal immune development and contribute to the recent epidemic of allergy and other immune disorders. Pregnancy can be complicated by new-onset or preexisting allergic disease, including rhinitis, urticaria, angioedema, or atopic dermatitis (AD). Asthma is the most common pulmonary disease encountered during pregnancy, occurring in 3 to 8% of pregnant women. Understanding the timing of events leading to allergic sensitization could help develop strategies to prevent the prevalence of diseases such as asthma, eczema, and hay fever.

Route of fetal allergen exposure

Exposure to allergens to the fetus can occur in two different ways:

  • 1.

    Through the fetal gut, by swallowing allergen in amniotic fluid. It has been possible to detect significant quantities of the major allergen of house dust mite, Der p 1, and ovalbumin in amniotic fluid.

  • 2.

    By direct transfer of allergens across the placenta mostly in a complexed form with IgG. The exposure to allergen complexed with IgG will occur optimally in the third trimester of pregnancy when there is active transport of IgG across the placenta.

There is evidence that maternal environmental exposures including dietary factors, ambient air pollution, microbial exposure, and cigarette smoke can modify neonatal immune responses and account for a rapid rise in respiratory disorders, including asthma onset and morbidity and reduced lung function. Conversely, improvements in children’s lung function and growth have been seen with a modest reduction in air pollution exposure.

Maternal dietary influences on immune development

Maternal nutrition is likely to play an important role in the development of the fetus and the immune system. Many dietary nutrients are shown to influence biological mechanisms and have immunomodulatory properties. This includes polyunsaturated fatty acids (PUFAs), antioxidants, and other vitamins.

A reduced intake of anti-inflammatory n-3 PUFA (found in oily fish) is implicated in the rise in allergic disease. Several studies have shown a protective relationship between maternal n-3 PUFA consumption in pregnancy and subsequent infant allergic disease ; some of them are not significant after allowing multiple comparisons.

Folate supplementation in pregnancy-induced hypermethylation (silencing) of regulatory genes in lung tissue was associated with the development of allergic airway disease and systemic allergic responses. This effect was also transmitted epigenetically to subsequent generations. Folic acid supplementation during pregnancy was associated with an increased risk of asthma and respiratory disease in infants. , In contrast, other studies have shown protective effects of folate in the postnatal period and allergic diseases.

Maternal antioxidants and vitamin intake are associated with differences in neonatal immune function and reduced risk of possible allergic outcomes, including recurrent wheezing and eczema. Higher maternal total intakes of antioxidants during pregnancy may decrease the chances of wheezing illnesses in early childhood. However, these findings are not consistent between studies, and concerns have been raised about the potential for antioxidants’ adverse effects on allergic diseases.

Increasing maternal vitamin D, E, and zinc intakes during pregnancy may decrease the risk of wheeze symptoms in early childhood. Low maternal dietary and total vitamin D intakes during pregnancy are associated with increased wheezing symptoms in children at 5 years. These associations were independent of maternal smoking status, intake of vitamin E, zinc, and calcium. Maternal plasma a-tocopherol (vitamin E) during pregnancy was positively associated with post-bronchodilator FEV (1) at 5 years, with a 7-mL (95% confidence interval, 0 to 14; P = .04) increase in FEV (1) per microgram/ml a-tocopherol. During pregnancy, maternal zinc intake was negatively associated with asthma ever (0.83, 0.71 to 0.78) and active asthma (0.72, 0.59 to 0.89). ,

Maternal microbial exposure

Several studies demonstrated that in utero (maternal) exposure to both pathogenic and nonpathogenic microbial products can inhibit the development of allergic phenomena in the offspring. Maternal exposure to high microbial burden in Germany and New Zealand farming environments is associated with altered expression of innate immune genes and reduced risk of allergic disease in the children, independent of postnatal exposure. Intranasal administration of Acinetobacter lwoffii to pregnant mice was associated with significant effects on the ontogeny of splenic CD4+ Th1 interferon-gamma production in the offspring of exposed mothers. This supports notions that microbial exposure may modify fetal gene expression and provides a potential epigenetic mechanism. Probiotics may reduce the risk of eczema, but there is extensive heterogeneity in study protocols and findings between studies. The role of probiotics in the prevention of allergic diseases is still unclear.

Maternal allergen exposure

Exposure to farm animals in early life, especially before birth, has shown to have a protective effect on allergies. However, exposure to pets in early life did not appear to either increase or reduce the risk of asthma or allergic rhinitis in children aged 6 to 10 years. A decreased risk of ADin children exposed to pets during infancy was observed. Prevention strategies for early allergies are mainly focused on allergen avoidance, and there is no clear evidence suggesting that changes in food or inhalant allergen exposure in pregnancy are responsible for the allergic disease. Also, there is insufficient evidence to suggest restrictive dietary recommendations will prevent allergic disease. On the other hand, several reports claim that any attempt to avoid or delay allergen exposure may increase the risk of allergic sensitization.

Antigen stimulates IgE bound to mast cells; the degranulation of released mediators increases vascular permeability and local inflammation. This results in the recruitment of eosinophils from the blood to the site of the parasitic infection. Eosinophils can also bind to IgE attached to the surface of the parasite, then release the contents of their granules to destroy the worm via an ADCC-type mechanism.

Maternal smoking

Maternal cigarette smoking in pregnancy has many adverse effects on the fetus, including effects on lung function and asthma risk. However, there is no clear evidence to the onset of allergic disease. ,

Other maternal exposures

Several medications (for example, paracetamol or acetaminophen) used in pregnancy are associated with an increased risk of childhood asthma. Some studies have indicated that acid-suppressive drugs in pregnancy are associated with an increased risk of developing childhood allergy. Several products of industry and agriculture are linked with immune disorders because of estrogenic (pro-Th2) properties. The effects of these and other modern environmental exposure on immune development is still unclear and difficult to investigate but should not be ignored.

Before pregnancy, all women with significant allergic conditions need to be assessed for a final diagnosis for better management. The available studies suggest that maternal avoidance of allergenic foods during pregnancy does not reduce the risk of allergic disease in the offspring, regardless of whether the infant is high risk or not. Thus several medical associations do not recommend maternal avoidance diets during pregnancy. ,

Biology of immunoglobulin E

IgE is one of the five classes of immunoglobulins. It is comprised of two identical light (L) and two identical heavy (H) chains and differs from the rest by the presence of epsilon (ε) heavy chain. IgE is a monomer and consists of four constant regions in contrast to other immunoglobulins that contain only three constant regions. Because of this extra region in IgE, its molecular weight is 190 kDa compared to 150 kDa for IgG.

IgE is the least abundant of all immunoglobulins (IgG > IgA > IgM > IgD > and IgE), present in circulation at very low concentrations (<1 μg/mL), which is approximately 300-fold lower than that of IgG. In contrast to IgG antibodies, which have a half-life of about three weeks, IgE is very short-lived in plasma (half-life, <1 day), but receptor bound IgE can remain fixed to mast cells in tissues for weeks or months. IgE is produced by IgE plasma cells, which are present in mucosal areas especially in the respiratory tract, where the secreted IgE mediates allergic reactions, and the gastrointestinal tract, where it may mediate expulsion of parasitic worm infections, specifically helminths. Antigen stimulates IgE bound to mast cells; the degranulation of released mediators increases vascular permeability and local inflammation. This results in the recruitment of eosinophils from the blood to the site of the parasitic infection. Eosinophils can also bind to IgE attached to the surface of the parasite, then release the contents of their granules to destroy the worm via an ADCC-type mechanism. IgE is an important part of the “first line of defense” against pathogens that enter the body across epithelial barriers. A deleterious effect of IgE can occur when it binds to normally innocuous antigens, such as pollen, triggering mast cell degranulation associated with allergic responses.

Synthesis

The concentration of serum IgE increases from birth until the age of 15 years and then decreases during adulthood. Furthermore, males tend to have a higher concentration of serum IgE than females.

Antibodies are produced by B cells. These cells are programmed to make IgM by default but can undergo “isotype switching” to produce IgE in specific situations. This switching requires cell surface interactions between B and T cells and from soluble factors from various cell types.

This “isotype switching” requires two types of signals:

  • Soluble factors like IL-4 and IL-13 released by many inflammatory cells, including Th2 cells, innate lymphoid cells, mast cells, and basophils. This interaction between the cytokines and their respective B-cell receptors activates the transcription at the specific epsilon germline locus via signal transducer and activator of transcription (STAT).

  • The interaction between the B cell CD40, a member of the tumor necrosis factor receptor superfamily and T cell CD40 ligand (CD154) results in DNA class-switch recombination. IgE expression is also triggered by nuclear factor-kB (NF-kB). STAT6 and NF-kB synergize to activate B cell activator protein, promoting the production of IgE.

B cell isotype-switching to produce antigen-specific IgE occurs primarily in mucosal lymphoid tissues, with the highest amounts of antigen-specific IgE production in tonsils and adenoids. However, some also occurs in peripheral tissues. The newly formed IgE then diffuses through the tissues and into the circulation.

Antigen-specific IgE production can occur locally within the bronchial and nasal mucosa, in addition to the lymphoid tissues and bone marrow. This phenomenon of local IgE production is termed “entopy” and may underlie some cases of chronic “nonallergic” rhinitis and severe asthma.

The genetic predisposition to develop allergic disease (atopy) is complex and not well understood. Genetic factors strongly influence serum IgE levels and its regulation of production. Genome-wide association studies have identified several loci that may be important for IgE regulation, including loci in the gene encoding the alpha chain of the high-affinity receptor for IgE (FC-epsilon-RI-a), STAT6, and in the gene RAD50/IL-13 cluster. Once an individual is producing IgE to a specific allergen, exposure to that allergen usually boosts the production of IgE to it. This explains the seasonal increases in pollen-sIgE serum levels seen in patients with pollen allergy and the increases in drug allergies where symptoms can resolve if a patient can avoid that drug for years.

Immunoglobulin E receptors

There are two types of receptors through which IgE elicits its functions. These are high- and low-affinity receptors on mast cells, basophils, and other cells leading to degranulation of mast cells and basophils and antigen presentation. Binding to IgE enhances the binding to receptors and circulating levels of IgE correlates with the numbers of receptors.

The high-affinity receptor for IgE is Fc-ε-RI. IgE binds to the alpha chain. Fc-epsilon-RI exists in two forms: Tetrameric form (αβγ 2) and trimeric form (αγ 2)

The low-affinity IgE receptor, Fc-epsilon-RII (CD23), is present on a variety of cells. The constitutively expressed form, CD23a, is present only on B cells. In contrast, the inducible form, CD23b, is present on B cells, T cells, dendritic cells, monocytes, macrophages, neutrophils, eosinophils, intestinal epithelial cells, and platelets.

Relationship between immunoglobulin E and allergic disease

The measurement of total IgE and allergen-sIgE has variable utility in the diagnosis and management of allergic disorders. Elevated total IgE is seen in some allergic disorders, as well as several nonallergic diseases (infections, atopic, inflammatory diseases, and neoplasms). There is no specific cutoff value that discriminates patients with the allergic disease from those without, and there is considerable overlap.

Elevated total IgE is associated with an increased risk of allergic disease. There is significant overlap between affected and unaffected individuals, such that the measurement is most useful in the context of population studies, rather than in the diagnosis or management of an individual. Allergen-sIgE is usually detectable several years before a person becomes reactive to that allergen, although only a subset of sensitized people will ever develop symptoms.

For an allergy to be considered a “true” allergy, there are two factors that need to be present:

  • Clinical features—Development of specific signs and symptoms on exposure to that allergen

  • Sensitization—Presence of allergen-sIgE

The exceptions to the definition of a true allergy are the non–IgE-mediated food-induced allergic disorders, which are rarely encountered in general practice, for example, food protein-induced enterocolitis syndrome.

Allergy tests

Allergy testing is indicated in symptomatic situations. Testing is utilized in the diagnostic setting for allergies, to determine whether symptoms are caused by IgE antibodies and exposure to an allergen, and can be divided into in vivo and in vitro based methodologies:

  • 1.

    In vivo testing—includes allergy skin testing such as the scratch, puncture, or prick test (epicutaneous), intradermal tests (intracutaneous), and patch tests, and food and bronchial challenges such as direct skin testing

  • 2.

    In vitro testing—includes various techniques to test the blood for the presence of total and specific IgE antibodies to an antigen.

Both in vivo and in vitro methods have benefits and limitations that are important to consider in clinical practice.

In vivo testing

Skin prick testing

In vivo skin testing evaluates the body’s natural immune response to direct contact with an allergen in question. Commonly used in vivo testing modalities include skin prick or puncture and intradermal testing. It is the most rapid and sensitive testing modality for the detection of IgE-mediated allergen disease. It is a bioassay that detects the presence of allergen-sIgE on a patient’s mast cells. When an allergen is introduced into the skin of a patient during testing, it encounters cutaneous mast cells. Binding of the allergen occurs if the patient’s mast cells are coated with IgE recognizing that specific allergen. If both IgE and allergen are present in sufficient quantities, then adjacent IgE molecules directed against the allergen may be crosslinked on the cell surface and initiate intracellular signaling.

These events lead to mast cell activation, the release of the contents of intracellular granules (degranulation), and the de novo generation of inflammatory mediators. Degranulation releases preformed vasoactive mediators and enzymes, such as histamine, tryptase, chymase, and carboxypeptidase. Histamine is the primary mediator of the wheal and flare response. Other mediators (e.g., prostaglandin D2) are also involved, as the size of the wheel does not correlate directly with the concentrations of histamine released.

The clinical result of these cellular events is a positive skin test or a transient “wheal-and-flare” reaction. This reaction consists of a localized central area of superficial skin edema (wheal) surrounded by erythema (flare). This pruritic reaction represents the next phase of the allergic reaction. Late-phase reactions (LPRs) may develop at skin test sites in some individuals. These consist of deep tissue swelling, warmth, pruritus, and erythema beginning one to two hours after testing and resolving in 24 to 48 hours. LPRs are mast cell mediated and IgE dependent, although they do not predict symptoms on exposure and are not used in the diagnosis of IgE-mediated allergy. ,

A positive test result is a raised wheal on the skin with surrounding erythema. The histamine control typically produces a wheal of at least 3 mm in diameter. Not all allergens are available for skin testing. It is crucial to make sure that individuals are not at risk for anaphylaxis, such as individuals who have uncontrolled asthma and those who have reduced lung function. People who experienced anaphylaxis within the past 30 days are not good candidates for skin prick testing (SPT) because of false-negative test results. Prior anaphylactic shock causes the skin to be unreactive, a condition that lasts approximately 2 to 4 weeks. If the skin test results in a positive test, the results are still accurate and useful for the diagnosis of an allergy. Individuals with certain skin conditions (dermographism, acute or chronic urticaria, cutaneous mastocytosis, atopic dermatitis) are not candidates for skin prick tests because of false-positive test results. Other relative contraindications include active angina and cardiac arrhythmias, older adults who are in poor health, and pregnant women.

Medicines that may interfere with SPT include antihistamines (H1-blockers), which decrease the reactivity of the skin and should be stopped at least 72 hours before skin prick testing. Topical corticosteroids should not be used in the area of the testing site for 2 to 3 weeks prior because they are shown to reduce skin reactivity. Oral or inhaled steroids do not appear to alter the reaction to SPT and can be continued. Other medicines—such as tricyclic antidepressants, phenothiazines, benzodiazepines, quetiapine, and mirtazapine—may also reduce the reactivity of the skin. SPT should generally only be requested if these medicines can be discontinued temporarily. Histamine receptor-2 blockers (e.g., ranitidine) work mainly in the stomach instead of the skin but have, in the past, been included among the medicines which should be avoided before SPT. Intradermal testing is a more sensitive, but less specific, testing method than percutaneous testing for the detection of IgE antibodies. The number of intradermal tests may also vary from patient to patient.

Patch testing (application testing)

Patch testing is indicated for the evaluation of possible allergic contact dermatitis (CD). It is a method of investigation with internationally defined rules and well-established foundations which are under continuous review and updating. The reading and interpretation of test results, whether positive or negative, is a complex process that requires training and experience.

Patch tests are indicated for:

  • Patients with a diagnostic hypothesis of CD,

  • Patients with other skin conditions that may be aggravated by CD (atopic dermatitis, seborrheic dermatitis and stasis, nummular eczema, psoriasis, and dyshidrosis),

  • Patients with chronic eczema without an established etiology, and

  • Suspected cases of occupational contact dermatitis.

A false-positive result will occur in the presence of impurities in the test preparation if irritation occurs. False-negative results may occur due to inadequate penetration of antigen, insufficient occlusion, short contact of the antigen with the skin, if the site of an application was previously treated with corticosteroids or exposed to UV radiation, or if the patient underwent systemic treatment with corticosteroids and/or immunosuppressant drugs. Upon suspicion of false-positive or false-negative results, the patient should be retested with at least 30 days between tests.

Allergen provocation/challenge tests

The allergen challenge test has been the mainstay of diagnosis of allergic diseases for a long time since it offers direct proof of the clinical relevance of an allergen for allergic disease symptoms and severity. The formal diagnosis of allergic disease should include taking a detailed clinical history and demonstrating sensitization to a traceable allergen by skin prick test or in vitro IgE testing.

Nasal allergen provocation test.

NAPT is a research tool and rarely indicated in the routine evaluation of allergic rhinitis (AR). With patient clinical history and confirmation by SPT or SIgE testing, the diagnosis of AR is made. NAPT can be useful if there is a discordance between sensitization and clinical history and the absence of systemic atopy. This is mainly because some nonallergic conditions can mimic AR, and differentiation is crucial.

NAPT’s foremost drawbacks are the widespread methodological variability in terms of how the test is applied and interpreted. Also, the risk of adverse effects on ear, nose, and throat, and lack of comparison with “typical” allergen exposure. NAPT’s response can be immediate, between 10 and 20 minutes, late after 6 to 8 hours. It can be delayed with an average of 24 hours and resolves after 56 hours. This is typically associated with conjunctival responses in the majority of the patients.

Bronchial allergen provocation test.

Many (30%) AR patients self-report bronchial symptoms suggestive of asthma and reports suggest the existence of a new asthma phenotype defined by the absence of systemic atopy and positivity to BAPT with the allergens. Airway hyper-reactivity (AHR) is measured by challenging the airways with various physical and chemical stimuli. The airway’s narrowing is measured by changes in FEV1 after gradually increasing the dose of provoking agents. Post FEV1 was established as a baseline value. A decrease in FEV1 after a single-step BAPT is expressed as a percentage change from baseline. Once a 20% decline in FEV1 is achieved, the provoking agent’s inhalation should be stopped. AHR is expressed as PC20 (provocative concentration) or PD20 (provocative dose) of an inducement that causes a 20% reduction in FEV1.

Conjunctival allergen provocation test.

CAPT represents the effects of introducing an allergen on the ocular conjunctiva to evoke an IgE-mediated allergic response of the ocular surface mucosa in an assumed sensitized patient. They are useful, particularly in polysensitized patients. CAPT can be applied as a choice to NAPT even if the patients with AR do not report ocular symptoms. It involves instilling 20 to 30 μL of fixed concentrations of an allergen extract in the inferior external quadrant of the ocular conjunctiva. The contra-lateral eye is used as a control with a diluent. A typical IgE-mast cell-dependent immediate response occurs with itching, tearing, redness, and conjunctival edema within 5 to 20 minutes. The response subsides typically within 30 minutes, and a late-phase effect may infrequently occur 6 to 10 and up to 24 hour after provocation. CAPT continues underused in clinical practice, although it is a reliable and straightforward procedure that can provide valuable clinical information.

Oral food challenge.

The OFC is a gold-standard diagnostic for food-related adverse reactions leading to appropriate food avoidance. It mainly serves two roles in managing food allergies: to confirm diagnosis of a specific food allergy and to determine if an identified allergy persists or has resolved.

OFC is useful for identifying foods causing reactions in the following situations

  • Serum IgE testing and/or skin prick test (SPT) results are not consistent with the patient history.

  • When the family/patient and physician agree that the risk estimated from the medical history and the test results is outweighed by the benefit of possibly adding a food to the diet.

  • Determining whether food allergens associated with chronic conditions such as atopic dermatitis (AD) or eosinophilic esophagitis will cause immediate reactions.

  • Expanding the diet in persons with multiple dietary restrictions.

  • Assessing the status of tolerance to cross-reactive foods.

  • Assessing the effect of food processing on food tolerability (e.g., fruits and vegetables that may be tolerated in cooked form in patients with pollen food allergy syndrome).

Patients must be in good health on the day of the OFC to minimize the risk of a severe reaction and to not confound the interpretation of the results. Reaction severity cannot be reliably predicted with allergen-sIgE levels or component-resolved diagnostic testing. An OFC is not recommended if the patient has concurrent illness, fever, active respiratory symptoms such as coughing or wheezing, has needed to use a short-acting b-agonist to relieve respiratory symptoms within the preceding 48 hours, or is taking a β-blocker, because any of these factors may increase the risk of a severe reaction or interfere with the ability to effectively treat a reaction. If allergic diseases such as asthma, AD, urticaria, and/or allergic rhinitis are active, then the OFC may be delayed because these symptoms may also affect interpretation of the OFC.

OFCs are generally performed by gradually feeding the test food under supervision, with personnel and emergency treatments available in the event of anaphylaxis. OFCs can be safely performed in the allergist’s office with special attention to ensuring staff are prepared to assess and treat allergic reactions and anaphylaxis, and there is quick and ready access to emergency medical services.

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