Key Points
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Allergen-specific IgE antibody is a marker of allergic sensitization and a risk factor for allergic disease but, alone, it does not make the definitive diagnosis of allergic disease. Confirmation of allergic sensitization with a positive IgE anti-allergen analysis increases the likelihood that the patient’s symptoms may be a result of an immediate-type hypersensitivity response.
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Quantitative IgE antibody levels to selected foods (milk, egg, fish and peanut) if above a predefined IgE antibody threshold may eliminate the need for tedious and expensive food challenges (DBPCFC). Caution, however, needs to be exercised as the predictive threshold levels vary among clinical studies, due to differences in study populations, protocols and statistical analyses employed.
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Food antigen-specific IgG and IgG4 antibody levels are not diagnostically useful as they do not correlate with the results of oral food challenges.
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Clinically successful aeroallergen immunotherapy is almost always accompanied by high (micrograms/mL) levels of allergen-specific IgG antibody in serum.
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Mast cell tryptase is a serine esterase that is used as a marker of mast cell activation during anaphylaxis. Immunoreactive tryptase levels in serum of healthy adults are typically <5 µg/L. Elevated mature tryptase levels (>10 µg/L) are detectable 1 to 4 hours after the onset of systemic anaphylaxis with hypotension.
The diagnosis of human allergic disease begins and ends with the patient’s clinical history and physical examination. When the clinical history identifies allergic symptoms that are in temporal relationship to a definable and relevant allergen exposure, immunoglobulin E (IgE) antibody sensitization is then confirmed with in vivo skin tests (puncture/intradermal, see Chapter 19 ) or in vitro blood tests (allergen-specific IgE antibody serological assays). If there is a mismatch between the history and these primary diagnostic tests for sensitization, then a secondary provocation test (open or placebo-controlled food challenge, nasal challenge, bronchial challenge) may adjudicate the veracity of the history-driven diagnosis. This chapter discusses the laboratory’s contribution to the diagnostic algorithm and analytes that serve as diagnostic confirmatory tests when there is high suspicion of allergic disease based on a clinical history.
Immediate (Type 1) Hypersensitivity Response
Prausnitz and Kustner first described the immediate-type hypersensitivity allergic reaction using an in vivo test in which serum from Kustner, who was allergic to fish, was injected into the skin of Prausnitz. An immediate wheal and flare reaction in the skin was then induced when extracted fish antigen was injected into the same skin site. A serum factor or atopic reagin was later shown to be a novel immunoglobulin (IgE).
Box 18-1 summarizes the immune system components that are involved in the induction of IgE antibody and elicitation of the effector mechanisms of type 1 hypersensitivity. Inhalation, skin or parenteral exposure to allergens is the initiating event, during which these foreign molecules are presented to antigen-presenting cells on mucosal surfaces. Antigen-presenting cells process antigenic epitopes to T helper cells that secrete cytokines (IL-4, IL-10, IL-13) which induce B cell lymphocyte proliferation. As allergen-specific IgE antibody is produced, it circulates and binds onto FcεR1 receptors on mast cells and basophils . Upon re-exposure, allergen cross-links receptor bound IgE, causing an influx in calcium, which triggers preformed mediator release ( histamine, proteases ) and newly synthesized mediators ( leukotrienes, prostaglandins ). The pharmacological effects of these mediators on blood vessels and airways produce a spectrum of clinical symptoms including hay fever, asthma, eczema and anaphylaxis. Released c ytokines (IL-4, IL-5, IL-6) from degranulating mast cells serve to enhance the inflammatory response and IgE production.
* Analytes in italics are routinely measured in the clinical diagnostic allergy laboratory and thus they are discussed in the text. Analytes that are underlined are considered research analytes and they are not routinely measured in the clinical immunology laboratory.
Antigen Presentation
Allergen (exposure, entry at mucosal surfaces or local lymph nodes)
Antigen presenting cells (processing and presentation)
T H 2 lymphocytes
Cytokines (promoters of IgE production: IL4, IL-10, IL-13; inhibitors of IgE production: IFγ)
B cell lymphocytes
IgE Production and Sensitization
IgE (allergen-specific IgE antibody)
Connective tissue fixed and mucosal mast cells with FcεRI receptors
Circulating basophils with FcεRI receptors
Mast Cell Activation and Mediator Release
Re-exposure to allergen induces calcium ion influx into mast cells
Mast cell releases preformed and newly synthesized mediators
Release of trypase
Exocytosis of preformed histamine
Synthesis of newly formed lipid mediators from arachidonic acid
Prostaglandin D 2
Leukotriene B 4 , C 4 , D 4
Humoral Immune Response
Chronic antigenic challenge (inadvertent or intentional [immunotherapy]) induces antigen-specific IgG and IgA antibodies in blood and secretions)
An investigation that employed engineered antibodies and allergens showed that the concentration, specific activity (specific IgE to total IgE ratio), affinity (tightness of binding) and clonality (epitope specificity) of the IgE antibody response independently impact on effector cell activation. The study concluded that higher levels of basophil activation occur with higher overall total serum IgE levels, higher Derp2-specific IgE to total IgE ratios, broader clonality and higher IgE antibody affinities. Future serological assays for IgE antibody need to monitor more effectively these four important humoral immune response parameters.
Allergens
Allergens are substances, usually glycoproteins, that are released from weeds, grasses, trees, animal danders, molds, house dust mites, parasites, insect venoms, occupational substances, drugs and foods. They are capable of inducing IgE antibody (sensitization) in atopic or genetically predisposed individuals. Each of these source materials may be extracted with a physiological buffer to produce a final product (extract) that contains a complex mixture of allergenic and nonallergenic material. With the advent of molecular cloning techniques in the late twentieth century, many clinically important allergenic components have been identified and purified out of these complex allergen extracts. A systematic allergen nomenclature for allergenic extracts and components has been adopted that involves the first three letters of the genus and first letter of the species and, for the allergen component, a number. This scheme has been established to identify each unique allergen extract and component specificity. For instance, Ara h 1 signifies the group 1 allergen in peanut ( Arachis hypogaea ) which is a carbohydrate-bearing 7S vicilin-like globulin.
Allergenic components are adopted into the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Nomenclature committee database once they have an established purity to homogeneity, physical-chemical characterization by molecular weight, isoelectric point and glycosylation pattern, nucleotide and/or amino acid sequence and immunoreactivity to IgE antibody. Allergenic molecules are further classified into protein families according to their structure and function. Different allergenic molecules often share common epitopes which can result in immunological cross-reactivity. Other allergenic molecules can serve as unique markers for a particular allergen specificity. Examination of the combined protein family (PFAM) database and structural database of allergenic proteins (SDAP) identified approximately 12 000 protein families, of which only approximately 2% or 236 PFAMs are known to contain allergenic proteins. Of these families, 31 protein families contain multiple allergenic proteins (homologs, orthologs). Thus, allergens comprise a small fraction of the total number of protein families and they possess particular biological structures and functions. They tend to be pervasive or abundant in nature and stable to processing (e.g. heat and digestion) as a result of multiple cysteine linkages. They tend to form aggregates or polymers and many tend to be plant defense related. Importantly, not every member of a protein family is allergenic or cross-reactive. Box 18-2 lists the nine principal allergen families that manifest cross-reactivity due to structural similarity. In addition, it presents their principal biological function in nature and illustrative members of these allergen families.
Profilin : An actin-binding protein in tree/grass/weed pollen and foods of plant origin that is involved in the dynamic turnover and restructuring of the actin cytoskeleton (12–15 kDa); sensitive to heat and digestion
Birch ( Betula verrucosa )
Bet v 2
Natural rubber latex ( Hevea brasiliensis )
Hev b 8
Mercury ( Mercurialis annua )
Mer a 1
Timothy grass ( Phleum pratense )
Phl p 12
Serum albumin : Protein in milk, blood and epithelia of animals that functions to transport hemin and fatty acids to muscle tissue and maintains oncotic pressure; sensitive to heat and digestion
Cow ( Bos domesticus )
Bos d 6
Dog ( Canis familiaris )
Can f 3
Horse ( Equus caballus )
Equ c 3
Cat ( Felis domesticus )
Fel d 2
Chicken ( Gallus domesticus )
Gal d 5
Pathogenesis related proteins : PR10 Family (Bet v 1 homologs)-present in pollens, pomaceous and stone fruits, vegetables and nuts which functions as a ribonuclease and carrier of steroids (17 kDa); most PR10 proteins are sensitive to heat and digestion
Birch ( Betula verrucosa )
Bet v 1−
Hazel pollen ( Corylus avellana )
Cor a 1.010−
Hazelnut ( Corylus avellana )
Cor a 1.040
Apple ( Malus domesticus )
Mal d 1
Peach ( Prunus persica )
Pru p 1
Soybean ( Glycine max )
Gly m 4
Peanut ( Arachis hypogaea )
Ara h 8
Kiwi ( Actinidia deliciosa )
Act d 8
Celery ( Apium graveolens )
Api g 1
Procalcin : present in weed/grass/tree pollens but not foods that functions to bind calcium and regulate calcium levels; moderately stable
Birch ( Betula verrucosa )
Bet v 4−
Timothy grass ( Phleum pratense )
Phl p 7
Nonspecific lipid transfer proteins : present in fruits, vegetables, nuts and pollen which functions to shuttle phospholipids and other fatty acids between cell membranes; stable to heat and digestion (7–9 kDa)
Peanut ( Arachis hypogaea )
Ara h 9
Hazelnut ( Corylus avellana )
Cor a 8
Walnut ( Juglans spp )
Jug r 3
Peach ( Prunus persica )
Pru p 3
Mugwort ( Artemisia vulgaris )
Art v 3
Olive pollen ( Olea europaea )
Ole e 7
Plane tree ( Platanus acerifolia )
Pla a 3
Lipocalin : present in furry animals; functions to transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids; stable protein
Cat ( Felis domesticus )
Fel d 4, 7
Dog ( Canis familiaris )
Can f 1, 2, 4, 6
Parvalbumin : present in fish and amphibians; binds calcium and is involved in calcium signaling in fast-contracting muscles; stable to heat and digestion
Cod fish ( Gadus morhua )
Gad c 1
Shrimp ( Crangon crangon )
Cra c 4, 6
Tropomyosin : Present in crustaceans, mites, cockroaches and nematodes and functions as an actin-binding muscle protein that regulates actin mechanics in muscle contraction; stable to heat and digestion
Anisakis – herring worm ( Anisakis simplex )
Ani s 3
German cockroach ( Blattella germanica )
Bla g 7
Dust mite ( Dermatophagoides pteronyssinus)
Der p 10
Shrimp ( Penaeus monodon )
Pen m 1
Storage proteins: present in seeds and nuts; function as nutrient storage (e.g. 2 S albumin) and are stable to heat and digestion
Peanut ( Arachis hypogaea )
Ara h 1, 2, 3, 6
Hazelnut ( Corylus avellana )
Cor a 9
Walnut ( Juglans spp )
Jug r 1, 2
Soybean ( Glycine max )
Gly m 5, 6
Since 1968, complex physiological extracts of allergenic materials have been used as reagents in serological assays for IgE antibody quantification in serum as they in theory contain all the principal allergenic components for that specificity. There has been increasing interest in the use of native and recombinant component allergens as serological reagents because component resolved diagnosis may better resolve genuine sensitization from cross-reactivity in polysensitized patients. In food allergy, components can be particularly useful for certain foods such as peanut as they can facilitate the assessment of risk for a severe versus more mild allergic reaction and thus reduce the need for an oral food challenge. By identifying the specific components to which an individual is sensitized, more targeted immunotherapy may also be conducted. Thus, instead of measuring IgE antibody to crude cat dander extract, clinically used singleplex and multiplex microarray assays can measure IgE antibody specific to component allergens produced by cats, namely Fel d 1 (uteroglobin), Fel d 2 (cat albumin), Fel d 3 (cystatin), Fel d 4 (lipocalin), Fel d 5 (cat IgA), Fel d 6 (cat IgM) and Fel d 7 (cat IgG). Use of component allergens allows one to dissect more effectively the IgE antibody response into allergen families that share structural homologies and thus cross-react with each other.
Possibly the most well-studied family of cross-reactive allergens is the pathogenesis related proteins (PR10 family) which are present in pollens, pomaceous and stone fruits, vegetables and nuts. These 17 kDa proteins function as ribonucleases and carriers of steroids. Most PR10 proteins are sensitive to heat and digestion. The group 1 allergen from birch tree pollen, Bet v1, has a number of homologs. These include allergenic proteins from alder tree pollen (Aln g 1), hazelnut pollen (Cor a 1), apple (Mal d 1), peach (Pru p 1), soybean (Gly m 4), peanut (Ara h 8), celery (Apr g 1), carrot (Dau c 1) and kiwi (Act d 8). A primary sensitivity to Bet v 1 may result in oral allergy symptoms after exposure to any of these structurally similar allergenic molecules. The chip-based microarray system discussed below is a comprehensive tool for identifying IgE antibodies in a given patient’s serum that cross-react with components from seemingly disparate allergen sources.
Diagnosis of Type 1 Hypersensitivity
The diagnostic algorithm for human allergic disease begins with a thorough clinical history and physical examination. A suggestive history is followed by in vivo skin testing, in vitro serological assays and/or provocation challenge tests as confirmatory measures for the detection of IgE antibodies ( Box 18-3 ). The inter-relationship between each of these components of the diagnostic plan is illustrated in this chapter using natural rubber latex as a model allergen system.
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Allergen-specific IgE antibody is a marker of allergic sensitization and a risk factor for allergic disease but, alone, it does not make the diagnosis of allergic disease. It is performed as a confirmatory test in support of a clinical history that strongly suggests an allergic disorder.
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Allergen-specific IgE antibody is measured by non-isotopic autoanalyzers that employ a two-stage noncompetitive immunoassay format. In the assay, allergen-specific antibodies are bound to a solid phase allergosorbent and bound IgE antibodies are detected with labeled anti-human IgE. A heterologous total serum IgE calibration curve is used to interpolate response levels into quantitative estimates of allergen-specific IgE.
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Quantitative IgE antibody results are reported in kU A /L, traceable to the World Health Organization IgE Reference Preparation (1 U = 2.4 nanograms of IgE).
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The multi-allergen screen is a qualitative assay that measures allergen-specific IgE antibody to multiple aeroallergens and/or food allergens in a single test. The multi-allergen screening assay produces qualitative (positive or negative) results that lead to subsequent investigation of the patient’s serum or skin for IgE antibodies specific for individual clinically defined allergen specificities.
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A competitive inhibition format of IgE antibody assays is used to define the relative potency of allergen extracts used in skin testing, to identify the extent of cross-reactivity of human IgE antibody for structurally similar allergens (e.g. vespid vs Polistes wasp venom allergens) and in Hymenoptera venom allergy to select appropriate venoms for immunotherapy.
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Quantitative IgE antibody levels to selected foods (milk, egg, fish and peanut) if above a predefined IgE antibody threshold may eliminate the need for tedious and expensive food challenges (DBPCFC). Caution, however, needs to be exercised as the predictive threshold levels vary among clinical studies, due to differences in study populations, protocols and statistical analyses employed.
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Food antigen-specific IgG and IgG4 antibody levels are not diagnostically useful as they do not correlate with the results of oral food challenges.
Clinical History
Latex allergy diagnosis begins with a comprehensive clinical history. A child may present with complaints of hives, rhinoconjunctivitis, asthma or anaphylaxis that are temporally associated with exposure to a product that contains natural rubber. The allergist probes the child’s general atopic and specific latex allergy history using questions designed to identify predisposing risk factors such as an atopic state (seasonal rhinitis, early-onset asthma, eczema, food allergy), the frequency, consistency and magnitude of latex exposure, the presence of concomitant food allergy and hand dermatitis. Exposure to rubber-containing products provides clues which strengthen the clinical suspicion of latex allergy. The rapid onset of allergic symptoms around toy balloons, dental dams or other dipped rubber products (latex gloves, rubber toys) that contain high levels of allergen is supportive. In contrast, respiratory or upper airway symptoms around latex paint that does not contain natural rubber diminish the likelihood of latex allergy. The type of exposure, time of onset, and duration and severity of the symptoms can help differentiate between an immediate type 1 (protein-allergen induced) and delayed type 4 (rubber chemical induced) hypersensitivity. Finally, a genetic predisposition for atopic disease or parental history of allergy, chronic infectious or acute viral illness, relative contribution of Th1/Th2 cells to the immune response and the nutritional status of the individual are other potential risk factors.
Diagnostic Laboratory Methods
Analytes that are measured in the clinical immunology laboratory to support the diagnosis and management of patients suspected of having allergic disease are summarized in Box 18-4 . Historically, total serum IgE was used as a diagnostic marker for allergic disease. However, the wide overlap in the total serum IgE levels between atopic and nonatopic populations caused it to be superseded by allergen-specific IgE as the single most important laboratory analyte in the diagnostic work-up for allergic disease. Since 2003, all patients receiving anti-IgE therapy (Xolair) must first have a total serum IgE to determine whether or not they are a candidate for the treatment. According to the Xolair indication, if the patient’s total IgE falls between 30 and 700 kIU/L, (IU – international unit of IgE which is equivalent to approximately 2.4 nanograms of IgE) then the clinician can use the total serum IgE level to compute the starting Xolair dose using package insert criteria.
