23 Diagnosis of allergic diseases

Harald Renz

23.1 Hypersensitivity reaction

The term “hypersensitivity reaction of the immune system” includes various pathological immune reactions that manifest clinically in the form of different illnesses. For the majority, the classification of these reaction types according to Coombs and Gell is still valid. Tab. 23-1 – Hypersensitivity reactions lists the essential pathogenic features and some typical clinical manifestations associated with these types of reactions.

This chapter will focus on:

• IgE-mediated immediate type hypersensitivity reactions (type I)
• Hypersensitivity reactions that cause extrinsic allergic alveolitis due to precipitating serum antibodies (type III)
• Substances (e.g., allergy provoking drugs) that exert their effects via a type IV reaction.

It is clear that the classification according to Coombs and Gell, while now only serving as a general scheme, still retains some value for educational purposes. It is a simplified classification that no longer represents the complex immunoregulatory processes that are now known to be involved. For these reasons, the presentation of diagnostic procedures that follows will focus as much as possible on the clinical entities or the allergen groups.

23.1.1 Atopic diseases

The term atopy describes the genetic predisposition of an individual to develop an IgE mediated allergic reaction during his or her lifetime (Tab. 23-2 – Definitions used in allergology). This definition does not indicate the atopic phenotype that will be expressed in an individual case, the time of life at which the disease will manifest itself, or the type of allergens against which the IgE mediated immune response will be directed. The genetic risk essentially depends on the allergic phenotype of the parents. The maternal allergic situation in particular plays a decisive role since it indicates the presence of possible underlying materno-fetal interactions influencing the development of atopy in the offspring. The degree of genetic risk is summarized in Tab. 23-3 – Genetic risk for atopy.

Numerous epidemiological surveys have indicated a significant increase in the prevalence of allergic diseases in the last few decades, especially in industrialized countries such as Western Europe, North America, and Japan. Indeed, about 20% of the population in these countries is afflicted by an allergic disease.

In general, an allergic reaction can affect any organ, resulting in different clinical manifestations. However, the following three characteristic clinical presentations can be defined that represent the vast majority of disease manifestations:

• Allergic rhino conjunctivitis /1/. This is the most common allergic disease with a prevalence of approximately 15%. It is often the precursor of what is known as the “step-up phenomenon,” which refers to the spread of the allergic reaction to the lower respiratory tract leading to allergic bronchial asthma.
• Allergic bronchial asthma. In many cases, this is a severe chronic disease process. The prevalence of asthma is about 5%.
• Atopic dermatitis, also known as neurodermatitis or endogenous eczema. It is the hallmark of atopy in the skin.

In addition, various other clinical manifestations are encountered (e.g., chronic sinusitis, otitis, and gastrointestinal involvement such as stomatitis, gastritis, enteritis, colic, and constipation). Urticaria and Quincke’s edema (angioedema) may also be caused by allergic reactions; the same applies to migraine.

The complexity of the clinical manifestations illustrates that the differential diagnosis and therefore the history and the in vitro and in vivo clinical laboratory tests are crucially important for confirming the presence of an allergic reaction or for identifying the cause.

The most severe form of an allergic reaction is anaphylactic shock, which often has a lethal outcome within minutes. Classical triggers are insect venoms, but food components may also result in such drastic reactions.

The significant rise in the prevalence of allergic diseases and asthma over the last few decades has been explained by the hygiene hypothesis. This hypothesis is based on the theory that exposure to allergens during the prenatal and postnatal periods has a crucial role in the maturation of a child’s immune response, leading to the development of tolerance to these allergens. The development of tolerance is thought to be favored by long term, high level exposure to microbial antigens, which takes place preferentially in the respiratory and gastrointestinal tracts.

The hygiene hypothesis is based on the premise that patterns of exposure to microbes have changed significantly in the last few decades /2/. Children with low levels of exposure are unable to develop sufficient tolerance, which paves the way for the development of excessive or misdirected immune responses to harmless environmental antigens. This hypothesis is supported by epidemiological studies that have identified sub populations with low allergy rates compared to the general population.

These sub populations include children:

• With older siblings
• Who attended day care in the first year of life
• Who were born and grew up in the former East Germany
• Who had frequent respiratory infections (trivial viral infections in particular)
• Who live on farms.

The hygiene hypothesis is supported by experimental data as well as epidemiological evidence. However, a molecular link has not yet been proven. It is clear, nevertheless, that environmental exposure contributes significantly to the development of allergies and asthma. Associations have also been reported between polymorphisms and allergic phenotypes such as total and allergen-specific IgE, bronchial hyper responsiveness, and atopic dermatitis.

During the different stages of life, various expressions of the allergic phenotype appear; this age dependent relationship is shown in Fig. 23-1 – Expression of allergic phenotype during different periods of life. Food-related allergic reactions play a predominant role early on in life, during infancy and early childhood. Clinical manifestations mainly center on atopic dermatitis and gastrointestinal symptoms. As the prevalence of food related allergies decreases with age, the significance of airborne allergens increases. This is also associated with a shift in organ involvement, manifesting itself as illnesses of the upper and lower respiratory tract. In many such cases, allergic rhinitis can be considered a precursor of bronchial asthma.

23.1.2 Allergens

Tremendous progress has been made in terms of allergen characterization based on molecular and immunoreactive properties. Increasing numbers of allergens have been identified, characterized, and purified. These are the prerequisites for a modern approach to the diagnosis and treatment of allergies. A distinction must therefore be made between allergens whose amino acid sequence has been completely determined, and whose T cell and B cell epitopes are ideally also known, and those allergens that have not yet been characterized to this degree.

The complexity of the situation has resulted in the introduction of a new allergen nomenclature, based on the Latin name of the allergen, that starts with the first three letters of the species name. For instance, Dermatophagoides pteronyssinus is the Latin name of the house dust mite. In the allergen nomenclature, it is referred to as Der p. The main allergens of the house dust mite are then labeled with Arabic numbers e.g., Der p 1, Der p 2, Der p 3. The most important allergen groups are summarized in Tab. 23-4 – Important allergy groups.

Besides allergen characterization, allergen standardization is becoming increasingly important. This is mainly because of the need for quality assurance, which is an integral part of the diagnosis and treatment of allergy. It is the task of the allergen nomenclature subcommittee of the International Union of Immunological Societies (IUIS) to compile this nomenclature in conjunction with the WHO and to make additions as necessary. It is critical that the highest possible purity should be used for every allergen in diagnostic tests. In order to guarantee comparability both between diagnostic in vivo and in vitro tests and between different lots from the same manufacturer, allergen quantification methods must adhere to the relevant international standards. This also applies to the various in vitro diagnostic procedures. A significant improvement in the comparability of test results using materials from different manufacturers has been achieved by the introduction of WHO IgE standards. The diagnostic specificity of the tests depends primarily on the origin of the allergen extracts used, their purity, and their quantification.

23.1.2.1 Diagnostic approach

The German Society for Allergology and Clinical Immunology has published a position paper on in vitro allergy testing that describes the whole area of allergy diagnostics /3/. The following information is a condensed version of this guideline.

Family and personal history

Family and personal history is the starting point of any diagnostic investigation. The aim is to narrow down possible triggers of the allergic reaction. To this end, a detailed analysis is required that should attempt to establish a relationship between clinical symptoms and allergen exposure. Useful tools include an allergy diary, pollen count, and indoor allergen analysis.

Detection of allergic sensitization

This is the second step in diagnosis (Fig. 23-2 – Approach to the diagnostic of allergies). Allergic sensitization implies that an interaction has taken place between the immune system and the corresponding allergen and that a specific immune response to the allergen has occurred, the result of which is the synthesis of allergen specific IgE antibodies. These antibodies can be detected indirectly in vivo by means of skin prick testing.

A positive immediate type reaction occurring within 15–20 minutes is the pathophysiological correlate of sensitization. Cell bound IgE has a much longer half-life (months to years) than that of plasma IgE (2–3 days). This is one of several reasons why results of skin reaction testing may deviate from those obtained by quantitative measurement of serum allergen specific antibodies.

The detection of elevated total IgE is neither sensitive nor specific for atopic disease. Total IgE measurement is therefore, with the exception of elevated cord blood IgE, inadequate in screening for allergy. This test has poor diagnostic sensitivity in the face of good clinical specificity.

23.1.2.2 Specification and estimation of potential allergens

Multi allergen specific IgE screening tests are useful for this. They are firmly established for the groups of food related allergens and for the most important inhalation allergens. The individual allergen composition of the multi tests must always be taken into consideration when interpreting the results of these tests. A negative result rules out allergic sensitization only to the allergens contained in the particular test cocktail. Following a positive screening result, or in parallel with the screening test, measurements of individual specific IgE antibodies can be undertaken.

Under certain circumstances, cellular tests (e.g., histamine release and the lymphocyte stimulation test) are a valuable addition to in vitro diagnostics.

In contrast, the determination of allergen specific IgG antibodies plays only a minor role in the investigation of allergies and shows a poor correlation with the disease process. In addition, the protective effect of allergen specific IgG/IgG₄ antibodies is somewhat questionable.

23.1.2.3 Diagnosis of sensitization

Sensitization to an allergen does not necessarily mean that it is the cause of clinical symptoms. This is most reliably determined by provocation tests. This is particularly true in food-related allergies, in which double blind placebo controlled provocation testing on an inpatient basis has established itself as the gold standard. Useful and partially standardized provocation test procedures have been described for various allergen groups and target organs. In general, such provocation tests should only be performed by clinicians experienced in their use since the development of anaphylactic shock is always possible.

23.1.2.4 Allergic inflammation

The reaction in the target organ results in the development of allergic inflammation, which is characterized by strong activation of effector cells, including eosinophils. Eosinophilia in peripheral blood is therefore typical of allergic inflammatory responses. The detection of eosinophilia, however, is not suitable as an allergy screening test.

As a result of eosinophilic activity, a number of soluble products of these cells can be detected, including eosinophil cationic protein (ECP) and eosinophil protein X (EPX) /4/. These mediators may be suitable for assessing the severity of allergic inflammation, especially in atopic dermatitis and bronchial asthma. The intra individual pattern of these parameters is important for assessing the results.

23.1.2.5 Antigen-specific IgG antibodies

Extrinsic allergic alveolitis is caused by a type III allergic reaction /5/. Antigen specific IgG antibodies in particular play a decisive role due to the formation of antigen-antibody complexes. During this process, complement fragments are generated by complement activation, especially via the classical pathway, and are involved in the development of inflammatory reactions. Pathomorphologically, this reaction correlates with the development of inflammatory necrosis.

Triggering allergens are found especially in dust containing organic particles, which, due to chronic exposure and inhalation, leads to activation of the immune system, resulting in pulmonary damage. The majority of these cases of alveolitis are occupational diseases. Since most of these dust particles, due to their size, will reach the more distal branches of the bronchial system, this disease tends to manifest itself both as a disorder of the smallest airways and as alveolitis.

23.2 Total IgE

23.2.1 Indication

• Diagnosing atopy: measurement of total cord blood IgE in the newborn (this is not useful as a screening test due to its limited diagnostic sensitivity)
• Diagnosing allergies: during infancy, differential diagnosis between atopic dermatitis and seborrheic dermatitis; differential diagnosis between allergic bronchial asthma, chronic rhinitis, and sinusitis
• As an aid to assessing the specific IgE titer
• Extended investigation of allergies: parallel to screening for specific IgE antibodies, as part of the differential diagnostic evaluation of diseases with a possible allergic component such as acute recurrent and chronic urticaria, recurrent angioedema (Quincke’s edema), gastrointestinal symptoms (e.g., gastritis, enteritis) unclear exanthemas, and suspected drug-induced allergies
• Extended investigation of allergies in the case of eosinophilic pulmonary infiltrations, allergic aspergillosis, allergic alveolitis (e.g., farmer’s lung and pigeon breeder’s disease), Wegener’s granulomatosis, and Churg-Strauss vasculitis
• Illnesses associated with eosinophilia or fever of unknown origin (drug related fever)
• Suspected presence of uncommon parasitic diseases in the case of blood eosinophilia of unknown origin and unsuccessful parasite detection such as filariasis, trichinosis, Capillaria philippensis, or tropical eosinophilia; possibly also for monitoring therapy
• Congenital immunodeficiency syndromes: congenital T cell deficiency syndromes, hyper-IgE syndromes, Wiskott-Aldrich syndrome
• Acquired immunodeficiency syndromes: HIV infection
• Graft-versus-host disease and severe burns.

23.2.2 Method of determination

Competitive or immunometric assays using enzymatic, fluorescent, luminescent, or radioactive labels.

23.2.3 Specimen

Serum, secretions: 1–2 mL

23.2.4 Threshold values

Total IgE threshold values

Data expressed in μg/L; values are 95th percentiles; 2.4 μg/L = 1 IU/mL, thresholds according to Ref. /6/.

23.2.4.1 Clinical significance of total IgE in cord blood

The interpretation of a result can only be made if contamination of the cord blood by maternal blood has been definitively ruled out. Elevation of cord blood IgE to ≥ 2.1 µg/L (0.9 IU/mL) is associated with a risk of developing atopy. On the other hand, lower values do not rule out future onset of atopy. For this reason, general cord blood screening for IgE is not recommended. The determination should be reserved for individuals at risk (e.g. those newborns with a family history of atopy). See also Tab. 18.9-5 – IgE increases in non atopic diseases.

23.2.4.2 Elevated total IgE as part of allergy investigation

The highest IgE values are found in atopic dermatitis. Concentrations may reach several tens of thousands of IU/mL /7/. In the case of extremely high IgE values, cellular immunodeficiency must be ruled out as part of the differential diagnosis. Generally, higher total IgE values are found during the pollen season i.e., at the time of allergen exposure. There is a certain correlation between chronic allergen exposure and the level of total IgE. Total IgE elevation, however, does not confirm underlying allergic sensitization, which can only be verified by appropriate in vitro and in vivo diagnostic tests.

Many patients with atopy, especially those with mild or seasonal symptoms, have a normal total IgE. Therefore, normal IgE values in individual cases do not rule out the presence of atopic disease.

23.2.4.3 Elevated IgE in association with immunodeficiency

A number of congenital immunodeficiencies, especially those of the cellular immune system, may be associated with elevated total IgE. Screening tests of the humoral immune system for the diagnosis of immunodeficiency include the determination of total IgE, together with IgG, subclasses, IgA and its subclasses, IgM, and IgD.

In HIV infection, especially during the later stage with pronounced depletion of CD4⁺T cells, an atopy-like syndrome develops, sometimes associated with excessively high IgE levels.

Diseases that are associated with immune activation and skin alterations are also frequently associated with elevated total IgE. These diseases include graft-versus-host reactions and conditions associated with severe skin burns.

23.2.5 Comments and problems

Method of determination

For measuring total IgE in cord blood, an assay with an detection limit of < 0.35 U/mL is required.

Reference interval

It is important to note the wide range of values. IgE production is detectable by the 11th gestational week. In approximately 50% of newborns, IgE is detectable in cord blood. The highest IgE levels in healthy individuals are measured early in life.

Influence factors

A number of factors, including lifestyle and living conditions, have an impact on IgE levels. Active and passive smoking may result in increased IgE.

23.3 Allergen-specific IgE

The detection of specific IgE antibodies indicates an underlying allergic sensitization. The latter may not always correlate with the clinical symptoms. To assess the clinical relevance of allergic sensitization, detailed correlation between the history and clinical presentation is required /8/; organ-specific provocation testing may need to be conducted.

23.3.1 Indication

• Suspected IgE mediated immediate reaction; detection of sensitization
• Inability to perform skin testing due to cutaneous reaction anomalies (e.g., as seen in eczema, dermatitis, factitious urticaria, or systemic glucocorticoid therapy)
• In cases where interruption of antiallergic therapy is not feasible.

23.3.2 Method of determination

Competitive or immunometric assays using enzymatic, fluorescent, luminescent, or radioactive labels /9/.

23.3.3 Specimen

Serum: 1–2 mL

23.3.4 Clinical significance

To interpret the results of allergen specific IgE testing, the following factors must be taken into consideration /10/:

• The various methods of determination are characterized by comparable clinical sensitivity and specificity for most of the allergen groups. In the case of discrepancies, differences are mainly due to the allergenic extract that is used for the antibody determination.
• Assays used to quantify specific IgE antibodies must be calibrated in accordance with WHO standards
• The detection of specific IgE antibodies does not necessarily correlate with positive skin test reactions. Mast cell bound IgE has a much longer half life (months to years) than serum IgE (2–3 days). Thus, a positive skin test is still detectable in individuals with pollen allergy after the end of the pollen season, whereas IgE antibodies may decline rapidly following allergen exposure.
• The specific IgE antibody level may not correlate with the severity of symptoms and the clinical presentation. Higher titers of allergen specific IgE antibodies are found in severe atopic dermatitis and in patients with severe allergic reactions who are receiving inadequate treatment.
• A decline in specific IgE antibody titers during treatment, including desensitization treatment, does not necessarily occur. Furthermore, a decline in specific IgE antibodies does not confirm the success of therapy.

23.3.4.1 Screening tests for specific IgE antibodies

Numerous screening tests are available for detecting specific IgE antibodies directed against food and inhalation allergens. A positive screening test only implies that allergic sensitization to one or more allergens is present. It must be followed up by a detailed analysis in order to narrow down and identify the allergens responsible for sensitization. At this stage, screening tests using reagent strips should be replaced by quantitative determination methods. For the interpretation of a positive screening test, the exact allergen composition of each test must be known. Examples of mixtures used in screening tests are provided in Tab. 23-5 – Allergen mixtures in screening tests.

23.3.4.2 Recombinant allergens

The majority of allergens are glycosylated proteins. In recent years, a large number of protein components for many of the main clinically relevant allergens have been characterized at the molecular level. These components are available in recombinant form for routine allergy diagnosis.

A distinction must be made between the following components:

• Major allergens: components to which more than 50% of allergic patients are sensitized
• Minor allergens: components to which less than 50% of patients are sensitized.

Sequence comparison has revealed the molecular relationships between various allergenic components and defined a small number of important protein families. Sensitization to individual members of a protein family often leads to cross allergies.

The clinical significance of sensitization to individual family members varies. The following examples of selected protein families illustrate this:

• Storage proteins (Tab. 23-6 – Protein family of storage proteins): these proteins are found mainly in seeds and are important for plant growth. Because they are usually stable and heat resistant, they can cause reactions even if the food containing them is cooked.
• Pathogenesis related protein family 10 (PR-10) (Tab. 23-7 – Protein family of pathogenesis-related protein family 10 proteins): because these proteins are heat labile, they can be tolerated in cooked food. Reactions to these components are often associated with an oral allergy syndrome and sensitization is commonly related to cross reactivity between fruit and vegetables, especially in Northern Europe.
• Nonspecific lipid transfer proteins (Tab. 23-8 – Protein family of non specific lipid transfer proteins). These proteins are resistant to heat and enzymatic degradation. Reactions can occur even after food has been cooked. Sensitization to nonspecific lipid transfer proteins is often associated with severe systemic reactions as well as oral allergy syndrome.
• Profilins (Tab. 23-9 – Protein family of profilins). The profilins are actin binding proteins that show significant homology and cross reactivity even between distantly related plant species. They are commonly acknowledged as minor allergens in plants and other foods. Although sensitization to profilins rarely produces clinical symptoms, exceptions have been described. Profilin sensitization is a risk factor for allergic reactions to multiple pollens and food allergens.

If indicated clinically, component-based in vitro testing should be performed, to alert patients to potential cross reactions while also providing an estimate of severity and risk level based on their individual sensitization profile.

Component-based in vitro testing is becoming increasingly prevalent and replacing diagnostic tests that use whole extracts (native allergens) /11/.

23.3.4.3 Pollen allergy

When selecting allergens for the determination of specific IgE antibodies in pollen related allergies, it is important to note that different groups of pollen may cause symptoms during different seasons of the year. Fluctuations occur from year to year that are often attributable to climatic conditions. The predominant pollen responsible for allergies, as listed in Tab. 23-10 – Predominant pollen involved in allergies, is also dependent on geographic factors. Accordingly, pollen seasons at higher, mountainous elevations are different from those in low lying regions. Therefore, when selecting pollen allergens, it is important to take local, seasonal, climatic, and individual circumstances into account /12/. The list of predominant pollens provided is only relevant for continental climate zones.

23.3.4.4 Food allergy

The clinical symptoms of food allergy can be manifold.

The most important symptoms and target organs are shown in Tab. 23-11 – Symptoms associated with food allergy.

The most important food allergens are listed in Tab. 23-12 – Important food allergens.

Approximately 40% of all IgE mediated food allergies are directed against chicken egg white and cow’s milk. Awareness of peanut allergy has also increased over the last few years.

Allergic cross reactions exist between many foods and other products. It is important to remember that in the case of beef allergy, cross reactivity may be present with all other cow’s milk products and veal. Manifold cross reactions also exist in the case of seafood allergies (e.g., involving fresh and salt water fish, shellfish, and crustaceans). Examples are listed in Tab. 23-13 – Cross reactions between allergens of animal food origin.

Many patients with pollen allergy tolerate certain foods poorly, especially during the pollen season, and have type I allergic symptoms. The reason for this is that certain allergy triggering proteins contained in pollen and other plant tissues are similar and close family relationships exist between different plants. Examples of this phenomenon include the “celery-carrot-mugwort” syndrome and the familial relationship between different types of pitted fruit (Tab. 23-14 – Cross allergies between pollen and food).

In the case of food allergies, concordance between the clinical presentation, results of skin testing, and determination of specific IgE is very poor, mainly due to the instability of many food allergens. The most reliable detection of IgE mediated sensitization is therefore achieved by skin prick tests using native foods. Double blind, placebo controlled, provocative food testing in an inpatient setting is the gold standard for the verification of an underlying food allergy /12/.

23.3.4.5 Indoor allergens

The most important indoor allergens are the various species of mites contained in house dust as well as allergens from domestic animals. Tab. 23-15 – Important indoor allergens summarizes the most important representatives. In this context, it is important to remember that a detailed history provides the basis for targeted IgE antibody determinations.

23.3.4.6 Insect venom allergy

The European and North American terminology for the most important allergy relevant insect species is presented in Tab. 23-16 – Insect venom allergens. The detection of an insect venom allergy is based on a positive skin test, the detection of specific IgE antibodies, and possibly an insect sting provocation test.

Therapy consists of specific desensitization. A decline in specific IgE antibodies or an increase in specific IgG antibodies is not sufficient, however, to confirm successful desensitization.

23.3.4.7 Bee venom allergy

The major allergen of bee venom is phospholipase A2 (Api m 1). It also contains hyaluronidase (Api m 2). Many, but not all individuals with bee venom allergy can be identified using Api m 1 component diagnostics; in case of doubt, positivity for Api m 2 antibody should also be tested.

23.3.4.8 Wasp venom allergy

The major allergen of wasp venom is phospholipase A1 (Ves v 1). Wasp venom also contains hyaluronidase (Ves v 2). However, this is considered to be a minor allergen. The combined use of Ves v 1 and Ves v 5 is the most rational and efficient diagnostic approach.

Double positivity

If patients test positive for venom from both bees and wasps, it is important to determine whether this indicates true sensitization to both venoms or is merely the result of clinically irrelevant cross reactivity. In most cases, this is due to nonspecific reactivity against cross-reactive carbohydrate determinants (CCD) with identical structures that are present on venom allergens from both bees and wasps. The production of IgE antibodies directed against these CCDs can lead to clinically irrelevant double positive results in in vitro testing. Commercial assays are available that can identify these antibodies against CCDs.

23.3.4.9 Latex allergy

In order to diagnose a latex allergy, the following factors must be considered:

• Latex has been recognized as an allergen for a relatively long time and was originally associated with type IV reactions and atopic dermatitis. In the last few years, several shock related incidents, especially in conjunction with abdominal and other major surgery, have been reported. Latex absorbed by powder on surgical gloves is considered to be the trigger for these allergic reactions. Several proteins (14 kDa and 21 kDa) have been identified as allergens. Patients with spina bifida and urogenital malformations who have undergone multiple surgical procedures or catheterizations are at particularly high risk of developing latex allergy. Sensitization correlates with the number of surgical procedures. Patients with atopy as well as individuals with certain occupations (e.g., operating theater staff and workers in the rubber industry) are also at high risk.
• The diagnosis of latex allergy is based on the detection of specific IgE antibodies, positive immediate type skin reactions, and provocation testing (i.e., blowing up balloons). Approximately 20% of sensitized individuals are also symptomatic.
• Cross-reactions occur with avocados, kiwis, and bananas
• Negative skin test results or negative specific IgE findings have a negative predictive value of almost 100%.

23.3.4.10 Microbial antigens allergy

Specific IgE antibodies can also be produced in response to microbial antigens. A significant proportion of patients with atopic dermatitis, for example, demonstrate IgE antibodies against enterotoxins of S. aureus. Detection of these antibodies correlates with disease severity, the total IgE level, and colonization with enterotoxin producing S. aureus strains /13/. These antibodies may also have diagnostic significance in the case of nasal polyposis. Diagnostic tests are available for specific IgE directed against bacterial enterotoxins, including the staphylococcal enterotoxins (SE)-A, SEB, SEC, SED, and TSST1.

23.3.4.11 Occupational allergens

An accurate medical history is an essential aspect of the diagnostic evaluation of occupational allergies. Detailed knowledge of possible and potential allergens in the work environment is an absolute prerequisite for identification /14, 15, 16/.

Many of the substances in question are low molecular weight chemical structures that provoke allergic reactions as haptens (i.e., they do not have allergenicity unless they bind to larger proteins). Such haptens cannot be used directly for skin testing. For the determination of specific IgE antibodies to such substances (e.g., isocyanate), the haptens must be coupled, for example, to human serum albumin. Coupled haptens are also available for the determination of specific IgE antibodies.

A comprehensive list of all possible allergen groups and their association with particular occupations or branches of industry is beyond the scope of this chapter.

Only a few examples for these allergen groups are mentioned here:

• Classical allergens such as those deriving from domestic animals, pollen, and various types of mites and insects
• Food, including herbs and spices
• Laboratory animals and enzymes (amylase, proteases)
• Isocyanate, alkaline amine compounds such as peparacin.

Failure to detect corresponding IgE antibodies does not rule out the presence of an occupational allergy. Other reaction types (e.g., IgG-mediated mechanisms and non immunological interactions) must also be taken into consideration.

23.3.4.12 Drug allergies

The clinical presentation of drug induced allergy can be manifold, ranging from type I allergic reactions including anaphylactic shock, through pseudo allergic reactions, to autoimmune reactions (vasculitides). This is why the history is also particularly important in this case.

The manifold clinical symptoms may be based on various immunological reactions that require specific tests /16, 17, 18/. Classical immediate type reactions require skin tests as well as the detection of specific IgE antibodies. Other immunological mechanisms require autoantibody and lymphocyte transformation tests in order to detect T lymphocyte mediated immunological reactions.

It is important to remember that drug metabolites may also trigger a reaction or that a drug may not exert an allergenic effect unless it forms a drug protein complex. This limits the diagnostic value of negative serological tests.

Drugs frequently associated with type I allergic reactions include penicillins, contrast media, and local anesthetics. Furthermore, cross reactivities may exist between benzylpenicillin and amoxicillin in patients who are allergic to penicillin.

The inability to detect specific IgE antibodies may be explained by a prolonged time interval between the last drug intake and the test (e.g., due to the relatively rapid decline in IgE antibodies in the absence of allergen exposure). This is another reason for the poor diagnostic sensitivity of IgE detection methods.

The detection of specific antibodies only provides a clue as to prior sensitization and may not necessarily be associated with clinical symptoms.

23.4 Allergen induced mediator release

Bioassays are used to detect the cellular release of histamine or leukotrienes in the event of contact with allergens.

23.4.1 Indication

• Diagnosis of immediate type allergic reactions; as a supplement to other diagnostic procedures, especially in the case of unclear or equivocal results
• Diagnosis of non IgE mediated immediate type pseudo allergic reactions
• Testing of drugs e.g., aspirin, drug additives, nonsteroidal anti-inflammatory drugs, and native foods as well as individual allergens that are not commercially available.

23.4.2 Method of determination

23.4.2.1 Allergen-induced histamine release

Principle: the test relies on the release of histamine from basophil leukocytes in peripheral blood. Allergen specific IgE antibodies bind to high affinity IgE receptors on the basophil surface. Cross linkage of these allergen specific antibodies as a result of allergen exposure induces the release of mediators from these effector cells. Patient leukocytes (including basophils) are separated from heparinized blood using dextran sedimentation. The leukocytes, as well as a positive control (anti human IgE) and a negative control, are incubated with the suspected allergen. The histamine released into the cellular supernatant is measured. The result is usually expressed as a percentage of the maximal histamine release.

23.4.2.2 Allergen induced leukotriene (LT) release

Leukotrienes are released from different cell populations during an allergic reaction. These cells include basophils, eosinophils, monocytes, and macrophages. As part of IgE mediated allergic reactions (basophils) as well as pseudo allergic reactions (basophils and eosinophils, monocytes), leukotrienes are released following stimulation by the relevant allergens.

Principle: patient leukocytes (including basophils) are separated from heparinized blood using dextran sedimentation. Following a brief preincubation with interleukin-3, which enhances the test sensitivity, the leukocytes, as well as a positive control (anti-human IgE) and a negative control, are incubated with the suspected allergen. In the case of a positive test, leukotrienes (especially LTC4, LTD4, and LTE 4) are released from the cells, either by IgE mediated or other mechanisms. By using monoclonal antibodies that recognize LTC4, LTD4, and LTE4 at almost identical sensitivity and specificity levels, the amount of leukotrienes released can be quantified by ELISA /19/.

23.4.2.3 Basophil activation test

Principle: the BAT starts with the assumption that there is a correlation between basophil responsiveness in samples of peripheral blood and the clinically relevant responsiveness of effector cells such as mast cells and basophils in vivo.

Basophil activation is measured using flow cytometry. Basophils are identified by the following marker combinations: CCR3 or CD123 positive/HLA-DR negative or IgE positive/CD203c positive. Of these, the only really specific marker is CD203c. In a second step, the up regulation of an activation marker is quantified. The marker CD63 has proved to be particularly useful for this purpose. Preformed CD63 exists intracellularly and is transported to the cell surface following activation. CD203c can also be used; however, this is already expressed as a specific marker on the basophil surface and is rapidly up regulated in response to activation. These factors must be taken into consideration when interpreting the test results.

The BAT should be performed no later than 6–12 months after the clinical reaction. Patients should not have taken antihistamines or glucocorticoids in the 24 hours before the test.

The major problem with this test is the relatively high proportion of non responders (5–10%). For this reason, a positive control must also be performed for the IgE mediated reaction (e.g., anti-IgE or anti-IgE receptor antibodies).

The BAT is used as a second-line test after skin testing for the diagnosis of allergies to neuromuscular blockers (anesthesia). With respect to possible cross-reactivity in particular, this test has a high diagnostic specificity in the face of rather poor diagnostic sensitivity.

In the diagnosis of allergies to beta lactam antibiotics, IgE measurement and the BAT show comparable results with respect to diagnostic sensitivity and specificity. The BAT is particularly useful in the event of discrepancies between the history, skin test results, and IgE measurement. It does not have a role in the diagnosis of allergies to nonsteroidal anti-inflammatory drugs.

23.4.3 Specimen

Heparinized blood: 10 mL

23.4.4 Reference interval

Allergen-induced histamine release

Reference intervals must be established by each laboratory with appropriate positive and negative controls.

Leukotriene production

The basal value in non allergic blood donors is 154 ± 8.3 pg/mL (x ± s).

23.4.5 Clinical significance

A positive result in one of the two assays is of great prognostic value for the presence of underlying sensitization that may also be clinically relevant. However, correlation with the anamnesis and clinical picture must always be present.

A positive result does not necessarily imply the presence of an IgE mediated reaction. Mediators may also be released by other, IgE independent reaction pathways. The leukotriene assay in particular detects not only the reactions of basophils but also those of other effectors of the immune response that may become involved in the pathophysiological process /19/. In general, however, false negative results are rarely seen in conjunction with plausible positive and negative controls.

Cellular procedures also have the advantage of detecting pseudo allergic reactions and have a wide range of application for unconventional reagents and substances.

23.4.6 Comments and problems

Method of determination

Both histamine release test and leukotriene release test place high demands on personnel and technical resources. Staff experienced both in performing the tests and interpreting the results are essential. These procedures are therefore not the method of first choice in the investigation of allergies. They play a role in more extensive diagnostic evaluations, especially in cases where there is poor correlation between other tests, the history, the clinical picture, and in vivo results.

Since the tests are characterized by high imprecision, it is crucial to perform positive and negative controls at the same time. Due to the complexity of these tests, many factors may cause interference, ranging from cell separation to impaired mediator measurements.

23.5 Eosinophil cationic protein (ECP)

The presence of eosinophil leukocytes, in addition to local infiltration by lymphocytes (in particular highly activated CD4⁺T cells), is critical for the immuno pathogenesis of allergic diseases. Eosinophils are the most important inflammatory cells in allergic inflammation; they are found in large numbers in the mucosa of the upper and lower respiratory tract. They are also detectable in bronchial lavage fluid. Products of these cells, such as ECP, are found in the skin of patients with atopic dermatitis. The essential characteristic of these cells is their ability to release mediators that exert cytotoxic effects as part of the inflammatory reaction, thus contributing significantly to the destruction of tissue /20, 21/.

There is growing evidence that the quantitative determination of these mediators may allow an assessment of the degree and extent of the inflammatory response occurring within the target organ of allergic disease. ECP represents a prototype of these cytotoxic proteins.

23.5.1 Method of determination

Whole blood samples are allowed to clot under standardized conditions (1 h at 20° ± 1 °C). This induces the release of ECP from pre-activated eosinophils by mechanisms not yet fully understood. ECP measurement is also possible in other biological fluids (e.g., lavage fluid). ECP in serum or lavage fluid is determined by immunoassay.

23.5.2 Specimen

Serum following standardized coagulation, other body fluids: 1 mL

23.5.3 Threshold value

Determined by fluorescence immunoassay

23.5.4 Clinical significance

Since ECP is not released from pre-activated eosinophils unless blood coagulation has started, ECP determination allows an assessment of the degree of cellular activation in peripheral blood. This may not necessarily correlate with the degree of activation within the target organ (e.g. skin, lung, or nose). Likewise, there is no correlation with the eosinophil count in the peripheral blood.

Basal values show large inter individual variation. The determination of ECP is therefore only useful in monitoring patients with severe allergic diseases.

Only longitudinal assessment provides useful information regarding the ongoing disease process. The determination of ECP is therefore suitable for monitoring disease activity and treatment in a certain group of patients e.g., atopic dermatitis /22/. This also applies to desensitization therapy, during which a decline in the concentration of ECP occurs.

23.5.5 Comments and problems

The major difficulty is the collection of blood samples under standardized conditions. Fluctuations in temperature and time are of particular concern. Using different blood collection equipment can also cause interference.

23.6 Quantification of indoor allergens

The extent of allergen exposure contributes significantly to the risk of developing allergic sensitization and associated symptoms. Allergen elimination represents an essential therapeutic step in the management of allergic diseases.

It is therefore important to perform a quantitative as well as a qualitative assessment of the allergen exposure of patients. In recent years, allergens and their components (the so called major allergens) have been progressively better characterized, thus facilitating the development of monoclonal antibodies directed against them. It is therefore currently possible to quantify the allergens contained in various specimens by using immunoassays /23/. These tests thus provide the basis for detailed environmental allergen analyses for the most important indoor allergens.

23.6.1 Indication

• Quantitative determination of exposure to important indoor allergens in patients with asthma, perennial allergic rhinitis, and atopic eczema
• Monitoring of measures for eliminating and avoiding allergens
• Research related aspects of the immunology and epidemiology of allergic diseases
• Quality control and standardization of allergen extracts.

23.6.2 Method of determination

ELISA using monoclonal antibodies directed against major allergens.

23.6.3 Specimen

Dust samples (e.g., acquired from mattresses, pillows, carpets, and upholstery).

23.6.4 Reference interval

Refer to Tab. 23-17 – Assessment of indoor allergens.

23.6.5 Clinical significance

Factors to consider when assessing measurements of important indoor allergens are listed in Tab. 23-17. The threshold values shown here can, at present, only be regarded as indicators for the development of allergic sensitization or the risk of an immediate type allergic reaction. Standardization of specimen collection (e.g., by vacuum cleaning a mattress for ten minutes) is an absolute prerequisite for test comparability.

Detailed standardization protocols have yet to be established. Allergen extraction is necessary before a specimen can be used in the ELISA; different manufacturers recommend various procedures for achieving this. Prior to allergen extraction, the specimen must be weighed in order to determine the concentration.

23.7 Allergen specific IgG

Type I reaction

The measurement of allergen specific IgG antibodies is of only minor significance in the diagnostic evaluation of immediate type allergic reactions. Although detection of these antibodies indicates the presence of immunological sensitization, it does not correlate with clinical symptoms. In addition, the relevance of these antibodies for the disease process has not been reliably established.

IgG₄ antibodies play a certain role in the pathogenesis of allergic diseases because they are controlled and regulated by immunological mechanisms similar to those that control and regulate IgE antibodies. There is a lack of conclusive data as to whether allergen specific IgG₄ antibodies are clinically more sensitive and specific than their IgE counterparts.

During desensitization therapy, especially in patients with allergies to insect venom, a decline in IgE antibodies is frequently associated with an increase in the corresponding IgG antibodies. Furthermore, IgG antibody measurement can only provide clues as to the success of desensitization therapy; increasing IgG antibody concentrations alone cannot be seen as proof of therapeutic success.

23.7.1 Method of determination

Ouchterlony double diffusion, immunoelectrophoresis, electroimmunodiffusion, passive hemagglutination, and immunofluorescence

23.7.2 Specimen

Serum: 2–3 mL

23.7.3 Clinical significance

The detection of specific IgG antibodies provides evidence for allergic sensitization but it is not an indication of clinical relevance. Its diagnostic value is increased by repeated determinations and evaluation of the IgG antibody pattern /24/.

The antigen composition is very important to the diagnostic usefulness of the test. If the allergen preparation does not contain the crucial antigenic determinants, a positive result will not occur. This is why the antigen selection is particularly important.

23.7.3.1 Extrinsic allergic alveolitis

Extrinsic allergic alveolitis is characterized by inflammation of the alveoli and pulmonary parenchyma whereas in bronchial asthma, an allergic reaction occurs in the airways /25/. It develops as a reaction to prolonged antigen stimulation in predisposed individuals. As a prerequisite for the development of the disease, the inhaled particles with a size of 3–5 μm must be able to reach the bronchioli or alveoli in order to initiate an immunological reaction. The immunological correlate of this process is a type III allergic reaction.

A crucial element for the initiation of this process is the synthesis of allergen specific IgG antibodies that form immune complexes with antigens, thereby activating the complement cascade. Supported by activated complement factors, such antigen-antibody complexes can be phagocytozed by macrophages, which are major participants in the development of the inflammatory reaction.

In addition, specific lymphocytes are stimulated and can be detected in the broncho alveolar lavage. These are mainly CD8⁺T cells and NK cells, in contrast to the CD4⁺T cells observed in allergic asthma.

Since many of the offending antigens are present in organic dusts and long term allergen exposure is a prerequisite for the development of allergic sensitization, it is not surprising that many of these clinical allergies are diagnosed in the context of occupational diseases. The range of triggers for extrinsic allergic alveolitis is also correspondingly wide (Tab. 23-18 – Selected allergens in extrinsic allergic alveolitis: occurrence and type of clinical presentation).

Extrinsic allergic alveolitis is a chronic disease in which the inflammatory process is initially reversible but advanced conditions such as pulmonary fibrosis can develop during the chronic stage.

The working group for extrinsic allergic alveolitis of the Guidelines for diagnosing extrinsic allergic alveolitis has defined diagnostic criteria for the disease. To firmly establish the diagnosis, the following three criteria must be fulfilled:

• Confirmed or probable exposure
• Respiratory or systemic symptoms
• Detection of antigen specific sensitization.

In addition, one of the following four criteria must also have been met:

• Objective evidence of impaired pulmonary function
• Abnormal findings on chest X-ray
• Positive inhalation provocation test
• Positive broncho alveolar lavage result.

These criteria demonstrate that crucial significance is attributed in the diagnostic evaluation to the detection of antigen specific sensitization (allergen specific IgG) /26/. At the same time, however, the detection of specific IgG antibodies in parallel to the detection of allergen specific IgE antibodies is only a reflection of corresponding sensitization. The clinical relevance of these findings must be established by performing appropriate additional investigations.

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