Section 2 Allergy Essentials

#### **Chapter 2**

## Fundamentals of Molecular Allergy: From Bench to Bedside

*Henry Velázquez-Soto and Maria C. Jimenez Martinez*

#### **Abstract**

This chapter describes the fundamentals of molecular allergy diagnosis and raises the concept of allergens, allergenic components, and recombinant allergens. In addition, the authors review quality aspects related to the laboratory methodology. In the last part of the chapter, the different singleplex and multiplex platforms currently used for molecular diagnosis are compared. Finally, the diagnostic systems' challenges, strengths, and pitfalls are discussed to understand their clinical impact. Undoubtedly, this chapter will be handy for the background knowledge for health personnel, allergists/immunologists, and clinical laboratory personnel to guide the selection of diagnostic tests for allergy as well as their interpretation and therapeutic approach.

**Keywords:** molecular allergy, laboratory tests, allergens, allergy diagnostic

#### **1. Introduction**

Allergies are one of the most prevalent diseases affecting almost one billion people worldwide [1]. The traditional approaches for identifying allergen-specific IgE have undergone a revolution as a result of the growing need for the best diagnostic techniques, technological advancements, and understanding of allergen structure and obtention. These technical and scientific advances are the fundamentals of molecular diagnosis and precision medicine in allergy [2, 3].

#### **2. Principles of laboratory testing for molecular allergy**

Immunoglobulin E (IgE) is one of the five immunoglobulin isotypes described in humans and is considered to mediate hypersensitivity type I reactions and be the main soluble molecule involved in allergy pathology [4]. This immunoglobulin has historically been recognized as a biomarker for allergic processes. Due to its feasibility to detect and measure in serum samples, several laboratory methods focus on the identification of total IgE (tIgE), and specific IgE (sIgE) [5].

Measurements of tIgE and sIgE are based on antigen-antibody reactions. For tIgE detection, an anti-IgE antibody (detection antibody) will bind to the fragment crystallizable region in the immunoglobulin E. For sIgE, the serum sample is incubated

with the allergen-coated surface before incubation with the detection antibody, thus allowing allergen-specific IgE to be detected. Finally, the reaction is detected according to the platform methodology: radiation, colorimetry, fluorometry, or chemiluminescence [6–8].

#### **2.1 Units and equivalences**

Serum IgE is usually found in very low concentrations ranging <1μg/mL. Most immunoassay systems now use a total calibration curve that is associated with the World Health Organization (WHO) IgE standard and reported in arbitrary units; for better comprehension, tIgE is reported in IU/ml or kIU/L, which is equivalent to 2.4 ng/ml; [9] while sIgE is reported in kUA/L (kUA/L kilo mass units of allergenspecific antibody per unit volume) [10].

#### **2.2 Methodologies for tIgE and sIgE determination**

The evolution of methods for IgE diagnostic comprises methods like Radio-Immuno-Sorbent-Test (RAST), Paper-Radio-Immuno-Sorbent-Test (PRIST), and Enzyme-Linked Immuno-Sorbent-Assay (ELISA), gave rise to more reliable, safe, and automatized methods. A deeper revision of these methodologies could be found in [6].

#### **2.3 Current methodologies used for sIgE determination**

#### *2.3.1 Enzyme-linked-immuno-sorbent assay (ELISA)*

ELISA protocols are based on colorimetric reactions. Allergen is bound to the plate, then the sample of the patient containing IgE is incubated, allowing it to react with the allergen, forming the first antigen-antibody complex. Then a secondary antibody linked to an oxidizing enzyme binds to the previously formed complex, and by addition of the substrate, the color begins to develop. Finally, the plate is read in a spectrophotometer to detect the absorbance, which is proportional to the sIgE concentration (**Figure 1**) [11].

#### *2.3.2 Immunoblot*

For immunoblot-based methods, antigens are bound to a polymeric membrane acting as the solid phase, allowing IgE to interact with the different allergens. Then a phosphatase alkaline-linked secondary antibody is added to the reaction. Finally, the substrate precipitates leaving colored marks in the spots where patient´s IgE reacted with allergen (**Figure 1**) [12].

#### *2.3.3 Chemiluminescence*

The method for chemiluminescence platforms is very similar to ELISA. Alkaline phosphatase, which is linked to the secondary antibody, produces chemiluminescence signals when it reacts with its substrate, the phosphate ester of adamantyl dioxetane. In this method, the intensity of chemiluminescence is proportional to sIgE concentration (**Figure 1**) [13].

*Fundamentals of Molecular Allergy: From Bench to Bedside DOI: http://dx.doi.org/10.5772/intechopen.107512*

#### **Figure 1.**

*Fundamentals of current methods for IgE detection. Different techniques are used to determine tIgE and sIgE in patients' samples; all of them are based on Ag-Ab reaction.*

#### *2.3.4 Fluoro-enzyme-immunoassay (FEIA)*

In FEIA techniques, the secondary antibody is coupled to galactosidase, which reacts with 4-methylumbelliferyl βgalactoside to generate fluorescence proportional to the amount of specific IgE in the sample (**Figure 1**) [14].

#### **3. Singleplex platforms for IgE determinations**

Singleplex platforms permit the determination and quantification of tIgE or sIgE. In the case of sIgE determinations, these instruments identify one allergen per reaction.

Singleplex devices usually include the following components: [2, 14]


#### **3.1 ImmunoCAP Phadia by ThermoFisher**

It was the first automated platform using FEIA as the operating principle, showing high concordance with RAST in its results. Phadia 250 has a processing capacity of 60 tests per hour and allows the use of native and recombinant allergenic components which are grouped within the ImmunoCAP line, such as grass pollens, weed pollens, tree pollens, microorganisms, animal proteins, and mites, among others [15, 16].

#### **3.2 Immulite by Siemens**

Immulite is an IgE detection platform based on chemiluminescence. This equipment determines a variety of allergens from animals, drugs, food, grasses, insects, mites, mold, parasites, trees, and weeds, among others. It also includes a panel of 26 recombinant allergenic components. Immulite 2000 is capable of processing up to 200 results per hour and with a sensitivity of up to 0.1 kU/L [17, 18].

#### **3.3 Hytec 288 by Hycor Biomedical**

Hytec 288 is an immunoassay instrument based on ELISA. This platform offers the determination of single allergens and allergen mixture from drugs, food, grasses/ weeds, animal proteins, among others. This equipment could perform up to 288 tests per run [19].

These three platforms are the leaders in the global market and exhibit excellent analytic sensitivity, precision, reproducibility, and linearity in total and allergenspecific IgE assays, but some variability in allergen-specific IgE quantitative estimates [16, 19].

#### **4. Multiplex platforms for IgE determinations**

Multiplex immunoassays allow for the identification of IgE sensitization repertoires against a diverse set of allergens. In contrast to singleplex platforms, the results are semiquantitative and not interchangeable. Characteristics of both platforms can be seen in **Table 1.**

#### **4.1 Immuno solid-phase allergen chip (ISAC), by Thermo Fisher**

The immuno solid-phase allergen chip (ImmunoCAP-ISAC) was the first multiplex platform designed and approved for IgE identification. This platform is based on the FEIA on-chip methodology, which can identify up to 112 allergenic components from 48 different allergen sources in approximately 4 hours. The ISAC system employs ISAC standardized units (ISU-E) ranging from 0.3 to 100 ISU-E, equivalent


**Table 1.**

*Comparison between singleplex and multiplex platforms.*

to 0.3–100 kUa/L, to categorize sIgE concentration into four groups: undetectable or very low (0.3 kUA/L), low (0.3 to 13 kUA/L), moderate to high (13 to 153 kUA/L), and very high (153 kUA/L) (**Figure 2**) [11, 20].

#### **4.2 EUROLINE by Euroimmune**

Euroline is a semiquantitative based immunoblot instrument. It provides precoated membranes for detecting sIgE from various allergen sources. These precoated membrane panels are tailored to the clinically relevant allergens in the regions where these are commercialized. Interestingly, this platform offers reagents for diminishing cross-reactive carbohydrate determinants (CCD), improving sensitivity. The number of allergens detectable in one membrane varies depending on the panel in use (8–45 allergens). The results can be obtained in a lapse of approximately two hours [21].

#### **4.3 Allergy Explorer (ALEX) by Macro Array Diagnostics**

The Allergy Explorer platform was the first to use an ELISA-based methodology to determine tIgE and sIgE levels for 117 extracts and 178 recombinant allergens at

#### **Figure 2.** *Immuno solid-phase allergen chip (ISAC). Multiplex immunoassay based on FEIA methodology.*

the same time. This device can block the determination of clinically irrelevant sIgE directed against CCD. The platform has manual and automated processing formats, with the capacity to analyze up to 50 patients in an approximate time of 4 hours. ALEX contains pre-designed panels by a group of symptoms or group of allergens, such as grass pollens, dander allergens, epithelium of animals, mites and cockroaches, molds, and yeasts.

The results of the tests are presented graphically, including the allergen's name, the specific allergen component or extract, the biological function, and the reported sIgE concentration in kUA/L. The final report includes a demonstration of possible cross-sensitization as well as interpretation and medical follow-up recommendations for the treating physician. ALEX employs a classification based on the concentration of sIgE obtained: Negative or uncertain (0.3 kUA/L), low (0.3 to 1 kUA/L), moderate (1–5 kUA/L), high (5–15 kUA/L), and very high (> 15 kUA/L) (See **Figure 3**) [22].

These three instruments evaluate the eight most common allergen families: Bet v 1-related protein (PR-10); Venom group 5 allergen family; Cupin Superfamily; EF-hand domain (Ca++ binding proteins); Expansin C-terminal domain; Lipocalin; Profilin; and Prolamin superfamily [20–22].

Although evaluated in different allergic diseases with patients sensitized to different allergens its performance, sensitivity and specificity have been reviewed and tested by different authors (**Table 2**) [8, 23, 24].

#### **5. Allergens, allergenic extracts, and allergen components.**

As mentioned above, laboratory diagnosis relies on antigen-antibody reactions, with the allergen defining the IgE specificity. Therefore, it is essential to emphasize the concepts of allergen, source, and obtention methods.

#### **5.1 Allergens**

Allergens are any molecule that binds to IgE antibodies [25]. Allergens are immunogenic antigens that induce a robust Th2 response, characterized by high IL-4 and IL-13 production with secretion of IgE [26].

**Figure 3.** *Allergy Explorer (ALEX). Multiplex immunoassay based on ELISA technique.*


#### **Table 2.**

*Comparison of multiplex platforms.*

#### **5.2 Allergen extracts**

Allergen extracts (AEs) are complex mixtures of allergenic and nonallergenic molecules, including proteins, lipids, saccharides, nucleic acids, lipids, low molecular weight metabolites, pigments, and salts. AEs are obtained from natural sources such as pollens, animals, and insects, using physical methods (grinding) or chemical methods (solvents). Based on their intended application, allergen extracts should be characterized and subjected to quality control. As a result, validated assays must be developed to ensure the presence of relevant allergens for diagnostic or therapeutic applications (**Figure 4**) [27, 28].

#### **5.3 Allergen components**

Allergen components are isolated proteins derived from a purified extract of a specific allergenic source. These allergens, whether native or recombinant, are generally homogeneous and subject to stringent quality control [14].

Recombinant allergens are the most effective approach for obtaining allergen components. These highly pure allergens are produced by biotechnology; the process begins with cDNA obtention from mRNA through reverse transcription. Then, the cDNA may be modified (point mutations, chimeras/hybrids, fragmentation, oligomerization) to obtain the most accurate allergen molecule. Subsequently, the cDNA is inserted into expression vectors, usually *E. coli*. or *P. pastoris*, to express the protein and obtain the recombinant allergen. The allergen is then isolated, purified, evaluated, and validated for its usage in diagnostic platforms or to be used as a hypoallergenic allergens for immunotherapy (**Figure 5**) [29].

#### **5.4 Structural importance of allergens**

#### *5.4.1 Proteins*

Proteins constitute the vast majority of allergens, but only a few allergens bind IgE antibodies in the serum of most allergic patients. These molecules are known as "major allergens." A major allergen is defined as an antigen that binds to IgE in 50%

#### **Figure 4.**

*Obtention of allergen extracts and allergen components from allergenic sources. Different techniques are used to obtain allergen extracts from diverse allergen sources. Most allergen extracts contain sensitizing allergen, allergenderived materials, non-allergenic components, and contaminants. Following the obtention of allergen extracts, allergen components are isolated and purified, and protein characterization is performed.*

or more of clinically allergic patients' serum. Other antigens that account for less than half of IgE binding are known as "minor allergens." Identifying major allergens has aided in understanding the immune response during allergic reactions, sensitization in atopy, and diagnostic applications.

#### *5.4.2 Carbohydrates*

Specific IgE antibodies for oligosaccharides are present in some patients, these antibodies cause numerous cross-reactions in vitro, given the designation crossreactive carbohydrate determinants (CCDs). However, in recent years, oligosaccharide epitopes have been implicated in allergic sensitization, acute allergic reactions, and not just cross-reactions; consequently, characterization and discovery of glycan allergens have been a challenge. Currently, there are about approximately 20 oligosaccharides found in pollens, venoms, nematodes, worms, and ticks that are distributed in five glycans groups and have been shown to be significant for allergic disease [30].

*Fundamentals of Molecular Allergy: From Bench to Bedside DOI: http://dx.doi.org/10.5772/intechopen.107512*

#### **Figure 5.**

*Obtention of recombinant allergens. Recombinant allergens are obtained by isolating the mRNA from the allergenic source. Then transcribed into cDNA and inserted in bacteria or yeasts to allow its expression. Finally, clinical validation is needed to be used for diagnosis in vitro or in vivo.*

#### *5.4.3 Major groups*

Group A. Cross-reactive carbohydrate determinants. Most CCDs are N-glycans, characterized by a basic structure of two GlcNAc with two or three terminal mannose residues. Allergens with these glycans are *Ole e* 1, *Api g* 5, *Bla g* 2 [30, 31].

Group B. Mammalian non-human oligosaccharides. The glycan structures described in this group are the disaccharide galactose-alpha-1,3-galactose, and the monosaccharide N-glycolyl neuraminic acid. These glycans are related to anaphylaxis and could be fond in red meat, tick bites, and some monoclonal antibodies [32, 33].

#### *5.4.4 Minor groups*

Group C. Oligosaccharides with O-linkage. O-glycans are oligosaccharides attached to serine or threonine residues on a protein and sometimes to tyrosine, hydroxylysine, or hydroxyproline. Examples of allergens expressing O-glycans are *Art v* 1, *Amb a* 4 [32].

Group D: Oligosaccharide Epitopes expressed on Schistosomes and other Helminths. These oligosaccharides have a single terminal galactose or N-acetylgalactosamine residue (GalNAc), keeping a molecular similarity to CCDs. Their clinical significance is still under study since alpha-1, 3-fucose epitope could be implicated with a paradoxical protective effect in asthmatic patients [34].

Group E. Short-chain galactooligosaccharides (GOS). GOS are usually produced by bacterial beta-galactosidase and occur naturally in milk processed with prebiotics. They are typically a chain of 2 to 6 galactose molecules attached to glucose and have been recognized in allergic reactions [30, 32].

#### *5.4.5 Lipids*

Lipid antigens are much less understood than carbohydrate antigens, they have been shown a direct effect on allergenic potential and cause allergic responses. For example, lipids delay the enzymatic digestion of Ara h 8 allowing this molecule to reach the intestinal immune system and favoring sensitization. Conversely, lipid-associated allergens such as Der p 2, Der p 5, and Der p 7 have been related to increased asthma symptoms and severe allergic reactions [35]. Thus, the application of sIgE determination against lipids is limited.

#### **6. Interpretation, clinical applications, and limitations for molecular allergy**

Even though the first cases of pollen-induced hay fever were documented in the early 1800s, it was not until 100 years later that a relationship to a serum factor called reagin was discovered (IgE) [4]. In the mid-1960s, allergy diagnosis was primarily relied on skin testing, and allergen extracts were far from standardized. However, developing recombinant allergens and starting allergen cloning between 1988 – 1995 created new opportunities for studying and diagnosing allergy disorders [29, 36, 37]. Molecular allergy is the practical application of these advances, allowing us to manage patients with high accuracy, and leading into the era of precision medicine.

#### **6.1 Singleplex** *vs***. multiplex immunoassays**

As previously discussed, singleplex assays allow detection of IgE antibodies specific to the allergens identified in the patient's clinical history. A multiplex platform, in contrast, enables defining a person's IgE reaction to the whole range of allergens arrayed on a chip.

The main benefit of the singleplex immunoassays is that it measures the allergenspecific IgE antibody level in kilounits per liter (kUA/L) based on a total IgE calibration system that can be traced back to a human reference preparation from the WHO. The assay has high precision and reproducibility, reporting values as low as 0.1 kUA/L (range, 0.1–100 kUA/L), without interference of allergen-specific IgG antibodies.

Compared to multiplex immunoassays, singleplex assays have fewer allergen molecules available, give an incomplete IgE reactivity profile with just one or a few tests, are more expensive if more than one measurement needs to be taken, and need a larger amount of serum [38]. In contrast, multiplex assays are semiquantitative and provide a comprehensive IgE pattern using only a small volume of serum, which could be useful in the evaluation of polysensitized patients; but are only available in laboratories with high-end machinery with highly trained personnel, delaying results by days or weeks **(Table 3)** [39, 40].

Molecular immunoassays have some advantages over *in vivo* assays, such as the ability to be performed regardless of extensive skin disease or medications used, minor pain or anxiety- provoking in children, little patient cooperation required, and no risk to the patient. The fact that the whole allergen of a fresh allergen is more sensitive than purified allergen components is one of the limitations of molecular diagnosis compared to *in vivo* evaluation, this is particularly important if the goal is to perform allergen-specific immunotherapy [39, 40]. In contrast, advances in molecular allergy have enabled the development of vaccines based on recombinant DNA technology and synthetic peptide chemistry that could be monitored with sIgE or sIgG determinations throughout treatment [41].

#### **6.2 Clinical allergy** *vs***. sensitization**

The majority of allergens, but not all, are sensitizing, which is defined as the capacity to induce allergen-specific IgE antibodies. Non-sensitizing allergens can only cause allergic symptoms if the individual has been sensitized to a cross-reactive allergen [3]. Cross-reactivity defines an antigen attribute intrinsically related to the allergen molecular characteristics that determine immune recognition by IgE.


**Table 3.**

*Variables to consider when the molecular diagnosis is selected for the clinician: Singleplex vs. Multiplex instruments.*

Identification of cross-reactivity is critical to detect patients with a high risk of anaphylaxis for example in peanut and tree nuts or seeds allergy. Other cases of cross-reactivity are latex and food, that is, banana, avocado, kiwi, and chestnut; and cross-reactivity between shellfish and insects due to chitins, specifically tropomyosin in dust mites

The precise point at which a sensitizing allergen causes clinical symptoms is determined by several factors such as quantity, exposure route, antigen structural characteristics, genetics, microbiota, innate or adaptative immune interactions, and microenvironment, among others. Thus, identifying an IgE-mediated mechanism is a critical step that directs avoidance measures and suitable pharmacological treatment. However, positive skin tests or specific IgE assay results do not always indicate that an allergen is causing symptoms; the clinical significance of allergen exposure and its relationship to symptoms must be established by examining the patient's medical history.

#### **6.3 Allergen extracts** *vs***. recombinant allergens**

Although diagnostic assays based on purified recombinant allergens are becoming more popular, extracts from natural allergen sources continue to be widely used. The composition of an allergenic extract has a significant influence on the results of any IgE-based immunoassay.

Allergen extracts used in some platforms are made up of a variety of allergens, some of which have little or no clinical significance, such as carbohydrate epitopes in peanut or timothy grass pollen, which might result in false positive findings [38]. The use of allergenic extracts allows to precisely detect the specificity of the IgE in a patient's sample, but also permits the evaluation of only clinically relevant components from allergenic sources.

In the other hand, protein characterization of allergens has been fundamental to understand IgE cross-reactivity data in the absence of allergen-antibody complexes. Some of the benefits of recombinant allergens include increased diagnostic accuracy, the ability to distinguish genuine sensitization from cross-reactivity, the ability to evaluate the type and risk of an allergic reaction, and the ability to select patients and suitable allergens for immunotherapy [29, 42].

### **7. Conclusions**

Personalized therapy based on genetic, immunologic, and functional endotyping, defined as the examination of a biological or pathological process, including therapeutic response through biomarkers determination, is part of the new treatment advances for allergy patients known as precision medicine. As previously discussed, a correct diagnosis is critical in these therapeutical approaches. In the case of molecular allergy, the choice of testing is influenced by several variables, including test accessibility, clinical history, technical constraints, type of allergen, immunoassay accuracy, single or multiplex platforms, and most importantly, the clinical question that the analysis pretends to resolve.

Finally, despite molecular diagnosis is an excellent tool for selecting the appropriate allergens for immunotherapy, minimizing potential test-related complications, evaluating polysensitization with difficult interpretation, and possibly predicting clinical outcomes. Unfortunately, the high cost, access limited in low-income countries, restricted availability due to regulatory affairs in others, and a lack of sufficient clinical studies with recombinant allergens keep molecular allergy out of reach for routine use, but with a promising future once these limitations are overcome.

#### **Acknowledgements**

Thanks to Jesús Mendoza for designing and drawing the chapter figures.

### **Funding**

This work was partially supported by the Conde de Valenciana Foundation and the Department of Biochemistry, Faculty of Medicine, UNAM.

### **Conflict of interest**

The authors declare no conflict of interest.

*Fundamentals of Molecular Allergy: From Bench to Bedside DOI: http://dx.doi.org/10.5772/intechopen.107512*

#### **Author details**

Henry Velázquez-Soto1 and Maria C. Jimenez Martinez1,2\*

1 Department of Immunology and Research Unit, Institute of Ophthalmology "Conde de Valenciana Foundation", Mexico City, Mexico

2 Faculty of Medicine, Department of Biochemistry, National Autonomous University of Mexico, Mexico City, Mexico

\*Address all correspondence to: mcjimenezm@facmed.unam.mx

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Aerobiology in the Clinics of Pollen Allergy

*Franziska Zemmer and Fatih Ozkaragoz*

#### **Abstract**

Diagnosis and treatment of pollen allergies is facilitated by the cooperation between the allergist and the aerobiologist. The selection of relevant allergens for in vivo diagnosis, the interpretation of results, the timing of trials, and treatments should be related to the local pollen season, abiotic variables, and the patient history. Meteorological aspects and flowering dynamics of plants condition the course of the pollen season each year. Pollen forecasting integrates weather data with long- and short-term pollen data. Crowdsourced patient symptoms are used to delineate pollen threshold loads in the forecast. Integrating aerobiological expertise warrants the success of allergy diagnostics and treatment.

**Keywords:** Pollen allergy, diagnostics, allergenicity, aerobiology, forecasting, pollen thresholds

#### **1. Introduction**

With the establishment of skin testing to diagnose for allergy, botanical knowledge on allergenic pollen sources became essential. The synergistic cooperation between allergists and botanists from those years shall be illustrated with an example from Ankara (Turkey), where in 1967 the botanist Kamil Karamanoglu and allergist Kemal Özkaragöz issued lists of allergenic plants, the way their pollen is carried (by wind or insects), their form of growth, and where and in which plant communities they grew [1]. Based on that knowledge, clinical trials were commenced to investigate the allergenic potential of pollen [2] based on the Thommen's postulates [3]. Subsequently, the first Turkish pollen calendar was published for Ankara [4].

Nowadays, pollen information is issued regularly by competent monitoring intuitions as preventive measure for the allergic population in many countries. According to biogeographic peculiarities, the vegetation cover differs from region to region giving rise to varying types of pollen allergens. Which allergens to use in test batteries grounds on the knowledge of the allergenic pollen flora of the area. Immunotherapy, the only treatment that may lead to longer-lasting relief of the burden, can only be successful by considering the geographic circumstances of the patient. That means to work with relevant and good quality pollen data, have information on the course and intensity of the pollen season, and be able to interpret this information. The aerobiologists' point of view on immunotherapy and the implementation of clinical trials has been elaborated, and standards for aerobiological tasks in clinical trials proposed [5].

This to prevent harm to patients, increase the quality and success of the trial, and to obtain comparable results (ibid).

Electronic health technology (eHealth) and mobile applications (mHealth) are, on one hand, a popular way to convey pollen information to the public. Registered users, on the other hand, provide data on their symptoms, which can be used to improve forecasting, monitor public allergy morbidity, and be part of studies on pollen allergy [6]. Pollen threshold loads for symptom development, for example, can be assessed this way along with aerobiological data [7].

In this chapter, we elaborate, firstly, on the allergist's perspective on the choice of allergens, emphasizing the need for diagnostics without harming the patient, and when serum-specific IgE testing is adequate. We delineate limitations of allergy tests and explain why not every pollen type causes an allergic response.

Secondly, we convey the aerobiologist's perspective on pollen forecasting, the role of pollen threshold loads obtained with crowdsourced patient symptoms, and support this with an example from Istanbul.

#### **2. The allergists' perspective**

Skin testing has been used to diagnose allergic disorders for more than 50 years. Back in the mid-twentieth century, pricking the skin with a solution of the allergen using a lancet was the only method but today there are several test devices capable of results that are more reliable and reproducible. Skin testing continues to be the main test to confirm an IgE-mediated immunologic reaction. Not only skin testing is the best indicator of the underlying allergic pathology, but it also remains to be the most inexpensive test with rapid results making it practical in office setting. The binding of specific IgE on tissue mast cells to the offending allergen is the unique attribute of skin testing. Alternatively, the patient can be challenged with the allergen by directly applying it to the mucous membranes, nose, bronchi, or even eyes, but the extra advantage of such procedures does not justify the risk and inconvenience. Allergen skin testing has been shown to exhibit reliable correlation with such mucosal challenges [8].

It is crucial, however, that the physician ordering or interpreting these tests be cognizant of the dynamics that can affect the outcome. Some important considerations as to when these tests are indicated and how they should be interpreted are outlined here.

#### **2.1 Choosing the allergens with clinical relevance**

Allergy testing without a clear indication or random testing for arbitrarily chosen allergens is not acceptable. The selection of allergens should be determined based on the patient's exposure history and correlated with their symptoms. The relevant allergens should be based on the medical history, age, and the environment, geography of the patient. Knowledge on the average and the course of the pollen seasons of the patient's geography is essential in this regard [9]. The tested allergens should be able to predict and/or confirm the clinical disease.

Most clinicians would order these tests for two main reasons: (1) the planning of avoidance of the allergen and (2) specific immunotherapy. Interdisciplinary collaboration among allergists, aerobiologists, and atmospheric scientists is important to identify the relevant allergens in the environment connecting the time of exposure to symptoms.

#### **2.2 Allergen sensitivity without allergy**

The test results should be validated by associating the exposure to allergens under natural conditions or controlled challenges with the particular allergen. There may be skin sensitivity without symptoms which may not have any validity to the current clinical problem of the patient. Routine use of arrays of skin tests or usual annual tests without a definite clinical indication is unwarranted. Nevertheless, some asymptomatic skin sensitization may be a risk factor for future organ sensitivity; hence, some clinicians would still value this coincidental sensitivity to monitor the patient for future development of clinical allergy. In a prospective trial of 15 asymptomatic patients with positive skin prick test to birch, 60% were later reported to develop true clinical allergy [10]. But we must be aware of the cascade effect in medicine which was brilliantly outlined by James Mold [11] referring to the detrimental process that once triggered proceeds to the inevitable conclusion of unnecessary tests, patient and/or physician anxiety ending up with wrong treatments, adverse effects and/or morbidity and not to mention the uneconomic medical expenses. Healthcare providers must guard against the vortex of this domino effect leading to the collision course of such preventable events. End result of such actions are infants being deprived of essential nutrients, needless anxiety for patients and caregivers, inappropriately prescribed more expensive medications possibly leading to antibiotic resistance, etc. Preventing cascade effects should be a part of the education curriculum of physicians and providers. This is not only important in the field of allergy and immunology but all specialties of medicine, especially in the technology era of healthcare services where more is unfortunately considered better.

#### **2.3 Serum-specific IgE testing**

In vitro serum IgE testing is sometimes safer than skin testing in patients with cardiovascular disease, or when severe anaphylactic reactions are expected. We also prefer to perform serum in vitro testing for patients who are unable to withhold their antihistamines or other medications interfering with tests. Another common medication that is problematic for skin testing is Omalizumab, which also interferes with many immunoassays, except the ImmunoCAP method, which usually remains accurate [12]. Skin testing on infants less than a year old may be challenging and results may not reflect true sensitivity [13]; thus, we prefer serum-specific IgE tests for infants as young as 6 to 8 weeks of age which only requires capillary blood collection [14].

On the flip side, caution is advised for commercial remote practice laboratories performing such serum tests. Some laboratories bypass the clinician and perform serum IgE tests based on the history submitted by the patients and start immunotherapy according to these ambiguous serum IgE test results. As with the skin tests, the interpretation of specific serum IgE levels require the same meticulous clinical history, physical examination, and, in some instances, challenges with natural or laboratory exposure to allergens. This, obviously, is not the typical practice of commercial laboratories [11]. The serum-specific IgE level per se may not reflect the clinical sensitivity due to the fact that clonality and affinity of the IgE antibody plays a role in translation of serum IgE production to clinically relevant allergic sensitization [15]. Thus, it is important to understand that, although an IgE-mediated immunological response is necessary to develop allergic disease, it is not sufficient.

#### **2.4 Limitations of allergy tests**

False-positive allergy test results may occur. Some tree pollens share cross-reactive carbohydrate determinants with other pollens or, for example, honey bee venom [16]. It is also not uncommon for a pollen-sensitive patient to be living in another area, not exposed to the same pollen. The co-sensitization should be differentiated from crosssensitization when testing with extract reagents with common epitopes. Another reason for getting negative reading on allergy tests is the fact that the pathogenesis of the organ sensitization may not involve IgE-mediated pathways. Alternative immunologic pathways or non-immunologic pathways may be at play. The clinician should be in close contact and consult other specialties as well, to fully understand the scope of the organ symptoms. Also, patients who experienced an anaphylactic event may have false-negative skin test results for up to 2–3 weeks after the episode. In vitro testing serum-specific IgE levels are not affected and can be performed in the postanaphylactic situation where testing cannot be postponed.

Serum-specific IgE test can also display false-positive or -negative levels based on the binding affinity/avidity of the offending allergen to the solid-phase system used for testing. The circulating levels of cross-reactive peptides and specific antibodies of another class, e.g. IgG or the high levels of nonspecific serum IgE levels, may also affect the readings. We do not perform IgG or IgG subclass antibody tests for food or other allergies. They have no clinical relevance. There have been reports of monitoring IgG4 during venom immunotherapy, but this is not validated [17].

#### **2.5 Not all pollens are created equal**

Most pollen types do not cause allergies and the ones that cause allergies do so in different potencies. Not all pollen types elicit an allergic response or in immunologic terms have the recognition moieties, the epitopes, that bind to specific receptors on B or T cells. Allergenicity is usually elicited by the peptidic epitopes. The glycan moieties of these glycoproteins affect the immunogenicity of these peptides. Glycans are in variable proportions in different pollens affecting allergenicity. Even when they do have these epitopes, the conformational shape of the pollen structure may limit the three-dimensional spatial alignment of the allergen to the IgE antibody binding sites. These are some of the factors that will alter the allergenicity of pollen intrinsically at the biochemical level.

Environmental factors also have effects on the allergenicity of pollen. Pollutants such as heavy metals, ozone, sulfur dioxide, nitrogen dioxide, and diesel exhaust particles may affect the allergenicity [18], by activating the immune system, referred to as the adjuvant effect [19]. Extended pollen seasons linked to climate change were recognized as factors for, not only increased pollen production but also increased allergen content of their grains [20, 21].

#### **3. The aerobiologists' perspective**

#### **3.1 Forecasting**

We see them in newspapers, on weather apps, and on specific pollen apps: warnings on allergenic particles currently airborne in a certain region. Issuing pollen forecasts is the inherent chore of an aerobiologist. Trained technicians count meticulously

#### *Aerobiology in the Clinics of Pollen Allergy DOI: http://dx.doi.org/10.5772/intechopen.107311*

every pollen grain captured on a slide on a number of vertical or horizontal transects under a light microscope at a magnification of 400 x so to cover at least 10% of the slide's surface to estimate the concentration of airborne pollen per cubic meter air per day [22]. This procedure, where pollen is sampled with a volumetric Hirst-type device on a weekly basis, and evaluated retrospectively, falls under the norm CSN EN 16868 [23]. Issuing warnings based on retrospective data makes forecasting essential. The longer the time series of daily or possibly bi-hourly data, the better will be the forecast. Curves of mean pollen concentrations for each pollen type help to assess the variability within years. For allergy pollen warnings, the aerobiologist uses weather forecasts, past year's data and ideally observes the phenology of plants shedding allergic pollen [24, 25]. Models like SILAM (System for Integrated modeling of Atmospheric composition) [26] and COSMO-ART (Consortium for Small-scale Modeling Aerosols and Reactive Trace gases) [27] can provide additional information to incorporate in pollen warnings in Europe.

#### **3.2 Pollen threshold loads**

Although forecasts are useful to help patients avoid exposure when levels are high, pollen and weather data alone do not bear the information, whether or not there is an actual risk for the allergic population to suffer symptoms of allergy. It has been shown that allergy morbidity to a specific pollen type may change over the pollen season [7, 28, 29]. With the inclusion of patient's symptom data, a dose–response relationship between exposure and symptoms can be estimated and the accuracy of pollen load thresholds determined. The focus hereby can be on the allergenic pollen type itself, for example, grasses [28, 30], ragweed [7, 31], or birch [32].

There are several ways to obtain patient symptoms, as reviewed in [33]. Practicable are, for example, questionnaire-based daily surveys for prospective clinical trials [29]. Items may include a four-point symptom scale (0 = zero; 1 = mild; 2 = moderate; and 3 = severe) related to the eyes, nose, bronchi, and medication use [ibid]. Additionally, general health is assessed on a ten-point scale [31]. Study participants may send their data to the research-coordinator daily or weekly. This way to gather symptom data is laborious but allows for the control of missing or hampered data [31]. The number of patients in exposure studies, however, is often limited in size ranging from 12 to 430 in 26 studies as reviewed in a Finnish study [34].

Another way to obtain self-reported patient data on symptoms is by means of electronic pollen diaries. Examples of crowdsourced data include the Dutch Allergieradar. nl, established in 2009 to "improve the hay fever forecasts and to decrease the amount of hay fever symptoms patients experience" [35]. The Europe-wide active pollendiary.com [36] coordinated by the Medical University of Vienna follows the same aim. Pollen data from adhering pollen monitoring networks and single stations are fed into the EAN (European Allergy Network). Registered users are encouraged to log their symptoms regularly over the hay fever period. The service is also available as an application "Pollen" that, as the website, provides personalized symptom forecasts based on previous five records [37]. The application is currently available in Austria, Germany, Switzerland, Sweden, Spain, Great Britain, and South Tyrol [38]. As emphasized in [37], the expertise of the aerobiologist is a pillar to generate personalized pollen information. The symptoms recorded translate into a 3-day allergy risk forecast for the respective region, where a sampling device is located. This personalized forecast includes, besides pollen and weather data, other risk factors for respiratory allergy like ozone, sulfur dioxide, nitrogen dioxide, and particulate matter.

#### **3.3 The power of crowdsourced data**

The development of pollendiary.com is a result of yearlong collaboration between a network of aerobiologists, data scientists, and sound management. Quality assurance is a main concern to avoid potential harm to hay fever sufferers due to inaccurate forecasts [39]. An evaluation of nine free applications showed that the accuracy of the grass pollen load forecast was 50% of six apps when compared to the actual grass pollen concentration in the location [39]. Web-based applications are an easy way to self-empower hay fever sufferers and to monitor symptom development in the allergic population. The number of hay fever morbidity data obtained between 2009 and 2019 by means of pollendiary.com across Europe was 240.000, with 190.000 logs from Germany and over 32.000 from Austria [6]. The solid number of crowdsourced data on symptoms enables the aerobiologist to provide more accurate forecasts. As a matter of fact, regional pollen concentrations alone cannot be a measure for the pollen allergenicity experienced by the population in a certain area. It is known that pollen concentrations do not exactly correlate with the allergen content in the air, as shown for ragweed in Turkey [40] or olive in Spain [41]. Pollen potency, the ratio between pollen and allergen concentrations per cubic meter, varies considerably in time and space [42, 43]. The origin of the allergen content in the air is at present not predictable as linked to ruptures of pollen that release micronic allergenic particles at varying weather conditions and altitude, resuspensions, long range transport via air currents, and the allergen content in the plants of origin [42]. Thus, the inclusion of locally experienced symptom data in pollen warnings is essential to issue the correct threshold loads for a particular region.

#### **3.4 An example from Istanbul**

The assessment of local thresholds is a pressing issue in allergology [44]. They are unique in each biogeographic area, as factors like pollen sources, their allergenicity, climate, pollution, and the genetic fingerprint at individual and population level are determining factors for symptom thresholds [33]. Crowdsourced symptom data, pollen, weather, and pollution data can be included in models that display the dose–response relationship experienced by the allergic population. Standardized pollen data as in the EAN and a uniform method for symptom data collection as in pollendiary.com allows for the calculation of threshold levels, for example, with nonlinear regression models [7, 45]. Anti-allergic medication can be an indicator for allergy morbidity. Here we show preliminary results of a study conducted with data (n = 725) from the European part of Istanbul (**Figure 1**).

In Istanbul anti-allergy medication use started at about 4 p/m3 , and increased linearly till about 11 p/m3 . Subsequently, the bending of the curve [46] suggests that the threshold for moderate medication use has been reached. Between 18 p/m3 and 30 p/m3 medication use was the most intense. After that it decreased to remain at a moderate level at >= 50 p/m3 . As few as about 4 p/m3 caused morbidity that patients sought to mitigate with drugs. In Istanbul the non-linear relationship in the doseresponse curve illustrates that symptoms do not necessarily aggravate at a grass pollen concentration higher than 25-30 p/m3 . Longer time series of data with more users than the ones presented here would yield more solid results. Promoting the use of an electronic hay fever symptom diary should be an integral part of a public pollen information system.

*Aerobiology in the Clinics of Pollen Allergy DOI: http://dx.doi.org/10.5772/intechopen.107311*

#### **Figure 1.**

*Preliminary effect curve of pollen concentrations (p/m3 ) (2014–June 2016) in Istanbul on medication use with thresholds. The green line denotes the start of medication use in unit increase in the log (y) abundance by unit increase in x. The yellow line denotes the threshold for moderate medication use. The red line marks the saturation threshold for intense medication use. The blue line marks zero effect. Note the relationship between the confidence intervals and the data logs.*

#### **4. Conclusions**

Aerobiological expertise plays an integral role in the clinics of allergic disease in the qualitative forecasting of the pollen season to support the allergist in the assessment of morbidity and the timing of treatment. Cooperation between the allergist and the aerobiologist help select the most relevant allergens for in vivo diagnosis, improve the interpretation of results, and select the most appropriate immunotherapy regimen tailored for the patient.

#### **Acknowledgements**

We thank Uwe Berger, MBA from the Aerobiology and Pollen Information Research Unit, Department of Oto-Rhino-Laryngology, Medical University of Vienna, for granting permission to use symptom data from pollendiary.com for the preliminary analysis of pollen thresholds.

#### **Author details**

Franziska Zemmer1 \* and Fatih Ozkaragoz2

1 Free University of Bolzano, Bolzano, Italy

2 Academic Allergy Asthma and Immunology Assoc, Houston, USA

\*Address all correspondence to: franziska.zemmer@unibz.it

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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