The REAL project was designed with a qualitative methodology to explore the perspectives of allergology experts across Spain. The recommendations emerged from discussions that aimed to align current scientific evidence with the practical realities faced in daily clinical practice in order to define recommendations in the management of polyallergic patients using AIT based on mixtures from different allergenic sources.

The scientific committee was composed of eight expert allergists with extensive experience in the diagnosis and management of polyallergy. Each member represented a Spanish region with distinct allergenic exposure patterns, ensuring that the project reflected the country's diverse sensitization profiles (See Supplementary Figure S1). The regions included:

Region 1: Cataluña and Islas Baleares

The first meeting with the scientific committee, held in September 2024, focused on identifying the key clinical aspects to consider for the use of AIT based on allergen mixtures from different allergenic sources, and focusing on how, when, and why such treatment should be used. The main benefits and barriers of multi-allergen immunotherapy were also discussed. The eight regional meetings were held between November and December 2024 following the Workmat® methodology. Each meeting was attended by 6–9 experts and was chaired by the scientific committee member representing the corresponding region. After every session, a report summarizing key outcomes and controversies was created. In January 2025, the scientific committee met to compare regional results, assess differences and similarities between the selected geographical areas, explore their causes and consolidate findings. Drawing on their in-depth understanding of the regional data, the committee members identified general trends and validated a set of recommendations applicable at the national level. No major discrepancies were observed between regions; minor variations reflected local clinical practice and the realities of managing polyallergic patients, which are discussed in the manuscript.

The Workmat® methodology is a structured, evidence-based approach designed to promote collaborative debate and the exchange of expert clinical perspective (28–30). It was implemented in the 8 regional meetings and enabled systematic and equitable data collection across groups.

During each session, experts worked in small groups to answer targeted questions and complete related exercises displayed on posters. The original materials used during the workshops are included in the Supplementary Material to allow readers to understand the structure of the exercises and the topics addressed in each session. Likert-type scales were used for scoring, ranging from 1 to 9, being 1 to 3 (not important/disagreement), 4 to 6 (neutral), and 7 to 9 (important/agreement). The percentage of experts scoring within each of these ranges was calculated to assess the level of agreement among experts to support the recommendation.

Each regional meeting followed a three-phase structure:

Presentation of evidence: The regional scientific committee member reviewed the most relevant data.

A total of 61 experts were invited to participate in the regional meetings. Demographic characteristics and clinical experience of participants can be found in Table 1. Experts were predominantly female (77.78%), working mostly in public centers (84.44%), and having more than 10 years of experience in the management of AIT (64.44%). Out of the regions represented on the panel, region 4 (Comunidad de Madrid, Castilla y León and Castilla-La Mancha) had the highest representation (n = 9; 20.00%), followed by region 6 (Bilbao, La Rioja, Navarra and Cantabria) (n = 7; 15.55%).

In general, the experts manage nearly 300 patients per month on average (range 60–900), with 60% being polyallergic, and most consultations were follow-up visits (58.23%) (Table 1).

All experts agreed that it is essential for the allergist to establish a correct and thorough diagnostic pathway to prescribe the most appropriate therapeutic option. The allergy diagnosis presumes a pragmatic method, based on three essential steps: clinical record of symptoms history, skin prick test (SPT), and specific immunoglobulin E (sIgE) (Figures 1, 2). A detailed medical history will always be the key starting point to understand the patient's background, symptoms, and allergy triggers. Experts considered that an accurate diagnosis is affected by other factors, such as the allergist's experience and the patient's environment. SPT and sIgE are effective, accessible, and cost-efficient tools, that are usually the first step in diagnosis and guide the choice of subsequent complementary tests. In this context, experts agreed that component-resolved diagnosis is a particularly valuable tool due to its usefulness. However, it is not always feasible, as it is not routinely used or available in all allergy patient care centers (Figure 1). The majority (62.5%) of experts consider component-resolved diagnosis useful when there is polysensitization (Figure 2). All experts agreed that the use of component-resolved diagnosis is not strictly required in all cases, but it can enhance diagnostic precision and optimize AIT in polysensitized patients. On the other hand, the experts evaluated the usefulness of nasal and/or conjunctival provocation tests, as well as those carried out in exposure chambers, but their availability is low due to their demand of time and additional resources (Figures 1, 2). The calendar of symptoms was considered very useful in the diagnostic process by the experts in the REAL Project, but their effectiveness depends upon their correct completion by the patient (Figure 1).

In addition, pollen level registries were considered helpful, although their viability depends on the availability of a robust and reliable monitoring system. Similarly, acarological maps could potentially support diagnosis, but their utility is currently limited due to the lack of updated information (Figure 1).

The REAL Project provides a qualified description of the pros and cons of selecting a particular therapy by keeping in focus the critical guiding factors chosen by the experts and based on the available evidence (Figure 3). When prescribing AIT in polyallergic patients, the presence of high symptom intensity or disease severity, as well as a clear impact on the patient's QoL were outlined as the most significant factors by 100% of experts, followed by patient preferences (87.5% of agreement), the adequate control of allergy with symptomatic treatments (87.5% of agreement), and the duration of such symptoms (62.5% of agreement). However, experts emphasized that neither allergy control nor symptom duration should limit the prescription of AIT, since the duration of symptoms throughout the year is not always correlated with disease severity, and symptom control does not preclude vaccination if symptoms are properly managed with medication (Figure 3). Avoiding exposure to a particular allergen is important, but not always achievable, so it was classified as less relevant.

Other factors that came up in the discussion were very relevant, including: the socio-economic conditions of each patient (habitat, profession, lifestyle), and the fact that age should not be considered a barrier for being treated with AIT. From the experts' perspective, AIT should therefore not be restricted to patients between the ages of 5 and 65 (data not shown).

The experts involved in the REAL Project discussed different topics related to the potential use of specific AIT prescriptions based on mixtures from different allergenic sources that are described below.

Polysensitization is an immunological event that is relevant from an epidemiological and clinical point of view (31) and is typically more common than monosensitization in general population surveys conducted worldwide using standard panels of allergens (32–35). Unsurprisingly, polysensitization accounts for 60%–80% of patients who consult an allergist (34, 36, 37). In 2022, a study conducted in Spain obtained a map of sensitization to allergenic sources based on clinical history, allergic symptoms, and SPT results. This study demonstrated that 79% of allergic patients from different regions of Spain showed sensitization to two or more allergenic sources (9).

Participants of the REAL Project highlighted that among patients with allergic symptoms, an increasing number of subjects are polysensitised and most importantly polyallergic. The severity of symptoms and the prevalence of asthma are also rising (31, 38). The burden of allergic symptoms is therefore increasing, with a negative impact on patients' overall health (39). Because of this growing trend of polyallergy, panelists emphasize the relevance of meeting patients' needs, which involves having an accurate identification of their sensitization profile and a proper diagnosis.

Importantly, Spain presents a unique and particularly complex allergenic landscape. Unlike other countries where a small number of allergenic sources may predominate, Spanish patients are frequently exposed to a wide range of allergens due to geographic, climatic, and environmental diversity (26, 27). This results in regional differences in the prevalence and intensity of sensitization patterns. This complexity of the allergenic exposome scenario makes the diagnostic process especially challenging and highlights the need for tailored strategies adapted to local sensitization profiles.

Given this context, the panelists emphasized the importance of accurately identifying each patient's sensitization profile and establishing a proper diagnosis. When sensitization to two or more allergens is present, it is important to identify the allergenic source(s) with the greatest impact on QoL. The allergists' main responsibility is to determine which sensitizing allergens are relevant concerning the clinical symptoms of the allergy. A detailed clinical history alone is still often sufficient for an etiological diagnosis, being frequently enough to identify the clinically relevant allergen or allergens responsible for the allergic respiratory disease. As an example, it has been reported that the clinical history alone had a predictive value of 82%–85% for seasonal allergens and at least 77% for perennial allergens, but when both sIgE and SPT are available these values may increase to 97%–99% (40).

In the present study, participant allergists agree that an adequate diagnosis requires correlating clinical findings with SPT and/or sIgE quantification, with component-resolved diagnosis recommended to distinguish genuine sensitizations from cross-reactivity in polysensitized patients. According to what has been published, panelists outline the limited value of SPT in recognizing some relevant allergens (41). However, SPT is commonly used in daily clinical practice because of its simplicity, sensitivity and positive predictive value in quickly identifying IgE sensitization to inhalant allergens (42).

The first step to properly approach polysensitization involves differentiating between genuine or primary sensitization and cross-sensitization. Component-resolved diagnosis may help allergists to identify clinically relevant allergens and distinguish genuine polysensitization (“co-sensitization”) from polysensitization due to cross-reactivity (“cross-sensitization”). Accurate identification of the allergens responsible for sensitization may help allergists develop personalized etiological AIT treatments (43, 44).

Following the identification of the most clinically relevant allergens based on clinical history and the complementary test of choice (SPT, sIgE, component-resolved diagnosis), the next decision will be how to treat a polyallergic patient. AIT is a recognized effective therapy for respiratory allergy with a unique potential ability to modify the natural course of the disease and induce long-term clinical benefits, that can persist for years after discontinuation of treatment (45). It is necessary then to know which factors may influence the choice of treatment. The allergist evaluates the clinical history based on the presence, severity, and duration of seasonal and/or perennial symptoms and their impact on Quality of Life (QoL). The causal relationship between the most troublesome clinical symptoms and the relevant allergens appears to be a key factor to the experts. Consistent with this, a previous Spanish Delphi consensus on the diagnosis and treatment of polyallergy—with the participation of 62 allergists— stated that AIT prescriptions should be based on scientific evidence and only given if relevant allergens have been identified that correspond to symptom severity (1).

Based on previously published experiences (16, 17, 46), this project shows that allergen mixtures based on different allergenic sources are a valid alternative when two or more allergens that may or may not overlap in time, have a comparable and substantial impact on the patient´s symptoms and QoL. Furthermore, these allergen mixtures, especially those at undiluted concentrations, offer several advantages over AIT with simultaneous individual extracts, as they allow the treatment of multiple clinically relevant sensitizations in a single application, simplifying the treatment without compromising its efficacy and saving both time and costs.

Allergists in the US believe that addressing as many sensitizations or allergies in a patient as possible offers significant clinical benefits (46). More recently, a survey was conducted online with allergy-practicing clinicians in 19 countries worldwide in 2016. A total of 1,029 eligible clinicians prescribing AIT provided information on their approach to AIT, including managing polyallergic patients; 98% of the responders reported the treatment of polyallergic patients. In this regard, country-specific differences were detected: 58% of those polyallergic patients received AIT treatment for a single allergen, while 42% were prescribed AIT with multiple allergens, including either mixture (24%) or single allergen AIT (18%) (22). These findings underscore the importance of maintaining a patient-centered approach in AIT preparations, in alignment with regulatory authority recommendations. It is essential that AIT products meet high-quality standards and are supported by evidence of efficacy and safety. Based on our discussions, 100% of the participants agreed to use up to three distinct allergens in a single extract when dealing with mixtures of different allergenic sources; and, if possible, choose to use mixtures in a single vaccine over several individual extracts.

In this regard, prescriptions of AIT with three or more different allergenic sources are rare in European countries other than Spain (47). In observational surveys in France, only 1.1% of AIT prescriptions contained three or more allergenic sources (48). The recommendation when using mixed AIT formulations is maintained at a maximum of three different allergenic sources, but it may change in the future due to the increase in the prevalence of polyallergy and the development of new treatments.

Nonetheless, when treating polyallergy there is a trend in some European countries towards prescribing AIT with a single allergen or with several allergens, but separately. The appropriate dose of each allergen in a mixture based on different allergenic sources should be at a therapeutic concentration to prevent the dilution of each allergen extract below the therapeutic dose. This approach aligns with the principles of the EMA, which emphasizes the use of the minimum number of allergens in mixtures with justified proportions of individual allergens (49). This restriction might be explained based on several premises. In addition to the aforementioned dilutional effect, there is a possibility of degradation due to the enzymatic activity of some extracts, and the difficulty in demonstrating the efficacy of many allergens (49, 50). Additionally, it was advised to minimize the number of allergens in the mixture according to the patient's polyallergic profile and the use of extracts with proven scientific evidence of their efficacy and safety.

In this context, the use of polymerized allergen extracts in the treatment of polyallergic patients is emerging as a promising therapeutic strategy, overcoming the limitations traditionally stated by the EMA with respect to the mixture of allergens. Polymerization of extracts significantly reduces the binding capacity of specific IgE to allergens (antigenicity) without altering the ability to induce an adequate immune response (immunogenicity) (4, 51, 52). This allows the concentration of allergens to be increased without increasing the risk of adverse reactions (1, 51). In addition, this process inactivates the enzymatic activity of the extracts, which makes it possible to combine allergens from different sources, even those with high enzymatic activity that, under normal conditions, would be incompatible. In summary, polymerized extracts allow the simultaneous administration of multiple allergens at optimal concentrations within a single formulation, eliminating the need for dilution (49, 53).

Published investigations of real-world data coming from almost 3,000 children and adolescents with rhinoconjunctivitis with/or without asthma suggest that AIT with mixtures of depigmented and polymerized extracts is efficacious and well tolerated (50).

This is particularly relevant in polyallergic patients, where the use of stable and standardized mixtures is crucial to maintain the effectiveness of the treatment over time and to avoid loss of potency due to extract incompatibility. Therefore, to provide optimal treatment, the panel of allergists recommended using polymerized allergen extracts—such as depigmented polymerized extracts—that have a very reduced proteolytic activity, avoiding allergen mixtures that may interact (53).

The route of administration of AIT should be customized for each patient using a patient-oriented approach (16). When deciding between the sublingual immunotherapy (SLIT) or the subcutaneous immunotherapy (SCIT) routes, there are many factors to consider, such as availability, treatment cost, adherence, clinical experience, and scientific evidence (54). All these factors have made SCIT the preferred route for our experts. Overall, numerous studies provide evidence supporting the effectiveness of SCIT in the management of allergic rhinitis with or without asthma. These studies have primarily focused on the relief of allergic symptoms and the enhancement of QoL, the decrease in exacerbations and improvement in lung function; and reducing the use of medications for symptomatic treatment (54–57). Similarly, numerous studies have demonstrated the short and long-term efficacy of SLIT in allergic rhinitis with or without asthma in both adult and pediatric patients (54, 58, 59), but this was not the best-considered route of administration in terms of adherence, where the experts were clearly in favor of SCIT.

The administration of AIT, including that with different allergenic sources, involves a two-phase scheme: an initiation phase using one of three protocols (i.e., standard, cluster, or rush); and a maintenance phase following a perennial, pre-seasonal, or pre/co-seasonal schedule. This study supports the use of polymerized extracts initially administered in a rush build-up dosing phase, which allows the maintenance dose to be reached more quickly without involving a greater number of adverse reactions (60), followed by a perennial maintenance schedule, which is the most commonly used to ensure continuous and effective treatment.

The use of polymerized allergen extracts has improved both the efficacy and safety of AIT in polyallergic patients; therefore, it is essential to conduct more individualized evaluations of the response to treatments, considering the type of extract and the mixtures.

The most relevant features to assess the response to AIT are the improvement of either nasal, conjunctival, and/or bronchial symptoms, the reduction in the amount of medication, and the enhancement of the patient's self-reported QoL. Prior studies indicate that physicians can prescribe mixed multi-allergen immunotherapy with confidence in polysensitized and polyallergic patients by focusing on clinical/QoL relevance and safety (5). Pfaar et al. conducted a 2-year multicenter, double-blind, placebo-controlled trial of SCIT with mixed depigmented and polymerized birch and grass pollen extract in 285 patients with allergic rhinoconjunctivitis who experienced symptoms during both birch and grass pollen seasons (61). Compared to placebo, SCIT resulted in a significant 19.4% reduction in the cSMS, with a good safety profile. An observational, retrospective, multicentric study of 40 pediatric patients with rhinitis/rhinoconjunctivitis, with or without controlled asthma, showed that an undiluted mixture of depigmented and polymerized grass/olive (Olea europaea) allergens was effective in both perennial and pre/co-seasonal schedules, and therefore suitable for polyallergic patients (17). In this report, the response of AIT revealed a significant improvement in symptoms, QoL, and global perception of the disease, whereas safety profiles were good and similar in both schedules.

It is known that as the number of patient sensitizations increases, the severity of associated respiratory diseases escalates (37). Single multiallergen AIT must demonstrate effectiveness while upholding a solid safety profile; in contrast, multiple monoallergen immunotherapy can be associated with poor treatment adherence and high costs. Another retrospective observational study investigated the effect of using a 2-pollen single undiluted multiallergen SCIT (duration, 1.8 years) in 97 patients (median age 32 years) in routine clinical practice in Spain (62). SCIT treatment, including combinations of Grass-mix with Olea europaea, Parietaria judaicaspp, Cupressus arizonica, Platanus acerifolia, or Salsola kali or Olea europaea with Parietaria judaica, Cupressus arizonica or Salsola kali, was effective and safe in both children and adults. This was a well-tolerated dosing scheme, with little systemic reactions (6 out of 97 cases), none of which led to treatment discontinuation, showing that it is a suitable option for the treatment of polyallergic patients.

AIT has therefore proven to be safe, although some factors are dependent on the patients and/or the extracts. Furthermore, the administration site may affect tolerability (57, 63). SLIT has shown less incidence of systemic reactions than SCIT: the incidence of SLIT systemic reactions is 0.0045% (64). The most common reactions are local and usually appear in the first days of treatment. The rate of systemic reactions in SCIT is between 0.1% and 1.56% per administration, and they usually occur within the first 30 min after application. The frequency of anaphylactic reactions in rush or ultrarush schedules without prior medication, as well as delays in the administration of SLIT, are risk factors for systemic reactions during the initiation phase (57).

Notably, the participating allergists consider that all formulations appear to be recommendable if they are stable, safe, and effective, including allergens that impact the QoL and symptoms, and do not have proteolytic activity. Only the lack of experience relative to a particular AIT mixture in a certain geographic region may condition to recommend its use to treat polyallergy. The provided combinations were deemed advisable for various reasons: (i) these common allergens are usually responsible for the symptoms observed in each particular geographical area; (ii) the use of these allergen mixtures in AIT makes it possible to provide a less expensive therapy, facilitating the prescribing process and enhancing treatment adherence; and (iii) these mixtures were highly believed to be safe for the patient.

In this context, it is important to note that certain allergenic sources, although not highly prevalent in other European countries, are frequently involved in the development of allergic symptoms in Spain—either nationally (e.g., Olea europaea, Parietaria judaica) or in specific regions (e.g., Salsola kali, Cupressus arizonica) (65, 66). These complex and region-specific sensitizations patterns are often managed through mixed allergen formulations, as long as they are stable and meet safety standards. Therefore, stability data on allergen mixtures is particularly relevant when considering the use of individualized combinations adapted to the sensitization profiles observed in different Spanish regions.

Although the absence of provocation data precludes establishing causal relationships or definitive therapeutic hierarchies, the collective experience of the experts provides a pragmatic and contextually valid framework for guiding clinical decision-making in the Spanish setting.

In this study, we gathered expert input on the use of such mixtures in polyallergic patients, providing practical guidance for their application. The recommendations derived from this project are based on expert judgement and real-world clinical practice rather than on data from provocation tests or other controlled experimental assessments.

While qualitative, non-randomized methods have inherent limitations, including potential selection bias and unequal regional representation, the consistency of expert opinions across Spain supports the validity of the findings. These recommendations reflect the Spanish clinical context, shaped by geographic and environmental diversity, regulatory frameworks, and the widespread use of polymerized extracts. Although direct extrapolation to other countries may be limited, the structured Workmat® methodology and the expert reasoning behind these recommendations may offer relevant insights for regions that face similarly complex polysensitization patterns, supporting the integration of evidence with routine clinical practice.