Allergy-mediated airway diseases are a major health concern due to their high prevalence, increasing incidence, and clinical manifestations affecting multiple organs. According to the World Health Organization (WHO), 500 million people worldwide suffer from rhinitis and 262 million have asthma. These two diseases significantly impair the quality of life of the inflicted individuals and their relatives (1).

Nowadays, allergy specialists have no tools to distinguish between sensitization and clinical allergy by just using in vitro testing (2, 3).

Disease confirmation, identification of the triggers (allergens), and severity assessment are key to determining the need and appropriateness of therapies aimed to modify the course of the disease, such as allergen-specific immunotherapy.

For an accurate diagnosis, quantification of sIgE is a decisive factor. However, the current criteria for determining positive/negative or clinically relevant/non-relevant sensitization/non-sensitization are influenced by a lack of evidence-based cut-offs (4).

The “clinically relevant sensitization” concept, generally considered as sIgE detectable loads in the context of consistent symptoms, is a must to discriminate which patients would benefit from allergen immunotherapy. Besides the traditional optimal cut-off based on sensitivity/specificity balance, a new diagnostics strategy based on the ratios of whole extract-specific IgE/total IgE and component-specific IgE/whole-extract specific IgE has recently been proposed to define the relevance of sensitization in the patient symptoms (5).

Differentiating mono from polysensitized patients is also relevant for their management. Among allergic pollens, olive tree pollen (Olea europaea) is one of the most important respiratory allergy triggers in the Mediterranean area and in regions of America, South Africa, Japan, and Australia (6). In Spain, it is considered the second cause of pollinosis, and the first in certain southern regions. To date, 14 allergenic proteins have been identified in olive pollen, from Ole e 1 to Ole e 12, Ole e 14, and recently Ole e 15 (7) (according to the nomenclature of the International Union of Immunological Societies (IUIS, International Union of Immunological Societies) (8, 9). In addition to olive, grass pollen is another relevant pollen in Mediterranean countries, and in fact, both share overlapping pollination periods, thus increasing the diagnostic complexity in areas where they coexist. Due to this overlap, it is difficult to determine whether symptoms are due to genuine sensitization or cross-reaction. Therefore, a component-resolved diagnosis (CRD) is needed to establish the genuine sensitizer(s).

Double sensitization to Poaceae and Oleaceae makes it more challenging when evaluating the indication of AIT. In this case, the specific allergenic component culprits of the two-pollen species must be identified to discriminate between genuine bisensitization and cross-reactivity, thus allowing the specialists to pinpoint the specific trigger(s) (i.e., grass and/or olive pollen) (3, 10–13).

In conclusion, our aim was to establish effective and efficient cut-off points for sIgE to genuine allergenic components and to the whole allergens (Poaceae and Oleaceae). By establishing these cut-off points, we could identify genuine sensitizers and ultimately predict the clinical reactivity to these pollens while increasing the probability of AIT success in highly exposed patients.

Diagnosing allergic asthma and/or rhinoconjunctivitis is a complex task. Although serological tests are helpful, they all require interpretation from an allergy specialist. Of 1,172 patients for whom allergy to two olive and/or grass pollen was suspected, 831 were confirmed as clinical allergic patients to one or both. Not all the 341 patients for whom allergy was ruled out tested negative for sIgE if considering 0.35 kU_A/L as the cut-off. Thus, revealing the lack of real validated cut-offs to assess the sensitization clinical relevance. The sIgE cut-off has been generally set to 0.35 kU_A/L, without considering other factors that could influence the immunogenicity of the allergen and the characteristics of the disease presentation. For the same reason, values below this cut-off have been shown to be relevant (25). This arbitrary cut-off has been under debate in different publications that, using different approaches, have tried to find cut-offs for sIgE aligned with clinical reality, both for diagnosis (26–28) and treatment (indication of AIT or provocation tests, both of them with side effects) (4, 29–31). Few studies have been published specifically looking for these cut-offs in the diagnosis of seasonal rhinitis and or asthma to date (32, 33). This work aimed to obtain cut-offs for sIgE in our population under high olive and grass allergenic pressure to help us in the decision to indicate AIT.

The simultaneous pollination of olive trees and grasses increases the diagnostic complexity in our area and therefore, more improved diagnostic tools are required, such as the establishment of evidence-based cut-offs and discrimination between mono vs. polysensitization. The proposed algorithm allows us to discriminate while minimizing the number of sIgE tests (Figure 1).

Cut-off evaluation analysis has revealed that the AUC of Lolium-sIgE shows a very good performance for this whole extract, and is very similar to the one corresponding to rPhl p 1,5-sIgE. The same trend can be seen when looking for the best cut-off. Likewise, the correlation between Lolium-sIgE and rPhl p 1,5-sIgE was excellent, as expected (14–17). Given the above, we understand that the allergy diagnosis can be made efficiently with either of the two Poaceae determinations without expanding the algorithm. Due to cross-reactivity of profilins and polcacins (34), we better recommend using the determination of rPhl p 1,5-IgE for the diagnosis, considering the cut-off above 1.65 kU_A/L in our population. When the best grass was obtained, the cut-off was applied to our patient samples, and good sensitivity (92.49%) and much better specificity (82.55%) were obtained compared to those obtained with the 0.35 kU_A/L cut-off (Table 7). Schäfer et al. evaluated the diagnostic value of SPT and sIgE in 75 children with hay fever and concluded that although at 0.35 kU_A/L NPV was similar for both methods (close to 100%), the PPV was very low for the in vitro assay. Increasing ImmunoCAP cut-off to 1.5 kU_A/L paired PPV from both methods (32). Van Hoeyveld et al., aimed to study sIgE cut-offs in pollen allergy, including grasses, using +LR as the main diagnostic tool. They concluded that for grass and birch pollen, the likelihood ratios for allergy increased with sIgE load. The likelihood ratio was <0.03 for specific IgE <0.1 kU_A/L, between 1.4 and 4.2 for sIgE between 0.35 kU_A/L and 3.5 kU_A/L, and very high (∞) for specific IgE >3.5 kU_A/L (33).

There are currently no consensus criteria for indication of AIT to grass pollen according to sIgE levels. In our daily practice, AIT is only prescribed in grass patients with Lolium-sIgE levels above 8.83 kU_A/L. This value is considerably higher than the diagnostic cut-off, considering that the prescription of grass AIT depends on several other factors. Thus, only patients with a considerable quality of life impairment due to allergic symptoms are selected for AIT. In our study, the severity of rhino-conjunctivitis and asthma correlated with Lolium sIgE levels, but paradoxically, not with those of rPhl p 1,5. It can be explained by the fact that severity has been shown to increase together with the number of recognized components, which may be represented in the Lolium extract (35, 36).

Unlike grasses, Olive pollen contains a greater diversity of genuine and relevant allergenic components in our region, and different sensitization profiles (Figure 2) need to be considered (37). The use of defined diagnostic algorithms is appropriate depending on the outcome that we want to achieve. If the clinical goal is merely confirmatory (confirmation of true clinical allergy), the determination of Olea sIgE is appropriate, using the cut-off of 6.50 kU_A/L to diagnose the allergy with confidence. However, if there is an additional objective such as immunotherapy prescription or patient phenotyping for any other purpose, it is essential to modify the algorithm. In this specific case, we recommend starting from the 2.41 kU_A/L cut-off.

The use of component-resolved extended diagnosis for olive pollen allergy depends, again, on the therapeutic goal (38). In cases where a most comprehensive diagnosis is required, it makes sense to implement an automated algorithm when established criteria are met. In our experience, it does not seem appropriate to test for components if Olea sIgE is less than 2.41 kU_A/L. When AIT is considered, the only determination of Olea sIgE in our population may be insufficient, even at levels above 6.50 kU_A/L, since Ole e 1, Ole e 7, and Ole e 9 display different sensitization profiles (Figure 2), as shown previously (12). Clinical symptoms and AIT response will differ depending on which one of them is the dominant sensitizer. For example, AIT is not recommended in patients with olive pollen allergy mainly driven by Ole e 7. Given the variability of the allergenic content of the different immunotherapy extract industrial batches and the AIT-related adverse event incidence, this must be considered (39). Diagnosis based on the ratio between sIgE to the molecular component and the sIgE to the whole extract may be appropriate to determine the main allergy driver component (5).

In our population, Ole e 1 sIgE above 10.45 kU_A/L was confirmatory for clinical allergy to olive pollen, but it was not a necessary condition. In other words, low or even undetectable levels of Ole e 1 sIgE did not rule out the diagnosis of olive pollen allergy, which could also be confirmed by Ole e 7 sIgE using the same diagnostic approach: values above 5.65 kU_A/L are also confirmatory. Both Ole e 1 and Ole e 7 have similar correlation coefficients with Olea whole extract. Ole e 9 showed a lower value because its presence is almost always associated with Ole e 1 sIgE.

When the best Olea sIgE cut-off was applied to our population, a good sensitivity (95.3%) and an increased specificity (83.7%) (Table 7) were obtained in comparison to those achieved with the arbitrary 0.35 kU_A/L cut-off.

The fact that we were unable to settle a cut-off value for Olea AIT indication reinforces the concept that this is a multiple factor-dependent decision that must be made on a case-by-case basis. A real-life example is represented by Ole e 9, which does not seem to influence the indication for olive AIT in our population. Nevertheless, the evidence of variability in Ole e 9 content from one immunotherapy batch to another requires an accurate characterization of Ole e 9 sensitized patients and only commercial immunotherapy extracts containing considerable amounts of the protein are valid for them (39).

Moreover, the Ole e 7 cut-offs for AIT prescription that we have calculated are biased by a particular prescribing habit in our group, which in clinical practice reduces the prescription of olive pollen AIT to patients exhibiting much higher levels of sIgE Ole e 7 than sIgE Ole e 1. The main reason is that a higher incidence of AIT systemic reactions in olive-allergic patients with exclusive or predominant sensitisation to Ole e 7 has been described (40). In fact, current EAACI recommendations do not recommend AIT treatment for patients with Ole e 7 sensitisation (41).

As also described by Moreno et al. (37), it should be noted that the final indication for AIT is conditioned by the sensitization complexity, meaning that it does not only depend on the different components involved but also the relative amount of sIgE to each of them. This issue was particularly relevant in the 58 patients who received double AIT (olive pollen extract plus grass pollen extract).

The main limitation of this study is the retrospective nature of data from clinical practice without a specific previous design. For this reason, there are notable data gaps, such as tIgE in some included patients. The analysis of the so-called ratios and the determination of cut-off values for these ratios could also have been of interest to complete our studies (5). Another limitation is that the sIgE values used in the ROC curves for diagnosis and immunotherapy probably conditioned the expert's decision, both in the diagnostic conclusion and the indication for immunotherapy. In addition, the response to specific immunotherapy was not assessed in this study, and therefore, there are no data to test the goodness of ROC curves to predict this response. Finally, implementing the quality-of-life scales to assess the severity of asthma/rhinitis could have introduced relevant information for our analysis.

Allergic profiles are local, so when talking about the generalization of the results (use of our established cut-off in other clinical settings), we must consider that this study was carried out in a single center in a specific geographical area; therefore, the safety of the data obtained when applied in other regions, still has to be determined. Nevertheless, the value of the differences in the variables between groups has been obtained with an explicitly stated confidence interval of 95%.

The main strength of the present study is that it thoroughly examines the use of likelihood ratios for the diagnosis and the immunotherapy indication in olive and grass pollen allergy. The reporting of sIgE sensitization in vitro test results using a Bayesian approach has recently gained interest. In contrast to the dichotomy of a single, often arbitrary cut-off point, the use of LRs helps to determine to what extent a test result changes the probability of a particular diagnosis or treatment indication (32, 42–45). Hence, the present study could help allergists in clinical decision-making. In any case, this work would reflect the need to establish different cut-off points for the standards used, which would interest other centers.

This study establishes different cut-offs (supported by robust data from a big patient cohort) that shorten the gap between sensitization and clinical relevance in the diagnosis of seasonal allergic rhinitis to olive or grass pollen, or both. It also provides cut-offs that, together with the clinical history and the sensitization profile, could help in clinical decision-making, such as the AIT prescription. Additionally, it suggests that simplified diagnostic algorithms would save analytical determinations while improving patient diagnosis and healthcare system efficiency.

Further prospective studies are needed in which the predictive value of sIgE for diagnosis could be compared with the expert's diagnosis, blinded to this factor and based on clinical and skin prick test results. Likewise, it could be helpful to include the evaluation of the response to immunotherapy and not exclusively the indication for immunotherapy.