Table 4 Diagnostic work-up

IN VIVO TESTS

 Skin tests for common aeroallergens and food allergens should be performed, especially if IA episodes are reported within about 2 h from meals or after outdoor activities or contact with animals. With new types of foods being launched on the market, such as insect products, carefully tracing the dietary clinical history is mandatory and PbP should be carried out [63] if no extract-based skin prick tests are available. In patients with known pollen allergies, severe forms of oral allergy syndrome should be considered, in particular in those with LTP sensitizations, which could lead to anaphylactic reactions [64]. While considering possible food allergies, it is important to carefully investigate hidden or uncommon allergens, sometimes used as decorations rather than declared ingredients. As pointed out by Bilò et al. [65], failing to identify food allergens usually depends on mislabeling and cross‐contamination

IN VITRO TESTS

Blood tests

  Tryptase

   The diagnosis can be supported by elevated acute serum tryptase level. Most centers rely on the calculation of a significant increased tryptasemia if the acute total tryptase level is at least 20% plus 2 ng/ml over the patient’s basal tryptase level. However, Mateja et al. [66] evaluated that an acute/baseline tryptase ratio of 1.685 has a sensitivity of 94.4% and a specificity of 94.4% for anaphylaxis diagnosis. Moreover, using a low/high clinical suspicion, the cut-off ratio was 1.868 when suspicion was low and 1.374 when suspicion was high. An online calculator (https://triptase-calculator.niaid.nih.gov) is freely available. Although tryptase levels above the defined normal value (e.g., > 11.4 ng/mL in most laboratories) can be a valuable source of information, some cases of anaphylaxis may not be associated with tryptase elevation. Some authors even argued that certain slight variations could be considered as a normal intra-individual fluctuation. Waters et al. [67] have highlighted the importance of obtaining different baseline tryptase values: among their case-series, the suggested formula (20% plus 2) lacks specificity compared to the above mentioned 1.685 threshold ratio (acute/basal tryptase levels)

The correct evaluation of tryptase (at baseline, possibly more than once, and during acute allergic events) [68] has gained more importance in the last years after the definition of HaT [69, 70]. If baseline levels are greater than 8 ng/mL, it is advisable to consider additional tests for HaT. The scientific debate on normal tryptase values is still ongoing, as some authors propose a 1–15 ng/mL normal values interval [71], while other authors argue for individual ranges [72]. Moreover, few specific data on the topic in the pediatric age have been collected so far

  Specific IgE and Component Resolved Diagnostics

   Heaps et al. [73] evaluated the use of an allergen microarray (ISAC®, ThermoFisher, Uppsala, Sweden) to gather more information in patients with IA. In 20% of cases, a “highly likely” allergen was identified, previously not detected with skin or specific IgE tests (although no provocation test was performed to confirm a cause/effect relationship). The Component Resolved Diagnostics (CRD) is a useful diagnostic tool both in routine evaluation (e.g., selection of allergen immunotherapy) and in selected cases, e.g. in IA or in patients with multiple allergies or concomitant diseases [74]. Cardona et al. [32] have recently pointed out that, with CRD, a percentage of IA could be resolved, as some of the most important allergens in the field could only be identified with this diagnostic technique (alpha-gal, omega-5-gliadin, lipid transfer proteins and oleosins). Other tests are available in addition to ISAC®, such as Immunolyte® (Siemens Healthcare Diagnostics Inc, Elangen, Germany), Alex2® (MADX, MacroArray Diagnostics GmbH, Wien, Austria), Euroline® (EUROIMMUN AG, Lubeck, Germany) and FABER® (Allergy Data Laboratories srl, Latina, Italy)

  Basophils Activation Test and Mast Cell Activation Test (MAT)

   Anaphylaxis could rely on different pathogenesis and it could be completely independent from the classical IgE mediated pathways. Activation and degranulation of MCs and basophils could occur through complement cascade, Mas-related G protein-coupled receptor X2 (MRGPRX2) pathway, or even independently from MCs and basophils [75]. A diagnostic aid could be provided by Basophils Activation Test (BAT) and Mast Cells Activation Test (MAT), unfortunately often available for research purposes only

  Basophils Activation Test

   Basophil Activation tests (BAT) could be helpful to confirm CRD or when standard workup turns out negative. Different allergens are available, including perioperative drugs [76] and Hymenoptera venom [77]

  Mast CellActivation Test (MAT)

   Another cellular test could be performed, especially when skin tests are not available or indicated [78]. MAT also has some advantages compared to BAT since it does not require fresh samples to be analyzed in a very short timeframe. Moreover, it could be performed with passively sensitized MCs – testing MCs responsiveness beforehand – this way overcoming the non-responders’ issue. MATs could be useful in IgE-mediated and non-IgE mediated reactions with reports such as with aeroallergens, foods and several type of medications [79]

 Other tests

 A complete evaluation should always include a panel of the Complement Pathway (C3, C4, C1‐INH functional and quantitative tests, anti-C1-INH antibodies), especially if angioedema is reported

  24 h urine collection

   A 24-h urine collection to include circadian variations should be considered, so as to exclude some anaphylaxis mimicries such as pheochromocytoma, carcinoid syndrome and medullary thyroid carcinoma. Tests may include histamine levels and its metabolites (such as N-methylhistamine or methylimidazole acetic acid), catecholamines and its metabolites (such as dopamine, adrenaline, noradrenaline, vanillylmandelic acid, 5-hydroxyindoleacetic acid), chromogranin A, prostaglandin D2 and leukotriene C4. However, their cutoff levels, specificity, and sensitivities are not well established [54]

  Evaluation for mast cellactivation diseases

   A specific mention must be made about the evaluation of MCAD, which includes mastocytosis and MCAS. To formalize a MCAD diagnosis, three diagnostic criteria should be fulfilled: clinical manifestations, MCs activation markers and response to therapy [80]. Once diagnosed, MCAD could be further classified as primary, secondary and idiopathic [81]. Primary MCAD recognizes a clonal origin (such as point mutation D816V in c-KIT and/or aberrant CD25 expression). In secondary MCAS, a non-clonal MCs population is responsible, as MCs are shown to be normal in quantity and function and activated by IgE mediated and non-IgE mediated pathways, such as MRGPRX2 or complement cascade, or physical factors as exercise. In idiopathic MCAS, in which AI has been proposed to be included [19], none of the abovementioned mechanisms could be demonstrated. The importance of a correct and prompt MCAD diagnosis has been addressed by several recent papers [82,83,84,85]. Giannetti et al. [86] have also provided specific indications and diagnostic algorithm for the pediatric age

  Bone marrow biopsy

   REMA o NICAS score could be used to determine when to perform bone marrow biopsy in patients with recurrent mast cell-mediated symptoms or recurrent IA episodes [48, 87,88,89]. However, it has been suggested that children, unless demonstrated involvement of spleen, liver, lymph nodes, and peripheral blood are present,do not need to perform a bone marrow biopsy [86, 90]

PROVOCATION TESTS

 Provocation tests

  After the identification of a possible specific allergen through clinical history or through a positive allergy test, a provocation test (PTs) could be necessary to confirm the diagnosis. PTs must be performed in hospital settings with specialized personnel and equipment [91]. In case of drug hypersensitivity, if a PT for the culprit drug is not indicated, an alternative drug should be identified [92]. PTs could be also associated with exercise to diagnose FDEIA [93]