Methodological considerations

6.1.1 STUDY POPULATIONS

In the cross-sensitization study we included all patients that were skin prick tested for birch pollen at the Skin and Allergy Hospital during the chosen study years 1997-2013. This resulted in a very large sample size, at over 100,000 individuals. The tested individuals had all had some clinical indication for skin prick testing, i.e. suspicion of an allergy or atopy. This makes our results generalizable to patients that need assessment of sensitizations and advice on the very common finding of nut sensitization. The findings of our study will help to assess sensitized patients in primary as well as in specialist care centers.

Our study population in Rovaniemi was markedly smaller than the Helsinki population, because the general population of Lapland is smaller and, in addition, we were not able to include all patients skin prick tested to birch pollen during the study years, because the archive had to be searched and saved manually. The Rovaniemi study population of nut-sensitized patients included mainly hazelnut-, almond-, and peanut-tested patients. Other species were very infrequently tested, which limited the geographical comparison to only these commonly tested nuts. We assessed overall birch pollen sensitization rates in Rovaniemi from a separately collected population of patients that were tested during the time when the results were reported in millimeters. For nut

sensitization, we also included qualitative results, which limited us to studying sensitization only from a qualitative point of view.

Our study is limited by the fact that we assessed sensitizations only. Clinical relevance was assessed only in a subgroup of patients and was based on patient-reported symptoms and no challenges were conducted. Some patients may possibly have experienced more severe symptoms when challenged to high doses of nut protein, however, any severe reactions were not confirmed by the challenge, either. Furthermore, our study population included subjects with the clinical suspicion of birch and nut allergies, and our data are thus subject to

overestimation of these co-sensitizations. Moreover, the findings of age-related changes can be regarded only as indicative and may be skewed by any possible differences in patient selection or changes in population rather than at an individual level. Our research extends to the year 1997 and was designed to study cross-sensitizations in skin prick tests. We were limited to skin prick tests, and the lack of serum samples, and due to the large sample size we, we were not able to analyze serum-specific IgE. In clinical use, however, component-resolved diagnostics is adapted to discriminate between cross-sensitization and true allergy.

In addition to skin prick testing, we studied the diagnostics of nut allergy in patients who were double-blind placebo-controlled challenged to peanut. This enabled us to compare laboratory measurement diagnostics to the gold standard double-blind placebo-controlled challenge. Our population was selected based on sensitization to peanut either in skin prick tests or in an in vitro IgE test for the whole peanut extract. This makes our results generalizable to patients referred to specialist centers or examined in a primary health care setting and who are peanut-sensitized. Our patients had either experienced symptoms from peanut previously or had never ingested peanut and were unaware of their allergy status. This setting reflects true nut allergy diagnostic dilemmas accurately, as many children, especially younger ones, have never ingested specific nut species.

Peanut oral immunotherapy was studied in severely allergic patients. The allergy was diagnosed with a double-blind placebo-controlled peanut challenge, and our inclusion criteria for OIT required patients to have at least a moderate-to-severe reaction in the challenge, in order to be eligible for immunotherapy.

Thus, our patients had a true and severe peanut allergy, and patients with only pollen-cross-reactive peanut sensitization were excluded. This study was designed to assess the efficacy and safety of peanut oral immunotherapy in moderate-to-severely allergic children with even anaphylaxis, as they would benefit the most from this treatment.

A major limitation of our study was that the study groups were not randomized but families chose themselves whether or not they wanted to have

immunotherapy. Also, subgroup analyses according to outcomes were limited in this study, because the study was powered to show the efficacy of

desensitization. Thus, we were unable to unravel factors that would predict success in OIT.

In the study of antibody changes during immunotherapy, we included all patients that received the intervention and control patients that continued to avoid peanut, i.e. the traditional treatment for nut allergy. Two patients from the avoidance group were excluded because of an unavailable serum sample.

The sample size in peanut oral immunotherapy was based on the primary outcome, which was the ability to pass the end-challenge after achieving the

maintenance phase. Thus, we were limited to this sample size when assessing our secondary outcomes.

6.1.2 CHALLENGES

Peanut challenges were conducted in a double-blind placebo-controlled fashion, which is the gold standard of food allergy diagnostics. The cumulative 1255 mg protein dose is sufficient to exclude reactivity to accidental amounts of peanut.

The study challenges were conducted according to PRACTALL guidelines,¹⁵⁶ which makes them generalizable to other study centers. The lowest dose of peanut protein in our challenge protocol was selected based on clinical demands and was set to 5 mg. Lower starting doses would have enabled us to examine the threshold dose for the Finnish population more accurately, however, our threshold results were in line with published thresholds from other populations.

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6.1.3 IGE MEASUREMENTS

IgE was measured both in singleplex ImmunoCAP and in multiplex microarray ImmunoCAP ISAC. In singleplex measurements, serum samples were diluted if the results exceeded the 100 kU/L limit, in order to measure even the highest IgE levels in our patients, some of whom had IgE levels counting several hundreds. We were able to study IgG4 in ImmunoCAPs as well, and this method was not a routine standard at the time of the study.

In the IgE microarray ImmunoCAP ISAC, we were able to screen our patients in relation to 112 allergens from a large variety of allergenic sources and protein families. As the microarray is semi-quantitative, we were unable to compare our results directly from the singleplex ImmunoCAPs to the microarray.

6.1.4 AIRWAY MEASUREMENTS

Airway inflammation in patients receiving peanut oral immunotherapy was measured by examining fractional exhaled nitric oxide and airway

hyperresponsiveness by methacholine challenges. The measurements were conducted before treatment and approximately one month after achieving the maintenance phase, i.e. after the build-up phase. Fractional exhaled nitric oxide is recommended for monitoring airway inflammation in patients with asthma

237, while the methacholine challenge is recommended for testing airway

hyperresponsiveness.²³⁸In our study, patients' asthma treatment was conducted according to clinical needs. As asthma is a risk factor for severe reactions in food-allergic reactions, we assessed possible asthma symptoms in all study visits. None of our patients was excluded from the study due to poor asthma control.