Clinical symptoms observed by Schocker et al. (2004) [951] in 26 patients included: Oral allergy syndrome in 7 of 26 patients, anaphylaxis (6/26), angioedema (6/26), urticaria (2/26) and food-dependent exercise-induced anaphylaxis in a single patient.

14 patients were reported with systemic symptoms (8 urticaria, 7 respiratory, 6 gastrointestinal, 2 angioedema and 2 OAS) by Beyer et al. (2002) [670].

31 patients were reported by Wensing et al. (2002) [666] all of whom showed oral allergy syndrome together with rhinoconjunctivitis (2/31), urticaria (2/31), urticaria, angio-oedema and anaphylactic shock (1/31) and nausea (2/31).

Clinical symptoms observed by Ortolani et al (2000) [448] include : 1) Systemic reactions: anaphylaxis, exercise-induced anaphylaxis, 2) Cutaneous symptoms: angioedema, atopic dermatitis, eczema, rhinoconjunctivitis, urticaria. 3) Gastrointestinal symptoms: angioedema of lips and tongue, diarrhoea, glottis edema, laryngeal edema, oral itching, swelling of lips, tongue and throat, vomiting, oral allergy syndrome, perioral erythema. 4) Respiratory symptoms: allergic rhinitis, asthma, bronchial obstruction, dyspnoea. 5) Other symptoms: migraine.

In a study of 172 patients who experienced anaphylactic reactions, 13% reacted towards tree nuts including hazelnut (Pumphrey and Stansworth, 1996 [479]).

Clark and Ewan (2003) [615] used a positive (histamine 10 mg/mL) and negative control (saline). SPT wheal diameters were listed as <3 mm, 3-7 mm and >8 mm.
Crespo et al. (2002) [834] used a positive (histamine 10 mg/mL) and negative control (saline) and the SPT was counted as positive if the wheal diameter minus the control was >3 mm.
Akkerdaas et al. (2003) [669] also used a positive (histamine 10 mg/mL) and negative control (glycerol) but counted the SPT positive if the wheal diameter was 0.25 of the positive control.

Asero et al. (2002) [667] studied 600 patients with Rosaceae (apple, pear, peach, cherry, apricot, plum, or almond) allergy and used positive SPT with commercial plum extract; negative SPT with a commercial birch pollen extract; in-vitro IgE reactivity to the 9-10 kDa fraction of peach peel or immunoblot with peach peel showing a single band at 10 kDa; and total inhibition of reactivity to whole peach extract (containing Bet v 1-related allergen, profilin, and LTP) by purified peach LTP on enzyme-linked immunoassay (ELISA) to select 20 patients monosensitised to nsLTPs.

Crespo et al. (2002) [834] studied 15 fruit allergic patients.

Akkerdaas et al. (2003) [669] studied 30 patients, 15 of whom had hazelnut allergy confirmed by DBPCFC.

Clark and Ewan (2003) [615] tested 1000 peanut or tree nut allergic patients and 63 reacted most strongly to hazelnut.

Asero et al. (2002) [667] found that all 20 patients monosensitised to nsLTPs gave positive SPT to the hazelnut extract.

Crespo et al. (2002) [834] reported that 6/15 fruit allergic patients had either a positive SPT or CAP-RAST test to hazelnut.

Akkerdaas et al. (2003) [669] found that the 9 extracts tested gave variable results, probably because they differed in which allergens they contained (the extracts were tested for Cor a 1, Cor a 8 and profilin, thaumatin-like protein and a 38-kD allergen). There were 6/30 false negatives.

Clark and Ewan (2003) [615] found that only 3% of the hazelnut tolerant individuals had a strongly positive SPT with hazelnut (≥8 mm) while (over all nuts) 98% of the allergic individuals had SPT ≥3 mm.

CAP-RAST (Pastorello et al. 2002) [671].

CAP and EAST (Müller et al. (2000) [1116] and Schocker et al. 2004 [951]).

Müller et al. (2000) [1116] used sera from 27 hazelnut allergic patients and 28 sera from children with positive CAP to both hazelnut and birch pollen.

Pastorello et al. (2002) [671] used sera from 65/67 patients with confirmed allergy by DBPCFC (Ortoloni et al. 2000 [448]) together with an additional 7 patients from the Milan region who had more severe reactions after ingestion of hazelnut (with anaphylactic reaction by case history).

Crespo et al. (2002) [834] used sera from 15 fruit allergic patients.

Beyer et al. (2002) [670] used sera from 14 patients with systemic hazelnut allergy.

Akkerdaas et al. (2003) [669] used sera from 14 of the 30 patients used for SPT.

Schocker et al. (2004) [951] used sera from 26 patients with a clear history of adverse reactions to hazelnut and without birch pollen allergy.

Lauer et al. (2004) [1117] used sera from 65 patients with a positive case history of immediate-type reactions to hazelnuts (39 of 65 of the patients were DBPCFC positive to hazelnuts).

Pastorello et al. (2002) [671] found that 12/65 patients had negative CAP IgE assys for hazelnut while all were CAP positive for Bet v 1 from birch pollen. The additional 7 patients who had more severe reactions after ingestion of hazelnut were all positive for hazelnut but only 2 were positive for Bet v 1 and another for Bet v 2 from birch pollen. These patients had IgE against the nsLTP, Cor a 8.

Crespo et al. (2002) [834] reported that 6/15 fruit allergic patients gave either a positive SPT or CAP-RAST test to hazelnut.

Beyer et al. (2002) [670] found that all 14 sera were positive for hazelnut, 0.75->100 kU/l, and 8 were also positive for hazelnut pollen.

Akkerdaas et al. (2003) [669] found that both the tested commercial extracts (7/9) and their own extract bound variable amounts of IgE from the 14 sera by RAST and RAST inhibition but that two extracts bound significantly more IgE from most sera.

Schocker et al. (2004) [951] found sera from 1/26 patients gave CAP class 5, 4/26 class 3, 14/26 class 2, 2/26 class 1 and 5/26 negative with hazelnut extract. 1/26 sera gave EAST class 3 with rCor a 8, 7/26 class 2, 6/26 class 1 and 12/26 negative. Only 1/26 sera gave class 3 EAST to rCor a 1.0401.

Pastorello et al. (2002) [671] used a 7.5% to 20% gradient 1D-SDS PAGE. Samples were boiled for 5 minutes in SDS with beta-mercaptoethanol.

Beyer et al. (2002) [670] used 2D separation using 7-cm immobilized pH 5-8 gradient strips (Biorad) with urea, CHAPS and tributylphosphine for 17 hours followed by iodoacetamide treatment and electrophoresis on a 4-12% gradient gel (Bis-Tris ZOOM, Ivitogren, Carlsbad, Calif.).

Schocker et al. (2004) [951] separated hazelnut extract (20 µg/cm total protein) and recombinant nsLTP (0.5 µg/cm) using NuPAGE-Bis-Tris gels (10%; Novagen, Groningen, The Netherlands) under nonreducing conditions.

Lauer et al. (2004) [1117] separated purified nCor a 11 and rCor a 11 (1.5 µg/cm), rCor a 1.04 (0.5 µg/cm), rCor a 2 (0.5 µg/cm) and rCor a 8 (0.5 µg/cm) by Tricine SDS/PAGE. Reducing conditions were used for nCor a 11 and rCor a 11 and rCor a 8 was treated under non-reducing conditions.

Pastorello et al. (2002) [671] transferred proteins to nitrocellulose paper (Amersham) or PVDF membranes (ProBlott, Applied Biosystem, Foster City, Calif.). The nitrocellulose was blocked, cut into strips and incubated overnight with sera. IgE was detected with iodine 125 labelled anti-human IgE (Pharmacia & Upjohn) diluted 21:5 in blocking solution and X-ray film at -70 degrees C for 4 days.

Beyer et al. (2002) [670] transferred proteins to Immobilon-P membrane (Millipore, Bedford, Mass). Proteins were visualized with Comassie Blue . Immunoblots were incubated with gentle agitation for 1 hour with 1:4 to 1:10 dilutions of sera in PBS-Tween with 1% (w/v) BSA and 10% (v/v) goat serum. After washing, alkaline phosphatase conjugated monoclonal anti human IgE in the same conditions was added for 45 minutes. After washing and adding 50 mM Tris at pH 9.6, allergens were revealed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium

Schocker et al. (2004) [951] transferred proteins onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Serum samples were pooled and diluted 1:20 in PBS containing 0.05% Tween 20. The bound IgE antibodies to hazelnut extract were detected by incubating the strips with iodine 125–radiolabeled sheep anti-human IgE (SH25-1-p7; Sanquin, Amsterdam, The Netherlands).

Lauer et al. (2004) [1117] transferred proteins on to nitrocellulose membranes (0.2 µm) by semi-dry electroblotting. The membrane was blocked in Tris-buffered saline with 0.3% (v/v) Tween 20 and incubated with 1:10 (v/v) diluted patients' sera. Bound IgE antibodies were detected by enzyme-coupled anti-human IgE followed by enhanced chemiluminescence (Pharmacia).

The amount of total protein transferred to the membrane as well as blocking method were generally critical for the result (Vieths, S., personal communication).

Müller et al. (2000) [1116] report the profile of IgE reactivity of sera from both groups (27 and 28 patients) against raw hazelnut extract. Bands were detected at 11-12, 18-19, 10-22, 24-24, 38-39, 43-44, 48-52 and >60 kDa (also cross-reactive carbohydrate at >50 kDa). 93% and 79% of sera in the two groups detected the 18-19 kDa band. 12 sera from hazelnut allergic patients were tested with raw and roasted hazelnut extracts. IgE from 11/12 sera reacted to a 17-18 kDa band in raw hazelnut extract, which completely disappeared using roasted hazelnut. IgE from 6/12 sera bound to a 48 kDa band from raw hazelnut extract. 4 of these 6 sera were CAP positive to roasted hazelnut and also detected proteins between 36 kDa and 90 kDa from roasted hazelnuts on immunoblots, including a 43 kDa band. Müller et al. also described a heat-stable hazelnut allergen with a mass below 14 kDa that was recognised by IgE from one patient.

Pastorello et al. (2002) [671] found 63/65 patients had IgE specific for the 18 kDa allergen, 62/65 for a 47 kDa, 36/65 for a 32kDa and 33/65 for a 35 kDa allergen. The IgE binding to the 18 Kda allergen was completely inhibited by birch pollen extract as was binding to one 14 kDa band. IgE binding to the higher molecular wt. allergens was also inhibited. The 7 extra patients from Milan all reacted to the 9 kDa allergen Cor a 8. This was not inhibited by birch pollen extract but was by peach extract and by peach LTP.

Beyer et al. (2002) [670] found that 12/14 patients (86%) had IgE recognising Cor a 9 at 40 kDa.

Akkerdaas et al. (2003) [669] identified an allergen running at 38 kDa in 6 of the commercial hazelnut extracts and in their own extract which bound IgE from pooled sera. A 2S albumin with cross-reactivity to Ber e 1 was also observed in some extracts.

Schocker et al. (2004) [951] found that 16/26 patients' sera displayed IgE reactivity to a 9 kDa protein in hazelnut extract. 2/16 sera detected binding above 20 kDa in addition to the 9 kDa protein. 1/26 sera contained only IgE antibodies to Cor a 1.04 at 18 kDa.

Lauer et al. (2004) [1117] found that rCor a 11 and nCor a 11 bound IgE from 43% and 47% of the 65 serum samples respectively. These authors also reported that 98.5% had IgE to the major allergen, rCor a 1.04; rCor a 2 was recognized by IgE from 15.4% of the sera, whereas no reactivity was found towards rCor a 8 (LTP). rCor a 1.04 and rCor a 2 had strong IgE reactivity, resulting in intensive bands, whereas often a weaker signal for Cor a 11 was observed.

Ground dried hazelnuts were used except Skamstrup Hansen et al. (2003) [665] who also used ground roasted hazelnuts.

Ortolani et al (2000) [448] gave doses in a pudding made from ground hazelnuts (20 g), water (100 mL), sugar (13 g), peppermint syrup (48 mL), rice flour (11 g), bitter cocoa powder (10 g), saffron (a pinch), and milled rice grains (1 tablespoon). The first dose of 1.4-1.5g of ground hazelnut was kept 30 - 60 sec. in the mouth before swallowing. This dose is doubled at 10 -15 min intervals until either reactive symptoms were observed or all the pudding had been consumed

Wensing et al. (2002) [666] used seven doses of 1mg, 3mg, 10mg, 30mg, 100mg, 300mg and 1g of hazelnut protein (corresponding to 6.4, 19, 64, 190, 640, 1900 and 6400mg of hazelnut meal, respectively). These doses were completed with a mixture of 0.67g/g dry oatmeal flakes, 0.33g/g brown sugar and 0.0033g/g salt up to a total weight of 15g per test meal. The placebo doses consisted of oatmeal flakes, brown sugar and salt alone. Just before administering, the test meal was mixed with water of a temperature of 40-50°C to obtain oatmeal porridge. All patients were noseclipped while eating the test meals.

Ortolani et al (2000) [448] tested 86 patients

Wensing et al (2002) [666] tested 31 patients

Crespo et al. (2002) [834] reported 3 positive challenges after excluding a patient with a history of anaphylaxis from 6 patients with positive SPT or IgE test.

Skamstrup Hansen et al. (2003) [665] tested 17 hazelnut allergic patients.

Schocker et al. (2004) [951] challenged 8 patients.

Wensing et al. (2002) [666] used 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300 mg and 1 g of hazelnut protein (corresponding to 6.4, 19, 64, 190, 640, 1900 and 6400 mg of hazelnut meal, respectively). 4 patients reacted to the lowest dose. The dose corresponds to the initial subjective reaction. From the dose response curve, 50% of a hazelnut-allergic population will suffer from an allergic reaction after ingestion of 6 mg.

Ortolani et al (2000) [448] found that of 86 patients, 67 (77.9%) had a positive DBPCFC, 8 were placebo responders and 11 were non responders. Of these 59 had OAS, 3 oral and gastrointestinal symptoms and 5 oral and systemic symptoms.

Wensing et al. (2002) [666] found 29/31 patients reacted and one additional patient reacted to both hazelnut and placebo. All reacters showed itching of the mouth as the first symptom, one showed generalized urticaria and lip swelling and another slight lip swelling at 1mg. dose. Some gastrointestinal symptoms, abdominal pain and nausea, were also reported. The challenge was continued until objective symptoms were observed or subjective symptoms persisted for 1 hour (7 patients). 1 patient's challenge was ended after subjective symptoms because of anxiety.

Skamstrup Hansen et al. (2003) [665] found that 5/17 patients reacted to roasted hazelnut with OAS while all 17 reacted to raw hazelnut (as Ortolani et al 2000 [448]). The median dose for reaction was more than doubled for roasted hazelnut.

Schocker et al. (2004) [951] used challenge to diagnose 7 patients reporting oral allergy syndrome and one reporting excercise induced anaphylaxis.