Study Population
Patients aged 18-45 years of both sexes who had a history of allergic diseases were enrolled in the study. Women of childbearing potential should have a negative blood pregnancy(β-HCG) test result. Patients were invited for a preliminary assessment to evaluate their serum sIgE concentration (UniCAP, Phadia, Sweden) of the eight investigated allergens. For Study 1, patients with the sIgE concentration of one of the eight allergens within 0.70 ~ 49.9 KUA/L were enrolled. For Study 2 patients were requested to have the sIgE concentration of at least two kinds of the eight allergens within 0.70 ~ 100 KUA/L. Besides, patients enrolled in Study1 should pass a SPT of the positive and negative control solution in the "Skin Prick Test Kit for Der.f.(Dermatophagoides Farinae) Detection" (produced by Zhejiang Wolwo Biopharmaceutical Co., Ltd.), the diameter of wheal was ≥3 mm for positive control solution and no wheal for negative control (redness was allowed).
Key exclusion criteria included the following: people with orthostatic hypotension, a history of halo needles or halo blood, intolerance to pricks, symptoms of angioedema, or contraindications to adrenaline; women during pregnancy or lactation; had severe allergic reactions or anaphylactic shock; with a history of asthma or in the stage of asthma attack; had used antihistamine drugs within 7 days; had used phenothiazine drugs or imipramine antidepressants within 30 days before the trial, or being treated with beta blockers or angiotensin converting enzyme inhibitors; undergone ultraviolet chemotherapy within 30 days; had used glucocorticoids in the forearm within 2 days; with skin scratches; skin infections, dermatitis, pathological changes such as trauma, scars or tattoos on both forearms; with a recent history of drug or alcohol abuse, and use of tobacco or nicotine containing products within 3 months before check-in.
All participants were provided with written informed consent before study initiation. The study protocol was reviewed by the independent Ethics Committee of the Third Xiangya Hospital of Central South University and the ethics committee of Tianjin Medical University General Hospital, and performed in accordance with the principles stated in the Declaration of Helsinki. The trial was registered on www.chictr.org.cn (ChiCTR1900023952, 06/19/2019, retrospectively registered).
Skin Test Material
Eight different SPT solutions were tested: artemisia annua pollen, platanus pollen, dog dander, betula platyphylla pollen, cat dander, humulus pollen, blattella germanica and ragweed pollen. The test products were provided in vials containing different concentrations of each allergen. Histamine phosphate (1.70 mg/mL) was used as positive control and glycerin saline solution (v:v=1:1) as negative control solution. All SPT solutions and positive and negative controls were manufactured by Zhejiang Wolwo Biopharmaceutical Co., Ltd.
Study Design
Study 1
Study 1 was conducted as an open-label, parallel study to investigate the effectiveness and tolerance of the subjects to the single allergen within a concentration range at two centers in China. Patients with serum sIgE concentration of the allergen within 0.70 ~ 49.9 KUA/L were enrolled into corresponding allergen groups. SPTs with single allergen and positive and negative controls were performed at the same time and subjects should be followed up to 24 h. After a washout period of 48 hours, the next concentration of SPT solution would be given. The test products contained three different concentrations of each allergen increasing in three-fold steps, and the concentration of each SPT solution were shown in Table 1.
Study 2
Study 2 was conducted as a single-center, open-label, parallel study to investigate the tolerance of the subjects to the combination of eight allergens at two different concentrations. Patients with the serum sIgE concentration of at least two kinds of the eight allergens within 0.70 ~ 100 KUA/L were enrolled. SPTs with both eight allergens and positive and negative controls were performed at the same time and subjects should be followed up to 24 h. Each allergen in Study 2 included two concentrations groups. The optimally diagnostic concentration in Study 1 was selected as Conc.1 for group 1, and the triple of optimally diagnostic concentration was Conc.2 for group 2. The two concentrations of each SPT solution were shown in Table 2. The test of Conc.1 group was started first, and if there was no event that meets the termination criteria, the test of Conc.2 group was performed.
Skin Prick Test
SPTs were performed on the volar sides of forearms. The investigated allergens as well as positive and negative controls were tested. The test areas were numbered by means of a suitable skin marker and had a minimum distance of 3 cm to each other. The skin was pricked lightly and quickly vertically through the drop of the SPT solution by means of a microlancette. For each prick a new microlancette was used.
The test solution was removed in 3 min after the SPT by laying an absorbent paper towel on the skin prick area and carefully pressing it on the skin. The wheal outlines were read after 15-20 min. The wheal outline was traced with a ballpoint pen, then taken off from the patient’s skin and documented by sticking them into the grid paper using a broad piece of translucent tape. The average diameter (Y) was recorded as: Y = (D + d) / 2, the longest diameter of the wheal was D and the mid-vertical line of the longest diameter was d. Then the original wheal area was preserved as a picture. For assessment of a positive SPT reaction, the wheal had to be ≥3 mm in diameter. A valid SPT result also required a positive histamine reaction (≥3 mm) and a negative saline control reaction (< 3 mm) [15].
Estimation of Sensitivity and Optimally diagnostic concentration
The estimation of sensitivity was carried out in Study 1. The number of patients with a positive SPT result at different concentrations of each allergen was counted to calculate the positive rate of each allergen at different concentrations, i.e. the sensitivity of the allergen. The sensitivity was estimated by the number of patients with a positive SPT result (diameter of wheal≥3 mm) divided by the number of all patients with a valid SPT result. And the optimally diagnostic concentration for each allergen was investigated by determination of sensitivity for each concentration of eight allergens based on safety. Besides, the diameter of wheal at different concentrations of each allergen was compared to evaluate the relationship between allergen exposure and allergic reaction.
Safety Assessments
Both Study 1 and Study 2 were included in safety analysis. Safety assessments included physical examination, vital signs, 12-lead ECGs, routine hematology urinary and chemistry laboratory measurements. Besides, adverse events (AEs) were monitored throughout the study and were evaluated by investigators in terms of intensity, duration, severity, outcomes, and association with the investigated allergen extracts.
The wheal or itchy that appeared in prick area within 24 hours after the prick test was not considered as an AE, while it would be recorded as an AE related to allergen if the above phenomenon still exists after 24 hours.
Statistical Analysis
Sensitivity and safety analysis were planned to be descriptive. The safety population consisted of all patients exposed to any investigated allergens. All patients with a valid SPT result in Study 1 were evaluated for sensitivity analysis. The sensitivity for each concentration of eight allergens was estimated by the number of patients with a positive SPT result divided by the number of all patients with a valid SPT assessment. A valid SPT assessment required a positive histamine reaction (≥3 mm) and a negative saline control reaction (< 3 mm), and a positive SPT result additionally required a wheal with the diameter≥3 mm. Related-Samples Friedman's Two-Way Analysis was used to evaluate the difference of wheal diameter between different concentrations of each allergen. SPSS Statistics 26 was used for statistical analysis, and the significance test level was 0.05.