A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Summary

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Abstract

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China's largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.

For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.

Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.

This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

Introduction

Grass pollen accounts for nearly 50% of immunoglobulin E (IgE)-related allergies and, because of its airborne nature, imposes a global risk of allergic airway diseases, particularly asthma and allergic rhinitis^1,2,3,4. Prevention against grass pollen allergies in a region relies on information about the local distribution of the pollen allergens. Unfortunately, the spectrum of the sensitizing allergens varies from place to place—an observation generally known as regional specificity.

In China, a vast East-Asian territory, regional specificity of grass pollen sensitization is further complicated by limited data currently available from the southern part of this country over the past decades⁵. Such a paucity in data has led to a difficulty in showing the overall grass pollen sensitization in this large country⁶. Additionally, the very limited data on Chinese southerners were largely obtained from self-report questionnaires, skin prick tests (rather than laboratory serum tests), total IgE measurements (rather than allergen-specific IgE (sIgE)), and sIgE detection to allergens (rather than to allergen component molecules).

The self-reporting of allergic symptoms can be unreliable when specific allergens are concerned. Skin prick tests, although widely accessible, involve the use of non-standardized, crude allergen extracts, which themselves may contain other sensitizing components that confound the outcome interpretation. The reliability of the skin prick test also critically depends on skillful manipulation by allergists or specialist nurses, recent histamine use, and patient compliance (sometimes very difficult for young children). Total IgE is useful for screening an allergy but does not sensibly determine the offending allergens in a patient. Moreover, almost all allergens contain a number of allergen component molecules. Sensitization to the major components (i.e., Der p 1 and Der p 2 from house dust mites) may suggest a "genuine" allergy (allergy to a specific allergen, rather than false positivity due to cross-reactions with other allergens), but sensitization to certain ones (i.e., the cross-reactive Der p 10, a tropomyosin) may not⁷.

Before component-resolved diagnostics (CRD) was introduced, these disappointing setbacks impeded allergen studies in China. CRD precisely detects the components of an allergen by using recombinant or purified allergen molecules, thereby making the measurement easily quantifiable and characterizable⁸. In past decades, allergy studies frequently employed crude extracts of allergens that inevitably contain a number of irrelevant components. Many of these components are inert, but occasionally, some could lead to confounding positive reactions. The use of recombinant or purified allergen molecules in CRD can circumvent this and therefore provide better test accuracy. When processed in microarrays, a CRD approach rapidly screens or identifies sensitization to hundreds of allergen molecules in individual subjects. A well-designed CRD study on grass pollen sensitization in southern China is currently lacking. Ideally, such a study should include a local population of considerable geographic coverage, account for grass pollens commonly reported in China, and address the technical aspects mentioned above. To reach a practical level of clinical relevance and to facilitate the recruitment of serum samples, early investigation on sensitization to grass pollens as inhalant allergens may as well be focused on patients with allergic respiratory disorders, rather than on the general population.

This work describes a method used to test serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens in patients from Greater Guangzhou (southern China's largest city and its outskirts) with allergic rhinitis and/or asthma using the CRD approach⁵. These findings on sensitization to these subtropical and temperate grass pollens in southern China add important evidence to that currently in the literature.

Protocol

The study protocol, including human serum sample use, was approved by the Ethics Committee, First Affiliated Hospital of Guangzhou Medical University. All participants offered written informed consent, either independently or via their parents (in the case of children). The protocol has been registered online (Chinese Clinical Trial Registry, Reg No.: ChiCTR-DCC-13004003, refer to http://www.chictr.org/cn/).

1. Identification of the Study Population

NOTE: The Allergy Information Repository of State Key Laboratory of Respiratory Disease (AIR-SKLRD) is the primary source of patient recruitment, because AIR is the most well-documented large database of patients for allergen tests in southern China^9,10. AIR has been storing serum samples of patients for nearly a decade. Briefly, these stored samples were previously prepared by centrifuging 5 mL of phlebotomized venous blood for 10 min at 3,000 x g and recovering the supernatant. The serum samples were placed in -80 °C refrigerators for long-term storage. Samples that met patient inclusion criteria were ready to use for all testing in the present study.

1. From the AIR database, retrieve data about patients with allergic rhinitis and/or asthma who underwent serum tests for grass allergen sIgE between January 2013 and June 2015, the pre-designed study period.
2. Select patients with mild to moderate allergic rhinitis and/or asthma according to the Allergic Rhinitis and Its Impact on Asthma (ARIA)¹¹ and the Global Initiative for Asthma (GINA)¹² guidelines. Assign them as the "study group."
3. Exclude patients with incomplete medical records, those lost to follow up, those who refuse to give informed consent regarding the use of their serum samples for scientific purposes, those with identified immunodeficiency, those currently on immunotherapy or immunomodulatory agents, or those found to have parasitic infections.
4. Select a contemporary cohort of patients with non-respiratory allergies (i.e., eczema, allergic dermatitis, and food allergy) according to the same exclusion criteria and assign them as the "control group."
5. Make sure that the serum samples were collected before any prescriptions or treatments were given to minimize confounding effects on the laboratory findings. Exclude any serum sample not fulfilling this requirement.
6. Make sure that all subjects are permanent residents from all parts of Greater Guangzhou (i.e., born or have lived there for more than 10 years).

2. Study Flow and Measurements of Interest

1. Retrieve the serum samples of eligible patients from the bio-bank. To test an allergen/allergen component, use 140 µL of serum (including 100 µL to fill up the dead space).
   NOTE: The maximum total serum volume needed for each patient in this study is: 3 specific allergens and 8 allergen components, sIgE = 140 µL * 11 = 1,540 µL.
2. When using this instrument, primarily test the serum samples for sIgEs to whole allergens of Bermuda grass, Timothy grass, and Humulus scandens. Follow the instructions in Section 3.
   NOTE: These pollens were selected for the measurements because Bermuda grass^13,14,15 and Humulus scandens¹⁶ have been widely reported in China, while Timothy grass^17,18,19 is a typical plant species that lives in environments ranging from tropical to temperate zones and is most-studied worldwide.
3. Secondarily, test the serum samples identified in step 2.3 to have sIgEs to whole allergens for the presence of sIgEs to allergen components of Bermuda [Cyn d 1 (g216)], Timothy [Phlp 1 (g205), Phl p 4 (g208), Phl p 5 (g215), Phl p 6 (g209), Phl p 7 (g210), Phl p 11 (g211), and Phl p 12 (g212)], and cross-reactive carbohydrate determinants [CCD (o214)]. Follow the instructions in Section 3.

3. Fully-automated Test Procedure using the Immunoassay Analyzing System

NOTE: The whole procedure of allergen sIgE testing, which is fully automated and has been described elsewhere^7,8,9,10, follows the manufacturer's protocol.

1. Start the fully-automated immunoassay analyzer (e.g., ImmunoCAP1000) and use it to perform the immunoassay throughout the study. Turn on the built-in Information Data Management (IDM) computer.
   NOTE: The immunoassay analyzer is routinely in stand-by mode.
2. With the "Primary Power" on, switch on the "System Power" and wait 3 min until the built-in software starts. Click on "Load Rinse Solution" and "Load Washing Solution."
3. Add 140 µL of serum to a vial for each allergen/allergen component test. Label each vial containing 140 µL of serum with an identification number unique to each patient.
4. From the IDM interface, execute the following steps by clicking on the menu.
   1. Check the "Request List" window on the IDM to make sure that "sIgE" is the test method of choice.
   2. Load the sample tubes into the sample racks and the quality-control tubes into the quality-control racks, with bar codes.
      NOTE: For each patient, testing was initially planned for sIgE to 3 specific allergens and 8 allergen components, or 346*11 (3,806) sample tubes. For each sample tube, 3 quality-control tubes that contain low, medium, and high levels of sIgE control (see the Table of Materials for details) were matched. Eventually, only 58 patients who tested positive in the primary test (step 2.3) were further examined in the secondary test (step 2.3). The loading of tubes can be automatically set on the computer console.
   3. Select "Load Reagents" in the "Assay Processing" screen. Complete the loading of the sample and quality-control racks, development solution, conjugate, calibrators, carrier, pipette tips, stop solution, and washing solution according to the "Loadlist" (see the Table of Materials for details).
   4. In "Load and Start," press "OK."
      NOTE: At the end of the measurement, the results appear on the IDM.
   5. From the IDM interface, select the data to be exported. Click on "Menu," "Approve," and "Save As" and export the sIgE measurement results as a spreadsheet file.
   6. Import the spreadsheet file to a statistical software (e.g., SPSS)¹⁰. Using a specific module for Spearman's coefficients from the SPSS menu, analyze the correlations in sensitization between these grass pollens (positive sIgE to whole allergen) and pollen components (positive sIgE to allergen molecules).

4. Definition of sIgE Reactivity

NOTE: For an undiluted serum specimen, the range of specific IgE measurement is 0.35 to 100 kU/L.

1. Dilute the samples with sIgE values >100 kU/L 1:5 and re-assay. Where the value remains higher than 100 kU/L, proceed with further dilutions until an end-point level up to 1,000 kU/L is reached.
2. Based on the threshold value of 0.35 kU/L, consider an sIgE level exceeding 0.35 kU/L to be positive^5,7,10. Rate the reactivity of the sIgE tests as: class 1 (≥0.35 and <0.70 kU/L), class 2 (≥0.70 and <3.50 kU/L), class 3 (≥3.50 and <17.50 kU/L), class 4 (≥17.50 and <50.00 kU/L), class 5 (≥50.00 and <100.00 kU/L), and class 6 (≥100.00 kU/L).

5. Statistical Analysis

1. Analyze the data with statistical software (e.g., SPSS)¹⁰. Report the quantitative data as the mean and standard deviation, the qualitative data as the median and interquartile ranges (IQR), and the categorical data as the proportion of positive results.
2. Use a chi-square test for the between-group comparison of proportions, an unpaired t-test for quantitative data, and a rank-sum test for qualitative data.
3. Use Pearson's test for correlation analyses between parametric data, with the correlation coefficients expressed as "r," and Spearman's test for non-parametric values, with the correlation coefficients expressed as "r_s." Consider statistical significance to be when P-values <0.05.

Results

The fully-automated immunoassay analyzer used in this study has a solid-phase structure covered with covalently coupling allergens or allergen components that react with the molecules of interest (sIgE) in serum specimens from the patient. With an automatic program, the analyzer washes off all non-specific IgEs with 150 µL of rinse solution and repeats this 5 times. Then it adds enzyme-labeled anti-IgE antibodies (50 µL) to generate a complex. The enzyme-labeled anti-IgE antibody (conjuga...

Discussion

Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. Such an automatic procedure has been found to substantially support bench research and clinical studies, enable the standardized detection of many allergens and/or allergen components, and identify probable allergen cross-reactivity that would not have been readily revealed before the advent of CRDs. Moreover, the whole testing procedure can be completed with strict quality control, allo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ImmunoCAP 1000 Instrument	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	12-3800-01
Phadia Information Data Manager Software Package	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	12-3801-11
Software Package, ImmunoCAP 1000	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	12-3801-12
g2 (Bermuda grass)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-4131-01
g6 (Timothy grass)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-4100-01
w22 (Japanese Hop)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-4452-01
g216 (nCyn d 1)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-4972-01
g205 (rPhl p 1)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5234-01
g208 (nPhl p 4)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5288-01
g215 (rPhl p 5b)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5338-01
g209 (rPhl p 6)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5289-01
g210 (rPhl p 7)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5290-01
g211 (rPhl p 11)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5291-01
g212 (rPhl p 12)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5292-01
o214 Cross-reacting carbohydrate determinants (CCDs)	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-5339-01
Specific IgE Conjugate 400	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9310-08
Specific IgE Calibrator Strip	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9459-08
Specific IgE Curve Control Strip	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9312-08
Specific IgE Anti-IgE ImmunoCAP	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	14-4417-01
ImmunoCAP Specific IgE Negative Control	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9445-01
ImmunoCAP Specific IgE Control L	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9528-01
ImmunoCAP Specific IgE Control M	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9529-01
ImmunoCAP Specific IgE Control H	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9530-01
Development Solution	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9439-08
Stop Solution	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	34-2271-58
Washing Solution	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9202-08
FluoroC	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9264-01
Maintenance Solution Kit	Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)	10-9476-01