Name: a component-resolved diagnostic approach for a study on grass pollen allergens in chinese southerners with allergic rhinitis and/or asthma
Uploaded: 2017-06-04T13:00:00.000+00:00
Duration: 6 min 34 s
Description: Watch this Scientific Journal Video about A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma at JoVE.com

We thank Yueming Su for his assistance in preparing the figure. This study was funded by the Guangdong Foundation of Science and Technology (Project No.: 2014A020212352), the&#160;Guangzhou Education Bureau (1201630044; 1201630393) and the National Natural Science Foundation of China (NSFC 81572063; NSFC 81601394).

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 China9,10. 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 &#176;C refrigerators for long-term storage. Samples that met patient inclusion criteria were ready to use for all testing in the present study.
<ol>
	<li>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.</li>
	<li>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 &#34;study group.&#34;</li>
	<li>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.</li>
	<li>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 &#34;control group.&#34;</li>
	<li>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.</li>
	<li>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).</li>
</ol>
2. Study Flow and Measurements of Interest
<ol>
	<li>Retrieve the serum samples of eligible patients from the bio-bank. To test an allergen/allergen component, use 140 &#181;L of serum (including 100 &#181;L 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 &#181;L * 11 = 1,540 &#181;L.</li>
	<li>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 grass13,14,15 and Humulus scandens16 have been widely reported in China, while Timothy grass17,18,19 is a typical plant species that lives in environments ranging from tropical to temperate zones and is most-studied worldwide.</li>
	<li>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.</li>
</ol>
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 elsewhere7,8,9,10, follows the manufacturer&#39;s protocol.
<ol>
	<li>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.</li>
	<li>With the &#34;Primary Power&#34; on, switch on the &#34;System Power&#34; and wait 3 min until the built-in software starts. Click on &#34;Load Rinse Solution&#34; and &#34;Load Washing Solution.&#34;</li>
	<li>Add 140 &#181;L of serum to a vial for each allergen/allergen component test. Label each vial containing 140 &#181;L of serum with an identification number unique to each patient.</li>
	<li>From the IDM interface, execute the following steps by clicking on the menu.
	<ol>
		<li>Check the &#34;Request List&#34; window on the IDM to make sure that &#34;sIgE&#34; is the test method of choice.</li>
		<li>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.</li>
		<li>Select &#34;Load Reagents&#34; in the &#34;Assay Processing&#34; 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 &#34;Loadlist&#34; (see the Table of Materials for details).</li>
		<li>In &#34;Load and Start,&#34; press &#34;OK.&#34; 
		NOTE: At the end of the measurement, the results appear on the IDM.</li>
		<li>From the IDM interface, select the data to be exported. Click on &#34;Menu,&#34; &#34;Approve,&#34; and &#34;Save As&#34; and export the sIgE measurement results as a spreadsheet file.</li>
		<li>Import the spreadsheet file to a statistical software (e.g., SPSS)10. Using a specific module for Spearman&#39;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).</li>
	</ol>
	</li>
</ol>
4. Definition of sIgE Reactivity
NOTE: For an undiluted serum specimen, the range of specific IgE measurement is 0.35 to 100 kU/L.
<ol>
	<li>Dilute the samples with sIgE values &#62;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.</li>
	<li>Based on the threshold value of 0.35 kU/L, consider an sIgE level exceeding 0.35 kU/L to be positive5,7,10. Rate the reactivity of the sIgE tests as: class 1 (&#8805;0.35 and &#60;0.70 kU/L), class 2 (&#8805;0.70 and &#60;3.50 kU/L), class 3 (&#8805;3.50 and &#60;17.50 kU/L), class 4 (&#8805;17.50 and &#60;50.00 kU/L), class 5 (&#8805;50.00 and &#60;100.00 kU/L), and class 6 (&#8805;100.00 kU/L).</li>
</ol>
5. Statistical Analysis
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Use Pearson&#39;s test for correlation analyses between parametric data, with the correlation coefficients expressed as &#34;r,&#34; and Spearman&#39;s test for non-parametric values, with the correlation coefficients expressed as &#34;rs.&#34; Consider statistical significance to be when P-values &#60;0.05.</li>
</ol>

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The authors have nothing to disclose.

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...

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 rhinitis1,2,3,4. 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&#8212;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 &#34;genuine&#34; 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&#39;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.

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

a component-resolved diagnostic approach for a study on grass pollen allergens in chinese southerners with allergic rhinitis and/or asthma

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&#39;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.

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 &#181;L of rinse solution and repeats this 5 times. Then it adds enzyme-labeled anti-IgE antibodies (50 &#181;L) to generate a complex. The enzyme-labeled anti-IgE antibod...

Watch this Scientific Journal Video about A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma at JoVE.com

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

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.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

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9.9K Views.  State Key Laboratory of Respiratory Disease. 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.

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

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Immunology and Infection

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...

Symptom Assessment of Patients with Allergic Rhinitis Using an Allergen Exposure Chamber

Allergen exposure chambers (AECs) are clinical facilities that allow the exposure of participants to allergenic and non-allergenic airborne particles. They provide stable particle concentrations under controlled environmental conditions. This is of great importance both for diagnostic purposes and for the monitoring of treatment effects. 
Here, a protocol and the technical prerequisites for performing a safe and effective allergen challenge in subjects sensitized to airborne allergens (i.e., house dust mite [HDM]) in the ALL-MED AEC are presented. With this method, triggering allergic symptoms corresponds to natural exposure. This can be used for an allergy diagnosis or as a plausible endpoint in clinical trials, particularly for allergen immunotherapy (AIT). A controlled environment (temperature, humidity, and carbon dioxide [CO2]) in the chamber must be maintained. Allergen particles must be dispersed evenly within the AEC at stable levels throughout the challenge. For this presentation, allergic rhinitis (AR) patients sensitive to HDM allergens were enrolled. AR symptoms were assessed by the following parameters: total nasal symptom score (TNSS), acoustic rhinometry (ARM), peak nasal inspiratory flow (PNIF), and nasal secretion weight. The safety of the procedure was assessed by the peak expiratory flow rate (PEFR) and the forced expiratory volume in the first second (FEV1). The allergic subjects developed symptoms within 120 min of the trial. On average, the most intense symptoms appeared after 60-90 min and, after reaching a plateau, remained stable until the end of the trial.

A protocol for conducting a challenge in an allergen exposure chamber (AEC) facility is presented. AECs have proven to be safe and effective tools for the induction of allergic symptoms or as an endpoint in the efficacy testing of allergen immunotherapy due to their ability to maintain stable particle concentrations and environmental conditions.

Allergen exposure chambers (AECs) are clinical facilities that allow the exposure of participants to allergenic and non-allergenic airborne particles. ...

Basophil Activation Test for Allergy Diagnosis

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined.
BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests&#160;to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

The basophil activation test is a complementary in vitro diagnostic test for the evaluation of IgE-mediated allergic reactions based on the detection of basophil activation in the presence of a specific stimulus through the measure of activation markers by flow cytometry.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and ...

A Traditional Chinese Medicine Characteristic Therapy for Bronchial Asthma: Moxibustion

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest tightness, or cough, as a result of increased airway reactivity. With high diurnal variation, these symptoms often occur or worsen at night or in the morning. By burning and roasting the Chinese medica above human acupoints, moxibustion is a type of treatment that stimulates the activity of the human meridians through drugs and heat stimulation to prevent and treat diseases. According to the principle of syndrome differentiation and treatment of traditional Chinese medicine, acupoints are selected on the corresponding parts, which has a definite effect. It is regarded as a characteristic traditional Chinese medicine therapy for bronchial asthma. This protocol elaborates the methods and steps of patient management, material preparation, selection of acupoints, operation, and postoperative nursing to ensure safe and effective moxibustion treatment to significantly improve the clinical symptoms and quality of life of patients with bronchial asthma.

This protocol presents a curative procedure of moxibustion in treating patients with bronchial asthma.

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest ...

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Traditional Chinese Medicine Ear Point Therapy for Wheezing in COPD Patients

Auricular Acupressure as an Adjuvant Treatment for Wheezing in Stable Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a major public health problem. Due to the restriction of expiratory airflow, it is characterized by emphysematous destruction of the lungs. Shortness of breath is one of the main clinical symptoms. Auricular acupressure is a clinical therapy characteristic of Chinese medicine that treats the disease by compressing ear points. Usually, the seeds of Vaccaria segetalis are used to stimulate ear points, which has the effect of regulating qi and alleviating wheezing. In this paper, we propose this characteristic therapy of traditional Chinese medicine (TCM) for the clinical symptoms of wheezing of lung and kidney qi deficiency type in stable COPD patients. Ear points are selected as the treatment protocol for Lung (CO14), Spleen (CO13), Kidney (CO10), Shen Men (TF4), and Ping Chuan (AT1.2.4i) points. The protocol describes a case study using auricular acupressure for a patient with chronic obstructive pulmonary disease to relieve wheezing symptoms.

Author Spotlight: Efficacy of Auricular Pressure Bean Therapy in Reducing Wheezing Symptoms

This protocol describes an effective therapy using auricular acupressure to relieve wheezing symptoms in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major public health problem. Due to the restriction of expiratory airflow, it is characterized by ...

Evaluating Acupoint Catgut Embedding Therapy for Allergic Rhinitis Management

Acupoint Catgut Embedding Therapy in Traditional Chinese Medicine for Managing Allergic Rhinitis

Acupuncture Point Embedding Therapy is a comprehensive treatment method that combines modern biomaterials with acupuncture techniques. Acupoint catgut embedding (ACE) not only avoids the adverse reactions associated with Western medicine but also incorporates the benefits of traditional acupuncture, extending the duration of its therapeutic effects. Allergic rhinitis (AR) is a condition known for its challenging treatment and tendency to persist. This randomized controlled trial included 128 AR patients to evaluate the effectiveness of ACE for AR management. Data were collected at baseline, four weeks after treatment, and eight weeks after treatment. Baseline analysis was performed, and the visual analog scale (VAS) was used as a clinical observation index for this method. The rhinoconjunctivitis quality of life questionnaire (RQLQ) served as a quality of life index. Specific IgE (sIgE), IL-4, IL-10, and IL-12 were used as laboratory observation indices. The results of the experiment validate the clinical effectiveness of ACE in treating AR. Although this technique is efficient and commonly used in clinics, its invasive nature poses risks of hospital-acquired infections. Based on this experiment, this protocol refines traditional techniques, providing detailed instructions on patient preparation, operation techniques, and postoperative care to ensure the safe and effective administration of therapy. By standardizing this therapy, ACE is anticipated to become an important non-drug treatment option for alleviating symptoms in AR patients, thereby significantly enhancing their quality of life.

This protocol details the procedure for acupoint burrowing and demonstrates its effectiveness in treating allergic rhinitis.

Acupuncture Point Embedding Therapy is a comprehensive treatment method that combines modern biomaterials with acupuncture techniques. Acupoint catgut ...

A Microfluidic Cartridge-Based Detection System for Allergen-Specific Immunoglobulin E

Source: Huang, Z., et al. <a href="https://app.jove.com/v/59100">Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE).</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a protocol for detecting serum-specific immunoglobulin E using a microfluidic cartridge-based chemiluminescence system. Serum IgEs interact with distinct allergens immobilized in the reaction zone of the cartridge, which are subsequently detected with peroxidase-conjugated anti-IgE antibodies and a chemiluminescent substrate, allowing simultaneous detection of multiple allergen-specific IgEs.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Basic information of the study ...