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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E., Monasterolo, G., Monasterolo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+IgE+antibodies+against+purified+grass-pollen+allergens+(Phl+p+1,+2,+3,+4,+5,+6,+7,+11,+and+12)+in+sera+of+patients+allergic+to+grass+pollen.">Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen.</a> Allergy. 52 (12), 1180-1185 (2001).</li><li>Zafred, D., Nandy, A., Pump, L., Kahlert, H., Keller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+and+immunologic+characterization+of+the+major+grass+pollen+allergen+Phl+p+4.">Crystal structure and immunologic characterization of the major grass pollen allergen Phl p 4.</a> J Allergy Clin Immunol. 132 (3), 696-703 (2013).</li><li>Chabre, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+variability+of+group+1+and+5grass+pollen+allergens+between+Pooideae+species:+implications+for+immunotherapy.">Molecular variability of group 1 and 5grass pollen allergens between Pooideae species: implications for immunotherapy.</a> Clin Exp Allergy. 40 (3), 505-519 (2010).</li><li>Mari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+test+with+a+timothy+grass+(Phleum+pratense)+pollen+extract+vs.IgE+to+a+timothy+extract+vs.+IgE+to+rPhl+p+1+rPhl+p+2,+nPhl+p+4,+rPhl+p+5,+rPhl+p+6,+rPhl+p+7,+rPhl+p+11,+and+rPhl+p+12:+epidemiological+and+diagnostic+data.">Skin test with a timothy grass (Phleum pratense) pollen extract vs.IgE to a timothy extract vs. IgE to rPhl p 1 rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data.</a> Clin Exp Allergy. 33 (1), 43-51 (2003).</li><li>Grote, M., Stumvoll, S., Reichelt, R., Lidholm, J., Rudolf, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+allergen+related+to+Phl+p+4,+a+major+timothy+grass+pollen+allergen,+in+pollens,+vegetables,+and+fruits+by+immunogold+electron+microscopy.">Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen, in pollens, vegetables, and fruits by immunogold electron microscopy.</a> Biol Chem. 383 (9), 1441-1445 (2002).</li><li>Andersson, K., Lidholm, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+immunobiology+of+grass+pollen+allergens.">Characteristics and immunobiology of grass pollen allergens.</a> Int Arch Allergy Immunol. 130 (2), 87-107 (2003).</li><li>Tripodi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiles+of+IgE+to+Phleum+pratense+in+children+with+grass+pollen+allergy:+implications+for+specific+immunotherapy.">Molecular profiles of IgE to Phleum pratense in children with grass pollen allergy: implications for specific immunotherapy.</a> J Allergy Clin Immunol. 129 (3), 834-839 (2012).</li><li>Stumvoll, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+structural+and+immunological+characterization+of+a+timothy+grass+(Phleum+pratense)+pollen+allergen+Phl+p+4,+with+cross-reactive+potential.">Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen Phl p 4, with cross-reactive potential.</a> Biol Chem. 383 (9), 1383-1396 (2002).</li><li>Wood, R. 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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, allowing for high-throughput methods, as required in a survey of a large geographical region. Although the testing procedure is mostly run by a computer console, several critical issues should be brought to attention. It is mandatory that the instrument is used in rigorous compliance with the user manual; any aberration could result in unnecessary test errors, mechanical damage, or personal casualty from laser beam or high electric voltage. Always ensure that a proper and correct calibration curve has been stored in the system, as this is linked to the accuracy of the test. Otherwise, the sIgE concentration of a specified calibrator should be measured prior to the serum specimen tests. Afterwards, the system may automatically compute and store the calibration curve for subsequent use. As with other laboratory techniques, it is also important to observe the safety of human serum manipulation to protect technicians from infections and to avoid contaminating the environment.
For the first time, by using a fully-automated immunoassay analyzer as a CRD approach, this study showed a considerable prevalence of Bermuda grass sIgE positivity (22.5%) in patients with allergic rhinitis and/or asthma in Guangzhou, southern China. Since Timothy grass and Humulus scandens are not common, even non-existant, in Guangzhou, the positivity rates of Timothy grass (13.6%) and Humulus scandens sIgE (7.0%) may be attributed to cross-reactivity among grass pollens.
By analyzing grass pollen allergens, it was clearly shown that all Timothy grass IgE-positive patients were also positive to the specific Timothy grass antigen Phl p 4. Very few (17.1%) tested positive to Phl p 1, which was surprisingly different from data in Europe, where the rate of positivity to Phl p 1 is much higher than that of Phl p 418,20,21. Since Phl p 1 is the major component of Timothy grass, the predominant rate of Phl p 4 positivity over Phl p 1 in this study population may also suggest probable cross-reactivity other than &#34;genuine&#34; sensitization, because Phl p 4 shares a structural similarity with CCD and a number of other allergens, including trees, vegetables, and tropical fruits19,22,23,24. The clinical relevance of this observation could be that the majority of Timothy grass-positive patients in this study should never have been advised to receive Timothy grass immunotherapy, which currently uses Phl p 125.
Several limitations of the study methods and techniques must be mentioned. First, since up to 3 different calibrator strips can be placed in a pipette/reagent Tray in the immunoassay analyzer, at the maximum, three test batches can be run at the same time. When a heavy workload is required, like in a greater regional survey, it still takes a significant amount of time to process. Second, the cross-reactivity with human IgA, IgD, IgM, and IgG is nonexistent at physiological concentrations when using the analyzer from this study, so other investigational approaches are needed when cross-reactivity with these immunoglobulins are concerned. Third, despite the sophistication of the device, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise when a definitive diagnosis is being made. Fourth, due to the late introduction of allergen CRD in China, several commercial products of subtropical grass pollen components are unavailable or have not been approved for research until recently. Therefore, using this technique as a CRD approach for allergens substantially relies on the commercial availability of product supplies from the manufacturer for specific use in a region. In tropical and subtropical regions with a ubiquitous complexity of plant pollens, a CRD study can be insufficient and difficult due to a lack of corresponding allergen products for the tests. Such a dilemma could greatly compromise the efficiency of this technique.
Nevertheless, to the latest of our knowledge, this is a pioneering study that uses the CRD approach to examine sensitization to grass pollen allergens in southern China. Further studies can continue in this vein and validate these findings.
So far, techniques using the fully-automatic immunoassay analyzer have been accepted as the gold standard for in vitro allergen sIgE tests26,27. Given the very limited data on allergen sensitization within the vast territory of China, large-sample studies using the fully-automated immunoassay as a CRD approach can fill the gaps and add to the evidence available in this field. It is important to note that such studies, usually multi-center and nationwide collaborations, may necessitate the networking of immunoassays based on a protocol for standardization.

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 border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP 1000 Instrument</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3800-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Phadia Information Data Manager Software Package</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3801-11</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Software Package, ImmunoCAP 1000</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3801-12</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g2 
			(Bermuda grass)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4131-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g6 
			(Timothy grass)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4100-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">w22 
			(Japanese Hop)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4452-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g216 
			(nCyn d 1)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4972-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g205 
			(rPhl p 1)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5234-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g208 
			(nPhl p 4)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5288-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g215 
			(rPhl p 5b)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5338-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g209 
			(rPhl p 6)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5289-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g210 
			(rPhl p 7)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5290-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g211 
			(rPhl p 11)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5291-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g212 
			(rPhl p 12)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5292-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:157px;">o214 
			Cross-reacting carbohydrate determinants (CCDs)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5339-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Conjugate 400</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9310-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Calibrator Strip</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9459-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Curve Control Strip</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9312-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Anti-IgE ImmunoCAP</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4417-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Negative Control</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9445-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control L</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9528-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control M</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9529-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control H</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9530-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Development Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9439-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Stop Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">34-2271-58</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Washing Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9202-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">FluoroC</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9264-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Maintenance Solution Kit</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9476-01</td>
			<td style="width:177px;"></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 antibody (conjugate) is a secondary antibody that binds to a specific IgE. Unbound anti-IgE antibodies are eliminated by washing with 150 &#181;L of washing solution (repeated 3 times). The bound complex is treated with a developing agent (50 &#181;L) for 9 min at 37 &#176;C. The developing agent is an enzyme-digesting agent that cuts off all bound anti-IgE antibodies. The latter emits fluorescence under alkaline condition. The reaction is stopped with the addition of alkaline stop solution. After this, the instrument compresses the solid-phase structure to squeeze out the eluate and to measure the fluorescence of the resultant eluate at a wavelength of 470 nm. The fluorescence is linearly correlated to the absolute level of the analyte molecules of interest in the eluate. A higher fluorescence value means more sIgE in the serum specimen. To interpret the test results, the system automatically transforms the responses for samples to concentrations using a calibration curve. The measurement data are electronically transferred to the IDM software, which integrates functions for the calculation of analytical results, the calculation of statistics, and result reporting.
Following the inclusion and exclusion criteria, the present study investigated serum from 346 patients. The study group included 78 subjects with allergic rhinitis alone, 92 with asthma alone, and 88 concomitantly with asthma and rhinitis. The control group included 88 patients with non-respiratory allergies. These patients were aged 1-87 years old and were confirmed to be permanent residents from all parts of Greater Guangzhou, the target region of study. All study groups and the control group were comparable in demographical and social characteristics, including gender, age, and family history. Of the patients in the study group, 22.5% tested positive to Bermuda grass, 13.6% to Timothy grass, and 7.0% to Humulus scandens. In the study group, the rate of sensitization to any grass pollen was much higher than in the control group (Table 1). All Bermuda grass sIgE-negative patients were also negative to Timothy grass and Humulus scandens. It is of interest to note that patients with allergic rhinitis and/or asthma demonstrated a remarkably higher rate of sensitization to Bermuda grass (n = 58, 22.5%) than those with other allergies (n = 4, 4.5%).
There were no patients with class-6 sIgE reactivity in this study. Among the patients with Bermuda grass class-1 sIgE reactivity, the likelihood of positive tests for Cyn d 1, Timothy grass allergens, Humulus scandens, or CCD was below 30%. In contrast, patients with class-4 Bermuda grass sIgE reactivity had a 67-100% chance of concomitant positivity to Cyn d 1, Timothy grass allergens, Humulus scandens, or CCD (Figure 1). Bermuda grass sIgE reactivity levels demonstrated a significant correlation, with chances of positivity to other grass pollen IgEs (rs = 0.669, P &#60;0.001).
Timothy grass sensitization was further assessed by sIgE to Timothy grass components. Among the 35 Timothy grass-positive patients, the most frequently detected allergen component was Phl p 4 (100%, 35/35), followed by Phl p 1 (17.1%, 6/35). Three patients each (3/35, 8.6%) were positive for any one of Phl p 5, 6, 7, 11, and 12 (Table 2). Serum Phl p 4 sIgE levels correlated strongly with total Timothy grass allergen and CCD sIgE levels (rs = 0.941 and 0.928, respectively, P &#60;0.001; Table 3). 41.4% (24/58) of Bermuda grass-positive patients and 68.6% (24/35) of Timothy grass-sensitized patients were sIgE positive to CCD. Spearman&#39;s rank correlation analysis showed a significant association in sensitization between CCD and other grass allergens (Table 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55723/55723fig1.jpg" /> 
Figure 1: Co-sensitization Among Patients Positive to Bermuda Grass sIgE. 
This figure shows the percentage of patients with different classes of sIgE reactivity to Bermuda grass (n = 58) and a concomitant sensitization to Timothy grass, Humulus scandens, CCD, and other grass allergen components.&#160; <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55723/55723fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Rate of positive tests [% (n/patients)]</td>
		</tr>
		<tr>
			<td></td>
			<td>Bermuda grass</td>
			<td>Timothy grass</td>
			<td>Humulus scandens</td>
		</tr>
		<tr>
			<td>Patients with AR and/or AS</td>
			<td>22.5% (58/258) &#8224;&#8224;&#8225;</td>
			<td>13.6% (35/258) &#8224;</td>
			<td>7.0% (18/258)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Patients with AR</td>
			<td>21.8% (17/78) &#8224;&#8224;</td>
			<td>10.2% (8/78)</td>
			<td>7.7% (6/78)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Patients with AS</td>
			<td>22.8% (21/92) &#8224;&#8224;</td>
			<td>14.1% (13/92) &#8224;</td>
			<td>5.4% (5/92)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Patients with AR and AS</td>
			<td>22.7% (20/88) &#8224;&#8224;</td>
			<td>15.9% (14/88)</td>
			<td>8.0% (7/88)</td>
		</tr>
		<tr>
			<td>Age</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; &#60;14 years or younger</td>
			<td>18.6% (8/43)</td>
			<td>14.0% (6/43)</td>
			<td>7.0% (3/43)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; &#62; 4 years&#160;</td>
			<td>23.3% (50/215)</td>
			<td>13.5% (29/215)</td>
			<td>7.0% (15/215)</td>
		</tr>
		<tr>
			<td>Sex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Male</td>
			<td>25.7% (35/136)</td>
			<td>13.9% (19/136)</td>
			<td>7.4% (10/136)</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Female</td>
			<td>18.8% (23/122)</td>
			<td>13.1% (16/122)</td>
			<td>6.6% (8/122)</td>
		</tr>
		<tr>
			<td>Patients with other allergy</td>
			<td>4.5% (4/88) **</td>
			<td>1.1% (1/88) **</td>
			<td>0*</td>
		</tr>
	</tbody>
</table>
Table 1: Patients with sIgE-positive Tests for Bermuda Grass, Timothy Grass, and Humulus scandens Based on Disease Entity and Demographic Details. A higher proportion of patients with allergic respiratory diseases were positive for Bermuda grass (&#967;2 = 14.35, P &#60;0.001), Timothy grass (&#967;2 = 10.88, P &#60;0.001), and Humulus scandens (&#967;2 = 5.14, P = 0.02) versus the other allergy controls (**P &#60;0.01, *P &#60;0.05). The proportion of patients with a positive Bermuda grass test was remarkably higher than the proportion for Timothy grass (&#8225;P &#60;0.05) and Humulus scandens (&#8224;&#8224;P &#60;0.01). The proportion of patients with a positive Timothy grass test was higher than that of Humulus scandens (&#8224;P &#60;0.05). The proportion of positive tests to Bermuda grass, Timothy grass, and Humulus scandens sIgE did not vary significantly between the minor and adult groups (&#967;2 = 0.07, P = 0.79; &#967;2 = 0.06, P = 0.80; &#967;2 = 0.11, P = 0.74; respectively). Rates of grass pollen sensitivities were not statistically different between males and females (&#967;2 = 1.338, P =0.25; &#967;2 = 0.063, P = 0.80; &#967;2 = 0.38, P = 0.54; respectively). Similarly, no significant differences in sensitivity rates were indicated between the rhinitis and asthma patients (&#967;2 = 0.030, P = 0.985; &#967;2 = 1.165, P = 0.558; &#967;2 = 0.528, P = 0.768; respectively).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Patients (%)</td>
			<td></td>
			<td>Phl p 4</td>
			<td>Phl p 1</td>
			<td>Phl p 5</td>
			<td>Phl p 6</td>
			<td>Phl p 11</td>
			<td>Phl p 7</td>
			<td>Phl p 12</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; 28 (80.0%)</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 4 (11.4%)</td>
			<td></td>
			<td>+</td>
			<td>+</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 1 (2.9%)</td>
			<td></td>
			<td>+</td>
			<td></td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; 2 (5.7%)</td>
			<td></td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160; Total</td>
			<td></td>
			<td>35 (100%)</td>
			<td>6 (17.1%)</td>
			<td>3 (8.6%)</td>
			<td>3 (8.6%)</td>
			<td>3 (8.6%)</td>
			<td>3 (8.6%)</td>
			<td>3 (8.6%)</td>
		</tr>
		<tr>
			<td>sIgEa (kU/L)</td>
			<td></td>
			<td>0.52, 0.06-2.02</td>
			<td>0.02, 0.01-0.10</td>
			<td>0.04, 0.03-0.24</td>
			<td>0.12, 0.03-0.34</td>
			<td>0.05, 0.02-0.27</td>
			<td>0.03, 0.02-0.36</td>
			<td>0.10, 0.04-0.28</td>
		</tr>
	</tbody>
</table>
Table 2: Timothy Grass Sensitization According to sIgE Positivity and Levels of Timothy Grass Components (N = 35). +, positive tests; a, sIgE levels (kU/L) are shown as the median and IQR. Of the 35 Timothy grass sIgE-positive patients, 80.0% were solely Phl p 4-positive, while 11.4% were positive to Phl p 4 or Phl p 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Allergen</td>
			<td>Bermuda</td>
			<td>Timothy</td>
			<td>Humulus scandens</td>
			<td>CCD</td>
		</tr>
		<tr>
			<td>Bermuda</td>
			<td>-</td>
			<td>0.709**</td>
			<td>0.682**</td>
			<td>0.565**</td>
		</tr>
		<tr>
			<td>Cyn d 1</td>
			<td>0.583**</td>
			<td>0.787**</td>
			<td>0.751**</td>
			<td>0.865**</td>
		</tr>
		<tr>
			<td>Timothy</td>
			<td>0.709**</td>
			<td>-</td>
			<td>0.921**</td>
			<td>0.856**</td>
		</tr>
		<tr>
			<td>Phl p 4</td>
			<td>0.645**</td>
			<td>0.941**</td>
			<td>0.886*</td>
			<td>0.928**</td>
		</tr>
		<tr>
			<td>Phl p 1</td>
			<td>0.616**</td>
			<td>0.681**</td>
			<td>0.712**</td>
			<td>0.507**</td>
		</tr>
		<tr>
			<td>Phl p 5</td>
			<td>0.46</td>
			<td>0.273</td>
			<td>0.590*</td>
			<td>0.624*</td>
		</tr>
		<tr>
			<td>Phl p 6</td>
			<td>0.445</td>
			<td>0.306</td>
			<td>0.583*</td>
			<td>0.569</td>
		</tr>
		<tr>
			<td>Phl p 7</td>
			<td>0.681*</td>
			<td>0.481</td>
			<td>0.632*</td>
			<td>0.531</td>
		</tr>
		<tr>
			<td>Phl p 11</td>
			<td>0.589*</td>
			<td>0.372</td>
			<td>0.620*</td>
			<td>0.568</td>
		</tr>
		<tr>
			<td>Phl p 12</td>
			<td>0.671*</td>
			<td>0.411</td>
			<td>0.565</td>
			<td>0.446</td>
		</tr>
		<tr>
			<td>Humulus scandens</td>
			<td>0.682**</td>
			<td>0.921**</td>
			<td>-</td>
			<td>0.853**</td>
		</tr>
	</tbody>
</table>
Table 3: Correlation coefficients&#8224; for positivity between CCD and the grass pollen allergens. &#8224; is presented as the correlation coefficient rs; Spearman&#39;s correlation analyses were performed for this non-parametric dataset. * P &#60;0.05, ** P &#60;0.01.

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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Nanyang Technological University

Research

JoVE Journal

Medicine

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

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Posterior Semicircular Canal Approach for Inner Ear Gene Delivery in Neonatal Mouse

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss. In this study, we describe the posterior semicircular canal approach as a viable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear. The easy anatomic identification of the posterior semicircular canal, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery.

In this study, we describe the posterior semicircular canal approach as a reliable method for inner ear gene delivery in neonatal mice. We show that gene delivery through the posterior semicircular canal is able to perfuse the entire inner ear.

Inner ear gene therapy offers great promise as a potential treatment for hearing loss and dizziness. One of the critical determinants of the success of ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

A Method for Zanba-Stir-Fried Tiebangchui Process Optimization

Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method

The dried root of Aconitum pendulum Busch., called Tiebangchui (TBC) in Chinese, is one of the most famous Tibetan medicines. It is a widely used herb in northwest China. However, many cases of poisoning have occurred because of TBC's intense toxicity and because its therapeutic and toxic doses are similar. Therefore, finding a safe and effective method to reduce its toxicity is an urgent task. A search through the Tibetan medicine classics shows that the processing method of TBC stir-fried with Zanba was recorded in the "Processing specification of Tibetan medicine of Qinghai Province (2010)". However, the specific processing parameters are not yet clear. Thus, this study aims to optimize and standardize the processing technology of Zanba-stir-fried TBC.
First, a single-factor experiment was conducted on four factors: the slice thickness of TBC, amount of Zanba, processing temperature, and time. With monoester and diester alkaloid contents in Zanba-stir-fried TBC as indexes, CRITIC combined with the Box-Behnken response surface method was used to optimize the processing technology of Zanba-stir-fried TBC. The optimized processing conditions of Zanba-stir-fried TBC were a TBC slice thickness of 2 cm, three times more Zanba than TBC, a processing temperature of 125 &#176;C, and 60 min of stir-frying. This study determined the optimized and standard processing conditions for the usage of Zanba-stir-fried TBC, thus providing an experimental basis for the safe clinical use and industrial production of Zanba-stir-fried TBC.

Author Spotlight: Optimization of Processing Technology for Tiebangchui with Zanba Based on CRITIC Combined with Box-Behnken Response Surface Method  

The present protocol describes an efficient and standard detoxification processing method for Zanba-stir-fried Tiebangchui using CRITIC combined with the Box-Behnken response surface method.

The dried root of Aconitum pendulum Busch., called Tiebangchui (TBC) in Chinese, is one of the most famous Tibetan medicines. It is a widely used herb in ...

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 ...

Acupoint Application Combined with Acupoint Massage for Treating Constipation in a Patient with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. Constipation that reduces the quality of life and triggers the risk of diseases is a common concomitant symptom in patients with COPD. Currently, western medical treatment does not achieve the desired results for patients. A high recurrence rate accompanies it, whereas traditional Chinese medicine (TCM) has a long history and rich experience in treating chronic diseases. Both acupoint application and acupoint massage are characteristic therapies of TCM, with minor side effects, high safety, simple operation, and outstanding advantages. They are effective in treating constipation for patients with COPD and are considered an ideal alternative therapy for patients with chronic constipation. The purpose of this article is to introduce the method of acupoint application combined with acupoint massage for the treatment of constipation in patients with COPD, including the selection of points, items, treatment time, and operation procedure.

Here we demonstrate acupoint application combined with acupoint massage for treating constipation in a patient with chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. ...

Warm Acupuncture and Moxibustion to Relieve COPD with Abdominal Distension

A Preliminary Study on Warm Acupuncture and Moxibustion for Treating Chronic Obstructive Pulmonary Disease with Abdominal Distension

Most patients with COPD have a combination of abdominal distension, which has been shown to adversely affect pulmonary symptoms, frequency of acute exacerbations, and quality of life in patients with COPD. Warm acupuncture and moxibustion have been shown to be effective in relieving symptoms in patients with COPD combined with abdominal distention. Warm acupuncture and moxibustion are highly effective, easy to perform, and inexpensive forms of traditional Chinese medicine treatments. The standardized practice of warm acupuncture and moxibustion is very important for the treatment of COPD combined with abdominal distension. The specific steps include selecting the appropriate acupoints for needling through syndrome differentiation treatment and selecting moxa sticks of appropriate length for moxibustion for about 30 min after the De-qi. The course of treatment lasts for one week. The following indicators are specifically assessed: the score of the COPD Assessment Test (CAT) and the abdominal distension visual analog scale (VAS). This article will clearly illustrate how to standardize the manipulation of warm acupuncture and moxibustion to relieve COPD combined with abdominal distention.

Author Spotlight: An Effective Traditional Chinese Medicinal Treatment for Chronic Obstructive Pulmonary Disease with Abdominal Distension 

Here, we present a protocol to properly manipulate warm acupuncture and moxibustion for treating chronic obstructive pulmonary disease (COPD) with abdominal distension.

Most patients with COPD have a combination of abdominal distension, which has been shown to adversely affect pulmonary symptoms, frequency of acute ...

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), ...