Unidad de Alergia de Clínica Dr. Lobatón, S.L., Cádiz, Spain; Cátedra Externa "Inmunología y Alergia"-UCA-Asociación para la Investigación en Ciencias de la Salud. Dr. Pedro Lobatón.
Cytometry B Clin Cytom. 2014 Jan;86(1):63-9. doi: 10.1002/cyto.b.21114. Epub 2013 Aug 7.
There is an increase in the incidence of pollen related allergy, thus information on pollen schedules would be a great asset for physicians to improve the clinical care of patients. Like cypress pollen sensitization shows a high prevalence among the causes of allergic rhinitis, and therefore it is of interest to use it like a model of study, distinguishing cypress pollen, pollen count, and allergenic load level. In this work, we use a flow cytometry based technique to obtain both Cupressus arizonica pollen count and allergenic load, using specific rabbit polyclonal antibody Cup a1 and its comparison with optical microscopy technique measurement.
Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labeled with AlexaFluor(®) 488 or 750 and analysed by Flow Cytometry in both an EPICS XL and Cyan ADP cytometers (Beckman Coulter(®) ). Optical microscopy study was realized with a Leica optical microscope. Bland and Altman was used to determine agreement between both techniques measured.
We can identify three different populations based on rabbit polyclonal antibody Cup a1 staining. The main region (44.5%) had 97.3% recognition, a second region (25%) with 28% and a third region (30.5%) with 68% respectively. Immunofluorescence and confocal microscopy showed that main region corresponds to whole pollen grains, the second region are pollen without exine and the third region is constituted by smaller particles with allergenic properties. Pollen schedule shows a higher correlation measured by optical microscopy and flow cytometry in the pollen count with a P-value: 0.0008 E(-2) and 0.0002 with regard to smaller particles, so the Bland and Altman measurement showed a good correlation between them, P-value: 0.0003.
Determination of pollen count and allergenic load by flow cytometry represents an important tool in the determination of airborne respiratory allergens. We showed that not only whole pollen but also smaller particles could induce allergic sensitization. This is the first study where flow cytometry is used for calculating pollen counts and allergenic load.
花粉相关过敏的发病率不断增加，因此有关花粉时间表的信息将是医生改善患者临床护理的宝贵资源。由于柏树花粉致敏在过敏性鼻炎的病因中占有很高的比例，因此将其作为研究模型具有重要意义，可以区分柏树花粉、花粉计数和变应原负荷水平。在这项工作中，我们使用基于流式细胞术的技术来获得柏树花粉计数和变应原负荷，使用特异性兔多克隆抗体 Cup a1，并将其与光学显微镜技术测量进行比较。
从 Burkard 孢子陷阱中采集空气样本，使用特异性兔多克隆抗体 Cup a1 研究 Burkard 旋风柏树花粉，用 AlexaFluor®488 或 750 标记，并用 EPICS XL 和 Cyan ADP 细胞仪（贝克曼库尔特（®））进行流式细胞术分析。光学显微镜研究在 Leica 光学显微镜上进行。 Bland 和 Altman 用于确定两种技术测量结果的一致性。
我们可以根据兔多克隆抗体 Cup a1 的染色识别出三种不同的群体。主要区域（44.5%）具有 97.3%的识别率，第二个区域（25%）具有 28%的识别率，第三个区域（30.5%）具有 68%的识别率。免疫荧光和共聚焦显微镜显示，主要区域对应于完整的花粉粒，第二个区域是没有外壁的花粉粒，第三个区域由具有变应原特性的较小颗粒组成。花粉时间表显示，光学显微镜和流式细胞术测量的花粉计数相关性更高，P 值分别为 0.0008E-2 和 0.0002，较小颗粒的相关性更好，P 值为 0.0003。
流式细胞术测定花粉计数和变应原负荷是确定空气中呼吸性过敏原的重要工具。我们表明，不仅是完整的花粉，而且还有较小的颗粒也可以引起过敏致敏。这是首次使用流式细胞术计算花粉计数和变应原负荷的研究。
