title: Determination of allergenic load and pollen count of Cupressus arizonica pollen by flow cytometry using Cup a1 polyclonal antibody.
abstract: Background: 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. Methods: Airborne samples were collected from Burkard Spore-Trap and Burkard Cyclone Cupressus arizonica pollen was studied using specific rabbit polyclonal antibody Cup a1, labelled 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 & Altman was used to determine agreement between both techniques measured. Results: 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: 0.0008E-2and 0.0002 with regard to smaller particles, so the Bland & Altman measurement showed a good correlation between them, p-value: 0,0003. Conclusion: 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. © 2013 Clinical Cytometry Society.
authors.affiliation: Unidad de I+D+i 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.
authors.forename: Francisco Moreno
authors.initials: FM
authors.lastname: Benítez
authors.forename: Antonio Letrán
authors.initials: AL
authors.lastname: Camacho
authors.forename: Alfonso
authors.initials: A
authors.lastname: Del Cuvillo Bernal
authors.forename: Pedro Lobatón Sánchez
authors.initials: PL
authors.lastname: de Medina
authors.forename: Francisco J García
authors.initials: FJ
authors.lastname: Cózar
authors.forename: Ma Luisa Espinazo
authors.initials: ML
authors.lastname: Romeu
keywords: Allergenic Load; Allergy; Cupressus arizonica; Flow Cytometry; Pollen Count