title: Assessing basophil activation by using flow cytometry and mass cytometry in blood stored 24 hours before analysis.
abstract: BACKGROUND
  Basophil activation tests (BATs) have promise for research and for clinical monitoring of patients with allergies. However, BAT protocols vary in blood anticoagulant used and temperature and time of storage before testing, complicating comparisons of results from various studies.
  
  
  OBJECTIVE
  We attempted to establish a BAT protocol that would permit analysis of blood within 24 hours of obtaining the sample.
  
  
  METHODS
  Blood from 46 healthy donors and 120 patients with peanut allergy was collected into EDTA or heparin tubes, and samples were stored at 4°C or room temperature for 4 or 24 hours before performing BATs.
  
  
  RESULTS
  Stimulation with anti-IgE or IL-3 resulted in strong upregulation of basophil CD203c in samples collected in EDTA or heparin, stored at 4°C, and analyzed 24 hours after sample collection. However, a CD63hipopulation of basophils was not observed in any conditions in EDTA-treated samples unless exogenous calcium/magnesium was added at the time of anti-IgE stimulation. By contrast, blood samples collected in heparin tubes were adequate for quantification of upregulation of basophil CD203c and identification of a population of CD63hibasophils, irrespective of whether the specimens were analyzed by means of conventional flow cytometry or cytometry by time-of-flight mass spectrometry, and such tests could be performed after blood was stored for 24 hours at 4°C.
  
  
  CONCLUSIONS
  BATs to measure upregulation of basophil CD203c and induction of a CD63hibasophil population can be conducted with blood obtained in heparin tubes and stored at 4°C for 24 hours.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Calif; Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Kaori
authors.initials: K
authors.lastname: Mukai
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Calif; Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gaudenzio
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Sheena
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Calif; Stanford Blood Center, Palo Alto, Calif.
authors.forename: Nora
authors.initials: N
authors.lastname: Vivanco
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Calif; Stanford Blood Center, Palo Alto, Calif.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Bendall
authors.affiliation: Human Immune Monitoring Center, Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, Calif; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Holden T
authors.initials: HT
authors.lastname: Maecker
authors.affiliation: Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, Calif; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Chinthrajah
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Calif; Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Mindy
authors.initials: M
authors.lastname: Tsai
authors.affiliation: Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, Calif; Division of Pulmonary and Critical Care Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, Calif.
authors.forename: Kari C
authors.initials: KC
authors.lastname: Nadeau
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, Calif; Sean N. Parker Center for Allergy and Asthma Research, Stanford University School of Medicine, Stanford, Calif; Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, Calif. Electronic address: sgalli@stanford.edu.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Galli
mesh_terms: D000293:Adolescent; D000328:Adult; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D000925:Anticoagulants / Q000494:pharmacology; D015549:Basophil Degranulation Test / Q000379:methods*; D001491:Basophils / Q000187:drug effects; D002648:Child; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D021183:Peanut Hypersensitivity / Q000097:blood / Q000276:immunology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D055815:Young Adult
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000925:Anticoagulants; C427944:ENPP3 protein, human; D007377:Interleukin-3; C026408:anti-IgE antibodies; D006493:Heparin; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases
keywords: Basophils; CD203c; CD63; EDTA; anti-coagulants; cytometry by time-of-flight mass spectrometry; heparin; peanut allergy; platelets