Optimizing of the basophil activation test: Comparison of different basophil identification markers.
BACKGROUND
Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement.


METHODS
Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used.


RESULTS
The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+/CD203c+/CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+/CD3- cells and CRTH2+/CD203c+/CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+-population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations.


CONCLUSIONS
As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen.
Department of Dermatology and Allergy Biederstein, Technische Universität München, Germany, Munich, Germany.
Bernadette
B
Eberlein
Rebekka
R
Hann
Stefanie
S
Eyerich
Davide
D
Pennino
Johannes
J
Ring
Carsten B
CB
Schmidt-Weber
Jeroen
J
Buters
D000328:Adult; D000485:Allergens / Q000737:chemistry / Q000276:immunology; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015549:Basophil Degranulation Test; D001491:Basophils / Q000187:drug effects / Q000276:immunology* / Q000473:pathology; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology; D016022:Case-Control Studies; D002478:Cells, Cultured; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood*; D016130:Immunophenotyping / Q000379:methods*; D008297:Male; D008875:Middle Aged; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000276:immunology; D010936:Plant Extracts / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D011058:Pollen / Q000737:chemistry / Q000276:immunology; D011755:Pyrophosphatases / Q000235:genetics / Q000276:immunology; D054397:Receptors, CCR3 / Q000235:genetics / Q000276:immunology; D011971:Receptors, Immunologic / Q000235:genetics / Q000276:immunology; D011982:Receptors, Prostaglandin / Q000235:genetics / Q000276:immunology; D012130:Respiratory Hypersensitivity / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D060149:Tetraspanin 30 / Q000235:genetics / Q000276:immunology
D000485:Allergens; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; C515350:CCR3 protein, human; D017252:CD3 Complex; C554586:CD63 protein, human; C427944:ENPP3 protein, human; D010936:Plant Extracts; D054397:Receptors, CCR3; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; D060149:Tetraspanin 30; D007073:Immunoglobulin E; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; C036293:prostaglandin D2 receptor
basophil activation test; basophil identification markers; multicolour flowcytometry