title: Assessment of a novel flow cytometry technique of one-step intracellular staining: example of FOXP3 in clinical samples.
abstract: BACKGROUND
  By measuring multiple parameters on a single-cell basis, flow cytometry is a potent tool to dissect the phenotypes and functions of cell subsets. However, because this technique may be time-consuming, particularly for intracellular staining, it could be problematic for its use in daily routine or in large cohorts. Recently, a novel reagent has been developed to perform intracellular staining in one step. The objective of our study was thus to assess this new method in comparison with the reference technique by focusing on FOXP3 staining in clinical samples.
  
  
  METHODS
  Peripheral blood was collected from 15 HIV-1-infected patients, 5 critically ill patients, and 5 healthy volunteers and stained using the two different methods. Different subsets of FOXP3 positive cells were investigated by flow cytometry.
  
  
  RESULTS
  When comparing results obtained with the two techniques, no statistical differences between the percentages of CD4+FOXP3+, CD4+CD25+FOXP3+, and CD4+CD25+CD127-FOXP3+ cells were observed. Besides, a strong correlation between percentages of CD4+FOXP3+CD25+CD127- lymphocytes measured with both techniques was found in patients (r: 0.843, P < 0.001, intra-class correlation coefficient: 0.820, P < 0.001). Importantly, flow cytometry stainings obtained with the one-step method were very robust with an excellent intra-assay precision, a better discriminative power and correct stability and reproducibility of the staining even after blood storage.
  
  
  CONCLUSIONS
  With a strong correlation between the percentages of FOXP3+ Tregs when compared with the reference method, a better staining quality, a shorter realization time and no need of isotype control, this one step procedure may represent an important improvement for a daily routine use of intracellular staining.
authors: Hospices Civils de Lyon, Hôpital Edouard Herriot, Laboratoire d'Immunologie, Lyon F-69003, France.
authors: Julie
authors: J
authors: Demaret
authors: Julien
authors: J
authors: Saison
authors: Fabienne
authors: F
authors: Venet
authors: Christophe
authors: C
authors: Malcus
authors: Francoise
authors: F
authors: Poitevin-Later
authors: Alain
authors: A
authors: Lepape
authors: Tristan
authors: T
authors: Ferry
authors: Guillaume
authors: G
authors: Monneret
mesh_terms: D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D016022:Case-Control Studies; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000276:immunology*; D015870:Gene Expression; D015658:HIV Infections / Q000175:diagnosis / Q000276:immunology / Q000473:pathology*; D015497:HIV-1; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008297:Male; D008875:Middle Aged; D015588:Observer Variation; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000473:pathology*
chemical_list: D015703:Antigens, CD; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors