title: The use of isotypic control antibodies in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets by flow cytometry. Are they really necessary?
abstract: OBJECTIVE
  Isotypic control reagents are defined as irrelevant antibodies of the same immunoglobulin class as the relevant reagent antibody in a flow cytometry panel. The use of the isotypic control antibody has been advocated as a necessary quality control measure in analysis of flow cytometry. The purpose of this study was to determine the necessity of an isotypic control antibody in the analysis of CD3+ and CD3+, CD4+ lymphocyte subsets.
  
  
  METHODS
  We performed a prospective study of 46 consecutive patient samples received for lymphocyte subset analysis to determine the need for the isotypic control. For each sample, a sham buffer (autocontrol) and isotypic control reagent were stained for three-color immunofluorescence, processed, and identically analyzed with Attractors software. The Attractors software allowed independent, multiparametric, simultaneous gating; was able to identically and reproducibly process each list mode file; and yielded population data in spreadsheet form.
  
  
  RESULTS
  Statistical analysis (Fisher's z test) revealed no difference between the CD3+ autocontrol and CD3+ isotypic control (correlation = 1, P < .0001) or between the CD3+, CD4+ autocontrol and the CD3+, CD4+ isotypic control (correlation = 1, P < .0001). The elimination of the isotypic control reagent resulted in a total cost savings of $3.36 per test. Additionally, the subtraction of isotypic background can artifactually depress population enumeration.
  
  
  CONCLUSIONS
  The use of an isotypic control antibody is not necessary to analyze flow cytometric data that result in discrete cell populations, such as CD3+ and CD3+, CD4+ lymphocyte subsets. The elimination of this unnecessary quality control measure results in substantial cost savings.
authors.affiliation: Department of Laboratory Medicine, Cleveland Clinic Foundation, OH 44195, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Sreenan
authors.forename: A
authors.initials: A
authors.lastname: Tbakhi
authors.forename: M G
authors.initials: MG
authors.lastname: Edinger
authors.forename: R R
authors.initials: RR
authors.lastname: Tubbs
mesh_terms: D000906:Antibodies; D017252:CD3 Complex / Q000032:analysis*; D015704:CD4 Antigens / Q000032:analysis*; D003365:Costs and Cost Analysis; D005434:Flow Cytometry; D006801:Humans; D016130:Immunophenotyping / Q000191:economics / Q000379:methods*; D016131:Lymphocyte Subsets / Q000166:cytology / Q000276:immunology*; D011446:Prospective Studies; D011786:Quality Control; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D017252:CD3 Complex; D015704:CD4 Antigens