title: Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses.
abstract: BACKGROUND
  Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation) and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt), and cytomegalovirus (CMV) antigens. These antigens were determined to be low (HER-2/neu), moderate (tt), and robustly (CMV) immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response.
  
  
  RESULTS
  Linear regression analysis indicates that both stimulation index (SI) (p = 0.011) and IFN-gamma secreting precursor frequency (p < 0.001) are significant indicators of antigen specific immunity. ROC curves plotted to assess the performance of tritiated thymidine incorporation and the ELISPOT assay indicate that SI is a significant indicator of low T cell response to the HER-2/neu vaccine (p = 0.05), and of moderate and robust responses to tt (p = 0.01) and CMV (p = 0.016), respectively. IFN-gamma precursor frequency is a significant indicator of a robust T cell response to CMV (p = 0.03), but not of moderate tt (p = 0.09), or low HER-2/neu (p = 0.09) T cell responses.
  
  
  CONCLUSIONS
  These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.
authors.affiliation: Center for Translational Medicine in Women's Health, 2nd Floor, 815 Mercer Street, Box 358050, University of Washington, Seattle, WA 98109 USA. vgoodell@u.washington.edu
authors.forename: Vivian
authors.initials: V
authors.lastname: Goodell
authors.forename: Corazon
authors.initials: C
authors.lastname: dela Rosa
authors.forename: Meredith
authors.initials: M
authors.lastname: Slota
authors.forename: Beth
authors.initials: B
authors.lastname: MacLeod
authors.forename: Mary L
authors.initials: ML
authors.lastname: Disis
mesh_terms: D000328:Adult; D000368:Aged; D000917:Antibody Formation / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology*; D049109:Cell Proliferation; D004273:DNA, Neoplasm / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D005260:Female; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology*; D007371:Interferon-gamma / Q000378:metabolism; D008875:Middle Aged; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000378:metabolism / Q000382:microbiology*; D013936:Thymidine / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D004273:DNA, Neoplasm; D000939:Epitopes; D014316:Tritium; D007371:Interferon-gamma; D013936:Thymidine