title: Residual subset population analysis in WBC-reduced blood components using real-time PCR quantitation of specific mRNA.
abstract: BACKGROUND
  Implementation of WBC reduction of the blood supply increases the importance of measurement of residual WBC subtypes responsible for immunologic and infectious complications of transfusion.
  
  
  METHODS
  Real-time RT-PCR assays were developed to detect mRNA encoding lineage-specific WBC markers. Primers and fluorescent probes were designed for CD45 (pan-WBC), CD3 (T-lymphocyte), CD19 (B-lymphocyte), CD14 (monocyte), and CD66 (granulocyte), and the specificity was assessed by comparison with flow cytometric analysis of enriched cell populations. WBC subsets were examined in WBC-reduced whole blood prepared with filters (WBF2, Pall; and RZ2000, Baxter) and in platelet concentrates prepared with other filters (Autostop, Pall; and PLX-5, Baxter) and apheresis (COBE Spectra LRS, Gambro).
  
  
  RESULTS
  All real-time RT-PCR assays were linear over >5 log concentration range, allowing pre-WBC-reduction and post-WBC-reduction comparisons. Sensitivity limits ranged from 10 cells per mL (CD45) to 200 cells per mL (CD19). Assay specificity was confirmed by the close correlation of real-time RT-PCR and immunophenotyping results by flow cytometry. For all subsets, >3.8 log and >3.1 log reduction was obtained during WBC reduction of whole blood and platelets, respectively.
  
  
  CONCLUSIONS
  Real-time RT-PCR assays are suitable for analysis of subset removal during WBC reduction. There was no significant difference between the two whole-blood filters or between platelet filtration and apheresis in the removal of any WBC subset.
authors.affiliation: Department of Hematology, Division of Transfusion Medicine, University of Cambridge, UK.
authors.forename: J
authors.initials: J
authors.lastname: Pennington
authors.forename: S F
authors.initials: SF
authors.lastname: Garner
authors.forename: J
authors.initials: J
authors.lastname: Sutherland
authors.forename: L M
authors.initials: LM
authors.lastname: Williamson
mesh_terms: D015703:Antigens, CD / Q000235:genetics; D001800:Blood Specimen Collection / Q000592:standards*; D019070:Cell Lineage; D006801:Humans; D016130:Immunophenotyping; D007937:Leukapheresis; D007958:Leukocyte Count; D016131:Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015703:Antigens, CD; D012333:RNA, Messenger