Residual subset population analysis in WBC-reduced blood components using real-time PCR quantitation of specific mRNA.
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.
Pennington
J
J
Department of Hematology, Division of Transfusion Medicine, University of Cambridge, UK.
Garner
S F
SF
Sutherland
J
J
Williamson
L M
LM
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
D015703:Antigens, CD; D012333:RNA, Messenger