title: Quantitation of residual WBCs in filtered blood components by high-throughput, real-time kinetic PCR.
abstract: BACKGROUND
  The effort to eliminate transfusion complications associated with WBCs has led to the widespread use of filters able to reduce WBC concentrations to <or=0.1 WBC per microL blood. This has necessitated sensitive QC methods to quantitate residual WBCs in filtered units. One fast, effective method is DNA amplification using real-time kinetic PCR (kPCR).
  
  
  METHODS
  Two methods of preparation of standards were compared and used for the optimization of quantitative kPCR. The first involved spiking genomic DNA cell lysate into a diluent, followed by a series of 1 in 10 dilutions. The second involved spiking serial 1 in 10 dilutions of WBCs into twice-filtered fresh whole blood. Two hundred fifty filtered frozen whole-blood samples were amplified in duplicate to show the kPCR assay's reproducibility. Another 359 filtered frozen whole blood samples were used to compare data from kPCR with data from a standard PCR protocol using (32)P-labeled probe and autoradiography. All specimens were amplified for conserved HLA DQ(alpha) sequences.
  
  
  RESULTS
  Standards prepared by both methods gave reproducible and equivalent results. Quantitation of standards representing a dynamic range of 8 x 10(o) to 8 x 10(5) WBCs per mL, yielded standard deviations ranging from 0.59 cycle to 1.04 cycles (a one-cycle increase is equivalent to a twofold increase in WBC concentration). The scatter graph of the 250 samples tested in duplicate by kPCR generated a slope of 1.0122 and an R(2) value of 0.9265. The comparison of kPCR and (32)P-probe hybridization results on 359 clinical samples gave a scatter-graph slope of 0.9428 and an R(2) value of 0.8718, indicating excellent agreement of the methods over a 4-log dynamic range.
  
  
  CONCLUSIONS
  kPCR is a high-throughput, sensitive assay that could prove useful in routine quality assurance of the WBC reduction process.
authors.affiliation: Molecular Biology Blood Centers of the Pacific, Irwin Center, Research Division, San Francisco, California 94118, USA. thlee@qpcr.com
authors.forename: Tzong-Hae
authors.initials: TH
authors.lastname: Lee
authors.forename: Li
authors.initials: L
authors.lastname: Wen
authors.forename: Vera
authors.initials: V
authors.lastname: Chrebtow
authors.forename: Russell
authors.initials: R
authors.lastname: Higuchi
authors.forename: Robert M
authors.initials: RM
authors.lastname: Watson
authors.forename: John J
authors.initials: JJ
authors.lastname: Sninsky
authors.forename: Michael P
authors.initials: MP
authors.lastname: Busch
mesh_terms: D003199:Computer Systems; D004247:DNA / Q000097:blood; D005374:Filtration; D006801:Humans; D007700:Kinetics; D007958:Leukocyte Count / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D004247:DNA