title: Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets.
abstract: BACKGROUND
  Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest.
  
  
  METHODS
  Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2(-ΔΔCt) of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples.
  
  
  RESULTS
  Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 10(6) WBCs/L.
  
  
  CONCLUSIONS
  The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.
authors.affiliation: Department of Laboratory Medicine, Chang Gung Memorial Hospital; Department of Medical Biotechnology and Laboratory Science, College of Medicine, Department of Pathology, School of Medicine, Molecular Medicine Research Center, Chang Gung University, Taoyuan County, Taiwan.
authors.forename: Ding-Ping
authors.initials: DP
authors.lastname: Chen
authors.forename: Chien-Feng
authors.initials: CF
authors.lastname: Sun
authors.forename: Hsiao-Chen
authors.initials: HC
authors.lastname: Ning
authors.forename: Chien-Ting
authors.initials: CT
authors.lastname: Peng
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Wang
authors.forename: Ching-Ping
authors.initials: CP
authors.lastname: Tseng
mesh_terms: D001771:Blood Banks / Q000592:standards; D001792:Blood Platelets / Q000166:cytology; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics*; D006801:Humans; D007958:Leukocyte Count / Q000379:methods / Q000592:standards*; D047589:Leukocyte Reduction Procedures / Q000379:methods / Q000592:standards*; D017713:Platelet Transfusion / Q000592:standards*; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D000094902:Blood Banking / Q000379:methods
chemical_list: D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)