title: A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants.
abstract: BACKGROUND
  GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs.
  
  
  METHODS
  To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR.
  
  
  RESULTS
  Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing.
  
  
  CONCLUSIONS
  This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.
authors.affiliation: Mackay Memorial Hospital Transfusion Medicine Laboratory, Tamsui, Taiwan.
authors.forename: Kate
authors.initials: K
authors.lastname: Hsu
authors.forename: Yen-Chun
authors.initials: YC
authors.lastname: Lin
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Chang
authors.forename: Yung-Syu
authors.initials: YS
authors.lastname: Chan
authors.forename: Hsueh-Ping
authors.initials: HP
authors.lastname: Chao
authors.forename: Ting-Ying
authors.initials: TY
authors.lastname: Lee
authors.forename: Marie
authors.initials: M
authors.lastname: Lin
mesh_terms: D001483:Base Sequence; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D060005:Genotyping Techniques; D006021:Glycophorins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008951:MNSs Blood-Group System / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013624:Taiwan
chemical_list: C409973:GYPB protein, human; D006021:Glycophorins; D008951:MNSs Blood-Group System; C068315:Miltenberger III-like molecule glycophorin variant