title: A new strategy to identify rare blood donors: single polymerase chain reaction multiplex SNaPshot reaction for detection of 16 blood group alleles.
abstract: BACKGROUND
  As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology.
  
  
  METHODS
  The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism.
  
  
  RESULTS
  We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA.
  
  
  CONCLUSIONS
  This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood.
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: Flavia Roche Moreira
authors.initials: FR
authors.lastname: Latini
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: Diana
authors.initials: D
authors.lastname: Gazito
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: Carine Prisco
authors.initials: CP
authors.lastname: Arnoni
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: Janaína Guilhem
authors.initials: JG
authors.lastname: Muniz
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: Rosangela
authors.initials: R
authors.lastname: de Medeiros Person
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: Fabricio Oliveira
authors.initials: FO
authors.lastname: Carvalho
authors.affiliation: FAMEMA - Faculty of Medicine of Marília, Marília, SP, Brazil.
authors.forename: Wilson
authors.initials: W
authors.lastname: Baleotti
authors.affiliation: INCTs-Unicamp Blood Centre, Campinas, SP, Brazil.
authors.forename: Lilian
authors.initials: L
authors.lastname: Castilho
authors.affiliation: Colsan - Blood Collection Centre, São Paulo, Brazil.
authors.forename: José Augusto
authors.initials: JA
authors.lastname: Barreto
mesh_terms: D000483:Alleles; D001782:Blood Donors; D001789:Blood Group Antigens / Q000032:analysis / Q000235:genetics*; D001788:Blood Grouping and Crossmatching / Q000191:economics / Q000379:methods*; D001938:Brazil; D003362:Cost-Benefit Analysis; D003365:Costs and Cost Analysis; D017931:DNA Primers; D046148:Donor Selection / Q000191:economics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide
chemical_list: D001789:Blood Group Antigens; D017931:DNA Primers