title: Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.
abstract: BACKGROUND
  Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT.
  
  
  METHODS
  SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR).
  
  
  RESULTS
  Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results.
  
  
  CONCLUSIONS
  The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors: Chiaki
authors: C
authors: Taira
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan. Electronic address: kmatsuda@shinshu-u.ac.jp.
authors: Kazuyuki
authors: K
authors: Matsuda
authors: Core Technology Development Center, Seiko Epson Corporation, Fujimi, Japan.
authors: Akemi
authors: A
authors: Yamaguchi
authors: Core Technology Development Center, Seiko Epson Corporation, Fujimi, Japan.
authors: Masayuki
authors: M
authors: Uehara
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors: Mitsutoshi
authors: M
authors: Sugano
authors: Laboratory of Clinical Chemistry and Immunology, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto, Japan.
authors: Nobuo
authors: N
authors: Okumura
authors: Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.
authors: Takayuki
authors: T
authors: Honda
mesh_terms: D000483:Alleles; D002648:Child; D046528:Chimerism; D005260:Female; D005500:Follow-Up Studies; D005838:Genotype; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods*; D019337:Hematologic Neoplasms / Q000628:therapy; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D018895:Microsatellite Repeats; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D013997:Time Factors; D018183:Transplantation Chimera / Q000235:genetics*; D014184:Transplantation, Homologous; D061349:Unrelated Donors; D055815:Young Adult
keywords: AS-qPCR; Droplet-AS-PCR; HSCT; SNP