title: Real-time biallelic polymorphism-polymerase chain reaction for chimerism monitoring of hematopoietic stem cell transplantation relapsed patients.
abstract: BACKGROUND
  An accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP).
  
  
  METHODS
  The allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients.
  
  
  RESULTS
  Allele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003-0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127 ± 0.193% of recipient DNA.
  
  
  CONCLUSIONS
  We conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation.
authors.affiliation: Department of Laboratory Medicine, Chang-Gung Memorial Hospital, Taoyuan County, Taiwan.
authors.forename: Ding-Ping
authors.initials: DP
authors.lastname: Chen
authors.forename: Ching-Ping
authors.initials: CP
authors.lastname: Tseng
authors.forename: Wei-Ting
authors.initials: WT
authors.lastname: Wang
authors.forename: Mei-Chia
authors.initials: MC
authors.lastname: Wang
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Tsai
authors.forename: Chien-Feng
authors.initials: CF
authors.lastname: Sun
mesh_terms: D000483:Alleles; D001483:Base Sequence; D046528:Chimerism; D017931:DNA Primers; D005819:Genetic Markers; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012008:Recurrence
chemical_list: D017931:DNA Primers; D005819:Genetic Markers