title: Enhanced ascertainment of microchimerism with real-time quantitative polymerase chain reaction amplification of insertion-deletion polymorphisms.
abstract: BACKGROUND
  The characterization of microchimerism (MC) by gene amplification has been limited by few allogeneic markers, ascertainment bias, and assay analytic performance. To address this, a panel of 12 MC assays based on insertion-deletion (InDel) polymorphisms had been optimized.
  
  
  METHODS
  The InDel assays were validated with comprehensive in vitro spiking studies at the stochastic limit of detection. Their ability was also determined to ascertain MC of unknown source genotype with both theoretical and actual donor-recipient pairs, and the assays were applied to a clinical population of 73 trauma patients who received transfusions where MC was previously characterized by HLA-based assays alone.
  
  
  RESULTS
  In the stochastic spiking experiments, all assays were sensitive to a single copy of target DNA, and no false-positive amplification occurred among 1128 samples studied. Among 219 theoretical donor-recipient pairs, informative alleles existed for 99.5 percent with both InDel and HLA compared to 91.3 percent with HLA alone. In the clinical population, 33 cases of MC were detected (9 more cases than by HLA-DR alone) in the nonleukoreduced (non-LR) group and 8 cases (1 more case than by HLA-DR) in the LR group for the short-term follow-up. Among 27 long-term follow-up samples, 8 cases were detected overall (3 more cases than by HLA-DR alone).
  
  
  CONCLUSIONS
  It is concluded that an InDel-based assay panel has excellent technical performance characteristics while also allowing for ascertainment of some MC cases not detectable with HLA alone. The tandem use of both the InDel and the HLA provides a powerful tool for the enhanced ascertainment of MC.
authors.affiliation: Blood Systems Research Institute, San Francisco, California 94118, USA. tlee@bloodsystems.org
authors.forename: Tzong-Hae
authors.initials: TH
authors.lastname: Lee
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Chafets
authors.forename: William
authors.initials: W
authors.lastname: Reed
authors.forename: Li
authors.initials: L
authors.lastname: Wen
authors.forename: Yunting
authors.initials: Y
authors.lastname: Yang
authors.forename: Jennifer
authors.initials: J
authors.lastname: Chen
authors.forename: Garth H
authors.initials: GH
authors.lastname: Utter
authors.forename: John T
authors.initials: JT
authors.lastname: Owings
authors.forename: Michael P
authors.initials: MP
authors.lastname: Busch
mesh_terms: D000328:Adult; D001782:Blood Donors; D046528:Chimerism; D005189:False Positive Reactions; D005260:Female; D005500:Follow-Up Studies; D006684:HLA-DR Antigens / Q000235:genetics*; D006801:Humans; D047589:Leukocyte Reduction Procedures; D008297:Male; D008875:Middle Aged; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D015203:Reproducibility of Results; D017384:Sequence Deletion; D014947:Wounds and Injuries / Q000150:complications / Q000235:genetics / Q000628:therapy
chemical_list: D006684:HLA-DR Antigens