title: Detection of HLA-DRB1 microchimerism using nested polymerase chain reaction and single-strand conformation polymorphism analysis.
abstract: For the detection of microchimerism, molecular methods detecting donor-specific HLA-DRB1 alleles in the recipient are most commonly used. Nested polymerase chain reaction sequence specific primer (nested PCR-SSP) methods widely used to increase the sensitivity of detection have been reported to give frequent false-positive reactions. We have developed a new method combining nested PCR with single-strand conformation polymorphism analysis (nested PCR-SSCP) and tested the 1 to 0.00001% level of microchimerism for 27 different HLA-DRB1 alleles. For most (26/27) of the HLA-DRB1 alleles tested, this method could detect 0.01 to 0.001% of microchimerism and its sensitivity was equal to or better than that of nested PCR-SSP tested in parallel. Its specificity was verified by visualizing particular DRB1-specific SSCP bands under test. Nested PCR-SSP indicated frequent false-positive reactions, mainly caused by nonspecific amplification of DRB3/B4/B5 alleles present in the major (recipient) DNAs. We have compared a real-time quantitative PCR for non-human leukocyte antigen (HLA) target (insertion/deletion marker) using a commercial kit (AlleleSEQR Chimerism assay), and its microchimerism detection sensitivity (around 0.1%) was 1 step (10 times) lower than that of nested PCR-SSP or -SSCP methods for HLA-DRB1 alleles. We validated that the newly designed nested PCR-SSCP affords good sensitivity and specificity and may be useful for studying microchimerism in clinical settings.
authors.affiliation: Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul 110-744, Korea.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Song
authors.forename: Hye Yoon
authors.initials: HY
authors.lastname: Chung
authors.forename: Shin Young
authors.initials: SY
authors.lastname: Joo
authors.forename: Eun Youn
authors.initials: EY
authors.lastname: Roh
authors.forename: Moon-Woo
authors.initials: MW
authors.lastname: Seong
authors.forename: Yunsu
authors.initials: Y
authors.lastname: Shin
authors.forename: Myoung Hee
authors.initials: MH
authors.lastname: Park
mesh_terms: D000483:Alleles; D046528:Chimerism; D005189:False Positive Reactions; D005240:Feasibility Studies; D059811:HLA-DRB1 Chains / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods / Q000639:trends; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
chemical_list: D059811:HLA-DRB1 Chains