title: A comparative study of HLA-DRB typing by transcription-mediated amplification with the hybridization protection assay (TMA/HPA) versus PCR/SSOP.
abstract: To evaluate alternative human leukocyte antigen (HLA)-DNA typing methods, we used a system of transcription-mediated amplification (TMA) with a probe hybridization protection assay (HPA) in a microtiter plate format developed by Chugai Pharmaceutics Ltd. (Tokyo, Japan) to perform intermediate-level DRB typing for 502 individual samples. Two hundred fifty-two samples submitted to our Clinical Immunogenetics Laboratory were prospectively tested concurrently with a locally developed intermediate-level DRB polymerase chain reaction/sequence-specific oligonucleotide probe (PCR/SSOP) assay in a double-blind fashion. In addition, 250 retrospective samples of archived frozen cells or DNA from clinical and research panels, previously typed by allele-level DRB1 PCR/SSOP, were chosen to include 66 distinct DRB1 alleles representing Caucasian, American Black, Asian, and Native American ethnic groups. Among the prospectively typed samples, except for four samples with a TMA/HPA microplate handling problem, a single TMA/HPA allele assignment (1/462 alleles = 0.2%) was discordant with PCR/SSOP. Among the 250 retrospective samples, a single HPA probe for codon 57 aspartic acid consistently cross-reacted with the codon 57 valine sequence of DRB1*0807. However, TMA/HPA identified six samples with previous PCR/SSOP typing errors, all of which involved identification of sequences at codons 67-71 in samples heterozygous for two DR52-associated DRB1 alleles. Assay turnaround time from sample preparation to results was 11 h for 24 samples or 6-7 h for 1-4 samples. In summary, we found the TMA/HPA DRB typing system to provide rapid, reliable, and accurate HLA-DRB typing results. The current TMA/HPA methodology could be improved by use of a molded plastic cold block to provide more consistent and secure microtiter plate cooling than the current water/ice slurry. Nevertheless, this methodology, based on a microtiter plate format but without the usual plate washing steps of the traditional ELISA, has superior potential for microplate handling and reagent distribution with a robotics system and a work surface incorporating microplate heating and cooling units.
authors.affiliation: Clinical Immunogenetics Laboratory, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Smith
authors.forename: K
authors.initials: K
authors.lastname: Matsubara
authors.forename: E
authors.initials: E
authors.lastname: Mickelson
authors.forename: A
authors.initials: A
authors.lastname: Marashi
authors.forename: L
authors.initials: L
authors.lastname: Regen
authors.forename: L A
authors.initials: LA
authors.lastname: Guthrie
authors.forename: J A
authors.initials: JA
authors.lastname: Hansen
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D005784:Gene Amplification; D006684:HLA-DR Antigens / Q000145:classification / Q000235:genetics / Q000276:immunology*; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D014158:Transcription, Genetic
chemical_list: D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D015345:Oligonucleotide Probes