PMID- 9328793 OWN - NLM STAT- MEDLINE DCOM- 19971212 LR - 20190905 IS - 0198-8859 (Print) IS - 0198-8859 (Linking) VI - 55 IP - 1 DP - 1997 Jun TI - A comparative study of HLA-DRB typing by transcription-mediated amplification with the hybridization protection assay (TMA/HPA) versus PCR/SSOP. PG - 74-84 AB - 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. FAU - Smith, A G AU - Smith AG AD - Clinical Immunogenetics Laboratory, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, USA. FAU - Matsubara, K AU - Matsubara K FAU - Mickelson, E AU - Mickelson E FAU - Marashi, A AU - Marashi A FAU - Regen, L AU - Regen L FAU - Guthrie, L A AU - Guthrie LA FAU - Hansen, J A AU - Hansen JA LA - eng GR - AI33484/AI/NIAID NIH HHS/United States GR - CA18029/CA/NCI NIH HHS/United States PT - Comparative Study PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - Hum Immunol JT - Human immunology JID - 8010936 RN - 0 (HLA-DR Antigens) RN - 0 (HLA-DRB1 Chains) RN - 0 (HLA-DRB3 Chains) RN - 0 (HLA-DRB4 Chains) RN - 0 (Oligonucleotide Probes) SB - IM MH - Alleles MH - Amino Acid Sequence MH - *Gene Amplification MH - HLA-DR Antigens/classification/genetics/*immunology MH - HLA-DRB1 Chains MH - HLA-DRB3 Chains MH - HLA-DRB4 Chains MH - Histocompatibility Testing/*methods MH - Humans MH - Molecular Sequence Data MH - Nucleic Acid Hybridization MH - Oligonucleotide Probes MH - Polymerase Chain Reaction MH - Transcription, Genetic EDAT- 1997/06/01 00:00 MHDA- 1997/11/05 00:01 CRDT- 1997/06/01 00:00 PHST- 1997/06/01 00:00 [pubmed] PHST- 1997/11/05 00:01 [medline] PHST- 1997/06/01 00:00 [entrez] AID - S0198885997000852 [pii] AID - 10.1016/s0198-8859(97)00085-2 [doi] PST - ppublish SO - Hum Immunol. 1997 Jun;55(1):74-84. doi: 10.1016/s0198-8859(97)00085-2.