A comparative study of HLA-DRB typing by transcription-mediated amplification with the hybridization protection assay...

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.