[Cause Analysis of HLA-B Allele Missed Detection Based on AlltypeTM FastplexTM Next Generation Sequencing].

OBJECTIVE

To investigate the missed detection of HLA-B allele in one case using AlltypeTM FastplexTM next generation sequencing (NGS), and evaluate the accuracy and limitations of NGS in HLA genotyping.

METHODS

HLA routine detection was performed using PCR-sequence-specific oligonucleotide probe (SSOP) and AlltypeTM FastplexTM NGS. After discovering a sample from a patient with Glanzmann's thrombasthenia that had inconsistent HLA-B locus results, the sample was retested using PCR-sequence-based typing (SBT) and AlloSeq Tx17 hybrid capture NGS technology to verify the detection results of AlltypeTM FastplexTM NGS.

RESULTS

The SSOP typing results showed that the patient's HLA-B locus was B*15:01, 40:01 , while the AlltypeTM FastplexTM NGS results showed that the patient was homozygous for B*40:01, 40:01 . The homozygous sample was retested with SBT and AlloSeq Tx17 hybrid capture NGS to confirm the NGS test results. The SBT result analysis showed that there were two possible combinations: B*15:01, 40:01 and B*15:07, 40:01, indicating that the SBT results were ambiguous. However, the AlloSeq Tx17 NGS results clearly showed B*15:01, 40:01 , which was consistent with the SSOP typing results, thus confirming that AlltypeTM FastplexTM NGS missed the B*15:01 allele at the HLA-B locus.

CONCLUSION

The NGS method based on amplicon library construction has the limitation of missing detection of HLA alleles. Especially for the detected homozygous samples, multiple experimental methods should be used for verification to ensure the accuracy of HLA genotyping.