[Cause Analysis of HLA-B Allele Missed Detection Based on Alltype^TM Fastplex^TM Next Generation Sequencing]

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

HLA routine detection was performed using PCR-sequence-specific oligonucleotide probe (SSOP) and Alltype^TM Fastplex^TM 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 Alltype^TM Fastplex^TM NGS.

The SSOP typing results showed that the patient's HLA-B locus was B*15:01, 40:01 , while the Alltype^TM Fastplex^TM 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 Alltype^TM Fastplex^TM NGS missed the B*15:01 allele at the HLA-B locus.

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.