P044 The use of HLA-DQ EXON 3 Data in SSO analysis can produce discrepant HLA typing results

Herein we document two cases of discrepant typing results obtained when data from both exon 2 and 3 is used to analyze LabType SSO results for HLA-DQ using the HLA Fusion software. The first case is an ASHI PT sample (HT-184) tested by LabType SSO and analyzed using HLA Fusion 4.1. For this sample, 39% of survey participants reported HLA-DQA1*05:05, 26% HLA-DQA1*05:02 and the remaining labs reported either a new allele, HLA-DQA1*05:01G or P. By sequence based analysis (SBT) the correct typing is HLA-DQA1*05:02,*01:01. When using LabType SSO and analyzing with HLA Fusion 4.1, including both exon 2 and 3, the typing obtained is HLA-DQA1*05:05/09/11 when bead #56 is excluded. The software flags bead #56 as a false positive bead. No typing is obtained without excluding bead 56. HLA-DQA1*05:05 was the most common response on the PT survey indicating several labs may be analyzing data using Fusion and excluding bead #56 from the analysis. The correct typing HLA-DQA1*05:02 can only be obtained when HLA-DQA1 exon 3 is excluded from the analysis despite the fact that the probe attached to bead 56 binds to DQA1*05:02 but not DQA1*05:05. The issue encountered with this sample stems from the fact that HLA-DQA1*05:02 does not have an exon 3 sequence in IMGT. As such, the probe pattern for exon 3 has not been established for this allele. The software automatically rules out alleles with no exon 3 sequences from the possible allele string when the analysis includes both exon 2 and 3 rather than flagging it as a possibility and indicating exon 3 sequence is not available for this allele. The second case is a patient typed by SBT as HLA-DQB1*06:11, *03:03. When the sample is tested using LabType SSO and analyzed using HLA Fusion 4.1 the typing obtained is HLA-DQB1*03:03, *06:112 N. The correct typing, HLA-DQB1*03:03, *06:11 can only be obtained when HLA-DQB1 exon 3 data is excluded from the analysis. Again this is because the sequence for exon 3 is not available in IMGT for HLA-DQB1*06:11:01. The two cases presented are representative of others we have encountered and reinforces the importance of software validation in the laboratory. Regardless of vendors and methodologies most software have limitations and impute some HLA typing data. As such, a variety of typing samples with specific allele combinations should be used in validation studies.