Body: A deceased donor of unknown ethnicity was being assessed for solid organ transplant. Initial typing by rSSO assay [Thermo Fisher] identified class II alleles DRB1*04,07; DRB4*01,01N; and DQB1*03:02G,03:03G. Follow-up high-resolution testing was performed by NGS [Werfen] and showed DRB1*04:04,07:01; DRB4*01:03N,01:03N; DQB1*03:02,03:03. Close review of genomic site 9985 at the end of intron 2 found only A nucleotides, verifying that an expressed DRB4*01 allele was not present. Additional testing was performed by real-time PCR [Thermo Fisher] and SSP [CareDx and Thermo Fisher], all showing only DRB4*01:03N alleles in concordance with the NGS results. Complete typing results by all methods is displayed in Table 1.
Surrogate flow crossmatching was performed against a serum with only DR53 antibody (MFI21079) and was negative, further confirming the absence of a DR53 antigen. Review of the original rSSO results found that a bead was flagged by the software as a possible false positive, and when this bead was adjusted to negative, DRB4*01 was no longer present and only the DRB4*01N remained.
Assuming one haplotype was the well-documented DRB1*07:01-DRB4*01:03N-DQB1*03:03, we sought to differentiate between homozygosity and hemizygosity of the DRB4*01:03N allele. Performance of a research-use-only NGS application by CareDx confirmed there were two copies of the DRB4*01:03N antigen (Figure 1), and neither haplotype contained a deletion of the DRB4 gene. It was therefore determined the donor’s two Class II haplotypes were the well-documented DRB1*07:01 – DRB4*01:03N – DQB1*03:03 and the uncommon DRB1*04:04 – DRB4*01:03N – DQB1*03:02.
Conclusion: Common strong haplotype associations can be a useful tool in the Histocompatibility Laboratory to resolve ambiguities, however less common haplotypes are being reported more frequently with advances in high-resolution typing technologies and must be considered. This case demonstrates the value of utilizing multiple testing platforms, and that caution and correlation should be used when interpreting test results.
