Genotyping Applications for Transplantation and Transfusion Management: The Emory Experience

Abstract

Current genotyping methodologies for transplantation and transfusion management employ multiplex systems that allow for simultaneous detection of multiple HLA antigens, human platelet antigens, and red blood cell (RBC) antigens. The development of high-resolution, molecular HLA typing has led to improved outcomes in unrelated hematopoietic stem cell transplants by better identifying compatible alleles of the HLA-A, B, C, DRB1, and DQB1 antigens. In solid organ transplantation, the combination of high-resolution HLA typing with solid-phase antibody identification has proven of value for highly sensitized patients and has significantly reduced incompatible crossmatches at the time of organ allocation. This database-driven, combined HLA antigen/antibody testing has enabled routine implementation of "virtual crossmatching" and may even obviate the need for physical crossmatching. In addition, DNA-based testing for RBC antigens provides an alternative typing method that mitigates many of the limitations of hemagglutination-based phenotyping. Although RBC genotyping has utility in various transfusion settings, it has arguably been most useful for minimizing alloimmunization in the management of transfusion-dependent patients with sickle cell disease or thalassemia. The availability of high-throughput RBC genotyping for both individuals and large populations of donors, along with coordinated informatics systems to compare patients' antigen profiles with available antigen-negative and/or rare blood-typed donors, holds promise for improving the efficiency, reliability, and extent of RBC matching for this population.

Figure 1

The different methods utilized for molecular HLA typing include: A) Sequence Specific Priming (SSP) – PCR products amplified using sequence-specific primers are examined by agarose gel electrophoresis, which displays pattern of positive and negative reactions to determine presence or absence of HLA alleles. Each lane identifies a unique allele or set of alleles and the reaction pattern(s) is analyzed using HLA genotyping software. B) Sequence-specific oligonucleotide probing (SSOP) – PCR amplified products are added to specific oligonucleotide probes affixed to solid-phase matrices (e.g., a microparticle or microwell plate) and binding is assessed colorimetrically or via multiplex flow cytometry. C) Sequence-based testing (SBT) –nucleic acid sequencing of the HLA alleles are performed by Sanger or Next Generation (massively parallel) sequencing to determine the nucleotide sequence of HLA genotypes.

Figure 2

In the beginning, the foundation of HLA testing was serologic in nature with an interdependence of antibodies to identify antigens, and antigens to identify antibodies. However, as time progressed, HLA laboratory testing took two distinct roads, one driven by the need for high resolution molecular characterization of HLA alleles, the other driven by the need to identify and classify HLA antibodies via serology. The directions these two paths took evolved from the clinical needs in stem cell and solid organ transplantation: Bone marrow/stem cell transplantation depends on accurate, reliable allele resolution to identify HLA-matched recipient-donor pairs while solid organ transplantation requires identification of HLA antibodies, specifically donor specific antibodies (DSA). Antigen testing led to high resolution HLA typing, which identifies individual HLA alleles; while antibody identification has progressed using solid-phase methods that now have the ability to identify allele-specific antibodies and characterize unique epitopes restricted to distinct HLA alleles. Eventually, the two roads of histocompatibility testing converged, with antigen and antibody testing coming full circle and once again each relying on the other. This convergence is exemplified in stem cell transplants, wherein donors expressing an HLA allele to which the recipient has a corresponding antibody must be considered before moving forward with a transplant. This requires high resolution antigen typing of the donor as well as high resolution antibody testing of the recipient to be performed. Similarly, since allele-specific HLA antibodies can be identified in solid organ transplant recipient, high resolution HLA typing of donors will be needed to determine compatibility.

Figure 3

Inverted orientation of the RHD and RHCE genes (top), RHD and RHCE locus structures of conventional RHD and RHCE genes, and the most frequently occurring variant haplotypes in individuals of African descent which complicate transfusion in SCD patients. The 10 coding exons of RHD and RHCE are shown as white and black boxes respectively. The location of nucleotide changes are designated by an asterisk (*). Rhesus boxes are shown as white and gray triangles with a resulting hybrid Rhesus box in individuals with the RHD deletion. The arrow (↓) indicates a 37-bp duplication in intron3/exon 4 junction and the hatched boxes represent exons encoding the untranslated region of the inactive RHD pseudogene (RHDψ) due to the nonsense mutation in exon 6 leading to a premature translation stop codon. Figure references: Ref #70, 73, 74.