Featured Article
Technology Spotlight
Please see our Clinical Diagnostics Assays section to find manufacturers that sell these products
Human leukocyte antigens (HLAs) are membrane-bound glycoproteins that have basic roles in immunity. These antigens are major impediments to organ and tissue transplantation, and are associated with many diseases, particularly autoimmune conditions. HLAs are encoded by two extremely polymorphic gene families, which are located on the short arm of chromosome 6 (6p21.3). The HLA genes are the human equivalent of the genes of the major histocompatibility complex (MHC), which is found in most other vertebrate species. There are two different, but structurally similar, classes of HLAs. The class I proteins—HLA-A, -B, and -C—are expressed ubiquitously on enucleated cells and present endogenous antigens to CD8+ killer T-cells. The class II proteins—HLA-DR, -DQ, and -DP—are expressed on a more restricted range of cells, including B-cells, monocytes, macrophages, dendritic cells, and activated T-cells, and present exogenous antigens to CD4+ helper T-cells.
HLA testing identifies an individual’s unique assemblage of human leukocyte antigens, and is essential for transplantation of bone marrow and solid organs, studies of disease association, and vaccine development and applications. HLA testing is also used in forensic investigations. The technology used for HLA typing has evolved in parallel with the understanding of the genetic complexity of the family of genes located on chromosome 6. Over 2000 alleles of the classical class I and II loci are now known.1
HLA Typing Methods
Initially, HLA polymorphisms were typed in complement-dependent cytotoxicity assays of T-cells and B-cells using panels of antisera collected from multiparous women or individuals who had received multiple blood transfusions. Although good serology is still useful as an adjunct to DNA-based typing, the approach has many technical drawbacks and shortcomings, including insufficient resolution for stem cell transplantation. The advent of recombinant DNA technology, which paved the way to identifying genetic differences among HLA alleles directly, has resulted in abandonment of classic serological typing methods by many laboratories. Today, several different DNA-based HLA typing methods are commonly used by clinical laboratories. All methods are based on PCR amplification of specific sequences in the HLA genes of interest. PCR primers and probes can be designed and validated by individual laboratories, but are also available commercially.
PCR-based approaches to typing vary, based on typing resolution levels—“antigen level” is low resolution, and “allele level” is high resolution. PCR using panels of sequence-specific primers (SSPs) amplify particular alleles or allele groups. The presence or absence of amplification products indicates the presence or absence of a particular allele. This is a rapid test, with low resolution similar to serologic testing, and is used to type deceased organ donors, where speed is important. However, PCR-SSP also can be used for higher-resolution testing. PCR using sequence-specific oligonucleotide (SSO) probes in a hybridization assay is used for low- or intermediate-resolution typing in screening tests to identify potential donors that may later undergo testing at higher resolution. A variant of this technique uses probes linked to fluorescently labeled beads in a flow cytometry assay (xMAP® technology, Luminex Corporation, Austin, TX). Direct sequencing-based typing (SBT) is for HLA typing at the highest resolution, and is used for the donors and recipients in an allogeneic stem cell transplant procedure, or in studies of disease associations.
- Life Technologies Corporation (Grand Island, NY) offers a wide range of HLA typing kits of various brands from Invitrogen™. Technologies include high-resolution SSP and SBT. There are kits for clinical applications and for research-use-only (RUO) applications.
- Texas BioGene Inc. (Richardson, TX) offers HLAssure™ SBT Typing Kits for high-resolution typing of HLA alleles using PCR techniques with SBT. The kits are CE-marked for in vitro diagnostics and for RUO. The company also offers the Morgan™ HLA SSP Typing Kits for low-to-medium-resolution typing of HLA alleles using PCR techniques with SSP. The kits are FDA-cleared and CE-marked and for RUO. The company has an HLA Outsource Typing Service Lab that is certified by the American Society of Histocompatibility and Immunology.
- R.O.S.E. Europe GmbH (Steinbach/Taunus, Germany) offers the low-resolution ROSE HLA ABDRDQ SSP Typing Kit, which uses 96-well plates and a universal PCR program, and provides results in less than 2 hr. The company also offers a range of high-resolution SBT Typing Kits that are CE-marked.
Reference
- Howell, W.M.; Varter, V. et al. The HLA system: immunobiology, HLA typing, antibody screening and crossmatching techniques. J. Clin. Pathol. 2010, 63(5), 387–90.
Please see our Clinical Diagnostics Assays section to find manufacturers that sell these products