HLA Typing: Introduction, Methods, and Applications

Introduction to Human Leucocyte Antigen Typing

Humans' major histocompatibility complex (MHC) proteins are encoded by the HLA (human leukocyte antigen) system. The human immune system is regulated by these glycoproteins that are part of the cell membrane. MHC is divided into two classes: MHC class I and MHC class II. The HLA gene complex is found on chromosome 6p21 in a 3.6 Mb region. With over 10,000 different HLA alleles identified to date, they are the most polymorphic gene family in the human genome. As a result, the ability to assemble an immune response can vary significantly between individuals within a cohort drawn from a single population Transplant rejection, autoimmune disease, vaccine pharmacogenomics, cancer, infectious diseases, and mate selection have all been linked to HLA genes.

HLA genotyping is the process of determining an individual's HLA class I and class II gene polymorphisms, which is required for transplant matching and disease association research. Due to high polymorphism in various genomic regions, unambiguous HLA genotyping is technically challenging. The introduction of next-generation sequencing (NGS) has altered the landscape of genotyping. Traditional HLA genotyping and Sanger sequencing assays have restrictions, and high-resolution HLA genotyping utilizing PCR and NGS is distinctively able to recognize these limitations in patients. It allows for reliable, simple, high-quality, and high-throughput analysis of the key HLA genes, with data phased to at least six digits. Another benefit is that because DNA frameworks are made up of single molecules, the phasing problem is solved.

Detection of HLA Antibodies and Its Applications

Antibodies to HLA have been discovered in about 20% of multiparous women and 30% to 50% of multi-transfused patients. The traditional serological cytotoxicity method, flow cytometry, and solid-phase method are the three primary approaches for detecting HLA antibodies.

Serological cytotoxicity method

Magnetic beads are utilized to segregate B lymphocytes from blood or spleen for HLA class II typing. HLA class I typing can be done with the remaining leucocytes. The lymphocytes are then placed in individual wells on Terasaki plates, which consist of various specific antibodies. The cells in that well will die if the specific antibody and HLA antigen attach. This method is unable to distinguish between HLA and non-HLA cytotoxic antibodies.

Flow cytometry

Freshly nucleated leucocytes are mixed with fluorescently labeled monoclonal antibodies in this experiment. Surface HLA antigens that bind to antibodies fluoresce, allowing flow cytometers to detect them as they pass through a laser beam.

Solid-phase technique

The reaction is established using PE-conjugated FC-specific antibodies after fluorochrome-dyed polystyrene beads coated with particular HLA antigens are cultured with the patient's serum. Although this technique is more delicate than serological cytotoxicity and ELISA, the therapeutic effect of increased sensitivity has yet to be determined.

Detection of HLA Polymorphism and Its Workflow

DNA-based methods have dominated in typing laboratories since the advancement of polymerase chain reaction (PCR), owing to their precision and reproducibility, as well as increased sensitivity and resolution. PCR-sequence specific primers (SSP), PCR-sequence-specific oligonucleotide (SSO), Real-time PCR, Sequencing-Based Typing (SBT), and next-generation sequencing (NGS) are all DNA-based methods.

Because of its accuracy, high throughput, and speed, NGS, also known as high-throughput sequencing, is gradually becoming the preferred method for HLA typing. The most significant benefit of NGS is that it eliminates ambiguities at a cost comparable to SBT and without the need for additional screenings DNA library preparation, targeted amplification of genes that code for HLA peptides using emulsion or solid-phase PCR, DNA sequencing-based typing, and sequence alignment against gene bank databases, such as the international ImMunoGeneTics project/human leukocyte antigen (IMGT/HLA) database, are all part of the NGS process. HLA typing based on high-throughput sequencing is commonly used in clinical diagnosis, treatment, and personalized medicine.

References:

1. Duke JL, Lind C, Mackiewicz K, et al. Determining performance characteristics of an NGS‐based HLA typing method for clinical applications. Hla. 2016, 87(3).
2. Carapito R, Radosavljevic M, Bahram S. Next-generation sequencing of the HLA locus: methods and impacts on HLA typing, population genetics and disease association studies. Human immunology. 2016, 77(11).
3. Gabriel C, Fürst D, Faé I, et al. HLA typing by next‐generation sequencing–getting closer to reality. Tissue antigens. 2014, 83(2).