Human Leukocyte Antigen (HLA) Testing

Email

Send Email

Recipient(s) will receive an email with a link to 'Human Leukocyte Antigen (HLA) Testing' and will have access to the topic for 7 days.

Subject: Human Leukocyte Antigen (HLA) Testing

(Optional message may have a maximum of 1000 characters.)

×


Introduction

  • The human leukocyte antigen (HLA) system, located on the short arm of chromosome 6 at position 6p21.3, is the major histocompatibility complex (MHC) in humans containing a cluster of genes related to immune system. The classical HLA spans 3.6 Mb and is subdivided into three regions: class I, class II, and class III. Each region contains numerous gene loci, including expressed genes, transcripts, and pseudogenes. Many HLA loci are among the most polymorphic genes within the human genome.

  • The class I region contains the genes encoding the “classical” class I HLA antigens, HLA-A, HLA-B, and HLA-C. Class I antigens, made up of an alpha chain and a beta chain (beta-2 microglobulin encoded on chromosome 15), are expressed on almost all cells of the body at varying density except erythrocytes and trophoblasts. This region also contains other HLA class I genes such as HLA-E, HLA-F, and HLA-G; MHC class I chain–related (MIC-A and MIC-B) genes; and a variety of other genes, not all of which are immune related.

  • The class II region contains the genes encoding the “classical” class II molecules, HLA-DR, HLA-DQ, and HLA-DP. Class II antigens are only expressed on B cells, dendritic cells, and monocytes and can be induced during inflammation on many other cell types that normally have little or no expression. The class II molecules also consist of an alpha and a beta chain both are encoded by genes within the MHC.

  • Between class I and class II is the class III region. This region does not contain any of the HLA genes. It does, however, contain many genes of importance in the immune response, for example, complement, tumor necrosis factor, and heat shock protein.

  • Clinical applications of HLA are associated with, but not limited to, the following: solid organ transplantation, stem cell transplantation, platelet transfusion, disease association, and drug sensitivity.

Testing Methods

HLA Typing

  • HLA typing was originally performed by serology. Due to the limited accuracy, the serologic HLA typing has largely been replaced by DNA PCR–based methods. Sequence-specific oligonucleotide probing (PCR-SSOP), sequence-specific primer amplification (PCR-SSP), and sequence-based typing (SBT) are commonly used in clinical HLA laboratories.

    • PCR-SSP: Multiple pairs of PCR primers, designed to anneal within DNA regions present in certain alleles or groups of alleles, are used for PCR amplification. If the corresponding alleles are present, their specific amplicons can be detected by gel electrophoresis. The size of the PCR products needs to be known for interpretation, and the electrophoresis migration must be run longer to separate all PCR fragments.

    • PCR-SSOP: Locus-specific PCR primers were selected to amplify the HLA locus of interest followed by the hybridization using sequence-specific oligonucleotide probes. The HLA genotype was assigned on oligonucleotide positive reactions. Currently, the Luminex platform is the most commonly used PCR-SSOP method. Oligonucleotide probes, covalently linked to a set of polystyrene carboxylated microbeads, are designed to specifically detect the nucleotide sequences at the polymorphic sites of the HLA specificities.

    • SBT: This is the only technique that directly detects the nucleotide sequences of an allele, thus allowing exact assignment. Sequence analysis software generates final allele assignment by comparing the nucleotide sequence obtained with a database of all known alleles.

HLA Antibody Analysis

  • The deleterious role of HLA antibodies has become more apparent with the development of sensitive assays identifying previously undetectable antibody specificities in the last few years, replacing the less sensitive lymphocyte target–based complement-dependent cytotoxicity (CDC) assays. Depending upon the clinical application needs, HLA antibody analysis by CDC, ELISA, flow cytometry, and Luminex platform can all be utilized, either individually or in combination, to characterize the HLA antibody.

T- and B-cell crossmatch

  • The crossmatch is used to determine if the recipient has antibodies against the potential donor. The test is performed between the patient's serum and the potential donor's T and B cells. Complement-dependent cytotoxicity (CDC), anti–human globulin–enhanced CDC (AHG-CDC), and flow cytometric crossmatches are commonly used in the lab. The sensitivity of the crossmatch methods varies a lot; CDC as the least sensitive and flow as the most sensitive. A positive crossmatch is a strong indication of HLA incompatibility.

Engraftment Monitoring

  • To provide clinicians with accurate information of the engraftment status by quantitatively determining the proportion of donor- and recipient-derived cells in the patient posttransplant. Short tandem repeats (STRs) are the most commonly used markers for this assay. STRs, also referred to as microsatellites, are short sequences of DNA, distributed throughout the genome that is repeated in tandem variable number of times. The number of repeats of different STR markers varies between individuals, giving a highly polymorphic system that can be used to uniquely identify donor-derived DNA from patient-derived DNA. With the exception of monozygotic twins, careful selection of a number of STR markers will enable most patient-derived DNA to be distinguished from donor-derived DNA.

Suggested Reading

Bontadini  A. HLA techniques: Typing and antibody detection in the laboratory of immunogenetics. Methods.  2012;56;471–476.
 
×