Introduction

The HLA (human leukocyte antigens) complex is located on the short arm of chromosome 6.[1][2][3] The HLA genes follow the principles of Mendelian genetics and the encoded antigens are co-dominantly expressed on the cell surface. In the absence of a recombination event, HLA genes are normally inherited en bloc from each parent due to their close proximity resulting in their close physical linkage. The HLA haplotype is a combination of linked HLA genes (HLA-A, -B,-C,-DR, -DQ,-DP) transmitted on a single parental chromosome.[2] HLA antigens are expressed on the surface of many cells and play a major role in self-recognition, evoking the immune response to an antigenic stimulus, and to the orchestration of cellular and humoral immunity.[1]  HLA complex is known to be highly polygenic as it is composed of many genes, which can divide broadly into three categories: Class I, Class II, and Class III.[2] Polymorphism is another feature of the HLA molecule. Polymorphism allows the presence of multiple variations of antigens or alleles. The HLA class I and class II antigens have the most highly polymorphic structural genes found in humans, which allows amino acids in any given HLA molecule to vary slightly from one person to the next. This variation generates distinct HLA types and also causes allograft rejection when tissues are transplanted.[4]

Development

The foundations of the HLA complex were laid down in 1958 by three scientists: Jean Dausett, Jon van Rood, and Rose Payne. They described the presence of alloantigens on leukocytes in human sera obtained from multiparous women or patients who had received multiple transfusions in three separate papers.[5][6][7] Among the three, Jean Dausett was given credit for the discovery of the first HLA antigen, and in 1980 he received the Nobel prize. Discovery of the HLA is the first milestone of the field of immunohistocompatibility. However, the impact of immunohistocompatibility testing in transplantation was established in 1969. Patel and Terasaki demonstrated in kidney transplantation recipient and donors pairs that there is a  strong correlation between a positive lymphocyte crossmatch and a hyperacute rejection as a common outcome.[8] This compelling data resulted in a mandatory prospective crossmatch before kidney transplantation to identify the antibodies reacting with donor lymphocytes that have the potential to cause a graft loss.

Biochemical

Structure

Class I and Class II molecules differ structurally. HLA Class I molecules are comprised of a polymorphic alpha chain, which is encoded by the Class I genes, and the B2 microglobulin chain, which is encoded by a gene on chromosome 15. The alpha chain is also known as the heavy chain and also has three domains: alpha 1, 2 and 3. The alpha1 and alpha2 domains contain the majority of polymorphic regions conferring HLA antigen specificity. HLA Class II molecules have one alpha chain and a beta chain.[3][9] Class II region contains DR, DQ, and DP. Each class II molecule has two genes, A and B encoding the alpha and beta chains.[9][10]

Function

The MHC genes that act as transplantation antigens are the classical HLA genes. The classical HLA class I genes (HLA-A, -B and -C) express on most of the somatic cells in the body. Classical HLA Class II genes (HLA-DR,-DQ, -DP) express on antigen presenting cells such as B- cells, activated T cells, dendritic cells, macrophages, and epithelial cells of thymic gland. If interferon is present, other types of cells can also express class II HLA molecules. The primary function is the distinction of self from non-self. Both class I and class II molecules present short peptides derived from pathogens to T cells to initiate the adaptive immune response. The HLA class I molecules display peptides that result from the degradation of cytosolic proteins to the cell surface where they can is recognizable by the CD8+T cells. HLA class II molecules present peptides that result from the degradation of endocytosed proteins to the cell surface where they are recognizable by the CD4+ T cells.[3]

Testing

HLA Typing:

HLA typing is defined polymorphisms of class I and class II loci.[11]  Serology was the first HLA typing methodology. The microcytoxicity method detects the presence of antigen by cytotoxic cell death induced by activation of complement. In this in vitro assay, T or B lymphocytes are isolated from an individual and are incubated with serum containing HLA antibodies of a known HLA specificity obtained from multiparous women.[12][13] The enhancements of molecular methodology eliminate the necessity of HLA typing by using serum with anti-HLA antibodies of all HLA antigens. Although the polymorphisms of the HLA are widely distributed throughout the gene, the vast majority of the polymorphisms congregate within exons 2 and 3. In the modern day, molecular methods are used to identify the specific nucleotide sequence polymorphisms distinctive of a specific HLA antigen. Subsequently, artificially synthesized commercially available DNA probes or primers are used to detect HLA polymorphisms.[13]

There are three different categories of DNA based molecular HLA typing methods available. All of these methods determine the HLA types based on polymorphisms identified by polymerase chain reaction (PCR) technology which uses a heat-stable DNA polymerase to amplify the target sequences of HLA genes rapidly.

1- Sequence-specific primer (SSP) typing: This technique uses sequence-specific primer pairs that target and amplify a particular DNA sequence where PCR primers are designed to bind only to a specific HLA allele or allele groups. Confirmation of the amplified alleles is by the presence or absence of DNA amplification by a particular primer pair that are visualized by agarose gel electrophoresis.[2][13] Sequence-specific primers used in this method are directed to specific targets. Therefore the presence of the amplification will correspond to the presence of the designated allele(s). Commercially available primer pair sets can define a full HLA type of an individual (HLA-A, -B,-C, -DR, -DQ, and -DP). Deceased donor typing is the most common application of this method as it provides rapid results. This technique allows for low-resolution HLA typing in the DNA based typing nomenclature (A*01).[2][14] Disadvantage SSP typing is not suitable for testing a large number of samples.[2]

2- Sequence-specific oligonucleotide probes (SSOP): This technique is frequently used in clinical histocompatibility laboratories. This method identifies HLA polymorphisms using oligonucleotide probes on a solid phase matrix (i.e., microbeads) in broadly generated, exon and locus-specific PCR products.[13] SSOP methodology provides low to high-level resolution (antigen level typing) by DNA based typing results. This methodology is suitable for testing a large number of samples in batches.

3- Sequence-based typing (SBT): This technique is performable on several different platforms. One is Sanger-based DNA sequencing, and the other platforms have their basis in next-generation sequencing. The most common application of SBT typing is to identify allelic level HLA typing for stem cell transplantation recipients and donors. An allele refers to a unique nucleotide sequence for a gene. By DNA based typing nomenclature, allelic resolution utilizes all the digits in a current allele name. (A*01:01:01:01). This methodology allows for confirmation of new allelic sequences.[2][13][14]

As HLA typing methods developed and molecular HLA typing replaced serologic methodology, HLA nomenclature has changed to reflect the complexity of the output of the advanced typing methodologies.[11]

HLA Antibody Testing:

Identification of HLA antibodies is important in a pre-transplant and post-transplant setting in solid organ transplantation. Currently, single antigen bead assays are the most common approach for HLA antibody identification. This test is a solid phase assay that utilizes microbeads coated with recombinant single HLA antigens. The analysis is performed on a specialized flow cytometry platform.[15] Interpretation of this assay is highly complex and performed by specially trained laboratorians.

Crossmatching:

Flow cytometric crossmatch is an essential test to determine the compatibility between a donor and a recipient. It assay is frequently used prior to transplantation of solid organs.[2]

Clinical Significance

HLA is an important barrier for hematopoietic stem cell (HPC) transplantation. Therefore, HLA matching and compatibility between the donor and the recipient is necessary for a successful HPC transplantation. HLA antigens also have a role in graft-versus-host disease (GVHD), a potentially serious complication of allogeneic stem cell transplantation. GVHD occurs when donor T cells react to host antigens on antigen-presenting cells (APCs) and attack host tissues, with sequential activation of donor T cells and monocytes/macrophages.[16]

In solid organ transplantation, HLA antibodies play an important role, and HLA matching is not as important. With patient exposure to foreign HLA antigens due to pregnancy or blood transfusions or transplantation, they can make antibodies against epitopes of those foreign HLA molecules; this is called HLA alloimmunization. Presence of HLA antibodies against graft can cause antibody-mediated rejection and graft loss.[17]

HLA alloimmunization can also cause platelet refractoriness. This condition creates difficulty in finding compatible platelet units, especially in transfusion-dependent HPC transplant patients. In platelet refractoriness HLA Class I antibodies especially antibodies against HLA-A and HLA-B play an important role.[18] HLA antibodies also play a role in febrile nonhemolytic transfusion reactions and transfusion-related acute lung injury (TRALI).[19]