Clinical Immunology & Serology CLS 311 PDF

Clinical Immunology & Serology CLS 311 Human Leukocyte Antigen Mr. Abdullah Abdali CLS Lecturer MS.c in Immunology and Immunotherapy  Introduction: Human Leukocyte Antigen (HLA) The HLA system includes a group of genes and their molecular products that are...

Clinical Immunology & Serology CLS 311 Human Leukocyte Antigen Mr. Abdullah Abdali CLS Lecturer MS.c in Immunology and Immunotherapy  Introduction: Human Leukocyte Antigen (HLA) The HLA system includes a group of genes and their molecular products that are involved in immune regulation and cellular differentiation. Cont'D Antigens of the HLA system also designated histocompatibility locus antigens, human leukocyte antigens, transplantation antigens and tissue antigens. The HLA gene products are glycoprotein antigens found on the surface membranes of all nucleated cells of the body, including; Solid tissues and most of the circulating blood cells; namely lymphocytes, granulocytes, monocytes and platelets. Mature non-nucleated red cells usually lack HLA antigens demonstrable by conventional methods, but immature, nucleated RBCs exhibit HLA reactivity.  Immunologic recognition of differences in HLA antigens is probably the first step in the rejection of transplanted tissue. The genes of the HLA system are present in the major histocompatibility complex (MHC) on the short arm of chromosome 6. HLA genes are highly polymorphic, meaning they vary greatly between individuals, making HLA typing essential in several medical contexts. Discovery of HLA  The MHC that encodes cell surface antigens was first discovered in skin graft rejection experiments with mice.  The presence of HLA in humans was first recognized when multiply transfused patients experienced transfusion reactions despite proper cross-matching.  It was discovered that these reactions resulted from leukocyte antibodies rather than antibodies directed against RBC antigens.  Major Histocompatibility Complex (MHC) The MHC is the most complex immunogenetic system presently known in humans. The MHC includes several loci closely linked. Each of these loci involves numerous alleles, having at least 10 to 40 alleles per locus that control the production of their corresponding antigens. Although HLA was originally identified by its role in transplant rejection, it is now recognized that; – Proteins encoded in this region are involved in immunologic recognition including: Interaction between different lymphoid cells and between lymphocytes and antigen- presenting cells (the products of HLA genes play a crucial role in our immune system). The HLA genes encode for three classes of MHC molecules: a. Class I major transplantation antigens: This class includes the main HLA-A, B, and C antigens. b. Class II immune response gene region antigens are encoded in the HLA-D region and can be subdivided into three families, called HLA-DR, DC (DQ), and -SB (DP). c. Class III molecules bear no clear relation to class I and II molecules aside from their genetic linkage. Class III molecules are involved in immunologic phenomena because they represent components of the complement pathways. The HLA genes encode for three classes of MHC molecules: Fig. 1: Major histocompatibility complex (MHC). Fig. 2: Example of the inheritance pattern of class I and class II HLA antigens. A complete set of alleles located in each parent’s chromosome is inherited as a unit by each child. There is a 25% chance that two children in a family will inherit the same sets and have identical HLA typing. Techniques for Detection of HLA Antigens: 1. Serological Techniques A. Microlymphocytotoxicity Assay (CDC): Principle: This classical method uses complement-mediated cytotoxicity to identify HLA antigens. Lymphocytes from the test subject are mixed with HLA-specific antisera and complement. If the HLA antigens on the lymphocytes match the antisera, the complement is activated, causing cell lysis. Detection: Lysis is detected using a dye exclusion test (e.g., trypan blue or eosin dye uptake). Positive Reaction: Cell lysis indicates the presence of HLA antigens matching the antisera used. Negative Reaction: No lysis indicates no match. Techniques for Detection of HLA Antigens: B. Flow Cytometry  Principle: Fluorescently labeled antibodies specific to HLA antigens are used to detect and quantify HLA molecules on the surface of cells.  Applications: Detects pre-formed HLA antibodies (in the recipient's serum). Useful for crossmatching in organ transplantation. Advantages: High sensitivity and specificity. Limitations: Requires sophisticated equipment and trained personnel.  Techniques for Detection of HLA Antigens: 2. Molecular Techniques A. SSP (Sequence-Specific Primers) PCR:  Uses primers specific to known HLA alleles.  Amplifies only specific HLA sequences.  Provides allele-level typing resolution.  Applications: Commonly used for donor-recipient matching. (identifying specific HLA alleles for transplantation.) B. SSO (Sequence-Specific Oligonucleotide) Hybridization:  PCR amplification is followed by hybridization with oligonucleotide probes specific to HLA alleles.  Advantages: analyze multiple alleles simultaneously (Higher throughput than SSP, useful for population screening)  Results are matched to predefined allele patterns Techniques for Detection of HLA Antigens: RT-PCR (Real-Time PCR):  Quantifies HLA gene expression and detects specific alleles.  Often used for high-sensitivity applications. 3. DNA Sequencing Techniques A. Sanger Sequencing:  Direct sequencing of HLA genes for high-resolution allele identification.  Time-consuming but provides detailed results. B. Next-Generation Sequencing (NGS):  High-throughput sequencing method for comprehensive HLA typing.  Allows for precise and unambiguous allele-level resolution.  Widely used in transplantation, population studies, and disease association research. Techniques for Detection of HLA Antigens: 4. Luminex-Based Techniques Principle: Beads coated with HLA-specific antigens are used to detect HLA antibodies in the serum. Advantages: High throughput, sensitivity, and specificity. Althaf, M.M., El Kossi, M., Jin, J.K., Sharma, A. and Halawa, A.M. (2017). Human leukocyte antigen typing and crossmatch: A comprehensive review. World Journal of Transplantation, [online] 7(6), pp.339–348. doi:https://doi.org/10.5500/wjt.v7.i6.339. Comparison of the techniques  Importance of HLA in Medical Contexts 1. Transplantation: 1. HLA compatibility is critical for organ and tissue transplantation (e.g., kidney, bone marrow). 2. Matching donor and recipient HLAs minimizes the risk of rejection and improves transplant success. 2. Disease Association: 1. Certain HLA types are linked to autoimmune diseases (e.g., HLA-B27 and ankylosing spondylitis) and other conditions. 3. Immune Response: 1. HLAs are essential in presenting antigens to T-cells, enabling the immune response to pathogens. 4. Pharmacogenomics: 1. Some HLA types predict adverse drug reactions, such as HLA-B*57:01 with abacavir hypersensitivity. Table 1: HLA Testing Applications  Hematopoietic progenitor cell transplants  Solid-organ transplants  Platelet selection for refractory patients  Disease association:  Ankylosing spondylitis—B27  Celiac disease—DQ2  Optimize certain drug therapy regimens  Abacavir sensitivity (for HIV treatment) and B*57:01 allele Thank You