DNA Based Tissue Typing and Human Leukocyte Antigens Comprehensive Study Guide

Molecular Composition of HLA Class I Molecules: * Class I molecules are membrane-bound proteins consisting of a heavy chain and a light chain. * The $\alpha$ (Alpha) Chain: This is a transmembrane polypeptide containing three extracellular domains: $\alpha_1$ , $\alpha_2$ , and $\alpha_3$ . * $\beta_2$ -microglobulin: This is a small, non-glycosylated protein that associates non-covalently with the $\alpha_3$ domain of the alpha chain to provide structural stability. * Spatial Orientation: The $\alpha_1$ and $\alpha_2$ domains form the peptide-binding groove where antigens are presented to T cells.
Molecular Composition of HLA Class II Molecules: * Class II molecules are heterodimers composed of two transmembrane polypeptide chains of similar size. * The $\alpha$ (Alpha) Chain: Consists of two extracellular domains, $\alpha_1$ and $\alpha_2$ . * The $\beta$ (Beta) Chain: Consists of two extracellular domains, $\beta_1$ and $\beta_2$ . * Spatial Orientation: The peptide-binding groove in Class II molecules is formed by the interaction between the $\alpha_1$ and $\beta_1$ domains.

Chromosomal Location: The MHC is located on the short arm of Chromosome $6$ .
Definition and Scope: The MHC locus is a large genetic region that encodes for the Human Leukocyte Antigens (HLA) as well as various other genes involved in immune function.
Biological Purpose: HLA molecules, which are the gene products of the MHC, serve as membrane proteins responsible for the critical biological task of recognizing non-self cells and tissues.

MHC Class I Region: * Gene Products: Includes HLA-A, HLA-B, and HLA-C. * Tissue Location: Present on the surface of all nucleated cells in the human body. * Function: Responsible for the identification and destruction of abnormal or infected cells (such as viral-infected or malignant cells) by cytotoxic T cells.
MHC Class II Region: * Gene Products: Includes HLA-D (often subdivided into DR, DQ, and DP). * Tissue Location: Specifically located on B lymphocytes, monocytes, macrophages, dendritic cells, activated T cells, endothelial cells, and Langerhans cells. * Function: Facilitates the identification of foreign antigens by helper T cells, triggering the adaptive immune response.
MHC Class III Region: * Gene Products: Includes complement components C2, C4, and Factor B. * Tissue Location: Found as plasma proteins circulating in the blood. * Function: Provides a defense mechanism against extracellular pathogens.
Cytokine Genes within the MHC: * Gene Products: Tumor Necrosis Factor-alpha (TNF- $\alpha$ ) and Tumor Necrosis Factor-beta (TNF- $\beta$ ). * Tissue Location: Found as plasma proteins. * Function: Regulate cell growth and differentiation processes.

Nature of Polymorphism: * HLA gene sequences are highly polymorphic, meaning they differ significantly between individuals. * Differences often occur at single base pairs, resulting in different sequences known as alleles.
WHO Nomenclature Standard: * The World Health Organization (WHO) Nomenclature Committee has established a standardized system to identify HLA alleles. * Example Structure (HLA-DRB1): * HLA: Indicates the gene region. * DR: Indicates the subregion. * B: Indicates the specific gene locus. * 1: Indicates the $\alpha$ - or $\beta$ -chain polypeptide type.
Need for DNA-Level Nomenclature: Because an increasing number of HLA alleles are being identified through genetic sequencing, a specific nomenclature for DNA sequences is required to manage the complexity of tissue typing.

Haplotype Inheritance: HLA alleles are inherited together in blocks known as haplotypes. Because individuals inherit one haplotype from each parent, every person (with the exception of identical twins) possesses a unique set of HLA alleles.
Allografts: In clinical transplantation, the moved organs are termed allografts, meaning the donor organ and the recipient are genetically distinct.
Compatibility and Matching: The success of an engraftment relies on HLA matching. Comparing alleles between donor and recipient reduces the risk of rejection.
Resolution: This term refers to the level of detail with which an allele is determined during the typing process.

Complement-Dependent Cytotoxicity (CDC) Test: * Lymphocytes are HLA typed by crossmatching them to Panel Reactive Antibodies (PRA). * Mechanism: A lymphocyte is mixed with specific antibodies and complement proteins. If the antibody recognizes the antigen on the cell surface, the complement is activated, leading to cell death. * Interpretation: A "Positive reaction" is indicated by a dead cell; a "Negative reaction" occurs if the cell remains viable because the antibody did not bind.
Bead Arrays and Flow Cytometry: * Recipient antihuman antibodies are assessed by crossmatching them to known lymphocyte antigens. * These antigens are conjugated to microparticles (beads). * Results are analyzed using flow cytometry to determine the presence and specificity of antibodies.

Restriction Fragment Length Polymorphism (RFLP): * Uses Southern blot analysis to identify HLA Class II alleles. * Initially focused on highly polymorphic loci such as HLA-B and HLA-DRB. * Specimens consist of whole-blood collected in anticoagulants; cell lines of known HLA types serve as reference samples.
Sequence-Specific Oligonucleotide Probe Hybridization (SSOP): * Uses immobilized probes to identify patient alleles through hybridization. * Bead-Array SSOP: Probes are immobilized on fluorescent beads for high-throughput analysis.
Sequence-Specific PCR (SSP-PCR): * Performed using primers that are specific to certain alleles. * Testing is typically conducted in a $96$ -well plate format, where each well contains primers recognizing different alleles.
Sequence-Based Typing (SBT): * Considered a high-resolution method. * Polymorphic regions are amplified via PCR and then directly sequenced. * Includes Next-Generation Sequencing (NGS) based typing, which utilizes MHC regions and long-range PCR.

Hierarchy of Resolution: * Low-Resolution: CDC (Serology), PCR-SSP, PCR-SSOP, PCR-RFLP. * Intermediate-Resolution: PCR-SSP, PCR-SSOP, SSP-PCR mixed with PCR-RFLP, or SSOP-PCR mixed with SSP-PCR. * High-Resolution: PCR-SSP, PCR-SSOP, SBT.
Common Methodological Discrepancies: * Epitopes vs. Sequence: DNA sequence changes do not always result in a change to the physical epitope recognized by antibodies. * Serology Limits: Serological methods may fail to recognize every allele that is detectable via DNA analysis. * Cross-Reactivity: Serological antibodies may react with multiple different alleles, leading to ambiguity. * Discovery of New Alleles: Retyping may yield different results if new alleles have been identified since the initial test was performed.
Resolving Ambiguities: Sequence ambiguities and discrepancies can often be resolved by using NGS or by combining different methods: * $q$ SSP-PCR followed by PCR-RFLP. * $q$ SSOP followed by SSP-PCR. * SBT results clarified by serology.