Modern Blood Banking Transfusion Practices Chapter 21 The

Modern Blood Banking & Transfusion Practices 6 th Edition Introduction § Human leukocyte antigen (HLA) testing is a specialized branch or division of immunology for human histocompatibility testing. § It supports specialties in transplantation, transfusion, and immunogenetics. § 1954: Jean Dausset observed that patients whose sera contained leukoagglutinins had received more blood transfusions than other patients. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Nomenclature § The HLA genetic region is a series of closely linked genes that determine surface antigens or receptors responsible for the recognition and elimination of foreign tissues. § It's also called the major histocompatibility complex (MHC). § The HLA complex contains an estimated 35 to 40 genes. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Nomenclature (cont’d) § These genes are located on three regions of the short arm of chromosome 6 § Class I genes § Class III genes Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices Nomenclature (cont’d) § HLA genes are highly polymorphic, and several alleles exist at each locus. § The antigenic specificities, defined by serologic reactivity, are designated by numbers following the locus symbol. Copyright © 2012 F. A. Davis Company 6 th Edition

Modern Blood Banking & Transfusion Practices 6 th Edition Nomenclature (cont’d) § Continued investigation using molecular techniques of DNA sequence analysis revealed HLA allelic variants that were not detectable by traditional serologic techniques. § This complexity necessitated the development of a new system of nomenclature for HLA genes. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Antigens and Antibodies § HLA antigen composition must be valuated in prospective donor-recipient pairs before organ transplantation and in candidates for platelet therapy refractory to random donor platelets. § § Presensitization to HLA antigens may cause rapid rejection of transplanted tissue or poor platelet survival following transfusion. HLA-antigen testing is also used in disease correlation, paternity testing, and anthropologic studies. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Antigens and Antibodies (cont’d) § Two codominant alleles for each locus § The physical linkage of the HLA genes results in all of the genes on a single chromosome typically being inherited together. § The entire set of A, B, C, DR, DQ, and DP genes located on one chromosome is called a haplotype, usually inherited as a unit. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Antigens and Antibodies (cont’d) § Genetic crossovers and recombination in the HLA region are uncommon (less than 1 %). § The HLA genotype represents the association of the alleles on the two chromosomes as determined by family studies, and the term haplotype refers to the allelic makeup of a single chromosome. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Crossing Over § The effect of rearranging the genes on the chromosome to produce new haplotypes in the general population § During meiosis, exchange of material between the paired chromosomes can occur. § During chromosomal replication, replicated chromosomes often overlay each other, forming x-shaped chiasmata. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Linkage Disequilibrium § An observed value of the joint frequency of codominant alleles that is significantly different from the expected frequency (the product of the individual allele frequencies) § Disequilibrium between the B and DR loci alleles may account for problems in correlating B locus serotyping with allograft survival and disease associations, with clinical significance for allograft survival. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Gene Products § Globular glycoproteins, composed of two noncovalently linked chains § Class I (HLA-A, HLA-B, and HLA-C) molecules: heavy chain with a molecular weight of 45, 000 daltons associated noncovalently with β 2 microglobulin § Class II (HLA-DR, HLA-DQ, and HLA-DP) molecules: two similar-sized chains of a molecular weight of 33, 000 (α) and 28, 000 (β) daltons associated noncovalently throughout their extracellular portions Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Gene Products (cont’d) § Class I molecules: present on all nucleated cells, dendritic cells, and platelets § Class II molecules: present only on B lymphocytes, activated T lymphocytes, macrophages, monocytes, and endothelial cells § Surface topography of these molecules contributes to their potential immunogenicity Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Function of the HLA System § § § Role in the adaptive arm of the immune system Recognition and elimination of foreign antigens Self and non-self discrimination Large degree of polymorphism seen in MHC Thought to have evolved to present a large range of foreign peptide antigens Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Antibodies to HLA § The majority of HLA alloantibodies are Ig. G and are divided into two groups. § Those that detect a single HLA gene product § Those that detect more than one HLA gene product Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Cross Reactivity § Cross-reactive HLA antigens share important structural elements with one another but retain unique, specific elements. § The majority of cross-reactive alloantibodies detect HLA specificities of allelic molecules coded by the same locus. § Most specificities can be grouped into major CREGs or currently defined major cross-reactive groups. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition The HLA System § The agglutination methods initially used to define HLA antigens have been succeeded by a precise microlymphocytotoxicity test. § Cytotoxicity techniques require only 1 to 2 μL of serum and are sensitive and reproducible. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition The HLA System (cont’d) § Immunomagnetic bead techniques positively select lymphocyte subpopulations for use in HLA typing, both for class I and II antigen. § Alternatively, B cells can be identified by fluorescent labeling or binding of labeled antiimmunoglobulin to the cell surface immunoglobulins on B cells only. § Serologic HLA testing uses a form of complementdependent microlymphocytotoxicity (CDC). Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Molecular Techniques § HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DR, HLA-DQ, HLA-DP) typing is performed in most laboratories by DNA analysis techniques. § Molecular typing for Class I and Class II alleles is required in hematopoietic stem cell transplantation. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices § HLA Molecular Techniques (cont’d) Sequence-specific oligonucleotides ( SSO), 6 th Edition involving PCR amplification of a chosen sequence using primers flanking that sequence § Sequence-specific primers (SSP), involving oligonucleotide primers designed to obtain amplification of specific alleles or groups of alleles § Sequence-based typing (SBT), involving terminalend incorporation of fluorescently-labeled nucleotides during PCR reactions Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Antibody Detection Techniques § Recipient lymphocytotoxic HLA antibodies to donor antigens are associated with accelerated graft rejection, poor response to platelet transfusion, and TRALI. § The unknown serum tested against a panel of cells or soluble antigen of known HLA phenotype § 30 cells for screening § 60 cells for accurate antibody identification Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Antibody Detection Techniques (cont’d) § Microlymphocytotoxicity methods with sensitivity chosen for the intended purpose of the assay. § Enzyme-linked immunoadsorbent crossmatch assays (ELISA) tests use purified HLA antigens to detect patient antibodies. § § Ig. G and Ig. M antibodies may be detected. ELISA can be used as a screening assay for the detection of anti-HLA antibodies as well as a method to determine antibody specificity. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Antibody Detection Techniques (cont’d) § Flow cytometric antibody screen detects antibody binding directly, for screening or for determination of antibody specificity. § Can distinguish between Ig. G and Ig. M antibodies § Can also detect noncomplement-fixing antibodies § Utilize T and B lymphocytes as targets or, in a newer technique, employ purified HLA antigens coated onto microparticles 2 to 4 μm in diameter Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition HLA Crossmatch Techniques § Lymphocytotoxicity is the most widely used technique. § Flow cytometry can be used to facilitate detection of low levels of antibodies in recipients. § Virtual crossmatch involves selecting donorrecipient pairs based on donor HLA type and recipient alloantibody profile. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Clinical Significance of the HLA System § The HLA system is of primary clinical importance in transplantation. § Recent clinical applications include paternity testing to identify the polymorphic genetic markers in a paternity trio. § HLA antigens are associated with disease susceptibility more than any other genetic marker, although not solely clinically useful as a diagnostic tool. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Clinical Significance of the HLA System (cont’d) § Platelet Transfusion: HLA Class I antigens are expressed variably on platelets. § HLA alloimmunization can result in refractoriness to random donor platelet transfusions. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Clinical Significance of the HLA System (cont’d) § Transfusion Related Acute Lung Injury (TRALI): the clinical syndrome of new acute lung injury (ALI) developing within a clear temporal relationship to transfusion, in patients without or with alternate risk factors for ALI § Exact pathophysiology of TRALI unclear Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Clinical Significance of the HLA System (cont’d) § Antibodies against HLA class I and class II molecules have been found in 50% to 89% of products associated with TRALI. § Suspected TRALI cases are typically evaluated by analysis of donor blood product for anti. HLA and anti-HNA antibodies via immunoassay or serologic techniques. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Clinical Significance of the HLA System (cont’d) § Evaluation determines whether the donor associated with the suspected causative blood product should be removed from the donor pool. Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices 6 th Edition Implications for Organ Transplantation § HLA testing can be part of these decisions § Determination of how best to select potential recipients § Regulation of immunosuppressive treatment § How best to precondition potential recipients so that their immune systems will accept a graft Copyright © 2012 F. A. Davis Company

Modern Blood Banking & Transfusion Practices Potential Applications for HLA Testing § § § Hematopoietic stem cell transplantation Kidney transplantation Heart transplantation Liver transplantation Lung transplantation Pancreas and islet cell transplantation Copyright © 2012 F. A. Davis Company 6 th Edition