Chapter 21

1. Chapter 21 The HLA System

2. 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.

3. 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.

4. Nomenclature (cont’d) • These genes are located on three regions of the short arm of chromosome 6 • Class I genes • Class II genes • Class III genes

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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 β2-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

13. 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

14. 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

15. Antibodies to HLA • The majority of HLA alloantibodies are IgG and are divided into two groups. • Those that detect a single HLA gene product • Those that detect more than one HLA gene product

16. 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.

17. 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.

18. 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 anti-immunoglobulin to the cell surface immunoglobulins on B cells only. • Serologic HLA testing uses a form of complement-dependent microlymphocytotoxicity (CDC).

19. 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.

20. HLA Molecular Techniques (cont’d) • Sequence-specific oligonucleotides (SSO), 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 terminal-end incorporation of fluorescently-labeled nucleotides during PCR reactions

21. 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

22. 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. • IgG and IgM 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.

23. 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 IgG and IgM 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

24. 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 crossmatchinvolves selecting donor-recipient pairs based on donor HLA type and recipient alloantibody profile.

25. 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.

26. 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.

27. 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

28. 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.

29. 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.

30. 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

31. Potential Applications for HLA Testing • Hematopoietic stem cell transplantation • Kidney transplantation • Heart transplantation • Liver transplantation • Lung transplantation • Pancreas and islet cell transplantation