Human Leukocyte Antigen Test
The human leukocyte antigen test, also known as HLA or tissue typing, identifies antigens on the white blood cells that determine tissue compatibility for organ transplantation (i.e., histocompatibility testing). There are six loci on chromosome 6 where the genes that produce HLA antigens are inherited: HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP.
Unlike most blood group antigens which are inherited as products of two alleles (alternative genes), many different alleles can be inherited at each of the HLA loci. These are defined by antibodies (antisera) that recognize specific HLA antigens, or by DNA probes that recognize specific oligonucleotide sequences within the HLA allele. Using specific antibodies, 26 HLA-A alleles, 59 HLA-B alleles, 10 HLA-C alleles, 26 HLA-D alleles, 22 HLA-DR alleles, nine HLA-DQ alleles, and six HLA-DP alleles can be recognized. This high degree of genetic variability (polymorphism) makes finding compatible organs more difficult than finding compatible blood for transfusion.
HLA typing, along with ABO grouping, is used to provide evidence of tissue compatibility. The HLA antigens expressed on the surface of the lymphocytes of the recipient are matched against those from various donors. HLA typing is performed for kidney, bone marrow, liver, pancreas or heart transplants. The probability that a transplant will be successful increases with the number of identical HLA antigens. HLA typing is not performed for blood transfusion or corneal transplants, or for a graft of autologous tissue such as skin or bone.
Graft rejection occurs when the immune cells (T lymphocytes) of the recepient recognize specific HLA antigens on the donor's organ as foreign. These antigens are referred to as Class II histocompatibility antigens. The T lymphocytes initiate a cellular immune response characterized by release of cytotoxins and other cytokines that result in graft rejection. The cytotoxic reaction of the T lymphocytes is directed against the Class I histocompatibility antigens on the surface of the organ. Alternatively, T lymphocytes present in the grafted tissue may recognize the host tissues as foreign and produce a cell mediated immune response against the recipient. This is called graft versus host disease (GVHD), and it can lead to life-threatening systemic damage in the recipient. HLA testing is performed in order to reduce the probability of both allograft rejection and GVHD.
HLA typing is also used along with blood typing and DNA tests to determine parentage (i.e., for paternity testing). The HLA antigens of the mother, child, and alleged father can be compared. When an HLA antigen of the child cannot be attributed to the mother or the alleged father, then the latter is excluded as the father of the child.
A third use of HLA testing called linkage analysis is based upon the fact that the region where the HLA loci are positioned, the major histocompatibility complex (MHC), contains many other genes located very close to the HLA loci. The incidence of crossing-over between HLA genes during gamete formation is generally less than 1%. Consequently, the HLA antigens from all six loci are inherited together and segregate with many other genes located within the same region of chromosome 6. Many of the MHC region genes are involved in immunological processes. Consequently, alleles that are known to increase the chance of developing various autoimmune diseases have remained associated with specific HLA alleles. For example, 2% of people who have the HLAB27 allele develop an arthritic condition of the vertebrae called ankylosing spondylitis. However, approximately nine out of 10 white persons who have ankylosing spondylitis are positive for HLA-B27. Because of this association the disease and this HLA type are linked. Family members of a person with ankylosing spondylitis who are HLA-B27 positive have a much higher likeli-hood of developing this condition than those who are not. Some notable autoimmune diseases that have a strong association with HLA antigens include Hashimoto's thyroiditis (an autoimmune disorder involving underproduction by the thyroid gland) associated with HLA-DR5; Graves' disease (an autoimmune disorder associated with
HLA testing is performed using white blood cells harvested from peripheral blood collected by venipuncture. The blood should be collected using either heparin or ACD anticoagulant. The nurse or phlebotomist performing the venipuncture should observe universal precautions for the prevention of transmission of blood-borne pathogens. If possible, this test should be postponed if the patient has recently undergone a transfusion. Any white blood cells from the transfusion may interfere with the tissue typing of the patient's lymphocytes.
The HLA gene products can be grouped into three classes. Class I consists of the products of the genes located on the HLA-A, HLA-B, and HLA-C loci. These HLA antigens are found on all nucleated cells. Class II molecules consist of antigens inherited as genes from the HLA-DR, HLA-DQ, and HLA-DP loci. These HLA antigens are normally found only on B lymphocytes, macrophages, monocytes, dendritic cells, endothelial cells, and activated T lymphocytes. Class III molecules consist of several proteins belonging to the complement system and cytokines produced by lymphocytes such as tumor necrosis factor. Class III molecules are not evaluated in histocompatibility testing.
Because the HLA loci are closely linked, the HLA antigens are inherited as a group of six antigens called a haplotype. Each person receives one haplotype from each parent. HLA antigens, like blood group antigens, are codominant, and a person expresses both the alleles when two different genes are inherited from each parent. Since crossing-over does not often occur, the probability of siblings having an identical haplotypes is one in four. Therefore, siblings provide the opportunity for the best matches. Unfortunately, they can donate bone marrow, a kidney, and a section of their liver, but cannot donate other solid organs. Approximately 85% of transplants are organs from cadavers, and because the HLA antigens are so highly polymorphic, the chance of identical haplotypes falls precipitously.
Histocompatibility testing consists of three tests, HLA antigen typing (tissue typing), screening of the recipient for anti-HLA antibodies (antibody screen), and the lymphocyte crossmatch (compatibility test). HLA antigen typing may be performed by serological or DNA methods. In the serological method, called the microcytotoxicity assay, lymphocytes are harvested from the blood by density gradient centrifugation. A solution of Ficoll-Hypaque is layered underneath the whole blood and the tube is centrifuged. Red blood cells and granulocytes are denser than the gradient and are forced to the bottom. The mononuclear cells are less dense than the gradient and are found at the top just underneath the platelets. The mononuclear cell layer is removed and washed. T-cells are removed by one of several methods, for example by binding to magnetic beads coated with T-cell antibodies. The B-cell enriched suspension is tested against a panel of specific antibodies to HLA antigens. The cells are added to wells of a microtiter tray each containing a different antibody. After incubating, rabbit complement is added to each well. Following a second incubation, a dye, Eosin Y, is added. Next, a formalin solution is added to fix the cells and stop any further immunological destruction. If the specific antibodies in the well recognize the HLA antigen on the lymphocytes, they will bind to the cells forming antigen-antibody complexes. The antigen-antibody complexes activate the complement proteins causing partial lysis of the cells. Eosin Y stains only those cells that are dead (i.e., unable to exclude the dye). The cells coated with antibodies can be identified by examining each well with an inverted phase contrast microscope. Cells that are stained pink are positive. The percentage of stained cells in each well is used to determine whether the cells are positive or negative for the HLA antigen. In general, when 60% or more of the cells are stained they are considered positive for the HLA antigen defined by the antibody in the well.
The alternative approach to HLA typing is DNA testing. In this method, white blood cells (granulocytes and lymphocytes) are separated from peripheral blood by lysis of the red blood cells and centrifugation. The DNA is extracted from the white cells and added to the wells of a microtiter tray. Each well contains an oligonucleotide primer complementary to a small segment of DNA. Each primer will hybridize with the DNA belonging to only one HLA allele. Therefore, if the primer attaches to the DNA, the corresponding HLA antigen coded by that allele was present on the cells. A master mix containing DNA polymerase and oligonucleotide triphosphates is added to each well and the plate is incubated in a thermal cycler that causes the DNA sequence framed by the primers to be amplified. The amplified products are detected by electrophoresis. The presence of a DNA band in the gel indicates a positive test for the respective HLA antigen.
A laboratory will perform HLA typing by either the serological or DNA method. In either case, HLA typing
The antibody screen is performed in order to detect antibodies in the recipient's serum that react with HLA antigens. The most commonly used method of HLA anti-body screening is the microcytotoxicity test with an antiglobulin phase. Leukocytes harvested from the blood of donors of known HLA types are added to the wells of a microtiter plate. Serum from the recipient is added to each well and incubated. The cells are washed to remove any unbound proteins, and antihuman immunoglobulin (AHG) and rabbit complement are added. If an antibody against an HLA antigen is present, it will bind to the cells. The antigen-antibody complexes will bind the anti-human immunoglobulin resulting in partial lysis by the complement. Eosin Y is added and the cells are examined under the microscope. The presence of pink-stained cells indicates the presence of anti-HLA antibodies. As for HLA typing, the percentage of cells stained in each well is used to determine whether the serum is positive or negative for HLA antibody. The antibody screen is reported as the percentage of panel reactive antibodies (PRA). For example, if 50 cells are tested and 10 wells show a positive test, then the PRA is 20%. The higher the number of different HLA antibodies the lower the probability of finding a compatible match. ELISA test kits that use HLA antigens bound to the wells of a commercially prepared microtiter tray are also available for HLA antibody screening.
The third component of a histocompatibility study is the crossmatch test. In this test peripheral blood lymphocytes from the donor are separated into B and T lymphocyte populations. Purified T cells are prepared by mixing the lymphocyte suspension with magnetic beads coated with monoclonal antibodies to the B lymphocytes. The B-cells bind to the beads that are then pulled to the bottom of the tube by a magnet. The supernatant cell suspension can be used for the T-cell crossmatch. Purified B lymphocytes are produced in like manner except that an antibody to the T-cells is used. In the crossmatch serum from the recipient is mixed with T-cells or B-cells from the donor. The T-cell crossmatch is performed by the same microcytotoxicity method as described above for the antibody screen (i.e., an AHG reagent is used). The B-cell crossmatch is performed by the same microcytotoxicity method as described previously for HLA typing. A positive finding indicates the presence of preformed antibodies in the recipient that are reactive against the donor tissues. An incompatible T-cell crossmatch contraindicates transplantation of a tissue from the T-cell donor.
The HLA test requires a blood sample collected in heparin or acid-citrate-dextrose (ACD). There is no need for the patient to fast before the test.
The patient may feel discomfort when blood is drawn from a vein. Bruising may occur at the puncture site or the person may feel dizzy or faint. Pressure should be applied to the puncture site until the bleeding stops to reduce bruising. Warm packs can also be placed over the puncture site to relieve discomfort.
Risks for this test are minimal, but may include slight bleeding from the puncture site, fainting or feeling lightheaded after venipuncture, or hematoma (blood accumulating under the puncture site).
HLA typing either by serological or DNA methods is reported as the phenotype for each HLA loci tested. The antibody screen test is reported as the percentage of panel
Tissue typing results for both donors and recipients and antibody screen results for recipients are submitted to the United Network for Organ Sharing (UNOS) database. The database searches all regional donors that are ABO compatible for an HLA identical match. If none is found, the database searches the national database for ABO compatible donors and scores the match. A point system is used based upon several parameters including the number of matching HLA loci, the length of time the recipient has been waiting, the recipient's age, and the PRA score.
Health care team roles
A physician specializing in transplantation medicine orders HLA typing tests. A nurse or phlebotomist draws the blood sample. Histocompatibility tests are performed by clinical laboratory scientists/medical technologists with specialized training in cellular immunology and DNA typing procedures. Results are interpreted by a physician who specializes in transplantation immunology.
Antigen—A molecule, usually a protein, that elicits the production of a specific antibody or immune response.
Autoimmune disorders—A disorder caused by a reaction of an individual's immune system against the organs or tissues of one's own body.
Cornea—The transparent outer layer of the eye. It covers the iris and lens.
Haplotype—A set of alleles of a group of closely linked genes which are usually inherited as a unit from one parent.
Lymphocyte—A class of white blood cell that is responsible for the immune response to antigens.
Phenotype—A trait produced by a gene. For example, the specific HLA antigen(s) inherited for the HLA-A locus is the phenotype for that gene.
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Mark A. Best