The human leukocyte antigen (HLA) test, also known as HLA typing or tissue typing, identifies antigens on the white blood cells (WBCs) that determine tissue compatibility for organ transplantation (that is, 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 (types of gene that occupy the same site on a chromosome), 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 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 (blood type) 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. Human leukocyte antigen typing is performed for kidney, bone marrow, liver, pancreas, and heart transplants. The probability that a transplant will be successful increases with the number of identical HLA antigens.
Graft rejection occurs when the immune cells (T-lymphocytes) of the recipient recognize specific HLA antigens on the donor's organ as foreign. The T-lymphocytes initiate a cellular immune response that result in graft rejection. 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. Human leukocyte antigen testing is performed to reduce the probability of both rejection and GVHD.
Typing is also used along with blood typing and DNA tests to determine the parentage (that is, 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 on the region where the HLA loci are positioned, the major histocompatibility complex (MHC), which contains many other genes located very close to the HLA loci. The incidence of crossing-over between HLA genes during fertilization of the egg by sperm 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. As a result, 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 ten white persons who have ankylosing spondylitis are positive for HLA-B27. Because of this association, the disease and this HLA type are linked. Thus, a person with ankylosing spondylitis who is also HLA-B27 positive would have family with a much higher likelihood of developing ankylosing spondylitis 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 overproduction by the thyroid gland), associated with HLA-B8 and Dw3; and hereditary hemochromatosis (excess iron stores), associated with HLA-A3, B7, and B14.
HLA testing is performed using WBCs. If possible, this test should be postponed if the patient has recently undergone a transfusion, because any WBCs 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 are not evaluated in histocompatibility testing.
Because the HLA loci are closely linked, the HLA antigens are inherited as a group of six antigens is called a haplotype. The probability of siblings having identical haplotypes is one in four. Therefore, siblings provide the opportunity for the best matches. They can donate bone marrow, a kidney, and a section of their livers, but they 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 decreases quickly.
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.
A laboratory will perform HLA typing by either the serological (blood fluid) or DNA method. In either case, HLA typing of HLA-A, HLA-B, HLA-DR, and HLADQ antigens is performed for solid organ transplants. HLA typing of HLA-C antigens is also included when tissue typing is performed for bone marrow transplants.
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 antibody screening is the microcytotoxicity test. If an antibody against an HLA antigen is present, it will bind to the cells. The higher the number of different HLA antibodies, the lower the probability of finding a compatible match.
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. In the crossmatch, serum from the recipient is mixed with T-cells or B-cells from the donor. 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. 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 having blood taken, or hematoma (blood accumulating under the puncture site).
HLA typing either by serologic (blood fluid) or DNA methods is reported as the phenotype for each HLA loci tested. The antibody screen test is reported as the percentage of panel reactive antibodies (PRA). The percent PRA is the number of wells reactive with the patient's serum expressed in percent. The crossmatch is reported as compatible or incompatible.
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.
American Association of Blood Banks. Technical Manual. 13th ed., Bethesda, MD: American Association of Blood Banks, 1999.
Beutler, E., et al., eds. William's Hematology, 6th ed. New York: McGraw-Hill, Inc. 2001.
Henry, J. B. Clinical Diagnosis and Management by Laboratory Methods, 20th ed. New York: W. B. Saunders Company, 2001.
National Institutes of Health. [cited April 5, 2003] http://www.nlm.nih.gov/medlineplus/encyclopedia.html .
Mark A. Best