HLA Testing

Medical Experts

Contributor

Lazar-Molnar

Eszter Lzr-Molnr, PhD, D(ABMLI), F(ACHI)

Associate Professor of Pathology (Clinical), University of Utah

Director, Histocompatibility and Immunogenetics Laboratory, University of Utah Health

Medical Director, Immunology, ARUP Laboratories

The human leukocyte antigen (HLA) complex is composed of cell surface proteins that regulate the immune system. All individuals have the same loci that code for HLA, but there are many different alleles that could be expressed at each locus (more than 34,000 in total have been assigned).^,

Laboratory testing for HLA is commonly used to determine transplant compatibility because different individuals may have very different HLA alleles, which may cause alloreactivity between recipients and donors.^, In addition to its role in transplantation, laboratory testing for HLA may be used in pharmacogenetics to determine the risk of an adverse reaction to specific drugs, to evaluate disease associations, in vaccine trials, or for platelet transfusion support. Most HLA testing is currently performed using molecular genetic methods (e.g., probe-based hybridization methods or massively parallel sequencing).

Quick Answers for Clinicians

What are the differences between low- and high-resolution HLA typing?

“Resolution” in human leukocyte antigen (HLA) typing refers to how much information is yielded by molecular typing methods (e.g., next generation sequencing). Low-resolution (“two digit”) typing provides information about the locus and allele group, which supplies the information for the first field in the standard nomenclature.^, In some cases, this information may correspond to a serologic equivalent, which is also considered low-resolution typing. Low-resolution typing may be used to rapidly type donors or recipients for solid organ transplantation. High-resolution (“allele level” or “two field”) typing distinguishes between specific HLA proteins, which provides the information for the second field in the standard nomenclature.^, Next generation sequencing and single molecule real-time sequencing technologies are able to provide information for the third and fourth fields in the standard nomenclature.

What is the panel-reactive antibody percentage, and how does it relate to HLA testing?

The panel-reactive antibody (PRA) percentage is an estimate of the percentage of the donor population that a specific organ recipient might be incompatible with due to preformed human leukocyte antigen (HLA) antibodies.^, It is calculated based on the recipient’s unacceptable antigens, which are determined by laboratory testing (e.g., the solid phase single antigen antibody assay). The calculation is complex and also takes into account HLA frequencies in the national donor pool.^, The Organ Procurement and Transplantation Network (OPTN) provides an online calculator that can be used to assist clinicians in calculating the PRA percentage.

What are donor-specific HLA antibodies, and how are they relevant to HLA testing?

Donor-specific human leukocyte antigen (HLA) antibodies are recipient antibodies directed against HLA antigens that are expressed by donated organs or tissues. The recipient may have developed these antibodies due to existing sensitization from a previous blood transfusion, pregnancy, or transplant, in which case they are referred to as preformed donor-specific antibodies (DSAs). These antibodies can also develop posttransplantation, in which case they may be referred to as de novo DSAs. DSAs have been associated with organ dysfunction and rejection; preexisting antibodies can cause hyperacute or acute rejection, whereas the development of these antibodies after transplantation suggests the onset of acute or chronic rejection. Laboratory testing for HLA antibodies and crossmatch testing (antibody identification testing not performed at ARUP Laboratories) before transplantation may help eliminate or minimize the risk of organ rejection by improving donor/recipient matching (i.e., ensuring that a transplant recipient does not receive an organ against which they have HLA antibodies). Historically, the cell-based complement-dependent cytotoxicity assay was used to test for HLA antibodies, but current guidelines recommend the use of solid phase assays, which are more sensitive and specific.

Indications for HLA Typing

HLA typing may be appropriate for:

Recipients and their donors who are being considered for hematopoietic stem cell or solid organ transplants
Patients who are being considered for treatment with specific drugs that have side effects associated with specific HLA alleles
Diagnostic support for diseases associated with HLA
Immunization/vaccination trials
Platelet transfusion support for sensitized patients

Laboratory Testing

HLA testing recommendations depend on the indications and needs of the patient. Laboratory testing includes HLA typing, antibody screening, and antibody identification (testing not performed at ARUP Laboratories).

Transplantation Testing

HLA testing is used in both solid organ and hematopoietic stem cell transplantation (HSCT) to determine compatibility. Greater numbers of matched HLA loci between donor and recipient have been correlated with better outcomes.^,^,

Solid Organ Transplantation

Within the United States, HLA testing for solid organ transplantation is regulated by the Organ Procurement and Transplantation Network (OPTN) policies. Refer to the OPTN policies for specific information about requirements for HLA antigens and equivalencies.

HLA Typing for Solid Organ Transplantation

Molecular HLA typing may be performed using a variety of genetic techniques, including polymerase chain reaction (PCR) followed by sequence-specific oligonucleotide probe hybridization (SSOP), or massively parallel sequencing (next generation sequencing). Requirements for molecular HLA typing and reporting, including the timing of testing and reporting, vary by donor or recipient status and the organ(s) to be transplanted:

For deceased donors, molecular typing of class I and II HLA loci (HLA A, B, Bw4, Bw6, C, DR, DR51, DR52, DR53, DQA1, DQB1, and DPB1) must be performed and reported.
For kidney, kidney-pancreas, pancreas, or pancreas islet candidates, molecular typing of a smaller subset of both class I and class II HLA loci (HLA A, B, Bw4, Bw6, and DR) must be performed and reported before registering on the wait list.

HLA Antibody Screening for Solid Organ Transplantation

HLA antibody screening using at least one solid phase immunoassay with purified (single) HLA antigens must be performed when requested by authorized individuals involved in the solid organ transplantation process.

Crossmatch Testing for Solid Organ Transplantation

A final crossmatch test and report are required before any transplant that involves a kidney. Any physical crossmatch must identify all class I and class II antibodies using donor B lymphocytes and must identify all class I anti-HLA antibodies using donor T lymphocytes. The technique used for the crossmatch must be sensitive and distinguish between B- and T-lymphocyte reactions. Crossmatching is performed in specialized histocompatibility laboratories; methods include complement-dependent cytotoxicity (CDC) or flow cytometry-based assays.

Hematopoietic Stem Cell Transplantation

HLA Typing for Stem Cell Transplantation

High-resolution molecular typing of class I HLA loci (HLA A, B, C) and some class II HLA loci (HLA DRB1 and DPB1) is recommended for all individuals receiving an allogeneic HSCT and their adult donors. Typing of other class II HLA loci may also be useful. If umbilical cord blood is used as the source of hematopoietic stem cells, high-resolution molecular typing of all class I HLA loci (HLA A, B, C) and HLA-DRB1 is recommended. Typing of DQB1 and DPB1 may also be useful.21851

Pharmacogenetics

HLA testing for specific alleles associated with sensitivities to severe, sometimes life-threatening side effects of certain drugs, including abacavir, allopurinol, and carbamazepine, is used to guide treatment with these drugs. See the following associated content for more information:

HLA-B*57:01 testing for abacavir sensitivity:
- HLA-B*57:01 for Abacavir Sensitivity Test Fact Sheet
- ARUP Consult Human Immunodeficiency Virus - HIV topic
- ARUP Consult Germline Pharmacogenetics - PGx topic
HLA-B*58:01 testing for allopurinol hypersensitivity:
- HLA-B*58:01 Genotyping, Allopurinol Hypersensitivity Test Fact Sheet
- ARUP Consult Gout topic
- ARUP Consult Germline Pharmacogenetics - PGx topic
HLA-B*15:02 testing for carbamazepine hypersensitivity:
- HLA-B*15:02 Genotyping, Carbamazepine Hypersensitivity Test Fact Sheet
- ARUP Consult Germline Pharmacogenetics - PGx topic

HLA genotyping may also be useful in immunization/vaccination trials. Testing for specific class I and/or class II antigens or alleles via genotyping may be appropriate to determine or rule out patient eligibility. The particular antigen or allele(s) to be tested should be specified at the time testing is ordered.

Disease Associations

Specific HLA antigens have been associated with a number of diseases. For a discussion of HLA testing in some of these diseases, please refer to the following related content:

ARUP Consult Ankylosing Spondylitis Genotyping (HLA-B27) Test Fact Sheet
ARUP Consult Celiac Disease topic
ARUP Consult Dermatitis Herpetiformis topic

HLA-DQB1*06:02 Genotyping for Narcolepsy

Narcolepsy is a chronic neurologic sleep disorder that manifests with excessive daytime sleepiness and difficulty in maintaining wakefulness. Narcolepsy is a rare disease with an overall prevalence of 0.02-0.06% in the U.S. and Europe.

There are two forms of narcolepsy. Type 1 is associated with cataplexy (sudden loss of muscle tone induced by strong emotions) and characterized by severely reduced or undetectable hypocretin 1 (orexin A) levels in the cerebrospinal fluid (CSF), a finding that has diagnostic value. Nearly all patients with type 1 narcolepsy carry the HLA-DQB1*06:02 allele; however, this allele is quite common in the general population (depending on ethnicity) and by itself is not causative of the disease. Homozygosity for the DQB1*06:02 allele, compared with heterozygosity, doubles the risk for narcolepsy. Narcolepsy type 2, which is not associated with cataplexy or decreased orexin levels, is also associated with the DQB1*06:02 allele, but only 30-50% of patients with narcolepsy type 2 are positive for the allele.

Due to its low positive predictive value, testing for HLA-DQB1*06:02 is not recommended for screening or initial diagnosis in cases of suspected narcolepsy, but it may be useful for ruling out narcolepsy type 1 in individuals with narcolepsy when clinical history and sleep studies are inconclusive.

HLA Testing for Immunization/Vaccination Trials

Platelet Transfusion Support

Because HLA antibodies are a potential cause of platelet refractoriness, HLA testing is useful in platelet transfusion support. Platelets do not express class II HLA; therefore, class I HLA typing is sufficient. HLA antibody screening is recommended, and if the screen is positive, antibody identification (testing not performed at ARUP Laboratories) may be useful to facilitate platelet selection. HLA testing may be included as a component of some panel tests for platelet refractoriness.