What is HLA (human leukocyte antigen) typing and when is it indicated?

What is HLA Typing and When is it Indicated?

HLA (human leukocyte antigen) typing is the laboratory identification of specific alleles in the highly polymorphic HLA gene complex on chromosome 6p21.3, which encodes the major histocompatibility complex (MHC) molecules that present antigens to T cells and determine immune compatibility between individuals. 1

Definition and Biological Basis

• HLA molecules are expressed on almost all nucleated cells and function as the primary system for distinguishing self from non-self in immune surveillance 2, 3
• The HLA system includes three classical class I loci (HLA-A, -B, -Cw) and five class II loci (HLA-DR, -DQ, -DP, -DM, -DO), with over 12,000 established alleles and more in discovery 1, 3
• HLA genes are codominantly expressed, meaning each individual inherits one set from each parent, resulting in two HLA haplotypes per person 4
• The locus is highly polymorphic due to extensive pathogen-driven natural selection and exhibits unique long-range linkage disequilibrium across the entire MHC region 1

Primary Clinical Indications for HLA Typing

Hematopoietic Stem Cell Transplantation (HSCT)

• High-resolution HLA typing of HLA-A, -B, -C, and -DRB1 is mandatory for unrelated donor selection, as mismatches at these loci markedly increase mortality, acute graft-versus-host disease, and transplant-related complications 5
• Mismatches at HLA-A and HLA-DRB1 are less tolerated than mismatches at HLA-B or HLA-C, making precise typing at these loci critical for survival outcomes 5
• Each additional HLA mismatch reduces survival in a dose-dependent manner: approximately 10% lower survival per mismatch in low-risk disease patients and approximately 5% lower in high-risk disease patients 5
• For cord blood transplantation, high-resolution HLA-DRB1 typing is the minimum requirement, with HLA-C typing strongly recommended 5

Solid Organ Transplantation

• For kidney transplantation, high-resolution HLA genotyping is recommended (Grade 1A) to accurately assess anti-HLA antibody specificity and identify donor-specific antibodies that predict antibody-mediated rejection and graft loss 5
• HLA-DQ mismatches significantly increase the risk of graft loss in both living and deceased donor kidney transplantation (HR 1.12), with HLA-DQ donor-specific antibodies being particularly strong, persistent, and resistant to treatment 6
• Patients with 2 HLA-DQ mismatches in their first graft have >50% chance of becoming highly sensitized if they require retransplantation 6
• For liver transplantation, high-resolution HLA-DRB1 genotyping is recommended (Grade 2C) for post-transplant donor-specific antibody assessment 5
• For heart and lung transplantation, high-resolution HLA genotyping (Grade 1B) is recommended for accurate anti-HLA antibody specificity assessment 5
• HLA matching has had the greatest clinical impact in kidney and bone marrow transplantation, where efforts are made to match at the HLA-A, -B, and -DR loci 3

Platelet Transfusion Management

• HLA typing is essential for evaluating platelet refractory patients who may have developed HLA antibodies causing platelet destruction 2
• Extended antigen matching protocols require HLA typing to prevent alloimmunization in chronically transfused patients 7

Disease Association Studies

• The HLA locus has the largest number of disease associations of any locus genome-wide, with specific alleles conferring risk for autoimmune and inflammatory conditions 1
• HLA-DRB1 polymorphisms modulate rheumatoid arthritis risk by changing the capability to present autoantigens or increasing autoreactive T cells during thymic selection 1
• HLA-C*06:02 is strongly associated with psoriasis, likely due to increased CD8+ T-cell-mediated inflammatory reactions 1

Pharmacogenomics

• HLA typing is critical for identifying patients at risk for severe drug hypersensitivity reactions before initiating certain medications 2

Cancer Immunotherapy

• Precise HLA haplotyping is essential for accurate neoantigen prediction in personalized cancer vaccine design and immunotherapy response prediction 1
• Loss or attenuated expression of HLA genes represents a mechanism of resistance to immunotherapies 1

HLA Typing Methodologies

Clinical-Grade Methods (Gold Standard)

• Sequence-specific PCR amplification remains the gold standard for clinical HLA typing 1
• Next-generation sequencing platforms (Illumina MiSeq, PacBio RSII) combined with PCR amplification provide high-resolution sequencing of the HLA locus 1
• CAP/CLIA-regulated HLA typing assays are expected to be robust and remain the gold standard, particularly for class II typing 1

Computational Methods

• Computational HLA typing using whole genome sequencing (WGS), whole exome sequencing (WES), or RNA-seq data provides a cost-effective alternative to clinical typing 1
• HLA class I typing algorithms (OptiType, Polysolver, PHLAT) have reached up to 99% prediction accuracy when compared to clinical typing results 1
• Class II HLA typing algorithms remain less reliable and require additional development, with PHLAT, HLA-VBSeq, and seq2HLA performing best with WES and RNA-seq data 1
• Use of clinical-typing results remains advisable for class II typing due to poorer consistency between computational tools and clinical assays 1

Historical Methods

• Serological methods and cellular techniques (T-cell cloning) were historically used but have shortcomings including problems with antisera standardization and technical expertise requirements 8
• Oligonucleotide probing provides a more rapid, sensitive typing method that can identify HLA-D region alleles differing by only a single nucleotide 8

Technical Considerations for Accurate HLA Typing

Resolution Levels

• High-resolution typing characterizes alleles at the protein sequence level and is essential for transplantation to identify allele-level mismatches that produce adverse effects comparable to antigen-level mismatches 5
• HLA alleles are named using four fields of digits (e.g., HLA-A*02:101:01:02), representing allele group, specific HLA protein, synonymous coding changes, and non-coding differences 1
• The first two fields are generally sufficient for peptide-MHC binding affinity prediction 1

Sample Selection

• Normal (peripheral blood) samples have the advantage of representing germline HLA alleles present in both tumor cells and antigen-presenting cells, with typically higher quality genomic DNA than tumor samples 1
• For hematologic malignancies, skin samples are often used instead of peripheral blood to avoid contamination with malignant cells 1
• Tumor DNA may be complicated by aneuploidy affecting the HLA loci, which can interfere with HLA typing but is important to observe 1

Data Type Considerations

• RNA-seq data often exhibit highly variable coverage across HLA loci, potentially leading to variable typing accuracy 1
• WGS data provide comprehensive breadth of coverage but generally at the expense of overall depth 1
• Exome data coverage varies depending on the exome reagent's design and capture efficiency for HLA regions 1
• Sufficient read coverage for each HLA locus should be evaluated when assessing HLA-typing confidence 1

Quality Control

• A consensus approach involving multiple computational tools has become common strategy to increase confidence in HLA typing results 1
• HLA-typing tools generally do a poor job of reporting typing confidence, requiring careful evaluation 1

Common Pitfalls and How to Avoid Them

• Do not rely solely on antigen-level typing for HLA-DRB1; allele-level mismatches produce adverse effects comparable to antigen-level mismatches 5
• Do not assume that matching HLA-DRB1 alone is sufficient; comprehensive matching of HLA-A, -B, -C, and -DRB1 is required for optimal HSCT outcomes 5
• Do not focus only on HLA-A, -B, and -DR while neglecting HLA-DQ matching in kidney transplantation, as HLA-DQ mismatches are increasingly recognized as critical for long-term outcomes 6
• Do not fail to consider both chains of the HLA-DQ molecule (DQA1 and DQB1) when assessing compatibility, as both contribute to immunogenicity 6
• Red-cell genotyping should be used rather than serologic phenotyping whenever feasible, as genotyping yields higher accuracy and remains reliable even shortly after transfusion 7
• Do not delay urgent transplantation; balance the cost and turnaround time of high-resolution typing against clinical urgency 5
• HLA typing tools are not designed for de novo typing; they determine which known alleles best explain sequence reads, making identification of somatic HLA mutations a separate exercise from HLA typing 1
• Do not overlook ethnic differences in HLA frequency when interpreting matching probability, as HLA allele frequencies vary significantly between populations 1, 6