Molecular Typing Education

With worldwide increases in HLA testing and advanced genetic technology, the number of known HLA allele variants has increased exponentially; as of October 2025, there were more than 44,000 known unique variants of HLA.¹

HLA is grouped into Class I and Class II, based on molecular aspects and functions. “Classical” HLA are the genes most commonly tested in clinical and research settings. Class I includes the A, B, and C loci; Class II includes DR, DQ, and DP.

Given the variation and complexity of HLA genes, specific nomenclature evolved over time. Initially named in the order of discovery (e.g. A1, A2, etc.), the standard naming of a full HLA sequence includes the gene name numerical designation to classify each variant resulting in a unique amino acid sequence, for example, HLA-A*02:01. Additional fields indicate any mutations which do not change the final protein sequence (HLA-A*02:01:01), any mutations in non-coding regions (HLA-A*02:01:01:01), and any variations which affect gene expression (HLA-A*01:04:01:01N).²

This level of extensive genetic detail is valued for some applications but may not be necessary for all settings. Therefore, the idea of resolution levels in HLA typing becomes important to consider. 

Low resolution HLA typing generally refers to classification of the protein and is sometimes referred to as a “first field” or antigen-level typing.

High resolution may include at least a “second field” allele-level typing, or unambiguous definition of the allele variants created by non-synonymous variation (mutations which result in an amino acid sequence change).

High resolution also may indicate further sequence definition to the third or fourth field, including synonymous mutations which do not change the amino acid sequence, and mutations in non-coding regions, which may affect gene expression.

Intermediate typing indicates that there is some information about the specific allele variant, but not all possibilities have been ruled out.

In certain circumstances, high resolution typing may include intermediate level typing with some ambiguity, if there is a clear indication of a result with high population frequency, if any additional alleles not ruled out occur only rarely.

The level of resolution of the technology employed can depend on the type of transplant or other testing application and the urgency of obtaining the result, among other factors.

Typing of Human Leukocyte Antigen (HLA) genes is an important component of organ and bone marrow transplantation, various HLA-associated diseases and pharmacogenetics to screen for drug hypersensitivity.³

In the solid organ transplant setting, HLA typing is performed for recipients and potential donors. For deceased donor organ donation, the emphasis is on rapid results. A traditional paradigm has focused on matching HLA at low resolution only to avoid hyperacute rejection, but outcomes may be improved with higher resolution typing and a closer HLA match. Confirmatory testing may be performed after the initial HLA typing to confirm donor identity or for other reasons. 

In stem cell transplant testing, high resolution typing for recipients and donors is desired to ensure the most successful replacement of a patient’s bone marrow. Additionally, avoiding graft-versus-host disease and optimizing the possibility of graft-versus-leukemia effects may influence the choice of donors. Confirmatory HLA typing is a recommended practice to confirm accurate identification of donors and recipients due to the potential consequences of a sample mix-up. This confirmatory testing may be high, intermediate, or low resolution.

HLA typing may also be done for manufacturing and identity confirmation of red blood cell and platelet transfusion units, cord blood and stem cell units, and for clinical and investigational cellular therapies.

Certain diseases are associated with particular HLA types, including celiac disease, multiple sclerosis, diabetes, rheumatoid arthritis, psoriasis, and many others. HLA typing can assist in diagnosis or exclusion of related pathologies.

Additionally, specific HLA types are associated with hypersensitivity reactions to certain medicines, so pharmacogenetic testing for HLA may be done before prescription of these drugs.

Various research applications for immunology, autoimmune diseases, transplantation, fertility, and cellular and genetic therapy may include HLA testing as well.

CDC

Complement-dependent cytotoxicity (CDC), or microlymphocytoxicity, was an early technology based on anti-sera binding to HLA proteins expressed on cells. The addition of complement triggers cell killing, and dyes allow the estimation of living and dead cells. While fundamental to the history of HLA studies, DNA techniques have largely replaced CDC in many laboratories due to their increased sensitivity and specificity.2,5

PCR

Polymerase chain reaction, or PCR, is one of the key techniques in molecular biology, amplifying a single DNA molecule into millions of copies in a short time. PCR serves as an initial step in multiple HLA typing technologies.

Genomic DNA is combined with a mix including forward and reverse primers for the gene(s) of interest, a heat-stable polymerase, and deoxynucleotide triphosphates (dNTPs), which can be incorporated into a forming DNA strand. Additionally, the mix includes buffer salts and magnesium ion co-factors. This mixture undergoes repeated heating and cooling in a thermal cycler instrument to denature the DNA into single strands, anneal primers, and elongate the newly forming strands of DNA. Through the cycles, the number of target gene copies increases exponentially, referred to as gene amplification.⁴

Once the target gene has been amplified, the resulting amplicon is ready for further steps to determine the specific gene classification.

 SSP

The SSP, or sequence-specific primer, methodology is based on the principle that completely matched oligonucleotide primers are more efficiently used by the polymerase in amplifying a target sequence than a mismatched oligonucleotide primer.⁵ Primer pairs are designed to have perfect matches only with a single allele or group of alleles. Under defined and validated PCR conditions, perfectly matched primer pairs result in the amplification of target sequences (i.e., a positive result) while mismatched primer pairs do not result in amplification (i.e., a negative result). 

After the PCR process, the amplified DNA fragments are separated by agarose gel electrophoresis. Applying an electrical current causes migration of the DNA through the gel, forming discrete bands separated based on fragment size.  Fluorescent dye included in the gel mix incorporates into the DNA, and bands can be visualized by exposing the gel to ultraviolet light. Interpretation of PCR-SSP results is based on the presence or absence of specific amplified DNA fragments. The pattern of positive and negative bands can be compared to known reactivity patterns to determine the HLA type.

RT-PCR  

Real-time PCR refers to technology that allows the identification and/or quantification of genes during or immediately after completion of the PCR cycle.⁶ For HLA typing applications, this utilizes similar SSP chemistry including sequence specific primers to amplify the targeted series of genes.

One common detection method utilizes fluorescent dye that infiltrates into double-stranded DNA, resulting in high fluorescence. After PCR, the DNA is heated a final time to dissociate fully, leading to a sharp decrease in fluorescence as the DNA becomes single stranded. 

Raw fluorescence, first derivative (-dF), and temperature data are utilized to produce “melt-curves” for each well. The specific temperature of dissociation will vary based on the length of the fragment, the guanine-cytosine content, and other amplicon-specific factors. Similarly to standard SSP, RT-PCR SSP can utilize melt curves to determine positive or negative amplification; software analysis may be used to interpret the pattern of positive and negative reactions to determine the HLA type. 

In addition to melt-curve technology, various types of genetic probes may be utilized in RT-PCR applications. These probes may bind with a fluorescence quencher to a specific segment of DNA, releasing the fluorophore as the probe is displaced by the polymerase amplifying the target gene. This fluorescence is then interpreted as positive for the gene of interest. Probes are available with various conformations and chemistries and may be used in combination with melt curves to expand allele coverage.

SSO

The next technology is often referred to as SSO or SSOP, sequence-specific oligonucleotide probes. This utilizes group-specific primers for PCR to amplify an entire region of interest. Specific identification of the genes present occurs by hybridizing unique probes to the amplicon.⁷

Early versions of SSO utilized gel electrophoresis and transfer of bands to a membrane, with subsequent application of probes to the DNA. This was referred to as “forward” SSO.

Reverse SSO utilizes a solid phase to immobilize the probes themselves, and amplified DNA is hybridized to the matching probes. This testing may be multiplexed to combine many assays in one well, allowing labs to batch-test large volumes of samples efficiently. 

First, target DNA is PCR-amplified using a group-specific primer. The PCR product is biotinylated, which allows it to be detected using R-Phycoerythrin conjugated Streptavidin (SAPE). The PCR product is denatured and allowed to re-hybridize to complementary DNA probes conjugated to fluorescently coded microspheres. A flow analyzer identifies the fluorescent signatures of the microspheres and the intensity of PE (phycoerythrin) on each microsphere. The assignment of the HLA typing is based on the reaction pattern compared to patterns associated with published HLA gene sequences.

SSO testing achieves an intermediate-level resolution, which may range from low to high depending on the allele.

SBT

Sequence Based Typing, SBT, or Sanger sequencing, was invented in 1977 and has long been used in HLA laboratories. It relies on PCR using dideoxynucleotide triphosphates (ddNTPs) tagged with radioactive or fluorescent markers, in addition to standard nucleotides.^2,7 During PCR, the DNA strands will end when one of the modified nucleotides is incorporated, referred to as chain termination. This results in various lengths of amplified DNA fragments ending with a marked nucleotide. 

Capillary electrophoresis is then used to separate the fragments by size, with the marker signatures indicating which nucleotide occurs at end of each fragment. From these readings, the sequence can be constructed with high accuracy. SBT technology can indicate variant positions but cannot define which alleles occur on which chromosome (referred to as phasing). SBT may result in a clear high-resolution result or may require additional rounds of testing for sequence clarification.

NGS

NGS, or Next-Generation Sequencing, is also referred to as Massively Parallel Sequencing (MPS). One feature of NGS is the capacity to pool samples from multiple individuals. Each DNA sample can be tagged with a “barcode” or unique identifier which can then be analyzed on an individual basis.⁸ This allows higher throughput and lower sequencing costs.

Various sequencing protocols may proceed through the key steps in different orders but are consistent in the main aspects.⁹ NGS may start with a long-range PCR step or begin directly from genomic DNA. The DNA is fragmented, and the desired fragment sizes are selected. Sequencing platform adapters are ligated and unique identifiers are added to each DNA sample. The presence of these unique tags allows samples to be pooled for sequencing. 

A library preparation PCR step may be used to increase the yield of the sample. The DNA concentrations will be quantified and adjusted if needed, and the samples pooled and prepared for the sequencer.

Various sequencing chemistries exist; these may rely on precise pH change to indicate addition of particular nucleotides or rely on clonal amplification with incorporation of fluorescent dyes to indicate addition of specific nucleotides. All NGS platforms perform sequencing of millions of small fragments of DNA in parallel which are referred to as reads. After sequencing, software analysis can demultiplex the data, that is, correlate which reads correspond to which DNA samples. The millions of reads can also be aligned with each other and/or aligned with a known reference sequence to interpret the specific typing results.

NGS has increased the efficiency of HLA typing by sequencing; however, like any technology, it does have limitations.  NGS typically takes more than 24 hours to complete and can be subject to sequencing errors.

Hybrid Capture

NGS by hybrid capture technology utilizes probe binding to enrich the targeted areas of the genome.¹⁰ Utilizing these probes allows multiple gene systems to be tested at once, for example HLA and ABO. 

Genomic DNA is fragmented and tagged with necessary adaptors for sample identification and further assay steps. A short PCR is performed to increase sample yield, and biotinylated probes are added to hybridize specific regions of interest. Streptavidin-conjugated magnetic beads are added, which bind to the regions of interest which can then be isolated using a magnet while the off-target DNA is washed away. An additional short PCR step increases the final yield of the library, and the samples can then be sequenced similarly to standard NGS.

Hybrid Capture may be less susceptible to PCR amplification bias since in this methodology the genes of interest are not amplified directly. This methodology may also help reduce hands-on time and  help provide a shorter turn-around time.

Nanopore Sequencing

Nanopore sequencing, sometimes called third-generation sequencing, utilizes biological nanopores in a membrane exposed to electrical current.¹¹ As a strand of DNA moves through the pore, specific changes in the ionic current can be translated to the nucleotide sequence. Real-time data analysis is possible which may support faster result reporting. 

Common library preparation procedures include an initial PCR step, ligation of adaptors and unique identifiers, optional pooling, and loading on the flow cell. A motor protein feeds single stranded DNA through the pore, with the resulting current changes interpreted by machine-learning models.

Nanopore sequencing reagents may be subject to higher rates of sequencing error,¹² but also may enable high-resolution typing with turnaround times suitable for deceased donor applications.

Comparison of molecular technologies

The choice of assay technique is influenced by the type of sample, the required turnaround time for results, and the desired resolution. Most laboratories employ a combination of techniques and tests to deliver comprehensive HLA typing services.