Single HLA Locus Reference Standard

Human leukocyte antigen (HLA) molecules are located on the short arm of chromosome 6 of humans. It is a polymorphic region of about 3.6M involved in immune response, containing more than 200 genes. Except for identical twins, it is almost impossible to find people with the same HLA. Everyone's HLA varies greatly, making it the most complex genetic system discovered so far. Currently, the IPD-IMGT/HLA database has included 38,008 alleles (version 3.55, 2024-01).

A single HLA locus genotyping standard refers to a biological sample (usually DNA or a cell line) whose genome sequence for a specific HLA locus (such as HLA-B) has been accurately identified and recognized by internationally recognized institutions using gold standard methods (such as sequencing).

Core Characteristics:

Locus-Specific: Responsible for typing results at a specific HLA locus.

Known and Unambiguous: The genotype is 100% certain and typically represents the most common or representative type at that locus.

High Accuracy: Typing results have been repeatedly validated across multiple platforms and laboratories and are considered the "truth" or "gold standard" for that type.

The class I region corresponds to genes such as HLA-A, -B, and -C, and encodes the alpha chain of class I molecules. The beta 2 microglobulin of class I molecules is encoded by genes in non-HLA regions. It also contains non-classical HLA-E, -F, and -G.

The class II region corresponds to genes such as HLA-DR, -DQ, and -DP, and encodes the alpha chain gene (A1) and the beta chain gene (B1). The alpha chain and the beta chain pair to form a functional HLA molecule. Most individuals carry an additional DRB gene (DRB3 or DRB4 or DRB5), which encodes a different beta chain that may pair with the alpha chain.

The class III region, in which genes encode complement system proteins and TNF family genes.

The typing test of HLA class I A, B, C loci and class II DR, DQ loci alleles plays a key role in the donor-recipient matching of hematopoietic stem cell transplantation, bone marrow transplantation, and other tissue and organ transplantation such as liver and kidney. The degree of HLA genotype matching between the donor and the recipient significantly affects the long-term survival rate of the transplant recipient. The higher the matching degree, the higher the long-term survival rate. HLA genotype mainly refers to the five loci HLA-A, B, C, DRB1, and DQB1. HLA genotyping can help establish a hematopoietic stem cell donor library and conduct HLA population genetic polymorphism analysis. In recent years, the China Bone Marrow Bank is actively advocating high-score entry, that is, 4-bit high-resolution typing tests on five gene loci such as HLA-A, B, C, DRB1, and DQB1, in order to fundamentally improve the efficiency of HLA matching.

At the same time, HLA-I class B loci such as B27 and B5801 genotyping also play an important role in personalized drug selection, such as auxiliary diagnosis of ankylosing spondylitis and guidance of gout medication. HLA-B*1502 has also been shown to be highly associated with Steven Johnson syndrome caused by taking the anti-epileptic drug carbamazepine in the Han population.

1.HLA typing covers a relatively complete set of 11 loci A B C DRB1/3/4/5 DPA1 DPB1 DQA1 DQB1, ultra-high resolution typing;

2.Whole genome DNA format, including exons and introns;

3.PCR-SBT or third-generation sequencing identification, accurate confirmation of typing sequence;

4.Human cell background, better simulation of clinical samples, and reproducibility;

5.Sustainable and stable supply, small batch-to-batch differences;

6.ISO90001 system certification, first-class product quality.

*Quality control and validation: This is the most crucial function. Laboratories test the standards every time they run an HLA typing experiment. If the test results of the standards differ from the known values, this indicates possible contamination, reagent failure, or operational error in the experimental process (from DNA extraction and PCR amplification to sequencing or hybridization). The results are unreliable and must be retested.

*Calibration and standardization: Results obtained by different laboratories, using different kit brands, and on different sequencing platforms may vary. Using a unified standard helps laboratories around the world calibrate their systems, ensuring that "A*02:01 reported by laboratory A" and "A*02:01 reported by laboratory B" refer to the exact same thing, thus achieving standardized and comparable results. This is crucial for international collaborations, such as bone marrow bank matching.

*New method/test kit evaluation: When developing a new HLA typing technology or commercial kit, performance validation using a known standard is essential to assess its accuracy, sensitivity, and specificity.

*Personnel training and assessment: used to train new technicians and assess their operation and result interpretation capabilities.

HLA typing itself is closely associated with a variety of diseases. Therefore, as a quality assurance standard, it is also indirectly related to the diagnosis, research, and treatment of these diseases.

1. Organ and hematopoietic stem cell transplantation:

This is the most directly relevant field. The key to transplant success lies in the HLA compatibility between the donor and recipient. Even the slightest typing error can lead to severe immune rejection or graft-versus-host disease (GVHD), which can be life-threatening. Therefore, the accuracy of HLA typing is the cornerstone of transplant success, and the role of standards is irreplaceable in this regard.

2. Autoimmune diseases:

Many autoimmune diseases are strongly associated with specific HLA types. For example:

* Ankylosing spondylitis (AS) is strongly associated with HLA-B*27.

* Rheumatoid arthritis (RA) is associated with types such as HLA-DRB1*04:01.

* Type 1 diabetes and celiac disease are also associated with specific HLA types. Accurate HLA typing aids in the diagnosis and risk prediction of these diseases.

3. Adverse Drug Reactions:

Certain drugs can cause severe or even fatal adverse reactions in carriers of specific HLA genotypes. For example:

*Severe skin reactions caused by allopurinol (an antigout drug) are associated with HLA-B*58:01.

*Carbamazepine (an anti-epileptic drug) is associated with HLA-B*15:02.

HLA genetic screening of patients before taking these medications can effectively prevent adverse reactions. Standards ensure the reliability of screening results.

4. Susceptibility/Resistance to Infectious Diseases:

Certain HLA types influence immune responses to viruses (such as HIV, HPV, HCV, and HBV), determining whether an individual is susceptible or able to effectively clear the virus.

1.Routine Quality Control in HLA Typing Laboratories:Any laboratory providing clinical HLA typing services (typically affiliated with a major hospital's blood transfusion department/transplant center or a third-party laboratory) must use standards as routine in-house quality control products.

2.External quality assessment (proficiency testing): Organized by national or international authoritative organizations (such as the China Bone Marrow Bank, ASHI, and EFI), blind samples (essentially standards) are distributed to laboratories to assess their typing capabilities. Only laboratories that pass the assessment are qualified to issue clinical reports.

3.Kits and Manufacturers: IVD (in vitro diagnostic) reagent manufacturers must use standards to verify the performance of their HLA typing kits during development and mass production.

4.Scientific Research: Standards are also used in research projects such as immunology, genetics, and disease association analysis to ensure the accuracy and reliability of research data.

5.Forensic Medicine and Paternity Testing: The HLA system is highly polymorphic and was once an important genetic marker. Although STR technology is now more commonly used, it is still used in certain special cases, and standards are also needed to ensure accuracy.