IMMUNOLOGY TEXTBOOK - Mercury Podillya 2013

IMMUNOLOGICAL RESEARCH METHODS

Molecular HLA Genotyping

Previously, when cellular Antigens of the HLA system served as the primary object of study, our understanding of the HLA Gene complex was largely shaped by indirect evidence. This included analyzing HLA antigen distribution in populations, conducting family studies, and observing reactions driven by HLA antigens. Today, advances in Molecular Genetics and immunochemistry allow us not only to perform high-resolution analyses of HLA antigens but also to examine the HLA genes themselves. Significant progress in this field accelerated with the discovery and Structure/175.html">Implementation of DNA segments essential for research, which ultimately unlocked vast opportunities for the rapid and precise analysis of HLA molecular polymorphism.

Molecular HLA genotyping using PCR detects various HLA alleles at the DNA level. This makes it possible to identify Class II HLA gene alleles that are either difficult or impossible to detect via serological typing alone. For instance, while serological techniques have identified 14 antigens in the DR locus, DNA typing reveals over 100 alleles. The HLA phenotype is recorded in numerical order of the HLA antigens according to standard nomenclature. For example: a subject's HLA-phenotype might be designated as A1, 2; B5, 12; DR2, 5; DQ3, 4.

The discovery of novel alleles has necessitated a revision of the HLA nomenclature, and four-digit designations are now standard (e.g., A0101 instead of A1). When multiple alleles are found that, under the former Classification, encoded different subtypes of a single antigen (for example, 12 such subtypes were identified in HLA-A2), they are designated as distinct alleles sharing the same initial digits—ranging from HLA0201 to HLA0212 or from HLAB2701 to HLAB2707, for instance.

If typing reveals only a single antigen for a given locus, this indicates that the individual is homozygous for that gene, meaning they inherited alleles of identical Specificity from both mother and father.

The aforementioned principles apply primarily to class I HLA, where only a single chain is polymorphic. In class II HLA, due to the potential polymorphism of both the beta and alpha genes, designated alleles are specified based on the chain that bears the variable DNA region determining specificity, such as DQA1-0501 and DQB1-0501.

Several methodologies exist for performing the Polymerase Chain Reaction (PCR).

SSP (Sequence-Specific Primer) - the most common and technically straightforward method, in which each allelic variant or group of alleles corresponds to a specific pair of primers. Amplification results are detected via gel Electrophoresis. The interpretation of HLA typing results comes down to a clear-cut yes/no regarding the presence or absence of PCR products.

SSO (Sequence-Specific Oligonucleotide) - involves the non-specific amplification of the target DNA region (locus) followed by specific Hybridization with labeled DNA probes.

SBT (Sequence-Based Typing) - a method used for HLA genotyping and de novo sequencing of target DNA (i.e., sequencing unknown DNA sequences). To perform SBT-based HLA genotyping, ConsenSys SBT reagent kits are used to read the sequences of amplified DNA obtained through genotyping with LABType SSO Reagents. ConsenSys SBT kits include sequencing primers and two wash buffers.

All HLA genotyping Methods involve three main stages: Stage I - DNA extraction, Stage II - amplification, and Stage III - detection of amplification results.

The analysis begins by drawing venous Blood and isolating leukocytes from the sample (Blood Cells that display Histocompatibility Antigens most abundantly on their surface). The HLA phenotype is determined using the polymerase chain reaction. PCR is a highly accurate method, with a reliability rate reaching 98%.

The in vitro DNA amplification reaction relies on the ability of nucleotide triphosphate molecules, in the presence of DNA polymerase and under optimal conditions (pH, Ionic strength, Temperature), to synthesize a complementary strand on a single-stranded DNA template. A fundamental requirement for this synthesis is the presence of an artificially synthesized oligonucleotide primer.

To enhance reaction accuracy and sensitivity, a pair of primers is typically used. The reaction is controlled by adjusting temperature conditions. At a specific temperature (depending on the Nucleotide Composition of the DNA), the double DNA helix denatures, or melts. Lowering the temperature reverses this process, allowing complementary strands to reassociate, or anneal. In the presence of excess primers in the solution, the probability of a primer annealing to the DNA template is significantly higher than that of the full complementary strand rejoining. Because the primer is shorter than the template, active extension (synthesis) of the complementary strand begins. The rate of elongation can reach several hundred (sometimes over a thousand) NUCLEOTIDES per second. Consequently, at the end of a cycle, The amount of DNA is doubled (specifically, the region flanked by the primers). The temperature is raised again—melting, annealing, synthesis—and the solution now contains 4 copies of the original template. The number of copies (amplicons) grows exponentially, and after 30–40 cycles, the solution contains anywhere from 10 million to 10 billion copies of the initial template. Furthermore, because a pair of primers is used, the amplicons will be of a strictly defined size. Such quantities of DNA can be visualized, for instance, through agarose gel electrophoresis with ethidium bromide staining.

SSP (Sequence-Specific Primers) technology is based on PCR utilizing allele-specific primers that capture the targeted HLA DNA segments, followed by detection via gel electrophoresis.

Stages of the technology:

1. DNA extraction from the test blood, Bone Marrow, or tissue sample (ranging from 15 min to 2 hours, depending on the method).

2. DNA amplification (45 min - 1.5 hours). The DNA is dispensed into the wells of a PCR plate, each containing primers of a specific sequence. The number of wells (PCR reactions) is thus determined by the number of loci being typed and the number of allelic variants within each locus. For example, low-resolution DR locus typing (screening HLA typing) typically involves determining about 24 specificities (24 wells of a PCR plate).

3. Electrophoresis (20-30 min). The amplicons are loaded into the wells of an agarose gel. In wells where the primer specificities matched the target DNA segments, a product band appears in the gel.

4. Result analysis. Using an interpretation table or software, technicians determine which HLA allele corresponds to the observed product band.

Advantages of SSP technology: it performs both low- and high-resolution typing and detects point allelic polymorphisms with an accuracy nearly comparable to sequencing.

The main drawback is low throughput. A 96-well thermal cycler can process only a single sample during low-resolution typing (the DR locus requires 24 tubes, the A locus 24 tubes, and the B locus 48 tubes). Consequently, typing a single sample for loci A, B, and DR—from DNA extraction to result interpretation—takes approximately 3 hours. Only 2-3 individuals can be typed per day.

SSP technology is recommended for HLA laboratories performing both low- and high-resolution typing with a moderate workload of up to 5-7 individuals per week, such as transplantation departments, bone marrow transplant clinics, and research institutes focusing on Genetic Disorders.

SSO (sequence-specific oligonucleotide) technology involves PCR amplification followed by detection via hybridization of amplicons with oligonucleotide probes.

Workflow stages:

1. Genomic DNA extraction, identical to the SSP technology Procedure.

2. Sample amplification takes place in a single PCR tube; rather than generating "specific" HLA amplicons, a total genomic DNA sample is produced (45 min – 1.5 h).

3. Dot-blot hybridization is The process of binding (conjugating) these now-specific DNA segments to oligonucleotide probes immobilized on specialized nylon strips. Unbound DNA fragments are washed away, and the conjugated fragments corresponding to HLA alleles are stained using peroxidase (1.5 – 2 h).

4. Result interpretation using specialized equipment.

Advantages of SSO technology: full automation capability; system sensitivity allows for the detection of point Mutations in HLA genes; specialized probe technology (enables the determination of polymorphisms of two or more linked genes on a single DNA strand); reduced repeat typing rates due to the absence of ambiguous results typical of conventional typing; post-hybridization stability (simultaneous Processing of multiple sample results); single-tube amplification for typing each locus (determining loci A, B, C, DRB1, DRB3, 4, 5, or DQB1 in one tube); high throughput; Minimization of genotyping errors through a single amplification process; detection time of less than 1 minute per sample; unlike the SSP method, the total assay time is approximately 2 hours.

The main limitation of SSO technology is that it cannot be used for high-resolution HLA typing. Its resolving power is restricted to low-to-moderate resolution. Therefore, if high-resolution typing is required, the laboratory must have an additional electrophoresis system.

SSO technology is recommended for: umbilical cord blood cryobanks; bone marrow donor registries; large transplant centers; oncology centers.

HLA genotyping by sequencing. SBT (sequence-based typing) technology represents The final stage of molecular analysis for a pre-extracted, amplified, and preliminarily tested DNA fragment. Sequencing involves determining The nucleotide sequence of a DNA fragment by generating a series of complementary DNA molecules that differ in length by a single base.

To perform SBT-based HLA typing, the laboratory must additionally be equipped with a Sequencer.

Workflow stages:

1. Hybridization of the test DNA fragment with a primer.

2. Enzymatic DNA Synthesis.

3. Denaturation of the resulting products using formamide (yielding unique oligonucleotide sequences that vary in length and incorporate the primer).

4. Four-lane Polyacrylamide gel electrophoresis (corresponding to the four types of nucleotides).

5. Analysis of results via autoradiography.

Advantages of SBT technology: the "gold standard" of HLA typing; comprehensive sequence information; high-resolution sequencing (up to the 4th digit); ability to detect novel alleles; adaptability to high-throughput laboratory workflows.

Abbott Molecular reagents enable a heterozygous sequencing strategy: high-resolution typing of HLA antigen alleles, including PCR amplification and fluorescent DNA Sequencing;

Features: 1 locus-specific PCR; typed loci: HLA-A, -B, -C, -DRB1, -DQB1, -DPB1; includes a primer set for "rough" sequencing; software performs analysis and suggests subsequent steps for confirmatory sequencing; HARPs (Hemizygous Ambiguity Resolution Primers) are used to resolve ambiguities.