IMMUNOLOGY TEXTBOOK - Mercury Podillia 2013

IMMUNOLOGICAL RESEARCH METHODS

Clinical Significance of HLA Typing and Genotyping

When selecting a donor for transplantation, the Sequential Stages of genotyping utilizing various technologies should be carried out as follows:

1) high-to-moderate-throughput genotyping of a large sample volume using rSSO technology to narrow down the pool of potential Donors;

2) SSP technology genotyping to resolve ambiguities arising from the SSO method;

3) high-resolution recipient and donor genotyping using LABType SSO HD and LABType SSO HLA Exon 4-7 Supplement kits to achieve allele-level resolution (comparable to the resolution yielded by sequencing);

4) determination of donor and recipient seropositivity levels.

Monitoring post-transplantation antibody levels and the Specificity profile of anti-HLA Antibodies.

It is essential to apply all available HLA typing technologies. SSO and SSP technologies do not compete with one another, but rather act as complementary Methods.

Indications for ordering the assay:

✵ HLA Class II Gene typing is used to diagnose specific forms of Infertility and recurrent Pregnancy loss, which may stem from a high degree of HLA class II gene Homology within a couple despite complete fertility in both partners.

✵ HLA class II gene typing is a mandatory investigation for donor Selection in organ transplantation.

✵ Certain allelic variants of HLA class II genes are associated with an elevated risk of various conditions, including type 1 Diabetes Mellitus, rheumatic diseases, autoimmune thyroiditis, and Susceptibility to infectious diseases, among others.

Indications for HLA typing and genotyping during pregnancy. Each gene can have thousands of variant forms known as alleles. Diverse combinations of these alleles account for the extensive multivariance of gene combinations. It is precisely the matching of alleles that indicates the genetic compatibility or incompatibility of two individuals. A child inherits one set of genes from the mother and one from the father.

Spousal mismatch regarding HLA Antigens and the distinction between the embryo and the maternal Organism are crucial factors required for the maintenance and successful gestation of a pregnancy. If the couple exhibits compatibility concerning HLA class II antigens, antibodies against these antigens fail to develop, leaving the fetus unprotected from maternal natural killer Cells. The presence of more than 3 shared HLA genes between husband and wife is established as one of the causes of recurrent miscarriages.

A resemblance between partners in terms of Histocompatibility Antigens results in the embryo bearing a "similarity" to the maternal organism. This causes insufficient antigenic stimulation of the woman's immune system, failing to trigger the reactions necessary for conception and pregnancy maintenance. A high number of matching Major Histocompatibility Complex (HLA) antigens between spouses leads the mother's body to fail in recognizing the embryo as a fetus; instead, it is perceived as an altered self-Cell, against which destructive mechanisms are deployed. Consequently, the maternal immune system suppresses embryonic implantation. In one out of every three cases, infertility or recurrent pregnancy loss is driven by the genetic traits of the couple.

Determining the couple's genotype for HLA class II antigens is of paramount importance for diagnosing immune-mediated forms of pregnancy loss. Phenotyping for HLA-DR and HLA-DQ antigens is highly recommended, particularly for HLA-DR, as these antigens are expressed on cells in vastly greater quantities and possess the highest immunogenic potency. Immunological incompatibility between partners can be established when there are three matches among the allelic Variants of the DRB1, DQA1, and DQB1 genes in the tested couple.

The Etiology of early pregnancy loss in the absence of karyotypic anomalies is most frequently immunological in nature. An increasing body of research points toward a close interrelation and mutual regulation between the endocrine and immune systems, which manifests in the endometrium during the Cytology/cytology/16.html">Early stages of implantation. Maternal sensitization to paternal fetal HLA antigens, spousal HLA resemblance, and the presence of specific antigens within the parental HLA phenotype lead to spontaneous miscarriages, severe gestational toxicosis (Preeclampsia), congenital fetal malformations, and reduced offspring resistance to adverse environmental factors.

To overcome the challenges associated with spousal HLA similarity, several therapeutic approaches are available: maternal immunization with a concentrated culture of paternal lymphocytes, which increases the antigenic load by 10,000-fold compared to baseline; and human immunoglobulin immunotherapy, which exerts immunomodulatory and immunostimulatory pharmacological effects.

Significance of HLA typing and genotyping in transplantology. Nowadays, donor and recipient matching based on HLA antigens using DNA typing has become a routine procedure in the immunological laboratories of transplant centers. Recent data indicate that the 1-year survival rate of cadaveric renal allografts reaches 90% when the donor-recipient pair is selected using DNA typing. In contrast, when the pair is matched via serological typing, this figure drops to 68%.

The association between HLA system antigens and disease susceptibility. Investigating the links between the HLA system and various pathologies holds substantial significance for Epidemiology, nosology, Diagnostics, prognosis, and Treatment (Table 18).

Table 18. HLA-associated diseases

Significance of HLA typing and genotyping in diabetes mellitus. Type 1 diabetes mellitus is a condition with a hereditary predisposition dictated by an unfavorable combination of normal genes, the majority of which govern various links in autoimmune processes.

Genes conferring susceptibility to type 1 diabetes are located across various Chromosomes, with more than 15 such genetic systems currently identified. Among them, the class II GENES OF THE HLA region, situated on the short arm of chromosome 6, have been the most extensively studied.

The risk of developing diabetes in siblings can also be estimated based on their degree of HLA identity with the affected individual: if they are fully identical, the risk is highest at approximately 18%; in semi-identical siblings, the risk is 3%; and in completely non-identical siblings, it is less than 1%.

Analyzing genetic markers helps stratify groups by their risk of developing diabetes, which dictates different approaches to early preclinical Diagnosis. Furthermore, genetic marker profiling significantly enhances the prognostic value of immunological and hormonal tests. Table 19 presents the HLA class II gene alleles associated with the risk of developing type 1 diabetes.

Table 19. HLA class II gene alleles associated with the risk of type 1 diabetes

Determining the HLA phenotype via DNA typing is equally crucial for resolving cases of disputed paternity. In this highly sensitive and delicate matter, The Use of PCR likewise increases the precision of the analysis.

Finally, PCR has valuable Structure/179.html">Practical Applications in identifying microbial DNA for the diagnosis of infectious pathologies. Recent experience clearly demonstrates the immense potential of PCR in this field.

Detection of specific pathogen DNA segments using Polymerase Chain Reaction with electrophoretic detection. Principle of the method. The method is based on identifying a specific microbial DNA fragment through the accumulation (Amplification) of copies of that target (target DNA) during the synthesis of new DNA strands.

Polymerase chain reaction consists of repeatedly cycled steps of target DNA Synthesis in the presence of a thermostable DNA polymerase, deoxynucleoside triphosphates (dNTPs), an appropriate buffer solution, and oligonucleotide primers that define the BOUNDARIES OF THE amplified target DNA region.

Each cycle comprises three stages operating at different Temperature regimes. In The First stage, DNA strands are separated at 94 °C; next, primers anneal to homologous sequences on the target DNA at 57-62 °C; and finally, new DNA strands are synthesized via primer extension in the 5'-3' direction at 72 °C.

Every cycle doubles the number of copies of the amplified region, allowing 35 cycles to generate a sufficient quantity of the DNA fragment—flanked by the chosen primer pair—for detection via gel Electrophoresis.

Performing a PCR assay involves 3 laboratory stages:

1) clinical sample Processing (DNA extraction);

2) PCR setup (amplification);

3) detection of amplification products (in this protocol, electrophoretic Separation of products in an agarose gel).

Required Equipment and Consumables

Stage 1 - DNA extraction from biological samples: Class II laminar flow cabinet; solid-block thermostat for 1.5 mL Eppendorf-type tubes maintaining temperatures up to 99 °C; high-speed centrifuge for 1.5 mL tubes (8,000–12,000 rpm); microcentrifuge-vortex (1,500–3,000 rpm); variable-volume pipette dispensers (5–50, 20–200, 100–1000 µL); vacuum aspirator (pump) with a trap flask; rack for storing 1.5 mL tubes; workstation rack for 1.5 mL tubes; disposable pipette tips up to 200 µL and up to 1000 µL; refrigerator with a freezer compartment for storing clinical specimens; disposable gloves; and a DNA/RNA Extraction Kit for Serum and Plasma (universal for all pathogens present in the analyzed sample).

Stage 2 - PCR amplification: PCR workstation with a UV lamp; programmable thermal cycler (amplifier); microcentrifuge-vortex (1,500–3,000 rpm, hereinafter vortex); variable-volume pipette (5–50 µL) for handling biological samples; variable-volume pipettes (0.5–10, 5–50, 20–200, 100–1000 µL) for preparing the working reagent mixture; disposable 0.5 mL (or 0.2 mL) polypropylene microtubes for amplification; disposable tips up to 200 µL and up to 1000 µL for preparing the working reagent mixture; disposable filtered tips (with aerosol barriers) up to 100 or 200 µL for biological samples; workstation rack for 0.5 mL (or 0.2 mL) tubes; tip racks for 200 µL tips; disposable gloves; waste container for used tips; refrigerator with a freezer compartment for storing stock Reagents; and a PCR reagent kit (specific to each pathogen).

Stage 3 - Amplification product detection: horizontal electrophoresis chamber; DC power supply with a voltage of at least 150 V; UV transilluminator; microwave oven for melting agarose; technical balance for weighing agarose; Water distiller; video documentation system for gel electropherograms equipped with a light-shielding cabinet or hood, connected to a personal computer; variable-volume pipette (5–50 µL) for loading samples onto the gel; variable-volume pipette (100–1000 µL); disposable tips up to 200 µL for sample loading; disposable tips up to 1000 µL; workstation rack for 0.5 mL (or 0.2 mL) tubes; agarose, ethidium bromide solution, 50x TAE buffer for gel preparation and electrophoresis (or a ready-to-use kit for electrophoretic detection); and a large-volume plastic container for deactivating buffer and gels.

Composition of PCR Reagent Kits

The reagent kit comprises three sets:

1. Sample preparation set (DNA extraction). DNA/RNA Extraction Kit for Serum or Plasma (Cat. No. 020201) (for 100 samples): 1.1. Denaturing solution, 45 mL; 1.2. Isopropyl alcohol, 30 mL; 1.3. Wash solution, 100 mL; 1.4. Carrier solution (tRNA), 300 µL; 1.5. Deionized water, 5 mL; 1.6. Chloroform, 12 mL.

2. PCR amplification set. These sets are available in various formats depending on the reaction volume and their level of pre-readiness.

3. Amplification product detection set. Ready-to-use kits and individual reagents.

Ready-to-use kits:

Kit No. 1 (Cat. No. 030106) (for 100-150 samples): agarose - 2x2 g, 50x TAE buffer - 25 ml, ethidium bromide solution - 30 µl.

Kit No. 2 (Cat. No. 030107) (for 120 samples): 2% agarose gel (40 wells) - 3 pcs, 50x TAE buffer - 25 ml.

Individual reagents: agarose (100 g/pack; 2 g/pack) (Cat. No. 030101); ethidium bromide solution (1 ml/pack) (Cat. No. 030104); 50x TAE buffer (200 ml/pack; 120 ml/pack) (Cat. No. 030102); 2% agarose gel for electrophoresis (40 wells) (1 pc/pack; 5 pcs/pack) (Cat. No. 030108).

Procedure

1. Selection of test material. The choice of clinical material for the study is determined by the most likely site of localization of the pathogen.

2. Collection, transport, and storage of samples. To obtain serum, venous Blood is collected in a dry disposable plastic tube and allowed to clot (30 min at room temperature until a complete clot is formed). Centrifuge the tube for 10 min at 3000 rpm at room temperature; transfer the resulting serum into a clean, dry 1.5 ml Eppendorf-type polypropylene tube using a filter tip.

To obtain plasma, venous blood is collected in a disposable plastic tube containing an anticoagulant solution (0.05 M EDTA solution or 4% sodium citrate solution at a ratio of 500 µl of blood to 50 µl of anticoagulant). Heparin is not recommended for use. Centrifuge the tube for 20 minutes at 3000 rpm at room temperature, transfer the resulting plasma (upper phase) using an individual filter tip into a dry disposable plastic tube, and use it for DNA extraction.

To obtain blood cell mass, collect 500 µl of venous blood into a disposable plastic tube with 50 µl of an anticoagulant solution (0.05 M EDTA solution or 4% sodium citrate solution). Heparin is not recommended for use. Centrifuge the tube for 5 min at 3000 rpm at room temperature. Discard the plasma (upper phase) using an individual tip. The resulting blood cell mass is used for DNA extraction.

Whole native blood must not be stored!

Uncooled samples of serum, plasma, and cell mass can be used for DNA extraction within 2 hours. Storage at +4...+8 °C is allowed for no more than 24 hours, and at -18...-20 °C for no more than 2 weeks.

Transport of processed samples to the laboratory must be carried out in an ice-filled thermos or thermal container within 12 hours.

3. DNA extraction from biological samples:

3.1. Add 3 µl of carrier and 450 µl of denaturing solution to clean 1.5 ml Eppendorf-type polypropylene tubes.

The denaturing solution contains phenol. Avoid contact with Skin and mucous membranes.

Number the tubes and place them accordingly in a rack.

3.2. Add 50 µl of the test serum or plasma (or 100 µl of blood cell mass) to the corresponding tubes using filter tips. Close the tubes tightly.

3.3. Mix thoroughly on a vortex mixer for 10 sec., and then incubate at room temperature for 10 minutes.

3.4. Centrifuge for 15 seconds at 12,000 rpm. High-speed centrifugation must be performed in a centrifuge with a lid to ensure tight pressure on the tube caps. After each centrifugation step, it is advisable to wipe the inner surface of the compression lid and the rotor surface with a disinfectant solution.

3.5. Add 100 µl of chloroform, close the tubes tightly, and vortex for 5 seconds.

3.6. Centrifuge for 5 minutes at 12,000 rpm.

3.7. Transfer up to 300 µl of the upper aqueous phase into a clean 1.5 ml Eppendorf-type polypropylene tube containing 300 µl of isopropyl alcohol, using filter tips. Mix on a vortex for 5 sec.

3.8. Centrifuge for 12 minutes at 12,000 rpm.

This procedure results in The formation of a translucent, loose pellet.

3.9. Remove the supernatant with a vacuum aspirator into a trap flask using disposable tips, leaving about 20 µl of liquid at the bottom of the tube. Perform this procedure with extreme care, gradually removing the supernatant only from the upper layer of liquid without disturbing the loose pellet. It is recommended to orient the tubes in the centrifuge rotor to mark the Location OF THE pellet.

3.10. Add 1 ml of washing solution to the tube containing the pellet. Close the tubes tightly, mix on a vortex, and centrifuge for 10 minutes at 12,000 rpm.

3.11. Remove the supernatant as completely as possible using a vacuum aspirator into a trap flask without disturbing the pellet. Air-dry the pellet for 20–30 minutes at room temperature, leaving the tubes open.

3.12. Add 50 µL of deionized water, close the tubes, incubate for 10 minutes at room temperature, and then mix by vortexing.

The purified DNA solution can be stored at -18...-20 °C for up to two weeks.

4. PCR Assay (Amplification)

4.1. Prepare and label 0.5 mL (or 0.2 mL) amplification tubes corresponding to the number of test samples to be analyzed for the target pathogen DNA. Prepare and label tubes for positive ("C+") and negative ("C-") controls. When using the HERPOL 1+2 kit, the reaction requires two positive controls.

Note that for OneStep reagent kits (ready-to-use master mixes pre-aliquoted into individual amplification tubes), labels should be applied to the tubes containing the master mix for the test samples and the negative control. Tubes containing the positive control are completely ready for placement in the thermal cycler. Amplification for OneStep kits starts from section 4.7.

4.2. 20–30 minutes before preparing the working amplification mix, take the PCR (amplification) reagent kit out of the freezer and thaw the contents (it is recommended to place the tube with Taq polymerase in an ice bath). Thoroughly vortex the tubes containing the reaction mixture and the fully thawed dilution buffer to mix the contents.

4.3. Prepare the amplification reagent mix using the kit components based on 1 sample: it is recommended to prepare a mix for at least 5 reactions to ensure accurate enzyme dispensing—17.5 µL of dilution buffer, 2.5 µL of reaction mixture, and 0.2 µL of Taq polymerase.

When preparing the working amplification mix, all components must be added using separate pipette tips.

4.4. After adding the Taq polymerase, which should be done last, thoroughly mix the solution by pipetting.

4.5. Add 20 µL of the working amplification mix to each of the prepared amplification tubes.

4.6. Add 1 drop (approx. 25 µL) of mineral oil to all tubes.

4.7. Add 5 µL of the processed sample (see DNA extraction section) into the corresponding reaction tube containing the working amplification mixture, beneath the mineral oil layer.

4.8. Add 5 µL of the appropriate positive DNA control into the positive control tubes, and 5 µL of the diluent into the negative control tube, using individual filter tips for each addition.

Please note that for One Step reagent kits (where the ready-to-use mixture is pre-aliquoted into individual amplification tubes), the positive control is fully prepared for placement in the thermal cycler (amplifier).

4.9. Close the tubes and centrifuge for 3–5 seconds at 3,000 rpm at room temperature (+18...+25 °C) using a microcentrifuge-vortex.

4.10. Transfer the tubes to a thermal cycler (amplifier) preheated to +93 °C (or +94 °C) and perform amplification According to the program specific to the kit type:

Attention: In One Step reagent kits (ready-to-use mixture pre-aliquoted into individual amplification tubes), the positive control sample is completely ready for placement in the thermal cycler (amplifier).

4.11. Close the tubes and centrifuge for 3–5 seconds at 3,000 rpm at room temperature (+18...+25 °C) using a microcentrifuge-vortex.

4.12. Transfer the tubes to a thermal cycler (amplifier) preheated to +93 °C (or +94 °C).

5. Perform amplification using the following programs: POLYHEP B (HBV), HERPOL (HSVII), HERPOL 1+2 (HSVI+II), EBARPOL (Epstein-Barr virus), CYTOPOL (Cytomegalovirus), etc.

6. Detection of amplification products. Separation of amplification products by horizontal gel electrophoresis.

6.1. Pour TAE buffer into the electrophoresis apparatus, prepared by diluting 50× TAE buffer 50-fold with distilled water.

6.2. Add 2 mL of 50× TAE buffer and 100 mL of distilled water to 2.0 g of agarose.

6.3. Melt the prepared mixture on a hot plate or in a microwave oven. Add 10 µL of a 1% ethidium bromide solution to 100 mL of the molten agarose. Mix thoroughly.

6.4. Cool the molten agarose to 50-60 °C and pour it into the gel casting tray. To form sample wells in the agarose gel, place a comb onto the tray using a bulldog-type clamp. After the agarose has solidified, carefully remove the comb from the gel and transfer the gel tray into the electrophoresis chamber.

6.5. Load 10-15 µL of the amplification product into the gel wells following the numerical order of the samples. Include positive and negative controls.

6.6. Connect the electrophoresis chamber to the power supply and set the voltage to achieve an electric field strength of 10-15 V/cm of gel. Perform electrophoretic separation of the amplification products from the cathode (-) to the anode (+). Monitor the electrophoretic separation visually by tracking the dye front. The dye band should migrate 1.5-2 cm from the origin.

Visualization of Electrophoresis Results

6.7. Remove the gel from the mold and place it on the Glass surface of a UV transilluminator.

CAUTION! Always wear gloves when handling agarose gels. Ethidium bromide is a potent mutagen.

6.8. Turn on the transilluminator and analyze the results. The analyzed DNA fragments will appear as glowing orange-red bands when exposed to UV radiation at a wavelength of 310 nm.

7. Data analysis

7.1. For kits with an internal control, the negative control sample (C-) must exhibit a single orange-red band corresponding to the internal control (IC) (Table 19). The appearance of a second band at the positive control level indicates contamination of the kit components.

For kits without an internal control, no bands should be present. The appearance of a band at the positive control level indicates contamination of the kit components.

7.2. For kits with an internal control, the positive control sample (C+) must exhibit two bands: 1) an orange-red band corresponding to the positive control (PC) (Table 20); 2) an orange-red band corresponding to the internal control. For kits without an internal control, a single band corresponding to the PC must be present.

7.3. Test samples:

- the absence of an orange-red band strictly at the level of the positive control (PC) indicates that the target pathogen DNA is absent in the test sample;

- the presence of a band with electrophoretic mobility matching the positive control indicates the presence of the target pathogen DNA in the test sample.

All negative samples must exhibit an orange-red band corresponding to the internal control (IC).

If an analyzed sample in kits with an internal control lacks both the positive control band and the internal control band, it means that the amplification reaction has failed (possible reasons include: the sample contains PCR inhibitors, improper thermocycler operation, or a protocol error during the assay). In this case, if the sample contains a high concentration of the target pathogen DNA, the internal control band may not be visible on the gel electropherogram (sample 3 in Table 21). This is a completely acceptable scenario, and such samples should be interpreted as positive.

8. Result Interpretation

Table 20. Control Samples

Table 21. Test Samples

9. Troubleshooting PCR Inhibition

9.1. If both the IC and PC bands are absent in the test samples (reaction inhibition), the sample must be re-analyzed starting from the DNA extraction stage.

9.2. Transfer 100 µL of the DNA solution extracted per step 4 into a new tube containing the "DNA-EXPRESS" reagent, and repeat the entire extraction procedure (steps 4.1 - 4.4), PCR setup (steps 5.1 - 5.10), and results analysis (steps 6.1 - 6.8).

9.3. If re-processing and re-analyzing the sample fails to yield the IC or PC band, it is recommended to collect a new biological specimen from the patient.

10. Storage and transportation conditions of reagents 10.1. The kit «DNA/RNA Extraction Kit from Serum or Plasma» must be stored at +2...+8 °C throughout its entire shelf life (6 months from the date of manufacture). Storage and transportation of this kit at room temperature for no more than 2 days is permitted.

10.2. Amplification kits (individual components) must be stored at a temperature of minus 18-25 °C throughout their entire shelf life (6 months from the date of manufacture). Storage and transportation of this kit at temperatures not exceeding 0 °C for no more than 2.5 days is permitted.

10.3. One Step amplification kits must be stored at a temperature of +2...+8 °C. The shelf life is 3 months from the date of manufacture.

10.4. Electrophoretic Detection Kit No. 1, agarose, 50x TAE buffer, and ethidium bromide solution may be stored at room temperature throughout their entire shelf life (indicated on the label).

10.5. Electrophoretic Detection Kit No. 2 and samples prepared in agarose gel must be stored at +2...+8 °C throughout their entire shelf life (6 months from the date of manufacture).