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Department of blood collection

DEPARTMENT OF MOLECULAR IMMUNOGENETICS

WORKING HOURS OF THE CENTER: 7.00-15.00h
PHONE: 033/567-313
FAX: 033/270-170
Responsible person : Manager Elma Fejzić, graduated biologist
LIST OF ANALYSIS AND WORK METHODS:

  • Typing of HLA Class I antigen
  • Typing of HLA class II antigen
  • Typing of HLA –B27 antigen
  • Panel of reactive antibodies HLA (PRA)
  • Cross reaction test (cross-mach)

DETAILED DESCRIPTION OF ACTIVITIES:
For a serological method, a 9 ml vacuum tube with anticoagulant heparin is required.
(Class I typing and typing of HLA-B27 antigens)
For a molecular method, a 5 ml vacuum tube with an anticoagulant EDTA is required.
(Typing of Class I and Class II)
(For a test panel of reactive antibodies, a 5 ml vacuum tube without anticoagulant is needed.)
HLA system
Human Leukocyte Antigen is the name for the major tissue compatibility complex (GKTP) in humans. It contains a large number of genes that are related to the immune system. They are located on the short arm of chromosome 6, section 6p21.3 and encode the synthesis of proteins that represent antigens with immunologically compThey are coded for the genes found on the short arm of chromosome 6. They are glycoproteins. They are found on all core and thrombocytic cells. Classical HLA class I antigens are divided into three locuses: HLA-A, HLA-B and HLA-C.etent cells.
HLA class I antigens
They are coded for the genes found on the short arm of chromosome 6. They are glycoproteins. They are found on all core and thrombocytic cells. Classical HLA class I antigens are divided into three locuses: HLA-A, HLA-B and HLA-C.
HLA class II antigens
Cells encoded are centromeric (section 6p21.3) on the short arm of chromosome 6. HLA class II antigens are organized in multiple subunits: HLA-DR, HLA-DQ, HLA-DP.
HLA typing represents a series of procedures and methods in serological and molecular diagnostics. They have a common and ultimate goal to determine individual HLA class I or class II HLA classes at the allelic or allele level. The purpose of these methods is to determine the degree of tissue compatibility between potential donors and recipients.
The typing of HLA genotypes can be divided into:
1. Type alleles or HLA typing at a lower level of low resolution, which is sufficient for alogenous transplantation of solid organs.
2. Type-allele or HLA typing at a higher level of gene resolution (HRT) is significant for alogenous bone marrow transplantation.
In clinical practice, HLA genotyping data is primarily used in transplantation immunology in the selection of organ donors and bone marrow, and in identifying predispositions or predisposing genes for a wide spectrum of diseases, particularly those with infectious and autoimmune etiology.
Methods:

HLA class I typing (A, B and C) is performed using a microlimphocytotoxic test (MLCT)
2. HLA typing class I (A, B and C) low resolution using polymerase chain reaction and qualitative detection of sequential specific PCR products by electrophoresis on agarose gel (methodology based on PCR-SSP-Sequence Specific Primers )
HLA typing of class I (A, B and C) low resolution, using asymmetric PCR amplification with different primers per sample, and biotin biotypes are mixed during PCR with the mix of spheres and bind with complementary probes during hybridization (methodology based on PCR-SSO -Sequence Specific Oligos) Luminex technology-fluoroanalysis on microspheres.
4. HLA class II typing (DRB1, DRB3, DRB4, DRB5 and DQB1) low resolution, polymerase chain reaction and qualitative detection of sequential specific PCR products by electrophoresis on agarose gel (methodology based on PCR-SSP- Sequence Specific Primers )
5. Low-resolution HLA typing of Class II (DRB1, DRB3, DRB4, DRB5 and DQB1) by using asymmetric PCR amplification with different primers per sample, and biotin biotypes are mixed during PCR with the mix of spheres and bind with complementary probes during hybridization (Methodology based on the PCR-SSO -Sequence Specific Oligos) Luminex Technology Microspheres Fluoroanalysis.
HLA and disease
Today, more than 100 diseases (eye diseases, ocular manifestations - reactive arthritis, Reiter syndrome, RA, celiac disease, and psoriatic arthritis) have been associated with the HLA genes.

DISEASE HLA ANTIGEN
RHEUMATOLOGY
Ankylosing spondylitis B27
Reiter syndrome B27
Acute frontal uveitis B27
Rheumatoid arthritis DR1, DR4
Systemic lupus erythematosus B8, DR3, DR2
GASTROENTEROLOGICAL
Gluten-sensitive enteropathy - celiac disease B8, DR3, DQ2, DQ3
Autoimmune hepatitis DR3, DR4
Ulcerative colitis B5, DR2
HEMATOLOGICAL
Idiopathic hemohromatography A3, B14
Hodgkin's disease A1, B5, B8, B18
Pernicious anemia DR5
ENDOCRINOLOGICAL
Diabetes mellitus type 1 DR3, DR4, DQ2, DQ3
Graves' disease A1, B8, DR3, DQ2
Hashimoto's thyroiditis DR5
NEUROLOGICAL
Myasthenia gravis B8, DR3
Multiple sclerosis B7, DR2, DQ6
Narcolepsy DR2, DQ6
Schizophrenia A28
RENAL
Goodpaster syndrome DR2
Polycentric kidney disease B5
SKIN
Psoriasis Cw6
Dermatitis herpetiformis DQ2

Anti-HLA antibodies

Anti-HLA antibodies are typical of the anti-HLA antibodies that are formed after contact with the immune cells. The cause of sensitization is blood transfusion, pregnancy, and transplantation of tissues and organs. Most commonly belong to IgG immunoglobulins and activate complement. Therefore, all patients who are waiting for transplantation are tested every four months for the presence of lymphocytotoxic antibodies and a percentage of panel of reactive antibodies (PRA) is determined. The presence of antibodies is demonstrated by complement-dependent microlimfocytotoxicity test (MLCT) with and without the addition of dithiothreitol (DTT) and Luminex flour analyzer with microspheres .
Cross-match (CROSS-MATCHING)
Testing the recipients of antibody specific cells for potential donor microcytotoxic test (recipient serum and donor lymphocytes)
Positive body- contraindicated transplantation