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  2. What Is the HLA System? The HLA system refers to a group of related genes that play an important role in the immune system. Together, the proteins made from these genes form something called the major histocompatibility complex (MHC). These proteins are attached to almost all of the cells of our body (excluding red blood cells).
  3. HLA stands for human leukocyte antigen HLA typing is a kind of genetic test used to identify certain individual variations in a person’s immune system. The process is critical for identifying which people can safely donate bone marrow, cord blood or an organ to a person who needs a transplant. HLA typing is also sometimes called HLA matching.
  4. PURPOSE OF HLA TYPING • The most common reason for HLA typing is to help determine which people can provide the safest tissue transplants (solid organ or hematopoietic stem cell transplantation). • Potential tissue recipients must have the typing, as must anyone who might potentially want to donate tissue. • HLA type included in a bone marrow registry, for stem cell transplantation. • HLA typing may also be performed on terminally ill or recently deceased people who will be serving as organ donors. • The best possible donors have HLAs that closely match the HLA patterns of the recipient.
  5. CONDITIONS THAT REQUIRING TRANSPLANTATION  There are many different health conditions that may need to be treated through a transplant.  For example, various types of blood cancers and genetic blood disorders are treated through stem cell transplantation (taken either from the bone marrow or from the peripheral blood).  A solid organ transplant might be necessary for any essential organ that has become severely damaged.  This might happen through trauma, infection, autoimmune disease, genetic illness, toxins, or many other disease processes.  For example, one might need a kidney, liver or lung transplant if one’s own organs are very functioning very poorly.
  6. THE PROCESS OF HLA TYPING HLA typing assesses the particular HLA genes that have inherited Because there are a number of different HLA genes, as well as different variations of these genes, there are very many different possible color combinations that together make up your specific HLA type. HLA typing also usually includes testing for antibodies targeted to specific HLA proteins. Antibodies are made by part of the immune system. If a person already has an antibody against an HLA protein it may attack that protein if it is transplanted. This may cause the transplant to fail. So generally, shouldn’t receive a transplant from someone if already have an antibody against one of their HLA proteins.
  7. CONT……. HLA typing also often includes something called lymphocyte cross matching. Lymphocytes are a type of immune cell. Lymphocyte cross matching checks to see if the recipient has an antibody against a protein on the donor’s lymphocytes. If so, that person generally shouldn’t receive a transplant from that particular person. These people are at high risk of a transplant that won’t be successful.
  8. Is HLA Typing the Same as Blood Typing? No. HLA is much more complicated than blood typing because there are many more HLA markers that make a person’s cells unique. There are only eight basic blood types, and many people can safely receive more than one type of blood (depending on their type). To receive only blood from a person, do not need to be an HLA match, because HLA is not present on red blood cells. To receive a solid organ transplant, the recipient must have a compatible blood type with the donor, as well as the best HLA match possible. For stem cell donations, one needs a very strong HLA match, but blood type is not as important as it is for solid organ transplants.
  9. How Are HLA Genes Inherited? Because the HLA genes are located close together on DNA, they are usually inherited as a group. HLA type is composed of the set of HLA genes you inherited from mother and the HLA genes you inherited from father. Biological parents always share half of their HLA proteins with their children. This is also called a “half match.” Conversely, a child always is a half match with their parents. The set of HLA alleles found on one chromosome is called haplotype. Determination of haplotype is important for identification of HLA identical siblings because sharing of antigen from different haplotypes is common.
  10. HLA TYPING IMPORTANT ROLE The primary target of immune responses to allogeneic transplants Critical for response to antigenic stimuli Implicated in genetic susceptibility to auto immune disease.
  12. SEROLOGICAL TEST Potential donor′s and recipient's WBC′s added to the different wells of microtitre plates Antibodies specific for HLA class I and II added After incubation complement is added Cytotoxicity is assessed by uptake or exclusion of dye
  13. SEROLOGY HLA Typing is done serologically by MICROCYTOTOXICITY (microlymphocytotoxicity) which tests for complement mediated lysis of peripheral blood lymphocytes with a standard set of typing sera. Viable peripheral blood lymphocyte are obtained by discontinuous density gradient centrifugation using Ficol/ Tryosil or Ficol / Sodium Metrizoate at a density of 1.077 at 19⁰C-22⁰C
  14. Microlymphocytotoxic test 3 stages • Viable lymphocyte are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound. • Rabbit serum as a source of complement is added, incubate. If antibody is bound HLA antigen on the cell surface it activates the complement which damages the cell membrane making it permeable to vital stains. • Results are visualized by adding dye usually a flurochrome eg. Ethidium Bromide although both trypan blue and Eosin Y have been used .
  15. • If the reaction has takes place the EB enters the cell and binds to the DNA • For ease double staining is normally used. A cocktail of Ethidium Bromide and Acridine Orange, quenched using Bovine Hemoglobin to allow simultaneous visualization of both living and dead cells. • White blood cells from potential donors and the recipient are added to separate wells of a microtiter plate. The example depicts the reaction of donor and recipient cells with a single antibody directed against an HLA-A antigen. • The reaction sequence shows that if the antigen is present on the lymphocytes, addition of complement will cause them to become porous and unable to exclude the added dye.
  16. Presence or absence of various MHC alleles determined by antibody mediated cytotoxicity
  17. Cont……. • Test is left for 10 minutes and then read using an inverted fluorescent microscope. • A mixture of T and B lymphocytes can be used for HLA class I typing. • B lymphocytes are required for HLA class II typing by serology. (Normal population 85-90% to T and 10-15% B cells) • This can be achieve using a number of methods
  18. In the past neuraminidase treated sheep red blood cell resetting and nylon wool have been used. Immunomagnetic bead separation is the current method of choice. It utilizes polystyrene microspheres with a magnet able core coated in monoclonal antibody for a HLA class II b chain monomorphic epitope. Positive selection
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  20. MIXED LYMPHOCYTE REACTION o It has been observed the lymphocytes from one donor, when cultured with lymphocytes from an unrelated donor, are stimulated to proliferate. o It has been established that this proliferation is primarily due to a disparity in the Class II MHC (DR) antigens and T cells of one individual interact with allogeneic class-II MHC antigens bearding cells (B cells, dendritic cells, Langerhans cells, etc…) this reactivity was termed with mixed leukocyte reaction (MLR) and has been used for studying the degree of histocompatibility.
  21. In this test, the lymphocyte (responder cells) are mixed with irradiated or mitomycin C treated leukocytes from the recipient, containing B-lymphocytes and monocytes (stimulator cells). The responder T cells will recognize the foreign class II antigens found on the donor and undergo transformation (DNA synthesis and enlargement: blastogenesis) and proliferation (mitogenesis) occurs The T cells that respond to foreign Class II antigens are typically CD4+ TH-1 type cells These changes are recorded by the addition of radioactive (tritiated, 3H)
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  23. Molecular HLA typing DNA based procedures has increased the accuracy of HLA typing and lead to the identification of serologically undetected alleles and many subtypes of serological specificities. DNA based methods of type for HLA alleles have therefore focused in the analysis of nucleotide variation occurring in both exon 2 and 3 of class I genes and exon-2 of class II genes.
  24. PCR BASED METHODS/ THREE CATEGORIES  Polymorphism is identified directly as part of the PCR process, although there are post amplification steps e. g (SSP)  Product containing internally located polymorphism that can be identified by a second technique e. g PCR sequence specific oligonucleotide probing (SSOP), PCR- RFLP,PCR followed by sequencing.  Conformational analysis
  25. STEPS OF MOLECULAR CLONING • Extraction of genomic DNA • Amplification of the genes of interest and • Detection of sequence polymorphism that define the alleles.
  26. DNA EXTRACTION Genomic DNA extracted from nucleated cells; the purity of the DNA extracted may be an important factor for successful results GENE AMPLIFICATION/PCR The specificity of the amplification can be locus specific e. g (HLA- A, HLA-B, HLA-DRB1), group specific… e.g. (DRB1-01, DRB1- 02) Or allele specific DRB1-0401, DRB1-0402 The primers are selected to amplify a single allele or a group of alleles For PCR, the specificity is determined by the sequence of the primers and amplification condition. Most typing scheme require conditions the avoid the co-amplification of pseudogene.
  27. Amplification of exon 2 (approximately 270bp ) of HLA class II is sufficient to achieve the highest resolution level. For typing HLA class I, both exon 2 and 3 and the intervening intron (fragment longer than 900bp ) are amplified by single pair of primers. Detection of sequence polymorphism that define the alleles • Sequence specific primers (SSP) group and alleles specific primers • Hybridization with sequence specific oligonucleotide probes (SSOP) • Sequence based typing (SSP)
  28. Sequence specific priming • The procedure relies on the specificity of primer extension that is matched or mismatched with the template at its 3’end. • A combination of 2 primers designed for each of 2 polymorphic sequence motifsin cis allows the identification of an allele or a group of alleles that are characterized by these 2 motifs. • The method is rapid and ideally suited for small numbers of samples. Because very few sequences are absolutely allele specific, SSP combine several primer to discriminate a particular allele unambiguously.
  29. Hybridization with sequence specific oligonucleotide probes (SSOP) The procedure relies on the locus-specific amplification of the genomic DNA segment comprising the polymorphic sites of HLA alleles. Amplified DNA is then immobilized on a solid support, usually a nylon membrane, and then hybridized with a battery of (SSOP) (direct hybridization). Fluorochromes are linked with the probes to allow their detection by chemiluminescence. Alternatively, SSO probes can be immobilized on a solid support, for example color-coded microspheres, and hybridized with labeled PCR product (reverse hybridization). The higher the number of probes the better the resolution level. Usually 50- 100 probes per locus are used for intermediate/high resolution typing, however, only the probe completely matched with the target sequence amplified will hybridize and give a positive signal.
  30. SEQUENCE BASED HLA TYPING • Sequence based HLA typing involves determining the nucleotide sequence of an amplified segment of HLA gene • SBT presents the advantages over the other procedure because of the relatively fast (24-48 hours), high level resolution • More reliable and specific method
  31. CONFORMATIONALANALYSIS Different mutation generates specific conformational changes in PCR products, these are identified by electrophoresis analysis. E.g.….. heteroduplex analysis, single strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE)
  32. CLINICAL SIGNIFICANCE OF HLA TYPING In organ transplantation In transfusion therapy Disease association Disputed paternity In cancer prevention Anthropological studies
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