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IMMUNOGENETIC TESTS

IMMUNOGENETIC TESTS. MHC = Major histocompability complex HLA = Human lymphocyte antigen. HLA class I: HLA-A, B and C Present on all nucleated cells. HLA class II : HLA-DQ, DP, DR Present on: B cells, monocytes, macrophage, dendritic cells and on activated T cells.

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IMMUNOGENETIC TESTS

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  1. IMMUNOGENETIC TESTS

  2. MHC = Major histocompability complexHLA = Human lymphocyte antigen HLA class I: HLA-A, B and C Present on all nucleated cells HLA class II : HLA-DQ, DP, DR Present on: B cells, monocytes, macrophage, dendritic cells and on activated T cells

  3. Solid organ and BM transplantation • Disease association Appropiate specimens: WB, Lymphocytes, biopsy specimens, buccal swabs, etc.

  4. Laboratory methods • HLA typing is performed at various resolution levels and the methods used also vary according to the resolution level. • Detection at Ag level low resolution • Detection at allel level  High resolution

  5. Anti-HLA antibodies • Tests for the present of circulating anti-HLA are critical for: • Organ transplants • Blood and platelet transfusions • Blood and plasma donors

  6. Anti-HLA antibodies in patient's serum may react with cells from a few or many individuals. • Sensitization is reported as the percent Panel Reactive Antibody (PRA) • Computerized analyses of lymphocytotoxicity reaction patterns on well-characterized cell panels identify the specific HLA-antigens against which antibodies are directed, and "safe" antigens, which are not recognized. • Autoimmune diseases, particular medications, or infections may cause false positive reactions. • Microwells or beads coated with HLA Ags may also used

  7. HLA typing methodologies • Serological methods • Cellular methods • RFLP • Sequence Based Typing (SBT) • Sequence Specific Oligotyping (SSO) • Sequence Specific Priming (SSP)

  8. Serologic methods • Microlymphocytotoxicity assay (Terasaki plates)

  9. Cellular HLA Typing Methods • MLC (mixed lymphocyte culture) Mytomicin C treatment  Stimulator cells

  10. Sequence specificprimers (SSP)

  11. Sequence specific oligohynridisation (SSO)

  12. Luminex

  13. Sequence based typing

  14. Human MHC class I chain-related gene A (= MICA) typing • The expression, structure and function of MICA differs considerably from classical HLA class I genes. • act as ligands for cells expressing a common activating natural killer cell receptor (NKG2D) • Highly polymorphic; functionally relevant in transplant rejection, autoimmune disease and cancer

  15. MICA antibodies have been implicated in solid organ rejection. • MICA genotyping uses luminex technology to type MICA alleles. • The test targets MICA sequence polymorphisms defined by DNA-typing methods. • MICA typing can be helpful in determining whether patients are matched or mismatched for MICA with their donors.

  16. KIR typing • Killer Cell Ig-Like Receptors (KIRs). • NK cells distinguish abnormal cells from healthy cells through variable inhibitory and activating KIRs. • The combinations of KIR and HLA class molecules balance the NK cell response between tolerance of healthy cells and killing of unhealthy cells • 16 KIR genotypes

  17. There are 14 KIR genes and two pseudogenes located in theleukocyte receptor complex (LRC) on chromosome 19q13.4. Human NK cells express various combinations of these 16 KIR genes with two common haplotypes: Group A, which has more inhibitory receptors and Group B, which has more activating receptors.

  18. Microchimerism Testing • Sequence Based Typing (SBT) • Sequence Specific Oligotyping (SSO) • STR and VNTR

  19. VNTR

  20. Clinical Indications for Chimerism Testing in Hematopoietic Cell Transplant • Routine post-transplant documentation of the donor/recipient origin of white blood cells in peripheral blood and/or marrow. Documentation of engraftment may include testing lineage-specific cell subsets, such as CD3 positive T-cells and CD33 positive myeloid cells. • Evaluate donor/recipient cells in patients with inadequate marrow function. • Define whether recurrent or new malignancy has originated from recipient or donor cells.

  21. Assess prognostic risks of rejection and recurrent malignancy. • Document the persistence of donor cells post-transplant in patients with recurrent disease or prior to donor lymphocyte infusion (DLI). • Evaluate whether graft rejection has occurred in recipients that are candidates for a second transplant. • Differentiate the origin of donor cells in recipients who have received a second transplant with a different donor or a transplant with double cord blood units. • Detect the presence of maternal derived cells in patients diagnosed with Severe Combined Immuno-Deficiency (SCID). • Verify genetic identity of putative identical twins.

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