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Histocompatability testiing
- 2. INTRODUCTION Histocompatibility is the compatibility between the tissues of different individuals so that one accepts a graft from the other without giving an immune reaction. Provides Human Leukocyte Antigen(HLA) typing. HLA plays important role because 1-Primary targets of immune responses 2-critical responses for antigenic stimuli 3-Implicated in genetic susceptibility to autoimmune disease.
- 4. HLA TYPING METHODS SEROLOGY used to be the ‘gold standard’. CELLULAR rarely used now. MOLECULAR being the fast method of choice.
- 5. FOR TRANSPLANTATION HLA typing methods depends on -relationship to the recipient -time constraints -specimen characteristics To prevent graft versus host disease(GvHD) A crossmatch is required to check the presence of donor- specific preformed antibodies in the patient’s serum.
- 6. Antibody screening used to detect alloantibodies. Lymphocytotoxicity test used for antibody screening,serologic HLA typing & crossmatching.
- 8. TISSUE TYPING Cell isolation:- -Lymphocytes are preferred -Isolated from -whole peripheral blood by density gradient separation -from buffy coat -in cadaveric testing -from lymph nodes & spleen. Performed on B lymphocytes for class II HLA,HLA-DR & -DQ antigens.
- 9. Magnetic beads for isolation of B & T lymphocytes -Beads with anti-CD2 or anti-CD8 for T cells -Beads with anti-CD19 for B cells. From whole blood,buffy coats or PBL preparation Requires less incubation time.
- 11. COMPLEMENT-DEPENDENT LYMPHOCYTOTOXICITY TEST 1 A plastic tray of 60-72 wells of 15µl capacity is taken 2 1µl of HLA antisera is added 3 1µl of test cells added 4 Incubation for 30mins at RT 5 5µl of complement added to each well 6 Incubation for 60mins. 7 A dye,Eosin Y followed by formalin is added
- 12. 8 Dead & live cells are counted by phase contrast microscope 9 Each well in the tray are viewed individually.Percentage of dead cells in each well is calculated & scoring is done.
- 15. CROSS MATCHING To detect the presence of antibodies in patient’s serum directed against the HLA antigens of the potential donor If present,recipient’s body will reject the transplant. Serologic methods,flow cytometry can be used for crossmatching.
- 16. PBL LYMPHCYTOTOXICITY CROSSMATCHING 1 PBL of donor are incubated with labelled anti-CD3 mAbs which bind to T cells. 2 Labelled anti-CD3 bound T cells are seperated from B cells in flow cytometer 3 Seperated stained T cells are incubated with recipient’s serum 4 After washing a flurochrome labelled anti-human IgG is added & incubated 5 Flow cytometer counts the number of T cells
- 18. CROSSMATCHING FOR AUTOANTIBODIES Autoantibodies against lymphocytes in the recipient’s serum may bind non-specifically to donor lymphocyte Gives a false positive crossmatch result Masks the presence of specific anti-donor antibodies Detected by auto cross match Recipient’s own lymphocytes & serum are combined in a standard cytotoxicity test.
- 19. THANK YOU
Hinweis der Redaktion
- Hla encodes the MHC complex.Its present in all the nucleated cells of our body.esp in wbcs.
- Cellular were used for Class II typing.
- HLA matching differs within families.Based on Mendelian inheritance 25% of offspring inherits the same HLA type.
- Free of RBC & platelet contamination.Platelets,amniocytes & fibroblasts can also be used.special isolation procedures as 80% are T cells in PB
- Approx 2000 lymphocytes are dispensed into each well.Abs bind with specific HLA antigens on the surface of lymphocytes.complement proteins causes death of lymphocytes that are coated with mAbs.No coating then no complement activation.
- Live cells-bright & refractile..Dead cells-swollen & dark.
- Flow cytometry is 30-250 times more sensitive
- 3-if abs present binds to T cells…..4-Flurochrome binds with HLA abs which are bound with donor’s T cells….5-Histogram is created.
- Donor is disqualified.