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2. HLA Typing 2

3. Introduction Each year many people worldwide are diagnosed with leukaemia or other fatal blood disorders. Bone marrow or stem cell transplantation offers the only possible curative treatment for many of these patients. Each year many people worldwide are diagnosed with leukaemia or other fatal blood disorders. Bone marrow or stem cell transplantation offers the only possible curative treatment for many of these patients. 3

4. HLA typing What is tissue typing? Matching of stem cell donor to a recipient is determined by comparing their tissue types, also known as their Human Leucocyte Antigen (HLA) types. An individual's HLA type is present on nearly all tissues in the body. The white cells from a blood sample are a convenient source of "tissue" that the laboratory can use to determine an individual's HLA type. What is tissue typing? Matching of stem cell donor to a recipient is determined by comparing their tissue types, also known as their Human Leucocyte Antigen (HLA) types. An individual's HLA type is present on nearly all tissues in the body. The white cells from a blood sample are a convenient source of "tissue" that the laboratory can use to determine an individual's HLA type. 4

5. GvHD Cells from the donor's immune system which are introduced along with the transplanted stem cells ("graft") can also recognise HLA mismatches and attack vital organs of the recipient's body ("host"). This is called graft versus host disease (GvHD). 5

6. Searching for a related donor An HLA type consists of two main groups: Class I antigens (HLA-A, -B, -C) and Class II antigens (HLA-DR, -DQ, -DP). There are six HLA antigens considered most important for determining compatibility: 1.two A antigens, 2.two B antigens, 3.two DR antigens (eg: A 3, 32; B 7, 37; DR 1, 15). An HLA type consists of two main groups: Class I antigens (HLA-A, -B, -C) and Class II antigens (HLA-DR, -DQ, -DP). There are six HLA antigens considered most important for determining compatibility: 1.two A antigens, 2.two B antigens, 3.two DR antigens (eg: A 3, 32; B 7, 37; DR 1, 15). 6

7. HLA classes 5/27/20167

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11. We inherit a set (or haplotype) of HLA-A, B and DR antigens from each parent. 11

12. How is HLA typing reported? HLA typing is reported as a series of numbers. For example, an HLA type will appear on the report as A 3, 32; B 7, 37; DR 1, 15. HLA typing is reported as a series of numbers. For example, an HLA type will appear on the report as A 3, 32; B 7, 37; DR 1, 15. 12

13. How is HLA typing performed? A 20-30 ml blood sample is required to perform HLA typing. The white cells are isolated from the blood and typing is performed by two different methods: Serological testing: where the white cells are used DNA testing: where DNA extracted from the white cells is used. A 20-30 ml blood sample is required to perform HLA typing. The white cells are isolated from the blood and typing is performed by two different methods: Serological testing: where the white cells are used DNA testing: where DNA extracted from the white cells is used. 13

14. Nomenclature HLA-Aidentifies HLA A locus HLA-A1 serologically defined antigen HLA-A* : asterisk denotes HLA alleles defined by molecular methods HLA-A*01 :2 digit resolution denotes a group of alleles corresponds usually to serological group – low resolution HLA-A*0101 : 4 digit resolution – sequence variation between alleles results in amino acid substitutions HLA-A010101 :6 digit resolution – non coding variation: sequence changes synonymous no amino acid substitution HLA-Aidentifies HLA A locus HLA-A1 serologically defined antigen HLA-A* : asterisk denotes HLA alleles defined by molecular methods HLA-A*01 :2 digit resolution denotes a group of alleles corresponds usually to serological group – low resolution HLA-A*0101 : 4 digit resolution – sequence variation between alleles results in amino acid substitutions HLA-A010101 :6 digit resolution – non coding variation: sequence changes synonymous no amino acid substitution

15. Nomenclature HLA-A01010101 8 digit resolution – sequence variation occurs within the introns or 5’ / 3’ extremities of the gene HLA-A01010102N Null allele Alphabetical suffix NNull allele LLow level expression AAberrant expression CMolecule present in cytoplasm only SSecreted molecule present only as soluble form HLA-A01010101 8 digit resolution – sequence variation occurs within the introns or 5’ / 3’ extremities of the gene HLA-A01010102N Null allele Alphabetical suffix NNull allele LLow level expression AAberrant expression CMolecule present in cytoplasm only SSecreted molecule present only as soluble form

16. SEROLOGY Complement Dependent Cytotoxicity (CDC) Viable peripheral blood lymphocytes are obtained by discontinous density gradient centrifugation using Ficoll at a density of 1.077 at 19º - 22ºC. Microlymphocytotoxic test : 3 stages Complement Dependent Cytotoxicity (CDC) Viable peripheral blood lymphocytes are obtained by discontinous density gradient centrifugation using Ficoll at a density of 1.077 at 19º - 22ºC. Microlymphocytotoxic test : 3 stages

17. Microlymphocyototoxic test (1) 1.Viable lymphocytes are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound. 2. Rabbit serum as a source of complement is added, incubate. If antibody is bound to the HLA antigen on the cell surface it activates the complement which damages the cell membrane making it permeable to vital stains. 1.Viable lymphocytes are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound. 2. Rabbit serum as a source of complement is added, incubate. If antibody is bound to the HLA antigen on the cell surface it activates the complement which damages the cell membrane making it permeable to vital stains.

18. Microlymphocyototoxic test (2) 3. Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Eosin have been used. 3. Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide although both Trypan Blue and Eosin have been used.

19. Microlymphocytotoxicity test (3) Test is left for 10 minutes and then read using an inverted fluorescient microscope. A mixture of T and B lymphocytes can be used for HLA typing. B lymphocytes are required for HLA Class II typing by serology. (Normal population 85-90% T and 10-15% B cells) Test is left for 10 minutes and then read using an inverted fluorescient microscope. A mixture of T and B lymphocytes can be used for HLA typing. B lymphocytes are required for HLA Class II typing by serology. (Normal population 85-90% T and 10-15% B cells)

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21. Molecular All commonly used molecular methods require good quality genomic DNA. There are numerous methods for extraction of DNA from whole blood. There are ‘in house’ methods based on Miller et al’s Salting Out which are cheap and easy but labour intensive. All commonly used molecular methods require good quality genomic DNA. There are numerous methods for extraction of DNA from whole blood. There are ‘in house’ methods based on Miller et al’s Salting Out which are cheap and easy but labour intensive.

22. Molecular Methods The application of molecular techniques to HLA typing began around 1987 when the Southern Blot technique was used to identify restriction fragment length polymorphisms (RFLP’s) associated with known serological DR/DQ and cellular Dw defined specifities. Around 1992 polymerase chain reaction (PCR) methods were developed. Most methods currently used have a PCR element within the technique. The application of molecular techniques to HLA typing began around 1987 when the Southern Blot technique was used to identify restriction fragment length polymorphisms (RFLP’s) associated with known serological DR/DQ and cellular Dw defined specifities. Around 1992 polymerase chain reaction (PCR) methods were developed. Most methods currently used have a PCR element within the technique.

23. Molecular Methods(2) PCR Three steps per cycle– denaturation, annealing and extension. The introduction of the Thermal Cycler revolutionised the use of PCR within the routine laboratory. PCR Three steps per cycle– denaturation, annealing and extension. The introduction of the Thermal Cycler revolutionised the use of PCR within the routine laboratory.

24. Molecular methods PCR SSOP ( Sequence Specific Oligonucleotide Probes) ‘ PCR SSOP ( Sequence Specific Oligonucleotide Probes) ‘

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30. Terasaki HLA Typing Trays 30

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