HLA Flashcards
Exam 4
Describe the genetics of the HLA antigens.
The HLA genes are a series of 35-40 closely linked genes on 3 regions of chromosome 6. These 3 regions code for the 3 different classes: Class I, II and III.
What is unique about the way HLA genes are inherited?
All the genes on that portion of the chromosome are usually inherited together since they are physically linked. This means that an entire haplotype will be inherited from the parent.
Describe the difference in structure between Class I and Class II HLA proteins.
Class I has one heavy chain and a B2-microglobulin, a heavy chain that folds into 3 domains , and only the heavy chain inserts into the membrane. The Class II proteins have two similar sized chains that each fold into two domains (a and b), and both chains insert into the membrane.
On what cells are class I HLA antigens located?
All nucleated cells, dendritic cells, platelets
On what cells are class II HLA antigens located?
B lymphocytes, activated T lymphoctyes, macrophages, monocytes, endothelial cells
What are Sequence Specific Oligonucleotides (SSO)?
PCR amplification that is performed with primers flanking a chosen region. The amplified DNA is denatured and hybridized with oligonucleotide probes for allele-defining DNA
What are Sequence Specific Primers?
Primers for PCR amplification target specific DNA sequences. These can identify an HLA allele. You can then look for the presence or absence of the amplification products by gel electrophoresis
What is sequence based typing?
Determine the exact nucleotide sequence of HLA genes by molecular cloning or PCR using labeled ddNTPs
Describe the procedure for microlymphocytotoxicity. It is used to identify HLA antibodies and for crossmatching. The se
rum (with antibodies) is incubated with leukocytes (from the donor or reagent) exogenous complement, and fluorescent dye. If the antibody is present, it will bind the leukocytes, activate the complement, and then form holes in the cell allowing dye to enter the cell. If there is no antibody present then the dye cannot enter. A >40% uptake of the dye is considered a positive reaction.
What are some disadvantages of cytotoxicity testing?
Need cells for testing which can be difficult to find regularly to keep fresh and in stock. Frozen lymphocytes are more fragile leading to false positives. Low titers may not be identifiable.
What are ways to increase sensitivity of cytotoxicity testing?
Increase incubation time, add a wash step, add DTT, add AHG reagent
Describe the process of flow cytometry and its use in HLA testing.
This is used for antibody detection. Serum (with antibodies ) is incubated with donor lymphocytes. The lymphocytes are washed and fluorophore-labeled anti-immunoglobulin is added. The flow cytometer uses a laser to emit fluorescence from the fluorophore if antibody has attached.
What is the multiplex single-antigen bead immunoassay (SAB)?
This uses recombinant individual HLA proteins. The serum (with antibodies) is tested against the proteins bound to the beads. Anti-Ig can then bind to the antibody and labels the microbead with a flurophore which can be detected by a flow cytometer.
If you are looking for an HLA match on a patient, what family member has the best chance of being a match?
Sibling
Why does platelet transfusion become an issue for a patient that has an HLA antibody?
Class I HLA antigens are expressed on platelets. If they have an antibody to the HLA antigen on the platelet, they will destroy the transfused platelets. This is called platelet refractoriness. The patient must receive HLA platelets negative for the HLA antibody they have created.
