HLA system Flashcards
What is the structure of Class I antigens
Heavy alpha chain (1, 2, and 3) with light chain Beta2-microglobulin
What cells are class 1 antigens located on?
Most nucleated cells including platelets.
not on corneas, neurons, epithelial cells or germinal cells
What are the class 1 antigens found on RBC’s?
Residual amounts are found on RBC’ membrane called Bg antigens (Bennett Goodspeed)
What HLA does Bga represent?
B7
What HLA does Bgb represent?
B17
B57 or B58
What HLA does BgC represent?
A28
A68 or A69
What HLA antigens are found on platelets?
mostly A and B with some C at low levels
What are the Class II HLA antigens?
DR, DQ, and DP
What is the structure of Class II HLA antigens?
Alpha and heavy chain
What cells are Class II antigens found on?
B lymphs, monocytes, macrophages, dendritic cells, intestinal epithelium, and activated T lymphs
What is the function of Class I antigens?
HLA class I molecule carries the viral peptide to the cell surface for presentation. CD8 cytotoxic T cells (Tc) recognize HLA Class I molecules on the surface of the cell. Tc cells attacks the infected cell, causing cell death
What is the function of Class II antigens?
Class II molecules present exogenous peptides which are degraded into peptides by enzymes in the infected cell. Class II molecules inside the cell insert the peptides into the peptide binding groove and transport the foreign peptide to the cell surface for presentation. CD4 T cells (Th) surveying the cells recognize HLA Class II molecules. T lymphocytes secrete cytokines to stimulate other cells (e.g. B cells to make antibodies).
What are class III HLA molecules?
Tumor necrosis factor (TNF)
Steroid enzyme 21-hydroxylase
Complement C2, Bf, C4A (Rogers), C4B -(Chido) = Absorbed onto RBC membrane
What the common public antigens?
Class I: HLA Bw4 and Bw6
Class II: HLA DR51, DR52 and DR53
What are splits?
HLA antigens historically believed to be one specific was split into multiple specifies because of more sensitive testing and molecular methods
What are GREG’s?
Cross reactive epitope group
HLA antigens that share common epitopes and have cross reactivity with serological testing
What is the nomenclature for HLA antigens?
HLA-DRB1*04:01 Locus = DR Protein = B1 chain * = typed by molecular method Serological allele = 04 (antigen) Variant of allele = 01 (subgroup)
What is a centimorgan (cM)?
It is a unit for measuring genetic linkage. It is defined as that distance between chromosome loci for which the expected average number of intervening chromosomal crossovers in a single generation is 0.01
HLA-A & HLA-B crossover rate is 0.8%
HLA-A & HLA-B distance is 0.8 Centimorgan
What is SSO or SSOP?
Sequence specific oligonucleotide probe is labeled and used to detect sequences in immobilized DNA (microbead or membrane)
What is rSSO?
Reverse Sequence specific oligonucleotide probe. Each probe is attached to different microbead and binds to target DNA after PCR.
Gives Low to high resolution HLA typing
What is SSP?
Sequence Specific Primers - specific primers are used to amplify a particular DNA sequence. Multiple PCR assays are required because each reaction is specific for one allele.
Amplified PCR products visualized on gel electrophoresis for pattern. Presence of product = present of allele amplified
What is DNA sequencing?
High resolution testing for HLA alleles
Fluorescently labeled nucleotides are added during PCR and measured using capillary electrophoresis. Provide DNA Sequence which is compared to known DNA sequence of HLA alleles to find match.
What is STR DNA analysis?
Short tandem repeats in DNA are used to detect chimerism in BMT and paternity testing.
DNA is amplified, cut, and electrophoresis is used to measure size of fragments
What is the HLA antigens show linkage?
B and C are closely associated and can predict what C is based on B
DR and DQ are closely associated
DP has recombination
What antigens are matched in solid organ transplantation?
ABO is most important
Match HLA-A, HLA-B, and HLA-DR
Low resolution DNA method
If PRA is negative= can mismatch solid organs
What HLA antigens are matched in BMT?
HLA-A, HLA-B, HLA-C, DRB1 (8/8)
HLA-A, HLA-B, HLA-C, DRB1, DQB1 (10/10)
High resolution DNA method
Avoid DRB1 and C mismatches
What is lymphocytoxicity?
HLA sera of known specificities added to micro plates + patients lymphocytes are added. (B cells for Class II)
Antigen-antibody reaction takes place.
Rabbit complement added; if enough antibody binds to antigens on lymphs, complement cascade will be activated and cause damage to the cell membrane
Dye (e.g. eosin) added. Damage to cells detected by addition of dye; intact cells exclude the dye; dead cells take in stain
What is Mixed Lymphocyte Culture (MLC)?
A “cellular cross-match” prior to transplantation, especially to prevent GVHD in HPC.
Lymphocytes from 2 individuals cultured together. Each cell population able to recognize the “foreign” HLA antigens of the other. As a response to these differences, the lymphocytes transform into blast cells, synthesize DNA and uptake radioactive thymidine.
The response is then measured.
Rarely used but may be used as a monitor of immune function
Microcytotoxicity for HLA antibodies?
The patient’s serum is tested against a “panel” of target lymphocytes with known HLA types.
Addition of goat anti-human kappa chain increases the likelihood of complement binding and subsequent cell injury
The percent of the panel cells to which the patient has formed cytotoxic antibodies is referred to as the panel reactive antibody (PRA) level.
Used to be gold standard.
What is the Luminex multiplex?
The Luminex assay involves testing patient sera against a panel of recombinant soluble HLA antigens, each bound to specific microbeads. Each microbead is labeled with a distinct set of fluorophores, enabling direct evaluation of antibody binding specificity. Anti-HLA antibody binding is detected by labeling with PE-conjugated anti-Ig and measured on a specialized flow cytometer
How is flow cytometry used in crossmatching?
The flow cytometry crossmatch is performed by incubating donor cells with recipient sera, followed by a fluoresceinated goat antihuman immunoglobulin. A phycoerythrin-labeled antibody to detect either T or B cells is used to discriminate between the two subpopulations of lymphocytes. Cells are analyzed, and results are expressed as positive or negative, based on the shift in fluorescence intensity of the test serum with respect to negative serum.
How are B and T cells separated in Flow cytometry?
CD3-PerCP = T-cells CD19-PE = B-cells
What is relative risk?
The degree of association between a given HLA type and disease is often described in terms of relative risk (RR), which is a measure of how much more frequently a disease occurs in individuals with a specific HLA type when compared to individuals who do not have that HLA type
What HLA antigen is associated with Celiac Disease?
DQ2 (RR>250)
What HLA antigen is associated with ankylosing spondylititis?
B27 (RR >150)
What HLA antigen is associated with Narcolepsy?
DQ6
What is the cause of febrile non-hemolytic transfusion reactions?
Caused by HLA antibodies as well as granulocyte and platelet antibodies.
Recipient antibodies react with transfused donor white cells carrying HLA antigens; release of IL-1, IL-8 and other cytokines; resulting in fever.
What is the mechanism of TRALI?
HLA antibodies in donor plasma react with and fix complement to the granulocytes of the recipient, leading to severe capillary leakage and pulmonary edema.
What is TA-GVHD?
Donor T cells (graft) proliferates in recipient (host)
Depends on host immunocompetence & HLA dissimilarity
90% - 95% fatality rate
What type of XM is required for heart transplant?
ABO most important. Unable to perform XM in time so if no HLA antibodies HLA mismatch is acceptable. Virtual XM if HLA antibodies present.
