HLA system Flashcards
1
Q
What is the structure of Class I antigens
A
Heavy alpha chain (1, 2, and 3) with light chain Beta2-microglobulin
2
Q
What cells are class 1 antigens located on?
A
Most nucleated cells including platelets.
not on corneas, neurons, epithelial cells or germinal cells
3
Q
What are the class 1 antigens found on RBC’s?
A
Residual amounts are found on RBC’ membrane called Bg antigens (Bennett Goodspeed)
4
Q
What HLA does Bga represent?
A
B7
5
Q
What HLA does Bgb represent?
A
B17
B57 or B58
6
Q
What HLA does BgC represent?
A
A28
A68 or A69
7
Q
What HLA antigens are found on platelets?
A
mostly A and B with some C at low levels
8
Q
What are the Class II HLA antigens?
A
DR, DQ, and DP
9
Q
What is the structure of Class II HLA antigens?
A
Alpha and heavy chain
10
Q
What cells are Class II antigens found on?
A
B lymphs, monocytes, macrophages, dendritic cells, intestinal epithelium, and activated T lymphs
11
Q
What is the function of Class I antigens?
A
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
12
Q
What is the function of Class II antigens?
A
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).
13
Q
What are class III HLA molecules?
A
Tumor necrosis factor (TNF)
Steroid enzyme 21-hydroxylase
Complement C2, Bf, C4A (Rogers), C4B -(Chido) = Absorbed onto RBC membrane
14
Q
What the common public antigens?
A
Class I: HLA Bw4 and Bw6
Class II: HLA DR51, DR52 and DR53
15
Q
What are splits?
A
HLA antigens historically believed to be one specific was split into multiple specifies because of more sensitive testing and molecular methods
16
Q
What are GREG’s?
A
Cross reactive epitope group
HLA antigens that share common epitopes and have cross reactivity with serological testing
17
Q
What is the nomenclature for HLA antigens?
A
HLA-DRB1*04:01 Locus = DR Protein = B1 chain * = typed by molecular method Serological allele = 04 (antigen) Variant of allele = 01 (subgroup)
18
Q
What is a centimorgan (cM)?
A
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
19
Q
What is SSO or SSOP?
A
Sequence specific oligonucleotide probe is labeled and used to detect sequences in immobilized DNA (microbead or membrane)
20
Q
What is rSSO?
A
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
21
Q
What is SSP?
A
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
22
Q
What is DNA sequencing?
A
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.
23
Q
What is STR DNA analysis?
A
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
24
Q
What is the HLA antigens show linkage?
A
B and C are closely associated and can predict what C is based on B
DR and DQ are closely associated
DP has recombination
25
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
26
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
27
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
28
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
29
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.
30
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
31
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.
32
How are B and T cells separated in Flow cytometry?
```
CD3-PerCP = T-cells
CD19-PE = B-cells
```
33
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
34
What HLA antigen is associated with Celiac Disease?
DQ2 (RR>250)
35
What HLA antigen is associated with ankylosing spondylititis?
B27 (RR >150)
36
What HLA antigen is associated with Narcolepsy?
DQ6
37
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.
38
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.
39
What is TA-GVHD?
Donor T cells (graft) proliferates in recipient (host)
Depends on host immunocompetence & HLA dissimilarity
90% - 95% fatality rate
40
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.
41
What type of XM and HLA match is required for Liver transplant?
ABO is most important. HLA matching not important because liver’s unique abilities to act as a sink for anti-HLA antibodies and to regenerate itself if destroyed by antibody
42
What type of XM and HLA match is required for Kidney Transplant?
Both recipient and donor are tested for HLA-A, -B, and –DR antigens; Donors are tested for HLA-C and –DQA and -DQB
PRA is positive must avoid HLA antibodies
Must perform XM with donor lymphs
43
What is a hyperacute graft rejection?
Occurs immediately due to antibody
44
What is an acute graft rejection?
Occurs after 5 days due to cellular mechanism
Acute GVHD typically presents anywhere from a few days to 100 days after transplantation. The skin, gastrointestinal tract and liver are most commonly involved
45
What is chronic graft rejection?
Occurs after 30 days due to cellular and antibody mechanisms
Chronic GVHD typically occurs months after transplantation. In addition to the acute GVHD symptoms chronic autoimmune typed of disorders such as biliary cirrhosis, Sjogren’s syndrome, and system sclerosis may develop.
46
What HLA antigens are matched in HSCT?
```
10/10 match is ideal: A, B, C, DR, DQ
HLA-A, HLA-B, HLA-C, DRB1 (8/8)
6/6 match: A, B, DR
High resolution DNA method
Avoid DRB1 and C mismatches
ABO not important
```
47
What is ASHI?
American Society of Histocompatibility and Immunogenetics; organization that governs the field of HLA
48
What is a Syngeneic Transplant?
A graft taken from an individual of the same genotype as the recipient, such as a monozygotic twin.
49
What is a Xenogeneic Transplant?
A graft transplanted from one species to another.
50
What is lymphocytotoxicity testing used for?
Used to detect Class I HLA-A-B-C and Class II DR-DQ antigens. Lymphocytes are used because they are readily available from blood and give reproducible results. Lymphocytes from lymph nodes and spleen may also be used. This test is also used for HLA crossmatching
51
What anticoagulant is used for HLA typing and what temperature is specimen stored at?
ACD is anticoagulant of choice but sodium heparin may be used. Specimens that require only DNA testing may be drawn in EDTA also. Specimens are stored at RT.
52
How is PRA measure by flow?
Testing the patient’s serum against a panel of 30 to 60 different target cells determines the PRA and often the specificity of the antibodies. In highly sensitized patients testing can include the use of beads or microparticles coated with single purified HLA antigens. Antibody coating is detected utilizing a fluorescent-labeled antihuman globulin and read on a flow cytometer for increased sensitivity.
53
What specimen is required for HLA antibody testing?
Plain red top tube with NO anticoagulant is required (serum must be used, not plasma). Specimens should be stored refrigerated until tested.
54
What is HPC peripheral blood process?
The donor is given a hematopoietic growth factor to provide for better HPC mobilization and recovery. Recovery is performed by apheresis machines. The product is taken to a laboratory where minimal processing will be performed. Volume reduction will often take place. A sample will be removed for bacterial culturing and for CD34 stem cell counts. The cryoprotectant dimethylsulfoxide (DMSO) is added to the product before controlled-rate freezing. The unit is placed in the vapor phase of liquid nitrogen for storage.
55
Why are T cells depleted?
Decreases the incidence or at least lessen severity of GVHD
56
How do you calculate relative risk
(# with HLA antigen & disease) X (# NO HLA antigen NO disease) /
(# with HLA antigen NO disease) X (# without HLA antigen & disease)
57
How can ABO incompatible Kidney be used in transplants?
IVIG, Plasmapheresis, and rituximab
58
What are the complications of a Major ABO mismatched BMT?
immediate hemolysis of RBC's at time of infusion, pure red cell aplasia, and delayed RBC engraftment due to recipient antibodies to graft
59
How can adverse effects of Major ABO Mismatched BMT be avoided?
Wash RBC's from BMT product, but may lose valuable stem cells
Transfusion donor type secretor plasma to bind recipients antibodies and dilute.
Use plasmapheresis to remove isohemagglutins from donor
60
What are the complications of minor ABO mismatched BMT's?
Recipients RBC's are hemolyzed by isohemagglutins produced by donor lymphs. Occurs 7-14 after transplant.
61
What is PLS?
passenger lymphocyte syndrome - Donor lymphs in solid organ or BMT produce antibodies that hemolysis recipients RBC's
Plasma reduction of BMT does not help
Rituximab may reduce incidence
62
What is the CD marker for HSC's?
CD34
63
When is autologous BMT used?
Used as rescue after antineoplastic therapy.
64
What screening tests and questions are required for auto BMT collection?
Health assessment is required
Question on infections disease is not required
Infectious disease testing is required because products are cryo-preserved and stored with other products presenting a risk.
65
When is allogenic BMT used?
To treat malignant disease when an graft vs tumor effect is needed or congenital blood disorders
66
What level of matching may be acceptable for a UCB?
Matching at 4 of 6 loci is sufficient for
HLA-A and -B at the antigen level and for HLADRß1 at the allele level
This is because UCB cells have naïve immune function and are privative in nature.
67
What drugs are used before BMT apheresis?
G-CSF - stimulate cells to proliferate
| plerixafor - helps mobilize cells to PB
68
What preservative is used in cryopreserved HSC's?
DMSO is a colligative cryoprotectant; it diffuses rapidly into the cell, reducing the osmotic stress on the cell membrane. DMSO prevents dehydration injury by moderating the nonpenetrating extracellular solutes that form during ice formation. It also slows extracellular ice crystal formation
69
What QC is routinely performed on HSC's?
Cell count and differential, viability, CD34+ cell enumeration, sterility testing, and colony forming unit assays
70
What are the advantages of using Cord HSC's?
higher proliferative and self-renewal capacity and their association with a lower incidence of graft vs host disease (GVHD), particularly acute GVHD, and less stringent HLA matching required
71
What is the optimal number of CD34 for HSCT?
2 x 10^6 CD34+ cells per Kg body wt is optimal for autologous transplants. More cells are often collected for allogeneic transplants.
72
What additive is used for cryopreservation of HSCT?
```
Dimethyl Sulfoxide (DMSO ) is most commonly used at a concentration of 10%. It is an intracellular cryoprotectant that prevents ice crystals from forming within the cells and disrupting the cellular membranes.
HES is an extracellular, non-penetrating cryoprotectant that is less commonly used.
```
73
How long does engraftment take?
Engraftment should occur within 100 days of transplant but usually happens much faster (10 to 30 days for PMNS, 15+ days for platelets). Once engraftment occurs, patients can leave isolation.
74
How is engraftment monitored?
It is evidenced by recovery of neutrophil, platelet, and red cell production (absolute neutrophil count > 500 cells/ul, platelets > 20K/ul).
75
What HLA antigen is Type 1 diabetes associated with?
DQ8
76
What HLA antigen is Multiple sclerosis associated with?
DR15 and DQ6
77
What is HLA antigen is rhuematoid arthritis associated with?
DR4
78
What is direct exclusion?
When child has a marker that father lacks
79
What is indirect exclusion?
When child lacks a marker that father should have passed on.
Ex) child appears homozygous for Fya and father appears homozygous for Fyb.
80
What is PI?
compares two mutually exclusive hypotheses that are expressed as a likelihood ratio. Probability of producing a child from the known parent with alleged parent and a randomly selected male of same ethnicity
PI greater than 100 is very strong
81
What is PP?
probability of parentage (PP) - is the PE expressed as a percentage.
82
What is PE?
The ability of a genetic marker to exclude a falsely accused man of paternity is known as power of exclusion.
a combined PE of 0.9925 = 99.25% of random individuals will be excluded
83
What is ionizing radiation?
When an atom is exposed to ionizing radiation there is enough energy to remove electrons from their orbits causing the atom to become charged or ionized
Examples: photons (x-rays, gamma rays), alpha and beta particles, neutrons
84
What is ionizing radiation?
When an atom is exposed to ionizing radiation there is enough energy to remove electrons from their orbits causing the atom to become charged or ionized
Examples: photons (x-rays, gamma rays), alpha and beta particles, neutrons
85
What are gamma rays?
A photon, similar to ordinary visible light but shorter in wavelength than ultraviolet light, hence the higher energy level
Produced following spontaneous decay of cobalt-60 or cesium-137
Can travel unimpeded for great distances
Can easily go right through people but can be blocked by lead
86
What is half life of Cesium 137?
The half life of Cesium 137 is 30.2 years
| The time required for one-half of the original atoms in a sample to decay
87
What is RFLP?
restriction fragment length polymorphism is DNA is DNA is cut with restriction endonuclease, electrophoresis, followed by southern blotting and hybridization of VNTR.
88
What is currently used for relationship testing?
Lab use PCR to analysis STR (short tandom repeats)
89
What is required when performing serological testing to determine paternity?
Test must be done in duplicate, ideally by two separate tech's
90
Which ABO system has the highest PE?
HLA-A and HLA-B = 0.87
MNSS = 0.31
Rh system = 0.27
91
When does GVHD occur?
incomplete histocompatibility between donor and recipient
Immunocompetent cells are present in the transfused product
the recipient is unable to reject the donor cells
92
What organs are affected in GVHD?
Spleen, Liver, GI, Bone marrow, lymph nodes, and skin
93
What are the signs and symptoms of GVHD?
Fever, Rash, Loss of appetite, nausea, Vomiting, Diarrhea, hepatitis, bone marrow suppression, and death
94
Who is at risk for GVHD?
```
Immunocompromised individuals:
-Bone marrow transplant patients
-Chemotherapy patients
-Radiation therapy patients
-Fetuses and newborns
-Heart, lung and liver transplant patients
Non-immunocompromised individuals:
-Recipients with same haplotype as the donor (genetically similar)
-Blood relatives of the donor
```
95
What is the treatment for GVHD?
Acute GVHD occurs with 100 days and treatment is usually unsuccessful
Patients with chronic GVHD may respond to immunosuppressive regimens including steroids and alkylating agents
Prevent is key
96
What is radiation?
Radiation is the process by which energy is emmitted or propagated in the form of rays, waves, or particles
97
What is radioactivity?
The spontaneous disintegration of radioactive substances (radioactive isotopes) in which the nuclei undergo breakdown (decay) and give off penetrating radiation (in the form of particles or rays)
98
What is RAD?
radiation adsorbed dose
RAD has been replaced by gray
1 Gy = 100 rads
quantity of ionizing radiation
99
What is REM?
Roentgen Equivalent Man
Accounts for difference in biological effectiveness
1 sievert (Sv) = 100 rem
100
What is ALARA?
ALARA is the acronym for As Low As Reasonably Achievable, a radiation safety principle for minimizing radiation doses and releases of radioactive materials by employing all reasonable methods.
