IBTS - HLA Flashcards
What is HLA
Human Leucocyte Antigen
Cell surface glycoproteins involved in antigen presentation to T cells
What is MHC
Major Histocompatibility Complex
The region of chromosome 6 which includes the genes coding for HLA proteins
What is the role of HLA antigens in the immune system
HLA type allows the immune system to discriminate from self and from non-self
What does class I MHC do?
Binds peptides derived from endogenously synthesised proteins (eg viral proteins in virus infected cells) they present the peptides to CD8+ T cells (primarily cytotoxic T cells)
What does class II MHC do?
Bind proteins from exogenous proteins (ie those in the external medium) they are internalised by professional antigen presenting cells and presented to CD4+ T cells (primarily helper T cells).
This ensures that cells harbouring infectious agents are recognised and attacked by Class I restricted CTLs, whereas Class II restricted helper T cells respond to soluble antigens and activate antibody producing B cells.
Talk about the inheritance of HLA
Each person inherits two copies of chromosome 6 - one from mom and one from dad
HLA genes are very polymorphic -> thousands of different HLA alleles
HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP genes involved
e.g. over 2000 HLA-A alleles
What HLA labs do we have in the IBTS
A Molecular Biology lab
- HLA and HPA genotyping
- SOO, SSP, Gel electrophoresis, NGS etc
Serology lab
- screening patient sera for anti-HLA and anti-HPA alloantibodies
Why would we need to carry out HLA typing
(6)
Haematopoietic stem cell transplant -> HLA matching
Platelet refractoriness -> HPA antibodies
TRALI -> HLA antibodies
Disease susceptibility e.g. ankylosing spondylitis
Drug sensitivity e.g. abacivar
Forensic investigations
Write about HLA and ankylosing spondylitis
Disease causing fusion of the vertebrae
HLA-B*27
90% of people with AS have the HLA*27 gene
Doesn’t mean you will have the disease but high association
Write about Abacivar and HLA
Abacivar is a reverse transcriptase inhibitor used in the treatment of HIV/AIDS
A single nucleotide polymorphism at HLA B*57:01 has been associated with hypersensitivity to this drug
Very rare but hypersensitivty can be fatal so patients are typed before administering the drug
Give three sources of cells for allogenic transplants
Bone marrow
Peripheral stem cells
Cord blood
Give four different types of donors used in transplants
HLA identical siblings - preferred
Unrelated but matched donor
Sibling/unrelated mm
Haploidentical
Why are HLA antigens so important in transplants
HLA antigens are the major antigens which determine the compatibility of transplanted tissues and organs
What occurs in HLA unmatched transplants
Graft versus host disease
When might you want GvHD
Want a degree of it when a bone marrow transplant is given to leukaemia patients
Want graft versus leukaemia affect
What is Graft vs Host disease
This is where the donor white blood cells recognise the host tissue as foreign and initiate an immune response
Acute or chronic
Unrelated donor transplants are associated with an increased incidence of acute GvHD and graft failure compared to matched donors
Talk about acute GvHD
Occurs in less than 100 days post transplant
Affects the skin, liver and GIT
Occurs in 40% of sibling transplant and 50% of unrelated donors
Talk about chronic GvHD
> 100 days post transfusion
Affects skin, liver, gut (as in acute) but also affects eyes, glands, CT and lungs
What is IUBMR
Irish Unrelated Bone Marrow Registry
What is the Irish Unrelated Bone Marrow Registry
A registry of donors who are prepared to donate to a matched patient
The IUBMR performs unrelated searches on behalf of the adult and paediatric services
Not ideal -> ideal donor is a HLA matched sibling -> 1 in 4 chance of a sibling being a match
Smaller family sizes means IUBMR being used more now
What is the gold standard resolution method for HLA typing
Next generation sequencing
Serology can be used but its very low resolution in comparison to NGS
What HLA typing methods can be used
SSO for HLA typing
SSP for HLA typing
Luminex for HLA antibody screening and ID
Sanger sequencing for high resolution HLA typing
NGS for high resolution HLA typing
How is DNA extraction carried out for HLA typing
Ez1 Biorobot
Magentic particle technology
Pre-filled, sealed reagent cartridges
Pre-programmed cards with different protocols
Complete automation of nucleic acid purification with no manual centrifugation steps
What is SSO
Sequence Specific Oligonucleotide
Also called Histo Spot
Histo Spot assay with Mr spot processor allows automated SSO typing
Low to medium resolution
Can type up to 96 samples at a time
What is SSP
Sequence Specific Primers
Works by targeting a particular DNA sequence
Several PCRs are preformed with a battery of SSPs which will amplify only alleles with sequences complementary to the primers
The presence of amplified material indicates the presence of the corresponding allele or group of alleles
The PCR product is seen as band on a gel
Advantages: particularly used for high resolution typing and is relatively fast
Disadvantages: laborious, small sample numbers
Talk about Sanger sequencing
Capillary based Sanger sequencing
Class I (A, B, C) Exons 2, 3 and 4
Class II (DRB1, DQB1) Exons 2 and 3
Group specific sequencing primers used to resolve ambiguities
Software processes the DNA sequence data and assigns a HLA type by camparison with a database
Why are anti-HLA antibodies important
Platelet refractoriness
Donor specific antibodies
Why are anti-HPA antibodies important
Platelet refractoriness (where HLA has been ruled out)
Neonatal alloimmune thrombocytopenia (NAIT)
Post transfusion purpura (PTP)
Define platelet refractoriness
The failure, in two consecutive transfusions of ABO identical platelets <3 days old, to achieve a corrected count increment (CCI) of 7.5 ay 15 minutes to 1 hour after the platelet transfusion
i.e. the platelet transfusion does nothing
What are the two types of platelet refractoriness
Non-immune mediated
Immune mediated
What is the corrected count increment for platelets
7.5 after 15 mins to 1 hour
What are the two types of platelet refractoriness
Non-immune mediated
- fever
- infection
- hypersplenism
- bleeding
Immune mediated
- antibodies!
What are the two main causes of immune platelet refractoriness
Anti-HLA
Anti- HPA
How do we screen for platelet refractoriness antibodies
Luminex technology
- flow cytometry based instrument
-
What are donor specific antibodies
Made when a stem cell transplant patient receives a HLA mismatched transplant
Could cause graft rejection
Can reduce antibody titre through plasmapheresis or may need to look at alternative donors
What are anti-HPA antibodies
Antibodies produced against antigens on platelets
Human platelet antigens are polymorphisms in platelet membrane glycoproteins that can stimulate production of alloantibodies once exposed to foreign platelets with different HPAs
What complications are caused by anti-HPA antibodies
Platelet refractoriness
- no response to platelets
- usually the cause when HLA antibodies ruled out
Neonatal alloimmune thrombocytopenia (NAIT)
Post transfusion purpura (PTP)
What is NAIT
Neonatal Alloimmune Thrombocytopenia
Severe thrombocytopenia caused by maternal alloimmunisation against human platelet antigens present on fetal cells (platelet version of HDFN)
What is the most common type of NAIT
HPA-1a -> seen in 80% of cases
HPA-5b seen in 5-15% of cases
