Organ Transplantation 21/03/23

Question 1

What is transplantation?

Answer 1

Transplantation is the act of transferring cells, tissues, or organs from one site to another.

Question 2

What is the history of transplantations?

Answer 2

-During WWII: treatment of burned airmen
-1954: Joseph Murray performed first successful kidney transplant from living donor (Nobel prize)
-1960s: introduction of immunosuppressive drugs
-1967: Dr Christian Barnard carried out the 1st human heart transplant
-Improved immunosuppression
-Increased numbers of tissues and organs being transplanted
-Databanks and transplant registry
-Ability to diagnose rejection episodes
-Future: xenogeneic transplantation

Question 3

What are the different types of transplants?

Answer 3

-Autograft
-Isograft
-Allograft
-Xenograft

Question 4

What is an autograft?

Answer 4

Tissue is derived from ‘self’, can be transplanted back to the same place or another site.

Question 5

What is an isograft?

Answer 5

In 1954 Joseph Murray bypassed the barrier of rejection by using the patient’s identical twin as the donor of a human kidney transplant - the first successful transplant.

Question 6

What is an allograft?

Answer 6

Tissue transferred from one individual to another (genetically non-identical, same species)
This is by far the most common form of transplant and includes:
✓ Kidney, Heart, Pancreas, Lung, Liver, Bowel (‘solid organ’)
✓ Islet
✓ Bone
✓ Cornea
✓ Skin
✓ Tendon
✓ Cartilage
✓ Stem cell

Question 7

What is a xenograft?

Answer 7

-Tissue transferred from one species to another (eg. Heart valves)
-Whole organ xenografting limited by potential for hyperacute rejection
-Current research aimed at making animals that are ‘humanised’ – lots more progress required
-‘Patient derived Xenografts’ are proving useful for cancer research

Question 8

What are immunological privileged sites?

Answer 8

Immunologically privileged sites are sites where grafts are not rejected, for example, the cornea.

Successful transplant of corneal allograft from cadaveric donor requires no assessment of HLA type and no administration of immunosuppressive drugs. Lack of rejection is due to naturally immunosuppressive environment in anterior chamber of eye and lack of blood vessels in the cornea.

Question 9

What is the biggest barrier to overcome with transplants?

Answer 9

The HLA.

Question 10

What is the HLA?

Answer 10

HLA is the Human Leucocyte Antigen.

-There are 6 ‘classical’ HLA loci, Class I (A,B,C) & Class II (DR,DQ,DP) each encoded by separate genes
-These molecules allow tissue to be recognised as ‘self’ or ‘non-self’ by the host immune system and therefore determine histocompatibility.

Question 11

What is the role and expression of HLA?

Answer 11

*The primary function of these antigens is to serve as recognition molecules in the initiation of an immune response so they are very polymorphic
*HLA antigens present peptides from foreign substances to effector cells of the immune system (mainly T-cells)
*HLA class I are expressed on nearly all cells and recognise pathogens that reside inside the cells (e.g. viruses)
*HLA class II are only found on immune cells and recognise pathogens that reside outside the cell (e.g.
bacteria

Question 12

What is the nomenclature for HLA?

Answer 12

-HLA-A defines the locus
-HLA-A24 Shows the serologically defined antigen
-HLA-A24 asterisk denotes that the allele has been defined by molecular methods (low resolution)
-HLA-A24:01 shows higher resolution, specific allele (required for HSCT)

Question 13

How are HLA inherited?

Answer 13

Parents have two HLA each so there can be a combination of 4 different haplotypes for offspring. This means there is a 25% of having an identical sibling.

Question 14

How is HLA typing serology done?

Answer 14

All potential organ recipients and donors must have their major HLA loci determined to minimise the chance of rejection. Also applies to bone marrow transplantation. ‘Terasaki trays’ used – plates with serum containing anti-HLA antibodies, patient cells and complement added, death occurs in
wells where antibody reacts with patient sample. Dyes show live (green) and dead (red) cells.

Question 15

How is HLA typing done with molecular methods?

Answer 15

All molecular methods require the extraction of high quality genomic DNA. In NHS laboratories this is frequently achieved using a semiautomated system that extracts genomic DNA from whole blood. It is also possible to isolate DNA from buccal swabs, saliva samples and fingernails.

Sequence specific primer (SSP) PCR is often the first step in determining HLA type. SSP tests consist of multiple different PCR primers specific for known HLA polymorphisms and are supplied in a kit format. Specific amplicons are produced if the primers are complementary to the sample DNA.

Question 16

How is HLA typing done with B27 and B57?

Answer 16

Individuals who are HLA-B27 (around 8%) have an increased risk for ankylosing spondylitis and other inflammatory disorders. Suspected AS cases will be screened for HLA-B27 as 95% of AS sufferers are B27 positive. HLA-B57 is associated with drug-induced inflammatory disorder. All HIV positive patients in the UK are screened for HLA-B57 prior to beginning Abacavir (reverse transcriptase inhibitor) treatment.

Question 17

What is anti-HLA antibody identification?

Answer 17

It was recognised over 40 years ago that recipients may have antibodies to antigens expressed on donor cells & this is a major risk factor for hyperacute rejection. These antibodies may arise from pregnancy, blood transfusion or previous transplantation. CDC crossmatch assays have been used successfully for several years, recipient sera is incubated with donor lymphocytes in presence of complement. Same principle as Terasaki trays: Complement Dependent Cytotoxicity.

Question 18

Why may people need transplants?

Answer 18

-Kidney failure
-Heart failure

Question 19

Where are donors sourced?

Answer 19

Living donors:
-Family members/friends can opt to donate kidneys or a part of their liver (subject to ABO/HLA compatibility)
-Altruistic donation also sometimes occurs
-Paired/pooled donations are an option when relatives do not match
Cadaveric donors:
-Those which are deemed to be ‘brain stem dead’ following appropriate testing
-Donation after circulatory death (DCD) or non-heart beating donors usually occurs after admittance to A&E

Question 20

What are the issues around consent?

Answer 20

-Recent opinion poll study data suggests that 90% of the UK population support organ donation
-In practice, only 68% provide consent in the event of a family member being declared brain dead
-The disparity is likely due to the effects of shock and grief in the situation
-We now have an ‘opt out’ system following Max and Keira’s law
-Even with ‘deemed consent,’ medics allow family input.

Question 21

How are transplants allocated?

Answer 21

-For kidney and pancreas, allocation is based on blood group match and HLA-A, B and DR, 000 mismatch are given priority
-Paediatric patients are always prioritised, then sensitization, waiting time, age match and location are also considered
-These factors should prevent a patient waiting for many years, avoid older organs being given to young patients and also reduce cold ischaemic time
-One HLA-A mismatch is considered, as is one HLA-B mismatch (100 and 010)
-For heart and lung, the main factors are ABO match and HLA-DR
-There are far fewer of these transplants and short CIT is essential
-The size of the heart relative to the donor is also important

Question 22

What is the pre-transplant crossmatch?

Answer 22

For all transplants, a crossmatch is always performed immediately prior to surgery. Recipients are screened prior to entry on the waiting list, but their antibody status may have changed (hyperacute risk). For heart and lung, logistics may prevent the pretransplant crossmatch, so a virtual crossmatch is performed instead.

Question 23

What is graft rejection?

Answer 23

-Can be hyperacute (should never happen!)
-Acute (sometimes happens despite best medical care)
-Chronic (always happens to some degree with solid organ)

Question 24

What is hyperacute rejection?

Answer 24

-Mediated by the humoral response
-Usually seen within minutes of transplantation
-Results from pre-existing donor-specific antibodies in the recipient or accidental ABO mismatch
-Antibodies activate the complement pathway, initiating the blood clotting cascade

Question 25

What is acute rejection?

Answer 25

-Caused by activation of T lymphocytes and occurs over several days
-Mismatched HLA causes cytotoxic T cell attack of endothelial cells
-The recipient can also form de novo DSAs, causing antibody-dependent, cell-mediated cytotoxicity
-These processes can be avoided through HLA matching and immunosuppressants

Question 26

What is chronic rejection?

Answer 26

-Multiple immune mechanisms, cell mediated and humoral
-End result: Vascular disease
-Alloantibodies bind to endothelial cells and recruit Fc-receptor bearing monocytes/MQ
-Inflammatory components in the vessel wall leads to damage, thickening of the vessel wall and narrowing of lumen, inadequate blood supply and damage
-Can involve lymphocytes, phagocytes, antibodies, complement

Question 27

What are immunosuppressive drugs?

Answer 27

-Allogeneic transplantation requires some degree of immunosuppression, by drugs, if the transplant is to survive
-In the 1950s, sub-lethal doses of total body irradiation (TBI) were combined with cortisone. This was a problem as patients died from infections
-Today, medics aim to find a balance between reducing rejection whilst maintaining a reasonable immune system

Question 28

What is the first successful xenograft?

Answer 28

Last January (2022), the first ever porcine to human heart transplant was successfully performed.
The patient was ineligible for the transplant waiting list and on ECMO bypass with only weeks to live, compassionate emergency approval was provided by the FDA. The heart was obtained from a genetically engineered pig, aimed at reducing acute rejection. It was successful but he only lived for 2 weeks.

Question 29

What are the barriers to xenografting?

Answer 29

Primate:
Advantage - concordant
Disadvantage - scare, endangered, ethical concerns, retrovirus

Monkey:
Advantage - concordant
Disadvantage - small size, retrovirus

Cow/horse/sheep:
Disadvantage - disconcordant, large size

Dog:
Advantage - size
Disadvantage - disconcordant, ethical concerns

Pig:
Advantage - partly concordant, size-compatible
Disadvantage - require genetic engineering

Question 30

What are the barriers to xenografting with pigs?

Answer 30

Genetic modification must be performed to avoid hyperacute, acute and chronic rejection. Given the relative advantages of porcine models, these have been most extensively studied and developed.

The key genetic modifications to date are:
1. Knock-out of porcine carbohydrate genes (GAL)
2. Induced expression of human complement and coagulation regulatory molecules

Question 31

What is donation after circulatory death?

Answer 31

DCD (also called non-heart beating donation) is the retrieval of organs from patients whose death is diagnosed and confirmed using cardiorespiratory criteria.

There are two types:
1. Controlled, takes place after death which follows the planned withdrawal of life-sustaining treatments
2. Uncontrolled, refers to organ retrieval after a cardiac arrest that is unexpected and from which the patient cannot or should not be resuscitated

Question 32

What is the DCD in the uk?

Answer 32

DCD has significantly increased in the past decade and now represents ~40% of all deceased organs.

Question 33

What is the islet?

Answer 33

Pancreatic islets, also called islets of Langerhans, are groups of cells in your pancreas. The pancreas is an organ that makes hormones to help your body break down and use food. Islets contain several types of cells, including beta cells that make the hormone insulin. 30% glucagon-producing α-cells, 60% insulin producing β-cells, with the remainder 10% made up of δ-cells (somatostatin-producing).

Question 34

How is islet transplantation done?

Answer 34

1. Donor pancreas - insulin producing islet in the pancreas
2. Ricordi chamber - key islet isolation device
3. Separated islets
4. Islets are introduced into the liver of the recipient
5. Transplanted islets secreting insulin in the liver

Question 35

What are the advantages of islet transplantation?

Answer 35

Can remove the need for insulin injection
Prevents hypos
Improves overnight control
Transplant surgery is easy, quick and minimally invasive
Potential for xenotransplantation?

Question 36

What are the disadvantages of islet transplantation?

Answer 36

Only 2% of the donor pancreas is islet material
Repeated transplants often necessary
Shortage of donors
Immunosuppression required – side effects

Question 37

What is the future of islet transplantation?

Answer 37

The use of induced pluripotent stem cells from mice could be used in the future. Authors generated mouse-rat chimaeric islets then transplanted them into a chemically-induced diabetic mouse model. The mice were normoglycemic for over a year with no immunosuppression.

Process:
1. Generation of autologous IPSCs
2. Interspecies blastocyst complementation
3. Removal of pancreata and isolation of islets
4. Transplantation of islets into diabetic mice

Question 38

What is a bone marrow transplantation?

Answer 38

Bone marrow transplantation is the transfer of bone marrow cells from one human to another. Bone marrow transplantation can be used to treat:
o Leukaemia
o Immunodeficiency/anaemias

Today, over 350 centres in Europe are performing more than 20,000 bone marrow transplants a year. Serious complications such as graft-versus-host disease.

Question 39

What are haematopoietic stem cells?

Answer 39

HSC can self-replicate & differentiate into any of the formed blood elements. HSC can be harvested
from cultures derived from bone marrow cells and are reinfused to reconstitute damaged bone marrow. HSC also found in small numbers in the peripheral blood.

Question 40

What are the sources of stem cells for transplant?

Answer 40

Patient - autologous
Matched sibling - only 25% chance of a match
Match unrelated donor - 50% chance of a match but outcome less favourable than sibling. UK centres only accept matches that are at least 9/10 at HLA-A/B/C/D/DR and DQ to reduce GVHD
Umbilical cord blood - restricted availability but better outcomes than MUD, less GVHD

Question 41

What is autologous HSCT?

Answer 41

Autologous - the transplanted stem cells have been previously taken from the patient
-Bone marrow or peripheral blood stem cells
-Less complicated – MHC compatible
-Allows for high dose chemo & radiotherapy pre-transplantation which will irreversibly damage the
bone marrow.

Question 42

What is the process of autologous HSCT?

Answer 42

1. Mobilise stem cells using growth factor and chemotherapy
2. Stem cells collected from peripheral blood
3. Stem cells frozen until required
4. Conditioning chemotherapy to suppress immune system
5. Stem cells thawed and re-infused
6. Support with blood products and antibiotics
7. Further follow up as outpatient for 2-3 months with regular blood tests and medication

Question 43

What are the differences between cord blood and bone marrow?

Answer 43

Cord Blood

-Collection is non-invasive, painless, and poses no risk to the donor
-Graft versus Host Disease (GVHD) is reduced to 10% due to the absence of antibodies in the stem cells
-Units are processed and ready for transplant
-Significantly less expensive

Bone marrow

-Collection is invasive and painful. Must be performed in a hospital surgical setting
-Due to the maturity of the stem cells, it requires a greater HLA match to perform a transplant
-Serious GVHD occurs in 60% of all unrelated bone marrow transplants
-Bone Marrow is dependent on donor participation

Question 44

What is graft-versus-host disease?

Answer 44

Occurs when immune cells from the donor (largely T-cells), attach the recipient’s tissues. GvHD is reduced by HLA matching as highly as possible.

Question 45

What is the graft versus leukaemia effect?

Answer 45

Occurs when immune cells from the donor (largely T-cells), eliminate residual leukaemia cells in the
recipient. GvHD and GvL often occur simultaneously.

Question 46

What is post-transplantation chimerism analysis?

Answer 46

*The level of engraftment post-transplant must be measured – gives an indication of outcome
*For malignancies, 100% donor engraftment is required to prevent relapse
*For anaemias and enzyme deficiencies, mixed chimerism may be enough to correct the disorder
*Chimerism is most often measured through STR analysis (STR = short tandem repeat)

Question 47

What organs determine the grade of reactions?

Answer 47

Liver
Skin
GI tract