Exam revision May 23 - Transfusion Science

Question 1

In what circumstances and for what reason would you perform a “Group and Save”?

Question 2

What mechanisms might cause the bone marrow to produce macrocytes and hyper segmented neutrophils?

Question 3

What does IAT stand for? Using a diagram explain the principle of an IAT test.

Answer 3

This test compares patient plasma against donor plasma and is used to identify clinically significant antibodies IgG ABO RH at 37oC before transfusion by observing agglutination.

Question 4

What do ewe mean by the term “clinically significant antibody” in transfusion science? List some below.

Question 5

With respect to transfusion-related antigen-antibody reactions what do we mean by the term “dosage”?

Question 6

In an antibody identification panel, what happens when we “enzyme treat” red cells? Name two antibodies that are enhanced by enzyme treatment and two antibodies where the reactions are no longer seen when enzyme treated panels are used.

Question 7

Explain how the results help you to narrow down your differential diagnosis (table of results as pic).

Question 8

List any potential sensitising events below and explain how these could have led to the production of alloantibodies.

Question 9

Why is it important to run both positive and negative controls on a DiaMed card?

Question 10

What is Christina’s ABO and Rh D type? (add pic of blood results)

Question 11

What antibodies area present on the antibody ID panel? Are there any that you are unable to exclude? (add pic of panel)

Question 12

What are the potential treatment options for Christina?

Question 13

Do you think Christina requires a transfusion? Using current guidelines to support your answer, explain your reasoning. In your answer, detail the type of component and any relevant specifications to be considered.

Question 14

Why is the RH blood group system clinically significant?

Question 15

Describe what happens in HDFN

Answer 15

Pregnancy – 20% of D- women will carry a ABO compatible D+ foetus – HDFN: haemolytic disease of the foetus newborn – and unless they are given injections of anti D-antibody to clear foetal cells from circulation before they are responded to in the 2nd or 3rd trimester, then the maternal cells will attack the foetus RBCs with their own anti-D antibody, causing haemolysis to the baby’s RBCs, anaemia, brain damage and death.
Woman = Rh D-
Babber = Rh D+ (gene from Dad allows expression of RhD+: annoying)
Immune system = Create IgG antibody to cross placenta (IgG can do this) and destroy the Rh D+ antigen which we do not recognise.
Medication = give prophylactic Anti-D antibody (human) which will coat foetal cells and cover the antigen to stop it being seen by maternal immune system. The cells can then be cleared from circulation and prevent an attack by immune system.

Question 16

Is there any interesting epidemiology around the Rh blood system?

Answer 16

Most people are D+ (people call this rhesus positive which is WRONG) so they have the D antigen present on their cells.
Japan – high in D neg but in 15% are D neg in UK.
D antigen is a very large protein which makes is highly immunogenic as it contains many epitopes. There are lots of foreign edges to it 😊 that the body will not recognise.
A study in the 50’s, using USA prisoner subjects, saw that >80% of people receiving RH D+ blood will make anti-D antibodies to it in response to a transfusion. 30% of patients infused with RH D- made anti-D antibodies in voluntary cases. Higher than the response rate to other blood group system antigens.
See slides as there is a whole slide on this which you need to know about - als odo not forget about the cyrpus link with pernicious anemia….

Question 17

Explain how the RH system in inherited using the Mendelian system

Answer 17

RH genes are on chromosome 1. There are two genes; the D gene and the CE gene (gives cC or eE).
One gene from each parent and the genes are co-dominant. It is mendelian inheritance.
Mendelian inheritance refers to certain patterns of how traits are passed from parents to offspring. These general patterns were established by the Austrian monk Gregor Mendel, who performed thousands of experiments with pea plants in the 19th century.
One gene from each parent; either a CE gene or a D gene.
The CE gene is gives a version of the protein which might have C or little c, E or little e.
The antigen expressed is dependent on the gene inherited. If we inherited the C, E and D this would be referred to as a triplet. If the D gene was lacking, there would only be a C and E and this would be portrayed by writing it as CEd – the small d represents that the d antigen is not present.

Question 18

Describe the structure of the RH proteins and how they creates different antigens.

Answer 18

The structure of RH antigens is complex. They span the RBC membrane 12 times and with a carrier molecule of 400+ amino acids. Its strong confirmation is important in maintaining the structure of the RBC and is also thought to have a role in transport as it can form a channel and allow molecules to enter the RBC.
The complex molecule contains confirmational proteins and folded parts of the protein will form different epitopes. So for eg; antigen G is formed when there is a serine residue at amino acid position 103.

This epitope is also found in the G and the C antigen so if someone is D+ they have the serine residue antigen on their RBC. If they ae C+ they will also be G+.
There are different point mutations (base changes) which give rise to the differences in the epitope on the antigen =
C: serine@103 little C: proline@103 E: Pro@226 or e: alanine@226.
The difference between being D+ and D = not having the 417 extra AA proteins. They are absent. This is generally in Caucasians.
RH genes are on chromosome 1. There are two genes; the D gene and the CE gene (gives cC or eE).

Question 19

Why is Rh no longer called rhesus blood group system?

Answer 19

A woman delivered a baby and needed a transfusion. She was given blood from her ABO compatible husband but had a severe reaction. When they looked at her plasma and tested it against donors. 80% of the donor blood agglutinated. Her blood group wasn’t named. It was declared “an interesting case”. However Landsteiner decided to test further and used a rhesus macaque monkey and injected it with rabbit and guinea pig blood cells. The rhesus monkey made an antibody to the rabbit and guinea pig cells. When this antibody was tested against human RBC’s, it caused agglutination or haemolysis with 85% of cases. It was thought that the woman who had reacted to the transfusion and the rhesus monkey were making an antibody to the same structure in the donor blood. This was later proved wrong.
It was the Rh antigen and the LW antigens (monkey reacted to) that they were reacting to. The LW antigen is abundant on RH D+ RBCs and the D antigen is the main antigen in the RH blood group system.
The heteroantibody was renamed LW (after Lansteiner and Wiener) and the human auto alloantibody was named anti-D.

Question 20

What is a blood group system?

Answer 20

What is a blood group system? A system which groups human blood into different types dependent on the presence or absence of antigens on the surface of RBCs which form part of the cell membrane. It can be a specific site on a protein, glycolipid of glycoprotein. Blood group systems increase every year.
Remember that O has strands, AB has strands with both types of protein and A has only one type of protein on its strands.

Question 21

Why is ABO the most clinically significant blood group?

Answer 21

There are 43 blood group systems recognised by the Society for Blood Transfusion and ABO is the most clinically significant., followed by RH (rhesus). This has two genes RHD and RHC. ABO was discovered in 1900 by Karl Landsteiner and he got the Nobel prize. He took cells from one person, mixed the cells with serum (clotted) and not plasma (anticoagulated blood) and they became agglutinated (clumped together). He assigned different antigens to different groups dependent on the reaction. He named them A B and O (meaning empty as they had neither of the antigens on their cells). He only saw three blood types as he didn’t use many people.
Sturli discovered the fourth blood group AB in 1902.

Question 22

Explain the simple Mendelian inheritance of a blood group.

Answer 22

A and B genes are autosomal and co-dominant. (autosomal = at least one gene on one inherited allele). O gene is recessive. Either A, B or O is inherited from each parent. The only way to be O is to have the O gene from BOTH parents.
The ABO genes are on chromosome 9 and H is on chromosome 19.
If the H gene is inherited: there will be a fucoseyltransferase (enzyme) which adds a fucose substrate group to produce the H antigen. This is a precursor and enables the build of the ABO antigens on top of that.
A gene inheritance, this makes a different enzyme galactosyltransferase which puts an N-acetylgalactoseamine onto the end of the H antigen to form the A antigen.
B gene inheritance gives the glycosyltransferase enzyme which puts a galactose group onto the end to make a B antigen.
To express A or B antigens on cells, the H gene needs to be inherited. If it is NOT inherited, the cells will look like group O cells.
Inheritance

Question 23

What is the difference between a phenotype and a genotype?

Answer 23

Phenotype – genes expressed Genotype – genes inherited.

Question 24

Explain homozygous and heterozygous and give examples of each in the ABO blood group system.

Answer 24

Homozygous: the presence of two identical genes on a particular locus (ch9:ABO and ch19: H)
All offspring would inherit the A and the O so would all end up typing as A as it is dominant. The genotype is AO.

Heterozygous: having two different alleles of a particular gene. Genetically AO but phenotyping as A.
This offsrping would inherit 50% group AB and 50% heterozygous group B.

Question 25

Explain why the ABO blood group system has naturally occurring antibodies.

Answer 25

ABO have naturally occurred antibodies. There are similar structures in nature that have similar antigens that look like A and B antigens (streptococci, influenza). From the age of about 3 months, our immune system is challenged with foreign materials (dirt, etc), and the antibodies are created in preparation. Lacking the antigen on cells (group A) and encounter a group A pathogen, the antibody will be made. If the antibody is seen in plasma before the age of three months, this is likely to have been from maternal origin and it has crossed the placenta. Anti A, Anti B, Anti A,B is made by group people and is a single antibody which reacts with A and B rather than a mix of two antibodies.
IgM antibodies for ABO start to develop from 3 months old. 5 years = normal adult levels which will stay the same until elderly and the immune system begins to drop off.

Question 26

Why is the ABO blood group clinically significant?

Answer 26

ABO are highly clinically significant because they are there even though there has not been a transfusion. Its waiting for an accident 😊 we have memory cells containing ABO antibodies. There is a strong response.
The other blood group systems will not have an antibody ready and waiting. It will have come as a response to the foreign material process. There will be a delay in reaction and a weaker response.
97% of people in the UK have blood groups other than AB. They have an antibody in the plasma and 55% have an antigen on the RBC.
ABO antibody will activate complement and create intravascular or extravascular haemolysis.
There are 9 MAJOR blood group systems and the rest are minor.
The most important point is: a difference in the amino acid sequence between one individual and another will stimulate an immune response. So they need to be taken into account with transfusions.

Question 27

Explain which antibody classes are used in the ABO blood group system.

Answer 27

IgM (10% of all Abs - heavy chains and Mu chains) very involved in clearing foreign material and is used for primary response as have TEN antigen binding sites. These will be present as can respond to environmental factors before there is even a transfusion. Anti-A and Anti-B antibodies are IgM antibodies.
IgG makes up majority heavy is gamma chains (75%) – involved in secondary response and is more tailored. Binds better to antigens. There needs to be a sensitising event for these to be present and this could be previous transfusion or pregnancy or a transplant.
To destroy foreign RBCs the complement system needs to be activated and this is a cascade of 9 proteins.
There are various ways to activate this; the classical pathway is used when transfusion takes place and the c1q complex is activated with antibody binding which is the first part of the cascade. This isn’t continually activated and can be switched off.
Antibody binding introduces a confirmational change in the c1q complex. This leads to production of a molecule which leads to further activation to the cascade. There is a ring of c5 and c9 proteins on the surface of the cell which makes a pore and this punches a hole to create lysis and kill the cell.
There are different amplification stages in the cell to promote more proteins. There can be a build up of c3 which will make the RBC attractive to phagocytes.

Question 28

How do RBCs die?

Answer 28

Intravascular haemolysis destruction – rupture of cell with complement activation which releases Hb into the plasma. This is mediated with ABO antibodies anti-A and anti-AB and happens within the circulation. This would happen to a patient with an ABO incompatible transfusion. The cells could be destroyed within circulation (within 20mins of transfusion with as little as 20mls of incompatible blood).
Extravascular – this is via the reticuloendothelial system (cells descended from monocytes which can perform phagocytosis of foreign particles) and this is used when antibodies have failed to activate complement to the terminal complex but c3 has been deposited on surface of cells which makes them a (delicious opsonised) target for phagocytes when they pass through the liver or spleen. Chomp. The cells will have chunks out of them and will be removed or less able to deform and travel through the blood vessels so they will be removed.
This may be associated with antibodies to the following blood antigens: Rh, MNSs, Lu, KELL

Question 29

How do RBCs agglutinate?

Answer 29

RBCs are biconcave discs with carbohydrate molecules on the cell surface. This gives them a net negative charge and they repel each other. In solution, they can attract positive ions (saline – sodium ions are pos charged). There will be a cloud of positively charged ions around the red cell and this stops the RBCs getting close together. This affects the antibodies that can cause agglutination as they will not be able to bind to an antigen on one cell and another cell and clump together. Using a normal ionic strength solution, IgM can cause agglutination as they can reach across the gap but IgG antibodies can’t. IgM antibodies include anti-A and anti-B. If IgG antibodies were being study, there would need to be enhancements to the solution to demonstrate the class of antibody present.

Question 30

Which is the most common blood group in the UK for the ABO blood group system?

Answer 30

Most common antigen is group O (45%), group A (43%), Group B (9%), Group AB (3%). My parents are both group A Rh+.

Question 31

If my parents are both A RH D+, which blood group will I be?

Answer 31

A D+?

Question 32

How are blood groups identified using agglutination?

Answer 32

Cells are tested with antibodies to see what antigens are present. If the antigen is present, the blood will agglutinate

Question 33

What are the differences when testing forward and reverse groups.?

Answer 33

If there is an antigen on the surface on the RBC, the plasma will agglutinate.
Testing the cells – this is the forward group which tests for the presence or absence of A and B antigens.
Testing the plasma (serum) – this is the reverse group which is testing for anti-A and anti-B antibodies in the plasma.

Question 34

What is the most common blood group in the UK?

Answer 34

In the UK population, O is the most common blood group (48%). Around 7% of the population have O -ve blood, yet this special group accounts for around 13% of all hospital requests. Why is this type in such demand? It’s down to antigens, or rather lack of them. O -ve blood lacks the A, B and D antigens, so it can safely be given to patients with any blood type, because it won’t trigger an immune response. O –ve is called universal for obvious reasons! To ensure we always collect enough we now offer O negative donors ‘priority’ appointments, so that we are able to better manage the high demand for this group.

OD pos - 38% most frequent - the universal donor
OD neg - 7%
AD pos - 37% Me :)
AD neg - 6%
BD pos - 7%
AB D pos - 2.5% - AB is the universal receiver
AB D neg - 0.5%

Question 35

What considerations need to be taken into account when choosing a testing technique?

Answer 35

What antibodies will it identify?
Specificity of antibody class. Can it detect IgG or IgM?
Sensitivity – will react even with a small amount of antibody in the plasma.
Speed – transfusions are needed quickly. IAT takes 30 mins and includes a 15 minute incubation.

Question 36

Why would an enzyme enhancement be used?

Answer 36

Can be added to allow to visualise the binding of IgG class antibodies.

he enzyme will reduce the charge off the RBC’s by removing certain proteins and carbohydrate residues, allowing the RBCs to come closer together and cause agglutination with the binding of IgG and associated antigens.

Which enzyme is used?

Papain or bromelain from papaya or pineapple. They remove carbohydrate and can remove protein so this needs to be carefully done as they can cleave above the MMS or Duffy enzymes with enzyme treating. This test can’t be used with these specificities as the antigen would be lost and the antibody would not be able to bind.
However, Rh carrier molecules are made stronger with IAT because the long chain proteins are removed and can bind to the Rh molecules.

Question 37

When would an IAT be used and how does it work?

Answer 37

A clinical significant antibody would be expected to react at 370C.
The test used for this is the Indirect Antiglobulin Test (IAT) and is designed to test 370C. If it doesn’t react at 37 then it’s not going to cause a reaction in the body of the patient. This test uses patient plasma against donor plasma and is used to identify clinically significant antibodies at 370C to detect IgG ABO RH blood group antibodies before transfusion. This is not on a tile – this is in a column. IAT is a gold standard technique.
The column holds capture technology which contains a blood group antigen which has been immobilised. Patient cells are added and if those cells have the antigen on their surface, they will stick to the well. A second antibody is then added.
IAT technique can screen, determine a patient blood group, or screen the presence of IgG antibodies in plasma (smaller than IgM). Cells cannot agglutinate when suspended in saline*.
The process of an IAT test (or Indirect Coombs test)
1) Cells have a blood group antigen on surface.
2) Plasma is added containing IgG class antibody.
3) Antibody binds to antigen.
4) Wash step removes unbound antibodies.
5) Incubate at 37o.
6) The antibody will bind to the corresponding antigen.
7) Second antibody is added; anti-human globulin which binds to the antibodies which have already bound to the antigen.
8) Cross links are formed between the antibodies and they clump together.

Question 38

What are the most common causes of blood transfusion complications?

Answer 38

Labeling errors.

Question 39

Name three other blood group systems and why is there a problem with detecting one of them?

Answer 39

ABO and Rh are the most important but there are also:

Kell system (anti-K)
Duffy system (anti-Fya )
Kidd system (anti-Jka) patients will make the antibody and the titer will rise but without stimulation the titer will fall and it can’t be detected.
Named after the first patient.
Compared to Rh these are less immunogenic 1000 less likely to stimulate antibody reaction and 6% of the population may be affected.

Question 40

Why can’t IgG antibodies bind RBCs together in certain solutions?

Answer 40

RBCs are biconcave discs with carbohydrate molecules on the cell surface. This gives them a net negative charge and they repel each other. In solution, they can attract positive ions (*in saline – sodium ions are pos charged). There will be a cloud of positively charged ions around the red cell and this stops the RBCs getting close together. This affects the antibodies that can cause agglutination as they will not be able to bind to an antigen on one cell and another cell and clump together. Using a normal ionic strength solution, IgM can cause agglutination as they can reach across the gap but IgG antibodies can’t. IgM antibodies include anti-A and anti-B. If IgG antibodies were being studied, there would need to be enhancements to the solution to demonstrate the class of antibody present.