3.10 - Blood Transfusion

Question 1

Introduction to blood groups

Answer 1

• RBC antigens are specific sites on different proteins and glycoproteins that form part of the RBC membrane
• an individual’s blood group refers to the combination of RBC antigens present
• RBC antigens differ depending on their specific sequence of oligosaccharides/amino acids but can be collated into different ‘blood group systems’
• blood group system - collection of one or more RBC antigens under the control of a single gene / cluster of closely linked homologous genes
• the ABO and Rh blood group systems are most clinically significant
• the clinical importance of a blood group system depends on the capacity of antibodies against the specific RBC antigens to cause haemolysis of RBCs - not all antibodies against RBC antigens cause haemolysis

Question 2

Antibodies against RBC antigens

Answer 2

Antibodies against RBC antigens are clinically significant if they can cause haemolysis resulting in either:

• haemolytic transfusion reactions (HTRs) - where incompatible RBCs are transfused i.e. the transfused RBCs have the antigen which corresponds to the antibody in the patient’s plasma
• haemolytic disease of the foetus and newborn (HDFN) - where the foetus has a different RBC antigen to mother (i.e. a RBC antigen inherited from father) and mother has produced an antibody to that RBC antigen that has crossed the placenta
  There are two types of antibodies against RBC antigens - naturally occurring antibodies and acquired antibodies

Question 3

Naturally occurring antibodies

Answer 3

• ABO antibodies (anti-A, anti-B) are ‘naturally occurring’ antibodies - their production is stimulated when the immune system encounters the ‘missing’ ABO blood group in foods/microorganisms
• this happens within first few months as sugars that are identical/similar to the ABO group antigens are found throughout nature
• ABO antibodies are mostly IgM antibodies that remain as IgM antibodies throughout life and do not class switch
• IgM = 5 Y-shaped units forming a pentameric structure
• interaction between pentameric IgM antibody and RBC antigens in vitro = easily visualised clumping (agglutination) of red cells - basis of ABO grouping
• IgM ABO antibodies can cause acute HTRs through activation of the complement system resulting in massive intravascular haemolysis - but they cannot cross the placenta to cause HDFN

Question 4

Acquired antibodies

Answer 4

• formed as a result of active immunisation (alloimmunisation - immune response to non-self antigens from members of the same species) to ‘non-self’ RBC antigens following exposure to RBCs from another individual
• exposure may arise due to incompatible blood transfusion, or during pregnancy when some foetal RBCs can enter maternal blood system (e.g. RhD negative mother carrying RhD positive foetus)
• they can potentially be produced against antigens of all other blood group antigen systems, which the individual lacks on their own RBCs - but not all alloantibodies are clinically significant
• acquired antibodies are usually IgG antibodies - in vitro, the interaction between IgG and RBC antigens cannot be directly visualised
• IgG antibodies generally do not cause massive intravascular haemolysis and death but do still cause haemolysis (mainly extravascular) –> delayed HTRs
• IgG can also cross placenta to cause HDFN

Question 5

ABO antigens and blood groups

Answer 5

There are 4 main blood groups within the ABO system:

• group A individuals express A antigen on RBCS
• group B individuals express B antigen on RBCS
• group AB individuals express both A and B antigens on RBCS
• group O individuals express neither A nor B antigens on RBCs

Question 6

How are ABO antigens formed?

Answer 6

• the A and B antigens are formed by adding specific monosaccharides onto a common glycoprotein and fucose stem (the ‘H’ antigen) on the RBC membrane and this is determined by the corresponding gene:
• the A gene codes for an enzyme that adds N-acetyl galactosamine (GalNac) to the common H antigen
• the B gene codes for an enzyme that adds galactose (Gal) to the common H antigen
• the A and B genes are codominant so presence of both genes results in formation of both A and B antigens
• the O gene produces an inactive enzyme so the H antigen remains unchanged = neither A nor B antigens formed - O gene is recessive
• group A - AA/OA; group B - BB/OB

Question 7

ABO antibodies

Answer 7

• individuals have naturally occurring antibodies in the plasma against any ABO antigen that they lack on RBCs (Landsteiner’s law - whichever ABO antigens are lacking, corresponding antibodies present)
• group A - A antigens, anti-B antibodies
• group B - B antigens, anti-A antibodies
• group AB - A and B antigens, no antibodies
• group O - neither antigen, anti-A and anti-B antibodies
• IgM ABO antibodies are reactive at 37oC and capable of fully activating complement = able to cause potentially fatal haemolysis (acute HTR) if incompatible blood is transferred
• small amount of IgG ABO antibodies present - can cross placenta but don’t usually cause HDFN as foetal RBCs have poorly developed ABO antigens which cannot support binding of IgG antibodies, and any IgG that crosses can be ‘mopped up’ by other cells containing ABO antigens

Question 8

ABO - selecting blood components for transfusion - RBCs

Answer 8

• to prevent HTRs, ABO compatible red cells should be selected for transfusion e.g. group A cells for group A patient
• in emergency situations where the group of the patient is unknown, group O cells can be given to any patient (universal donor) as they lack both A or B antigens, so there is no risk of acute HTR occurring even if a patient has anti-A/B antibodies
• ABO antibodies are found in plasma, not RBC transfusions

Question 9

ABO - selecting blood components for transfusion - platelets

Answer 9

• platelets of the same ABO group as the patient should be selected for transfusion where possible to:
  1. reduce risk of a poor response to the platelet transfusion due to anti-A/B antibodies causing destruction of transfused platelets (low risk as expression of ABO antigens on platelets is low)
  2. reduce risk of haemolysis by anti-A/B antibodies in transfused unit of platelets (suspended in plasma) - only occurs in platelets which have plasma with high levels of anti-A/B antibodies, units of platelets tested for this and labelled ‘high-titre negative’ if they do not have high levels
• therefore if platelets of the same ABO group as the patient are not available, platelets of other ABO groups can still be given if ‘high-titre negative’
• ABO antibodies are only found in plasma donations, not RBC donations

Question 10

ABO - selecting blood components for transfusion - fresh frozen plasma (FFP) / cryoprecipitate

Answer 10

• FFP / cryoprecipitate of the same ABO group as the patient should be selected for transfusion where possible to reduce risk of haemolysis
• as for platelets, there is only risk if the plasma contains high levels of anti-A/B antibodies
• if FFP / cryoprecipitate of the same ABO group is not available, other ABO groups can be transfused as long as ‘high-titre negative’
• group AB plasma contains no antibodies and can be considered universal plasma donor

Question 11

Rh antigens

Answer 11

• the Rh system consists of at least 45 antigens and after the ABO system, is the next most clinically significant
• the most important antigen is D - individuals are classed as either RhD positive or RhD negative depending on the presence of the D antigen on their RBCs
• blood group - ABO group and RhD type e.g. A positive = ABO A, RhD positive
• the D antigen is inherited as one gene (RHD) and the alleles are either ‘D’ or ‘d’ - D is dominant and codes for the D antigen, d is recessive and codes for no D antigen
• RhD positive - Dd / DD; RhD negative - dd
• relative frequencies of RhD + vs - varies in different ethnic groups

Question 12

Rh antibodies

Answer 12

• individuals who are RhD negative can make IgG anti-D antibodies following exposure to RhD positive RBCs either through transfusion or pregnancy (alloimmunisation)
• D positive - antigen D - no antibody
• D negative - no antigen - can develop anti-D antibody if exposed to RhD positive RBCs
  Anti-D antibodies are clinically significant as they can cause:
• delayed HTRs (extravascular haemolysis) - if RhD positive red cells are transfused –> anaemia, high bilirubin, jaundice
• HDFN - if RhD negative mother carries a RhD positive foetus as IgG anti-D antibodies can cross the placenta and haemolyse RhD positive foetal RBCs
• prevent formation of anti-D antibodies by ensuring RhD negative patients receive RhD negative RBCs and platelets
• to prevent pregnant women who are RhD -ve from forming anti-D antibodies if carrying RhD +ve foetus, they are given anti-D immunoglobulin during pregnancy - works by destroying any RhD positive foetal RBCs in maternal circulation before anti-D antibodies can be made

Question 13

Rh - selecting blood components for transfusion - RBCs

Answer 13

• red cells for transfusion should be the same RhD type as the patient e.g. RhD negative RBCs for RhD negative patients
• RhD positive patients can be transfused positive or negative RBCs
• group O RhD negative RBCs are used as emergency blood when a patient needs emergency transfusion and their blood type is unknown - precious source as only 6-7% of donors are O-

Question 14

Rh - selecting blood components for transfusion - platelets

Answer 14

• platelets for transfusion should be the same RhD type as the patient
• platelets do not express D antigen but units of platelets can contain small numbers of RBCs / fragments that can cause alloimmunisation in RhD negative patients
• if RhD positive platelets are given to RhD negative patient, alloimmunisation can be reduced by giving anti-D immunoglobulin (mainly women <50yo to prevent risk of HDFN)

Question 15

Rh - selecting blood components for transfusion - FFP / cryoprecipitate

Answer 15

• FFP / cryoprecipitate of any D type can be transfused regardless of the patient’s RhD type
• these plasmas contain components but not RBCs

Question 16

What other red cell antigens are there and what happens if a patient forms an antibody to them?

Answer 16

• dozens of others including Rh group C, c, E, e - others are Kell, Duffy, Kidd
• 8% of patients form an antibody to one or more of these and if that happens we must use corresponding antigen negative blood or there is a risk of delayed HTRs occurring
• e.g. O- patient who also has an anti-E antibody should be transfused O- RBCs which are negative for E antigen

Question 17

Pre-transfusion compatibility testing - what is it necessary to test to provide ABO and Rh compatible blood to a patient?

Answer 17

• necessary to check the patient’s ABO and RhD blood group and perform an antibody screen on their plasma to check for any acquired alloantibodies
• these tests are collectively known as ‘group and screen’
• automated analysers with computer control of specimen identification and result allocation

Question 18

Determining ABO grouping

Answer 18

• to determine ABO group we identify the ABO antigens on their RBCs (forward group) and the presence/absence of ABO antibodies in their plasma (reverse group)
• forward group - a sample of patient’s RBCs is tested against reagent anti-ABO antibodies, interaction between reagent anti-A/B with ABO antigens will result in clumping (agglutination) which can be seen
• reverse group - patient’s serum is mixed with reagent A and B RBCs, interaction between patient’s ABO antibodies and antigens on reagent will result in agglutination which can be seen

Question 19

Determining RhD grouping

Answer 19

• to determine RhD type, we identify the presence / absence of the D antigen on their RBCs by testing a sample of the patient’s RBC against reagent anti-D antibodies
• interaction with reagent anti-D antibodies will result in agglutination which can be seen

Question 20

What happens in an antibody screen?

Answer 20

• performed to detect presence of any acquired antibodies the patient may have developed
• patient’s serum is tested against panels of RBCs which together are known to express all of the clinically relevant RBC antigens
• any interaction between an antibody in serum and an antigen on RBC will result in agglutination (positive antibody screen)
• in this case, agglutination cannot be directly visualised and needs addition of another reagent (anti-human globin) before it can be seen
• if antibody screen negative - any donor blood that is ABO and RhD compatible can be given
• if antibody screen positive - antibody must be identified with use of large panel of RBCs and donor blood units that lack corresponding antigen is then chosen for cross-matching with recipient’s plasma prior to transfusion
• in emergencies where O- is acquired, a cross-match does not need to be performed

Question 21

What are the criteria for donor selection?

Answer 21

• volunteer, unpaid donors between 17-65
• no disease that might make blood donation hazardous for them e.g. cardiovascular/neurological disease
• no disease that makes blood donation hazardous for recipient e.g. risk of viral/bacterial/parasitic infections, certain diseases or drugs
• donor education and self-exclusion of those at high risk of having contracted bloodborne diseases are essential to ensure those in early infectious stage do not donate
• all potential donors complete a ‘donor health check questionnaire’ and then have a ‘health screening interview’ with a nurse to check eligibility

Question 22

What two tests are done on blood donations?

Answer 22

1. group and screening for ABO and RhD blood group along with other Rh groups e.g. C, c, E, e and K (for some, further testing is done e.g. Fya, Fyb) - every donation tested to ensure no strong clinically significant RBC antibodies are present in donor plasma = transfusions containing plasma only contain ABO antibodies
2. infection screening - vital to reduce risk of transfusion-transmitted infections
   - testing alone insufficient to reduce infection risk - cannot test for all infections etc
   - therefore, screening of donors prior to donation to identify those ineligible to donate is vital
   - minimum mandatory infection screen on all donations - HIV, hepatitis B, hepatitis C, hepatitis E, HTLV, syphilis
   - if infection screen positive, further confirmatory testing performed - if also positive, donation discarded

Question 23

How is blood collected from donors?

Answer 23

• 450 ml blood collected in sterile plastic bag containing anticoagulant

1. whole blood donation - not efficient (RBCs, plasma, platelets all together) - patients usually only need one of these components

• split one unit of blood by centrifuging whole bag, then squeeze each layer into satellite bags and cut free (closed system)

2. apheresis - donor connected to apheresis, blood from donor passes through machine which separates out particular component required and returns remainder to donor

• allows specific components to be collected e.g. RBCs from a patient with a rare blood group
• allows donors to maximise donations as a larger volume can be collected of specific components e.g. can donate a whole unit of platelets

Question 24

What are red cells given for?

Answer 24

• red cell transfusions to increase haemoglobin and thus restore oxygen carrying capacity of blood in patients with anaemia or blood loss
• red cells for transfusion known as ‘packed red cells’ - during manufacture and processing, majority of plasma is removed and red cells suspended in small volume of additive solution = maintain viability of RBCs
• one unit = increases Hb concentration by 10g/L
• important to check response to transfusion clinically and by checking Hb level post-transfusion
• packed red cells have a shelf life of 35 days and must be stored in a fridge at 4oC

Question 25

What are platelets given for?

Answer 25

• those with bone marrow failure
• problem with platelets e.g. thrombocytopenia
• massive bleeding or DIC
• platelet dysfunction - cardiac bypass and patients on anti-platelet drugs

Two methods for producing platelets for transfusion - pooled platelets - separated from 4 whole blood donations to form one unit, and apheresis platelets - single donor donates one unit

• one unit = increases platelet count by 10 x 10^9/L
• shelf life of 7 days and stored at room temperature
• require constant agitation to ensure they are continuously oxygenated within their gas permeable membranes
• check for response to transfusion both clinically and checking platelet count post-transfusion

Question 26

What is FFP given for?

Answer 26

• fresh frozen plasma contains all coagulation factors, fibrinogen, plasma proteins, electrolytes, anticoagulants
• given to those with clotting factor deficiencies - prolonged APTT or PT - treatment for bleeding/reduce risk of bleeding
• also given for reversal of warfarin (anticoagulant) e.g. for urgent surgery
• check for response to transfusion both clinically, and by checking PT and APTT following transfusion
• once collected it is frozen - maintains activity of clotting factors - when needed for transfusion, thawed in water bath and stored at 4oC (shelf life 3 years at -25oC)

Question 27

What is cryoprecipitate given for?

Answer 27

• made from FFP –> contains fibrinogen, factor VIII, VWF, factor XIII
• made by slow thawing of FFP overnight until no precipitate - precipitate then re-suspended in small volume of plasma and frozen to maintain activity of clotting factors
• transfusions given to treat bleeding or reduce risk of bleeding when fibrinogen level is low (e.g. due to a major haemorrhage or DIC)
• check for response to transfusion both clinically, and by checking fibrinogen level post-transfusion
• when needed for transfusion, thawed in water bath and stored at 4oC and used within 4 hours of thawing (shelf life 3 years at -25oC)

Question 28

Plasma-derived medicinal products

Answer 28

Licensed medicinal products manufactured from pooling of thousands of human plasma donations by a process called fractionation:

• human albumin solution (HAS) - to replace plasma volume in patients with plasma loss, replaces plasma in plasma exchange, initiates diuresis in patients with low albumin
• immunoglobulin (Ig) solutions - normal Ig contains antibodies to viruses that are common in the population, intramuscular normal Ig may be used to protect susceptible contacts against infection, high dose IV Ig used as replacement therapy in patients with severe Ig deficiency, specific Ig - made from selected donors with high antibody levels to the target of treatment
• clotting factor concentrates - single factor concentrates are available for the management of most inherited coagulation deficiencies (now haemophilia A/B treated with recombinant synthesised factor concentrates which carry no risk of viral/prion transmission). Prothrombin complex concentrates (PCCs) contain factors II, VII, IX, X = used to reverse effect of warfarin by replacing these deficient coagulation factors. Fibrinogen concentrates provide an alternative to cryoprecipitate for replacing fibrinogen