L1 Blood Blood transfusion 1

Overview

This lecture covers the molecular basis of the ABO blood group and Rh blood group systems and why they matter clinically, other clinically significant blood group systems, the laboratory testing used to establish compatibility before transfusion (direct and indirect antiglobulin testing), the main blood components and their clinical indications, and the massive haemorrhage pathway used when a patient is bleeding faster than blood can be replaced.

ABO blood group system

• ABO group is defined by which antigen is on the red cell surface: Group A has the A antigen, Group B has the B antigen, Group AB has both, Group O has neither.
• A person makes ABO antibodies against whatever antigen(s) they lack: Group A has anti-B, Group B has anti-A, Group AB has no ABO antibodies, Group O has both anti-A and anti-B.
• Biochemistry: a common precursor glycan chain on the red cell membrane is converted to the H-antigen by addition of L-fucose (alpha-L-fucosyltransferase). H-antigen is then converted to B-antigen by addition of D-galactose (alpha-galactosyltransferase), or to A-antigen by addition of N-acetylgalactosamine (alpha-N-acetylgalactosaminyltransferase).
• Inheritance: A and B alleles are co-dominant, O is recessive. A+A -> A; A+B -> AB; A+O -> A; B+B -> B; B+O -> B; O+O -> O.
• ABO antibody characteristics: ubiquitous from 6 months of age, present in plasma, mostly IgM with some IgG (IgM predominant), active over a wide thermal range (4-37C), and IgM ABO antibodies activate complement very efficiently.
• Complement activation: antibody (e.g. anti-A) binds antigen, C1 binds, then C2/C4 and C3/C5 enzymatic steps generate C3a/C3b and C5a/C5b, which assemble the Membrane Attack Complex (C6-C9) in the target membrane, lysing the cell and releasing haemoglobin.
• Consequences of complement activation in ABO-incompatible transfusion (e.g. Group A donor cells into Group O recipient): haemolysis releases haemoglobin, which binds haptoglobin and albumin (haptoglobin falls) and appears in urine (haemoglobinuria); C3a/C5a cause vasodilation and hypotension; fibrin deposition and DIC deplete coagulation factors and platelets, causing bleeding. Haemoglobinuria, hypotension and fibrin deposition all converge on renal failure.
• Acute haemolytic reaction symptoms: systemic chills and fever, increased heart rate, hypotension and uncontrollable bleeding, constricting chest pain, heat sensation at the transfused vein, haemoglobinuria and hyperbilirubinaemia, lumbar pain. In vitro, mixing incompatible plasma and red cells causes visible agglutination (clumping).

Rh and other blood group systems

• Rh system has 5 major antigens: D, C, c, E, e. Red cells for transfusion are routinely matched only for RhD.
• “Positive”/“negative” in a blood group refers to presence/absence of the RhD antigen (e.g. O Pos = group O with RhD antigen; A Neg = group A without it).
• Unlike ABO antibodies, Rh antibodies are not ubiquitous; anti-D forms only after exposure to RhD antigen via transfusion or pregnancy, so the aim is to avoid ever sensitising a patient.
• RhD-negative individuals, particularly women up to 55 years of age, should receive RhD-negative red cells: RhD antigen is highly immunogenic, with a 70% chance of stimulating antibody production if a D-negative person is transfused with D-positive red cells.
• Anti-D causes severe haemolytic disease of the fetus and newborn (maternal Rh antibodies cross the placenta and coat fetal red cells).
• For multi-transfused individuals, C, c, E and e antigens are also matched.
• RhD antigen is a membrane protein (Rh 30,000) that spans the red cell membrane multiple times; the RHD gene has 10 exons, each encoding a membrane-spanning region.
• Transfusion rule: D-positive patients can receive either D-positive or D-negative red cells, but D-negative patients should only receive D-negative red cells.
• Kell system: the K antigen is also immunogenic, with a 10% chance that a K-negative person exposed to K-positive red cells makes anti-K, another cause of severe haemolytic disease of the fetus and newborn; women under 50 therefore receive K-negative red cells. In NZ, 9% of people are K positive and 91% K negative.
• Other clinically significant systems (Duffy, Kidd, Ss, others) only require matching once a patient has already formed an antibody against them, since antibodies in these systems are not ubiquitous but arise from prior pregnancy or transfusion. Patients with these “unexpected” antibodies, especially multiple antibodies, take longer to provide compatible blood for.

Compatibility rules and population frequencies

• Red cell transfusion rule: donor red cells must lack the antigens for which the patient has corresponding plasma antibodies (Group O is the universal red cell donor; Group AB patients can receive from all groups).
• Plasma transfusion rule: donor plasma must lack the antibodies for which the patient has corresponding red cell antigens (Group AB is the universal plasma donor). RhD is not relevant for plasma since it contains no viable red cells; in an emergency, group A plasma may be given irrespective of the patient’s blood group.
• ABO compatibility is not essential for platelet transfusions or cryoprecipitate transfusions, though blood banks normally provide ABO-identical or compatible cryoprecipitate.
• In extreme circumstances RhD-positive blood may be given to RhD-negative individuals (except women of child-bearing age or patients with anti-D), only in consultation with a Transfusion Medicine Specialist.
• NZ blood group frequencies: Group O 47%, Group A 38%, Group B 11%, Group AB 4%; RhD negative 18% overall (full breakdown: 38% O+, 9% O-, 32% A+, 6% A-, 9% B+, 2% B-, 3% AB+, 1% AB-).

Pre-transfusion sample and testing pathway

• When a patient may need transfusion, the clinical team requests a “group and screen” and collects a 6 mL EDTA sample (pink-top tube).
• The laboratory checks sample and request-form labelling, then routinely performs ABO and RhD grouping (to select compatible donor blood) and an antibody screen (to check for unexpected non-ABO antibodies).
• A baseline sample should be sent before first transfusion when a patient will need multiple/ongoing transfusions (allows accurate extended typing) or is on certain drug therapies (e.g. daratumumab for multiple myeloma, or clinical trial drugs) that interfere with accurate blood group and antibody screen testing.
• NZ Blood Service specimen labelling requirements: all details must be hand-labelled at the patient’s side, including given name(s), family name, NHI and date of birth, date and time of collection on both sample and form, and the signature of the person labelling the tube.
• The person collecting the sample must sign a mandatory declaration certifying they collected the sample from the named patient, confirmed identity by direct enquiry and/or wristband inspection, and labelled and signed the sample by hand immediately at the bedside in the patient’s presence. Failure to complete the declaration can lead to sample rejection.

Laboratory antibody detection techniques

• Direct agglutination of red cells occurs with IgM antibodies but not with IgG antibodies (IgG alone coats cells without visibly clumping them).
• Indirect antiglobulin test (IAT): Step 1, incubate red cells with IgG antibody (e.g. RhD-positive cells with IgG anti-D) so cells become sensitised but not agglutinated; Step 2, add anti-human globulin (AHG), which cross-links the bound IgG and produces visible agglutination. Used to detect antibodies not already bound in vivo.
• Direct antiglobulin test (DAT): used when red cells already have IgG bound in vivo (e.g. a RhD-positive baby with maternal IgG anti-D on its cells). AHG is added directly to the cells, producing agglutination; it is a single-step test because no incubation step is needed.
• Labs use different testing platforms: tubes, cards/gel technique (agglutinated cells are trapped at the top of the gel, non-agglutinated cells pass to the bottom), and fully automated blood-typing systems.

Blood donation and component production

• Blood is collected either as whole blood (WB) or by plasmapheresis (PP, an apheresis procedure collecting plasma directly).
• Regular whole blood donors can donate every 3 months; plasma donors can donate every 2 weeks.
• Production pathway: whole blood is filtered (leucodepleted), then centrifuged to split into red cells and plasma. Red cells are combined with resuspending fluid to give resuspended red cells; plasma is frozen to give fresh frozen plasma (FFP).

Blood components: indications, thresholds and storage

• Red cells (source of oxygen-carrying haemoglobin): one unit is expected to raise haemoglobin by about 10 g/L in a 60 kg individual; haemoglobin should be checked after the first unit before charting another. Indications by Hb level (Transfusion Medicine Handbook, Table 4.10): <70 g/L usually indicated (a lower threshold may be acceptable if asymptomatic or specific therapy such as cobalamin/iron is available); <80 g/L likely appropriate in acute coronary syndrome (maintain Hb 80-90 g/L); 70-100 g/L likely appropriate during surgery with major blood loss or impaired tissue oxygen delivery; >90 g/L not likely appropriate in the critically ill under a restrictive transfusion policy (associated with reduced mortality), except sepsis with impaired tissue oxygen delivery, subarachnoid haemorrhage, ischaemic stroke, or cerebral ischaemia complicating traumatic brain injury; >100 g/L not likely appropriate unless a specific indication exists.
• Platelets (for clotting): indicated for bleeding/massive haemorrhage/massive transfusion, chemotherapy-induced bone marrow failure when platelet count is <5-20 x10^9/L, surgery/invasive procedures when platelet count is <50-80 x10^9/L, and some platelet function disorders.
• Fresh frozen plasma (source of clotting factors, used when INR >1.5; INR is a measure of clotting time relative to a normal control): indicated for coagulation factor deficiencies when the specific factor concentrate is unavailable, massive blood transfusion, reversal of warfarin effect (may not reverse other oral anticoagulants), plasma exchange for thrombotic thrombocytopenic purpura, liver disease, and DIC.
• Cryoprecipitate (source of fibrinogen; prescribe when fibrinogen <1.5 g/L, or <2 g/L in postpartum haemorrhage): indicated for massive bleeding, DIC, fibrinogen deficiency, and bleeding associated with uraemia.
• Storage and shelf life: red cells at 4±2C (35 days); FFP below -25C (2 years); platelets at 22±2C (7 days); cryoprecipitate below -25C (2 years).

Massive haemorrhage pathway (MHP)

• Massive haemorrhage is defined as blood loss exceeding the circulating blood volume within a 24-hour period.
• The MHP is a structured process aiming to maintain haemoglobin levels and avoid coagulopathy by smoothing communication so blood reaches the patient quickly, transfusing red cells and plasma in a 1:1 ratio, and adding platelets and cryoprecipitate at the right time.
• The Adult MHP is triggered by massive bleeding plus shock signs, heart rate >120, or systolic BP <90, escalating to Code Red (trauma, ABC score >=2, senior clinician approval), Standard MHP, or Obstetric MHP; tranexamic acid (2 g or 1 g) is given and a group and screen sent to Blood Bank.
• An initial Stat Pack is issued (Red Stat Pack: 2 RBC + 2 FFP; Stat Pack: 2 RBC; Obstetric Stat Pack) and the patient reassessed for ongoing bleeding/shock; if full MHP is activated, packs escalate (e.g. Standard Pack 1: 3 RBC + 3 FFP; Obstetric Pack 1: 3 RBC + 3 cryoprecipitate), cycling through Pack 2 (4 RBC, 4 FFP, 3 cryoprecipitate) and Pack 3 (4 RBC, 4 FFP, 1 platelet) alternately until bleeding slows, then MHP stops and targeted transfusion begins (guided by ClotPro if available).
• 1 g calcium (10 mL calcium chloride or 30 mL calcium gluconate) is given with every pack, ideally through separate IV access.
• Clinical targets: INR <1.5, APTT <40, fibrinogen >2 g/L, platelets >75x10^9/L, ionised Ca2+ >1.1 mmol/L; if a target component is not given, a default dose is used instead (4 U FFP, 3 U cryoprecipitate, 1 U platelets, 30 mL 10% calcium gluconate).
• Code Red uses an ABC score (pending mechanism = 1 point, SBP <=90 twice = 1 point, HR >=120 twice = 1 point; positive if score >=1); Code Red activation requires senior clinician approval and identifies the highest-risk trauma patients needing a multi-service response.
• Obstetric haemorrhage management addresses the Tone, Trauma, Tissue and Thrombin causes of bleeding; the initial 1 g tranexamic acid dose is repeated after 30 minutes if bleeding is significant and ongoing.

One slide (36) is a truncated website screenshot of the FFP indications table; its visible content duplicates the fuller indications list already captured above from slide 33.

Self-test

1. Define the ABO blood group of a person whose red cells carry neither the A nor the B antigen, and state which ABO antibodies they would have.
2. Describe the enzymatic steps that convert the precursor glycan chain into the A and B antigens.
3. Explain why ABO IgM antibodies are particularly dangerous in an incompatible transfusion, referencing their thermal range and complement activity.
4. Describe the sequence of events, from complement activation to renal failure, that follows an ABO-incompatible red cell transfusion.
5. A Group O patient with no RhD antigen is accidentally transfused with Group A, RhD-positive red cells. Predict which symptoms of an acute haemolytic reaction they might show.
6. Distinguish the antigen-matching rule for red cell transfusion from the rule for plasma transfusion.
7. Why must RhD-negative women up to 55 years of age receive RhD-negative red cells rather than RhD-positive red cells?
8. Distinguish the direct antiglobulin test (DAT) from the indirect antiglobulin test (IAT), including when each is used and why one needs an incubation step and the other does not.
9. List the details that New Zealand Blood Service requires to be hand-labelled on a transfusion sample at the patient’s bedside.
10. Describe the circumstances under which a baseline transfusion sample should be sent before a patient’s first transfusion.
11. What haemoglobin threshold generally indicates red cell transfusion, and what different threshold applies in acute coronary syndrome?
12. List the four main blood components used clinically, the substance each mainly supplies, and one condition-specific threshold or trigger for using each.
13. Describe the ratio of red cells to plasma used during the massive haemorrhage pathway, and the accompanying medication given with every pack.
14. Distinguish the Kell blood group system from the ABO and Rh systems in terms of when antibody matching is required.

Answers

Reveal answers

1. Group O; they have both anti-A and anti-B antibodies in their plasma.
2. The precursor chain is first converted to the H-antigen by addition of L-fucose via alpha-L-fucosyltransferase. The H-antigen is then converted to the B-antigen by addition of D-galactose via alpha-galactosyltransferase, or to the A-antigen by addition of N-acetylgalactosamine via alpha-N-acetylgalactosaminyltransferase.
3. ABO IgM antibodies are active across a wide thermal range (4-37C, so they react at body temperature) and activate complement very efficiently, driving formation of the Membrane Attack Complex and rapid intravascular haemolysis on exposure to incompatible antigen.
4. Complement activation proceeds via C1 binding, then C2/C4 and C3/C5 steps generating C3a/C3b and C5a/C5b, assembling the Membrane Attack Complex (C6-C9) which lyses red cells, releasing haemoglobin. Released haemoglobin binds haptoglobin and albumin (haptoglobin falls) and causes haemoglobinuria; C3a/C5a cause vasodilation and hypotension; fibrin deposition and DIC deplete coagulation factors and platelets causing bleeding. Haemoglobinuria, hypotension and fibrin deposition all converge on renal failure.
5. Systemic chills and fever, increased heart rate, hypotension and uncontrollable bleeding, constricting chest pain, heat sensation at the transfused vein, haemoglobinuria and hyperbilirubinaemia, and lumbar pain.
6. For red cells, donor cells must lack the antigens for which the patient has corresponding plasma antibodies. For plasma, donor plasma must lack the antibodies for which the patient has corresponding red cell antigens.
7. RhD is highly immunogenic (70% chance of stimulating antibody production if a D-negative person receives D-positive cells), and any resulting anti-D can cause severe haemolytic disease of the fetus and newborn in a future pregnancy, so exposure must be avoided in this group.
8. The IAT detects antibody not already bound to the cells: cells are first incubated with the antibody (sensitising them without agglutination), then anti-human globulin (AHG) is added to cause agglutination — two steps. The DAT is used when IgG is already bound to the cells in vivo (e.g. a D-positive baby with maternal anti-D), so AHG is added directly without a prior incubation step — one step.
9. Given name(s), family name, NHI and date of birth, date and time of collection on both the sample and the form, and the signature of the person labelling the tube.
10. Before first transfusion in patients expecting multiple/ongoing transfusions (to allow accurate extended typing), or in patients on drug therapies that interfere with blood group/antibody screen testing, such as daratumumab or clinical trial drugs.
11. Transfusion is usually indicated below 70 g/L; in acute coronary syndrome the threshold is higher, with transfusion likely appropriate below 80 g/L and a target of maintaining Hb 80-90 g/L.
12. Red cells supply oxygen-carrying haemoglobin (transfuse when Hb <70 g/L, or higher threshold in specific settings); platelets supply clotting cells (transfuse when count <50-80x10^9/L for surgery or <5-20x10^9/L with chemotherapy-induced marrow failure); fresh frozen plasma supplies clotting factors (used when INR >1.5); cryoprecipitate supplies fibrinogen (prescribed when fibrinogen <1.5 g/L, or <2 g/L in postpartum haemorrhage).
13. Red cells and plasma are given in a 1:1 ratio; 1 g of calcium (as calcium chloride or calcium gluconate) is given with every pack, ideally through separate IV access.
14. In ABO and Rh(D), matching is always performed on every transfusion because their antibodies are ubiquitous (ABO) or the antigen is highly immunogenic (RhD). Kell antibody matching is only required once a patient has already formed anti-K, since Kell antibodies are not ubiquitous and only arise after prior exposure via transfusion or pregnancy.