Clinical & Laboratory Transfusion Practice Notes

Learning Objectives

• Blood groups

• Testing in the Transfusion laboratory

• Antibody screens & antibody ID

• Red Cross Lifeblood

  • Donors

  • Components and Testing

• Clinical Practice

  • What do we transfuse

  • When do we transfuse

• Risks of transfusion

Blood Group Systems

• 45 blood group systems

• > 350 different antigens identified

• Examples:

  • Knops

  • Duffy

  • Lutheran

  • ABO

  • Gerbich

  • li

  • Diego

  • Yt

  • Cromer

  • Indian

  • LW

  • Kell

  • Rh

  • MNS

Role of Blood Group Antigens Example of Blood Group System

• Membrane transporters: Rh, Kidd

• Membrane bound enzymes: Kell

• Structural proteins: MNS

• Chemokine receptors: Duffy

• Cell adhesions molecules: Lutheran

• Complement regulation: Cromer, CD59

• Carbohydrate structures: ABO, H

Pretransfusion Testing

• Purpose:

  • To select blood components of appropriate ABO & RhD type that will not cause harm to the patient & will have acceptable survival when transfused providing maximum benefit to the patient

• If properly performed pretransfusion tests will:

  • Ensure patient is issued the designated blood component

  • Verify in most cases ABO compatibility between the component & the recipient

  • Detect most clinically significant unexpected antibodies

The Transfusion Process

• Decision to transfuse

• Consent

• Documentation

• Pre-transfusion sample collection & testing

  • ABO RhD blood group

  • Antibody screen +/- antibody ID

  • Crossmatch

    • Serological

    • Electronic

• Administration

• Monitor Response

• Review patient transfusion history

Fact

• International haemovigilance programs show most transfusion reactions are caused by human error:

  • Wrongly identified patient

  • Mislabeled blood sample

  • Mismatched sample and paperwork

  • Incorrectly tagged blood unit

  • Wrong blood component issued

  • Blood product issued to wrong patient

  • Wrong patient transfused

Pretransfusion Compatibility Testing

• 1. ABO Group

• 2. RhD Group

• 3. Unexpected plasma antibodies (Ab screen)

• 4. Crossmatch (Electronic or Immediate spin Vs IAT)

• Patient Sample - Tests Performed:

  • ABO group + RhD group + antibody screen

  • Group & screen

  • Group & save

  • Type & screen

  • Antibody Identification

  • Crossmatch

  • IAT Crossmatch

    • patient’s plasma against donor red cells

  • Electronic Crossmatch

    • Computer aided (Compatibility tables)

ABO Antigens & Antibodies

• Absence of A and/or B antigens results in antibodies against the missing antigens

• ABO antibodies develop during infancy due to exposure to environmental A, B and H like antigens

ABO & RhD Blood Grouping

• ABO grouping is the MOST IMPORTANT test

  • Error → fatal transfusion reaction

  • ABO discrepancies MUST always be resolved BUT group O can be issued if in doubt/emergency

• Rh blood group is SECOND MOST IMPORTANT

• ABO grouping:

  • test patient’s RBCs against anti-A & B

  • test patient’s plasma against A1 & B RBCs

• RhD grouping:

  • test patient’s RBCs against anti-D

ABO Grouping

Reaction of cells with Reaction of serum with reagent red cells

• Group A

• Group B

• Group AB

• Group O

• Anti-A

• Anti-B

• Anti-A,B

• A1 Cells

• B Cells

Blood Group Frequencies

• Majority of data derived from Caucasian populations – significant differences found in antigen frequency between ethnic groups (e.g., Europeans, Asians, African, Polynesian)

• Transfusion implications

Antibody Screening

• Needs to detect a range of clinically significant antibodies – develop due to:

  • Previous red cell transfusion

  • Pregnancy

• Clinically significant Ab causes reduced red cell survival or destruction

• Antibody Screening

  • ~5% of transfused patients will form an antibody

  • >2-40% of transfusion dependant patients produce antibodies

Antibody Screening

• Group O red cells used to prevent interference from naturally occurring anti-A or anti-B

• 3 cells from individual donors (not pooled)

• Screening cells selected so that the following antigens are present on at least one of the cell samples:

  • D, C, E, c, e, M N, S, s, P, Lea , Leb, K, k, Fya , Fyb , Jka& Jkb [Lu, Co, Cw]

• Indirect Antiglobulin Tests used to detect clinically significant IgG antibodies:

  • Column agglutination (manual or automated)

  • Tube (manual)

• Positive antibody screen needs to be further investigated to identify antibody specificity

Antibody screen flowchart

• Negative AND no history of antibodies

  • Group & Hold, or Crossmatch

  • Immediate spin or computer crossmatch

• Antibody Screen Positive OR history of antibodies

  • Crossmatch

    • Antigen negative blood

    • IAT crossmatch

  • Antibody identification panel

    • Confirmatory tests

      • Antibody exclusion/confirmation

      • Antigen typing

    • Advanced investigations

      • Alternative cell panels

      • Selected cell panels

      • Variation of technique

      • Enhancement of reactivity

      • Special techniques

      • Dilution/titration

      • Neutralisation/inhibition

      • Absorption-elution studies

      • Reference laboratory

• Clinical decision based on assessment of risk, degree of urgency and available time.

• Single unit crossmatch

What is a clinically significant antibody?

• An antibody is considered clinically significant if it has been associated with:

  • Reduced RBC survival or destruction in vivo

  • Haemolytic transfusion reactions (acute or delayed)

  • Haemolytic disease of the fetus & newborn

  • Haemolysis in vitro

  • In vitro macrophage response to antibody coated RBCs

Indirect Antiglobulin Test (IAT)

• Used for both antibody screen, antibody identification and crossmatching – detects antibody in plasma

• Plasma ± Additive solution

• 10-30 min incubation @ 37°C

• Centrifugation

• Agglutination

• Cassette contains pre-dispensed AHG

• No Agglutination

Direct Antiglobulin Test (DAT)

• Antibody on red cells: Test patient RBCs with AHG (in vivo sensitisation)

• Antibody in serum/plasma: Test patient plasma against reagent RBCs, and add AHG

Blood Component Request: Required Pretransfusion Patient Testing

• Red cells requested

  • Patient Testing

    • Antibody screen

    • ABO & RhD Group

    • Crossmatch

      • ABO RhD compatible

• Plasma or platelets requested

  • Patient Testing

    • ABO & RhD Group only

Final check

• Negative antibody screen and no history of clinically significant antibodies:

  • Immediate spin crossmatch = ABO compatibility check OR Electronic (Computer) crossmatch = ABO RhD compatibility check

• Positive antibody screen:

  • Clinically significant antibody

    • IAT crossmatch at @ 37°C with antigen negative red cells

  • Clinically insignificant antibody

    • IAT crossmatch compatible red cells at @ 37°C

• Crossmatch A crossmatch does not:

  • Guarantee normal survival of red cells

  • Prevent the development of an antibody

  • Detect all antibodies

  • Prevent delayed transfusion reactions

  • Detect ABO/RhD errors due to wrong blood in tube

Antenatal Testing

• Haemolytic disease of the fetus & newborn (HDFN)

• Transfer of maternal IgG antibodies via the placenta

Australia – Blood Donation

• Australian Red Cross LifeBlood

• ~3% of the eligible population give blood

• 80% receive a blood component during their lifetime

The Blood Process - Donation to Transfusion

• Decision to donate and registration

• Interview and Health Check

• Collection

• Processing

• Testing

• Quarantine

• Release

• Request and Crossmatch

• Issue

• Transfuse

• DISCARD

  • Blood that is positive for a TTI, damaged or incorrectly stored

Donation Process – Whole Blood

• Whole Blood

• Red Cells

• Fresh Frozen Plasma

• Platelets

• Platelet Rich Plasma

• Cryoprecipitate

Donation Process - Apheresis

• Apheresis

• Fresh Frozen Plasma

• THAWED PLASMA

• CSL Fractionated Blood Products

  • Intragam

  • Albumex 4

  • Albumex 20

  • Prothrombinex VF

  • Biostate

• Platelets

Donor Testing

• ABO & RhD group

• Antibody screen

• Human immunodeficiency virus (HIV) 1 & 2 Abs + HIV1 RNA

• Hepatitis B virus (HBV) surface antigen & HBV DNA

• Hepatitis C virus(HCV) Abs & HCV RNA

• Human T-cell lymphotropic virus (HTLV 1 & 2) Abs

• Malaria & CMV when necessary

• Syphilis serology

Blood Components

• What Do We Transfuse?

  • Red cells

    • Universal Donor

  • Plasma

    • Universal Donor

  • Platelets

  • Cryoprecipitate

  • Volume

  • Storage

  • Dose

  • Handling

  • Expiry

  • Clinical indications

Blood Components

• Red Cells: contain haemoglobin which carries oxygen

• Platelets: play a role in clotting and bleeding

• Plasma: contains proteins - albumin, globulins and clotting factors

• Cryoprecipitate: contains Factor VIII, fibrinogen, Factor XIII, von Willebrand Factor and fibronectin

Red Cells

• Volume:

  • 1 unit = 210 - 230mL

• Storage: 35 - 42 days @ 2-6°C

  • Transfuse at 20-24°C (37 °C) within 30 minutes of removing from fridge & over maximum 4 hours

• Dose: 1 unit of RCs raises average adult Hb by ~ 10g/L (1g/dL)

  • Transfuse one unit & reassess patient

• Handling: Transport in a validated shipper

• Cross-matched red cells are:

  • Valid 72 hours for pregnant women or patients who have been transfused in the last 3 months

Red Cells

• Before transfusing, ask:

  1. Could the patient compensate for existing anaemia?

  2. How rapidly and how much is patient bleeding?

  3. Is there an increased demand for $O_₂$?

  4. What is the state of tissue perfusion?

• Never transfuse on Hb or Hct alone

Red Cells

• Indications

  • Clinically significant anaemia

  • With symptomatic deficit of oxygen carrying capacity

  • Critical bleeding

• Contraindications

  • Hb ≥100 g/L

  • If anaemia can be treated with specific medications:

    • Iron

    • Vitamin B12

    • Folic Acid

Universal Red Cell Donor

• Universal Red Cell Donor - O RhD Neg or O RhD Pos

Red Cells Compatibility

• Group AB can donate RED CELLS to other AB, but can receive RED CELLS from all others

• Group A can donate RED CELLS to A and AB

• Group B can donate RED CELLS to B and AB

• Group O can donate RED CELLS to all blood types. It is the universal red cell donor.

Red Cell Compatibility

Plasma (FFP)

• Volume:

  • 1 unit = 250 - 300mL

  • Factor VIII ≥0.7IU/mL

  • Stable clotting factors 80-100%

• Storage:

  • 12 months @ <-25°C

• Dose:

  • Varies depending on indication

• Handling:

  • Snap frozen as soon as possible after collection

  • Thawed @ 37°C before use

• Expiry:

  • 5 days after thawing, if stored at 2-6°C

Plasma

• Indications

  • Bleeding or risk of major blood loss combined with coagulation factor deficiency

  • Plasmapheresis in TTP

  • Isolated factor deficiency when no suitable factor exists

• Contraindications

  • Hypovolaemia by itself

  • Routine in major surgeries

  • Immunodeficiency

  • Sepsis in newborns

  • Diet support / recuperation / parenteral nutrition

Universal Plasma Donor

• Universal Plasma donor - Group AB

Plasma Compatibility

• Group AB can donate PLASMA to all blood types. It is the universal plasma donor.

• Group B can donate PLASMA to B’s and O’s.

• Group A can donate PLASMA to A’s and O’s.

• Emergency use Group O can donate PLASMA to other O’s, but can receive plasma from all others.

Plasma Compatibility

Platelets

• Volume:

  • Pooled (4-6 units): 350 - 380mL

  • Apheresis: 100 – 400mL

• Storage:

  • 7 days @ 20-24°C, agitated gently & continuously

• Dose:

  • 1 pool or apheresis unit expected to raise platelet count by 20-40x$10^9$/L in a 70Kg adult

• Transfusion:

  • Transfuse at 20-24°C within 30 minutes of removing from storage & over maximum 4 hours

Platelets

• Indications

  • To treat bleeding:

    • Active bleeding with thrombocytopaenia

    • Massive blood loss, platelet count <50 x$10^9$/L

  • Prophylaxis:

    • Thrombocytopaenia & plt count <10x$10^9$/L

    • Higher thresholds may be relevant in certain situations (neurosurgery)

    • Neonates require higher counts

• Contraindications

  • Not given to patients with platelet destruction

    • ITP

    • TTP

    • HIT

    • Unless life threatening haemorrhage

Cryoprecipitate

• Clotting Factors: Fibrinogen FVIII FXIII vWF Fibrinectin

• Volume:

  • WB: 30-40 mL/bag

  • Apheresis: 55-65 mL/bag

• Storage: 12 months @ <-25°C

• Handling: Thaw at 37°C before use

  • Use immediately

• Dosage: Dependent on clinical situation, patient size & laboratory tests

  • Generally 30-40mL for each 10kg patient body weight

  • WB dose: 10 bags

  • Apheresis dose: 4 bags

• Expiry: 6 hours after thawing, stored @ 20-24°C

Cryoprecipitate

• Indications

  • Treatment of:

    • Fibrinogen deficiency

    • Dysfibrinogenaemia with clinical bleeding, trauma or DIC

• Contraindications

  • Do not use cryoprecipitate in treatment of:

    • Haemophilia

    • Von Willebrand’s disease

    • Factor XIII deficiency

    • Fibronectin deficiency

    • Unless no other treatment is available

When Do We Transfuse ???

Patient Blood Management

• Aim to optimise, conserve and manage the patient’s own blood to minimise or avoid exposure to allogeneic blood

• Changing from a product focus to a patient focus

• Patient specific team approach

• Results in improved patient outcomes

• An approach to safe, quality patient care…BUT one size doesn’t fit all

Patient Blood Management Guidelines

• Patient blood management guideline for adults with critical bleeding 2023

• Patient Blood Management Guidelines: Module 2 Perioperative

• Patient Blood Management Guidelines: Module 3 Medical

• Patient Blood Management Guidelines: Module 4 Critical Care

• Patient Blood Management Guidelines: Module 5 Obstetrics and Maternity

• Patient Blood Management Guidelines: Module 6 Neonatal and Paediatrics

Five Drivers Shifting the Paradigm from Product-Focused Transfusion Practice to Patient Blood Management

• Scarcity

• Cost

• Quality, Adverse efficacy & outcome

• Infectious risk

• safety issues

• Harness

• Optimise ent's

• optimise

• Minimise physio ed cell blood loss logical tolerance

Patient Blood Management Principles

• Optimise red cell mass

• Minimise blood loss & bleeding

• Harness & optimise physio- logical reserves

• Anaemia Iron deficiency Blood loss & bleeding Transfusion

• Planned, team approach, involving patient, combination of strategies

Three Pillars of Patient Blood Management

Points to Consider

1. Is the patient symptomatic?

2. Is the problem acute or chronic?

3. What is the aim of treatment?

4. Can I reduce blood loss?

5. Are there any other options or alternative treatments?

6. Has a cross-match been performed?

7. What are the specific clinical and laboratory indications for transfusion?

8. What are the risks of transmitting disease or adverse event?

9. Are benefits > risks?

10. Will the patient be monitored?

11. Have I recorded reasons for transfusion and administration instructions?

12. Does the patient understand and consent?

Hazards of Transfusion

• Identification

  • Patient

  • Samples

  • Lab errors

  • Transport

  • Administration

• Biological

  • Degeneration

    • Microaggregates

    • Cell debris

    • Products of storage

• Physical

• Chemical

• Transmitted

  • Viral

  • Bacterial

  • Protozoal

  • ?Prion

  • ? unknown

• Technical Or Clerical

• Volume over

• overload (TACO)

• Alloantibodies Active (Recipient) Cellular Plasma Proteins Passive (Donor) Leukoagglutinins

• Allergic reactions

• TA-GVHD

• Immunomodulation

• TRALI

• Physiological

• Clinical presentation

• IMMUNOLOGICAL

• INFECTIOUS

• STORAGE RELATED

Hazards of Transfusion

• Immune-mediated vs non-immune

• Immediate vs delayed

• Infection vs non-infectious

• Non-infectious but potentially fatal

  • Red cell incompatibility esp. ABO

  • Allergy/anaphylaxis, TA-GVHD, TRALI

• Risk depends on:

  • Context

  • Component/product

  • Balanced with risk of not receiving a transfusion

Hazards of Transfusion

Residual Risk Estimates

• Transfusion Transmitted Infections per Unit (Australia)