Blood group tests

Overview

>40 blood group systems, ~350 antigens. Only a handful matter clinically: ABO, RhD, Rh (C/c/E/e), Kell, Kidd, Duffy, MNS

ABO - the only system with naturally occurring antibodies

Group	Antigen on RBC	Antibody in plasma	AU frequency
O	None	Anti-A and anti-B	~40-49%
A	A	Anti-B	~31-38%
B	B	Anti-A	~8-11%
AB	A and B	None	~2-3%

Landsteiner's law: the plasma contains antibody against whichever ABO antigen the red cells lack - without prior exposure (from cross-reactive gut bacterial and food carbohydrates)
These antibodies are IgM -> complement-fixing -> intravascular haemolysis

Universal donor / recipient

Universal donor / recipient - and the reversal for plasma

	Red cells	Plasma / FFP
Universal donor	O RhD-negative (no A, B or D antigen)	AB (no anti-A or anti-B)
Universal recipient	AB RhD-positive	O

*Red cells and plasma are opposite. Give the cells the patient's antibodies cannot see; give the plasma that contains no antibody against the patient's cells.*

RhD

RhD-positive ~83% of Australians (higher in Asian populations, lower in some European)
No naturally occurring anti-D - it must be acquired by transfusion or pregnancy
Highly immunogenic: ~80% of RhD-negative recipients seroconvert after one RhD-positive unit
*Therefore: RhD-negative females of childbearing potential must receive RhD-negative red cells and platelets*

Alloimmunisation

Alloimmunisation rate ~1-3% per red cell unit in the general population; up to 30% in multiply transfused sickle cell and thalassaemia patients
Commonest clinically significant alloantibodies: anti-D, anti-E, anti-K, anti-c, anti-Jk-a, anti-Fy-a
Anti-K accounts for a disproportionate share of severe haemolytic disease of the fetus and newborn
Group frequencies vary by ancestry - the Australian donor pool is antigenically mismatched to some recipient populations, which is why alloimmunisation is high in haemoglobinopathies

Why some antigens matter more than others

Feature	Consequence
Immunogenicity	D >> K > c > E > Fy-a > Jk-a. A single exposure to D immunises most RhD-negative people
IgM vs IgG	IgM (ABO) = intravascular, immediate, lethal. IgG (all others) = extravascular, delayed, usually mild
Placental transfer	Only IgG crosses -> haemolytic disease of the fetus and newborn from Rh, Kell, Duffy; ABO HDFN is mild because anti-A/B is mostly IgM
Antigen expression on fetal marrow	Anti-Kell suppresses erythropoiesis directly -> anaemia out of proportion to haemolysis, low reticulocytes
Evanescence	Kidd antibodies fall below detection -> delayed haemolytic transfusion reactions

Duffy and malaria

Fy(a-b-) phenotype is near-universal in West African populations
*Duffy is the erythrocyte receptor for Plasmodium vivax*** -> Duffy-null confers resistance
The same ACKR1 promoter variant produces the benign ethnic (Duffy-null) neutrophil count

Bombay phenotype

Absent H antigen (FUT1 null) - the precursor for A and B
Types as group O but has anti-H as well as anti-A and anti-B
*Will haemolyse ordinary O blood - can only receive Bombay blood*

Group and screen

1. Forward (cell) grouping - patient red cells + known anti-A, anti-B, anti-D reagents

2. Reverse (serum/plasma) grouping - patient plasma + known A1 and B cells

*Forward and reverse must agree. Discrepancies from: newborns (no reverse group before ~4-6 months - antibodies not yet made*), elderly, hypogammaglobulinaemia, recent transfusion, ABO subgroups, cold agglutinins, myeloma rouleaux

3. Antibody screen (indirect antiglobulin test)

Patient plasma + screening cells of known phenotype, incubated at 37 C, + anti-human globulin
Detects free antibody in the plasma - the antibody that would attack transfused cells
Positive -> antibody identification panel -> antigen-negative units required

Crossmatch

Type	Method	When
Electronic/computer	Algorithmic ABO/RhD match, no serology	Negative antibody screen + two concordant historical groups + validated system. Minutes
Immediate spin	Patient plasma + donor cells, room temperature	ABO compatibility only
Full serological (IAT)	Patient plasma + donor cells + anti-human globulin at 37 C	Positive antibody screen, or history of an antibody. ~45-60 min, often longer

Direct vs indirect antiglobulin test

Direct vs indirect antiglobulin test - keep these apart

	Direct (DAT/Coombs)	Indirect (IAT)
Tests	Patient's red cells	Patient's plasma
Detects	Antibody/complement already ON the cells	Free antibody in plasma
Used for	AIHA, HDFN, haemolytic transfusion reaction, drug-induced haemolysis	Antibody screen, crossmatch, antenatal screening

Antenatal testing

Group, RhD type and antibody screen at booking, repeated at 28 weeks
Non-invasive fetal RhD genotyping on maternal plasma cell-free DNA - avoids unnecessary anti-D
Kleihauer-Betke or flow cytometry to quantify fetomaternal haemorrhage and calculate additional anti-D

Emergency release

A. Emergency release

Time	Product
Immediate	O RhD-negative red cells
	O RhD-positive is acceptable for males and post-menopausal females - conserves scarce O-negative stock
~10-15 min	Group-specific, uncrossmatched
~45 min	Fully crossmatched

*Group O RhD-negative supply is a finite national resource* - switch to group-specific blood as soon as the group is confirmed

Sample and identification requirements

B. Sample and identification requirements

Label the tube at the bedside, from the patient's wristband, in the patient's presence
*Never pre-label; never label away from the bedside* - misidentification here is the cause of nearly all fatal transfusions
Two independently collected samples confirming the group before non-emergency transfusion of a first-time patient
Sample validity typically 72 hours if transfused or pregnant within the last 3 months

Anti-D prophylaxis - RhD-negative pregnant women

C. Anti-D prophylaxis - RhD-negative pregnant women

Routine antenatal prophylaxis at 28 and 34 weeks (625 IU each in Australia)
Within 72 hours of delivery of an RhD-positive infant (625 IU, plus more if the Kleihauer indicates a large bleed)
After any potentially sensitising event: miscarriage, termination, ectopic, CVS/amniocentesis, external cephalic version, abdominal trauma, antepartum haemorrhage
Not required if fetal cell-free DNA genotyping confirms an RhD-negative fetus
*Anti-D prophylaxis has near-eliminated Rh haemolytic disease; it does nothing for anti-Kell or other antibodies*

Special matching requirements

D. Special matching requirements

Extended Rh and Kell matching for sickle cell disease, thalassaemia, and women of childbearing potential
Antigen-negative units for any historically identified antibody, for life
Antibody card issued to the patient; entry on hospital and shared transfusion records

When the group is unclear

E. When the group is unclear

Recent ABO-mismatched transplant, massive transfusion, cold agglutinins, warm autoantibody pan-reactivity -> discuss with transfusion medicine
Warm autoantibody: DAT positive, all screening cells reactive -> adsorption studies to unmask underlying alloantibodies. Do not delay life-saving transfusion for this

Associated conditions

Haemolytic disease of the fetus and newborn - anti-D, anti-c, anti-K
Acute and delayed haemolytic transfusion reactions
Autoimmune haemolytic anaemia (DAT positive, pan-reactive screen)
ABO-incompatible solid organ and stem cell transplantation; passenger lymphocyte syndrome
Duffy-null phenotype - P. vivax resistance and benign ethnic neutrophil count
Bombay phenotype
Paroxysmal cold haemoglobinuria - Donath-Landsteiner antibody, anti-P
Group O and lower von Willebrand factor levels (mildly increased bleeding tendency, decreased VTE risk)
Group A and increased VTE and pancreatic cancer risk

Antibody persistence

*An identified alloantibody is permanent in significance, even once serologically undetectable* - antigen-negative blood is required for life
Antibody titres fall (evanescence, classically Kidd) -> the reason delayed haemolytic transfusion reactions occur despite a compatible crossmatch
Multiply transfused patients accumulate antibodies -> progressively harder and slower to crossmatch, with real delays in an emergency

Practical failure points

Wrong-blood-in-tube from labelling away from the bedside
Historical antibody records held at another hospital and not retrieved
Assuming a negative antibody screen means the patient has never had an antibody
Delay in switching from emergency O-negative to group-specific blood

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