Blood Grouping and Rh Typing

Blood grouping defines the ABO type and the RhD status of red cells, and is the first step in transfusion, antenatal care and transplantation.

The ABO group is determined by antigens on red cells (forward group with anti-A and anti-B reagents) and by the corresponding naturally occurring antibodies in plasma (reverse group with A1 and B reagent cells), and the two must agree.

RhD status is determined with anti-D reagent, with weak D testing for weak reactions. The ABO and RhD results define compatible red cells and plasma and direct anti-D prophylaxis in pregnancy.

ABO incompatibility is the commonest cause of fatal transfusion reactions, usually from a sampling or identification error and not a laboratory error. About 45% of people worldwide are group O, but this varies by region. Group A is about 40% in many European populations, group B about 10% and group AB about 4%. In parts of sub-Saharan Africa and Latin America, group O exceeds 50%. RhD negativity is about 15% in people of European ancestry, 3 to 8% in people of African ancestry, and under 1% in East Asians.

Indications. Pre-transfusion testing, antenatal booking and any sensitising event, neonatal haemolytic disease, solid organ and stem cell transplantation, major haemorrhage, and unexplained anaemia or jaundice in the newborn.

Specimen. EDTA blood, labelled at the bedside with full identification. In most services a second independent sample, or a documented historical group, is needed before group specific red cells are issued.

Methods. Tube agglutination, column agglutination in gel or glass beads, solid phase red cell adherence, and automated microplate systems. Reactions are graded from 0 to 4+.

ABO forward grouping. Patient red cells with anti-A and anti-B.

ABO reverse grouping. Patient serum or plasma with A1 and B reagent red cells. Anti-A and anti-B are naturally occurring IgM and appear from about 3 to 6 months of age.

RhD typing. Patient red cells with anti-D (IgM or blend). A negative result is followed by a weak D test in some settings (indirect antiglobulin test), mainly for donors and in some countries for patients.

Reporting. The group is reported as A, B, AB or O with RhD positive or negative.

Compatibility.

For RhD, a negative recipient receives RhD-negative red cells. An RhD-positive recipient can receive either. Group O RhD-negative red cells are used in emergencies when the group is unknown, particularly in females under about 50 years.

Forward and reverse mismatch.

• Weak or missing antibodies: infants under 4 to 6 months (reverse group unreliable), older adults, hypogammaglobulinaemia, immunosuppression, chronic lymphocytic leukaemia, and after stem cell transplant.
• Weak subgroup antigens: A2 and A2B can carry anti-A1 (about 1 to 8% of A2 individuals), and weak A or B subgroups can give a weak forward reaction.
• Cold agglutinins, rouleaux and cold reactive antibodies add unexpected reverse reactions, and warm saline washing or warming of the sample resolves them.
• Acquired B phenotype: weak B-like reactivity in group A patients with gram negative sepsis, colon carcinoma or bowel obstruction.
• Mixed field reactions: recent transfusion of non-identical cells, stem cell transplant, chimerism (including twins), and foetomaternal haemorrhage.
• Bombay phenotype (hh): no A, B or H antigens on the forward group, and anti-A, anti-B and anti-H in the plasma. It appears as group O, but the patient can only receive Bombay red cells. Frequency is about 1 in 10 000 in parts of India, and about 1 in 1 million in Europeans.
• Polyagglutination, autoantibody coating of red cells, and Wharton's jelly contamination in cord samples cause false forward reactions.
• Cis-AB and the B(A) phenotype give unusual patterns and need molecular testing.

Weak D and partial D. Weak D types 1, 2 and 3 do not form anti-D, so these patients can receive RhD-positive blood and need no prophylaxis. Other weak D types and partial D can form anti-D after exposure. Molecular RHD genotyping identifies the type, and until it is known, patients who are women of childbearing potential receive RhD-negative blood. Anti-D immunoprophylaxis follows local policy by type.

RhD immunisation risk. Transfusion of RhD-positive red cells to an RhD-negative recipient causes anti-D in about 20 to 30% of those with an intact immune system.

Pregnancy. An RhD-negative woman needs anti-D immunoprophylaxis at about 28 weeks, after any sensitising event, and after delivery of an RhD-positive baby. Foetal RHD genotyping from maternal plasma cell free DNA can identify RhD-negative foetuses and avoid unneeded prophylaxis.

Haemolytic disease of the newborn. ABO incompatibility (group O mother, group A or B baby) is the commonest cause, usually mild, and RhD disease is more severe. The baby's group and direct antiglobulin test are checked.

Critical findings needing immediate action.

• Unresolved ABO discrepancy in an urgent need for blood: issue group O red cells and resolve the discrepancy afterwards.
• A suspected ABO-incompatible transfusion (fever, rigors, hypotension, loin pain, haemoglobinuria, bleeding): stop the transfusion, check patient and unit identity, keep the line open with saline, and call the transfusion laboratory and a senior clinician at once.
• Group O mother with an infant who has early jaundice: check the infant's group and direct antiglobulin test.

Pitfalls.

• Wrong blood in tube and labelling errors are the most dangerous failures, and the main reason for a second group sample.
• A group from another hospital without confirmation is not used for group specific issue.
• Recent transfusion, stem cell transplant or neonatal status changes the interpretation.
• Rouleaux and high protein concentrations can cause false agglutination, and saline replacement resolves it.
• Weak D testing is reagent dependent.
• Infants have fewer antigen sites, so forward reactions can be weak.
• An RhD result in a patient who has recently received RhD-positive red cells is unreliable.

Special groups.

• Neonates: forward grouping only, the reverse group is unreliable before 4 to 6 months. Maternal antibodies can be present in the infant's serum.
• Transplantation: ABO-incompatible transplants need antibody titres and special desensitisation methods (planned under specialist care), and group O donors are used for plasma depleted red cells.
• Sickle cell disease and thalassaemia on regular transfusion: matching for Rh (C, c, E, e) and Kell reduces alloimmunisation, and extended phenotyping is needed.
• Duffy null phenotype is very frequent in people of West African ancestry and is relevant to antigen matching.

Follow up tests. Antibody screen and identification, direct antiglobulin test, crossmatch, extended red cell phenotyping or genotyping, RHD genotyping for weak D, foetal RHD genotyping from maternal plasma, Kleihauer-Betke test or flow cytometry after a sensitising event, neonatal bilirubin and haemoglobin.