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Chapter 6 The ABO Blood Group System

Introduction

The ABO blood group system is the most clinically significant of all the blood group systems. This is

mainly due to the vast majority of the population carrying pre-formed ABO antibodies; this is the

only blood group system where if you lack the antigen you will make the corresponding antibody

without deliberate immunisation. As we learned in chapter 4 (antibody mediated red cell

destruction) ABO antibodies can cause intravascular haemolysis. If we were to randomly transfuse

red cells to a group of people without ABO grouping either the donors or the recipients there is a

high probability that some would receive ABO incompatible blood and potentially undergo an acute

haemolytic transfusion reaction. As such it is of paramount importance that both the blood donor

and blood component recipient are ABO grouped correctly and compatible blood components are

transfused.

Remember: the transfusion of ABO incompatible blood is avoidable and is classified as an NHS

NEVER EVENT.

In UK transfusion laboratories ABO grouping is a computer controlled automated process with as

little human intervention as possible. This is the best way to reduce human error and create safer

transfusion practices. ABO incompatible transfusions caused by an error in ABO typing are extremely

rare. Unfortunately automation has also led to a decrease in manual skills; transfusion of the wrong

ABO group due to a technical grouping error in the 21st century will invariably be the result of a

human performing a manual test. For this reason, wherever possible ABO grouping should be

automated. However there will be occasional cases where manual ABO grouping is required. This is a

straightforward process but one that requires robust procedures and careful checking at all stages.

This chapter aims to enable you to confidently understand and perform manual ABO grouping.

By the end of this chapter you will be able to:

Describe the basic genetic background of the ABO system

Investigate family trees based on ABO grouping results

Describe the biochemistry of ABO antigen production

List the ABO transferases, explain their action and list their products

Describe and discuss the different antigens of the ABO system with respect to

expression

development at birth

common subgroups

some rare subgroups

List the frequencies of the four major ABO blood groups in the UK population

List some of the frequency variations found in different ethnic populations

Explain and discuss the relationship of the H blood group system to the ABO blood group

system

Outline the mechanism leading to the Oh (Bombay) blood group including

inheritance patterns

The ABO Blood Group System

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frequency

antigen expression

antibodies present in serum and their implications for blood transfusion

Describe and discuss ABO antibodies with respect to

their production

immunoglobulin classes

methods of detection

ability to bind complement

clinical significance

selection of blood components for transfusion

Outline secretor and non-secretor status for ABH antigens

Including inheritance and frequency in the UK population

Explain and discuss ABO grouping procedures including

Forward and reverse grouping

Reagent requirements

Automated and manual techniques using different technologies

Interpretation of results

Describe, explain, interpret and resolve the mechanisms and causes of common anomalous

ABO grouping results including

Unexpected forward grouping results

Unexpected reverse grouping results

HISTORY

The ABO blood group system was discovered by Karl Landsteiner in 1901. It was the first blood group

system to be discovered. Landsteiner had mixed the sera and red cells from his co-workers and

discovered that they reacted in different ways. He named three groups A, B and O. In 1902

Decastello and Sturli discovered the additional AB phenotype. From his experiments Landsteiner

deduced that the lack of an A or B antigen resulted in the production of the corresponding antibody

by the individual. This is known as ‘Landsteiner’s Law’. The ABO system is the only system where this

phenomenon occurs.

Table 6-1: Basic ABO blood group system. This table forms the basis of ABO

compatibility/ incompatibility rules.

Landsteiner’s Law: Healthy adults lacking the A or B antigen on their red cells will have the

corresponding antibody present in their serum. See table 6.1


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ABO BLOOD GROUP FREQUENCIES

The frequencies vary dependent upon the population studied. Table 6-2 gives the approximate

frequencies for selected populations.

Table 6-2: Blood group frequencies for selected populations. Note that even within the UK small variations exist. It is also important to remember that variations exist within sub-populations and ethnic groups of different countries across

the world.


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ABO GENETICS

The ABO gene locus is on chromosome 9. At its most basic, one of three allelic gene options are

possible; A or B or O. A and B are codominantly expressed; O is amorphic. Table 6-3 describes the

allelic gene products.

Table 6-3: Allelic gene products and resultant antigens. Note that the gene product is not the antigen itself but an

enzyme.

The gene product is an enzyme; A or B transferase, which requires the presence of H antigen in

order to act. This means the ABO genetic pathway is also dependent on inheritance of the H gene of

the H blood group system. The H blood group system is separate and independent from the ABO

system, residing on chromosome 19. The H antigen is high prevalence occurring at a frequency of

99.9% in all populations. Most people will be homozygous for the H gene. Inheritance of two

amorphic h alleles resulting in the null phenotype Oh (Bombay) will be described further on. Table 6-

4 expands on the presence of H antigen.

Table 6-4: The A/B transferase converts H antigen to A/B antigen. Note that in group O people H antigen remains

unchanged as the O allele does not produce a transferase.

As each individual inherits chromosomes in pairs a number of combinations are possible which

together produce the person’s ABO genotype, which determines their ABO phenotype. Table 6-5

shows the possible different genetic backgrounds.

Table 6-5: Basic genetic backgrounds leading to O, A, B and AB phenotypes. Note that as O is amorphic inheriting a single

A or B allele produces sufficient A or B transferase to convert H antigen to A or B antigen respectively.


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Note that there are 6 possible basic ABO genotype combinations but because the O gene is

amorphic there are only 4 observable phenotypes:

The O phenotype can only be produced by an OO genotype

The AB phenotype can only be produced by an AB genotype

The A phenotype can be produced by the genotypes AA or AO

The B phenotype can be produced by the genotypes BB or BO

If the A allele is present A transferase acts on H antigen to produce A antigen.

If the B allele is present B transferase acts on H antigen to produce B antigen.

These alleles are codominant, so if a person inherits the A and B allele then both A and B

transferases are produced and will act on H antigen independently converting some to A and some

to B antigen. This produces the AB phenotype.

Note that the A and B transferases are very efficient but still do not convert all the H antigen present

to A or B antigen. Therefore group A, B, and AB people will still express some H antigen (this is

discussed in more detail later on in this chapter).

Inheriting two copies of A or B alleles does not result in stronger expression of A or B antigen

compared to an AO or BO genotype i.e. the ABO antigens do not show ‘dosage’ as seen for some

other blood group antigens. For example, group A red cells produced by the homozygote AA

genotype do not react more strongly with anti-A than group A red cells of the heterozygote AO

genotype.

Molecular genotyping is the only conclusive way to distinguish between the genotypes AA/AO and

BB/BO. Informative family studies may be used as an alternative method but caution should be used

as they are still subjective without DNA analysis.

The following few pages give examples of simple ABO inheritance. It is recommended you work your

way through them to give yourself some practice. Head to chapter 5 if you need to refresh your

genetics knowledge.


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Figure 6-1: Example of simple ABO inheritance.

Note that during meiosis paired chromosomes separate to form the gametes (sex cells). When a zygote (fusion of two gametes) forms the ABO blood group that will eventually be expressed depends on the ABO alleles inherited from both parents. Remember that a gamete contains 23 single chromosomes rather than 23 pairs of chromosomes.

It is possible for a group O person to have a group A or B child. In this scenario there is a 50:50 chance of the offspring being group A or group B because the father is group AB.


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Action/ reflection point: the following diagram gives the maternal, paternal and

possible offspring ABO phenotypes but only some of the genotype information. Your task is

to work out the missing information.


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Action/ reflection point: Your next task is to work out the possible inheritance

patterns in the questions below. On the next page figure 6-2 provides a series of

pictures to assist you. Photocopy the page and cut out the shapes to form your own

inheritance diagrams as shown in figure 6-1. Once each one is completed you may

find it useful to photograph them for your records. Answers are provided in the on-

line supporting material.

Work out the genotypes and phenotypes for the possible offspring of a

1. group B phenotype and group O phenotype mating

2. group A phenotype and group B phenotype mating

3. group A phenotype and group O phenotype mating

4. 2 group A phenotypes

Remember to include all possible genetic backgrounds! You may need to photocopy

more than one page or recycle the pictures as you move to the next pattern.


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Figure 6-2: Materials for action/ reflection point on the previous page.


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Common ABO subgroups: The A1 and A2 alleles

Individuals designated as group A actually consist of two main subgroups, known as A1 and A2,

resulting from the transferase produced by the A1 and A2 alleles respectively. Monoclonal anti-A

used in the laboratory does not differentiate between these subgroups and unequivocal positive

reactions are simply recorded as group A phenotype. This is because it is safe to give group A1 blood

to group A2 people and vice versa.

Approximately 78% of group A people will be A1; 22% A2. This also applies to group AB people.

The A transferase produced by individuals with the A2 allele is far less efficient at converting H into A

antigen. A1 individuals have approximately 1,000,000 A antigens per red cell whereas A2 individuals

have only around 250,000 A antigens per red cell. This is still a large number when compared to

other blood group systems (see table 1-2 in chapter 1) and A2 red cells are fully capable of causing an

acute transfusion reaction if given to a group O recipient (for example). As such group A blood

donation packs are labelled simply as ‘A’ rather than indicate the subgroup.

The A1 allele is dominant to the A2 allele. When present with a B allele both the A1 and A2 alleles

behave codominantly and both are expressed when present with an O allele. Therefore, the

phenotype A1 may be produced by the A1A1, A1A2 or A1O genotypes, whereas the phenotype A2

results from the presence of either the A2A2 or A2O genotypes. Group AB people can also be

subdivided into group A1B and A2B. This extends the phenotype possibilities from four to six and the

possible genotypes from six to ten. See table 6-6 below.

Table 6-6: Genotypic backgrounds of the common A1 and A2 phenotypes.

At this point you may be wondering why knowledge of common A subgroups is required at all in

routine testing. The simple answer is that A2 people can make anti-A1 and this can cause problems in

routine ABO grouping of both donors and patients.

Approximately 2% of A2 and 25% of A2B people produce a ‘naturally occurring’ anti-A1 antibody. As

implied by the name this antibody reacts with group A1 red cells but not group A2 red cells. It is not

produced in response to an A2 recipient having received A1 red cells. This defies Landsteiner's Law,

since the antibody is effectively directed against an antigen which the person still has, albeit in a


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smaller number. Anti-A1 is normally reactive at 4-22oC but not 37oC and is therefore not considered

to be clinically significant.

Group A reagent red cells provided for the reverse grouping test are purposefully selected to be A1

phenotype to ensure weaker examples of anti-A are detected. The occasional ‘problem’ with using

A1 reagent cells is that individuals who have produced an anti-A1 will give an unexpected reaction in

their reverse group i.e. as this test is performed at room temperature a reaction with the A1 cells

may be observed. As you are probably aware any unexpected reaction in routine grouping must be

investigated before a valid ABO group can be assigned. As such anti-A1 is a nuisance antibody that

can lead to a delay in blood provision if technicians are unaware of its occurrence or how to resolve

the unexpected grouping results. ABO antibodies and grouping techniques are discussed further on

in this chapter.

ABO BIOCHEMISTRY – Production of the A and B antigens

As outlined above the A and B alleles produce the A and B transferases respectively. These enzymes

act on a particular substrate, H antigen (also referred to as H substrate), converting it into a specific

product, A or B antigen. See figure 6-3.

Figure 6-3: Overview of the ABO genetic pathway

Figure 6-4 shows the pathway for the OO genotype.

Figure 6-4: As the O allele produces no functional transferase H antigen remains unchanged.


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As the production of A and B antigens is dependent upon the presence of H antigen we need to

extend this genetic pathway ‘backwards’; figure 6-5.

Figure 6-5: Genetic pathway expanded to show the interaction of H and ABO systems. Precursor substance is an extensive carbohydrate chain that is present on all red cells. The diagram above shows A/B transferase converting H to A or B antigen. The diagram below shows H remaining unchanged due to the amorphic O allele.

Remember: The H and ABO genetic loci are on separate chromosomes so H and ABO represent

separate, though biochemically related, blood group systems. It is therefore incorrect to refer to the

‘ABH blood group system’.

ABH transferase activity in more detail

The H allele, either expressed as the homozygote HH or heterozygote Hh, produces H transferase.

This enzyme adds a single terminal carbohydrate molecule, L-fucose, to the precursor substance.

This additional sugar moiety* converts the precursor substance to H antigen.

*moiety is a term used in chemistry to indicate a distinct part of a large molecule.

The A and B genes, via their enzyme products, each add a different terminal carbohydrate molecule

to H thus converting it to A or B antigen.

A transferase adds N-acetyl-D-galactosamine to H

B transferase adds D-galactose to H

The OO genotype produces no functional transferase and therefore H is left unchanged.


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The different carbohydrates added by the A, B or H transferases are termed IMMUNODOMINANT

sugars. This is the part that is antigenic.

Table 6-7 shows the ABH gene products and their resultant immunodominant sugars.

Table 6-7: ABH gene products and resultant added carbohydrate. Note that is acceptable to refer to the enzymes simply as A, B, or H transferase however it is important that you know the name of the immunodominant sugar that is added

by each specific transferase.

Figure 6-6 shows the basic structure of precursor substance and the ABH antigens.

Figure 6-6: Basic structure of the precursor carbohydrate chain showing the different terminal immunodominant sugars attached by the action of the H, A and B transferases. Note that the addition of L-fucose by H transferase to the starting

precursor substance allows the A/ B transferase to then add their respective immunodominant sugar. The ‘remainder’ of the precursor chain is essentially a long repeating carbohydrate structure of which there are at least 5 types. ABH

antigens found on the red cell membrane are derived from type 2 precursor substance.

ABH antigens are referred to as histo-blood group antigens because, if produced, they are also found

on platelets, white cells and most other tissues of the body


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Secretor and Non-secretor status

ABH antigens are also present in soluble form in all body fluids in people who are ‘secretors’. The

secretor gene Se codes for a transferase that converts soluble precursor substance into soluble H

antigen. Therefore if the Se gene and A/B genes are inherited the individual will have H and A/B

antigens present in their bodily fluids. Approximately 80% of the general population carry the Se

gene. Because of this red cells should be washed, or at least suspended in saline before blood

grouping to avoid any soluble plasma antigens from inhibiting the anti-A and/or anti-B reagents. See

table 6-8.

Table 6-8: Genetic background of secretor and non-secretor status showing soluble ABH antigens in bodily fluids.

Note that this can cause some confusion as this soluble H antigen is not reliant on the H transferase

discussed above!

For the production of red cell H antigen, H transferase produced by the H gene is required

For the production of soluble H antigen, secretor transferase produced by the Se gene is

required

ABO antigen development

A and B antigens are detectable as early as 5 weeks gestation. However they do not fully develop

until after birth meaning they are expressed weakly on cord cells in comparison to adult cells. This is

possibly a result of inadequate type 2 precursor substance availability. Monoclonal anti-A and anti-B

used in ABO grouping however, will give unequivocal positive reactions with group A, B and AB red

cells from full-term neonates and therefore forward grouping of neonates is rarely problematic. A1

individuals may appear serologically to be A2 phenotype and therefore anti-A1 typing on neonatal (<4

months) red cells is not recommended.

Amount of H antigen in different ABO groups

Since the H antigen remains unchanged for group O people it is therefore present in maximal

amounts on their red cells. In contrast groups A, B and AB have little H antigen present, having

converted most of it to A and/or B antigen. When both A and B genes are present, very little H

remains unconverted.

For the A subgroups; the A1 transferase is more effective at converting H than the A2 transferase.

Therefore, A2 individuals have less A antigen and more H antigen on their red cells than A1

individuals. Figure 6-7 shows the amount of H antigen present on a red cell by ABO group.


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Figure 6-7: Group O red cells have the most H antigen whereas group A1B cells have most of H converted to A and B antigens. The rarer A subgroups such as A3 and Ax will slot between the O and A2.

The H deficient phenotype, Oh (Bombay phenotype)

In the genotype hh where the H gene is absent, precursor substance remains unchanged because of

the lack of H transferase. As H antigen is required for A/ B transferases to work, A/ B antigens cannot

be produced even though the A/ B genes may be present and functional. See figure 6-8.

Figure 6-8: Genetic pathway for Oh genotype. Note that A and B alleles may be present and functional but, as both transferases require H antigen to add their immunodominant sugars, A and B antigen cannot be expressed.

This leads to total absence of A, B and H antigens and is known as the Oh or Bombay phenotype.

Remember that as 99.9% of the world’s populations carry the H gene, hh is an extremely rare

phenotype. Although originally reported in some individuals from Mumbai, Oh has been identified in

many other populations including Taiwan, Japan and Europe.

Oh individuals still carry ABO genes and, although not expressed, they are capable of being

transmitted normally to their offspring. Therefore if their offspring inherits H gene from their other

parent their ABO group will be expressed normally. See figure 6-9.


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Figure 6-9: Inheritance pattern of Oh. Note that the parents of the Oh individual are both carriers for the h allele. There is a 1 in 4 chance of any offspring being hh. The Oh individual has inherited both the A1 and B alleles, however without H transferase to produce H antigen the A and B antigens cannot be manufactured. Phenotypically their red cells will group as O.

The Oh individual is homozygous for h and therefore all their offspring will inherit an h allele. Half will inherit the A1 allele and half the B allele. The partner of the Oh individual is homozygous for both H and O alleles. Therefore all offspring will inherit an H and an O allele.

The two offspring resulting from this pairing are shown to be group A1 and group B. Although both parents are phenotypically O the A1 and B genes have been transmitted to the children by the Oh parent and, because they have a functional H transferase, the A and B gene products can produce A and B antigens.

Note that both children are carriers for the h gene.

If the ABO genes present in an Oh person are known, then the phenotypes may be written as OhO,

OhB, Oh

A, OhAB depending on the genes detected. The Oh person in figure 6-9 is Oh

AB. See table 6-9.

Table 6-9: Possible genetic backgrounds of the Oh phenotype

Other ABO Subgroups

Various subgroups of group A and B exist caused by genetic variations that result in inefficient

transferase enzyme production and/ or activity. The A/ B antigens are still produced but in much

smaller amounts. These are rare in the UK population.

A3

This phenotype has a frequency range given as approximately 1 in 1,000 to 1 in 180,000 of group A

people dependent upon the population tested.

Classically, group A3 is identified by a ‘mixed-field’ reaction (small agglutinates in a field of

unagglutinated cells) with anti-A and anti-A,B antisera (both polyclonal and monoclonal types). This

effect is believed to be due to the fact that only 4% of red cells produced by A3 people have enough

A antigen on them to cause agglutination. Group A3 people produce normal anti-B and occasionally

produce anti-A1.


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Ax

This phenotype has a frequency of 1 in 40,000 to 1 in 77,000 of group A people (again results are

dependent on the population tested). Classically Ax red cells are not agglutinated by sera from group

B people (polyclonal anti-A) but are agglutinated by sera from group O people (polyclonal anti-A,B).

Mixed-field reactions are not observed. Monoclonal anti-A used in ABO grouping is designed to

directly agglutinate Ax red cells. Group Ax people produce normal anti-B and also typically anti-A1.

The subgroup phenotypes A3 and Ax are not expressed when these alleles are in combination with an

A1 or A2 gene.

Subgroups of B

These are much rarer than subgroups of A and are usually found only in populations where the

group B frequency is high. It is unlikely you will encounter one during routine testing in the UK due

to the low frequency of group B in the general population.

ABO ANTIBODIES

Development and production

As discussed in chapter 1 on immunology, ABO antibodies are produced in response to common

chemical structures, identical to the A and B antigens, which are present widely in our general

environment. This is reflected in the way the antibodies develop after birth. Children of less than 3

to 4 months usually have little or no antibody present in their serum due to their underdeveloped

immune system and lack of antigenic exposure. Any antibody detected in neonatal samples (<4

months) is likely to be of maternal origin, resulting from placental transfer of IgG antibody. As such

reverse grouping of neonates is not recommended. Between 5 to 10 years of age, as a result of

plenty of environmental exposure, ABO antibody strength is usually comparable to that of a healthy

adult. Levels of ABO antibodies remain relatively stable during adult life and can decline in very old

age (>80 years, note that you should not expect the reverse group in ‘older’ people to be weak but

consider it a possibility when investigating a discrepant ABO grouping result).

The ABO antibodies that can be produced are:

Anti-A

Anti-B

Anti-A,B

Anti-A1

Anti-A,B is a cross-reacting single antibody produced only by group O people. It is capable of reacting

with either the A or B antigen. Note that it is not a mixture of anti-A plus anti-B (that reagent is

named anti-A+B) and it cannot be separated out into anti-A and anti-B. Anti-A,B recognises a

structure that is common to both the A and the B antigenic determinants.

Anti-A1 can be produced by any subgroup of A other than A1 individuals.

Remember: The ABO blood group system is unique in that ABO antibodies are routinely

made without requiring stimulation by transfusion, pregnancy or transplantation


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Clinical Significance

Anti-A, anti-B and anti-A,B are by far the most clinically important blood group antibodies capable

of causing both intravascular and extravascular red cell destruction. See also chapter 4: Antibody

Mediated Red Cell Destruction.

This is due to the following combination of factors. Anti-A, anti-B and anti-A,B:

are present in large amounts as both IgM and IgG class antibodies

Levels remain stable throughout adult life

Titre can vary considerably between individuals; for anti-A between 8 to 2048, for anti-B

between 8 – 256

IgG levels can be stimulated further by immunisation with A/ B antigens (e.g. through

pregnancy, ABO incompatible platelet transfusion)

They have high avidity for their respective antigens

are active at 37oC and activate complement

If an ABO incompatible transfusion is given the high density of A/B antigens per red cell will

enhance the antibody-antigen reactions in vivo

Anti-A,B is produced by group O individuals only. It is typically extremely strong (‘high titre’) and

capable of complement activation and subsequent intravascular haemolysis. In addition it is known

that group O also make more IgG anti-A, anti-B and anti-A,B than IgM type whereas antibodies made

by group B and A people make more IgM type than IgG. This goes some way to explaining why

severe cases of haemolytic disease of the fetus/ newborn (HDFN) due to ABO antibodies are usually

only seen in group O mothers (carrying an A/ B child). For further details see chapter [] HDFN.

Anti-A1 is usually only reactive below 25oC and considered a nuisance as it can interfere with ABO

grouping. It is very rarely active at 37oC.

A side note on anti-H produced by A1 and A1B phenotype individuals

Although this antibody does not belong to the ABO system its production is related and therefore is

of relevance in this section.

The conversion of almost all H present in some group A1 and A1B people can actually result in

production of a naturally-occurring anti-H. This is a cold reacting clinically insignificant antibody that

may be detected during ABO grouping i.e. in the reverse group. This type of anti-H reacts most

strongly with group O red cells, weakly with A2, and perhaps not at all with B and A1, phenotype red

cells. Note that production of this form of anti-H is not due to lack of the H gene or functional H

transferase.

Antibodies produced by the Oh phenotype

Group Oh people lack the H antigen completely and produce a naturally occurring anti-H that is

active at 37oC and is clinically significant. They also produce anti-A and anti-B in accordance with

Landsteiner’s Law. These individuals will react with all ABO groups except Oh.


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ABO GROUPING

The uniqueness of naturally occurring antibody production in the ABO system (Landsteiner’s Law)

allows two methods to be routinely used together for ABO grouping. This complementary testing

regime in the form of a ‘forward’ and ‘reverse’ group gives an added level of confidence in the

results. It also enables the identification of rarer and more unusual ABO groups through discovery of

unexpected reactions.

Both the 2012 BCSH guidelines for pre-transfusion compatibility procedures in blood transfusion

laboratories and the guidelines for the blood transfusion services in the UK (8th Edition) identify that

full ABO grouping comprises:

A forward group using monoclonal anti-A and anti-B grouping reagents

This test is used to identify any antigens present on the red cells

A reverse group using A1 and B reagent red cells

This test is used to identify any ABO antibodies present in the plasma.

Table 6-10 shows expected results for routine ABO grouping. Note: the final column has been left

blank for you to fill in.

Table 6-10: Expected full grouping reactions for the ABO blood groups. Note the controls used for the anti-A and anti-B reagents. The reverse group cannot be controlled per se but is always checked against the forward group for confirmation.

Standard ABO grouping is performed at 18-22oC, ‘room temperature’.

Protocols

Full ABO grouping must be performed on all samples from new/ unknown patients and donors.

Abbreviated ABO grouping comprising a forward group only can be adopted for all known/ historical

patients provided there is a secure and fully automated grouping system, with automated data

‘ABO grouping is the single most important serological test performed on pre-transfusion

samples and the sensitivity and security of testing systems must not be compromised’

Key Recommendation from the BCSH Guidelines for pre-transfusion compatibility procedures in

blood transfusion laboratories (2012)


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transfer, in place. All new/ unknown patients and donors must be tested twice in order to confirm

the ABO group result. Transfusion of ABO group specific blood components should not take place

until the initial grouping result has been confirmed using a second sample taken separately from the

first sample.

UK guidelines recommend that ABO grouping requiring manual intervention of any sort must include

a full ABO grouping method even if the patient has a historical ABO group recorded.

Anomalous ABO grouping results are any that do not fit with those shown in table 6-9. In these cases

the anomaly must be resolved before a valid ABO group can be assigned to the sample tested. In

cases where the forward and reverse group do not match up, you MUST NOT interpret the forward

group results as the group of the individual tested. Neither the forward nor the reverse group are

valid in such cases. Any abnormal grouping results must be recorded, but not interpreted, i.e. you

should simply state that the results are anomalous and require further investigation. Do not be

tempted to assign even a tentative blood group as this has the potential to lead to an incompatible

blood transfusion. There are many circumstances that may give rise to anomalous ABO grouping

results (see below).

Action/ reflection point: The following are awaiting anomalous ABO group

investigation; what interim action is required to ensure the safety of the blood

supply chain/ patient?

A blood donation unit, processed and awaiting blood group labelling?

A patient awaiting blood transfusion?


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Extended ABO grouping

Additional reagents and tests are available to help resolve ABO anomalies and include

For the forward group

Anti-A1

Anti-A,B

Anti-H

AB serum

Reagent control

For the reverse group

A2 cells

O cells

An autocontrol (sample cells tested against sample plasma)

Their use will be highlighted and discussed where applicable in the following sections.

Causes and resolution of anomalous ABO grouping results

Note: You MUST always follow your own laboratory procedures for investigating unexpected ABO

grouping results.

The following examples are listed to help you understand the importance of correct ABO grouping

and recognise some common reasons for unexpected reactions.

Whenever ABO grouping does not give the expected outcome the first step is to repeat the test. This

is to confirm that the reactions obtained are genuine. This should be done in the exact same manner

using the exact same sample. As routine ABO grouping is fully automated wherever possible, it is

likely that the anomaly is genuine. However, the test should still be repeated before further

investigations proceed. It may be necessary to obtain and test a fresh blood sample. Table 6-11 gives

an example of a useful initial ABO anomaly checklist.

Table 6-11: Possible checklist used when an ABO grouping anomaly is encountered. Once this list has been checked further investigations may commence.


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In all cases the patient or donor record should be checked for information that may help to explain

the unexpected reaction. Useful information includes patient diagnosis, age, historical results,

medication, transfusion and pregnancy history. In addition clerical errors should be ruled out as a

routine part of the investigation. Examples include sample mislabelling, transcription errors,

misidentification of the individual, incorrect data entry. Checking for possible equipment failure is

also recommended e.g. equipment calibration or overdue maintenance.

Action/ reflection point: Outline your laboratory procedure for investigating

unexpected ABO grouping results.


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A note on technician error

When manual ABO grouping is performed it is important to recognise that a technical error is more

likely to be the cause of unexpected ABO result than a ‘weird and wonderful’ ABO group. This is not

to say it is unlikely that you will encounter unusual ABO groups but it is important that you have

ruled out any possibility of a technical error first. Steps must be taken to investigate the reason to

prevent repeat mistakes. If repeat results are different from the original results possible reasons for

the original error must be investigated and resolved.

Common causes of incorrect ABO grouping results due to technician error include:

Wrong sample selected and tested

Sample tubes and/ or tests mislabelled or not labelled

Transposition of samples

E.g. forward group and reverse groups are from 2 different people

Transposition of reagents

E.g. A1 and B reagent red cells added to the wrong tests

Failure to follow Standard Operating Procedure (SOP) or manufacturer’s instructions e.g.

Incorrect red cell suspension used

Incorrect incubation time or temperature used

Incorrect diluent used

Incorrect centrifugation speed

Reagents or plasma not added to test

Results read in the wrong order

Wrong results recorded on worksheet

Use of expired reagents and/or kits

Genuine ABO anomalies

In cases where repeat testing shows a genuine ABO anomaly then one, or more, of the following

four broad categories (table 6-12) is usually evident. Each category is discussed in more detail below.

Table 6-12: Categories of possible ABO anomalies that may be detected in the forward or reverse grouping test.

Additional antigens detected

Unexpected positive reactions with anti-A or anti-B are very rare when using monoclonal grouping

reagents. However, unexpected mixed-field reactions may be detected in the following conditions

Post transfusion of non-identical ABO red cells

e.g. of group O blood into a group A patient


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Post bone marrow transplant (BMT) or haemopoietic stem cell transplant (HSCT)

e.g. group B recipient receives group O transplant

Weak expression of A or B antigen

e.g. A3 subgroup

Chimerism

Genetic material from 2 eggs and 2 sperm has fused in the womb into one person. They

may produce 2 red cell lines each with a different blood group.

This is a very rare event

Wharton’s jelly contamination

This occurs when cord blood samples are incorrectly taken. Wharton’s jelly is found in

the umbilical cord and causes red cells to clump. Washing the sample may solve the

problem.

Table 6-13 lists examples of investigations that can help to determine the cause of the mixed-field

reaction. Note that they are given as examples only. You must follow your own laboratory procedure

whenever you encounter any such anomalous result.

Table 6-13: Examples of investigation of various ABO grouping anomalies due to mixed-field reactions detected in the

forward group.


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Whenever mixed-field reactions are detected they should be recorded as such preferably indicating

the strength of the positive reaction seen e.g. ‘2mf’ indicates a weak positive reaction with a large

amount of negative cells present; ‘4mf’ indicates a strong positive reaction with fewer negative cells.

Rarer causes of unexpected antigens in ABO grouping

Cold autoimmune haemolytic anaemia can occasionally lead to forward grouping problems. Those

patients with very strong, high titre, cold reacting autoantibody leading to in vivo coating of red cells

can cause spontaneous agglutination of their red cells in vitro. This is usually rectified by warm-

washing (heat normal saline or PBS to 37oC) the red cells before testing.

The following conditions should not cause problems when using monoclonal anti-A and anti-B

reagents.

Acquired B (see below)

Polyagglutinability of red cells

Red cells spontaneously agglutinate causing a false-positive reaction

Cryptantigens, such as T and Tn, are concealed antigenic structures carried on all red

cells. They can become exposed in certain conditions (e.g. bacterial infections,

leukaemia) and can cause red cells to spontaneously agglutinate. However, monoclonal

anti-A and anti-B reagents are designed not to react with ‘cryptantigens’ and therefore a

washed red cell sample should not give any ABO grouping problems.

Acquired B

This phenomenon occurs in group A individuals where their red cells appear to have changed blood

group. The condition is associated with gastrointestinal bacterial diseases and colon cancer patients

due to the action of bacterial enzymes. The A immunodominant sugar, N-acetyl-D-galactosamine is

converted by these bacterial enzymes to galactosamine. With some anti-B reagents, both polyclonal

and monoclonal, this change is similar enough to the B immunodominant sugar, D-galactose, to give

a positive reaction. It can also be seen in vitro due to blood sample bacterial infection. Not all of the

A antigen is converted so the patient appears to be a group AB with anti-B in their reverse group.

The anti-B produced by patients with acquired B does not react with the acquired B antigen,, but

does react with normal B antigen. It is vitally important that group A people with acquired B antigen

are not incorrectly grouped as AB as this could lead to transfusion of incompatible AB or B red cells.

Monoclonal anti-B reagents used for blood grouping in the UK are designed not to react with the

acquired B antigen. This means that the acquired B antigen will not be detected by routine ABO

grouping methods.

Missing antigens

Unexpected negative reactions with anti-A or anti-B or very weak expression of antigens is

sometimes found in:

Some malignant diseases such as leukaemia, where ABO antigens can become very weak or

even disappear. Expression will return to normal during disease remission.

Red cells from a fetus or newborn infant

Note that most of the time ABO antigens are easily detectable from birth with

monoclonal reagents


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Gestational age rather than date of birth can be important to note when ABO grouping

premature babies. For example a preterm baby grouping as O may actually turn out to

be group A in a few months’ time as the baby develops. If their blood group is initially

recorded as O then this will cause problems for future investigations as their historical

group will not match their current group result.

An infant who has received intrauterine transfusions (IUT) will not give any apparent

anomaly however their blood group result at birth will not be their own! As group O

blood is almost exclusively used for IUT, the baby may appear to be group O at birth and

not their expected group. This can also cause problems in the patient’s blood group

record.

Additional antibodies

Unexpected positive reactions with A1 and/or B cells are the most common cause of anomalous ABO

grouping reactions. As the reverse group is performed at 18-22oC (‘room temperature’) any cold

reacting antibody present in the patient’s plasma can also be detected.

Remember: A1 and B reagent red cells used for reverse grouping also carry other blood group

system antigens!

Cold reacting alloantibodies

Anti-A1, anti-M, and anti-P1 are the most commonly encountered specificities resulting in

unexpected reverse grouping reactions. For example, if your patient happens to have an anti-P1 then

it may react with the A1 and/or B cells if they happen to carry the P1 antigen.

As highlighted earlier approximately 2% of A2 and 25% of A2B people produce anti-A1 (as well as

some of the rarer subgroups of A). This will react with the A1 cells used in the reverse group. See

table 6-14.

Table 6-14: Complete the results where possible. Indicate if further work is required with ‘yes’ or ‘no’.


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Samples 1, 2, 4 and 7 are straightforward and can be resulted. However results for samples 3, 5 and

6 all show discrepancies in the reverse group.

Anti-H produced by group A1 or A1B people typically reacts strongly with group O cells and slightly

weaker with A2 cells, but will not react with A1 cells (see above section on ABO antigen production).

As routine reverse grouping no longer includes O or A2 cells this nuisance antibody is not often

encountered.

Action/ reflection point: List the possible cause or causes of the discrepancies

in table 6-13 and how you could investigate/ prove your theory.

Remember: in the section on extended ABO grouping you were presented with a list

of additional reagents that can be helpful in solving ABO anomalies. Also, if you

suspect an irregular antibody is present how will you identify it?


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Note that anti-H in Oh phenotype does not appear as an additional antibody in the reverse group

but will be detected in the antibody screen. As Oh individuals also make anti-A and anti-B they

appear to be normal group O phenotype in routine grouping. Table 6-15 shows extended typing that

will distinguish between group O and Oh. Table 6-15: Expected extended grouping reactions distinguishing between group O and group Oh phenotype. Note that

without the addition of anti-H or the O reagent red cell both groups will appear the same phenotypically. The ‘auto’ column indicates the patient’s own cells and plasma have been tested against each other.

Photograph showing anti-H reactivity strength against various ABO groups. Direct agglutination method used.

Note that even though most H antigen is converted to A antigen in the A1 phenotype an unequivocal positive reaction is still seen with commercial anti-H.

Cold reacting autoantibodies

Patients with cold autoimmune haemolytic anaemia can sometimes be difficult to ABO group using

routine methods. The most common specificity of cold autoantibody is anti-I. As more than 99% of

adult red cells carry the I antigen, the presence of anti-I in the patient’s plasma may react with the

A1 and B cells if the antibody has high thermal amplitude. However, they will rarely be reactive at

37oC. This means the discrepancy is likely to be resolved by increasing the reaction temperature at

which ABO grouping is performed to 37oC. Changing the parameters of any test requires careful

control. In this case full ABO grouping is required and will entail forward and reverse grouping with

controls all performed at 37oC. Human polyclonal anti-A and/ or anti-B will react at 37oC but a longer

incubation period may be necessary.

Note that most healthy adults also produce auto anti-I but it typically reacts only at around 4oC and

rarely reacts at temperatures higher than 15oC. If reactive at room temperature any grouping issues

can be resolved as above.


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Transfusion of plasma components

Unexpected ABO antibodies can originate from recent transfusion of non ABO group specific plasma

or if the patient has undergone a plasma exchange procedure. For example a group A patient

transfused with a group O plasma component may result in unexpected anti-A detected in their

reverse group. It is always worth checking the transfusion history of your patient! Note that this can

happen with platelets as well as Fresh Frozen Plasma (FFP) and cryoprecipitate.

These antibodies are passively acquired, limited in amount and transient; once the plasma treatment

is stopped they will eventually become undetectable.

Treatment with Intravenous Immunoglobulin (IVIg)

IVIg is a human derived product manufactured from pooled plasma of multiple healthy donors that

is used for a wide range of conditions. Recipients of IVIg may undergo short (≤3 months) or long

term treatment. By its very nature IVIg contains a large mixture of different antibodies and therefore

IgG anti-A, anti-B and anti-A,B will also be present. Although IgG in nature these ABO antibodies are

still capable of direct agglutination and may cause unexpected reactions in the reverse group.

Missing antibodies

Unexpected negative reactions with A and/or B cells can occur in the following conditions

Some disease conditions

E.g. Hypogammaglobulinemia (inability to make immunoglobulin molecules at all)

Age of the individual

see ‘development and production of ABO antibodies’ earlier

Post ABO incompatible bone marrow or stem cell transplant

Individual has different blood group but may not make corresponding ABO antibody

Transfusion laboratories are not always informed when a patient has undergone

transplantation

Transfusion of plasma components and plasma exchange therapy

E.g. transfusion of AB plasma components

In some cases lowering the reaction temperature to 4oC will allow detection of weak ABO antibodies.

As described earlier, any manipulation of the test parameters requires full ABO grouping to be

performed at the adjusted temperature, including controls, to ensure the result is valid.

In cases where the ABO group anomaly cannot be resolved a sample should be sent to a

reference laboratory for further testing.

If blood is urgently needed then group O blood should be used.

Never select ABO group specific based on the forward group results alone.


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ABO ANTIBODIES AND IMPLICATIONS FOR TRANSFUSION

It must be noted that the implications for transfusion are different dependent on whether you are

transfusing red cell components and/ or plasma components. This is because group A, B and AB red

cells carry large amounts of A/B antigens and O, A and B plasma will contain ABO antibodies. This is

true for both the recipient and the component to be transfused. You must also be mindful that

platelets carry ABO antigens and platelet components also contain residual donor red cells.

High Titre (HT) ABO antibodies

ABO antibodies are present in all group O, A and B blood components at variable concentrations.

Even concentrated red cells will carry a small amount of donor antibody. All UK donations are tested

for the presence of high titre (HT) anti-A/B in accordance with current guidelines for the blood

transfusion services in the UK. A component labelled as ‘HT negative’ has been tested by diluting the

donor’s plasma to a maximum of 1 in 128 and found to be nonreactive with AB red cells. This will be

discussed further in chapter [] pre-transfusion testing and chapter [] blood donation testing.

Selection of blood components for transfusion by ABO group

ABO group identical components should be selected for the recipient wherever possible. This is the

best, and easiest, way to avoid complication.

Table 6-16: Compatible blood components based on the recipient ABO group. Note that compatible alternatives to the recipient’s group are given. They should only be used for transfusion when ABO-identical is unavailable.

Blood components for transfusion should be selected to match the ABO group of the

recipient wherever possible.


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Careful management of the blood supply in both collection from donors and ordering for patients is

designed to make selection of group identical the norm for most recipients. However, occasionally,

this may not be possible due to a number of reasons such as limitations in the donor supply, urgency

of requirement, additional special requirements.

The transfusion of non-ABO identical plasma components needs careful consideration as all

donations except group AB contain anti-A and/or anti-B and/or anti-A,B. Mismatched ABO group

plasma components may be given provided the criteria in table 6-17 are met. However this should

not become standard practice and must only be applied when absolutely necessary.

Table 6-17: Criteria for selection of suitable plasma components when ABO-identical is unavailable.

Further details on factors affecting blood component selection can also be found in chapter [] pre-

transfusion testing and chapter [] blood components.

The outdated concept of the ‘universal donor - universal recipient’

Due to the absence of antigens on the red cells of a group O and the absence of antibodies in the

plasma of a group AB, these groups were termed ‘universal donor’ and ‘universal recipient’

respectively. This concept is no longer acceptable in terms of selecting components for patients.

Patient blood management systems should be designed to allow ordering and receipt of group

specific ABO components for all patients wherever possible.


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Test Yourself - CHAPTER 6

1. Complete the table showing the expected reactions for the different ABO blood groups

2. What are the possible ABO genotypes of the offspring from the following matings?

a. Group B x Group AB

b. Group B x Group A2

c. Group B x Group A1

3. What possible genotypes produce the A1; A2; A3 and Ax phenotypes?

4. Which ABO blood group has the most H substance and which has the least?

5. List the subgroups of A that can produce anti-A1

6. Outline why expression of A and B antigens is dependent on the H gene

7. Outline why the ABO blood group system is the most clinically important

8. Interpret the results in this table making notes as necessary

9. Name the immunodominant sugars of the ABO blood group system

10. What is meant by secretor and non-secretor status?


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11. List the broad categories of ABO grouping anomalies. Give three examples from each category.

How might they be resolved?

12. Complete the following tables. Make a note where HT negative components will be required.


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13. The following table represents the ABO blood grouping results from testing 10 different blood

samples. All reagents and techniques have been checked and are working correctly (i.e. the

results depicted are not the result of clerical or technical error).

Provide an ABO blood group (phenotype) interpretation of each of these results and give reasons

for your decision. Note any unusual result and state any further work you would like to perform.

Remember if you are happy to state the blood group then you are implying that you are happy to

select ABO group specific blood for transfusion!

Assignment

Visit the Serious Hazards of Transfusion (SHOT) website and select one of the SHOT reports (any year

you wish). Look for reports of ABO grouping errors. How many, if any, were there? What was the

outcome? Do you agree with the findings?


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Further reading

To be added

References

For ABO genetics/ biochem/ subgroups see Daniels

Mollison

AABB

Illustrations

All images are created by the author.

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