ABO blood group system and discrepancy by dr princy.pptx

Blood groups (ABO and Rh groups)
Dr. Princy R Vaghasiya
1st
year resident doctor
Department of pathology
Zydus medical college and hospital, Dahod

• In 1900, Karl Landsteiner discovered the ABO blood groups and classified
human blood into A, B and 0 groups.
• A fourth blood group AB was discovered by Landsteiner’s associates, Von
Decastllo and Sturli in 1902. This marked the beginning of the whole
subject of blood group serology and made blood transfusion practicable.
• The four groups are determined by the presence or absence of blood
group antigens (agglutinogens) on the red blood cells, and accordingly, an
individual’s group is A, B, AB, or O (O denotes the absence of A and/ or B
antigens).
• ABO antigens are also present on lymphocytes, thrombocytes, organs,
endothelial cells, and epithelial cells. In addition, it has been shown ‘that
corresponding to antigens-A and B; there are naturally occurring
antibodies anti-A and anti-B (agglutinins) in the plasma/ serum of
individuals whose red cells lack the corresponding antigen.
ABO blood group system

• Antigens of the ABO system are detectable at 5 to 6 weeks of gestation.
• Newborns demonstrate weaker antigens, but ABO antigens are fully developed by
two to four years of age.
Table: The ABO antigens and corresponding antibodies

Genetics
• In 1924 Bernstein discovered that ABO antigens follow simple Mendelian
genetics.
• ABO system gene is located on chromosome 9 occupied by one of the
three allelic genes A, B or O.
• The coding region of ABO is organized into seven exons; exons 6 and 7
constitute 77% of it (Figure 1).
• The expression of the A and B genes is codominant.

Figure 1: Genomic organization of the ABO gene, showing the seven coding exons with codons
and nucleotides in each exon.

• In the laboratory, the presence or absence of A or B antigens and their
corresponding antibodies is used to determine the phenotypic expression
of the inherited genes.
• Since the O gene does not produce an antigen, it cannot be detected
directly, so the lack of A and B antigens on the cells indicates O blood
group.
Inheritance of ABO blood groups
• The child of the parents in example 1 would
test either A or B, and in example 2; the child
would be AB, A, B or O. (Figure).
• The presence of O genes could not be
determined in the laboratory. But the fact that
the child inherited the O gene could be
determined only by family studies.

Table: Phenotypes and genotypes in ABO blood group system

Biochemistry
• Expression of A, B, and H genes does not result in the direct production of
antigens. Rather, each gene codes for producing an enzyme known as a
transferase (Table).
• Each transferase catalyzes the transfer of a carbohydrate molecule to an
oligosaccharide chain. The attached carbohydrate provides antigenic
specificity.
Table: Summary of ABH gene and corresponding transferase enzymes.

• ABH antigens can be expressed on different oligosaccharide chains
attached to either a protein or a lipid molecule.
• The oligosaccharide is a carbohydrate molecule linked either in simple linear
forms or in a complex structure with a high degree of branching.
• Type 1 and type 2 chains differ in the manner in which terminal galactose
joins the subterminal N-acetyl glucosamine. The basic precursor substance
(Figure 2) has short chains of sugars (oligosaccharides).
• In the type 1 chain, the carbon-1 of galactose is linked to carbon-3 of N-
acetylglucosamine (1-3) linkage (Figure A).
• In type 2 chain, the carbon-1 of galactose is linked to carbon-4 of N-
acetylglucosamine (1-4) linkage (Figure B).
• In the red cell membrane, both glycolipids and glycoproteins with ABH
activity are present.
• In the plasma, only glycoproteins in the soluble form are found.
• Body serous and mucous secretions contain only glycoproteins in insoluble
form.

Figure 2 : Basic precursor chains.
A. Type-1
B. Type-2 oligosaccharide chains
(Gal: D-Galactose, GNAc: N-Acetylglucosamine, R-Rest of the molecule
attached to the protein/lipid)

• The basis precursor substance (oligosaccharide) is converted by an enzyme
L-fucosyl transferase (a product of H gene) to H substance by adding the
sugar L-fucose to the terminal D-galactose of the precursor substance.
• The H substance is partially converted by the specific transferases, namely
N-acetylgalactosaminyl transferase and D-galactosyl transferase (the
products of A and B genes) to A and B antigens by the attachment of N-
acetyl-galactosamine and D-galactose respectively, to H substance.
• The O gene is an amorph (no gene product), and group O cells contain only
H substance. Some H substance remains unconverted. Thus, all A and/or B
cells normally contain some H substance along with A and/ or B antigens.
• The amount of H substance on red cells in order of diminishing quantity is, O
> A2 >A2 B >B > A1 > A1 B The expression of A and B genes is dependent
on H gene expression.
• Most individuals are homozygous for the H Gene (i.e., HH). Since its allele h
is anamorphic gene, it has no observable effects on precursor substance.
The blood group resulting from homozygous hh condition is called the
Bombay (Oh ) blood group.

Figure: Simple diagram of the formation of
A, B and H specific structures

Secretor status
• A, B and H antigens are present on red cells and widely distributed
throughout the body tissues except in the central nervous systems.
• A, B and H substances are also found in the secretions of 80% of the
population.
• The ability to secrete A, B and H substance is determined by the presence
of the secretor gene (Se) in either the homozygous SeSe or heterozygous
Sese state, which is inherited independently of the ABO and Hh genes.
• Normally all secretors secrete H, in addition to A and/or B substance.
(Table: Secretor status)

ABO subgroups
• ABO subgroups represent phenotypes showing weaker and variable
serologic reactivity with human polyclonal anti-A, anti-B, and anti-AB
reagents.
• Some unusual ABO genes affect the activity of the gene products and may
result in subgroups of A and B.
• The weaker serologic reactivity of ABO subgroups is attributed to the
decreased number of A and B antigen sites on their red blood cells.

 Subgroups of A
• Subgroups of A are phenotypes that differ quantitatively or qualitatively
from the A antigen carried on the RBCs and found in the saliva of
secretors.
• A1 and A2 , the two major subgroups of A, constitute 99% or more of
group A people tested.
• The cells of approximately 80% of all group A (or AB) individuals are A1 (or
A1 B), and the remaining 20% are A2 (or A2 B) and weaker subgroups.
• It is not necessary to classify group A patients or donors as A1 or A2
except when the individual’s serum contains anti-A1.
• Anti-A1, reacting at 22°C or lower, has no clinical significance, but it is
clinically significant if it reacts at 37°C; Anti-A1 causes discrepancies
between ABO cell and serum grouping and also cause crossmatch
incompatibility. Any patient of group A2 , or A2 B having anti-A1 reactive at
37°C should be given A2 or A2 B group blood.

Table : Quantitative and qualitative differences in A1 and A2

 Weak subgroups of A
• Subgroups weaker than A2 occur infrequently.
• They are characterized by the declining number of A antigen sites on red
cells and a reciprocal increase in H reactivity.
• Adsorption and elution techniques may be necessary for the detection of
antigens on the surface of red cells.
• Weaker variants of A are mainly A3 , Ax , Am and A-intermediate.

• Classification of weak A subgroups is based on:
1. Degree of agglutination with Anti-A, Anti-A1 and Anti-AB.
2. Degree of H reactivity on the red Cells
3. Presence or absence of Anti-A1 in the serum
4. Presence of A and H substance in the saliva of secretors
5. Adsorption and elution studies
6. Family (pedigree) studies

 Subgroups of B
• Subgroups of B exist e.g., B3 , Bx and Bm, but they are rare and even less
common than subgroups of A.
• Widespread use of monoclonal Anti-A and Anti-B reagents has lessened
the problems of weak A or B subgroups because monoclonal antibodies
can agglutinate cells with weak or aberrant antigen expression.

 Bombay phenotype (Oh )
• The Bombay blood group was first reported in 1952 by Bhende in Bombay,
India.
• The frequency in India is around 1:7600.
• More than 130 Bombay phenotypes have been reported in various parts of
the world.
• It is inherited as an autosomal recessive trait. It lacks the H gene and is
homozygous for its allele h (hh).
• This group is characterized by the absence of A, B and H antigens on the
red cells.
• The serum of these persons contains anti-A, anti-B and anti-H.
• They are non-secretor of A, B and H substances in saliva.
• Bombay blood group patients can only be transfused with blood from
another Bombay group

 Para-Bombay phenotype
• The para-Bombay phenotype RBCs either completely lack H antigens or
have small amounts of H antigen present.
• The genetic basis for the para-Bombay phenotype is either a mutated H
gene (FUT1) with or without an active Se gene (FUT2) or a silenced H
gene with an active Se gene.
• In laboratory testing, red cells from para-Bombay individuals may (or may
not) have weak reactions with anti-A and anti-B reagents.
• The notations Ah , Bh and ABh describe Para-Bombay A, Para-Bombay B,
and Para-Bombay AB blood group, respectively.

 B(A) and A(B) phenotype
• The B(A) phenotype is an autosomal dominant phenotype characterized
by weak A expression on group B red cells.
• Serologically, red cells are strongly reactive with anti-B and weakly reactive
with monoclonal anti-A (<2+), and they possess a strong anti-A in their
sera.
• In general, the agglutination is weak with fragile, easily dispersed
agglutinates. Testing the sample with polyclonal anti-A or a different
monoclonal anti-A should resolve the discrepancy.
• An A(B) phenotype has also been described with monoclonal anti-B. The
A(B) phenotype was associated with elevated H antigen and plasma H-
transferase activity.
• It is hypothesized that the increased H precursor on these cells may permit
the synthesis of some B antigen by the A-glycosyltransferase.

 Antibodies of ABO system
• The ABH system is unique for the
presence of naturally occurring
antibodies against missing ABH
antigens.
• These antibodies begin to appear
during the first few (3-4) months of life,
probably from exposure to ABH
antigen like substances in the
environment.
• These antibodies reach a peak by 10
years of age and then gradually
decreases. (Additional situations that
exhibit reduced ABO antibody levels
are summarized in Table.)

• Anti-A or anti-B antibodies are usually naturally occurring and are mostly
IgM.
• However, some IgG and IgA antibodies are also present.
• IgG anti-A and anti-B are found more commonly in group O individuals
than in A or B individuals.
• O group individuals may have a high titer of anti-A and anti-B as they are
both IgM and IgG and are referred to as high titer O group individuals.
High titer antibodies in O groups are important in two situations:
1. O group donors as Apheresis platelets donor.
2. In pregnancy, if the mother is of group O and the baby is of group A or B,
the chances of ABO HDN is more as IgG type of anti-A or anti-B can
easily cross the placenta.
The IgM antibodies activate complement and often strongly react in vivo and
in vitro. Soluble blood group substances neutralize the IgM antibodies.

Anti-A (Anti-A + Anti-A1)
• The antibody anti-A is found in group B and group O individuals.
• Anti-A of group B serum appears from simple studies to contain separable
anti-A and anti-A1 and reacts well with A1 and A2 cells but not as well with
weaker subgroups of A.
Anti-B
• The antibody anti-B is found in group A and O individuals and react almost
with all B group cells but less effectively with weaker variants of the B
group.
Anti-AB
• The antibody anti-AB is found in group O individuals, and it reacts with
both A and B cells.

Anti-A1
• This is found in 1-8% of A2 and 22- 30% of A2B individuals.
• Anti-A1 lectin is manufactured from seeds of Dolichos biflorus and is
available commercially.
Anti-H
• Anti-H very rarely occurs as cold reactive agglutinin in individuals with very
low levels of H antigen on their cells and have little clinical significance.
• However, anti-H found in the Bombay group (Oh ) can often be potent and
reacts strongly at 37°C.
• It is an IgM antibody capable of binding complement and causing RBC
lysis. Anti-H is manufactured from the lectin of Ulex europaeaus.

 Clinical significance of ABO antibodies
• ABO antibodies are capable of causing both Hemolytic Disease of the
Fetus and Newborn (HDFN) and Hemolytic Transfusion Reactions (HTR).
• ABO compatibility is also significant in solid organ transplantation.
• In ABO-incompatible organ transplant, pre-and post-transplant ABO
antibody titer and plasmapheresis to reduce the titer of the incompatible
antibody will assist in achieving a positive outcome.

 ABO system and disease association
• ABH antigens, besides being found on red cells, are widely distributed
throughout the body. Disease association with a particular blood group
may light their biological role.

 ABO grouping discrepancy
Discrepancies are detected when forward and reverse grouping fails to tally
each other and hence require further investigations. Common sources of
clerical and technical errors resulting in ABO discrepancies are related to
one or more of the following:
1. Clerical issues
• Mislabeled specimen or testing tubes.
• Improper recording of test results
2. Clinical issues
• These discrepancies occur due to problem inherent to the patient/donor.
To solve this, essential information such as age, the patient’s/donor’s
medical condition, medication, recent transfusion history and pregnancy
must be considered.

3. Technical issues
• Not following manufacturer’s instructions
• Deleted procedural step.
• Missed or under interpreted weak reactions
• Incorrect interpretation of serological reactions
• Missing or incorrect reagents in test samples
• Equipment malfunction, centrifuge time or incorrect speed
• Contaminated antisera or cells
• Incorrect cell suspension

 General approach for solving discrepancy
• When investigating ABO group, always remember that RBC and serum grouping
reactions are strong (3+ to 4+), and the weaker reactions (<2+) typically
represent the discrepancy.
• When a blood group discrepancy is encountered, all results must be recorded,
but the interpretation of the ABO type must be delayed until the discrepancy is
resolved.
• Obtain fresh blood sample from the donor unit or patient to rule out discrepancy
due to contamination or unidentified samples.
• Repeat the test before additional investigations are carried out.
• Repeat cell grouping with fresh antisera (a different lot if possible) as appropriate.
• Perform a direct anti-globulin test on the cells to detect if cells are coated with
antibodies in case of previous transfusion or AIHA.
• Quality assurance of reagents, correct technique, careful observations, and
interpretation of results resolve many problems.

 ABO discrepancies may be arbitrarily divided into four
major categories
Group I discrepancies
• These discrepancies are
associated with unexpected
reactions in the reverse grouping
due to weakly reacting or missing
antibodies.
• Common causes are:
a. Newborns
b. Elderly patients
c. Patients with leukemia or lymphoma
d. Patients on immunosuppressive drugs
e. Patients with congenital or acquired
agammaglobulinaemia or
immunodeficiency diseases.
f. Patients whose existing ABO antibodies
may have been diluted by plasma
transfusion or exchange transfusion.
g. ABO subgroups
h. Substances in plasma or serum

Resolution
• The discrepancy can be resolved by enhancing the reaction with
prolonged incubation (30-45 min) at room temperature or at 40C for 15-30
min. It can also be resolved by increasing the cell : serum ratio.
Substances in plasma or serum
• Blood group substances (A and/or B substance) may be in excess amount
in any individual’s blood.
• Red cell suspension in the serum or plasma in such individual may
neutralize the antibodies in the testing reagents, and they fail to react with
the corresponding antigen(s) on the red cells.
• This type of discrepancy is resolved by washing the red cells three times
with normal saline.

Group II discrepancies
This is due to weak or missing antigens resulting in unexpected reactions in
the forward grouping.
Common causes are:
a. Subgroups of A or B
b. Leukemia and Hodgkin’s disease
c. The “acquired B”
d. Antibody coated red cells (DAT Positive)

Resolution
• This can be resolved by enhancing the reaction with prolonging incubation
(30-45 min) at room temperature or at 40 C for 15-30 min.
• Use Anti-AB and Anti-A1 antisera for subgroups detection. Adsorption-
elution and saliva testing may be required for weak subgroups.
Acquired B antigen
• Acquired B antigen is usually found in A1 individual with diseases of the
lower intestinal tract:
Carcinoma of the colon or rectum
Intestinal obstruction
Gram-negative septicaemia

• In A1 individual with acquired B antigen, cells agglutinate with anti-A and
anti-B, but the reaction with anti-B is a weak or mixed field. Anti-B present in
patients’ serum does not react with autologous cells. Acquired B antigen
should be suspected when the red-cell group appears as AB, and the serum
has Anti-B.
• To identify an acquired B antigen:
1. Observe the strength of agglutination with anti-A and anti-B. The reaction
with anti-A is usually much stronger than with anti-B.
2. Test the patient’s serum with his cells: Anti-B in the patient’s serum does
not agglutinate red cells with acquired B antigen.
3. Test the saliva for the presence of A and B substance. If the patient is a
secretor, A substance is present but not B.
4. Use acidified anti-B antisera. It will agglutinate only true B antigens and
not acquired B antigens (acidify anti-B typing reagent to pH 6.0 by adding
1 or 2 drops of 1 N HCl to 1 mL of anti-B antisera, and measure with a pH
meter.

Antibody coated red cells (DAT positive)
• Red cells may be coated with antibodies, and a direct antiglobulin test may
be positive.
• When red cells are coated with antibodies, they mask the antigens on the
surface of the red cells and give false-negative results.

Resolution:
• When the cells are coated with IgM, non-coated red cells can be obtained
by
1. Wash the cells with warm (37°C) saline.
2. 45°C elution technique, followed by warm washing.
3. Treatment of IgM coated red cells with dithiothreitol or 2-mercaptoethanol
also provide non-agglutinated samples.
4. Gentle elution to remove the adsorbed antibody. Repeat the direct
antiglobulin test and ABO grouping.
• When the cells are coated with IgG, non-coated red cells can be obtained
by
5. IgG immunoglobulin may be partially removed by 45°C elution.
6. Treatment of IgG-coated red cells with chloroquine diphosphate may be
used to remove IgG immunoglobulin from red cells.

Group III discrepancies
• These discrepancies between forward and reverse groupings are caused
by protein or plasma abnormalities and result in the rouleaux formation or
pseudo agglutination.
• This can be due to:
a. Elevated levels of globulin
b. Elevated levels of fibrinogen
c. Plasma expanders
d. Wharton’s jelly in cord blood samples

Resolution:
• Rouleaux is a stacking of erythrocytes that adhere in a coin like fashion, giving
the appearance of agglutination.
• It can be resolved by washing the cells with normal saline 3-4 times.
• If the serum/reverse grouping is affected, perform saline replacement
technique:
 Reagent cells and patient serum centrifuged to allow antigen and antibody to
react.
 Serum is removed and replaced by an equal volume of saline (saline
disperses cells).
 Tube is mixed, centrifuged, and re-examined for agglutination, rouleaux
disperses but not true agglutination.
Wharton's jelly:
• Wharton’s jelly in the cord blood may interfere with serologic testing and may
cause a discrepancy in ABO & Rh typing. This discrepancy is resolved by
properly washing cord blood red cells three times with normal saline.

Group IV discrepancies
• Discrepancies between forward and reverse groupings are due to
miscellaneous problems such as:
a. Autoantibodies
b. Alloantibodies
c. Mixed field agglutination as in Chimera
d. Cis-AB

Mixed-field agglutination as in chimera
A chimera is an individual with two separate cell populations, e.g. A cells
and O cells. It is very rare.
It can be artificial (transient) or permanent. Artificial (transient) chimera
may occur due to,
• Transfusion of group O red cells to group A or group B.
• Fetomaternal hemorrhage
• Bone marrow transplantation when the ABO group of the donor is different
than that of the recipient.
• Intrauterine transfusion

• Exchange transfusion One of these cell populations will live only for a short
time, making this type of chimera a transient phenomenon. Permanent
chimera may occur when two cell populations exist throughout life, e.g.
In twins, when an exchange of blood occurs in utero due to vascular
anastomosis, both cell populations grow, and both are recognized as self.
Dispermy (when two sperm fertilize one egg) and result in two cell
populations.
Resolution:
• In such cases, cell typing can give a mixed field pattern of agglutination.
• Patient history and perhaps cell separation studies are the best ways to
readdress these types of discrepancies.

Rh blood group system
• The RH blood group system is the most important protein blood group
system.
• It comprises 54 antigens numbered from Rh1 to Rh61 with 7 numbers
obsolete.
• The Rh system is highly immunogenic and complex, with numerous
polymorphisms and clinically significant alleles.
• The five major antigens of the Rh system are D, C, E, c and e. They are
coded by two closely linked genes RHD and RHCE. However, the
expression “Rh-positive” and “Rh-negative” has come to indicate the
presence and absence of D antigen, respectively.

Discovery of Rh system
• In 1939, Levine and Stetson described an antibody in the serum of a group
O mother who delivered a stillborn fetus and subsequently developed
symptoms of hemolytic transfusion reaction when transfused with her
husband’s group O blood.
• They noted that the responsible antibody causing hemolytic transfusion
reaction developed in the mother through an antigenic factor from the
fetus. The antibody was not named at that time.

• A year later, in 1940, Landsteiner and Wiener immunized rabbits and
guinea pigs with red cells of rhesus monkeys. The serum of immunized
rabbits contained an antibody named anti-Rh, which agglutinated the red
cells in approximately 85% of white people tested. Its antigenic
determinant was called the Rh factor.
• The antibody discovered by Levine and Stetson in the mother was
subsequently re-examined and found identical activity as the anti-RH
antibody found by Landsteiner and Wiener. This work led to the discovery
of the Rh system.
• In late 1940, Wiener and Peters demonstrated that an antibody similar to
anti-Rh, responsible for hemolytic transfusion reactions in patients
transfused with ABO compatible blood.

• Later, the evidence established that the animal anti-rhesus of Landsteiner
and Wiener was not identical to the human antibody called an anti-Rh
antibody, but by that time, the Rh blood group system had already
received its name.
• The anti-Rhesus formed by animals demonstrated by Landsteiner and
Wiener was renamed anti-LW in their honor for first reporting it.
• In 1943 and 1944, Fisher and Race discovered four additional antigens in
the Rh system and thus established five Rh antigens, viz. D, C, E, c and e
and their corresponding antibodies anti-D, anti-C anti-E, anti-c and anti-e.
• A subsequent discovery has brought the number of Rh related antigens
over 50, many of which exhibit quantitative variations. But in most
Transfusion Medicine settings, five principal antigens D, C, E, c and e and
their corresponding antibodies account for more than 99% of clinical
issues involving the Rh system.

Nomenclature of Rh system
• Currently there are four systems of nomenclature for Rh blood group
namely Fisher-Race, Weiner, Rosenfield and ISBT nomenclature.

1. Fisher Race: dce Terminology
• It is still the most commonly used terminology for Rh.
• In the 1940s Fisher and Race postulated the theory of three closely linked genes
D, C, and E and their alleles d, c, and e.
• The allele d has not been identified; the notation d indicates the absence of D
gene.
• In Fisher’s terminology, the same letter designation is used for both gene and
gene products (antigens) except that by convention, the symbols for the gene are
always printed in Italics.
• According to the Fisher Race theory, a person inherits a set of Rh genes from
each parent (haplotype) that constitutes the person’s genotype (e.g., DCe/DCe).
• The phenotype is determined by the antigens expressed on the red cells (e.g.,
DCe). Placing parenthesis around in phenotype e.g. (D) indicates weakened
antigen expression.

Comparison of Weiner and Fisher- Race nomenclature

2. Wiener: The Rh-hr terminology
• Wiener favoured the concept of multiple allelic gene at one locus resulting
in a complex gene, and he gave the Rh-Hr terminology.
• He believed that the immediate gene product is a single entity
agglutinogen (haplotypes) on the surface of red cells and that each
agglutinogen has a number of factors (antigens) each of which is
recognized by its own specific antibody.
• For example, the gene complex R1 gives rise to the agglutinogen
(haplotypes) Rh1 which possesses the three blood factors (antigens) Rho ,
rh’ and hr’’.

• In Wiener’s terminology, the gene complexes were designated by a single
italic letter R and r with superscript.
• The gene product agglutinogen (haplotypes) were designated by Roman
type Rh and rh with subscripts. The symbol for individual factors were
Roman characters in boldface type with Rho representing D and rh’, rh’’,
hr’ and hr’’ representing C, E, c and e factors (antigens) respectively.
• The shorthand phenotype notation mostly used employs single letters R
and r in Roman type with subscripts. Thus, R1 indicates C, D and e
antigens; R2 indicate c, D and E antigens and so on.

3. Rosenfield: numeric terminology
• As the blood group system expanded, it became difficult to assign names
to new antigens using existed terminologies.
• Rosenfield and his associates proposed a system that assigns a number
to each antigen of the Rh system in the order of discovery or recognized
relationship to the Rh system.
• This system has no genetic basis but simply demonstrates the presence or
absence of the antigen on the red cell.
• A minus sign preceding a number designates the absence of the antigen
on the red cell.

• If an antigen has not been typed for, its number will not appear in the
sequence. An advantage of this nomenclature is that the red cell
phenotype is thus described.
• For the five major antigens,
D is assigned Rh1,
C is Rh2,
E is Rh3,
c is Rh4 and
e is Rh5
• For red cells that type D+ C+ E+ c negative, e negative, the Rosenfield
designation is Rh: 1, 2, 3, -4, -5.
• If the sample was not tested for e, the designation would be Rh: 1, 2, 3, -4.
All Rh system antigens have been assigned a number

4. International Society of Blood Transfusion committee:
updated numeric terminology
• With the discovery of newer antigens and a need for uniform terminology,
the International Society of Blood Transfusion (ISBT) came up with the
universal numeric terminology.
• This nomenclature is both machines- and eye- readable and is based on
the genetic basis of the blood group.
• Each authenticated antigen is assigned a six-digit number where the first
three digits indicate the blood group system and the last three digits the
antigen specificity.

• 004 is the number assigned for the Rh blood group system by ISBT.
• It is followed by three digits depicting each antigen under this system. E.g.,
004001 represents D antigen.
• In 2008 the ISBT committee recognized RH-associated glycoprotein
(RHAG) as a new blood group system and assigned the number 030.

Genetics
• Now it is well known that Rh proteins are coded by two closely linked genes
RHD and RHCE, present on the short arm of chromosome 1p36.11.
• Like ABO, Rh genes exhibit codominant expression.
• RHD codes for the presence or absence of D antigen and RHCE encodes
the CE antigen in four combinations (CE, Ce, cE, ce), respectively. Both
RHD and RHCE genes have 10 exons and are 97% identical.
• Another gene important for the expression of Rh antigens is RHAG. It is
located on chromosome 6p21.
• It determines the successful expression of Rh antigens by coding for the Rh-
associated glycoprotein (RhAG). It forms complexes with the Rh protein
within the RBC membrane.
• Mutations in this gene can cause absent/ decreased expression of RhD and
RhCE proteins.

Rh antigens - phenotype and genotype
• The phenotype denotes the expression of antigens on red blood cells,
which can be determined with anti-sera.
• The genotype denotes the gene complex that codes for the antigen in a
particular individual.
• The phenotype of an individual can be reported by detecting common Rh
antigens on red cells by five antisera: anti-C, anti-c anti-D anti-E and anti-
e.
• However, an individual’s genotype with respect to Rh cannot be predicted
precisely by serological methods and is largely based on the frequencies
of antigens, gene complexes, and individual gene.

• An individual inherits one combination from each parent, giving rise to
genotype like CDe/cde (R1 r) etc.
• Such thirty-six possible genotype combinations could occur.
• The number of phenotypes would, however, depend upon the type of anti-
sera used for testing.
• Rh system antibodies show dosage phenomenon.

Biochemistry of Rh antigens
• Rh antigens are found only on red cells and are an integral part of the red
cell membrane.
• They are highly hydrophobic, non-glycosylated proteins that span the red
cell membrane 12 times with few extracellular loops.
• The proteins encoded by both genes (RHD and RHCE) differ by 32-35
amino acids.
• The protein coded by RHCE gene carries both C/c and E/e antigens.
• The amino acid position 103 determines C or c expression. Similarly,
amino acid position 226 determines the E or e expression.

• The predicted 12-transmembrane domain model of RhD and RhCE polypeptide.
• C/c and E/e polymorphisms are depicted with the most important C/c site arrowed.
• Differences in RhD and RhCE polypeptides marked in the diagram. (Representational presentation
only)

• Currently, more than 275 alleles of RHD and 50 RHCE alleles have been
discovered.
• Newer antigens are formed due to single nucleotide polymorphisms in the
gene arrangement.
• The RHAG protein is 38% identical to RhD/RhCE protein.
• Rh proteins have an important structural role in the erythrocyte membrane.
• The Rh complex is linked to the membrane skeleton through CD47-protein
4.2 interactions and Rh/RhAG-ankryin interactions.

D antigen
• An individual is designated as Rh-positive or Rh-negative depending on
the presence or absence of D antigen on the red cells.
• In India, 94.61% of the population is Rh(D)-positive, and 5.39% is Rh(D)
negative, but this percentage varies slightly in different parts of the country.
In Caucasians, 85% of the population is Rh(D)-positive, and 15% is
Rh(D)–negative.
• The D antigen is made of various epitopes that were originally defined by
anti-D antibodies made by sensitized individuals.
• D epitopes are highly conformational, and the amino acid changes have
resulted in altered D antigens.
• Altered D antigens are classified into Weak D, Partial D, Del, and non-
functional RhD.

Weak D
• Red cells with a reduced amount of D antigen requiring an Indirect
Antiglobulin Test (IAT) for detection are termed as Weak D (Du).
• Du red cells are agglutinated by some anti-D sera and not by others, but
mostly these react by the AHG technique.
• Weak D is caused by SNP(single nucleotide polymorphism) that alter the
amino acid sequences in the intracellular or transmembrane portion of Rh
protein, affecting the insertion of protein in the membrane
• There are two grades of weak D.

• Red cells of the higher grade of weak D are agglutinated by certain anti-D
sera, while red cells of the lower grade are detectable only by AHG test
(IAT).
• The incidence of weak D in India is reported to be between 0.3-0.5%
(Bhatia, 1985).
• A 2017 review estimates 0.2%-1% of routine RhD blood typing result in a
“serological” weak D phenotype.
• Among the various types of weak D, type 1, 2 and 3 are the most
common, accounting for almost 90%.

Clinical significance of weak D (Du)
• Donor Typing
- Weak D is much less antigenic in comparison to D; however, Du red cells
may be destroyed if transfused to a person already having anti-D.
- Du red cells, if transfused to D negative patients, can sensitize them,
which can cause HDN later (D negative females of reproductive age
group).
- Hence, weak D testing has to be done on all D negative donor units before
transfusion. Those that turn out positive should be labelled Rh(D)-positive.

• Recipient typing
- Weak D testing is currently not required for a recipient.
- The weak D recipients are classified as Rh(D) negative and safely transfused
with Rh(D) negative blood.
• Weak D testing in Infants
- Weak D positive infant can suffer from HDN if the mother possesses anti-D
antibodies.
- Weak D testing in newborns plays an important role in determining who
should receive anti-D immunoprophylaxis with Rh immunoglobulin (RhIG).
- Newborn infants of Rh(D) negative mothers are tested for D, and weak D
and RhIG is recommended for the mother of D positive or weak D positive
infants to prevent potential immunization.

Partial D
• Partial D individuals type as D positive but can make anti-D when they are
exposed to conventional D antigen.
• Earlier known as “category D”, these variant forms are caused due to
changes in the extracellular part (epitopes) of Rh proteins.
• The majority of partial D phenotypes are caused due to exchange of
genetic material from RHCE to RHD. This can result in the loss of D
epitopes on Rh protein forming new antigens.
• DVI is the most common form of partial D found in Europeans.

Del (D-Elution)
• These red cells have an extremely low level of D antigens that can be
detected only by adsorption and elution studies and not by any of the
routine serological methods (not even IAT).
• These are caused due to mutations in the RHD gene that causes
decreased expression.

D-Negative phenotype
• The frequency of “Rh-negative” individuals varies among different ethnic
groups. In India, the overall D negative prevalence is 5%, but it varies from
region to regions, such as 2.55% in Maharashtra, 6.49% in Uttarakhand
and 0.53% in Sikkim.
• In Whites, the prevalence of Rh-negative phenotype is 15%, whereas it is
lesser in Africans (6%) and Asians (1%) In Caucasians, D negative
phenotype is because of RHD gene deletion, while in Africans and Asians,
it is because of gene rearrangements and mutations.

Other Rh antigens
- C/c and E/e Other common Rh antigens are C, c, E and e.
- These are less immunogenic than the D antigen.
- D is considered to be the most potent immunogen, followed by c and E. The
order of immunogenicity is as follows,
D > c > E > C > e
- More than 50 RHCE alleles have been identified, which give rise to
compound and variant C/c and E/e antigens.
- Altered C and altered e are most commonly encountered resulting from
nucleotide changes in the RHCE gene.
- Individuals with altered C/c/E/e antigens might type positive for the antigen
and still make antibodies to them.

Variants of Rh antigen
- About 100 Rh antigens have been discovered so far.
- A variant of C/c viz. Cw, Cu, Cx, Cv and of E/e viz. Ew, Eu, es and
compound antigens viz. G (present on cells with C and D), f (combination
of c and e) etc., have been described.
- The commonest among C/c variants is Cw.

G antigen
- G antigen is found on red cells with either D or C antigen.
- Antibody to G antigen can appear as anti-D plus anti-C and cannot be
separated.
- Its significance lies in the fact that anti-G antibody in a D-Negative woman
who delivered D-(C+) infant can mimic anti-D. Such mothers can still make
anti-D when exposed to D positive cells and hence are candidates for
RhIG prophylaxis.
- Adsorption elution studies can distinguish Anti-D, -C and -G.

RH null syndrome
- The absence of all Rh antigens characterizes Rh null.
- “Regulator type” Rh null is caused by mutations in the RHAG gene. In this
type, normal Rh genes are present. However, they are unable to express
themselves.
- “Amorph type” Rh null is caused by nucleotide changes in the RHCE gene
and deletion of the RHD gene.
- Rh null red cells exhibit membrane abnormality (stomatocytes), resulting in
their reduced survival.

Rh antibodies
• Rh antibodies are clinically the most significant after anti-A and anti-B
antibodies. Almost all Rh antibodies result from immunization by
pregnancy or blood transfusion.
• They are immune (IgG) antibodies except, very rarely, for a few examples
of anti-E and anti-Cw that occur without any stimulus, and which may be
saline (IgM) antibodies.
• Rh(D) is considered to be the most antigenic, followed by c and E.
Although few Rh antibodies react in saline, most react best in high protein,
antiglobulin or enzyme tests. Even saline-reactive anti-D usually react to
higher dilution by AHG test.

• The reactivity of Rh antibodies can be enhanced by enzyme treatment of
red cells. Rh antibodies are reactive at 37oC and do not bind complement
(though it can occur in rare cases) when they combine with corresponding
antigens. Hence Rh antibodies cause extravascular hemolysis.
• Rh antibodies are the major cause of hemolytic disease of fetus and new-
born (HDFN) and lead to the destruction of transfused Rh-positive red cells
(HTR).
• Anti-c causes the most severe HDN following anti-D, among the Rh
antibodies.
• Rh immunization persists for many years. Even if the level of circulating
antibodies falls below the detectable threshold, subsequent exposure to
antigen results in rapid secondary response and antibody formation.

Rh phenotyping
• Rh typing generally refers to determining the D antigen status, whereas
the term “Rh phenotyping” refers to the testing of the red cells for the
presence or absence of all five principal Rh antigens in order to determine
the phenotype.
• Commercial antisera specific for the five major Rh antigens are available
that can be used for testing on slide/tube/ gel card method.

Rh genotyping
• Genotyping employs molecular techniques in order to determine the gene
sequence of the antigen.
• Since the RHD and RHCE genes are closely linked, the exchange of
genetic material between them is possible, giving rise to a variety of novel
alleles and antigens.
• These novel antigens can be picked up and assigned to a blood group
system with the aid of genotyping.

Clinical significance of Rh
• Transfusion
Rh(D) typing is a critical component of pretransfusion testing. When Rh
antibodies are detected, it is necessary to provide an antigen-negative
crossmatch compatible unit for transfusion.
Extended phenotyping and matching for Rh and Kell would be desirable if
resources permit.
Rh antibodies are IgG class and can cause clinically significant hemolysis
when transfused with antigen-positive blood. They are reactive at 37ºC.

• Hemolytic disease of the fetus and newborn
HDFN caused due to Rh antibodies is very severe because these antigens
are well developed on fetal cells, and the antibodies are IgG type that can
readily cross the placenta.
RhIg immunoglobulins have been successful in preventing HDFN in
susceptible D negative mothers.
RhIg needs to be given to unsensitized Rh(D) negative mothers who
deliver Rh(D) positive fetus.
This has to be given within 72hrs of the delivery. Hence, that makes typing
the infants for D antigen necessary.

The red cells from an infant suffering from HDN are coated with
immunoglobulin. The infant’s red cells may be so heavily coated with
antibody that all antigen sites are occupied, leaving no antigenic site to
react with anti-D.
The “blocking” phenomenon should be suspected if the infant’s cells have
a strongly positive DAT, and the cells do not react with anti-D.
The antibodies coated on the infant’s red cells are removed by the heat
elution method, and the red cells free from antibody are used for Rh typing
and cross-matching.

Reference
• TRANSFUSION MEDICINE TECHNICAL MANUAL – 3rd
Edition (2022)

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