Rhesus Blood Group System

THE RHESUS BLOOD GROUP
SYSTEM
6/10/2020 1Alick Mwambungu-BTS

Rhesus Monkey

Introduction
• The Rh system is the second important blood group
system after the ABO in transfusion medicine.
• The principal antigen is D, and the terms Rh positive
and Rh negative refer to the presence or absence of
D antigen.
• Rh antigens, especially D, are highly immunogenic
and can cause hemolytic disease of the newborn
(HDN) and severe transfusion reactions.

History
The D antigen (RH1)
• In 1939, Levine and Stetson reported that a patient, who had
delivered a stillborn infant and then suffered a severe reaction
to transfusion of her husband’s blood, had an antibody that
agglutinated the red cells of 85% of ABO-compatible donors.
• In 1940, Landsteiner and Wiener found that guinea pigs and
rabbits injected with rhesus monkey red cells made an
antibody that not only agglutinated rhesus monkey red cells,
but also the red cells of 85% of people.
• The human and animal antibodies were originally thought to
be the same, and the human antibody was called anti-Rhesus.

History
• It was later realized that the human antibody (now
called anti-D of the Rh system) does not identify the
same antigen as the rabbit and guinea pig rhesus
antibody,
• This was an error arising out of a phenotypic
association between the antigens.
• Levine suggested that the antigen defined by the
original rhesus antibody should be called LW in
honour of Landsteiner and Wiener.
• The blood group system originally identified by the
human antibody is now called the Rh system.

History
The expansion to include C, E, c and e (RH2 to RH5)
• By the end of 1943, four antisera detecting genetically related
antigens were available to Fisher and Race, who noticed that
two of them appeared to give antithetical results.
• They proposed that the antigens recognized by these two
antisera were allelic and called them C and c.
• They gave further letters, D (the original Rh antigen) and E, to
the antigens recognized by the other two antisera and
postulated that each had an alternative, which they called d
and e.
• Anti-e was found in 1945. Anti-d has never been found as no d
antigen exists.

The RH Genes
• Two genes, designated RHD and RHCE, encode the
Rh proteins.
• one encodes the D antigen and the other encodes CE
antigens in various combinations (ce, cE, Ce, or CE)
• Rh-positive individuals have both genes, whereas
most Rh-negative people have only the RHCE gene.
• Each gene has 10 exons and is the result of a gene
duplication on chromosome 1p34– p36.
• The RhD and RhCE proteins encoded by the two
genes differ by 32 to 35 amino acids.
•

The RH Genes
• Large number of amino acid differences between RhD
and RhCE proteins explains why RhD is so antigenic when
encountered by the immune system of someone who is
Rh negative and has only RhCE.
• The RHCE gene encodes C and c antigens, which differ by
four amino acids: Cys16Trp encoded by exon 1 and
Ile60Leu, Ser68Asn, and Ser103Pro encoded by exon 2 .
• E and e differ by one amino acid, Pro226Ala encoded in
exon 5
• The single gene, RHAG, located at chromosome 6p11–
p21.1 encodes RhAG.
• RHAG is 47% identical to the RH genes and also has 10
exons.

The RH Genes

Rh antigens
• The Rh antigens are expressed on polypeptides.
• The Rh polypeptides span the red cell membrane
exposing six extracellular loops on which are expressed
the Rh antigens.
• These polypeptides are associated in the membrane
with an Rh glycoprotein to form tetramers
• RhAG has a similar conformation, spanning the
membrane 12 times, but is glycosylated
• Two Rh polypeptides and two Rh glycoproteins, form
the Rh core complex
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Rh antigens

Rh antigens
• The Rh glycoprotein is essential for the
formation of this Rh core complex.
• Mutations in the genes controlling the
expression of the Rh polypeptides or the Rh
glycoproteins can occur.
• Can result in the Rhnull phenotype, in which no
Rh antigens are expressed.

Rh antigens
• Red cell defects seen in the Rhnull phenotype
include :
• Abnormal cation transport across the red cell
membrane
• and red cell morphological abnormalities, in the
form of stomatocytes (red blood cells that exhibit
a slit
• or mouth-shaped pallor rather than a central
pallor), which may cause a mild, compensated
haemolytic anaemia

Stomatocytes

Rh antigens
• Over 50 Rh Ags have been identified
• But most Rh problems are due to D, C, E, c or e
• Gene D is dominant to its allele d, because gene d is
an amorph which makes no detectable antigenic
product
• The alleles C and c, and E and e are codominant and
if both alleles are present, both will be expressed

Prevalence of Rh antigens
• D found in 85% of the population
• C found in 70% of the population
• E found in 30% of the population
• c found in 80% of the population
• e found in 98% of the population
• (d) which has never been identified but refers
to the 15% of the population who has no D
antigen

Characteristics of Rh Ags
• Fewer Ag sites than ABO.
• Unlike the ABO system the Rh Ags are not soluble
and are not expressed on the tissues.
• They are well developed at birth and therefore can
easily cause HNDB if baby has Rh Ag that mother
lacks.
• They are very good immunogens.
– This is especially true to D, which is the most immunogenic
after A and B antigens.

Clinical Significance
• The D Ag is, after A and B, the most important
red cell Ag in transfusion practice:
• Individuals who lack the D Ag do not have anti-D
in their serum.
• The Ab is produced through exposure to the D
Ag usually as a result of transfusion or
pregnancy.
• The immunogenicity of D is greater than that of
virtually all other RBC Ags studied.

Clinical Significance
• It has been reported that >80% of D negative
individuals who receive a single unit of D positive
blood can be expected to develop anti-D.
• The blood of all potential recipients is routinely
tested for D so all D negative recipients can be
identified and transfused with D negative blood.

Rh Antibodies
• Unlike ABO Abs that are mainly IgM, Rh Abs are
commonly IgG.
• NOT naturally occurring and therefore are formed by
immune stimulus due to transfusions or baby's RBCs
during pregnancy.
• The most common Ab to form is anti-D in Rh
negative individuals.
• D is considerably more immunogenic than the other
Rh antigens,which have the following order of
immunogenicity: c > E > e > C.

Dosage Effect
• Rh antibodies will react more strongly with
homozygous cells than with heterozygous
cells.
• Anti-E will react strongly with E+E+ cells and
more weakly with E+e+ cells
• This is Dosage effect!

Precaution!
To prevent problems due to anti-D:
• Always try to give Rh-negative individuals Rh-
negative blood
• Give Rho immune globulin to Rh-negative
mothers to prevent the formation of anti-D
during pregnancy.

Rh Inheritance and Nomenclature
Fisher-Race Theory
• Fisher and Race believed that the Rh system
consisted of three closely linked genes or alleles:
• D at one locus, C or c at the second, and E or e at
the third, as reﬂected in the DCE terminology
• The D gene is dominant to the d gene, but Cc and
Ee are co-dominant.
• According to Fisher-Race the loci are so close
together that crossing over cannot occur.
• The three genes on one chromosome will always
be inherited as a complex.

Fisher – Race Theory
• An offspring of the DCe/dce individual will
inherit either DCe or dce from the parent, but
not a combination such as dCe.
• Should such a combination occur it would
indicate crossing over - never been proven in the Rh
system of man.

Fischer – Race Theory
• With exception of the amorph d, each of the
allelic genes mentioned so far controls the
presence of its respective Ag on the RBC.
• DCe determines the presence of the antigens D, C
and e on the RBCs.
• If the same gene complex were on both of the
paired chromosomes D, C and e would be the
only Rh Ags demonstrable on the cells.
• If one chromosome carried DCe and the other
DcE, the Ags present would be D, C, c, E and e.
• Each Ag (except d) is recognizable by testing the
red cells with a specific antiserum.

Fischer – Race Theory
• The Rh gene complex possesses closely linked
genes (antigens) which could be assembled in
eight different ways: CDe, cDE, cde, cDe, cdE,
Cde, CDE & CdE.
• Because of the strong antigenic characters of
D, all individuals who lack the D antigen are
said to be Rh negative regardless of whether
the C or E antigen or both are present.

Wiener Theory
• Postulated by an American Dr. Alexander Wiener.
• Wiener's theory states that Rh antigens were the
products of a single gene coding for an “agglutinogen”
composed of multiple “blood factors.”
• Wiener differentiates the E and C antigen with “e” and “c”
antigen by the rh and hr symbol.
For example, one gene R1 produces one agglutinogen
(antigen) Rh1 which is composed of three "factors": rh',
Rh(o), and hr’’.

Wiener Theory
• The gene R1 has been inherited on one
chromosome and the gene r at the same locus on
the other chromosome.
• The gene R1 determines the agglutinogen Rh1 on
the red cell and this agglutinogen is made up of
at least three factors: Rho (D), rh'(C) and hr" (e).
• The gene r determines the agglutinogen rh on the
red cell distinguished by its factors
• hr' (c) and hr" (e).

Wiener Theory
• Note: The main difference between the Fisher-Race
and Wiener theories is that whereas the Fisher-Race
theory has three closely linked loci, the Wiener
theory has only one gene locus at which multiple
alleles occur.
• According to Wiener's hypothesis, each gene
produces a structure on the red cell called an
agglutinogen, and each agglutinogen can be
identified by its parts or factors that react with
specific Abs

Wiener Theory
• Wiener believed the gene has a number of alleles
resulting in the presence of various Rh antigens.
• According to Wiener there were 8 alleles,
• Ro
• R1
• R2
• Rz
• r
• r'
• r"
• ry

6/10/2020 33
Haplotype (Fisher System) Wiener System
CDe R1
cDE R2
CDE Rz
cDe Ro
Cde r`
cdE r"
CdE ry
cde rAlick Mwambungu-BTS

Translating from Wiener to Fisher-Race
It is easier to do the conversions if you
remember the following:
• R always refers to D whether it is Ro, R1, R2, or
the very rare Rz.
• r always refers to the lack of D
• o refers to having no C or E
• 1 or ' always refers to C
• 2 or " always refers to E
• The very rare haplotypes that have both a C
and E are given letters from the end of the
alphabet z and y.

Tippet theory
• In this model, suggested by Tippet in 1986, the Rh system
is controlled by two closely linked loci, RHD and RHCE.
• The RHD locus carries the gene for the RHD polypeptide,
which expresses all the D antigen epitopes.
• The RHCE locus carries the genes for the RHCE
polypeptide, which expresses both the C/c and E/e
antigens.
• The genes which encode the C/c and E/e antigens are co-
dominant alleles.
• RHCE exists in four allelic forms and each allele
determines the expression of two antigens in Ce, ce, cE
or CE combination.

6/10/2020 36
Rh Gene Complexes, Antigens, Possible Combinations and Percentages
Haplotypes Genes Present
Antigens
Present
Phenotype Percentage
R1 RHD RHCe DCe R1 42%
r RHce dce r 37%
R2 RHD RHcE DcE R2 14%
Ro
RHD RHce
(more common in
Blacks)
Dce Ro 4%
r' RHCe dCe r' 2%
r" RHcE dcE r" 1%
Rz RHD RHCE DCE Rz <1%
ry RHCE dCE ry <1%
Alick Mwambungu-BTS

Incidence of most common genotypes
Ags Present
Genotype Incidence(%)
Fisher DCE
Wiener
Haplotypes
Whites Blacks
1 D, C, c, e DCe/ce R1r 31.1 8.8
DCe/Dce R1Ro 3.4 15
Dce/Ce Ror 0.2 1.8
2 D, C, e DCe/DCe R1R1 17.6 2.9
DCe/Ce R1r' 1.7 0.7
3 ce ce/ce rr 15 7
4 DCcEe DCe/DcE R1R2 11.8 3.7
DCe/cE R1r" 0.8 <0.1
DcE/Ce R2r' 0.6 0.4
5 DcEe DcE/ce R2r 10.4 5.7
DcE/Dce R2Ro 1.1 9.7
6 Dce Dce/ce Ror 3.0 22.9
Dce/Dce RoRo 0.2 19.4
7 DcE DcE/DcE R2R2 2.0 1.3
DcE/cE R2r" 0.3 <<0.1

Weak D Phenotype
• Most D positive rbc’s react macroscopically
with Reagent anti-D at immediate spin
– These patients are referred to as Rh positive
– Reacting from 1+ to 3+ or greater
• HOWEVER, some D-positive rbc’s DO NOT
react at Immediate Spin using Reagent Anti-D.
• These require further testing (37oC and/or
AHG) to determine the D status of the patient.

Further testing of Patients Cells for
Weak D Status
• If negative at Immediate Spin, patient cells and anti-D
reagent are incubated at 37o C for 20 min’s.
• After incubation, Centrifuge, observe for agglutination.
If positive, report as Rh Positive.
• If negative wash three times and add AntiHuman
Globulin. Centrifuge. If NEGATIVE add CC cells and
report as Rh Negative if CC cells agglutinate. If
POSITIVE report as Weak D Positive.
• Patients/Recipients who require AHG testing to
determine the presence of the D antigen, and have the
D antigen are designated “Weak D Positive”.

Weak D Mechanism’s
There are three mechanisms that account for
the Weak D antigen.
1. Genetically Transmissible
2. Position Effect
3. Partial D (D Mosaic)

Genetically Transmissible
• Occurs when an amino acid substitution within
the RH polypeptide is located within the RBC
membrane or the cytoplasm
• Integration of the RhD protein into the
membrane will be hindered, leading to
quantitative weakening of the D antigen.
• There is usually no qualitative change, and
hence no anti-D immunization.

Genetically Transmissible
• The RHD gene codes for weakened expression of D
antigen. In this mechanism;
– D antigen is complete, there are just fewer D Ag
sites on the rbc. Quantitative!
– Common in Black population (usually Dce
haplotype). Very rare in White population.
• Agglutinate weakly or not at all at immediate spin
phase.
• Agglutinate strongly at AHG phase.
• Can safely transfuse D positive blood
components.

Position Effect
(Gene interaction effect)
• C allele in trans position to D allele
– Example: Dce/dCe, DcE/dCE
In both of these cases the C allele is in the trans position
in relation to the D allele.
• D antigen is normal, C antigen appears to be
crowding the D antigen. (Steric hindrance)
• Does NOT happen when C is in cis position
– Example: DCe/dce
• Can safely transfuse D positive blood components.

Position Effect
C in trans position to D:
D c e / d C e
C in cis position to D:
D C e / d c e
Weak D
NO weak D

Partial D (D Mosaic)
• The RhD protein traverses the erythrocyte
membrane several times, leaving only part of
the protein exposed at the surface .
• If an amino acid is substituted in a portion of
the RhD protein which is located at the outer
surface of the erythrocyte membrane
• single epitopes of the D antigen can be lost or
new antigens can be formed.

Partial D (D Mosaic)
• This may lead to missing one or more PARTS
of the D antigen
– D antigen comprises many epitopes:
• PROBLEM
– Person types D positive but forms allo anti-D that
reacts with all D positive RBCs except their
OWN.

Partial D: Multiple epitopes make up D antigen.
Each color represents a different epitope of the D
antigen.
The difference between Patient X and Patient Y is a single epitope of the D
antigen. The problem is that Patient Y can make an antibody to Patient X even
though both appear to have the entire D antigen present on their red blood cell’s
using routine anti-D typing reagents..
X.
Y.
Patient Y lacks one
D epitope.

Weak-D Determination:
Donor Blood
• When testing Donor Blood for the D
antigen, testing is required through all
phases.
– Weak-D testing is REQUIRED
• We need to know the D Status of all Donor
Blood. Why?
– Main problem is Rh Negative women of child
bearing age and paediatric patients.
• Donor RBCs are labelled Rh positive if any
part of the D antigen is present on the red
blood cell membrane.

Recipient Blood
Controversy
• AABB Standards state that you do NOT have to
perform complete D typing of recipient blood.
• Most weak-D patients can receive D positive blood
without forming anti-D.
• Partial D is very rare, BUT these patients are
capable of making allo anti-D even though they are
Weak D positive.
– So, some blood banks ONLY perform immediate spin D
and if it is negative they do NO further D testing and label
the patient (recipient) Rh (D) negative and transfuse Rh
Negative blood components.

Molecular Rhesus Blood typing
• The distinction between weak partial D from other forms
of weak D is crucial since the individuals with partial D
may form anti- D, if given D+ positive blood.
• This can only be achieved by molecular techniques
• RhD genotyping of transfusion recipients with a
serological weak D phenotype can conserve inventories
of RhD-negative RBCs without compromising transfusion
safety.
• Also, RhD genotyping of pregnant women when a
serological weak D is detected can avoid unnecessary
injections of Rh immune globulin without compromising
the safety of their pregnancy or the foetus.

Application of Rh System
• Paternity testing of blood group Ags is based
on a process of exclusion.
• Since RHD and RHCE are closely linked and Ce,
ce, cE are produced by a single gene, there are
limited combinations that the father can
provide.

Application of Rh system
• HDN predictability by testing father's Rh
genotype.
• This helps predict likelihood of HDN due to D
when mom has anti-D.
–The most common Rh genotype of the
father will indicate whether baby has O%,
50%, or 100% probability of being D
positive.
• If father is also D negative (ce/ce), baby will be
D negative as well and there is a 0%
probability of the baby suffering from Rho
HDN.

Application of Rh System
• If father's Rh genotype appears to be either,
R1r, R2r or Ror, the baby has a 50% probability
of being D positive and suffering from Rho
HDN.
• On the other hand if a father's Rh genotype
appears to be any of the following, R1R1, R2R2,
R1R2, RoRo, R1Ro, or R2Ro, baby has a 100%
probably of getting a D gene from his father
and therefore being D positive and suffering
from Rho HDN.

Prevention of Rh Incompatibility
• Premarital counseling? Ambitious?
• Proper matching of blood particularly in
women before childbearing.
• Blood grouping and Rh typing a must for every
woman, before 1st pregnancy.
• Proper management of unsensitized Rh
negative pregnancies.
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Rhesus Blood Group System

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