Rh Blood Group

System
Group 10: Tambasacan, Tomaro, Villanueva,
Valdez, Yabes
Introduction
• The second most important blood group system, in transfusion.
• Rh system antigens are very immunogenic.
• Rh antibodies produced only after exposure to foreign RBCs.
• Once present they can produce significant hemolytic disease of the
newborn.
• Rh positive and Rh negative are routinely used by public and by
experts in the field referring to blood type.
History
• 1930s, only ABO blood group system is recognized, thus blood transfusion is
only based in this system. Despite ABO matching there is continued result of
morbidity and mortality.
• End of 1930s, Levine and Stetson, hemolytic transfusion reaction in obstetrical
patient.
• One year later, Landsteiner and Wiener, antibody made by guinea pigs and
rabbits transfused with Rhesus macaque monkey.
• Rh primary cause of hemolytic disease of the newborn.
• Mid-1940s, 5 antigens made up Rh system
• Today, 57 different specificities of Rh blood group system.
Terminology
 Fischer-Race: DCE Terminology
• 1940s – antigen of the system produced by 3
closely linked sets of alleles.
• Each gene produce a product or antigen
• Antigen of the System: D, d (not an antigen),
C, c, E, e
• A person inherits a set of Rh genes from each
parent
• Individual’s Rh phenotype is reported as DCE
rather than CDE, (postulate: C/c locus lies
between D/d and E/e loci).
Fischer-Race: DCE Terminology
• Represents the easiest way to think about 5 major Rh
system antigens.
• Rh antigens continues to grow, Fisher- Race terminology
is becoming too limiting.
• Rare instances: individual fail to express an allelic antigen
at one or both Rh loci (may lack E and e, or CcEe
antigens).
• Deletion phenotype: DC- or Dc- or D-
• Person no Rh antigen = Rhnull (Phenotype: _/_)
• Weakened Rh antigen (D), (C), and (e).
 Wiener: Rh-Hr Terminology
• Rh gene produce at least 3 factors with in
Fisher–Race Wiener
agglutinogen.
Dce R0
• Fisher scale can be converted to Weiner
nomenclature. DCe R1

• Agglutinogen in Weiner nomenclature DcE R2

represents presence of single haplotype DCE RZ

expressing 3 different antigens. dce r
dCe r'
dcE r″
dCE ry
Rosenfield and Coworkers: Alphanumeric
Ternimology
• 1960’s Rosenfield ad associate
proposed to assigns a number to
each antigen of the Rh system Rh1 D
Rh2 C
• Only denotes the presence of antigen
Rh3 E
or its absence in the Rh system.
Rh4 c
• Numeric system suitable for
Rh5 e
electronic data processing.
• Similar nomenclature for other blood
groups (Kell, Duffy, Kidd, Lutheran,
and others).
 -Founded in 1935
-Universal Language for Nomenclature
Rh Terminologies
• Relationship Testing
-Determining the probable or predicted genotypes was
useful for parentage studies.

• Zygosity Testing
-Confirmation if the father possesses one or two copies of the
RHD gene.
MOLECULAR GENETICS
Theories of Genetic Control
• Weiner
1 gene = 1 product with separately recognizable factors

• Fisher and Race

Rh locus = 3 distinct genes

Tippett:
1 Rh genes + RHD + RHCE = Rh antigens
Rh Genes
• Chromosome 1
*RHD = P/A of RhD protein
*RHCE = RhCe, RhcE, Rhce, or RhCE proteins

• Chromosome 6
Rh-associated glycoprotein (RhAG) = coexpressor

Rhnull = No Rh antigen
Rh-Positive Phenotypes
 Rh Negative Phenotypes

 Mostoften found in:

European = No Rh antigen, but has 2 RHCE genes
African (66%) = No Rh antigen,
Asian = Mutation=
 Biochemistry
The product of RH genes are
nonglycosylated proteins.
Rh antigens:
• Reside on transmembrane
proteins
• They are an integral part of
the RBC membrane.
RHD and RHCE
The gene products of RHD and RHCE
Remarkably similar: both encode for
proteins composed of 416 amino acids
that traverse the cell membrane 12
times.
Amino acid position 103 is important in
determining C or c expression, and
position 226 differentiates E from e.
RHD: codes for the presence or
absence of the RhD protein
RHCE: codes for either RhCe, RhcE,
Rhce, or RhCE proteins
RHD and RHCE
RhD and RhCE proteins and RhAG are exclusively on red blood cells.
• Transmembranes
• Play a role in maintaining the structural integrity of red blood cells
• Transporters (based on their structure).
• Westhoff and colleagues showed they may have a role in transporting
ammonia.
• An alternative hypothesis is that they may be CO2 transporters.
Predicted model of the RhCE, RhD,
and RhAG proteins in the RBC
membrane.

C/c differ by several amino acids, but

only Ser103Pro on loop 2 is predicted
to be extracellular.
E/e differs by Pro226Ala on loop 4, and
CW and CX antigens are located on
the first extracellular loop of RhCE.
The antigens V and VS result from a
Leu245Val change located in the
predicted eighth transmembrane
region of RhCE. RhAG does not carry
Rh antigens
Antigen characteristics
• Integral to RBC membrane; passing
through the RBC wall 12 times.
• Found only on RBCs– are not soluble or
expressed on other cells
• Well developed at birth– can cause
hemolytic disease of the fetus and
newborn (HDFN). • The five common Rh antigens are
• Highly immunogenic D, C, E, c, and e.
• D antigen does not have an allele.
• C and c are alleles and E is allelic to
e.
D Antigen
Other Common Rh Antigens (C, E, c, e)
• Exposure to less than 0.1 mL of Rh- Most potent to least postent antigens:
1. D antigen
positive RBCs can stimulate
2. c antigen
antibody production in an Rh-
3. E antigen
negative person. 4. C antigen
• In the general population: 5. e antigen
• 85% Rh-positive
• 15% Rh-negative
Other Common Rh Antigens (C, E, c, e)
• C, c, E, and e antigens are
inherited through the RHCE gene
• The alleles are codominant,
therefore specificities from both
haplotypes are expressed as RBC
antigens.
Weak D: Variations of D Antigen Expression
When: Rh + RBC samples typed for the D antigen; expectation: strong positive
reactivity with anti D reagents

Weak D causes: Variations in the quantity or specificity of D antigen epitopes.

• Serologic weak D is noted when:

• Initial anti-D testing is negative
• Less than or equal to 2+ strong (detectable at the indirect antiglobulin testing (IAT)
phase)
Weak D: Variations of D Antigen Expression
• Individuals with RBCs carrying
weaker D antigen (historically called
Du type) can produce anti-D if they Weak D three categories:
are missing epitopes of the D 1. Position effect
antigen. 2. Quantitative
• “Weak D”: used to identify individuals 3. Partial-D antigen (missing one or
whose D was not detectable at more alleles)
immediate spin, without defining the
nature of the weakened expression.
Weak D: Variations of D Antigen Expression
1. Position Effect: C in Trans to D
• Also called gene interaction effect
• The allele carrying RhD is trans (or in the opposite haplotype) to the allele
carrying C.
• Example: Dce/dCe
• This interference with D expression does not occur when the C gene is
inherited in the cis position to RHD, such as DCe/dce.
Weak D: Variations of D Antigen Expression
1. Position Effect: C in Trans to D
• Weak D antigen testing: required for blood donors and newborns of D
negative mothers.
• Blood group reagents: approved by the FDA; contain both monoclonal IgM
and IgG antibodies.
• Monoclonal IgM: detect D antigen during immediate spin.
• IgG antibodies: detect D antigen in the antiglobulin phase of testing.
 Weak D: Variations of D Antigen Expression
2. Weak D: Quantitative Changes Due to
Fewer D Antigen Sites
• Results from inheritance of RHD genes
that code for a weakened expression of
the D antigen.
• On a molecular level, mutations in the
RHD gene occur = causing changes or
deletions in amino acids present in the
transmembrane or intracellular region of
the RhD protein = causing
conformational changes in the protein
 Weak D: Variations of D Antigen Expression
2. Weak D: Quantitative Changes Due to
Fewer D Antigen Sites
• Wagner, Gassner, and others described
the classification of weak D RBCS based
on single nucleotide polymorphisms
(SNP).
Del
• A phenotype occurring in individuals whose red blood cells possess an
extremely low number of D antigen sites that most reagent anti-D are unable
to detect.
• To detect the D antigen:
1. Adsorb and elute anti-D from the individual’s red blood cells
• Incubate anti-D with the RBCs in question at 37°C
• Then, elute the anti-D off the adsorbed red blood cells
2. Molecular studies: detect a mutant RHD gene that alters expression of the
RhD protein.
In the transfusion setting:
• Patients are typed for D: immediate spin or
other routine methods; negative result is
interpreted as Rh negative without any
further testing.
• Donors who initially type as D-negative must
be tested further to determine if they are
really weak D.
• Sandler and others suggest further tests to
assess the patient’s true D status
Weak D: Variations of D Antigen Expression
3. Partial D or D Mosaic
• D antigen expression can be weakened when one or more D epitopes within
the entire D protein is either missing or altered.
• The D antigen is not complete: one or more epitopes are missing.
• In Individuals with Partial-D antigens:
• may type weaker than expected
• may not react at all in routine procedures
• others show normal typing.
Weak D: Variations of D Antigen Expression
3. Partial D or D Mosaic
Wiener and Unger postulated:
• D antigen is made of antigenic subparts, genetically determined, that could be
absent in rare instances.

Tippett and Sanger worked with RBCs and sera of partial-D individuals to classify
these antigens.
• Seven categories were recognized, designated by Roman numerals I through VII.
• In some cases, the loss of D epitopes can result in a new antigen forming.
Weak D: Variations of D Antigen Expression
3. Partial D or D Mosaic
Partial-D antigens can be classified on a
molecular level and are attributed to hybrid
genes resulting from portions of the RHD
gene being replaced by portions of the
RHCE gene.

The resulting protein contains a portion of

RHD and RHCE in various combinations,
depending on the hybrid gene’s makeup.
Weak D: Variations of D Antigen Expression
3. Partial D or D Mosaic
• Anti-D made by individuals expressing partial D can cause hemolytic disease
of the fetus and newborn (HDFN) or transfusion reactions, or both.
• Monoclonal anti-D (MAb-D): Used to classify partial-D antigens; by Tippett and
coworkers.
• Commercial monoclonal anti-D panel: Currently available; does not define all
partial D and weak D types clearly
• A combination of serologic typing and molecular analysis: often required to
accurately categorize partial-D types
 D Epitopes on RhCE Protein
• Like RhD proteins that can express some
RhCE protein, resulting in the partial-D
phenotypes, RhCE protein can express
RhD epitopes detected by some
monoclonal anti-D.

• The Crawford (ceCF) phenotype, RH34, is

found in individuals of African descent. It
results from a specific amino acid change
in the RHce gene, resulting in an RhD
epitope on the Rhce protein.31
Detection of RH Antibodies and
Antigens
RH ANTIBODIES
• Although the Rh system was first recognized by
saline tests used to detect IgM antibodies, most Rh
antibodies are IgG immunoglobulins and react
optimally at 37°C or after antiglobulin testing in
any method used for antibody detection.
RH ANTIGENS
• Rh antigens are highly immunogenic; the D antigen is the most potent
• While the D antigen is most immunogenic, c antigen is the next most likely Rh antigen
to elicit an immune response, followed by E, C, and e.
• IgG1 and IgG3 are of the greatest clinical significance because the reticuloendothelial
system rapidly clears RBCs coated with IgG1 and IgG3 from the circulation.
• Because Rh antibodies are primarily IgG and can traverse the placenta and
because Rh antigens are well developed early in fetal life, Rh antibodies formed
by Rh-negative pregnant women cross the placenta and may coat fetal RBCs
that carry the corresponding antigen. This results in the fetal cells having a
positive direct antiglobulin test;
RH TYPING REAGENTS
• Saline anti-D has the advantage of being lowprotein-based and can be used to
test cells that are already coated with IgG antibody, as in patients who have
warm autoantibodies binding to their RBCs
• In the 1940s, high-protein anti-D reagents were developed that consisted
primarily of IgG anti-D.
• Potentiators of bovine albumin and macromolecular additives such as
dextran or polyvinylpyrrolidone were added to the source material to
optimize reactivity
In the late 1970s, scientists chemically modified the IgG anti-D molecule by
breaking the disulfide bonds that maintain the antibody’s rigid shape.41 This
allows the antibody to relax and to span the distance between RBCs in a low-
protein medium.
CLINICAL CONSIDERATIONS
Rh antigens are highly immunogenic. The D antigen is the most
immunogenic antigen outside the ABO system. When anti-D is detected, a
careful medical history will reveal RBC exposure through pregnancy or
transfusion of products containing RBCs. Circulating antibody appears
within 120 days of a primary exposure and within 2 to 7 days after a
secondary exposure.

Rh-mediated hemolytic transfusion reactions, whether caused by primary

sensitization or secondary immunization, usually result in extravascular
destruction of immunoglobulin coated RBCs. The transfusion recipient may
have an unexplained fever, a mild bilirubin elevation, and a decrease in
hemoglobin and haptoglobin.
HEMOLYTIC DISEASE OF THE FETUS AND
NEWBORN
• Erythroblastosis fetalis, the mother is Rh negative and the father Rh positive. The
baby has inherited the Rh-positive antigen from the father, and the mother
develops anti-Rh agglutinins from exposure to the fetus’s Rh antigen. In turn, the
mother’s agglutinins diffuse through the placenta into the fetus and cause red
blood cell agglutination
• Levine and Stetson1 postulated that the antibody causing the transfusion
reaction also crossed the placenta and destroyed the RBCs of the fetus, causing
its death. The offending antibody was subsequently identified as anti-D
• HDFN caused by Rh antibodies is often severe because the Rh antigens are well
developed on fetal cells, and Rh antibodies are primarily IgG, which readily cross
the placenta.
Rh Deficiency Syndrome: Rhnull and Rhmod
RHnull Syndrome
• Individuals who lack all Rh antigens
on their RBCs
2 TYPES
REGULATOR AMORPHIC
• Mutation occurs in the RHAG gene Mutation in each of the RHCE genes inherited
• This results in no RhAG protein from each parent and the common deletion of
expression and subsequently no RhD the RHD gene found in most D-negative
or RhCE protein expression on the individuals.
RBCs, even though these individuals The RHAG gene is normal.
usually have a normal complement of
RHD and RHCE genes.
Rh Deficiency Syndrome: Rhnull and Rhmod

RHnull Syndrome
• Individuals with RHnull syndrome demonstrate a mild
compensated hemolytic anemia, reticulocytosis,
stomatocytosis, a slight-to-moderate decrease in
hemoglobin and hematocrit levels, an increase in
hemoglobin F, a decrease in serum haptoglobin, and
possibly an elevated bilirubin level.
Rh Deficiency Syndrome: Rhnull and Rhmod

RHmod Syndrome
• Rare individuals exhibit a severely reduced expression of all Rh antigen
• Have a partial suppression of RH gene expression caused by mutations in the
RHAG gene.
• Rhmod individuals exhibit features similar to those with the Rhnull syndrome;
however, clinical symptoms are usually less severe and rarely clinically
remarkable.
Unusual Phenotypes
and
Rare Alleles
Cw
• was originally considered an allele at the C/c locus.
• Cw results from a single amino acid change most often found on the RhCe
protein.
• Cw is found in about 2% of Caucasians and is very rare in African Americans.
• Anti-Cw has been identified in individuals without known exposure to foreign
RBCs and after transfusion or pregnancy.
 f (ce)
• The f antigen is expressed on the RBC when both c
and e are present on the same haplotype. It has
been called a com-pound antigen.
• The antigen f was included in a series of these
compound anti-gens, which were previously referred
to as cis products to indicate that the antigens were
on the same haplotype.
 f (ce)
• Phenotypically, DCE/dceand DcE/DCecells will react the same
when tested with the five major Rh antisera: D+C+E+c+e.
However, when tested with anti-f, only the DCE/dce shows
positive reactivity, confirming the former genotype.
• Anti-f is generally a weakly reactive antibody often found with
other antibodies.
• In case of transfusion, f-negative blood should be provided. Anti-f
is not available as a reagent. It is not necessary to give blood
that is negative for both c and e antigens. It is adequate to
provide either c-negative or e-negative blood since all c-negative
or e-negative individuals are f-negative
 Rh i (Ce)
• Similar to f, rhi was considered a compound antigen
present when c and e are on the RhCe protein.
• Therefore anti-rhi would only react with cells from an
individual with a haplotype of DCeor Ce.
• Antigens cE (RH27) and CE (Rh22) also exist, but anti-
bodies produced to these antigens are not commonly
seen
 G

• G is an antigen present on most D-positive and all

C-positive RBCs
• In Antibody identification testing, anti-G reacts as
though it were a combination of anti-C plus anti-D
because most C-positive and D-positive cells are
G+.
• G was originally described in an rr person who
received D+C–E–c+e+ RBCs.
 Rh17 (Hr0)
• Rh17, also known as Hr0, is an antigen present
on all RBCs with the “common” Rh phenotypes
(e.g., R1R1, R2R2, rr).
• In essence, this antibody is directed to the entire
protein resulting from the RHCEgenes.
• When RBCs phenotype asD– (i.e. D+C-E-c-e-)
the most potent antibody they make is often one
directed against Rh17 (Hr0), which would react
with all cells except D–.
 Rh23, Rh30, Rh40, and Rh52
• Rh23, Rh30, and Rh40 are all low-prevalence antigens associated with a
specific category of partial D.
• These low-prevalence antigens result from the formation of the hybrid proteins
seen in individuals with partial-D phenotypes.
• Rh23 (also known as Wiel and Dw) is an antigenic marker for category Va
partial-D.
• Rh30 (also known as Goa or Dcor) is a marker for partial DIVa.
• Rh40 (also known asTar or Targett) is a marker for partial DVII.
• Rh52 or BARC is associated with some partial-DVI types.
 Rh33 (Har)

• The low-prevalence antigen Rh33 is most often found in whites and is

associated with the rare variant haplotype called R0 Har.
• R0Har gene codes for normal amounts of c, reduced amounts of e, reduced f,
reduced Hr0, and reduced amounts of D antigen written as (D)c(e).
• The D reactions are frequently so weak that the cells are often typed as Rh-
negative.
• As previously discussed, R0Har or DHAR results from a hybrid gene RHCE-
RHD-RHCE in which only a small portion of RHD is inserted into the
RHCEgene.
 Rh32
• Rh:32 is a low-prevalence antigen associated with a variant of the R1 [D(C)
(e)]haplotype called R = N(pronounced “R double bar N”).48The C antigen
and e antigen are expressed weakly.
• The D antigen expression is exaggerated or exalted.
• This gene has been found primarily in African Americans.
Rh43 (Crawford)
• Rh43, also known as the Crawford antigen, is a low-prevalence antigen on a
variant Rhce protein.
• The Crawford (ceCF) anti-gen is of very low prevalence found in individuals
of African descent.
 e Variants

• Like the variant D antigen seen in individuals possessing a hybrid or mutated

RHDgene, some individuals of African or mixed ethnic backgrounds possess e
antigen that exhibits similar qualities as those described for partial-D
phenotypes—that is, an individual may have a phenotype of e-positive but
produce antibodies behaving as anti-e.
• Individuals who possess two altered RHCE genes may have a phenotype of
e-positive but produce antibodies behaving as anti-e.
 V and VS
• V and VS are antigens of low
prevalence in the Caucasian population
but are more prevalent among African
Americans. • It results from a single amino acid change
• These antigens can be used as that occurs at position Val245 in the Rhce
predictors of an individual’s ethnic protein.
background because of this difference • Most hr B-individuals with r’s genotype
in prevalence. are VS+.
• The V antigen, also historically referred • The Val245 mutationin this haplotype is
to as ceS, is found in about 30% of found in the hybrid RHDIIIa-RHCE-
randomly selected African Americans. RHDIIIa gene described previously.
• The VS antigen, also known as eS , • Most V+ individuals are also VS+.
occurs in 32% of African Americans.
Landsteiner Wiener Blood Group System
• the Landsteiner Wiener (LW) antigen begins with the time when Rh antigens
were first recognized.
• The antibody produced by injecting rhesus monkey RBCs into guinea pigs and
rabbits was identified as having the same specificity as the antibody Levine
and Stetson described earlier.
• The antibody was given the name anti-Rh, for anti-rhesus, and the blood group
system was established.
• Many years later, it was recognized that the two antibodies were not identical;
the anti-rhesus described by Landsteiner and Wiener was renamed anti-LW in
their honor.
Landsteiner Wiener Blood Group System

• Phenotypically, there is a similarity between the Rh and LW systems.

• Anti-LW reacts strongly with most D-positive RBCs, weakly with Rh-negative
RBCs (and sometimes not at all), and never with Rh null cells.
• A weak anti-LW may be positive only with D-positive RBCs, and enhancement
techniques may be required to demonstrate its reactivity with D-negative cells.
 END

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