LECTURE
THE RH BLOOD GROUP SYSTEM
INTRODUCTION
Rh Blood Group
- highly complex, and the alloimmunization to
Rh blood group antigens can complicate
transfusion and pregnancy.
- term Rh refers to a specific red blood cell
(RBC) antigen (D) and to a complex blood
group system currently composed of 61
different antigenic specificities.
- Rh is the second in importance only to ABO
blood group system in terms of transfusion, as
the Rh system antigens are very immunogenic.
- Rh antibodies are produced only after
exposure to foreign red blood cells.
➢ they can produce significant hemolytic
disease of the fetus and newborn (HDFN)
as well as hemolytic transfusion reactions.
Rh-positive
- an individual’s red blood cells possess one
particular Rh antigen, the D antigen.
Rh-negative
- the red blood cells lack the D antigen.
HISTORY
• 1939 - Levine and Stetson described a hemolytic
transfusion reaction in an obstetrical patient.
• Landsteiner and Wiener described an antibody
made by guinea pigs and rabbits when they were
transfused with rhesus macaque monkey RBCs.
➢ antibody agglutinated 85% of human RBCs
and was named anti-Rh after the rhesus
monkey.
• The name Rh was retained for the human-
produced antibody, and anti-rhesus formed by the
animals was renamed anti-LW in honor of those
first reporting it (Landsteiner and Wiener).
• Mid-1940s - five antigens were defined in the Rh
system.
• 1980s - molecular testing further defined the
structure of the RH genes.
TERMINOLOGY
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Phenotype
- defined by the serologic detection of antigens
using specific antisera.
- Therefore an Rh phenotype represents the
results for serologic testing of RBC for D, C, c,
E, and e antigens.
Genotype
- an individual’s actual genetic makeup.
RH Genotype
- refers to the actual RH genes inherited by the
individual from his parents. Serologic results
may not exactly correspond with the genetic
expression.
Fisher-Race: DCE Terminology
- In the mid-1940s Fisher and Race defined the
five common Rh antigens and postulated that
the antigens of the system were produced by
three closely linked genes (D/d, C/c, and
E/e).
- Each gene was responsible for producing one
antigen on the RBC surface.
- Each antigen and corresponding gene were
given the same letter designation (when
referring to the gene, the letter is italicized).
- Fisher and Race named the antigens of the
system D, d, C, c, E, and e.
- Now it is known that there is no d antigen, but
some references continue to use the letter d
with Fisher-Race terminology as a placeholder
for D-negative individuals.
- According to the Fisher-Race theory, an
individual inherits a set of RH genes from each
parent (i.e., one D or d, one C or c, and one E or
e).
Haplotype
- combination of genes inherited from one
parent.
Genotype
 LECTURE
- pairing of maternal and paternal haplotypes
determines the offspring’s genotype.
- written as two haplotypes separated by a /
(i.e., DCe/Dce)
• There are rare phenotypes that involve deletions
of specific genes, and in those cases the deletion is
represented with a dash.
• an individual having only D and no C/c and E/e,
the Fisher-Race haplotype is written as D–.
• Placing parenthesis around (D), (C), and (e)
indicates weakened antigen expression.
Wiener: Rh-Hr Terminology
- Wiener believed there was one gene
responsible for defining Rh that produced an
agglutinogen containing three Rh factors.
- The agglutinogen may be considered the
phenotypic expression of the haplotype.
- original Wiener nomenclature named the five
common Rh antigens as Rho, rh’, rh”, hr’, and
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hr”, but these terms are no longer used in
favor of a modified form of Wiener notation.
- In the modified Wiener nomenclature, an
agglutinogen is described by a letter and
symbol assigned based on the factors present.
- uppercase R denotes the presence of the D
antigen.
- lowercase r indicates the absence of D
antigen.
- presence of the C antigen is indicated by a 1
or a single prime (‘).
- c antigen is implied when there is no 1 or ‘
indicated.
- E antigen is indicated by a 2 or double prime
(“).
- e antigen is implied when no 2 or “ is
indicated.
- R1 is the same as DCe
- r′ denotes Ce
- Ro is equivalent to Dce
- presence of E is indicated by the Arabic
number 2 or double prime (“).
- Lowercase e is implied when there is no 2 or
“ indicated.
- R2 is the same as DcE
- r” denotes cE
- r is equivalent to ce
- When both C and E are present, the letter z
or y is used.
- Rz denotes DCE
- ry represents CE.
- genotype for the Rhnull that arises from an
amorphic gene at both Rh loci is written as rr˭
and pronounced “little r double bar.”
- Modified Wiener terminology allows one to
convey Rh antigens inherited on one
chromosome or haplotype and makes it easier
to discuss a genotype.
- Fisher-Race nomenclature may be converted
to Wiener nomenclature and vice versa.
- It is important to remember that an
agglutinogen in Wiener nomenclature actually
represents the presence of a single haplotype
expressing three different antigens.
 LECTURE

- In the Wiener nomenclature, there is no

designation for the absence of D antigen.

Rosenfield and Coworkers: Alphanumeric - A minus sign preceding a number designates

Terminology the absence of the antigen.
- In the early 1960s, Rosenfield and associates - If an antigen has not been phenotyped, its
proposed a system that assigned a number to number will not appear in the sequence.
each antigen of the Rh system in order of its - five major antigens
discovery or recognized relationship to the Rh
system. ➢ D is assigned Rh1
- simply demonstrates the presence or absence ➢ C is Rh2
of the antigen on the RBC. ➢ E is Rh3
- Each antigen is assigned a number. ➢ c is Rh4

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 LECTURE
➢ e is Rh5
- The numeric system is well suited to electronic
data processing.
- Its use expedites data entry and retrieval.
- Its primary limiting factor is that there is a
similar nomenclature for numerous other
blood groups, such as Kell, Duffy, Kidd, and
more.
- when using the Rosenfield nomenclature on
the computer, one must use both the alpha
(Rh:) and the numeric (1, 2, –3, etc.) to
denote a phenotype.
International Society of Blood Transfusion
Committee: Updated Numeric Terminology
- Its mandate was to establish a uniform
nomenclature that is both eye- and machine-
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readable and is in keeping with the genetic
basis of blood groups.
- adopted a six-digit number for each
authenticated antigen belonging to a blood
group system.
- first three numbers represent the system,
and the remaining three the antigenic
specificity.
- Number 004 was assigned to the Rh blood
group system, and then each antigen assigned
to the Rh system was given a unique number
to complete the six-digit computer number.
- When referring to individual antigens, an
alphanumeric designation similar to the
Rosenfield nomenclature may be used.
- The alphabetic names formerly used were left
unchanged but were converted to all
uppercase letters (e.g., Rh, Kell became RH,
KELL).
- Therefore, D is RH1, C is RH2, and so forth.
➢ Note: There is no space between the RH
and the assigned number.
- The phenotype designation includes the
alphabetical symbol that denotes the blood
group, followed by a colon and then the
specificity numbers of the antigens defined.
- A minus sign preceding the number indicates
that the antigen was tested for but was not
present.
- The phenotype D + C – E + c + e + or DcE/ce
or R2r would be written RH:1, –2, 3, 4, 5.
- When referring to a gene, an allele, or a
haplotype, the symbols are italicized.
- A haplotype is followed by a space or an
asterisk, and the numbers of the specificities
are separated by commas.
➢ The R1 haplotype or DCe would be RH
1,2,5 or RH*1,2,5.
 LECTURE

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 LECTURE

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 LECTURE

SUMMARY ➢ Rh: 1 = D antigen

1. Fisher-Race ➢ Rh: 2 = C antigen
➢ DCcEe ➢ Rh: 3 = E antigen
2. Weiner ➢ Rh: 4 = c antigen
➢ Rh0, rh’, rh”, hr’, hr” ➢ Rh: 5 = e antigen
- Modified ➢ Rh: 6 = ce antigen
➢ R = D antigen
➢ r = d antigen
➢ 1 or ‘ = C antigen
➢ No 1 or ‘ = c antigen
➢ 2 or “ = E antigen
➢ No 2 or “ = e antigen
➢ R0 = Dce
➢ Rz = DCE
➢ R1 = DCe
➢ R2 = DcE
➢ ry = CE
➢ r’ = Ce
➢ r” = cE
➢ r = ce
Rz ry

R1 r’

R2 r”

3. Rosenfield
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GENETICS
I. RH GENES
RHD and RHCE
- two closely linked genes located on
chromosome 1 that control expression of Rh
proteins.
➢ RHD codes for the presence or absence of
the RhD protein.
➢ RHCE codes for either RhCe, RhcE, Rhce, or
RhCE proteins.
- codominant, which means that all products
inherited typically produce antigens
detectable on RBCs.
- each have 10 exons and are 97% identical.
- Each gene has a number of alleles, most of
which have been identified through molecular
testing techniques.
- website for the National Center
Biotechnology Information (NCBI)
➢ which maintains a database for human
blood group mutation.
II. RH-ASSOCIATED GLYCOPROTEIN (RHAG)
- resides on chromosome 6.
- polypeptide is very similar in structure to the
Rh proteins, with the difference being that it is
glycosylated (carbohydrates attached).
- forms complexes with the Rh proteins.
- termed a coexpressor and must be present for
successful expression of the Rh antigens.
- does not express any Rh antigens.
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LECTURE
- When mutations in the RHAG gene occur, it can
result in missing or significantly altered RhD
and RhCE proteins, affecting antigen
expression.
- have the Rhnull phenotype.
- assigned as a separate blood group system.
III. RH-POSITIVE PHENOTYPES
- inherit one or two RHD genes, which result in
expression of RhD antigen and are typed Rh-
positive.
- two RHCE genes are inherited, one from each
parent.
- Numerous mutations in the RHD gene have
been discovered that cause weakened
expression of the RhD antigen detected in
routine testing.
➢ generally termed weak D or weak
expression of D.
for
IV. RH-NEGATIVE PHEOTYPES
- so called because the RBCs lack detectable D
antigen.
- most common Rh-negative phenotype results
from the complete deletion of the RHD gene
➢ the individuals possess no RHD gene but
have inherited two RHCE genes.
AFRICAN ETHNICITY
- RHD pseudogene or RHD ψ
➢ occurs in 66% of individuals of African
ethnicity.
➢ unusual form of the RHD gene.
➢ an RHD-negative allele whose sequence is
identical to the RHD gene except for
missense mutations in exon 5 and exon
 LECTURE

6, a nonsense mutation in exon 6, and a - The product of RH genes is nonglycosylated

37-bp insertion at the intron 3/exon 4 proteins.
boundary. ➢ no carbohydrates are attached to the
➢ Individuals with this gene do not produce protein.
RhD protein. - Rh antigens reside on transmembrane
proteins and are an integral part of the RBC
ASIAN ETHNICITY membrane.

- Del - gene products of RHD and RHCE are

➢ Another mutation has been described in
Asians that alters the RHD gene, causing an
individual to type as D-negative.
V. MOST PROBABLE GENOTYPES
- Determining most probable genotypes was
used in the past for parentage studies, also
known as relationship testing, and for
population studies.
- remains important for the student to be able to
predict the most probable genotype based on
gene frequency and ethnicity.
remarkably similar in that both encode for
proteins composed of 416 amino acids that
traverse the cell membrane 12 times.
- proteins differ by only 32 to 35 amino acids.
- Amino acid position 103 is important in
determining C or c expression, and position
226 differentiates E from e.
- Only small loops of Rh proteins are exposed on
the surface of the RBC and provide the
conformational requirements for many
serologic differences between the Rh blood
types.
- RhD and RhCE proteins and RhAG are
exclusively on red blood cells.
➢ play a role in maintaining the structural
integrity of red blood cells.
➢ Based on their structure, it appears they
may also be transporters.

• A1 cells possess approximately 1 × 106 A antigens,

whereas RBCs possessing a double-dose
expression of the K antigen have 6,000 K sites.
• The greatest number of D antigen sites are on cells
of the rare Rh phenotype D– (refer to the
“Deletions” section).
• R2R2 cells possess the largest number of D antigen
sites.

RH ANTIGENS
I. BIOCHEMISTRY

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 LECTURE
II. ANTIGEN CHARACTERISTICS
- Rh antigens are proteins integral to the RBC
membrane, passing through the RBC wall 12
times.
- The antigens are found only on RBCs, and are
not soluble or expressed on other cells.
- The Rh antigens are well developed at birth,
and as such can cause hemolytic disease of the
fetus and newborn (HDFN).
- Rh antigens are also immunogenic, and
exposure to foreign antigens through
transfusion or pregnancy can cause an
immune response with the production of
corresponding antibodies.
- The five common Rh antigens are D, C, E, c, and
e, and their specific corresponding antibodies
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account for the majority of problems due to Rh
antibodies.
- The D antigen does not have an allele, but C
and c are alleles and E is allelic to e.
III. D ANTIGEN
- Rh antigens are highly immunogenic; the D
antigen is the most potent.
- most Rh-negative individuals lack the entire
RhD protein.
- Exposure to less than 0.1 mL of Rh-positive
RBCs can stimulate antibody production in an
Rh-negative person.
- Eighty-five percent of the general population
is Rh-positive, with 15% typing as Rh-
negative.
- Anti-D was the most common cause of HDFN
until Rh-immune globulin was introduced.
IV. OTHER COMMON RH ANTIGENS (C, E, c, e)
- 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.
- C, c, E, and e antigens are inherited through the
RHCE gene.
- alleles are codominant, therefore specificities
from both haplotypes are expressed as RBC
antigens.
WEAK: VARIATIONS OF D ANTIGEN EXPRESSION
- When Rh-positive RBC samples are typed for
the D antigen, they are expected to show
strong positive reactivity with anti D
reagents.
 LECTURE
- some individuals have RBCs that possess
variations in the quantity of D antigen or the
specificity of D antigen epitopes, resulting in
weakened expression of the D antigen when
tested with serologic methods.
- Serologic weak D is noted when initial anti-D
testing is negative, or less than or equal to 2+
strong, but detectable at the indirect
antiglobulin testing (IAT) phase.
- Advances in Rh testing methodology and
reagents have resulted in detecting more weak
D types without the need for IAT, but there are
still circumstances in which weak D testing is
necessary. Individuals with RBCs carrying
weaker D antigen (historically called Du
type) can produce anti-D if they are missing
epitopes of the D antigen.
- generic term weak D was used to identify
individuals whose D was not detectable at
immediate spin, without defining the nature of
the weakened expression.
- Weak D types can be separated into three
categories
➢ position effect
➢ quantitative
➢ partial-D antigen (missing one or more
alleles).
I. POSITION EFFECT: C IN Trans to D
- The first mechanism that may result in
weakened expression of D antigen.
- result in weakened expression of D antigen
was originally described as a position effect or
gene interaction effect.
- The allele carrying RHD is trans (or in the
opposite haplotype) to the allele carrying C.
- The Rh antigen on the RBC is normal, but the
steric arrangement of the C antigen in
relationship to the D antigen appears to
interfere with the expression of D antigen.
- This interference with D expression does not
occur when the C gene is inherited in the cis
position to RHD, such as DCe/dce.
- It is not possible to serologically distinguish
genetic weak D from the position effect weak
D.
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II. WEAK D: QUANTITATIVE CHANGES DUE TO
FEWER D ANTIGEN SITES
- The D antigens expressed appear to be
complete but fewer in number.
- 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, thus
causing conformational changes in the protein.
- Mutations for these weak D types occur
because SNP affects amino acid insertion of the
protein on the RBC membrane.
- When these changes occur, normal RhD
antigen expression is altered because there
are fewer antigen sites overall.
- Individuals with weak D phenotype rarely
make anti-D, since the RhD antigens are
present as expected, but protein changes occur
“inside” the red blood cell.
- On a molecular level changes in the RHD gene
occur, causing changes in amino acids present
in the transmembrane region of the RhD
protein.
- Mutations in the RHD gene causing this altered
ex pression have been categorized with types
1 and 3 being the most common.
III. Del
- 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.
- Adsorbing and eluting anti-D from the
individual’s red blood cells is often the only
way to detect the D antigen.
 LECTURE
- This procedure requires incubating anti-D
with the RBCs in question at 37°C followed by
eluting the anti-D off the adsorbed red
blood cells.
- The 37°C incubation provides time for the
anti-D to bind to the few D antigen sites
present on these red blood cells.
- Molecular studies can detect a mutant RHD
gene that alters ex pression of the RhD
protein.
• In the transfusion set ting, patients are generally
typed for D with immediate spin or other routine
methods, and a 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.
• patients who initially type as D-negative should
have RHD genotyping performed to as sess their
true D status.
- The goal of this further testing would be to
improve accuracy in Rh typing, conserve Rh-
negative units for those who truly need them,
and prevent unnecessary administration of Rh
immune globulin in women with weak D
serotypes.
IV. PARTIAL D or D MOSAIC
- The third mechanism in which D antigen
expression can be weakened is when one or
more D epitopes within the entire D protein is
either missing or altered, termed partial D
(sometimes called “D Mosaic”).
- The D antigen is not com plete because one or
more epitopes are missing. The sero logic
detection of the D antigen varies greatly for
partial-D individuals.
- Wiener and Unger postulated that the D
antigen is made of antigenic subparts,
genetically determined, that could be absent in
rare instances.
- If an individual lacks one (or more) pieces, or
epitopes, of the total D antigen, alloantibody
can be made to the missing epitope(s) if
exposed to RBCs that possess the complete D
antigen.
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- Seven categories were recognized, designated
by Roman numerals I through VII.
➢ Category I is now obsolete, and a few of the
categories have been further subdivided.
- the loss of D epitopes can result in a new
antigen forming.
- 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.
- If an individual with a hybrid RhD-RhCe-RhD
protein is exposed to red blood cells
possessing normal RhD protein, they will
make antibody to the portion of the RhD
protein they are missing.
- Anti-D made by individuals expressing partial
D can cause hemolytic disease of the fetus and
newborn (HDFN) or transfusion reactions, or
both.
- Once anti-D is identified, Rh-negative blood
should be used for transfusion.
- the anti-D produced is an alloantibody
because it is directed against an antigenic
epitope that the patient lacks.
➢ patient may type as D-positive, the serum
antibody will not react with the patient’s
own cells.
- The identification of a person with a partial D
routinely occurs after the person begins
producing anti-D unless there are discrepant
Rh (D) typings.
• partial-D antigens using monoclonal anti-D (MAb-
D).29 While partial D phenotypes are not common,
category VI is the most often encountered partial
D phenotype.
 LECTURE
V. D EPITOPES ON RhCE PROTEIN
- RhCE protein can express RhD epitopes
detected by some monoclonal anti-D.
- This can cause discrepant RhD type results
with historical information on a patient or
donor.
- rare individuals who possess these unusual
proteins and when typed with anti-D will show
positive reactivity even though the D epi tope
is on the RhCE protein.
- R0Har (RH33) results from a hybrid gene
RHCE-RHD-RHCE, in which only a small
portion of RHD is inserted into the RHCE gene.
➢ If this hybrid gene is paired with a normal
RHD gene, the individual will type Rh-
positive.
➢ if paired with a D-deletion, the individual
may type D-positive, depending on the
anti-D reagent being used.
- These individuals should be classified as RhD-
negative since they essentially lack the RhD
protein.
- The Crawford (ceCF) phenotype, RH34, is
found in individuals of African descent.
RH ANTIBODIES
I. CHARACTERISTICS OF Rh ANTIBODIES
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- 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 antibodies are usually produced following
exposure of the individual’s immune system to
foreign RBCs, through either transfusion or
pregnancy. Rh antibodies may show dosage,
reacting preferentially with RBCs possessing
double-dose Rh antigen.
- IgG1, IgG2, IgG3, and IgG4 subclasses of Rh
antibodies have been reported.
➢ IgG1 and IgG3 are of the greatest clinical
significance because the
reticuloendothelial system rapidly clears
RBCs coated with IgG1 and IgG3 from the
circulation.
- IgM Rh antibodies are formed initially,
followed by a transition to IgG.
- An individual with low-titer Rh antibody may
experience an anamnestic (secondary)
antibody response if exposed to the same
sensitizing antigen.
- Most commonly found Rh antibodies are
considered clinically significant.
- antigen-negative blood must be provided to
any patient with a history of Rh-antibody
sensitization, whether the antibody is
currently demonstrable or not.
- Rh antibodies do not bind complement.
➢ For complement to be fixed (or the
complement cascade activated), two IgG
immunoglobulins must attach to an RBC
antigen in close proximity to each other.
➢ Rh antigens (to which the antibody would
attach) are not situated on the RBC surface
this closely.
➢ when an Rh antibody coats the RBCs,
intravascular, complement-mediated
hemolysis does not occur.
- RBC destruction resulting from Rh antibodies
is primarily extravascular.
II. Rh ANTIBODIES IN PREGNANCY
 LECTURE
- 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 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.
➢ the coated fetal cells are removed
prematurely from the fetal circulation
which can result in anemia. HDFN due to
anti-D can be prevented with the use of Rh-
Immune Globulin, but complications due
to other Rh antibodies cannot be
prevented.
- Until the discovery of Rh-immune globulin,
anti-D was the most frequent cause of HDFN.
TESTING FOR RH ANTIGENS AND ANTIBODIES
I. Rh TYPING REAGENTS
- The reagents may be high-protein based or
low-protein based, saline based, chemically
modified, monoclonal, or blends of
monoclonals.
- The goal is to use a reagent anti-D that will
allow for typing individuals’ RBCs as quickly
and accurately as typing for ABO.
- Saline reactive reagents, which contain IgM
immunoglobulin, were the first typing
reagents available to test for the D antigen.
- Saline anti-D has the advantage of being low-
protein based and can be used to test cells that
are already coated with IgG antibody, as in
patients who have autoantibodies binding to
their RBCs.
- If a high-protein–based reagent was used
when typing these antibody-coated RBCs, a
false-positive reaction would be obtained
because the RBCs would agglutinate on their
own without the addition of anti-D.
- The primary disadvantages of saline typing
reagents are their limited availability, cost of
production, and lengthy incubation time.
- Because saline anti-D is an IgM
immunoglobulin, it cannot be used for weak D
typing.
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- (1940s) high-protein anti-D reagents were
developed that consisted primarily of IgG anti-
D.
➢ Human plasma containing high-titer D-
specific antibody was used as the raw
material.
➢ Potentiators of bovine albumin and
macromolecular additives, such as
dextran, were added to the source material
to optimize reactivity in the standard slide
and rapid tube tests to allow for direct
agglutination of red blood cells using an
IgG anti-D.
➢ this media causes RBCs to be in closer
proximity to each other and allows IgG
anti-D to crosslink and cause direct
agglutination.
➢ These reagents are commonly referred to
as high-protein reagents.
- To assess the validity of the high-protein Rh
typing results, a control reagent was
manufactured and had to be tested in parallel
with each Rh test.
- If the control reacted, the test result was
invalid and had to be repeated using a
different technique or reagent anti-D.
- The major advantages of high-protein anti-
D reagents are reduced incubation time and
the ability to perform weak D testing and slide
typing with the same reagent.
➢ This type of anti-D reagent is also
polyspecific.
- More than one clone of anti-D is produced by
immunized human donors and is therefore
able to recognize multiple epitopes on the RhD
protein.
- Chemically modified Rh typing reagents alter
the IgG anti-D molecule by breaking the
disulfide bonds that maintain the antibody’s
rigid shape.
➢ This allows the antibody to relax and to
span the distance between RBCs in a low-
protein medium.
➢ can be used for both slide and tube testing
and do not require a separate,
manufactured Rh control as long as the
samples type as A, B, or O.
 LECTURE
- When samples test AB Rh-positive or when the
Rh test is performed by itself, a separate
saline control or 6% to 8% albumin control
must be used to ensure the observed reactions
are true agglutination and not a result of
spontaneous agglutination.
- As monoclonal antibody production became
available, Rh monoclonal antibodies were
produced.
➢ These reagents are derived from single
clones of antibody-producing cells. The
antibody-producing cells are hybridized
with myeloma cells to increase their
reproduction rate, thereby maximizing
their antibody-producing capabilities.
II.
III. Rh TESTING REQUIREMENTS FOR
PRETRANSFUSION, OSTETRIC PATIENTS,
AND BLOOD DONORS
- For blood donors, it is critical to make every
effort to identify the presence of the D antigen,
because it is so highly immunogenic.
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➢ Because the D antigen is composed of
many epitopes and the monoclonal Rh
antibodies have a narrow specificity,
monoclonal anti-D reagents are usually a
combination of monoclonal anti-D
reagents from several different clones to
ensure reactivity with a broad spectrum of
Rh-positive RBCs.
➢ Reagents that are a blend of IgM and IgG
anti-D are routinely used to maximize
visualization of reactions at immediate
spin testing and to allow indirect
antiglobulin testing for weak D antigen
with the same reagent.
➢ AABB Standards require that patients
must be tested for the D antigen, but
additional testing for weak D is optional.
- All discrepancies must be fully resolved before
the unit can be labeled and released for
transfusion.
- Rh typing is typically performed with a
method designed to screen for the D antigen at
 LECTURE
the immediate spin phase (if tube testing) or
other screening method.
- Donors who test D-negative on the initial test
must have additional testing performed to
explore the possibility of a weak D phenotype.
➢ Even the smallest amount of D antigen
may trigger an immune response in a
patient exposed to the donated RBCs.
➢ The unit of blood is labeled as Rh-positive
or Rh-negative.
- In the transfusion service, patients are tested
for the D antigen as part of routine testing,
usually in combination with performing the
ABO typing.
➢ Patients who type as negative on the initial
test for the D antigen are often interpreted
as Rh-negative and given Rh-negative
units for transfusion.
➢ required to repeat the Rh typing on all red
blood cell units labeled as Rh-negative.
➢ This testing is a safety measure to verify
that the unit of blood is truly Rh-negative
and prevent transfusion errors due to
testing or labeling errors.
- Pregnant women are tested for the D antigen
to see if they are candidates for Rh-immune
globulin.
➢ Testing for weak D phenotype is optional.
➢ Women who test Rh-negative on initial
screening may be interpreted as Rh-
negative and treated as such for the entire
pregnancy.
IV. DETECTING AND IDENTIFYING Rh
ANTIBODIES
- Patients who are pregnant or anticipating the
need for transfusion must be tested for the
presence of all clinically significant antibodies,
including the Rh antibodies.
➢ The method must include incubation at
37°C, which culminates in an antiglobulin
test.
➢ If Rh antibodies are suspected, then a
complete antibody identification workup
should be performed to identify the
antibody specificity (or specificities)
present in the serum.
16 | P a g e
- Rh antibodies react stronger with cells that
have a double dose of the corresponding
antigen.
- Identification of Rh antibodies can be aided by
the use of special techniques and reagents.
- Rh antibody reactivity can be enhanced by
adding reagents such as albumin, low ionic
strength solution (LISS), and polyethylene
glycol (PEG) to the test system.
- Another method to enhance the reactions is to
treat the reagent red blood cells with
proteolytic enzymes such as ficin.
V. SELECTING COMPATIBLE BLOOD FOR
TRANSFUSION
- Rh antibodies are clinically significant, so units
that are negative for the corresponding
antigens must be selected for transfusion.
- Commercial antisera is available to test for the
five common Rh antigens, as well as some of
the other less common types.
- The search for compatible units can be aided
by recalling the frequencies of the Rh antigens.
- Using commercial antisera to screen the
available ABO/Rh-compatible units in house
can be accomplished easily.
- The e antigen is present in up to 99% of the
general population, so screening available
units in house may not yield a compatible unit.
CLINICAL CONSIDERATIONS
- Determining an individual’s RhD type and
testing to detect and identify RH antibodies
has been reviewed.
- It is important to understand the clinical
implications when Rh incompatibility exists.
I. TRANSFUSION REACTIONS
- When Rh antibodies are detected, a careful
medical history will reveal RBC exposure
through pregnancy or transfusion of products
containing RBCs.
- Circulating antibody can appear as soon as 10
to 14 days of a primary exposure and within
1 to 7 days after a secondary exposure.
 LECTURE
- 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.
- The direct antiglobulin test (DAT) is usually
positive, and the antibody screen may or may
not demonstrate circulating antibody.
- When the DAT indicates that the donor cells
within the recipient’s circulation are coated
with IgG, elution studies may be helpful in
defining the offending antibody specificity.
- If antibody is detected in either the serum or
eluate, subsequent RBC transfusions should
lack the implicated antigen.
- It is not unusual for a person with a single Rh
antibody to produce additional Rh antibodies
if further stimulated.
- Even after the Rh antibodies are no longer
detectable in the patient’s serum, RBCs must
be selected that lack the corresponding
antigens to prevent transfusion reaction.
II. HEMOLYTIC DISEASE OF THE FETUS AND
NEWBORN
- Anti-D was a frequent cause of fetal death
before the implementation of Rh-immune
globulin therapy.
- 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-immune globulin, a purified preparation of
IgG anti-D, is given to D-negative woman
during pregnancy and following delivery of a
D-positive fetus.
- Rh-immune globulin is effective only in
preventing RhD HDFN.
- No effort has been made to develop immune
globulin products for other Rh antigens (e.g., C,
c, E, e). Passively acquired anti-D in the serum
may be noted in mothers who receive
antepartum Rh-immune globulin, so it is
critical to obtain an accurate medical history
17 | P a g e
to establish the reason for the presence of the
anti-D.
- When present, RhD HDFN may be severe and
may require aggressive treatment.
Rh DEFICIENCY SYNDROME: Rhnull AND Rhmod
- Rare individuals have Rh deficiency or Rhnull
syndrome and fail to express any Rh antigens
on the RBC surface.
- Rhnull syndrome is inherited in one of two
ways—amorphic and regulator.
➢ Individuals who lack all Rh antigens on
their RBCs which can be produced by two
different genetic mechanisms.
- Other rare individuals exhibit a severely
reduced expression of all Rh antigens, a
phenotype called Rhmod.
- In the regulator-type Rhnull syndrome, a
mutation occurs in the RHAG gene.
➢ This results in no RhAG protein expression
and subsequently no RhD or RhCE protein
expression on the RBCs, even though these
individuals usually have a normal
complement of RHD and RHCE genes.
➢ These individuals can pass normal RHD
and RHCE genes to their children.
- In the second type of Rhnull syndrome (the
amorphic type), there is a mutation in each of
the RHCE genes inherited from each parent as
well as the deletion of the RHD gene found in
most D-negative individuals.
➢ The RHAG gene is normal.
- It should be noted that Rhnull individuals of
either regulator or amorphic type are negative
for the high-prevalence antigen LW and for
FY5, an antigen in the Duffy blood group
system.
- S, s, and U antigens found on glycophorin B
may also be depressed.
- 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.
 LECTURE
- Rhnull individuals, if exposed to normal Rh cells
through transfusion or pregnancy, can
produce a potent antibody, anti-Rh29, which
reacts with all cells except for those that are
Rhnull.
- When transfusion of individuals with Rhnull
syndrome is necessary, only Rhnull blood can
be given.
- Individuals of the Rhmod phenotype have a
partial suppression of RH gene expression
caused by mutations in the RHAG gene.
- When the resultant RhAG protein is altered,
normal Rh antigens are also altered, often
causing weakened expression of the normal
Rh and LW antigens.
- Rhmod individuals exhibit RBC abnormalities
similar to those with the Rhnull syndrome;
however, clinical symptoms are usually less
severe and rarely clinically remarkable.
UNUSUAL PHENOTYPES AND RARE ALLELES
- While the antibodies directed against these
antigens are less commonly encountered, they
can be clinically significant and, in some cases,
cause transfusion reactions, HDFN, or both.
I. Cw
- originally considered an allele at the C/c locus.
- It is now known that the relationship between
C/c and Cw is only phenotypic and that Cw is
antithetical to the high-prevalence antigen
MAR.
- results from a single amino acid change most
often found on the RhCe protein.
- Anti-Cw has been identified in individuals
without known exposure to foreign RBCs and
after transfusion or pregnancy.
- Anti-Cw may show dosage.
- Commercial anti-Cw reagent is not readily
available, but because of the low prevalence of
the Cw antigen RBCs compatible at the
crossmatch in the antiglobulin phase may be
selected for transfusion.
- Anti-Cw may not always be detected on
routine antibody screens because the low
frequency of the antigen means that some
18 | P a g e
screening cell sets may not have a Cw-positive
cell included.
II. 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 compound
antigen.
➢ f is likely a single entity resulting from
conformational changes in the RHCE
protein.
- The antigen f was included in a series of these
compound antigens, which were previously
referred to as cis products to indicate that the
antigens were on the same haplotype.
- It is now known they are expressed on the
RHCEe protein.
- Phenotypically, DCE/dce and DcE/DCe cells
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. It has been
reported to cause HDFN and transfusion
reactions. In case of transfusion, f-negative
blood should be provided.
- It is adequate to provide either c-negative or e-
negative blood since all c-negative or e-
negative individuals are f-negative.
III. rhi (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 DCe or Ce.
 LECTURE
- Antigens cE (RH27) and CE (Rh22) also exist,
but antibodies produced to these antigens are
not commonly seen.
IV. G -
- G is an antigen present on most D-positive and
all C-positive RBCs.
- The antigen results from the amino acid serine
at position 103 on the RhD, RhCe, and RhCE
proteins.
- 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+.
- originally described in an rr person who
received D+C–E–c+e+ RBCs. Subsequently, the
recipient produced an antibody that appeared
to be anti-D plus anti-C, which should be
impossible because the C antigen was not on
the transfused RBCs.
- Anti-G versus anti-D and anti-C is important
when evaluating obstetric patients. If the
patient has produced anti-G and not anti-D,
then she is considered a candidate for
Rh immune globulin.
- Elaborate adsorption and elution studies,
usually performed in an immunohematology
reference laboratory, may be valuable in these
situations to discriminate anti-D from anti-C
from anti-G.
- For transfusion purposes, it is not necessary to
discriminate anti-D and anti-C from anti-G, as
the patient would receive D-negative and C-
negative blood regardless if the antibody is
anti-D, anti-C, or anti-G.
V. 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 RHCE genes.
- When RBCs phenotype as D– (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–.
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VI. 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 D w) is an
antigenic marker for category Va partial-D.
- Rh30 (also known as Go a or D cor) is a marker
for partial DIVa.
- Rh40 (also known as Tar or Targett) is a
marker for partial DVII.46 Rh52 or BARC is
associated with some partial-DVI types.
VII. Rh33 (Har)
- The low-prevalence antigen Rh33 is most
often found in whites and is associated with
the rare variant haplotype called R0Har.
- 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 RHCE gene.
VIII. 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”).
- The 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.
IX. Rh43 (Crawford)
- Rh43, also known as the Crawford antigen, is
a low-prevalence antigen on a variant Rhce
protein.
 LECTURE
- The Crawford (ceCF) antigen is of very low
prevalence found in individuals of African
descent.
X. e Variants
- an individual may have a phenotype of e-
positive but produce antibodies behaving as
anti-e.
- These variant types result from multiple
mutations in the RHCE gene.
- Individuals who possess two altered RHCE
genes may have a phenotype of e-positive but
produce antibodies behaving as anti-e.
- The Rh antigens hrB (Rh31) and hrS (Rh19)
are rarely encountered in routine blood
banking.
- They are normally present in individuals who
possess normal RhCe or Rhce protein but are
lacking in individuals with normal RhcE or
RhCE proteins (i.e., e-negative).
- Several antigens, most notably hrB and hrS , are
lacking on the Rhce proteins because of a
variant RHCE gene.
- If individuals with these variant genes who are
hrB -negative or hrS -negative are immunized,
they may produce anti-hrB or anti-hrS .
- Individuals with an anti-hrB or anti-hrS require
RBCs that are lacking in the specific antigens,
which are exceedingly rare.
- the variant RHCE gene associated with the hrB -
negative phenotype is usually found with a
variant RHD-CE-D hybrid gene.
- The RHCE is inserted in the middle of the
RHDIIIa gene.
- The resulting protein lacks D antigen but
possesses an unusual form of the C antigen.
- The hybrid gene occurs in 22% of individuals
of African Americans.
- This haplotype that includes the RHDIIIa-
RHCE-DIIIa hybrid gene and variant RHCE
genes is referred to as r’s[(C)ces].
XI. V and VS
- V and VS V and VS are antigens of low
prevalence in the Caucasian population but
are more prevalent among African Americans.
20 | P a g e
- These antigens can be used as predictors of an
individual’s ethnic background because of this
difference in prevalence.
- The V antigen, also historically referred to as
ceS , is found in about 30% of randomly
selected African Americans.
➢ (It is most often found in individuals with
Gly263 amino acid change in Rhce; several
other mutations also occur in this gene.)
- The VS antigen, also known as eS , occurs in
32% of African Americans.
- It results from a single amino acid change that
occurs at position Val245 in the Rhce protein.
- Most hrB -individuals with r’s genotype are
VS+.
- The Val245 mutation in this haplotype is found
in the hybrid RHDIIIa-RHCE RHDIIIa gene
described previously.
- Most V+ individuals are also VS+.
XII. Deletions
- The deletion phenotype is indicated by the use
of a dash (–), as in the following example: D–.
- This phenotype results from individuals
possessing normal RHD gene(s) and hybrid
RHCE-RHD-RHCE in which the Rhce protein is
replaced with RhD.
- This helps explain the exalted D, as there are
extra D antigens recognized on the resulting
hybrid protein.
- The antibody made by D–/D– people is called
anti-Rh17 or anti-Hr0.
- A variation has been recognized within the
deletion D–, called D••.
- The D antigen in the D•• is stronger than that
in DC–, or Dc–, samples but weaker than that
of D– samples.
- A low-prevalence antigen called Evans (Rh:37)
accompanies the Rh structure of D•• cells.
- Some deletion phenotypes are missing E/e
only and are indicated as Dc– or DC–.
- Transfusion of individuals with a D– or D••
phenotype is difficult, as blood of a similar
phenotype or even the rarer Rhnull blood, all
lacking Rh17, would be required.
 LECTURE

LANDSTEINER WIENER BLOOD GROUP SYSTEM

- 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.
- Anti-LW reacts strongly with most D-positive
RBCs, weakly with Rh-negative RBCs (and
sometimes not at all), and never with Rhnull
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.
- Anti-LW shows equal reactivity with cord cells
regardless of their D type.
- This is an important characteristic to
remember when trying to differentiate anti-
LW from anti-D.
- Anti-LW more frequently appears as an
autoantibody, which does not present clinical
problems.

• One method is to treat the reagent panel cells with

0.2 m dithiothreitol (DTT) and test the patient
serum against the treated cells.
• LW antigens are denatured with DTT treatment
while D antigens are unaffected.
• Another strategy is to test the patient serum
against Rh-positive and Rh-negative cord blood
cells.
• Anti-D will react only with the Rh-positive cord
cells whereas the LW antibodies will react with all
cord cells tested, regardless of Rh type.

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