Rh system; the “Rh factor” became recognized as the D

Rh BLOOD GROUP SYSTEM antigen, now commonly known as Rh(D) antigen.

The clinical significance of the highly immunogenic

RhD antigen was quickly realized, in cases of transfusion
INTRODUCTION: and in the diagnosis and prevention of erythroblastosis
fetalis commonly called hemolytic disease of the fetus
• ISBT Number: 004 and newborn (HDFN).
• Number of antigens: 61
• Genes: RHD, RHCE
• Location of gene: chromosome 1 (locus: 1p36.11) Rh BLOOD GROUP TERMINOLOGIES:
• There are five (5) main Rh red cell antigens that
involve the most clinically significant transfusion There are four (4) terminologies used to describe the Rh
complications: system.
o Rh1: D ○ Rh4: c o Two are based on postulated genetic theories of Rh
o Rh2: C ○ Rh5: e inheritance:
o Rh3: E ▪ Fisher–Race: DCE Terminology
▪ Wiener: Rh–Hr Terminology)
• Rh is the second in importance only to the ABO blood o The third common terminology used describes only
group system in terms of transfusion, as the Rh the presence or absence of a given antigen
system antigens are very immunogenic. ▪ Rosenfield: Alphanumeric Terminology
o The fourth was established by the ISBT
• The terms Rh-positive and Rh-negative are used to ▪ International Society of Blood Transfusion (ISBT)
describe the presence or absence of the Rh(D) Committee on Terminology for Red Cell Surface
antigen, respectively. Antigens.
o Rh-positive: RBCs possess the D antigen
o Rh-negative: RBCs lack the D antigen Fisher–Race: DCE Terminology
• In the mid-1940s, Fisher and Race named the five
• Weak D antigen is a phenotype where the D antigen major antigens of the Rh blood group system—D, d,
is weakly or partially expressed on red blood cells, C, c, E, and e antigens.
and this antigen cannot be detected by routine • Test RBCs are allowed to react with antigen-specific
methods. Rh grouping reagents: Anti–D, Anti–C, anti–E, Anti–
o Weak D as a blood recipient: As recipients, c, and Anti–e.
patients with the weak D phenotype are • They postulated that the antigens of the system were
considered to be Rh(D)-negative, and usually produced by three closely linked genes (D/d, C/c, and
receive only Rh(D)-negative red cells. E/e).
• In the Fisher–Race designation, “D” indicates the
o Weak D as a blood donor: As donors, weak D presence of the Rh(D) antigen, and “d” indicates,
red cells are considered to be Rh(D)-positive when used, the lack of the D antigen. Since there is
because, even though the D antigen is weak, it is no actual “d” antigen, this is often dropped to avoid
present. If weak D red cells were transfused to confusion.
Rh(D)-negative patients, the patients might • According to the Fisher-Race theory, an individual
undergo alloimmunization to produce anti-D. inherits a set of RH genes from each parent (i.e., one
D or d, one C or c, and one E or e).
o The combination of genes inherited from one
HISTORICAL PERSPECTIVE: parent is called a haplotype (i.e., DCE)
• The pairing of maternal and paternal haplotypes
In 1939, Philip Levine and Rufus Stetson described determines the offspring’s genotype (the RH genes
the clinical importance of the Rh blood group system inherited from each parent).
through a case of an immunized pregnant woman. The o The genotype is written as two haplotypes
patient experienced an adverse reaction following the separated by a slash [/] (i.e., DCe/Dce).
transfusion of her husband’s blood after giving birth to a
stillborn infant. Her serum agglutinated about 80% of ABO
Fisher–Race Haplotype Terminology
compatible human red blood cell samples.
Anti–D Anti–C Anti–E Anti–c Anti–e Designation
Shortly after, Karl Landsteiner and Alexander + + 0 0 + DCe
Wiener (1940) described animal studies that discovered 0 + + 0 0 Ce
immune antibodies made in rabbits by injecting blood + 0 + + 0 DcE
from the rhesus monkey reacted with about 85% of 0 0 + + 0 cE
human red blood cells; the same red blood cells that + + + 0 0 DCE
reacted with the antibody made by the woman above.
0 + + 0 0 CE
Believing these observations described the same blood
+ 0 0 + + Dce
group, Landsteiner and Wiener named the Rh blood
0 0 0 + + ce
group after the rhesus monkeys (Macaca mulatta).
Scientists have since learned that the Rh blood group +, positive (agglutination); 0, negative (no reaction)
antigen described by Levine and Stetson is not the same
as the one found in rhesus macaques, and was later
named "LW" in honor of Landsteiner and Wiener. Wiener: Rh–Hr Terminology
• Alexander Wiener developed his own theory and
The immune case reported by Levine and Stetson shorthanded the Fisher-Race terminology scheme.
was called “erythroblastosis fetalis” to describe the fetal • In the modified Wiener nomenclature, an
symptoms that result from the mother’s exposure to an agglutinogen is described by a letter and symbol
antigen absent on her red blood cells but present in her assigned based on the factors present.
husband and inherited by the fetus. Over time, o The uppercase “R” denotes the presence of the
researchers recognized other antigens that make up the D antigen.
 o The lowercase “r” indicates the absence of the Summary of the Major Antigens of the Rh Blood
D antigen. Group System in Four Nomenclatures
o The uppercase “C” denotes the presence of the
C antigen and is indicated by a number 1 or a
Fisher–Race Wiener Rosenfield ISBT Number
single prime (').
o The lowercase “c” denotes the presence of the c D Rho Rh1 004001
antigen and is implied when there is no number 1 C rh' Rh2 004002
or single prime (') indicated. E rh'' Rh3 004003
o The uppercase “E” denotes the presence of the
c hr' Rh4 004004
E antigen is indicated by a number 2 or a double
prime (''). e hr'' Rh5 004005
o The lowercase “e” denotes the presence of the e
antigen and is implied when no number 2 or Summary of the Rh Haplotypes in
double prime ('') is indicated. Three Nomenclatures
o Finally, when both C and E antigens are present,
the letter lowercase “z” (subscript) or “y” Fisher–Race Wiener Rosenfield
(modified)
(superscript) is used.
▪ Rz denotes DCE DCe R1 Rh: 1, 2, -3, -4, 5

Rh-positive
Haplotypes
▪ ry represents CE
DcE R2 Rh: -1, 2, -3, -4, 5
• The Fisher-Race nomenclature may be converted to Dce Ro Rh: 1, -2, 3, -4, -5
Wiener nomenclature and vice versa.
DCE Rz Rh: -1, -2, 3, 4, -5
• In the Wiener nomenclature, as with the Fisher–Race Ce r' Rh: 1, 2, 3, -4, -5

Rh-negative
Haplotypes
designation, there is no designation for the absence
of D antigen – lowercase “d” is often dropped. cE r'' Rh: -1, 2, 3, -4, -5

ce r Rh: 1, -2, -3, 4, 5

Rh(D)-positive Haplotypes Rh(D)-negative Haplotypes
CE ry Rh: -1, -2, -3, 4, 5
R1 = DCe r' = dCe → Ce
R2 = DcE r'' = dcE → cE Summary of the Rh Genotypes in
R0 = Dce r = dce → ce Three Nomenclatures
Rz = DCE ry = dCE → CE
Fisher–Race Wiener Rosenfield
(modified)
Rosenfield: Alphanumeric Terminology
• In the early 1960s, Rosenfield and associates DCe/ce R1r Rh: 1, 2, -3, 4, 5
Rh-positive Genotypes

proposed a system that assigned a number to each DCe/DCe R1R1 Rh: 1, 2, -3, -4, 5
antigen of the Rh system in order of its discovery or
recognized relationship to the Rh system. DCe/DcE R1R2 Rh: 1, 2, 3, 4, 5
DcE/ce R2r Rh: 1, -2, 3, 4, 5
• Each antigen is assigned a number.
DcE/DcE R2R2 Rh: 1, -2, 3, 4, -5
o RH1: D ○ RH4: c
o RH2: C ○ RH5: e Dce/ce Ror Rh: 1, -2, -3, 4, 5
o RH3: E Dce/Dce RoRo Rh: 1, -2, -3, 4, 5

• A minus sign (–) preceding a number designates the DCE/ce Rzr Rh: 1, 2, 3, 4, 5
absence of the antigen. An advantage of this ce/ce rr Rh: -1, -2, -3, 4, 5
Rh-negative Genotypes

nomenclature is that the RBC phenotype is thus Ce/ce r'r Rh: -1, 2, -3, 4, 5
concisely described (i.e., the presence and absence
of Rh antigens). Ce/Ce r'r' Rh: -1, 2, -3, -4, 5
cE/ce r''r Rh: -1, -2, 3, 4, 5
Rosenfield Terminology
cE/cE r''r'' Rh: -1, -2, 3, 4, -5
Phenotype reaction with antisera:
Designation Ce/cE r'r'' Rh: -1, 2, 3, 4, 5
D C E c e
y
CE/ce rr Rh: -1, 2, 3, 4, 5
+ + 0 0 + Rh: 1, 2, -3, -4, 5
0 + 0 0 + Rh: -1, 2, -3, -4, 5
+ 0 + 0 0 Rh: 1, -2, 3, -4, -5
0 0 + + 0 Rh: -1, -2, 3, 4, -5
+ + + 0 0 Rh: 1, 2, 3, -4, -5 GENETICS:
0 + + 0 0 Rh: -1, 2, 3, -4, -5
+ 0 0 + + Rh: 1, -2, -3, 4, 5 • RHD and RHCE are two closely linked genes located
0 0 0 + + Rh: -1, -2, -3, 4, 5 on chromosome 1 that control the expression of Rh
proteins.
+, positive (agglutination); 0, negative (no reaction) o The RHD gene codes for the presence or
absence of the RhD protein.
• If an antigen is not phenotyped (np), its number will o RHCE gene codes for either RhCe, RhcE, Rhce,
not appear in the sequence. or RhCE proteins.
• RHD and RHCE are codominant, which means that
D C E c e Designation all products inherited typically produce antigens
0 + 0 0 np Rh: -1, 2, -3, -4 detectable on RBCs.
• Another gene important to Rh antigen expression is
RHAG, and it resides on chromosome 6. RHAG
controls the expression of the Rh-associated
glycoprotein (RhAG).
• RhAG is termed a co-expressor and must be present
for the successful expression of the Rh antigens.
However, by itself, this glycoprotein does not express
any Rh antigens.
o In rare instances, individuals express no Rh
antigens on their RBCs. These individuals are
said to have the Rhnull phenotype.
BIOCHEMISTRY:
• The products of RH genes are non-glycosylated
proteins. This means no carbohydrates are attached
to the protein.
• RhD and RhCE proteins and RhAG are exclusively on
red blood cells.
o Rh antigens reside on transmembrane proteins
and are an integral part of the RBC membrane
— traverse the cell membrane 12 times.
ANTIGEN CHARACTERISTICS:
• Rh antigens are proteins integral to the RBC
membrane, passing through the RBC wall 12 times.
• The antigens are found exclusively on RBCs, and
are not soluble or expressed on other cells.
• The Rh antigens are well developed at birth.
o Alloimmunization with the D antigen (during
pregnancy) is the most common cause of
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 account
for the majority of problems due to Rh antibodies.
• Alleles:
o D antigen does not have an allele.
o C and c antigens are alleles.
o E is allelic to e.
• The alleles are codominant, therefore specificities
from both haplotypes are expressed as RBC
antigens.
D Antigen:
• Highly immunogenic — the most potent Rh antigen.
• Alloimmunization: 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.
• 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.
D>c>E>C>e
Most immunogenic Least immunogenic
WEAK EXPRESSION OF THE Rh(D) ANTIGEN:
Mechanisms of Weak Expression:
Position Effect: C in Trans to D
• The first mechanism that may result in weakened
expression of D antigen was originally described as a
position effect or gene interaction effect.
• The terms "cis" and "trans" are from Latin, which
means "this side of" and "the other side of,"
respectively.
• The allele carrying RHD is trans (or in the opposite
haplotype) to the allele carrying C.
For example:
Dce/dCe
• This interference with D expression does not occur
when the C gene is inherited in the cis position to
RHD.
For example:
DCe/dce
• Molecular studies would differentiate the two types.
Practically speaking, this is unnecessary because the
D antigen is structurally complete. These individuals
can receive D-positive RBCs with no adverse effects.
Weak D: Quantitative Changes Due to Fewer D
Antigen Sites
• The second mechanism results from the inheritance
of RHD genes that code for a weakened expression
of the D antigen.
• The D antigens expressed appear to be complete but
fewer in number.
Rh Phenotype Del
• Del is 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.
• Adsorbing and eluting anti-D from the individual’s red
blood cells is often the only way to detect the D
antigen.
• This phenotype occurs most often in individuals of
Southeast Asian descent, occurring in up to 30% of
that population. It is rare in Caucasians.
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 complete because one or more
epitopes are missing.
CHARACTERISTICS OF RH ANTIBODIES: CAUSES OF ERROR IN HEMAGGLUTINATION
REACTIONS AND THEIR CORRECTIVE ACTIONS:
• Most commonly found Rh antibodies are considered
clinically significant. FALSE-POSITIVES
Likely cause: Corrective action:
• Most Rh antibodies are IgG immunoglobulins and
react optimally at 37°C or after antiglobulin testing Adjust suspension and
Cell suspension too heavy
(Coombs’ test) in any method used for antibody retype
detection. Wash with warm saline and
Cold agglutinins
retype
• Rh antibodies are usually produced following Test incubated too long or Follow manufacturer’s
exposure of the individual’s immune system to foreign drying (slide) instructions precisely
RBCs, through either transfusion or pregnancy — as
Use saline-washed RBCs,
a result of Rh(D) alloimmunization, anti-D Rouleaux
and retype
alloantibody is formed.
Use saline-washed RBCs,
Fibrin interference
• Rh antibodies may show dosage, reacting and retype
preferentially with RBCs possessing double-dose Rh Contaminating low- Try another manufacturer’s
antigen. For example, anti-E may show 3+ positive incidence antibody in the reagent or use a known
reactivity with E+e– RBCs versus 2+ positive reagent serum antibody
reactivity with E+e+ RBCs.
Polyagglutination
o In blood banking, the dosage effect is a Bacterial contamination of Open new vial of reagent
significant difference in antibody reaction reagent vial and retype
depending on the quantity of the target antigen Repeat test; read vial label
present on a target red blood cell. Incorrect reagent selected
carefully
• In addition, Rh antibodies are enhanced when testing Repeat test using shorter
Centrifugation too long
with enzyme-treated RBCs. centrifugation time
Rpm (revolutions per Repeat test using lower
• IgG1, IgG2, IgG3, and IgG4 subclasses of Rh minute) too high rpm
antibodies have been reported.
o IgG1 and IgG3 are of the greatest clinical
significance because the reticuloendothelial
system rapidly clears RBCs coated with IgG1 and FALSE-NEGATIVES
IgG3 from the circulation — as in the case of
extravascular hemolysis. Likely cause: Corrective action:
o IgA Rh antibodies have also been reported but Immunoglobulin-coated Use saline-active typing
are not routinely tested for in the blood bank. cells (in-vivo) reagent
Saline-suspended cells
• Rh antibodies are less effective (than IgM) in binding (slide)
Use unwashed cells
complement. For complement to be fixed (or the
complement cascade activated), two IgG Failure to follow
Review directions and
immunoglobulins must attach to an RBC antigen in manufacturer’s directions
repeat test
close proximity to each other. precisely
Omission of reagent Always add reagent first
o Therefore, when an Rh antibody coats the RBCs, manufacturer’s directions before adding cells
intravascular, complement-mediated hemolysis
does not occur. Resuspension too vigorous Resuspend all tubes gently
Read vial label carefully
Incorrect reagent selected
and repeat testing
Refer sample for further
Variant antigen
investigation
Reagent deterioration Open new vial
Repeat test, read vial label
Incorrect reagent selected
carefully
Repeat test using longer
Centrifugation too short
Rh Hemolytic Disease of the Fetus and Newborn: centrifugation time
• Because Rh antibodies are primarily IgG and can Repeat test using higher
traverse (cross) the placenta and because Rh Rpm too low
centrifugation speed
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; in HDFN, the coated fetal cells are
removed prematurely from the fetal circulation which
can result in anemia.
TESTING FOR RH ANTIGENS/ANTIBODIES:
Rh TYPING REAGENTS:
Types: high-protein-based, low-protein-based, saline-
reactive, chemically modified, monoclonal, or blends of
monoclonal reagents.
SALINE-REACTIVE REAGENTS:
▪ Contain IgM; was the first typing reagent
available to test for the D antigen.
▪ Advantage: low-protein-based and can be used
to test cells that are already coated with IgG
antibodies, as in patients who have
autoantibodies binding to their RBCs.
▪ Disadvantages: 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.
HIGH-PROTEIN-BASED REAGENTS:
▪ Developed in the 1940s; consisted primarily of
polyspecific IgG anti-D (can recognize multiple
epitopes on the D antigen).
▪ Potentiators added: bovine serum albumin
(20% to 24% BSA) and dextran – to optimize
reactivity in the standard slide and rapid tube
tests to allow for direct agglutination of Rh-
positive RBCs (brings antibodies and RBCs in
closer proximity).
▪ Major advantages: reduced incubation time and
ability to perform weak D testing and slide typing
with the same reagent.
▪ Disadvantage: they are reliable, but the
presence of potentiators and higher protein
concentration increases the likelihood of false-
positive reactions.
CHEMICALLY MODIFIED REAGENTS:
▪ This reagent alters the IgG anti-D molecule by
breaking the disulfide bonds that maintain the
antibody’s rigid shape.
▪ Advantages:
▪ It can be used for both slide and tube
testing and does not require a separate,
manufactured Rh control as long as the
samples phenotype as group A, B, or O.
o ABO phenotype AB, Rh-
positive: separate saline control
or 6% to 8% albumin controls
are used to ensure the observed
reactions are true agglutination
reactions.
▪ Low-protein content; thus, fewer false-
positive reactions are obtained.
▪ Because of its lower-protein base and
ready availability, the chemically
modified anti-D replaced the need for
saline (IgM) anti-D reagents.
▪ Because these reagents are not human-
derived, they lack all potential for
transmitting infectious diseases.
▪ Disadvantage:
▪ Narrow range of specificity (Rh antigens
have multiple epitopes), but blends of
monoclonal anti-D reagents are now
commercially available.
Rh TESTING REQUIREMENTS FOR
PRETRANSFUSION, OBSTETRIC PATIENTS, AND
BLOOD DONORS
▪ BOTH ABO and Rh blood groups must be
performed on all blood donors (and collected blood
units) and blood transfusion recipients, and obstetric
patients.
▪ For BLOOD DONORS:
o It is mandatory to include the Rh (D) testing
because the D antigen is highly immunogenic.
o An initial Rh-negative test result must be further
investigated for the presence of a weak D
antigen.
o Red cells that have the weak D phenotype
should, for most transfusion purposes, be
regarded as Rh-positive.
▪ For BLOOD RECIPIENTS/PREGNANT WOMEN:
o Patients are tested for the D antigen in
combination with the ABO typing.
o Rh-negative and Weak D phenotype as a
blood recipient: As recipients, patients with the
weak D phenotype are considered to be Rh-
negative, and usually receive only Rh(D)-
negative red cells.
o Giving Rh-negative RBCs to Rh-positive patients
is not a safety risk.
o It is not required that a weak D test be
performed because even if the patient truly was
a weak D phenotype.
o Pregnant women are tested for the D antigen to
see if they are candidates for Rh-immune
globulin.

MONOCLONAL REAGENTS:
▪ Monoclonal: produced from a single clone of
antibody-producing cells.
▪ Hybridoma technology: plasma cells are fused
(hybridized) with myeloma cells to increase their
reproduction rate, thereby maximizing their
antibody-producing capabilities.
▪ Advantages:
▪ Can be used for multiple modalities
(slide, tube, microwell, column
agglutination)
▪ IgG and IgM blend now available: to
maximize visualization of reactions at
immediate spin testing and allow indirect
antiglobulin testing for weak D antigen
with the same reagent.