The Rh blood group system is complex, with over 60 antigens identified. The RhD antigen is the most immunogenic after ABO. Individuals are Rh-positive if they possess the D antigen and Rh-negative if they lack it. Weak D variants exist where D antigen expression is reduced, increasing the risk of alloimmunization. The Rh system was initially thought to involve three genes (D, C, E) but genetic studies show it involves two closely linked genes, RHD and RHCE, as well as an associated glycoprotein gene RHAG. Weak D can arise from position effects, quantitative or partial D antigen expression. Ongoing research continues to uncover the genetic complexity of the Rh system.

1. The Rh Blood
Group System
Course: Adv. Blood Banking
Reference:
Harmening DM (2012). Modern blood banking and transfusion
practices, 7th ed. Philadelphia: F.A. Davis Company. Chap7

2. Introduction
• The term Rh refers to a specific red blood cell
antigen (D) and to a complex blood group
system currently composed of 61 different
antigenic specificities.
• Although Rh antibodies were among the first
to be described, researchers continue to
explore the complexities of the Rh blood
group system from its serology to its mode of
inheritance, genetic control, and the
biochemical structure of the Rh antigens

3. • Rh is the second in importance only to ABO
blood group system in terms of transfusion, as
the Rh system antigens are very immunogenic.
• Unlike ABO antibodies that are routinely
found in individuals who lack the
corresponding antigen, Rh antibodies are
produced only after exposure to foreign red
blood cells.
• Once present, they can produce significant
hemolytic disease of the fetus and newborn
(HDFN) as well as hemolytic transfusion
reactions

4. • The terms Rh-positive and Rh-negative are
used to describe the presence or absence of
the D antigen.
• Rh-positive indicates that an individual’s red
blood cells possess one particular Rh antigen,
the D antigen.
• Rh-negative indicates that the red blood cells
lack the D antigen.

5. History
• In 1939 Levine and Stetson described a hemolytic
transfusion reaction in an obstetrical patient.
• After delivering a stillborn infant, the woman required
transfusions.
• Her husband, who had the same ABO type, was
selected as her donor. After transfusion, the recipient
demonstrated classic symptoms of an acute hemolytic
transfusion reaction (AHTR).
• Subsequently, an antibody was isolated from the
mother’s serum that reacted at both 37oC and 20°C
with the father’s RBCs.
• It was postulated that the fetus and the father
possessed a common factor that the mother lacked

6. • While the mother carried the fetus, she was exposed to
this factor and subsequently produced an antibody
that showed positive reactivity when tested against the
transfused RBCs from the father.
• The antibody specificity was not identified, but it was
found to react with 80% of cells tested and was
suspected to be a maternal antibody to a factor on the
fetal cells.
• A year later Landsteiner and Wiener described an
antibody made by guinea pigs and rabbits when they
were transfused with rhesus macaque monkey RBCs.
• The antibody agglutinated 85% of human RBCs and
was named anti-Rh after the rhesus monkey

7. • A subsequent investigation by Levine and coworkers
demonstrated that the agglutinin causing the hemolytic
transfusion reaction, and the antibody described by
Landsteiner and Wiener appeared to define the same blood
group.
• Many years later it was recognized that the two antibodies
were different.
• However, 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
• By the mid-1940s, five antigens were defined in the Rh
system.
• In the 1980s, molecular testing further defined the
structure of the RH genes. The Rh blood group system
continues to be explored, and at present, over 60 different
Rh antigen specificities have been identified.

8. Terminology
• There are four terminologies used to describe the
Rh system.
• Two are based on postulated genetic theories of
Rh inheritance.
• The third common terminology used describes
only the presence or absence of a given antigen.
• The fourth was established by the International
Society of Blood Transfusion (ISBT) Committee on
Terminology for Red Cell Surface Antigens.

9. • It is important to distinguish between phenotype
and genotype before exploring the Rh
nomenclature.
• The phenotype of a given RBC is 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. A genotype is an individual’s actual
genetic makeup.
• An 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.

10. 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.
• Fisher and Race named the antigens of the
system D, d, C, c, E, and e

12. • 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).
• The combination of genes inherited from one
parent is called a haplotype. For example, if one
parent has the genes D, C, and e, then the
haplotype is written as DCe.
• The pairing of maternal and paternal haplotypes
determines the offspring’s genotype
• The genotype is written as two haplotypes
separated by a / (i.e., DCe/Dce).

13. Nomenclature of the Rh System:
Fisher-Race: The DCE Terminology

16. Fisher-Race: The DCE Terminology
• According to Fisher-Race proposal, each person
inherits a set of Rh genes from each parent (one
D or d, one C or c, and one E or e)
• The combination of the inherited haplotypes
determines one’s genotype and dictates one’s
phenotype
• The designation “d” does not represent an Ag
but the absence of D Ag
• In rare instances, a person may fail to express C
or c, E or e, or CcEe;

17. • The genotype for the Rh + exhibiting a
deletion phenotype may be –De, -DE,
CD- or cD-or -D-
• The person expressing no Rh Ags is said
to be Rhnull phenotype is - -/- - .
• Weakened expression of all Ags also
been reported; Rhmod

18. Wiener: Rh-Hr Terminology
• In his early work defining the Rh antigens, 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. Each
factor is an antigen recognized by an antibody.
• The original Wiener nomenclature named the
five common Rh antigens as Rho, rh’, rh’’, hr’, and
hr’’, but these terms are no longer used in favor
of a modified form of Wiener notation.

21. Wiener: The Rh-Hr Terminology

22. Rosenfield & Coworkers:
Alphanumeric Terminology
• It was proposed in 1960 in which a
number is assigned to each Ag
• DCEce: D is assigned Rh1, C Rh2, E Rh3,
c Rh4, e Rh5
• For the RBCs that type D+, C+, E+, c-, e-,
Rosenfield designation is Rh: 1, 2, 3, -4, -5

31. Proposed Genetic Pathways of the Rh System
• There are two closely linked genes located on
chromosome 1 control the expression of Rh
• RHD gene codes for presence or absence of D
polypeptides
• RHCE gene codes for either RHCe, RHcE, RHce, or
RHCE
• Another gene RHAG that resides on chromosome 6
and produces an Rh-associated GPs that forms
complexes with the Rh polypeptides within the
membrane

32. Proposed Genetic Pathways of the Rh System
• Rh-associated glycoprotein (RHAG gene)
must be present for the successful
expression of the Rh Ags and thus called
a coexpressor gene
• The genes are inherited as codominant
alleles. Offspring inherit one Rh haplotype
from each parent
• Rh Ags are transmembrane polypeptides
and are an integral part of the RBC
membrane traversing it 12 times

40. Weak D: 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.
• However, 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

41. • For many years, the 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.
• Now it is known that weak D can arise from several
mechanisms. Weak D types can be separated into three
categories: position effect, quantitative, and partial-D
antigen (missing one or more alleles).
• It has been shown that up to 2% of individuals with
European ancestry possess some altered form of D
antigen.
• Altered D antigen occurs more often in individuals of
African descent, but the exact prevalence is not known
• Finally, to further complicate matters, there are rare
individuals who possess D epitopes on their RhCE
protein.

42. Weak D or Du Variants
• Rh + cells when typed react strongly with anti-D
reagents
• With certain RBCs, antiglobulin testing is needed to
demonstrate the presence of D Ag. These are called
Du
 Genetic Weak D: The D Ags are complete but low in
number due to mutations in the Rh polypeptides. Rare in
whites but frequent in blacks
 C Trans: The allele carrying D is trans to that carrying C
as in Dce/dCe. This position effect results in weakened
expression of the D Ag
 Partial D (D mosaic): One or more of the D epitopes is
either missing and/or is altered

43. • The anti-D made by individuals expressing
partial D can cause HDNB & HTRxs
• Once anti-D is identified, Rh – is used for
transfusion.
• Del is a phenotype occurring in individuals
whose RBCs 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 cells is often the only way to
detect the D antigen.

44. Determination of D status
• Blood for Transfusion is considered +ve if either the D or the
weak D test is positive
• All Rh –ve results must be confirmed by Du testing????
• For transfusion recipients, the application of the Du test is
controversial. Those with genetic weak D and D trans have
complete D Ag & thus can be transfused safely with Rh +
blood without fear of production of anti-D. On the other hand,
the number of recipients with homozygous D mosaic is so
low and their chance to be sensitized is so low too. This
doesn’t Justify the use of Rh –ve blood for them
• Policy regarding transfusion of weak D recipients is
established individually within each transfusion service

45. • In obstetric patients, determining the D or weak
D status is critical
• All Rh –ve and weak D +ve obstetric patients
are candidates for RhIg (Rh Immune globulin) to
prevent developing anti-D
• When the mother is Rh-ve and the NB is
negative, the weak D status of the NB must be
determined
• When cells are coated heavily with IgG anti-D,
they may give negative results with anti-D.
Elution and identification of the Abs are needed
to verify the Rh status of the NB
Rh Antibodies

46. Rh Antibodies
• Rh antibodies are mostly IgG reacting optimally at 37 C
• They can cross the placenta and cause HDNB. It is often
severe bc the Rh Ags are well developed on fetal cells
• Rh Ags are highly immunogenic: D > c > E > C > e
• Less than 0.1 mL of Rh +ve cells can stimulate the IR
• Rh Abs are predominantly of subclass IgG1 & IgG3.
However, they don’t bind complement!!!!!!!!! Why?
• Rh Abs persists for years. Taking good history is essential
• They appear in primary exposure (IgM) within 120 days
and within 2-7 days after secondary exposure
• In Rh-mediated HTRxs, DAT is usually positive while Ab
screen may be negative

47. Rh Antibodies
• In Rh-mediated HTRxs 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.
• To prevent susceptible (D-negative) mothers from
forming anti-D, 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. No effort has been made to
develop immune globulin C, c, E, e

48. Rh Antigen Typing Reagents
• Saline anti-D (IgM): It is of low-protein based & thus can be
used to type IgG coated RBCs. Disadvantages: Low
availability, costly, long incubation, & not used for Du
• High-protein anti-D: Due to the presence of potentiators and
high protein concentration, it may give false positive results.
Control is a must!!! It requires short incubation and used for
Du and slide method
• Chemically modified IgG anti-D: A disulfide bond is broken. It
is of lower protein content, Used for slide & tube methods and
for Du
• Monoclonal Abs and Blend IgG-IgM anti-D: Can be used for
slide, tube, microwell, automation, & Du testing. They don’t
transmit infectious diseases

49. Rh Antigen Typing Reagents
• Monoclonal Abs and Blend IgG-IgM anti-D:
• D Ag 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.
• Some companies also blend IgM and IgG anti-D
to maximize visualization of reactions at
immediate spin testing and to allow IAT for weak
D antigen with the same reagent

51. Rh Deficiency Syndrome: Rhnull & Rhmod
• The individuals who lack all Rh-Ags are said to
have Rhnull syndrome, produced by two
mechanisms:
1. Regulator-Type: Mutation in the RHAG gene (no
expression) while the RHD & RHCE genes are normal
2. Amorphic-Type: There is a mutation in each of the
RHCE genes and deletion of the RHD gene while the
RHAG gene is normal
These individuals demonstrate a mild compensated mild
HA, retics, stomato, low Hb, low Haptoglobin, increased
Hb F. For transfusion, only Rhnull blood is given
• Rhmod is a severely reduced expression of all Rh
Ags with less severe symptoms due to suppression of the
RHAG gene

52. Unusual and rare phenotypes
• Cw: It is rare and found in 2% of population
• f (ce)
• rh1 (Ce)
• G: present on most D & C positive RBCs. Obstetric
pts with anti-G are candidates for RhIg
• Rh17 (Hr0), 23, 33, 32, ……etc
• V & VS
• E Varients
• Deletion: D- that lacks Ee & Cc

53. blood
is
meant
to
circulate