RH System PP

The Rh System
Immunohematology
Objectives
 Compare the three theories of
inheritance of the Rh antigens.
 List the antigens and antibodies of
the system using both Wiener and
Fisher-Race nomenclature.
 Convert haplotype from Fisher-Race
nomenclature into Wiener, and vice
versa.
Objectives
 Discuss key characteristics of

antigens and antibodies in the Rh
system.
 Compare major characteristics of
the Rh system to the ABO system.
 List the three theories of the weak D
antigen.
DISCOVERY
 1939 – Levine and Stetson working with a
woman with a fetus was suffering from
Hemolytic Disease of the Fetus and
Newborn (HDFN).
 1940 – Landsteiner and Wiener working
with guinea pigs and rabbits that had
been injected with red cell from Rhesus
monkeys.
 Source of term “Rh factor”.
 85% of human red cells were agglutinated by this
antibody.
Inheritance
 Genes located on chromosome 1.

 Alleles are co dominant.
Rh Inheritance: Wiener
 Wiener proposed
one gene that Rh0 rh
Rh1 rh’
produced an Rh2 rh”
“agglutinogen” with Rhz rhy
3 distinct
specificities.
Rh Inheritance: Fisher-Race
 3 closely linked
genes. D
 Each responsible for C or c
expression of one E or e
antigen.
 The major antigens
of the Fisher Race
system are:
 D
 C and its allele c
 E and its allele e.
NOTE:
There is no d antigen!
d is used to denote the absence of
D antigen.
Rh Inheritance: Tippett
 Using molecular
techniques, Tippett RHD
showed in 1993 that Rh RHCE

inheritance comes from 2
genes.
 One gene controls
production of the D antigen
(RHD).
 The second gene controls
production of C/E antigen
combinations (RHCE).
Haplotypes
 Both Wiener and
D
Fisher-Race
Ce ce
nomenclature
systems propose
haplotypes- genes
that are inherited as
a unit.
 A person inherits one
haplotype from each
parent.
Antigens
Wiener Fisher-Race Rosenfield
Rho D Rh:1
rh’ C Rh:2
rh” E Rh:3
hr’ c Rh:4
hr” e Rh:5
Haplotypes
Wiener Fisher Race
 Ro  Dce
 R1  DCe
 R2
 DcE
 DCE
 Rz
 dce
 r
 dCe
 r’
 dcE
 r”
 dCE
 ry
Antigens
 Integral transmembrane proteins.

 Found only on red cells.
 Expression is enhanced with enzyme
treatment.
 May show variability of expression.
 Well developed at birth.
 D is highly immunogenic.
Weak D
 Weakened  3 main causes of
expression of the D weak D:
antigen.  Inheritance
 Detected only when  Gene interaction
using an indirect  Partial D (aka
antiglobulin test. mosaic)
 May stimulate
production of anti-D.
Weak D - Inheritance
 Associated with inheritance of Ro.

 More commonly seen in Blacks.
 The D antigen is normal, but decreased
amounts of D antigen is found on the
RBCs.
Weak D – Gene Interaction
 C inherited on
chromosome D
opposite D. C
 C in trans
position.
 D antigen is
normal.
 Fewer antigens
per RBC.
Weak D – Gene Interaction
Position of C and D Position of C and D

antigens when C is antigens when C is
inherited in cis position inherited in trans position
 When C is inherited in trans position,

expression of the D antigen is reduced due
to steric hindrance from the C antigen.
Weak D – Partial D
 Formerly known as
the mosaic model.
 Portion of D antigen
is missing.
 Patient can make
anti-D directed at
portion of antigen
that is missing.
Other Rh Antigens
 Cw – Low frequency antigen related to
C/c.
 G – Found on cells that are positive for
either C or D.
 Anti-G reacts as if it were anti-D plus anti-C.
 ce – Compound antigen.
 Formed when c and e are inherited on the
same chromosome.
 Reacts with anti-f.
Deletions
 Deletions – missing all or part of the

RHCE gene
 E/e “disappears” more frequently (DC -)
 C/c “disappears” next (D- -)
Rh Null
 No Rh antigens on the RBC
 Amorphic:
 Both parents have one haplotype that is a total
Rh deletion, for example Dce/---
 Each parent passes the deletion on to the
offspring. (---/---)
 Regulator:
 Rh-associated glycoprotein gene (RHAG)
missing
 RHD and RHCE are normal
 Creates a deformity in the RBC membrane
leading to Rh Null Disease
Rh Null Disease
 Compensated
Hemolytic Anemia
 Stomatocytes
 Increased
reticulocytes
 Increased HGB F
 Can only receive red
blood cells products
from other Rh Null
individuals.
Antibodies
 Immune
 May react at 37oC
 React best in antiglobulin phase
 Clinically significant
Antibodies
 Do not activate complement

 May show dosage
 Enhanced by enzymes
 Often appear in combinations
ABO vs. Rh
Trait ABO Rh
Antigen Composition Glycolipid, Glycoprotein
glycosphingolipid or
glycoprotein
Ag Location on Cell Outer surface Transmembrane
Membrane
Ag Location in Body Red cells, platelets, Red cells only
lymphocytes,
endothelial and
epithelial cells, and in
secretions
Fully Developed at Birth? No Yes
Effect of Enzymes Enhanced Enhanced
ABO vs. Rh
Trait ABO Rh
Antibody Class IgM (some IgG) IgG
Natural or Natural Immune

Immune Ab
Ab Activate Yes No
Complement?
Reaction phase IS AHG
Clinically Yes Yes

Significant?
Rh Typing
TYPING SERA
 The Rh typing sera

in routine use is
anti-D.
 Anti-D anti-sera
contains antibody to
multiple D epitopes.
TYPING SERA
 Originally,
anti-D was in a high protein
medium that would cause
spontaneous agglutination in patients
whose cells were coated with antibody
(Positive DAT).
 A protein control (Rh-hr control) was run
in parallel on the patient’s cells.
TYPING SERA
 More commonly used today is an anti-D

in a low protein medium, which does not
cause spontaneous agglutination, and
therefore does not routinely require a
protein control.
 Saline based anti-D has also been used
to avoid problems with spontaneous
agglutination.
Routine Testing
Tube Method
2 - 5% cells
in saline
ID ID
D
Centrifuge at
3500 rpm.
Read, grade,
record.
Weak D
D determination may include a test for
weak D.
 Incubate at 37 C for 15 to
o
ID 30 minutes.
D
 Wash with saline (x3) to
remove unbound antibody.
 Add 2 drops of AHG
reagent.
 Centrifuge, then read for
agglutination.
Populations Requiring the Weak D Test
 Donors.
 Rh negative infants born to Rh negative
mothers.
 Any one who historically was typed as
Rh positive, but currently is typing as Rh
negative.
TYPING SERA
 Other Rh typing serum
includes anti-C, anti-E,
anti-c and anti-e.
 These may be high
protein reagents
(requiring a protein
control) or monoclonal
reagents.
Phenotype/Genotype
 Phenotype: Type for presence of D, C,
c, E and e antigens.
 Determine most probable genotype
based on phenotype results.
 Example: A patient’s phenotype is
D+, C+, c 0, E 0, e+
Determine the possible genotypes.
Phenotype/Genotype
 R1, r and R2 are the most common

haplotypes.*
 Ro r’ and r” are “mid-range” in frequency.
 Rz and ry are rare.
* In Caucasians
ABO & Rh Testing
Gel Method
 For the forward grouping, a 3-5%
suspension of red cells is made in a
diluent.
 10-12.5 uL of the cell suspension is
added to the microtubes containing
>A, >B, >D, and a control.
 For the reverse grouping, 50 uL of
a 0.8% suspension of A1 and B cells
is added to the buffered gel
microtubes, along with 50 uL of
patient’s serum or plasma.
 The reaction card with the
microtubes is centrifuged for 10
minutes.
 Read the card for agglutination.
Courtesy Ortho-Clinical Diagnostics Raritan, NJ
You are ready to perform ABO and Rh
determinations in the lab!

RH System PP

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RH System PP

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The Rh System

 Discuss key characteristics of

 Genes located on chromosome 1.

showed in 1993 that Rh RHCE

Wiener Fisher-Race Rosenfield

 Integral transmembrane proteins.

 Associated with inheritance of Ro.

Position of C and D Position of C and D

 When C is inherited in trans position,

 Deletions – missing all or part of the

 Do not activate complement

Natural or Natural Immune

Clinically Yes Yes

 The Rh typing sera

 More commonly used today is an anti-D

 R1, r and R2 are the most common

 Rz and ry are rare.

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