Transcript The Rh System Immunohematology

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.
Objectives



Evaluate reactions of Rh typing, using
conventional reagents.
Explain the principle of the weak D test.
Discuss situations when weak D testing
would be appropriate.
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.
Presence of D = Rh positive
Absence of D = Rh negative
May be missing the D gene (whites), or
have an amorph at that location (blacks
and other ethnicities).
Inheritance
Genes located on chromosome 1.
 Alleles are co dominant.

Rh Inheritance: Wiener

Wiener proposed
one gene that
produced an
“agglutinogen” with
3 distinct
specificities.
Rh0
Rh1
Rh2
Rhz
rh
rh’
rh”
rhy
Rh Inheritance: Fisher-Race

3 closely linked
genes.


Each responsible for
expression of one
antigen.
The major antigens
of the Fisher Race
system are:



D
C and its allele c
E and its allele e.
D
C or c
E or e
NOTE:
There is no d antigen!
d is used to denote the absence of
D antigen.
Rh Inheritance: Tippett

Using molecular
techniques, Tippett
showed in 1993 that Rh
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).

RHD
RHCE
Haplotypes

Both Wiener and
Fisher-Race
nomenclature
systems propose
haplotypes- genes
that are inherited as
a unit.
 A person inherits one
haplotype from each
parent.
D
Ce
ce
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

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Ro
R1
R2
Rz
r
r’
r”
ry
Fisher Race





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Dce
DCe
DcE
DCE
dce
dCe
dcE
dCE
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
expression of the D
antigen.
 Detected only when
using an indirect
antiglobulin test.
 May stimulate
production of anti-D.

3 main causes of
weak D:
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Inheritance
Gene interaction
Partial D (aka
mosaic)
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
opposite D.

C in trans
position.
D antigen is
normal.
 Fewer antigens
per RBC.

D
C
Weak D – Gene Interaction
Position of C and D
antigens when C is
inherited in cis position

Position of C and D
antigens when C is
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.
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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. (---/---)

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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
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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

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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,
glycosphingolipid or
glycoprotein
Glycoprotein
Ag Location on Cell
Membrane
Outer surface
Transmembrane
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
Antibody Class
IgM (some IgG)
IgG
Natural or
Immune Ab
Ab Activate
Complement?
Reaction phase
Natural
Immune
Yes
No
IS
AHG
Yes
Yes
Clinically
Significant?
Rh
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.
o

ID
D
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Incubate at 37 C for 15 to
30 minutes.
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
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Courtesy Ortho-Clinical Diagnostics Raritan, NJ
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.
You are ready to perform ABO and Rh
determinations in the lab!