the rh system immunohematology. objectives compare the three theories of inheritance of the rh...
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The Rh System
Immunohematology
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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.
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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.
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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.
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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.
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Presence of D = Rh positiveAbsence of D = Rh negative
May be missing the D gene (whites), or have an amorph at that location (blacks and other ethnicities).
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Inheritance
Genes located on chromosome 1.Alleles are co dominant.
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Rh Inheritance: Wiener
Wiener proposed one gene that produced an “agglutinogen” with 3 distinct specificities.
Rh0 rhRh1 rh’Rh2 rh”Rhz rhy
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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
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NOTE:There is no d antigen!
d is used to denote the absence of D antigen.
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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
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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
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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
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Haplotypes
Ro
R1
R2
Rz
r r’ r” ry
Dce DCe DcE DCE dce dCe dcE dCE
Wiener Fisher Race
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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.
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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: Inheritance Gene interaction Partial D (aka
mosaic)
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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.
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Weak D – Gene Interaction
C inherited on chromosome opposite D.C in trans
position.D antigen is
normal.Fewer antigens
per RBC.
D
C
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Weak D – Gene Interaction
When C is inherited in trans position, expression of the D antigen is reduced due to steric hindrance from the C antigen.
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
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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.
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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.
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Deletions
Deletions – missing all or part of the RHCE gene E/e “disappears” more frequently (DC -) C/c “disappears” next (D- -)
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Rh NullNo Rh antigens on the RBCAmorphic:
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
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Rh Null Disease
Compensated Hemolytic Anemia
Stomatocytes Increased
reticulocytes Increased HGB F Can only receive red
blood cells products from other Rh Null individuals.
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Antibodies
ImmuneMay react at 37oCReact best in antiglobulin phase
Clinically significant
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Antibodies
Do not activate complementMay show dosageEnhanced by enzymesOften appear in combinations
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ABO vs. Rh
Trait ABO RhAntigen Composition Glycolipid,
glycosphingolipid or glycoprotein
Glycoprotein
Ag Location on Cell Membrane
Outer surface Transmembrane
Ag Location in Body Red cells, platelets, lymphocytes, endothelial and epithelial cells, and in secretions
Red cells only
Fully Developed at Birth? No Yes
Effect of Enzymes Enhanced Enhanced
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ABO vs. Rh
Trait ABO Rh
Antibody Class IgM (some IgG) IgG
Natural or Immune Ab
Natural Immune
Ab Activate Complement?
Yes No
Reaction phase IS AHG
Clinically Significant?
Yes Yes
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Rh Typing
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TYPING SERA
The Rh typing sera in routine use is anti-D.
Anti-D anti-sera contains antibody to multiple D epitopes.
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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.
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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.
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Routine TestingTube Method
ID
D
ID
Centrifuge at 3500 rpm. Read, grade, record.
2 - 5% cells in saline
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Weak D
Incubate at 37oC for 15 to 30 minutes.
Wash with saline (x3) to remove unbound antibody.
Add 2 drops of AHG reagent.
Centrifuge, then read for agglutination.
IDD
D determination may include a test for D determination may include a test for weak D.weak D.
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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.
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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.
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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.
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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
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ABO & Rh TestingGel 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
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You are ready to perform ABO and Rh determinations in the lab!