1. The Rh System
Immunohematology

2. 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.

3. 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.

4. 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.

5. 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.
The antibody discovered by Levine and Stetson is what we now call anti-D. Landsteiner and Wiener believed that their Rh antibody was the same as Levine and Stetson’s antibody in the OB patient, and for many years, literature used the term “Rh” when referring to what we now call “D”. It was later discovered that the antibody of Landsteiner and Wiener was NOT the same as anti-D, but by that time Rh and D were synonymous. The term Rh is still used when referring to the D antigen to this day. The antibody discovered by Landsteiner and Wiener has been renamed LW in their honor. LW is closely related to the Rh system.

6. 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).

7. Inheritance
Genes located on chromosome 1.
Alleles are co dominant.

8. Rh Inheritance: Wiener
Wiener proposed one gene that produced an “agglutinogen” with 3 distinct specificities.
Rh0 rh
Rh1 rh’
Rh2 rh”
Rhz rhy
One gene product with multiple antigenic determinants.

9. 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
Inherited as a unit. Each gene produces one specific antigen. Genes and antigens share the same name.

10. NOTE: There is no d antigen!
d is used to denote the absence of D antigen.

11. 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
The CE gene locus has the following major alleles: CE, Ce, cE, and ce.

12. 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
The genes pictured represent 2 haplotypes. The one on the left is DCe. The one on the right is dce.

13. Antigens Wiener Fisher-Race Rosenfield Rho D Rh:1 rh’ C Rh:2 rh” E
The Wiener nomenclature is sometimes referred to as Rh-Hr.
The Rosenfield nomenclature system is useful in data entry.

14. Haplotypes Ro R1 R2 Rz r r’ r” ry Dce DCe DcE DCE dce dCe dcE dCE
Wiener
Fisher Race Ro R1 R2 Rz r r’ r” ry
Dce
DCe
DcE
DCE
dce
dCe
dcE
dCE

15. 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.
Approximately 50 antigens in the Rh system.
Each Rh protein runs through the membrane 12 times. Lack of Rh antigens disrupts other membrane proteins.
Over 80% of Rh negative people who are exposed to Rh positive blood will form anti-D.

16. 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)

17. 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.

18. Weak D – Gene Interaction
C inherited on chromosome opposite D.
C in trans position.
D antigen is normal.
Fewer antigens per RBC. D C
This is sometimes termed the positional effect.

19. 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.

20. 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.

21. 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.
Low frequency antigens occur in <10% of the general population.
Anti-G will react with D positive and C positive cells. The specificities cannot be separated.
When two genes are inherited on the same chromosome, we say they are in cis- position.

22. Deletions Deletions – missing all or part of the RHCE gene
E/e “disappears” more frequently (DC -)
C/c “disappears” next (D- -)
The genotype D- - expresses huge amounts of D antigen, since there is no competition from other Rh antigens (15,000 copies vs. 110,000 copies).

23. Rh Null No Rh antigens on the RBC Amorphic: Regulator:
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
The glycoprotein expressed by RHAG forms a complex in the RBC membrane with the other Rh antigens. This is necessary to have normal expression of Rh antigens. (Not a precursor substance!) RHAG is found on chromosome 6.

24. Rh Null Disease Compensated Hemolytic Anemia Stomatocytes
Increased reticulocytes
Increased HGB F
Can only receive red blood cells products from other Rh Null individuals.
Red cells will have shortened survival due to membrane deformity. There will be a slight decrease in H & H. There also may be decreased haptoglobin levels and increased bilirubin levels (from RBC destruction).

25. Antibodies Immune Clinically significant May react at 37oC
React best in antiglobulin phase
Clinically significant
Usually IgG.
Clinically significant antibodies are responsible for decrease red cell survival. Able to cause HTR and HDFN.

26. Antibodies Do not activate complement May show dosage
Enhanced by enzymes
Often appear in combinations
Antibodies that show dosage react better with cells having homozygous antigen expression than cells with heterozygous antigen expression.
Common combinations of Rh antibodies are anti-cE, -Ce, and -DC.

27. 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, lymphocytes, endothelial and epithelial cells, and in secretions
Red cells only
Fully Developed at Birth? No
Yes
Effect of Enzymes
Enhanced

28. 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?

29. Rh Typing
Usually, testing for the D antigen is performed at the same time as the ABO forward grouping.

30. TYPING SERA The Rh typing sera in routine use is anti-D.
Anti-D anti-sera contains antibody to multiple D epitopes.

31. 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.
Commercial Rh-hr control contains everything that is in the anti-D reagent, except the anti-D.

32. 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.
Low protein reagents require a control only when cells agglutinate with all antisera (AB +). The control may be Rh-hr control, 6% bovine albumin or saline.

33. Routine Testing Tube Method
2 - 5% cells in saline ID D ID
Centrifuge at 3500 rpm. Read, grade, record.
Conventional reagents tend to require 2 drops of reagent whereas monoclonal reagents usually require just 1 drop.

34. Weak D D determination may include a test for 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. ID D
In the weak D test, the incubation phase allows the anti-D in the reagent to bind to any D antigen present on the cells. The washing step removes unbound antibody. The addition of the AHG reagent following the washing step forms “bridges” between cells sensitized with anti-D, creating visible agglutination.

35. 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.
Donors of blood, organs and tissues.
Remember that even a weakened form of the D antigen may stimulate production of anti-D. So in infants born to Rh negative mothers, we want to be certain not to overlook a mother who would be a candidate for Rh Immune Globulin, if her infant truly is Rh positive.
Variations in the formulation of anti-D reagent may cause a discrepancy between current results and the patient’s previous history.
Each institution may have additional requirements on when to perform the weak D test.

36. 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.

37. 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.
When determining possible genotypes, remember that you have 2 haplotypes, one from each parent.
In this example:
D has no allele so if a patient is D positive, it is impossible for us to tell if D was inherited from just one parent or both parents. So you have 2 possible genotype combinations; one with D from each parent and the other with D from only one parent.
Of the antithetical pair C/c, only C is present, so both haplotypes must possess C. The same is true for E/e…only e is present so both haplotypes must possess e.
For this example then, we have two possible genotypes: DCe/DCe or DCe/dCe.
It is often easier to speak of genotypes using Wiener terminology. In this example, we have R1R1 or R1r’

38. 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.
Frequencies vary between races. Ro is the most common haplotype in Blacks.
Rz and ry occur in less than 1% of the population.
From our example on the previous slide, R1R1 would be the more probable genotype than R1r’
* In Caucasians

39. ABO & Rh Testing Gel Method
For the forward grouping, a 3-5% suspension of red cells is made in a diluent.
uL of the cell suspension is added to the microtubes containing >A, >B, >D, and a control.
For the reverse grouping, 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

40. You are ready to perform ABO and Rh determinations in the lab!