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 positiveAbsence 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 rhRh1 rh’Rh2 rh”Rhz 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

Ro

R1

R2

Rz

r r’ r” ry

Dce DCe DcE DCE dce dCe dcE dCE

Wiener Fisher Race

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: 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

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

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 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

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

ImmuneMay react at 37oCReact best in antiglobulin phase

Clinically significant

Antibodies

Do not activate complementMay show dosageEnhanced by enzymesOften appear in combinations

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

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

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 TestingTube Method

ID

D

ID

Centrifuge at 3500 rpm. Read, grade, record.

2 - 5% cells in saline

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.

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 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

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