Rh Typing and Weaker Variants of Rh System

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Discovery of D Antigen
The first human example of the antibody against the D antigen
was reported in 1939 by Levin and Stetson.
In 1940 Landsteiner and Wiener described an antibody
obtained by immunizing guinea pigs and rabbits with the red
cells of Rhesus monkey, which agglutinated approximately 85%
of humans tested and the corresponding determinant was called.
Rh Factor
After A, B, O antigen, the D antigen of the Rh system has the
most significant implications in transfusion practice.
The Rh system also contains other clinically significant
antigens (C, E, c and e) and also less significant antigens (variant
forms, cis products and G antigens).
Rh system is most complex genetically of all blood group
types since it involves 45 different antigens on the surface of
red cells. There are two possible alleles at each locus: c or C;
d or D; and e or E.
• According to Fisher and Race system, there are 3 pairs of
genetic alleles, namely Cc, Dd and Ee. One haplotype
consisting of c/C, d/D, e/E is inherited from each parent
and the resulting Rhesus type of the individual depends on
their inherited genotype. Now it is known that d does not
exist and sometimes it is used in Fisher Race terminology to
denote, the absence of D. The most common and important
antigens are D, C, E, c, and e. D is the most immunogenic
and in less than 3% of the individuals with D, C, E, c, and e
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Rh Typing and
Weaker Variants of Rh System
58

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Rh Typing and Weaker Variants of Rh System
59
antigens become immunogenic. Hence pre-transfusion
testing for the other antigens is not routinely performed.
• In 1962, according to Rosenfield, the nomenclature is based
on serological findings. As per Weiner, the Rh antigen
product is from single gene.
• Persons whose red cells lack the D antigen, do not regularly
have the corresponding antibody.
• Formation of anti-D antibodies almost always results from
exposure of D-negative person by transfusion or pregnancy,
to red cells with D antigen.
• The D antigen has greater immunogenicity. D negative
persons, who receive a D positive transfusion, are expected
to develop anti-D antibodies.
Genetics of Rh Blood Group
Despite its genetic complexity, the inheritance of the trait
usually can be predicted by two alleles D. And the Rh locus is
on 1p36.11. The Rh antigens are encoded by two genes RHD
and RHCE. RHD encodes for D antigen, whereas RHCE
encoded for Cc and Ee antigens. The d antigen does not exist
and generally used to denote absence of D antigen. The genes
(RHD and RHCE), both encode similar polypeptides of
417 amino acids with 12 membrane spanning domains.
Individuals who are homozygous dominant (DD) and
heterozygous (Dd) are Rh positive. Those who are homo-
zygous recessive (dd) are Rh negative (i.e they do not have
the Rh antigen).
Serologic Testing for Rh Antigen
• To determine whether a person has genes that encoded D,
C, c, E and e, the red cells are tested with antibody to each of
the antigens.
• If the red cells express both C and c or both E and e, it can be
assumed that the corresponding genes are present in the
individual.
• If the red cells carry only C/c or only E/e the person is
assumed to be homozygous for the particular allele.

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Essentials of Blood Banking and Transfusion Medicine
Routine Rh typing for donors and patients involve only the
D antigen.
Tests for the others Rh antigens are performed only for defined
purpose such as:
a. Identifying unexpected Rh antibodies.
b. Obtaining compatible blood for a patient with an Rh antibody.
c. Investigating disputed parentage.
d. Isolating a panel of phenotypic cells for antibody identifica-
tion.
Procedure
Now, monoclonal anti-D reagents have become widely
available. Slide tests produce optimal results only when a high
concentration of red cells and antibodies are combined at 37°C.
(Refer to chapter on Blood Grouping).
Tube test for Rh testing
1. Place one drop of anti-D serum (polyclonal/monoclonal) in
a clean test tube. (Refer to Fig. 4.6 ABO Blood Grouping and
Rh typing by tube technique.)
2. Add one drop of 2–5% suspension of the red cells to be tested.
3. Mix gently and centrifuge for 30–45 sec at 1000 rpm.
4. Gently resuspend the cell button and examine for agglutina-
tion. Grade the reaction.
Interpretation
• Agglutination in anti-D tube indicates that the red cells in
the tube are D-positive.
• If it has a smooth suspension, it is D-negative.
Matrix gel system for Rh typing: Anti-D (monoclonal IgM
antibodies) gel card for testing Rh-D antigen are in use.
If Rh negative by above methods it has to be further tested for
Du variant and if found negative it has to be labeled as Rh negative.
Weak or partial D
• Weak D expression primarily develops from single point
mutation of RhD gene, reflected in the form of reduced
number of D antigenic sites on the red cell membrane.

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61
• Red cells with partial D lack some antigenic sites on extra-
cellular portion of the RBC membrane.
Du: Weaker variant of D
• Du, the term was coined by Stratton in the year 1946.
• Donors/patient’s with Du phenotype are Rh positive.
• Du antigen is less immunogenic than D antigen and can
cause sensitization when given to D-negative individuals.
• With weak expression of D, most D positive red cells show
clear cut macroscopic agglutination after centrifugation with
reagent anti-D and can really be classified as D positive.
• For some D positive red cells, demonstration of the D antigen
requires prolonged incubation with anti-D reagent or addi-
tion of anti-globulin serum after incubation with anti-D.
• These cells were classified as Du/weak D
• But use of monoclonal anti-D reagents have picked up many
D positive compared to the older reagents.
Significance of weak D in blood transfusion
• Transfusion to D negative recipients with weak expression
of the D antigen, such red cells could elicit an immune
response to D.
• Du patients should receive Rh negative blood.
Du Test Procedure
Weak D expression can be recognized most reliably by anti-
human globulin after incubation of the test red cells with anti-
D. If the original test with anti-D was performed by tube testing,
continue with the procedure.
1. Mix and incubate the tube for 15–30 min at 37°C in water
bath.
2. Centrifuge for 15–60 sec × 1000 rpm.
3. Gently resuspend the cell bottom and examine them for
agglutination.
4. If the test red cells are strongly agglutinated in the anti-D,
record the sample as D positive.
5. If no agglutination, wash the cells for 3 times.

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Essentials of Blood Banking and Transfusion Medicine
6. Add 1 drop of anti-IgG (AHG).
7. Mix centrifuge, view for agglutination under micro-
scope.
Note: Du testing can also be performed with Matrix gel
system.
Rhnull phenotype: This is a rare variant of Rh system, when red
cells do not express Rh antigens due to abnormal RHAG gene,
which results in absence of Rh gene expression, RHD and RhC
genes. These patients have red cell membrane abnormalities
having stomatocytosis and haemolysis.