PRACTICAL ASPECTS OF RH GROUPING
Rh grouping in routine use for donors and patients
involves testing for Rh (D) antigen only
However tests for other important Rh antigens e.g.
C,c,E and e may be done for Rh genotyping.
The method of Rh grouping mainly depends on the
type of reagents available, for which the
manufacturers’ instructions have to be strictly
REAGENTS FOR RH (D) GROUPING
I Polyclonal human anti-D serum (IgG)
Potentiating or enhancing substances such as
albumin, enzymes and AHG reagent are used to
bring about agglutination with human IgG anti-D.
Anti-D serum (IgG) for saline or rapid tube test
(high protein medium) This contains
macromolecular additives and give reliable results.
Anti-D for saline tube test 2 types
Anti-D IgM Anti-D IgG - Chemically modified
II MONOCLONAL ANTIBODIES
IgM anti-D monoclonal reagent
IgM and IgG anti-D monoclonal reagent
Blend of IgM monoclonal + IgG polyclonal reagent
These antibodies are highly specific, react equally well
at 20°C as well as 37°C and are reliable for slide
and rapid test tube technique.
IgM anti-D monoclonal reagent cannot be used for Du
testing by indirect antiglobulin test (IAT) while IgM
+ IgG monoclonal reagent and blend of IgM
monolconal and IgG polyclonal can be used for Du
III CONTROLS FOR RH (D) GROUPING
Known 0 Rh (D) positive and 0 Rh (D) negative cells
may be used as controls with monoclonal anti-D
Alternatively, AB serum or diluents control
provided with the anti-D reagent or 22% bovine
serum albumin may be used as negative control
with the test cells
RH (D) GROUPING
In most of the blood transfusion laboratories,
Rh (D) grouping is performed along with the ABO
grouping and same techniques as used for ABO
grouping may also be employed for Rh typing
This technique may be used in emergency Rh (D)
typing if a centrifuge is not available.
The slide test is not recommended for routine test
as it may not pick up weak reactions, thus giving
a. Saline Agglutination test for Rh (13) Typing
1. Prepare 2-5% washed red cell suspension of test sample.
2. Place 1 drop of anti-D in cleaned tube labelled D.
3. Place 1 drop of 22% bovine albumin/control reagent in another tube
4. Add 1 drop of 2-5% test cell suspension to each tube.
5. Mix well and centrifuge at 1000 rpm for 1 minute (in case of using IgG
anti-D, incubate at 37°C for 10mm. and centrifuge (spin tube method) or
incubate at 37°C for 60 minutes (sedimentation method).
6. Resuspend the cell button and look for agglutination. All negative
results must be confirmed under microscope
Positive test : Agglutination in anti-D and smooth suspension in control
Negative test : Smooth suspension in all the tubes (test and control)
Test is considerable invalid if both test and control tubes show a
In such case, the test should be repeated using saline IgM anti-D.
For all microscopically negative reactions in donor grouping, Du testing
should be performed, hereas some workers suggest that if the two anti
D reagents used are potent and specific, it is not necessary to perform
ALBUMIN TECHNIQUE FOR RH (D) TYPING
Albumin increases the dielectric constant of the
medium and thus reduces the zeta potential. Due to
this effect, the electrical repulsion between the red
blood cells is less and the cells agglutinate. Mostly
22% bovine albumin is used, as higher concentrations
can cause rouleaux formation.
ENZYME AGGLUTINATION TECHNIQUE FOR RH
Proteolytic enzymes such as papain, trypsin, bromelain
and ficin digest the cell membrane partially and
expose Rh antigens to react with IgG antibodies.
When the membrane is partially removed, it brings
about a loss of negative electric charge on the red
cell which is responsible for keeping the cells a set
distance apart, hence small IgG molecules are able to
span the gap between cells and bring about
RH(D) GROUPING IN HAEMOLYTLC DISEASE OF
In haemolytic disease of the newborn, the baby’s red cells may
be coated with immunoglobulin and a saline reactive Rh
antiserum is usually necessary for testing.
When the cells are heavily coated with antibody, no free
antigenic sites remain for reaction, resulting in a negative
test. This is suspected when the infant’s cells show a positive
direct antiglobulin test (DAT) and a negative test with anti-D
In such instances it is recommended that the antibody should
be eluted by gentle elution (heating at 45°C for 30 minutes) to
expose the antigenic sites before testing.
Rh –ve persons mistyped, & transfused with Rh +ve
blood have 70% chance of becoming immunized
False +ve reactions can be identified by testing an Rh
control with the patient’s red cells each time an Rh
typing is performed
CAUSES OF FALSE POSITIVE REACTIONS
1. Positive direct antiglobulin test
2. Polagglutinable red cells
3. Cold agglutinins or Rouleaux formation
1- POSITIVE DIRECT ANTIGLOBULIN TEST
The presence of Ab on patient’s red cells can cause
a false +ve reaction with slide and tube anti-D
High protein medium reduces zeta potential
allowing red cells to move closer
The cell bound Ab can cross link cells and cause
2- POLAGGLUTINABLE RED CELLS
Rh –ve red cells that are polyagglutinable due to T
or Tn activation
Agglutination occurs if anti-T or anti-Tn present in
the anti-D reagent
Most anti-D reagents do not contain these
3- COLD AGGLUTININS OR ROULEAUX FORMATION
Rh typing is performed using serum suspended red
If individual’s serum contains cold agglutinin or
abnormal protein, false positive reactions can
False negatives are not readily identifiable, but can
occur in the following instances:
The most common is the result of too heavy cell
suspension due to too many cells for the amount of
antibody in the antisera.
They may also rarely be caused by extremely strong
positive DAT. In this case a the patient's D antigen sites
are coated in vivo and there are no sites left for
commercial anti-D to attach to. This can be fixed by
heating cells gently to elute off antibody without
damaging cells, then re-test.
RESOLVING RH PROBLEMS
Erroneous Results in Rh Grouping
1. Perform clerical checks for validity of labels and requisition
2. Obtain a fresh blood sample of patient.
3. Check patient rçcords for history, diagnosis, pregnancy,
medication and previous transfusion.
4. Check equipment and reagents for proper quality control.
5. Check the antisera and controls.
6. Perform alternative procedures such as washing of red cells with
warm saline, enzyme treatment of red cells, absorption / elution