Transcript Weak D

Practical
Blood Bank
We a k D t e s t i n g ( D u )
Weak expression of the RhD antigen (Du)
 The term DU is widely used to describe cells
which have :
 a quantitative reduction in the expression of their
RhD antigen.

Or qualitative variation in RhD antigen expression,
these are referred to as partial D.
 There are 5 phenotypes D (D+, D-, Weak D,
partial D)
Weak D:


all D antigen epitopes are present but are underexpressed
It is typically caused by a single amino acid switch in the
transmembrane region of the RhD protein.
Partial D

some D antigen epitopes are missing
Rhnull phenotype

The Rh antigens are thought to play a role in maintaining
the integrity of the RBC membrane -The absence of the
Rh complex alters the RBC shape, increases its osmotic
fragility, and shortens its lifespan, resulting in a hemolytic
anemia that is usually mild in nature.

Rh antigens may also be involved in the transport of
ammonium across the RBC membrane
When Du Test should be done
1. When weak or 1+ reactions are found. Microscopic
readings should only be done if mixed field
agglutination* is suspected.
2. When Rh typing discrepancies are found between
current and previous results.
3. When Rh negative neonates born to Rh negative
mothers. If the weak D testing is positive, the neonatal
Rh type would be reported as "D positive" and the
mother would be a candidate for Rh Immune Globulin
(RhIG).
PRINCIPLE
 To test for a weak expression of the D antigen.
 Red cells that react weakly or not at all in direct
agglutination tests with anti-D may react with anti-D by
the indirect antiglobulin test (IAT).
 Red cells that fail to react 2+ in direct agglutination tests
with anti-D are incubated with anti-D at 37° C and
examined for agglutination. The red cells are washed to
remove unbound antibody (IgG anti-D), then tested with
anti-IgG.
Specimens

Clotted or anticoagulated whole blood
Reagents & Equipments
o
o
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37oC incubator
Wash bottle with normal saline
Coombs serum - either polyspecific or anti-IgG
Coombs control cells (IgG coated control cells ).
All reagents, equipment, and supplies used in the Rh testing
procedure
Recommended techniques
SLIDE TECHNIQUE
1. Add to a clean, labeled slide:
 One drop of anti-D IgM and IgG blend.
 One drop of the test red cells suspension.
2. Mix well by gently and continuously rocking the
slide for approx. 30 seconds and incubate the slide
for 5 minutes at room temperature, with occasional
mixing, we can put the slide on source of light as
heat source (Box Lamp)
3. Examine macroscopically for agglutination. A
diffuse light source may aid reading.
2. TUBE TECHNIQUE – IMMEDIATE SPIN
1. Prepare a suspension of test washed red cells 2-3%
or 1.5 - 2% in LISS.
2. Place in a small, labelled test tube:
 1 volume of anti-D IgM & IgG blend.
 1 volume of suspended red cells.
3. Mix well.
4. Centrifuge immediately for 10 seconds at 1000g or
for a suitable alternative force and time.
5. Agitate the tube gently to dislodge the cell button
and examine macroscopically for agglutination.
6. Apparently negative tests which are to be tested
for DU should be further tested by the DU test
method.
3. TUBE TECHNIQUE – LISS
1. Place in a small, labelled test tube:
 1 volume of Anti-D blood grouping reagent
 1 volume of red cells suspended 1.5-2% in LISS.
2. Mix well and incubate for 15-20 minutes at 37°C.
3. Centrifuge immediately for 10 seconds at 1000g or
for a suitable alternative force and time.
4. Agitate the tube gently to dislodge the cell button
and examine macroscopically for agglutination.
4. DU TEST METHOD
(indirect antiglobulin test (IAT). )
After reading the immediate spin results, re-incubate
the test for a further 20 minutes at 37°C before
completing the DU test method described below.
OR
 After reading the LISS tube test, complete the DU
test, without further incubation, following the
procedure given below.

DU TEST METHOD
1.
Wash the test 4 times with a large excess of PBS (e.g. 4 ml
of PBS per 12 (or 10) x 75mm glass tubes).
NOTE: Allow adequate spin time to sediment the red cells
2.
3.
4.
5.
6.
make sure that most of the residual saline is removed at
the end if each wash to leave a ‘dry’ cell button.
Add two drops of anti-human globulin reagent to each
tube.
Mix thoroughly.
Centrifuge at 1000g for 10 seconds or for a suitable
alternative force and time.
Agitate the tube gently to dislodge the cell button and
examine macroscopically and microscopically for
agglutination.
Add 1 drop of IgG-coated control cells to the tube(s)
with negative results. Centrifuge, resuspend cells, read
macroscopically and record results. Agglutination (2+)
shall be present or the test shall be repeated.
Procedural Notes

Tests
should
be
read
immediately
after
centrifugation. Delay may cause bound IgG to
dissociate from red cells and either leave too little
IgG to detect or neutralize AHG reagent causing
false negative results.
Interpretation


A negative result in the immediate spin phase but
agglutination in the D tube following incubation
(with no agglutination in the DC tube) indicates a
positive test for weak D.
Lack of agglutination is a negative test and the
patient is considered truly D negative. Agglutination
in the DC tube invalidates the test.