PATHOLOGY AND LABORATORY MEDICINE
DIVISION OF TRANSFUSION MEDICINE
STANDARD WORK INSTRUCTION MANUAL
Weak D Typing
Approved By: Dr. Antonio Giulivi
Date Issued: 2004/04/05
Date Revised: 2009/12/31
1.0
Document No: RT.003
Category: Routine Testing
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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 a grade 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.
2.0
Scope and Related Policies
2.1
When testing potential blood component recipients, the test for
weak D is unnecessary.9.1
2.2
Testing for weak D should be performed on infants who type as Rh
negative if their mother is Rh negative and has no evidence of Rh
alloimmunization.9.1 Also for infants whose mother’s blood type is
unknown.
2.3
Obstetrical patients that type as Rh positive or weak D positive
should be designated as Rh positive. Patients whose red cells type
as Rh negative should be designated as Rh negative.
2.4
For apparent Rh negative obstetrical patients testing for weak D
may be performed as per facility policy.
2.5
A control system, appropriate to the anti-D reagent in use, shall be
included. If this control is positive, the Rh typing must be repeated
with an appropriate anti-D reagent and control.9.1
2.6
If an Rh typing problem is detected and transfusion is necessary
before resolution, for pre-menopausal females and children Rh
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Weak D Typing
negative blood products must be issued until the problem is
resolved. Other patients, in the absence of a known anti-D, may
receive Rh positive blood in emergency situations when there is a
shortage of Rh negative blood.
3.0
Specimens
EDTA anticoagulated whole blood
4.0
Materials
Equipment: Cell washer
Serologic centrifuge
Block for test tubes
Waterbath/Heating block at 37° C
Microscope
5.0
Supplies:
Test tubes – 10 x 75 mm
Serologic pipettes
Reagents:
Anti-D reagent
Control for Rh typing (suitable to anti-D reagent used)
Anti-IgG
IgG coated control cells
Normal Saline
Quality Control
5.1
See QCA.001 – Quality Control of Reagent Red Cells and Antisera.
5.2
Before performing and reporting a “weak D typing” an accurate
transfusion and pregnancy history should be obtained.
5.3
Test for weak D should not be performed in the following situations:
5.3.1 If the direct antiglobulin test (DAT) is positive on the red cells
tested.
5.3.2 If the patient has been transfused within the last three
months with cellular donor unit(s) of different Rh type.
5.4
Test for the weak D antigen should be done in the following
situations:
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Weak D Typing
5.4.1 When weak or grade 1 reactions are found. Microscopic
readings should only be done if mixed field agglutination is
suspected.
5.4.2 When Rh typing discrepancies are found between current
and previous results.
5.4.3 On 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).
6.0
5.5
If the test for weak D is performed on an obstetrical patient and
result is weakly positive and/or mixed field, there must be an
investigation to determine if the result is due to a large fetomaternal hemorrhage. If a feto-maternal hemorrhage had occurred
the test for weak D cannot be interpreted. The patient’s physician
must be notified.
5.6
For other causes of mixed field refer to NRT.004 – Rh Type
Problem Solving.
Procedure
6.1
Check the suitability of the specimen(s) to ensure that the
specimen label information matches the request form. PA.002 –
Determining Specimen Suitability steps 6.1 – 6.4.
6.2
Perform a patient history check. See PA.003 – Patient History
Check.
6.3
Label tubes as per established procedure. See PA.004 – Labelling
of Test Tubes and Block Set Up for Compatibility Testing.
6.4
Add the appropriate reagent to the labelled test tubes according to
the manufacturer’s insert.
6.4.1 Add 1 drop of anti-D to tube labelled D.
6.4.2 Add 1 drop of control reagent to tube labelled DC.
6.5
Check the specimens for abnormal appearance. See PA.002 –
Determining Specimen Suitability step 6.5.
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Weak D Typing
6.6
Compare the patient name or identification number on each tube
with the corresponding information on the request form (or
computer screen) to ensure they are the same.
6.7
Prepare a 3% patient red cell suspension. It is not necessary to
pre-wash the red cells, however, if a discrepancy is found the cells
should be washed and the tests repeated.
6.7.1 Label a test tube with the first 3 letters of the patient’s family
name; transcribe this from the specimen tube, not from the
request form. A pre-printed label may be used (ensure the
information coincides exactly with the information on the
specimen label).
6.8
Add 1 drop of the 3% patient red cell suspension to the
corresponding tubes labelled D and DC.
6.9
Mix and centrifuge tubes at 3400 rpm for 10-15 seconds.
6.10
Grade and record results. See PA.006 – Reading and Recording
Hemagglutination Reactions. If the cells are strongly agglutinated
(grade 2 or greater) in the anti-D tube and negative in the control
tube, record the interpretations as “D positive” and do not proceed.
6.11
Mix and incubate the tubes at 37° C for 15 minutes.
6.12
Check and record the temperature of the waterbath or heating
block on form QCA.006F.
6.13
After 15 minutes of incubation, if a 37° C reading is desired:
6.13.1 Centrifuge tubes in a serological centrifuge at 3400 rpm for
10 – 15 seconds (according to the manufacturer’s
directions).
6.13.2 Compare the patient name on each tube with the
corresponding information on the request form (or computer
screen) to ensure they are the same.
6.13.3 Read macroscopically. See PA.006 – Reading and
Recording Hemagglutination Reactions.
6.13.4 Grade and record results. See PA.006 – Reading and
Recording Hemagglutination Reactions. If the cells are
strongly agglutinated (grade 2 or greater) in the anti-D tube
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Weak D Typing
but negative in the control tube, record the interpretation as
“D positive” and do not proceed with the antiglobulin phase
of the test. If there is no reaction in either tube, proceed to
step 6.14.
6.14
Proceed to the antiglobulin phase. See PA.005 – Cell Washing
Automated and Manual.
6.15
Interpret the Rh type.
6.15.1 If patient cells react as described in 7.0 – Reporting, record
the Rh typing on the request form or in the computer as per
established procedure.
6.15.2 If the patient cells do not react as described in 7.0 –
Reporting, a discrepancy exists and must be resolved
before reporting the Rh type. See NRT.004 – Rh Typing
Problem Solving and Procedural Notes 8.2.
7.0
6.16
If a previous Rh typing result was found, compare it with the current
Rh result. If results are discrepant, see NRT.004 – Rh Typing
Problem Solving.
6.17
When the procedure is complete, perform a clerical check.
For each Rh type, check that:

The patient name and identification number are identical on all
specimens and the request form or computer screen

The patient name is the same on all the test tubes and the
request form or computer screen

The test results have been interpreted correctly
6.18
Initial or sign and record the completion time and date on the
request form or in the computer.
6.19
Verification of results must be recorded. See 7.0 Reporting.
Reporting
7.1
Anti-D
>2
The control must be negative. Interpret results as follows:
37° C
Control
0
Anti-D
NT
IAT
Control
NT
Interpretation
Rh positive
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Weak D Typing
<2
0
0
0
0
0
>1
0
<1
0
0
0
0
0
>1
>1
Rh positive
Rh negative
Unable to determine.
Additional investigation is
required. See step 7.2.
Unable to determine.
Additional investigation is
required. See step 7.2.
NT: Not Tested
7.1.1 Reactions that are a grade 2 or stronger after 15 minutes
incubation at 37° C indicate the presence of the D antigen.
Report as Rh positive.
7.1.2 Reactions that are a grade 1 or stronger by IAT indicate the
presence of the D antigen. Report as Rh positive.
7.1.3 No reaction (negative) by IAT indicates the absence of the D
antigen. Report as Rh negative.
7.2
If the control is positive or if the positive reaction with anti-D is
weaker than grade 1 by IAT:
7.2.1 Do not report the Rh type result.
7.2.2 Additional investigation is required. See NRT.004 – Rh
Typing Problem Solving.
8.0
Procedural Notes
8.1
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.
8.2
In cases where the result of the weak D typing cannot be reported
or is unresolved:
8.2.1 If cellular blood components are required STAT, before the
discrepancy is resolved, for pre-menopausal females and
children Rh negative blood products must be issued. Other
patients, in the absence of a known anti-D, may receive Rh
positive blood in emergency situations when there is a
shortage of Rh negative blood.
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Weak D Typing
8.2.2 If unable to determine the Rh type and the patient is a
neonate delivered by a mother who is Rh negative, a
Kleihauer-Betke test should be performed on the post
delivery maternal specimen to determine the amount of Rh
Immune Globulin (RhIG) required.
9.0
References
9.1
Standards for Hospital Transfusion Services, Version 2 –
September 2007, Ottawa, ON: Canadian Society for Transfusion
Medicine, 2007: 5.3.3.2, 5.3.3.3.
9.2
Roback JD, ed. American Association of Blood Banks Technical
Manual, 16th ed. Bethesda, MD: American Association of Blood
Banks, 2008: 447-448, 889-890.
9.3
Judd WJ, Johnson ST, Storry JR. Judd’s Methods in
Immunohematology, 3rd ed. Bethesda, MD: American Association
of Blood Banks, 2008: 15-20.
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