IMMUNOHAEMATOLOGY 411

Lorraine Thompson 00025062

LAB #2

24 th March, 2015

Title: Tube Test for the Determination for Weak D

Introduction: The objective of this lab is to determine if the weak expression of D
antigen is present using an indirect antiglobulin test. The Weak D test is performed in
order to detect weak expression of the D antigen on red cells, when the cells being
tested have failed to react with anti-D at the immediate spin phase. The red cells in
question are incubated at 37 oC with anti-D and then tested with anti-human globulin
(AHG) reagent. If the D antigen is present on the cells, the anti-D will sensitize the cells,
resulting in agglutination at either the 37 phase or the AHG phase. Lack of agglutination
at both phases indicates the D antigen is not present on the red cells. Weak D testing is
used to ensure Rh negative donors are truly D negative. It is done on all prenatal
patients and candidates for Rh immune globulin. After the A and B antigens, the D (Rh)
antigen is the most important red cell antigen in transfusion practice. The D antigen is
highly immunogenic and will stimulate antibody production in 80% of D negative
recipients receiving D positive red cells. It is necessary to detect weakened expression of
the D antigen in certain populations in order to prevent the formation of anti-D in Rh
negative individuals. Some red cells possess the D antigen but it is expressed so weakly
that the cells are not agglutinated directly by anti-D sera. An indirect antiglobulin test is
necessary to identify patients with the Weak D phenotype. There are three theories
behind the weak expression of the D antigen: 1) Amount of D epitopes on the red cells.
2) Position of the D allele to the C allele. 3) Inheritance of Partial or Altered parts of the D
antigen. It may or may not be done routinely on Rh negative candidates for transfusion,
depending on the policy of the transfusing institution. If routine weak D testing is done,
weak D positive patients should receive Rh positive blood.
Apparatus/ Materials:
3 EDTA Blood samples of
unknown blood type
Monoclonal or polyclonal AntiA
Monoclonal or polyclonal AntiB
2%-5% A cells reagent
commercially prepared
2%-5% B cells reagent
commercially prepared
Anti-D reagent
Rh control reagent or gamma
clone control
Anti-IgG
Check Cells
0.9 Saline

10% Bleach solution

Beaker
Test tubes
Test tube rack
Pipettes
37oC Incubator
BD Sero-Fuge
Sharpie permanent maker

Method:

IMMUNOHAEMATOLOGY 411
Lecturer: Ms. M. Carrillo

Page 1

IMMUNOHAEMATOLOGY 411
Lorraine Thompson 00025062

LAB #2

24 th March, 2015

1. Labelled test tubes to perform ABO type test and Rh test for 3 Patients EDTA
samples
2. Prepared a washed, 2-5% suspension of patient cells, performed an ABO type and
Rh type
3. Recorded the ABO, D and DC results. If the Rh test is negative, continue with step 4
.
4. Incubated the D and DC tubes at 37oC for 15 minutes.
5. Centrifuged both test tubes and read for agglutination. If the Rh test is negative,
continue with step 6.
6. Washed both tubes once with saline and decant the supernatant completely and
blot the rims of test tube dry to make a dry cell button.
7. Added two drops of Anti-IgG to the dry cell buttons mix gently and centrifuge.
8. Re-suspended gently and examine each cell button for agglutination.
9. Recorded results in the appropriate column on the worksheet
10.
Confirmed all negative results by adding one drop Check cells to all tubes
showing no agglutination and centrifuge 15 seconds.
11.
Gently re-suspended and examine the cell buttons for agglutination.
Agglutination should be present in this step or the test is invalid.
12.

Read, graded and recorded results.

Results:
Patient sample
Patient sample
Patient sample
Weak D:
Patient sample
Patient sample
Patient sample
background

#1 was determined to be AB Rh positive

#2 was determined to be O Rh positive
#3 was determined to be A Rh negative
#3 incubated for15 minutes at 37 oC showed no agglutinations present
#3 with added Anti-IgG showed no agglutinations present
#3 with added Check Cells showed very small agglutinates, turbid

Discussion:
A negative result in the immediate spin phase and no agglutination in the D tube
following incubation indicate a negative test for weak D. No agglutination was present in
the DC tube. Lack of agglutination is a negative test and the patient is considered truly D
negative. Agglutination in the DC tube invalidates the test. Check cells were added to
confirm all the negative results and agglutination occurred. When adding Check cells if
no agglutination is present at this stage of the procedure, the test is invalid and the
antiglobulin phase must be repeated. In the results obtain there is a possible source of
IMMUNOHAEMATOLOGY 411
Lecturer: Ms. M. Carrillo

Page 2

IMMUNOHAEMATOLOGY 411
Lorraine Thompson 00025062

LAB #2

24 th March, 2015

error that may have occurred due to the cells being washed only once and not 3 times.
This is a potential source of interference in the final result due to this.
Conclusion: The objective was not achieved as the weak D expression was not present.
The results after incubation was graded 0 as no reaction was observed from Patient
sample #3 with an A Rh negative blood type. When Anti-IgG was added no reaction was
observed and this was graded as 0. Addition of the Check cells produced a 1+
Agglutinate. I was expecting a 2+ Agglutinate result but I believe this occurred due to
the fact that the sample was only washed once. A true weak D should give at least a 2+
positive result. Weaker results may be due to mixed field agglutination in an Rh negative
individual who received Rh positive blood, or vice-versa. It was learnt that check cells
are used to verify negative results and the sample was truly Rh negative with no D
antigen present.
Reference:
Weak D Testing. (n.d.). Retrieved March 26, 2015.
Blood Bank. (n.d.). Retrieved March 26, 2015.

IMMUNOHAEMATOLOGY 411
Lecturer: Ms. M. Carrillo

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