Activity No. 10

ST.
ALEXIUS COLLEGE
Gen. San. Drive, City of Koronadal, South Cotabato, Philippines 09506, Tel.: (083) 228-2019, Fax: (083) 228-4015, Email: st.alexiuscollege@yahoo.com
Activity No. 10
Weak D (Dᶸ) Typing
I. Introduction
In Rh grouping, reaction slide/tube with no agglutination is not right away
reported as Rh negative. A test for weak D antigen is necessary to be performed
before a medical technologist reports the result as Rh negative. This determines
the presence or absence of Dᶸ factor of Weak D antigen on red cells. Some red
cells express the D antigen so weakly that most anti-D reagents do not directly
agglutinate the cells. Weak D expression can be recognized most reliably by an
Indirect Antihuman Globulin Test (IAT) after incubation of the test red cells with
anti-D.
II. Objectives
At the end of the activity, the student must be able to:
1. perform Dᶸ typing among Rh negative individuals.
2. determine the presence of weak D variant in red cell sample.
3. determine the variation of Rh (D) antigen.
4. identify the confirmatory test to be done when sample is Rh negative.
III. Materials
Centrifuge
Parafilm
Pasteur Pipette
Test Tube Rack
Test Tubes
IV. Reagents/Samples
22% Bovine Albumin
Laboratory Manual in Immunohematology (MT 313)

1
ST. ALEXIUS COLLEGE
Anticoagulated Blood Sample (EDTA)

Anti-D Typing Sera
Antihuman Globulin Sera (Coomb’s Reagent)
Normal Saline Solution (NSS)
V. Procedure
1. Prepare 5% Red cell suspension to be tested.
2. Label another set of tubes as U (Unknown) and NC (Negative Control)
3. Place 1 drop of Anti-D to tube labelled “U” and 1 drop of 22% of Bovine Serum
Albumin to tube labelled “NC”
4. Add 1 drop of 5% red cell suspension to each tube.
5. Mixed gently and cover all tubes with parafilm then incubate both tubes for 15
minutes at 37 ⁰C water bath.
Note: Unknown tube with agglutination is regarded as Rh positive.
Unknown tube without agglutination goes to the next procedure.
6. Wash the cells 3 times with NSS.
7. Aspirate the NSS completely after washing.
8. Add 2 drops of Antihuman Globulin Reagent (AHG) or Coomb’s Reagent to all
tubes and mix gently. Cover with parafilm.
9. Centrifuge for 15-30 seconds at 3,400 rpm.
10. Gently dislodge cell button and examine for agglutination.
11. Interpret and record results.
Note: Absence of agglutination confirms the blood group to be Rh
negative.Presence of agglutination means presence of weakly reacting D.
12.. For each negative tube, add 1 drop of well mixed check cells.
Note: Check cells or Coomb’s cells are RBC’s coated with IgG. A positive
result is expected after addition of check cells. This implies that the test
has been properly performed. Negative result with these cells indicates an
improperly performed test and the test should be repeated.

2
ST. ALEXIUS COLLEGE
PERFORMANCE ASSESSMENT SHEET

Name: _________________________________________ Date: __________________
Procedure Rating Comments

1. Wear laboratory gown
2. Wash hands and wear gloves
3. Assemble equipment and materials for Weak D typing
4. Prepare 5% Red cell suspension to be tested.
5. Label another set of tubes as U (Unknown) and NC
(Negative Control)
6. Place 1 drop of Anti-D to tube labelled “U” and 1 drop
of 22% of Bovine Serum Albumin to tube labelled “NC”
7. Add 1 drop of 5% red cell suspension to each tube.
8. Mixed gently and cover all tubes with parafilm then
incubate both tubes for 15 minutes at 37 ⁰C water bath.
9. Wash the cells 3 times with NSS.
10. Aspirate the NSS completely after washing.
11. Add 2 drops of Antihuman Globulin Reagent (AHG)
or Coomb’s Reagent to all tubes and mix gently. Cover
with parafilm.
12. Centrifuge for 15-30 seconds at 3,400 rpm.
13. Gently dislodge cell button and examine for
agglutination.
14. Interpret and record results.
15. Clean and return the equipment to proper storage.
16. Clean the work area with surface disinfectant.
17. Safely disposed the gloves in a pathologic waste
container and properly washed hands with disinfectant.
Rating: 4= Excellent 3= Very Satisfactory 2= Satisfactory 1=Poor
Level of Competency:
Instructor: Date:
3
ST. ALEXIUS COLLEGE
REPORT SHEET
Activity No. 10
Name: Rating:
Date Performed: Date Submitted:
Results:
Illustrate your final result obtained.
Unknown Negative Control
Reaction: _____________ Reaction: _____________

4
ST. ALEXIUS COLLEGE
Review Questions:
1. Enumerate and describe the three mechanisms by which the Dᶸ phenotype occurs?
2. What is the role of Rh in Hemolytic Disease of Newborn?
3. What is Rh null and Rh mod? Differentiate the two.
4. What test should you apply to detect presence of Weak D?

5
ST. ALEXIUS COLLEGE
5. What is LW antigen? Give its significance.
6. Enumerate and describe the rare alleles and unusual phenotypes of Rh Blood
Group?

6

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ST.

Laboratory Manual in Immunohematology (MT 313)

Anticoagulated Blood Sample (EDTA)

Laboratory Manual in Immunohematology (MT 313)

PERFORMANCE ASSESSMENT SHEET

Name: _______________________ Date:

Procedure Rating Comments

Rating: 4= Excellent 3= Very Satisfactory 2= Satisfactory 1=Poor

Unknown Negative Control

Reaction: _ Reaction: _

Laboratory Manual in Immunohematology (MT 313)

2. What is the role of Rh in Hemolytic Disease of Newborn?

3. What is Rh null and Rh mod? Differentiate the two.

4. What test should you apply to detect presence of Weak D?

Laboratory Manual in Immunohematology (MT 313)

5. What is LW antigen? Give its significance.

Laboratory Manual in Immunohematology (MT 313)

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