ST.

ALEXIUS COLLEGE

Activity No. 6
ABO Reverse Grouping

I. Introduction
Another way to determine ABO blood type in the laboratory is through
reverse grouping. Reverse grouping is synonymous to Backward Typing, Indirect
Typing and serum Typing. Reverse ABO blood typing identifies the antibodies
present in a patient's serum or plasma by using red cells of known antigenic
specificity. These antibodies are usually naturally occurring IgM antibodies. The
principle follows that whenever blood group antigen is present on the red cell, the
opposite antibody is present on the serum.

II Objectives
At the end of the activity, the student must be able to:
1. determine the antibodies present from the serum sample of an individual by
using known red cells.
2. be acquainted with the importance of reverse grouping in ABO typing.
3. identify the common sources of technical errors that will result in ABO
discrepancies.

III. Materials
Centrifuge Test Tube
Pasteur Pipette Test Tube Rack

IV. Reagents/ Samples

Normal Saline Solution (NSS)
Serum or Plasma Sample to be Tested
5% RCS of Known Group A cells

Laboratory Manual in Immunohematology (MT 313)

 ST. ALEXIUS COLLEGE

5% RCS of Known Group B Cells

V. Procedure
1. Prepare 5% RCS of Known A and Known B cells.
2. Label 2 tubes with “A” and “B”.
3. Add 1 drop of Known A to tube A and 1 drop of known B to tube B.
4. Place 2 drops of serum or plasma sample to each tube.
5. Mix gently and centrifuge for 15-30 seconds at 3,400 rpm.
6. Gently dislodge the cell button and examine for hemolysis or agglutination.
7. Grade each reaction and record results.
Note: Weak reactions will be enhanced if the tubes are left more that 5 minutes at room
temperature before centrifugation. Avoid excessive shaking which may disperse weak
agglutination. Hemolysis indicates the presence of antibodies, thus, mean a positive test.

Guide in the Interpretation of Reverse ABO Blood Typing

Patient’s Blood Known A Known B Known AB Known O
Type cells Cells cells cells
A 0 + + 0
B + 0 + 0
AB 0 0 0 0
O + + + 0
[+] with agglutination [-] no agglutination

● Agglutination in B and AB cells, no agglutination in A cells demonstrates the

presence of Anti-B in the serum, thus, indicating that the individual is blood type
A.

● Agglutination in A and AB cells, no agglutination in B cells demonstrates the

presence of Anti-A in the serum, thus, indicating that the individual is blood type
B.

Laboratory Manual in Immunohematology (MT 313)

 ST. ALEXIUS COLLEGE

● Agglutination in tubes with A, B, and AB cells, demonstrates the presence of Anti

A and Anti-B in the serum, thus, indicating that the individual is blood type O.

● No agglutination in tubes with A, B, and AB cells demonstrates the absence of

Anti-B in the serum, thus, indicating that the individual is blood type AB.

PERFORMANCE ASSESSMENT SHEET

ABO Reverse Grouping

Name: _________________________________________ Date: __________________

Procedure Rating Comments

1. Wear laboratory gown
2. Wash hands and wear gloves
3. Assemble equipment and materials for ABO Reverse
grouping.
4. Prepare 5% RCS of Known A and Known B cells.
5. Label 2 tubes with “A” and “B”.
6. Add 1 drop of Known A to tube A and 1 drop of known
B to tube B.
7. Place 2 drops of serum or plasma sample to each
tube.
8. Mix gently and centrifuge for 15-30 seconds at 3,400
rpm.
9. Gently dislodge the cell button and examine for
hemolysis or agglutination.
10. Grade each reaction and record results.
11. Clean and return the equipment to proper storage.
12. Clean the work area with surface disinfectant.
13. 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:

Laboratory Manual in Immunohematology (MT 313)

 ST. ALEXIUS COLLEGE

Instructor: Date:

Laboratory Manual in Immunohematology (MT 313)

 ST. ALEXIUS COLLEGE

REPORT SHEET
Activity No. 6
ABO Reverse Grouping

Name: Rating:
Date Performed: Date Submitted:

Results:
Illustrate the ABO Reverse grouping reactions of the different blood type.

Patient’s Blood Type A tube with Known A cells B Tube with Known B Cells

AB

Laboratory Manual in Immunohematology (MT 313)

 ST. ALEXIUS COLLEGE

Review Questions:

1. When hemolysis is present in the final solution, what does it indicate? Explain.

2. What is the importance of serum typing?

3. Can plasma be used in this experiment? Justify. What is the disadvantage?

Laboratory Manual in Immunohematology (MT 313)

 ST. ALEXIUS COLLEGE

4. When do ABO discrepancies occur? How can this be resolved?

Laboratory Manual in Immunohematology (MT 313)