1. ABO/Rh Tube Test Procedure
This PowerPoint presentation will provide the steps involved in the ABO/Rh test tube procedure

2. ABO Forward Typing
This test is used to detect the presence or absence of A and/or B antigens on an individual's red blood cells.
An individual's ABO group is determined by testing the red blood cells with reagent anti-A and anti-B sera.
Agglutination of the individual's red cells by the appropriate antisera signifies the presence of the antigen on the red cells
No agglutination with the antisera signifies its absence.

3. ABO Antigens by Blood Group
ABO Group
Antigens present on the red cells
Group A
A antigen
Group B
B antigen
Group O
There are no ABO antigens present on the red cells
Group AB
Both A and B antigens

4. ABO Reverse Typing
This test is used to detect ABO antibodies in an individual's serum, and is used to confirm the ABO
Individuals will produce potent, naturally occurring antibodies directed against the ABO antigens they lack.
The outcome of the serum typing (Reverse Typing) is compared with the outcome of the cell typing (Forward Typing) to ensure the accurate ABO determination.
A final interpretation of the blood group is made based upon the test tube reactions.

5. ABO Antibodies by Blood Group
ABO Blood Group
Antibodies in the serum/plasma
Group A
B antibody present
Group B
A antibody present
Group O
Both A and B antibodies present
Group AB
Neither A or B antibodies present

6. Rh (D) Typing
This test is used to detect the presence or absence of D (Rh) antigens on an individual's red blood cells.
A person can be either D (Rh) Pos or D (Rh) Neg depending on the presence or absence of the D (Rh) antigen
Control must be run in parallel with Rh grouping tests performed on patient’s samples, to avoid incorrect designation of Rh Neg, patient as Rh Positive.

7. ABO/Rh Tube Testing of a patient specimen
ABO Grouping
Determination of the patient’s correct ABO group is the most critical pre-transfusion serologic test.
It is essential that the ABO/Rh tube testing procedure be performed accurately and according to the standard operating procedure

8. Steps of the ABO/Rh Tube Test
Locate/obtain all of the necessary laboratory equipment
Obtain suitable patient specimen for testing
Label test tubes and set up test tube rack
Dispense appropriate volumes of reagents/patient specimen
Mix reagents/patient specimen
Grade test tube agglutination reactions
Interpret ABO/Rh

9. Laboratory Equipment Required
Specimen centrifuge
ABO/Rh reagents
Test tubes-12x75 mm; 10x75 mm
Test tube rack
Test tube serofuge
Disposable plastic pipettes
Sharps container for disposal of test tubes

10. Patient Specimen Requirements
Specimen collected in EDTA tube or a clotted tube
No hemolysis present
Adequate specimen volume
Appropriately labelled with patients full name, date of birth and hospital unique number

11. Label Test Tubes and Rack Set-up
Test tubes must be labelled with the last initial and first initial of the patient you are testing and the reagent you are using
For patient Adam Sandler the test tubes would be labelled as follows:
Test Tube label
Reagent
Grouping
SA-A
Anti-A reagent
Forward grouping
SA-B
Anti-B reagent
SA-D
Anti-D(Rh) reagent
SA-DC
Anti-D(Rh) control
SA-RA
A1 cells
Reverse grouping
SA-RB
B cells
SA-3%
None
Patient’s 3% cell suspension

12. Test Tube Rack Set-Up
Each laboratory may have a slightly different test tube rack set-up
For this learning module we will set up the test tube rack as follows:
SA-DC SA-3%
SA-D
SA-B SA-RB
SA-A SA-RA
**Please note: Rack set-up is slightly different than the set-up in the following video. Most laboratories do not use the anti-AB reagent and therefore we will not use this reagent in the ABO/Rh testing procedure.**

13. Dispensing Reagents/Patient Specimen
Specific reagent/patient specimen volumes need to be added to each test tube
Avoid cross-contamination of reagents or specimens.
Follow standard operating procedure for the volumes required

14. Mixing Reagents/Patient Specimen
Gently mix reagents/patient specimen in the test tubes
Test tube rack can be gently agitated to mix all test tubes at once
Mixing of reagents/patient specimen ensures adequate antigen/antibody uptake to promote optimal agglutination

15. Serofuging Test Tubes
Centrifuge test tubes in the serofuge for the time and RPM stated in the standard operating procedure.
Add a balance tube if required.

16. Dislodging Cell Button
Gently resuspend each cell button individually and examine for agglutination after serofuging
Grasp the tube lightly at the top, and hold vertically so that the cell button is facing toward you when viewed in the mirror.
VERY GENTLY shake the tube (shake from the wrist) to dislodge the cell button. Continuously observe the cells as they come off the cell button.
When the cell button is about one-half broken up, gently tip the tube horizontally and observe for agglutination.

17. Additional Notes for Dislodging Cell Button
Over shaking can break up fragile agglutinates, resulting in a false negative reaction.
Failure to completely resuspend the cell button may result in a mixed-field reaction being missed.

18. Grading Test Tube Reactions
Macroscopic
4+ One solid aggregate or clump of cells.
3+ Several large aggregates, clear background.
2+ Small to medium sized aggregates, clear background.
1+ Small aggregates, turbid reddish background.
+W Tiny aggregates, turbid reddish background.
MF Mixed Field – Any degree of agglutination in a sea of unagglutinated cells.
Hem - Hemolysis is interpreted as a positive reaction and may be graded as complete or partial. Both hemolysis and agglutination may be recorded on the same tube.
Ø Negative - no agglutination, smooth reddish background.

19. Grading Reaction Chart

20. ABO Interpretation Chart
Forward Type Results
Reverse Type Results
Blood Group Interpretation
Anti-A
Anti-B
Anti-D(Rh)
Anti-D control
A1 Cells
B cells +
A POS
A NEG
B POS
B NEG
O POS
O NEG
AB POS
AB NEG
Note- Do NOT use “+” as your interpretation of your agglutination reaction results. In the following chart “+” indicates a positive reaction for agglutination, record your GRADED reactions according to the grading reaction chart in previous slide.

21. Please watch the two videos on the ABO/Rh tube testing technique located in this section.
These videos will show you the steps involved in the ABO/Rh tube test and the techniques required to accurately perform the testing procedure.