Exp. No.: 2
ABO System Typing Technique (1) – Direct (Forward) Method
(Cell grouping)
Background
The ABO system antigens are found on all the cells of the body tissues, mainly on RBCs surface. There are three
types of antigens belong to ABO system; which A, B & H. The RBCs of each individual will have or lack A
and/or B Ags, attached to the precursor (H Ag). In the majority of cases, accordingly the blood group for the
individuals in regard to ABO system can be A, B, O or AB.
Principle
ABO antibodies are of the IgM class and react preferentially at 22C (RT) or below. Incubation at warm
temperatures may cause a false negative reaction. Enhancement of weak reactions may be obtained by RT
incubation or incubation at 4C.
Direct (Forward) Method
The agglutination of RBCs (hem-agglutination) test that used to detect A and B antigens on RBCs surface by
using reagents as anti-A, anti-B or (Anti-A,B sometimes) is called Direct (forward) method; according to the
tools that employed in the test either slides or tubes, this method can be classified into:

Direct (Forward) slide Method
 Direct (Forward) tube Method
Both depending on the same principle; meaning antigen and antibody reactions. Both can give the same results
(in the majority of cases), but the difference reside in the sensitivity and RBCs suspension involved in the
reaction.
Materials
EDTA Blood samples, Anti-A, Anti-B, Anti-A,B (optional-from group O serum, act as an additional check),
slides, normal saline, wooden sticks, 2%-5% cells suspension of A1 cells, B cells and test tubes. Monoclonal antiA and anti-B reagents have replaced polyclonal reagents.
Objectives
1. To train the students to be competent on preparing proper RBCs suspension
2. To be able to identify the principle of direct (forward) method
3. To be able to interpret the test result that they had
Procedure
Before starting the procedure be sure to understand the following notes
Note 1:
The reagent manufacturer’s instructions must be consulted before tests are performed; some manufacturers
recommend performing slide tests with whole blood, whereas others specify the use of RBCs suspensions of
lighter concentrations prepared in saline.
Note 2:
The anti-sera that you will be supported are manufactured from human sources.
Usually all anti sera types (Anti-A, Anti-B & Anti-A,B), are universally known and labeled to be easily
recognizable according to their color as follow:
Anti-sera
Color
Source
Anti-A
Blue
Group B donor
Anti-B
Yellow
Group A donor
Anti-A,B (Optional)
Colorless (clear)
Group O donor
Note 3 :
Positive and negative controls should be included with every test or batch of tests. The anti-A reagent should be
tested against group A (positive control) and B (negative control) cells, and the anti-B reagent should be tested
against group B (positive control) and group A (negative control) cells.

Direct (Forward) Slide Method
Label a clean glass slide with A and B.
Place 1 drop of anti-A on the glass slide.
On the same glass slide mentioned above leave a space then place 1 drop of anti-B.
Add to each drop of reagent on the slide 1 drop of well-mixed 50% RBCs suspension to be tested.
(Consult the reagent manufacturer’s instructions to determine the correct cell concentration to be used).
5. Mix the reagents and RBCs thoroughly, using a clean applicator stick for each reagent.
6. Gently tilt the slide continuously for up to 2 minutes. Do not place the slide over a heated surface!
Why?
7. Read, interpret and record the results of the reactions.
1.
2.
3.
4.

Interpretation
1. Strong agglutination of RBCs in the presence of any ABO typing reagent within 2 min. represents a
POSITIVE result.
2. A smooth suspension of red cells at the end of 2 minutes is a negative result.
3. Samples that give weak or doubtful reactions should be retested using TUBE METHOD.

Direct (Forward) tube Method
1. Label two plastic or glass tubes with A and B.
2. Place 1 drop of anti-A in a clean, labeled test tube.
3. Place 1 drop of anti-B in a clean, labeled test tube.
4. Add to each tube 1 drop of a 2% to 5% suspension (in saline) of the unknown red cells to be tested.
The equivalent amount of red cells can be transferred to each tube with clean dropper.
5. Mix the contents of the tubes gently and centrifuge them at calibrated spin time (2000 rpm for 2 min).
6. Gently re-suspend the cell buttons and examine them for agglutination.
7. Read interpret and record the test results.

Interpretation
1. Agglutination of tested red cells and either hemolysis or agglutination is a POSITIVE test results.
2. A smooth cell suspension after re-suspension of the cell button is a NEGATIVE test result.
Reporting the results (tube Method):
The result will be expressed as the following table
ABO Type
Anti-A reaction
Anti-B reaction
O
0
0
A
+4 or +3
0
B
0
+4 or +3
AB
+4 or +3
+4 or +3
Technical Notes
1. Positive reactions characteristically show 3+ to 4+ agglutination by reagent ABO antibodies.
2. Clotted samples should not be used when red blood cell testing is being carried out. Why?
3. Use medium-sized (75 × 10 or 12 mm) disposable plastic or glass tubes.
4. Slide testing imposes a greater risk of exposure to infectious samples.
5. In blood group serology, tube tests are generally done at 37°C, room temperature, or both. There is
some advantage in using a 20°C water bath rather than relying on “room temperature,” which in
different countries and seasons may vary from 15°C (or less) to 30°C (or more).
6. Personnel should follow safety measures detailed in the facility’s procedures manual.
7. Slide testing is NOT suitable for detection of ABO antibodies in serum/plasma.
8. Grading Serological Reactions in Tube Testing:
4+ Complete agglutination – no unagglutinated RBCs
3+ Strong reaction – a few detached masses of agglutinated RBCs; no unagglutinated RBCs
2+ Moderate reaction – large agglutinates in a sea of smaller agglutinates; few unagglutinated RBCs
1+ Weak reaction – many agglutinates of up to 20 RBCs with some smaller agglutinates and
unagglutinated RBCs
Conclusions/Comments/Questions
Answer the following questions:
1.
2.
3.
4.
5.
In direct method, we detect ……
How many Ag types do we have in ABO system?
Can we use heavy suspension of RBCs in tube method? Why?
Prepare 3% RBCs suspension; feel free to select the volume.
Which of the following is/are known to us before starting forward methods (RBCs Ags or Anti-sera)?