Reverse Blood Grouping

Several blood classification systems have been described in humans. Among them, the ABO blood classification system is the most important system for screening, transfusion and transplantation at present. The ABO blood classification system divides blood into 4 groups (A, B, AB, O) according to the presence or absence of A and B antigens on the surface of red blood cells and the natural anti-A and anti-B present in the serum. In normal human blood, there is a reciprocal relationship between antigens and antibodies as described below (Figure 1)

By cross-matching red blood cells and blood serum from different people, Landsteiner discovered that the blood of two people in contact agglutinates, which is due to the contact of the blood with the blood serum. Accordingly, he established the law to describe the relationship between red blood cell antigens and antibodies present in the serum for the ABO blood system:

1. If an agglutinogen is present in the red blood cells of a blood, the corresponding agglutinin must be absent in the plasma.
2. If an agglutinogen is absent in the red blood cells of a blood, the corresponding agglutinin must be present in the plasma.

This law and the Determination a blood group is illustrated in Figure 1.

ABO grouping can be performed in two ways
Forward grouping/Cell grouping: Red blood cells are tested for A and B antigens using known anti-A and anti-B sera.
Reverse grouping/Serum grouping: Serum is tested for anti-A and anti-B anibodies using known A and B red cells.

• Forward grouping tests for antigens on red blood cells.
• Reverse grouping tests for antibodies in the serum.

Reverse Blood Grouping/Serum Grouping:

Reverse blood grouping is a procedure to confirm ABO blood group based on the presence or absence of anti-A and anti-B in the serum using known A and B red blood cells. This is a cross-check for forward typing. Performing both forward and reverse grouping helps to verify accuracy. Due to the lack of synthesized immunoglobulins, anti-A and anti-B in newborns and very young infants, this procedure is not performed on infants younger than 4 months.

Principle

The method of reverse blood group determination is based on the principle of direct hemagglutination. A person’s erythrocytes contain blood group antigens on the membrane surface. When these antigens are treated with the corresponding antibodies, an antigen-antibody reaction occurs and agglutination is formed.

Blood Group Identification through Reverse Grouping:

Blood Group	Antibodies in Plasma	Reaction with A1 Cells	Reaction with B Cells
A	Anti-B	No reaction	Agglutination
B	Anti-A	Agglutination	No reaction
AB	None	No reaction	No reaction
O	Anti-A, Anti-B	Agglutination	Agglutination

• Anti-A antibodies: Present in the serum of individuals from groups B and O.
• Anti-B antibodies: Present in the serum of individuals from groups A and O.
• Agglutination confirms the presence of the corresponding antibodies, while the absence of agglutination indicates their absence.

Requirements

Specimen:

Serum is a sample intended for reverse blood typing. No special preparation of the patient is required prior to collection. The sample should be tested as soon as possible after collection, but samples may be stored at 2-8°C in case testing is delayed. Storage may result in weaker than normal reactions.

Reagent Red Cells Used in Reverse Grouping

Blood Group	Reagent Red Cells	Expected Antibodies in Plasma	Expected Agglutination Reaction
A	B Cells	Anti-B	Agglutination with B cells
B	A1 Cells	Anti-A	Agglutination with A1 cells
AB	None	None	No agglutination with A1 or B
O	A1 and B Cells	Anti-A, Anti-B	Agglutination with both A1 and B cells

Cell Suspension:

Although red blood cell reagents for serum grouping are commercially available, most laboratories prepare their own red blood cells for A and B testing from individuals known to belong to groups A and B. Prepare the grouped cell suspension as follows:

• Label the tubes A and B.
• Tube A: Place 1 drop of red blood cells from each of the 3 group A samples.
• Tube B: Place 1 drop of red blood cells from each of the 3 group B samples.
• Add normal saline and suspend the cells. Centrifuge the tubes for at least 1 minute at 1000 rpm. To prepare a 5% red blood cell suspension, add 1 drop of red blood cells to 19 drops of saline. Prepare a 20% suspension for the slide method.
• Test the prepared pooled cells by adding the antisera (anti-A, anti-B) used.

Reverse Blood Group Procedure:

Reverse blood typing can be performed by two methods: the tube method and the slide method. The tube method is preferred over the knife method.

Tube Method

1. Label two test tubes A and B.
2. Add two drops of the patient’s serum to be tested to each tube.
3. Add one drop of cell suspension A and B to the corresponding test tubes.
4. Mix well and centrifuge both tubes at 1000 rpm for 1 minute.
5. Carefully remove the tubes and completely resuspend the cells and examine them macroscopically for clumps and, if negative, microscopically.
6. Record the reactions and interpret the results.

Slide Method

1. Divide a clean slide into two halves, designating the left and right sides as A and B.
2. Add a drop of Patent Serum to be tested to both sides.
3. Add a drop of A and B cell suspension (20%) to the corresponding sides.
4. Using a clean applicator, mix the serum and cell suspension on both sides separately and spread them into a smooth, round circle.
5. Agitate the slide gently for 2 minutes and look for clumps.
6. Record reactions and interpret results.

Results Interpretation of Reverse Grouping:

• If agglutination is seen only with A cells, the patient’s blood type is B
• If agglutination is seen only with B cells, the patient’s blood type is A
• If agglutination is seen with both A and B cells, the patient’s blood type is O
• If agglutination is not seen with both A and B cells, the patient’s blood type is AB.

Interpretation of Reverse Grouping Results

Patient’s Plasma	Agglutination with A1 Cells	Agglutination with B Cells	Blood Group
Plasma from Group A	No	Yes	A
Plasma from Group B	Yes	No	B
Plasma from Group AB	No	No	AB
Plasma from Group O	Yes	Yes	O

• The serum of people with agammaglobulinemia may not contain detectable ABO antibodies.
• Natural anti-A and anti-B antibodies are only formed 3 to 4 months after birth and are therefore not suitable for newborns and infants. At this age, antibodies are usually of maternal origin.

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Possible References Used

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