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Reverse Blood Grouping Principle, Procedure, Result

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Table of Contents

The ABO blood grouping is divided into two methods such as Forward grouping/Cell grouping and Reverse grouping/Serum grouping. In Forward grouping/Cell grouping, the Red blood cells are examined for A and B antigens utilizing known anti-A and anti-B sera. While in Reverse grouping/Serum grouping, the Serum is examined for anti-A and anti-B antibodies by using known A and B red cells.

Reverse Blood Grouping

Reverse grouping is defined as using the reagent cells with known ABO antigens and testing the patient’s serum for ABO group antibodies.

  • It is a type of laboratory technique used to confirm ABO blood group based on the presence or absence of anti-A and anti-B in serum by utilizing known A and B red cells.
  • This test is performed along with forward grouping to check the accuracy.
  • The reverse Blood Grouping test is not applicable for infants below 4 months of age, because of the absence of synthesized immunoglobulins, anti-A and Anti-B in newborns and very young infants.

Aim

To check the accuracy of the Forward Blood Grouping test.

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Reverse Blood Grouping Principle

The reverse blood grouping procedure is based on the principle of direct hemagglutination. The erythrocytes of a person contain blood group antigens on the surface of the membrane. When these antigens are allowed to be treated with corresponding antibodies, antigen-antibody reactions occur and form agglutination.

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Requirements for Reverse Blood Grouping

  • Serum from the patient.
  • Group A and group B red cell Suspension.

Preparation of Cell Suspension

Most of the laboratories prepare their own A and B test red cells from persons known to be group A and group B. Make pooled cell suspension as follows:

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  1. Take two test tubes and label them as A and B.
  2. Then in Tube A, Place 1 drop of red cells each from 3 of A group samples and in Tube B place 1 drop of red cells each from 3 of B group samples.
  3. Add normal saline to the tubes to suspend the cells.
  4. Centrifuge them for at least 1 minute at 1000 rpm.
  5. Prepare 5% red cell suspension by adding 1 drop of RBC to 19 drops of saline. And also prepare 20% suspension for the slide method.
  6. Test the prepared pooled cells by adding the antisera (Anti-A, B) in use.

Procedure

There are present two methods for Reverse Blood Grouping test such as; Tube Method and Slide Method.

Tube Method

  1. Take two test tubes and label them as A and B.
  2. Add 2 drops of the Patient’s serum to each test tube.
  3. Add 1 drop of each of A and B cells suspension to the corresponding test tubes.
  4. Mix well and then centrifuge them at 1000 rpm for 1 minute.
  5. After centrifugation completely resuspends the cells.
  6. Examine the tubes macroscopically for the positive and negative results.

Slide Method

  1. Take a clean slide and divide it into two halves by usign a marker. Label the left side as A and the right side as B.
  2. Add one drop of the patient’s serum on both sides of the slide.
  3. Add one drop each of A and B cells suspension (20%) to the corresponding sides.
  4. Take a sterile Toothpick/inoculum loop to mix the serum and cell suspension on both sides separately and spread into a smooth round circle.
  5. Keep the slides in steady condition for 2 minutes.
  6. After 2 minutes observe the slides for agglutination.

Results and Interpretation

  • Blood group A: If agglutination occurs with B cells only.
  • Blood group B: If agglutination occurs with A cells only.
  • Blood group AB: If agglutination does not occur with both A and B cells.
  • Blood group O: If agglutination occurs with both A and B cells.
Reverse Blood Grouping Principle, Procedure, Result
Reverse Blood Grouping Principle, Procedure, Result

Limitations

  • The Serum collected from the patients with agammaglobulinemia should not contain detectable ABO antibodies.
  • This method is not suitable for newborns and infants, because they lack anti-A and anti-B, which is only formed 3-4 months after birth.

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