# Reverse Blood Grouping – Principle, Procedure, Result > Reverse grouping is defined as using the reagent cells with known ABO antigens and testing the patient’s serum for ABO group antibodies. Canonical URL: https://biologynotesonline.com/reverse-blood-grouping-principle-procedure-result/ Author: Sourav Pan Last updated: April 14, 2024 ![Reverse Blood Grouping – Principle, Procedure, Result](https://biologynotesonline.com/wp-content/uploads/2021/07/Reverse-Blood-Grouping.jpg) 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. ## 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. ## 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: - Take two test tubes and label them as A and B. - 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. - Add normal saline to the tubes to suspend the cells. - Centrifuge them for at least 1 minute at 1000 rpm. - 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. - 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 - Take two test tubes and label them as A and B. - Add 2 drops of the Patient’s serum to each test tube. - Add 1 drop of each of A and B cells suspension to the corresponding test tubes. - Mix well and then centrifuge them at 1000 rpm for 1 minute. - After centrifugation completely resuspends the cells. - Examine the tubes macroscopically for the positive and negative results. ### Slide Method - 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. - Add one drop of the patient’s serum on both sides of the slide. - Add one drop each of A and B cells suspension (20%) to the corresponding sides. - Take a sterile Toothpick/inoculum loop to mix the serum and cell suspension on both sides separately and spread into a smooth round circle. - Keep the slides in steady condition for 2 minutes. - 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. ## 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.