There are various blood grouping systems in humans, with the most common one being the ABO blood grouping system. It is the basis for blood screening, transfusion, and transplantation.

Aside from the usual ABO blood grouping system, another grouping system called reverse blood grouping also called reverse typing. (1, 2)

What is Reverse typing?

Reverse typing is the process of checking the patient’s serum level for the presence of ABO antibodies. A few drops of serum are placed in a test tube containing a red blood cell sample.

The test tube is shaken, ensuring that the mixture is mixed thoroughly in a centrifuged manner. The sample mixture is observed in a well-lit white background to check for hemolysis in the supernatant fluid.

The sample (cell button) is further observed for agglutination as such indicates a positive reaction. (2, 3, and 4)

Principle

Reverse blood grouping is based on the direct hemagglutination principle. Human erythrocytes have blood group antigens found on the cell’s membrane.

When a particular antigen is treated with antibodies, an antigen-antibody reaction takes place, forming agglutination. (3, 4)

Requirements

  • Specimen – The specimen used for reverse blood grouping is serum. Once blood is extracted from the patient, the serum is collected and tested right away. The specimen can last up to two to eight hours, but a delay in testing could result in weaker reactions.
  • Cell suspension – Lab testing can use cell suspensions in the form of readily available cell reagents. Still, many laboratories prefer preparing their own cell suspension in the form of A and B test red cells.
    • Tube A – In test tube A, put a drop of red blood cells from each three of the A group sample.
    • Tube B – A drop of red blood cells is placed from three of the B group samples.

Suspend the cell sample by adding normal saline. Shake the tube in a centrifuge manner for about a minute at 1000 pm.

Create a 5% red cell suspending by adding. A drop of red blood cells to 19 drops of normal saline. For the slide method, create a 20% suspension. The pooled cells are checked by adding anti-A, A (antisera). (4, 5, and 6)

Procedure

The image above shows the reverse blood grouping method using the test tubes images and pictures

Picture 1 : The image above shows the reverse blood grouping method using the test tubes.

Method 1 – Test Tube Method

  • Two test tubes are labeled with A and B.
  • Each test tube containing the specimen is added with two drops of serum.
  • Place a drop of the A and B cell suspension to the right test tube.
  • Mix each test tube in a centrifuge manner, ensuring that each test tube is mixed for a minute at 100 rpm.
  • Remove the test tube and resuspend the cells.
  • Check for signs of agglutination. If there’s no visible agglutination, examine the sample once more under the microscope.
  • Take note of the reaction and interpret the result accordingly. (6, 7, and 8)
The image above shows the reverse blood grouping procedure using the slide method pciture and image

Picture 2 : The image above shows the reverse blood grouping procedure using the slide method.

Method 2 – Slide Method

  • Get a clean slide and mark it in two halves. Label the left side as A and the right side as B.
  • Add a drop of serum on each side (A and B cell suspension (20%).
  • Get a clean applicator and gently but thoroughly mix the serum and cell suspension. Spread the mixture into a smooth round circle.
  • Rock the slide for a few minutes (two minutes) and watch for signs of agglutination.
  • Examine the slide for reactions and interpret the results accordingly. (6, 8, and 9)

Interpreting Results

The image shows the possible result for the reverse blood grouping method. image

Picture 3 : The image shows the possible result for the reverse blood grouping method.

  • Agglutination in A cell only – It indicates that the patient’s blood group is B.
  • Agglutination in B cell only – It indicates that the patient’s blood group is A. (5, 6)

Reverse Blood Grouping Limitations

  • Detection of ABO antibodies is difficult in people with a condition called agammaglobulinemia, an inherited immune disorder characterized by low antibody concentration in the blood caused by a lack of specific lymphocytes in the lymph and blood.
  • Naturally occurring antigens A and B formed three to four months after birth. Therefore, reverse blood grouping cannot be performed in newborns and infants because their antibodies are from maternal sources. (8, 9, and 10)

Here is the video for better understanding of this proceedure – https://www.youtube.com/watch?v=b4DR53-Th5A

Frequently Asked Questions (FAQs)

What is the significance of reverse blood grouping?

Reverse blood grouping is important as it helps detect and resolve ABO discrepancies. This method makes it easy to detect red blood cells with A and B blood group antigens. (3)

Why is serum used during reverse blood typing?

The serum is used during reverse blood typing because it contains antibodies reactive to reagent red blood cells that are coated with antigens A and/or B. The type of reagent red blood cells that cause agglutination will depend on the type of antibodies found on the patient’s serum. Thus, confirming the patient’s blood type. (6, 9, and 10)

Why are the possible reasons for false blood typing results?

 Many conditions are leading to false blood typing results, such as:
• Effects of medications
• Antibodies
• Preservatives
• Disease condition
• Cross-contamination while doing the testing (2, 8, and 10)

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