ABO and Rh Typing: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Applications, and Keynotes

Introduction of ABO and Rh Typing

ABO blood grouping and Rh typing are the most common blood group systems. There are nearly 300 blood group systems so far discovered. Other than ABO and  Rh are MNS, Lutheran, Kell, Duffy, etc. Karl Landsteiner discovered ABO ( A, B and O)  in 1900. Two years later, Decastello and Sturli added AB and finally ABO system gets completed. In 1940, Karl Landsteiner and Winner discovered Rh factor in red blood cells (RBCs)  of the Rhesus monkey (Macca rhesus).

Principle of ABO Blood Grouping and Rh Typing

ABO blood grouping and Rh typing work on the principle of agglutination. The normal red blood cells possessing antigens will clump in the presence of corresponding antibodies.

Methods of ABO and Rh Typing

  1. Slide
  2. Tube

Note: Here we are concerned with the slide method of ABO blood grouping and Rh typing.

ABO Blood Grouping-Tube Method
Fig. ABO Blood Grouping-Tube Method

Requirements for ABO and Rh Typing

  • Clean and grease-free Glass slides
  • Applicator sticks
  • Pasteur pipette/droppers
  • Centrifuge
  • Antisera- A (blue color)
  • Antisera-B (yellow color)
  • antisera-D
  • Normal saline
  • Test tube ( 10×75 mm)
  • Specimen: Blood

Procedure of ABO Blood Grouping and Rh Typing

  1. Preparation of 10% RBCs suspension in normal saline-Mix 5 drops ( 50µl each) of sedimented RBCs with 2 ml of normal saline. Centrifuge at 1500 RPM for 1 minute and discard the supernatant. Put 2 ml of normal saline into the sedimented RBCs and mix it well. This preparation gives a 10% suspension of RBCs.
  2. Take a glass slide in which place a drop of antisera-A on one half and a drop of antisera-B on the other half.
  3. Take another glass slide and place a drop of antisera-D at the centre.
  4. Using a pipette or dropper add a drop of the RBCs suspension to all separate antisera.
  5. Mix each cell and antisera using separate applicator sticks.
  6. Tilt the glass slide back and forth for up to 2 minutes.
  7. Observe for agglutination or clumping.

Result and Interpretation of Blood Grouping

For the ABO blood group system

Clumping on  antisera-A but not B: Blood group A

Agglutination or clumping on antisera -B but not A: Blood group B

Clumping on both antisera A and B: Blood group AB

No clumping on both antisera-Aand B: Blood group O

For Rh typing

Clumping on antisera-D: Positive

No clumping on antisera-D: Negative

ABO and Rh Typing-Slide Method
Fig. ABO and Rh Typing-Slide Method

Applications Blood Grouping and Rh Typing

It is applicable to the following fields-

  1. Prior to blood transfusion
  2. In the case of hemolytic disease of the newborn (HDN)
  3. Determination of paternity in medico-legal cases
  4. Relationship of blood groups, susceptibility to various diseases e.g. blood group A is more prone to gastric carcinoma whereas blood group O is more prone to systemic lupus erythematosus.


  • Blood obtained from finger-prick may be tested directly by the slide method quickly without making 10% RBCs suspension to avoid clotting and drying.
  • A test that shows no agglutination within 2 minutes is considered negative.
  • Never interpret peripheral drying as fibrin stands as clumping.
  • Rh factor is found in 85% of people.
  • Blood group % in US population  are  A-41%, B-9%, AB-4% and 0-46%.
  • Blood group % in Indian population are A-27%, B-31%, AB-8%  and  0-34%.
  • Blood group% in Nepales population are A-33%, B-27%, AB-12%  and  0-28%.

Further Reading

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732084/
  2. https://en.wikipedia.org/wiki/ABO_blood_group_system
  3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497328/
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741336/
  5. https://nios.ac.in/media/documents/dmlt/hbbt/Lesson-07.pdf
  6. https://www.labpedia.net/blood-banking-part-1-blood-groups-abo-and-rh-system-blood-grouping-procedures/
  7. https://rfums-bigtree.s3.amazonaws.com/files/resources/abo.pdf
  8. https://www.jstor.org/stable/40793065?seq=1#metadata_info_tab_contents

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