Determination of Blood Group: ABO and Rh Systems

“Blood group” refers to the system that comprises specific red blood cell (RBC) antigens from a series of allelic or closely-linked genes in the same chromosome. Blood type is the particular reaction pattern when testing the antisera within a given system.     

Karl Landsteiner discovered the blood grouping system in 1901, which helped understand the reason for clumping, sometimes observed when the blood of two individuals was mixed. He gave the concept of the two clinically most crucial blood grouping systems; ABO and Rh system of blood grouping. Screening of blood groups of donors and receivers before transfusion is critical as incompatibility during a transfusion can lead to the death of the receivers. Besides ABO and Rh systems, there is numerous other blood grouping system; some of them are the Lewis system, H-antigen system, Lutheran grouping system, and MNS grouping system. 

Blood Grouping System

At present, there are 33 types of blood groups. Among which ABO system and Rh blood grouping system are most commonly used. Some of the blood grouping systems are discussed below:

ABO Blood Group System

It is the most important type of blood grouping system for screening blood because people above six months possess clinically significant anti-A or anti-B antibodies in their serum. The ABO blood group relies on the three genes, A, B, and O, located on chromosome 9. The genotype and phenotype of all the genes are as follows:

Genotype Phenotype
AA or AOBlood group A
BB or BOBlood group B
ABBlood group AB
OOBlood group O
  1. The person with AO or AA genotype has A antigen in their RBC (red blood cell) and antibodies against B (anti-B) in their serum. 
  2. The person with BO or BB genotype has B antigen in their RBC and anti-A in their serum. 
  3. The person with genotype AB has both A and B antigens in their RBC. But neither anti-A nor anti-B in the serum. 
  4. The person with the OO genotype has neither A nor B antigen in their RBC. But has anti-A and anti-B in their serum.   

Rh Blood Group System

The Rhesus or Rh system is the second most crucial blood grouping system. The C,c, D,d, E, and e are responsible genes in the Rh system. The six genes help in obtaining thirty-six different combinations of genotypes. But the D gene plays a crucial role in the Rh grouping. RBC of any individual may or may not have an Rh factor or immunogenic D-antigen. The presence of antigens and antibodies determines a person as Rh positive or Rh negative.

  1. Rh positive: Has D-antigen in the RBC.
  2. Rh negative: Lacks D-antigen in the RBC.

Unlike the ABO system, anti-D does not circulate in those that lack D-antigen but forms in the body if transfusion of Rh positive blood group occurs in Rh negative person. 

Lewis Blood Group System

In the Lewis blood grouping system, the Lewis antigen is formed in the tissues and secreted in body fluids which are then absorbed by the RBC membrane. Since the Lewis antigen is secreted as bodily secretion, it is also known as secretor antigens. It has been the cause of retarded growth in children and can sometimes lead to transfusion reactions.    

MNS Antigen Blood Group System

Landsteiner and Levine described MNS antigen system in 1927. This system is based on two genes, glycophorin A and glycophorin B. An autosomal locus on chromosome 4 and a pair of alleles LM and LN control this blood group. The anti-M and anti-N are usually IgM and are rarely associated with transfusion reactions.  

Lutheran Grouping System

The Lutheran grouping system has four allelic antigens representing single amino acid substitution in the Lutheran glycoprotein at chromosome 19. It is not clinically significant as antibodies against the blood group are rare.  

Determination of ABO Group Blood

The determination of the ABO group is called blood grouping, blood typing, or blood matching. Two methods can do the ABO grouping; 

  1. Cell grouping- where the RBCs are tested for antigens A and B using anti-A and anti-B sera.
  2. Serum grouping- where the serum is tested for anti-A and anti-B antibodies using known A and B antigens. It is also known as a reverse grouping.  

Principle of ABO Blood Grouping

The immunological reaction- agglutination is the basis for performing ABO blood grouping. Agglutination occurs when the collection of antigen and its corresponding antibodies mixes, and separated particles are seen in clumps or masses. In the case of ABO grouping, agglutination occurs when A or B antigen is mixed with anti-A or anti-B antibodies, respectively.

GroupAntigen in RBCAntibody in serum
ABA and B both No antibody
ONo antigenanti-A and anti-B

Requirement for ABO Blood Grouping

  1. Blood sample of patient and donor
  2. Monoclonal anti-A and anti-B antisera. (Anti-A is coded blue color, and anti-B is coded yellow color)
  3. Tile/Tube

Procedure for ABO Blood Group Determination

Tube method

  1. Take two glass tubes with 1 volume (1 drop) of 3-5% RBC of patients.
  2. Place 1 ml anti-A serum in the first tube and anti-B serum in another tube.
  3. Mix the content by gently taping at the base of the tubes.
  4. Leave the tubes at room temperature for five minutes and centrifuge at the lowest setting for one minute.
  5. Then observe the tubes for agglutination. 

Tile method

  1. Place a drop of anti-sera A on one end of a clean, grease-free tile and one anti-sera B on another side.
  2. Then place the drop RBC suspension on both the antiserum and mix thoroughly. Rock the slide slightly for 2 minutes.
  3. Observe the agglutination (clumps) with the naked eye and confirm using the microscope.    

Result Interpretation

Tube method

  1. If the agglutination occurs in the tube with antiserum A, the blood group is A.
  2. If the agglutination occurs in the tube with antiserum B, the blood group is B.
  3. If the agglutination occurs in both tubes, the blood group is AB.
  4. If no agglutination occurs, the blood group is O.

Tile method

  1. If agglutination occurs in the end with antiserum A, the blood group is A.
  2. If agglutination occurs in the end with antiserum B, the blood group is B.
  3. If agglutination occurs in both ends, the blood group is AB.
  4. If there is no agglutination, the blood group is O.

Determination of Rh Factor

Rh or Rhesus factor is present in the RBC as antigens. Landsteiner and Wiener first discovered it in the “Rhesus” monkey. Since antigen D is more antigenic than other Rh antigens, agglutination reaction to antiserum D gives a positive test result.


Among all the genes responsible for producing Rh factor, the D gene secreting antigen D in the RBC of humans is more prominent. So, during the determination of the Rh factor, the presence of the D antigen is considered Rh-positive. In contrast, the absence of D antigen after agglutination reaction with antiserum D is Rh negative. 


  1. Patient’s blood
  2. Monoclonal antisera D (color-coded red)
  3. Tile/Tube


Like ABO typing, the Rh factor determination is carried out in tile as well as tube. 

The procedure is similar to the ABO grouping system.

Tube method

  1. Take 1 volume (1 ml) of blood in a glass tube.
  2. Mix an equal volume of antisera D in the same tube.
  3. Centrifuge at low speed for 1 minute.
  4. Observe the agglutination with the naked eye or for confirmation under a microscope.

Tile method

  1. Place a drop of the patient’s blood at the end of the tile.
  2. Gently place a drop of antisera D on top of the blood drop.
  3. Mix thoroughly using a toothpick or mixer.
  4. Observe agglutination after 2 minutes.


  1. If agglutination is seen, Rh is positive.
  2. If agglutination is absent, Rh is negative.

Importance of Blood Grouping

Performing blood grouping before transfusion is essential to avoid the complication of transfusion reactions. The following are some of the importance of blood grouping:

  1. Knowing your blood type: In case of an emergency, knowing your blood group can be critical because you may require a blood transfusion or be eligible to donate to a suitable person. Also, it helps in donating blood if you are fit and healthy, are 17-66 years old, and weigh at least 50 Kg.
  2. Cross-matching during transfusion: Transfusion should only be performed between compatible persons. If the blood group is not compatible, it may lead to complications as blood can hemolyze and cause the person’s death.   
  3. During pregnancy: Rh factor plays a critical role in the pregnancy, so blood grouping is always advised for pregnant mothers. Rh-negative mothers should be given Rh-negative blood; if the child is Rh-positive, there is a risk of Rh sensitization. Rh sensitization can lead to a variety of complications in pregnancy. The pregnant woman is provided with Rh immunoglobulin (RhIg) at 28 weeks to avoid the risk, which stops the production of Rh antibodies for the rest of the pregnancy.   

Transfusion Reactions Due to Incompatibility

Transfusion reactions are adverse reactions in the recipient’s body due to the transfusion of incompatible or mismatched blood. A reaction occurs between the donor’s RBC and the recipient’s plasma during the incompatible transfusion. It happens only when the amount of agglutinins against the donor’s RBC is high in the recipient’s plasma. The reaction can be mild with hives and slight fever or severe, leading to renal failure, shock, and death. So, it is categorized into two subheadings; non-hemolytic and hemolytic transfusion reaction.

Non-hemolytic Transfusion Reaction

A non-hemolytic transfusion reaction can occur after a few minutes to hours (due to ABO incompatibility) or a month (due to Rh incompatibility). These are very mild reactions symbolized by common symptoms like fever, difficulty breathing, and severe itching. 

Hemolytic Transfusion Reaction

Hemolytic transfusion reaction may be acute or delayed in case of ABO incompatibility. During an acute hemolytic reaction, rapid hemolysis occurs in the donor’s RBC. It shows the symptoms like fever, chills, increased heart rate, shortness of breath, nausea, vomiting, red urine, low blood pressure, bronchospasm, rigor, and back pain, and some may develop pulmonary edema and congestive cardiac failure. 

Delayed hemolytic transfusion reaction occurs after 1-5 days of incompatibility of the ABO blood group. The hemolysis of RBC results in a large amount of hemoglobulin in the plasma. It leads to; jaundice, cardiac shock, and renal shutdown.

During Rh incompatibility, the delayed transfusion reactions begin when the incompatible blood is transfused for the second time and can lead to the same complications as in ABO transfusion reactions.


  • Cheesbrough, M. (2006) “Blood transfusion practice,” in District Laboratory practice in tropical countries Part 2. second. Cambridge: Cambridge University Press, pp. 348–369. 
  • Mitra, Ranadhir & Mishra, Nitasha & Rath, Girija. (2014). Blood groups systems. Indian journal of anaesthesia. 58. 524-8. 10.4103/0019-5049.144645.
  • NHS. (2023). Blood groups. NHS choices.

Ashma Shrestha

Hello, I am Ashma Shrestha. I had recently completed my Masters degree in Medical Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology.

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