Blood Grouping Principle and Procedure

Blood grouping

• The concept of Blood grouping was first discovered by Karl Landsteiner in 1901, who was an Austrian-American immunologist and pathologist. He received Nobel Prize in Physiology or Medicine in 1930 for this discovery.
• His discovery helps us to determine blood groups and thus opened a way for blood transfusions that can be carried out safely. 
• There are present two types of Blood grouping as forward grouping and Reverse grouping.
• The forward blood grouping is defined as using a known source of antibodies to detect the antigens on the red blood cells. 
• While Reverse grouping is defined as using the reagent cells with known ABO antigens and testing the patient’s serum for ABO group antibodies.
• The ABO and Rh are the major, clinically significant, and the most important of all the blood group systems
• The ABO blood groups are classified into four groups based on the presence or absence of two inherited antigenic substances on the surface of red blood cells (RBCs).
• The four major ABO blood groups are  “A” group, “B” group, “AB” group, “O” group.
• Blood group A contains antigen-A and antibody-B, Blood group B contains B-antigen and antibody-A, Blood group AB contains antigen-A and antigen-B and no antibodies, and Blood group O has no antigen and contains antibodies anti-A and anti-B.
• The Rhesus system (Rh) is the second most important blood group system in humans. The most significant and immunogenic Rhesus antigen is the RhD antigen. 
• The Rh factor is present on the surface of RBC in most people. It is a type of antigen and those who have it are called Rh+. Those who lack this antigen are called Rh-. 
• The Rh antibodies are absent in the blood of those persons who have Rh-. But they can produce Rh antibodies if they get blood from a Rh+ person, whose Rh antigens can induce the formation of Rh antibodies (as the immune system is triggered by the presence of an unknown antigen in the system).
• The  Rh+ person can receive blood from an Rh- person without any problem.
• The Rh system consists of two allelic genes such as; RhD and RhCE

The objective of Blood Grouping

To determine the blood group and Rh factor of an individual

Blood Grouping Principle

The ABO and Rh blood grouping system is based on agglutination reaction. When red blood cells carrying one or both the antigens are exposed to the corresponding antibodies they interact with each other to form visible agglutination or clumping. The ABO blood group antigens are O-linked glycoproteins in which the terminal sugar residues exposed at the cell surface of the red blood cells determine whether the antigen is A or B. Blood group A individuals have A antigens on RBCs and anti-B antibodies in serum. Similarly, blood group B individuals have B antigens on RBCs and anti-A antibodies in serum. Blood group AB individuals have both A and B antigens on RBCs and neither anti-A nor anti-B antibodies in serum. Whereas, blood group O individuals have neither A antigens nor B antigens, but possess both anti-A and anti-B antibodies in serum. The Rh antigens are transmembrane proteins in which the loops exposed on the surface of red blood cells interact with the corresponding antibodies. 

Material Required

• Anti A Sera
• Anti B Sera
• Anti RhD Sera
• Cavity slide
• Disposable Mixing Stick
• Blood Lancet
•  70% Alcohol/ Spirit
• Cotton 

ABO blood Grouping Procedure

The Blood Grouping test can be done by two methods such as Slide or tile method and the Tube method.

Slide or tile method

1. Dangle the hand down to increase the flow of blood in the fingers
2. Clean the fingertip to be pierced with spirit or 70% alcohol (usually ring or middle finger).
3. With the help of the sterile lancet, pierce the fingertip and place one drop of blood in each of the cavities.
4. Add one drop of antiserum into each cavity as shown below:

5. Mix each blood drop and the antiserum using a fresh mixing stick.
6. Observe agglutination in the form of fine red granules within 30 seconds. Anti RhD takes a slightly longer time to agglutinate compared to Anti A and Anti B.

Note: Proper care should be taken while disposing of the lancet and mixing sticks

Tube method

1. Take five test tubes and add one drop of anti-serum A, B, and AB in first three tubes.
2. Add one drop of the serum of the patient in tubes 4 and 5.
3. Add one drop of known RBC in A1 and B in tubes 4 and 5.
4. Add one drop of RBC 5% suspension of the patient in tubes 1, 2, and 3.
5. Add one drop of the patient serum in tubes 4 and 5.
6. Incubate at room temperature for 60 to 90 minutes.
7. OR centrifuge for 15 seconds.
8. Now, observe microscopically for agglutination or lysis using optical help from the following table 2.

Result of ABO blood Grouping

Slide Method

S. No.	Anti A	Anti B	Anti RhD	Blood Group
Slide 1	Agglutination	No agglutination	Agglutination	A +ve
Slide 2	No agglutination	Agglutination	Agglutination	B +ve
Slide 3	Agglutination	Agglutination	Agglutination	AB +ve
Slide 4	No agglutination	No agglutination	Agglutination	O +ve

Tube Method

Blood grouping tube method interpretations:

Tube 1	Tube 2	Tube 3	Tube 4	Tube 5	Blood group
anti-A	anti-B	anti-AB	A1- red blood cells	B-red blood cells
Negative	Negative	Negative	Positive	Positive	O
Positive	Negative	Positive	Negative	Positive	A
Negative	Positive	Positive	Positive	Negative	B
Positive	Positive	Positive	Negative	Negative	AB

Rh Blood Grouping Procedure

The Rh Blood Grouping can be done by both the slide method and tube method.

The slide method

1. Take two slides, one for the patient and one for the control.
2. Add one drop of Anti-Rh D on the patient slide and one drop of albumin on control slide.
3. Add one drop of 40% – 50% RBC suspension on both slide.
4. Mix them thoroughly on both slide.
5. Observe the agglutination on the slides.

The Tube method

1. Take two tubes, one for the patient and one for the control.
2. Add one drop of Anti-Rh D in patient tube and one drop of albumin in control tube.
3. Add one drop of 2% – 5% patient’s RBC suspension on both slide.
4. Mix thoroughly and then centrifuge them.
5. Gently resuspend the sample.
6. Observe for the agglutination.

Rh Blood Grouping Result

Result for Slide Method

• Positive Result: If the patient sample shows agglutination and the control shows suspension.
• Negative Result: If both patient and control sample shows suspension.

Result for Tube Method

• Rh-D Positive: Agglutination in patient’s tube; and smooth suspension in control tube.
• Rh-D Negative: Smooth suspension in both tubes.

Interpretation

• If agglutination is observed when blood is mixed with Anti A reagent, then the individual is said to have blood group “A”.
• If agglutination is observed when blood is mixed with Anti B reagent, then the individual is said to have blood group “B”.
• If agglutination is observed when blood is mixed with Anti A and Anti B reagent, then the individual is said to have blood group “AB”.
• If no agglutination is observed when blood is mixed with Anti A and Anti B reagent, then the individual is said to have blood group “O”.
• If agglutination is observed when blood is mixed with Anti RhD reagent, then the individual is said to have “+ve” Rh factor.
• If no agglutination is observed when blood is mixed with Anti RhD reagent, then the individual is said to have “-ve” Rh factor.

Application of blood grouping

• The blood grouping is used during the blood transfusion.
• It also used for paternity disputes.
• Used to detect hemolytic disease of newborn.
• Susceptibility to various diseases Ex: O group- peptic ulcer.
• Used for part of Health check-up and job, driving licensing, etc.

Precautions

• Read the entire procedure before preceding with the experiment.
• During the experiment wear gloves.
• Make sure the slide is clean and dry prior to use.
• Avoid touching the antisera reagent dropper to the blood sample.
• The result of the reaction should be interpreted immediately after mixing.
• To prevent a false result avoid the intermixing of the antisera reagents while performing the experiment.

References

• https://himedialabs.com/TD/HTI008.pdf
• https://vlab.amrita.edu/?sub=3&brch=69&sim=192&cnt=1
• https://www.labpedia.net/blood-banking-part-1-blood-groups-abo-and-rh-system-blood-grouping-procedures/
• http://www.histopathology.guru/academics/undergraduate-academics/rbc-bleeding-disorders-transfusion-medicine/blood-grouping/
• https://www.apollodiagnostics.in/blog/blood-types-and-blood-group-testing
• https://www.labtestsguide.com/blood-group-test-procedure
• https://www.healthline.com/health/how-to-find-out-your-blood-type#_noHeaderPrefixedContent