Blood Grouping by Agglutination Test

 

Perform Blood Grouping by Agglutination Test

Objective- To determine the ABO blood group and Rh factor of a blood sample by observing the agglutination reaction with specific antisera.

Theory

Blood grouping is based on the presence or absence of specific antigens (A and B) on the surface of red blood cells (RBCs). The ABO blood group system classifies blood into groups A, B, AB, and O depending on these antigens. The Rh system classifies blood as Rh-positive or Rh-negative based on the presence or absence of the D antigen.

When RBCs are mixed with anti-A, anti-B, or anti-D (anti-Rh) sera, agglutination (clumping) occurs if the corresponding antigen is present on the RBC surface. This agglutination is visible to the naked eye and is used to determine the blood group.

Requirements

• Fresh blood sample (usually capillary or venous)
• Anti-A serum
• Anti-B serum
• Anti-D (Rh) serum
• Clean glass slides
• Sterile lancet or needle (for capillary blood)
• Micropipettes or pipettes
• Normal saline (0.85% NaCl)
• Clean applicator sticks or disposable toothpicks

Procedure

1. Label three spots in glass slides as Anti-A, Anti-B, and Anti-D.
2. Place a drop of each respective antiserum on the labeled slides.
3. Add a small drop of blood sample to each drop of serum.
4. Mix each serum and blood drop thoroughly using a separate clean applicator stick.
5. Observe the mixtures for visible agglutination within 1-2 minutes at room temperature.

Observation Table

Test Mixture	Agglutination (+) / No Agglutination (–)	Interpretation
Blood + Anti-A	+ / –	Presence/absence of A antigen
Blood + Anti-B	+ / –	Presence/absence of B antigen
Blood + Anti-D	+ / –	Presence/absence of Rh factor

 Result

• Agglutination with Anti-A only: Blood group A
• Agglutination with Anti-B only: Blood group B
• Agglutination with both Anti-A and Anti-B: Blood group AB
• No agglutination with Anti-A and Anti-B: Blood group O
• Agglutination with Anti-D: Rh-positive
• No agglutination with Anti-D: Rh-negative

Discussion

Blood grouping by agglutination is a reliable, rapid, and simple method to determine ABO and Rh blood groups. This test is critical for safe blood transfusions and organ transplantation. Improper grouping may lead to transfusion reactions. Hence, it is important to perform the test carefully, interpret results accurately, and confirm if needed.

Conclusion

The qualitative agglutination test allows identification of ABO and Rh blood groups by detecting specific antigens on red blood cells. Positive agglutination confirms the presence of corresponding antigens and helps guide safe transfusion practices.

Precautions

• Use fresh blood samples to avoid false results.
• Avoid contamination between samples and reagents.
• Use separate applicators for mixing each test.
• Interpret agglutination carefully and repeat if uncertain.
• Always confirm blood group with a second test if possible.

Reference

Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries, Part 2. Cambridge University Press.