Rh Phenotyping using Tube Method


Apart from the ABO blood group system, the Rh system is also important because anti-RhD can cause hemolytic transfusion reactions (highly immunogenic) and are normally IgG, thus able to cross the placenta and lead to hemolytic disease of the newborn (HDN). The Rh system was discovered in 1939 by Karl Landsteiner and Alexander Wiener. 

The Rh system consists of two homologous transmembrane proteins, both encoded by genes on chromosome 1, that span the red cell plasma membrane 12 times. They appear to be used for the transport of carbon dioxide and/or ammonia across the plasma membrane. The Rh blood group locus is composed of 2 related structural genes, RhD and RhCE, which encode the membrane proteins that carry the D, Cc and Ee, antigens. The RhD gene may be either present or absent, giving the Rh D+ or Rh D- phenotype, respectively. Alternative RNA splicing from the RhCE gene generates 2 proteins, which encode the C, c, E or e antigens. Rh antibodies rarely occur naturally; most are immune. Anti-D is responsible for most of the clinical problems associated with the system and a simple subdivision of subjects into Rh D+ and Rh D- using anti-D is sufficient for routine clinical purposes. Anti-C, anti-c, anti-E and anti-e are occasionally seen and may cause both transfusion reactions and hemolytic disease of the newborn. Anti-d does not exist. 

While routine blood group testing often focuses on the RhD antigen, also known as the “D” positive or negative status, there’s a whole world of Rh phenotyping waiting to be explored. Beyond D, several other Rh antigens like C, c, E, and e play crucial roles in blood compatibility. Antibodies against these antigens can cause hemolytic reactions if incompatible blood is transfused. Luckily, commercially available reagents like anti-C, anti-c, anti-E, and anti-e can help identify these additional Rh phenotypes.

Rh phenotyping can be performed using various techniques, including tube agglutination tests and gel card tests. While tube tests were the traditional method, gel cards have become increasingly popular due to their ease of use, speed, and improved sensitivity.

Principle of Rh Phenotyping

At the heart of the protocol lies the concept of antigen-antibody interaction, similar to ABO RhD blood group typing. Rh proteins, like D, C, E, and e, adorn the surface of red blood cells, acting as antigens. If your body lacks a specific antigen, say D, it may develop antibodies against it (anti-D). Transfusing incompatible blood bearing that antigen triggers a violent immune response, potentially leading to life-threatening hemolytic reactions.

Red blood cells are first suspended in a solution containing antisera, each targeting a specific Rh antigen (e.g., anti-D, anti-C, anti-E). If a cell possesses the specific antigen, it binds to the corresponding antibody, forming visible clumps called agglutination. The absence of agglutination indicates a lack of the antigen

If a potential incompatibility is detected, red blood cells are subjected to individual tests with each antisera. This detailed analysis confirms the presence or absence of specific Rh antigens, allowing for a precise determination of the individual’s Rh phenotype.

Image illustrating the principle of forward blood group testing, highlighting antigen-antibody interactions
This image depicts the key principle involved in forward blood group testing, a process that determines an individual’s blood type. It illustrates how antibodies in the serum react with antigens on red blood cells, causing them to clump together or “agglutinate.”


  • Polypropylene/glass tubes 10 mm x 75 mm
  • Serofuge
  • Centrifuge
  • Pasteur pipettes
  • Commercially prepared anti-D, -C, -E, -c and -c sera
  • Patient’s EDTA whole blood
  • Normal saline


  1. Label each test tube with the corresponding antibody (Anti-D, Anti-C, etc.). Include a control tube with no antibody.
  2. Using a Pasteur pipette, add one drop of blood into each test tube.
  3. Add two drops of the appropriate antisera to each respective tube.
  4. Gently flick or rotate the tubes to mix the blood and reagents thoroughly.
  5. Place the tubes in a 37°C incubator for 30 minutes to allow for optimal antigen-antibody reactions.
  6. After incubation, centrifuge the tubes for 1 minute at 1000 RPM.
  7. Gently resuspend the cell button by tapping the tubes.
  8. Examine each tube under a light source to look for visible clumping or agglutination of red blood cells.


Positive results Rh phenotyping are indicated by agglutination of tested blood. A negative result is observed when there is a clear supernatant after resuspension in the red cell agglutination test.

  • Agglutination in a tube: Indicates the presence of the corresponding antigen on the red blood cells.
  • No agglutination: Indicates the absence of the corresponding antigen.
  • Control tube: Should show no agglutination, confirming the validity of the test.

Assessment of red cell agglutination test grading

SymbolAgglutination scoreDescription
4+ / Complete (C)12Macroscopically visible cell button with a clear supernatant.
3+10Macroscopically visible large clumps of cell button with a clear supernatant.
2+8Macroscopically visible small clumps of cell button with a clear supernatant. 
1+5Just macroscopically visible fine granular clumps of the cell button and the supernatant is turbid and reddish. 
or weak (w)3Only microscopically visible fine granules of the cell button and the supernatant is turbid.
00No agglutination seen. The supernatant is clear and reddish in color.
MF (mixed field)MFA mixture of agglutinated and unagglutinated red cells seen. 
HComplete hemolysis of the patient sample. The supernatant is grossly red with no evidence of red cells. 
This image depicts the red cell agglutination test score for forward blood group typing, a crucial step in determining an individual's Rh phenotyping. The agglutination patterns observed in the test tubes provide clear and distinct indicators of the blood group, ensuring accurate blood compatibility assessments for transfusions.
This image showcases the red cell agglutination test score, a pivotal tool in determining an individual’s Rh blood type. The distinct agglutination patterns observed in the test tubes provide definitive clues to the blood group, ensuring accurate blood compatibility for transfusions.

Disclaimer: This protocol is intended for informational purposes only and may need to be modified depending on the specific laboratory procedures and patient circumstances. Always consult with a qualified healthcare professional for guidance. See additional information.


  1. American Association of Blood Banks (AABB). Technical Manual, 21st Edition, 2023.
  2. Dean L. Blood Groups and Red Cell Antigens [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2005. 
  3. Harmening DM. Modern Blood Banking & Transfusion Practices (FA Davis Company). 2018.
  4. Li HY, Guo K. Blood Group Testing. Front Med (Lausanne). 2022 Feb 11;9:827619. doi: 10.3389/fmed.2022.827619. PMID: 35223922; PMCID: PMC8873177.

Related Diseases

Flow Cytometry Immunophenotyping of Blood

Flow Cytometry Immunophenotyping of Blood

Introduction Immunophenotyping using flow cytometry is a powerful technique used to identify and characterize cells based on the specific proteins they express on their surface or within their cytoplasm. These proteins, called antigens, act as unique markers that...

Hematopoietic cell surface markers

Hematopoietic cell surface markers

Introduction to Hematopoiesis Hematopoiesis, pronounced "hee-ma-toh-poy-ee-sis," is the remarkable process by which your body creates all the different types of blood cells including the red blood cells, white blood cells and platelets. . It's like having a...

Agarose Gel Electrophoresis Preparation for PCR

Agarose Gel Electrophoresis Preparation for PCR

Introduction Unraveling the mysteries of life often requires peering into the microscopic world of DNA. Conventional Polymerase Chain Reaction (PCR) is a powerful technique that allows us to amplify specific DNA sequences, making them visible and easily analyzed for...