Red blood cells (RBCs) or erythrocytes are small, biconcave shaped cells (fig 1) circulating in the blood stream making up around 50% of the blood volume. They carry out important function of transporting oxygen to the cells and carbon dioxide to the lungs.

Fig 1: Coloured SEM images of normal RBC (Avsievich et al., 2019.)

With respect to the surface antigens, red blood cells in humans may differ from each other.  There are around 410 different blood cell antigens in the humans including ABO, Rh, Kidd, MNS and Lewis (see fig 2). These antigens trigger immunologic response when introduced into an incompatible recipient.

(Just for info: Read this review paper on different types of Red cell antigens)

Fig 2: Model of RBC membrane components that carry blood group antigens (Reid et al., 2012).

The most important red cell antigens are ABO and Rh. ABO blood group type is very important when in case of blood transfusion. Rh or Rhesus antigen is immunogenic and can cause red cell haemolysis. There are around 49 different kinds of Rh antigens. It  is clinically important, especially in the cases of the hemolytic disease in the newborns.

Anti- Rh antibodies are IgG (mostly subclasses IgG1 and IgG3, less often IgG2 and IgG4) and IgM. Based on their capacity to agglutinate these antibodies can be described as the Complete or Incomplete antibodies.

Complete antibodies: These antibodies are capable of causing agglutination. This includes the IgM antibodies, which can directly agglutinate red cells in saline, due to their pentameric structure (Fig 3).

Fig 3: RBCs agglutination by the pentameric IgM.

Incomplete or blocking antibodies: These antibodies cannot directly bring about agglutination of the red cells. The incomplete antibodies, includes IgG that cannot bring negatively charged RBCs together, due to their short Fab arms and repulsion between red cells. However, IgG sensitises the RBCs and can lead to hemolysis.

The incomplete antibodies can bind and block the antigenic sites. This blocking prevents agglutination by other agglutinators, including IgM, as well. Due to it’s ability to not agglutinate the red cells, anti-Rh IgG antibody can remain ‘hidden’.

Fig 4: Monomeric IgG are unable to agglutinate RBCs due to distance between the repelling negatively charged RBCs.

Complement components:

Both IgM and IgG (IgG1 and IgG3 subtypes) antibodies can fix complement and cause hemolysis. Hence the blood may show presence of complement components (such as C3b).

When an Rh negative person is exposed to Rh positive RBCs during either transfusion, pregnancy or organ transplantation, anti-Rh antibodies and complement components like C3b are synthesized. Hence utmost care should be taken before and after these processes and the blood should be checked for compatibility.

A test, called the ‘Coombs test‘, has been designed for identifying the compatibility of blood from two individuals. Coombs test is also known as Antiglobulin test and was discovered by Robin Coombs, Arthur Mourant and Rob Race in the early 1940s.

In Coombs test, presence of the incomplete IgG antibodies and the complement component against the Rh antigen are detected.

Anti-IgG antibody (see in fig 5) against the Fc portion of anti-Rh (incomplete) antibody is used to cross link and agglutinate the RBCs. Anti-IgG antibody is an anti-human immunoglobulin, made by injecting human IgG into animals, which produce polyclonal antibodies specific for human IgG. Similarly antibodies against human complement system factors are synthesized. Now-a-days, monoclonal antibodies are used. The solution of the anti IgG and anti-C3b called Coombs reagent.

(Just for info: Read our post on Monoclonal Antibody Production.)

Coombs reagent is added to the sample with RBC and IgG and observed for agglutination. Based on the source of the anti-Rh IgG or the sample used, the test are of two different types: The Direct and Indirect Antiglobulin test.

1. Direct Coombs Test or Direct antiglobulin test (DAT):

Source of anti-Rh IgG: Surface of RBCs.

DAT is used to detect the RBCs bound antibodies or sensitisation. The antibodies against the complement component (C3) to may also be used to detect their presence on the surface of RBCs. It is a one step process as shown in the fig 5.

Fig 5: Positive DAT: Agglutination of RBCs bound with incomplete IgG by anti-IgG antibodies.


The DAT have three components:-


-Antibodies bound to the RBC antigen (anti-Rh IgG)

-Anti-human IgG


The RBCs are washed with isotonic saline solution. To these RBCs, Anti-human serum specific for IgG (or Coombs reagent) is added. The mixed sample is observed to for agglutination, both macroscopically and microscopically.


This test can help to detect immunologic (e.g., autoimmune) and non-immunologic (e.g., drug-induced) hemolytic anemias.

• Immunologic Anaemia

DAT helps analyse autoimmune hemolysis. In autoimmune disorders an individual produce antibodies against his own RBCs and result into Autoimmune Hemolytic Anaemia.

• Non immunologic Anaemia:

Some drugs can cause the immune system to recognize own red cells antigens as foreign, resulting into synthesis of antibodies attacking the body’s own red blood cells leading to hemolysis.

• Diagnose Hemolytic disease of the newborn (HDN):

The direct Coombs test detects if maternal anti-Rh antibodies have already bound fetal RBCs. On this test Fetal RBCs are mixed with anti-Ig antibodies and agglutination is observed.

(Just for info: know more about autoimmune hemolytic anemia.)

2. Indirect antiglobulin test (IAT):

The indirect Coombs’ test detects free antibodies against human RBCs in the patient’s serum*. This is a two step process (see fig 6).

(*serum is the fluid part of the blood without the clotting factors and the blood cells.).


The components of indirect Antiglobulin test are

– Recipients/patient’s serum

– Donors RBCs

– Anti-IgG antibodies


In the indirect antiglobulin test (IAT), the serum is collected from the patient/ recipient. This serum is incubated with the normal Rh+ve blood cells. If the serum contains the anti-Rh antibodies, the RBCs and anti-Rh antibodies complexes are formed. When anti-IgG antibodies against anti-Rh antibodies are added, the RBC complexes are cross linked and the agglutination occurs (see fig 6).

Fig 6: Positive IAT- Agglutination of normal RH +ve RBCs by anti-IgG antibodies due to presence of anti-Rh antibodies.


• Crossmatching:

The indirect Coombs’ test is used in crossmatching. The IAT should be performed before blood transfusion to avoid anti-Rh antibodies in Rh-ve recipient destroying donors Rh+ve RBCs, and leading to haemolytic transfusion reactions. A positive IAT test result means the recipient’s blood is incompatible with that of the donor’s.

(Just for info: know more about haemolytic transfusion reactions)

• Prevent HDN:

The indirect Coombs test helps in detecting anti-Rh antibodies in the (Rh-ve) mother’s serum, which could bind and lead to lysis of fetal (Rh+ve) RBCs, hence causing HDN.

In this test, mother’s serum is incubated with Rh-positive RBCs. If mothers serum have anti Rh antibodies, agglutination takes place on addition of anti-Ig antibodies.

Detecting maternal anti-Rh before binding fetal RBCs can help take preventive measures which include administrating anti-Rh antibodies.

~ Hence Coombs is an important diagnostic test in the medical field.

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Blood Cell Antigens and Antibodies. Fiona A.M. Regan, in Dacie and Lewis Practical Haematology (Twelfth Edition), 2017

Avsievich T, et al. Sci Rep. 2019.Mutual interaction of red blood cells influenced by nanoparticles. Sci Rep. 9(1): 8430.

Reid et al. (2012) The Blood Group Antigen FactsBook. 3rd Edition. Academic Press 2012

Dean L. Blood Groups and Red Cell Antigens [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2005. Chapter 4, Hemolytic disease of the newborn.