Direct Antiglobulin (Coombs) Test (DAT)

Introduction

The direct antiglobulin test (DAT), also known as the Coombs test, is a crucial tool in diagnosing immune-mediated red blood cell (RBC) destruction. This test detects the presence of antibodies or complement proteins bound to the surface of RBCs, indicating a potential autoimmune reaction or incompatibility with an external antigen. 

Red blood cells (RBCs) carry a net negative charge due to sialic acid residues on their surface. This negative charge creates a repulsive force, preventing them from directly sticking together. However, the presence of antibodies and complement proteins can bridge this gap and cause RBCs to clump together, a process called agglutination.

There are two main types of antibodies involved in agglutination:

  • Agglutinating antibodies: These antibodies have two antigen-binding sites, allowing them to bind to two different RBCs simultaneously. This strong binding overcomes the repulsive force and leads to visible clumping.
  • Non-agglutinating antibodies: These antibodies typically have only one antigen-binding site and are weaker than agglutinating antibodies. While they can bind to RBCs, they cannot overcome the repulsive force and form visible clumps. These non-agglutinating antibodies are mostly of the IgG class, although some IgM antibodies can also fall into this category.

Non-agglutinating antibodies are often called “sensitizing antibodies” because they mark the RBCs for destruction by the spleen or phagocytes. While they may not cause visible clumping, the bound RBCs are still vulnerable to clearance. This is why the direct antiglobulin test (DAT) is essential for detecting these sensitized RBCs, even if agglutination is not evident.

Principle of DAT

Agglutination of red blood cells in the direct antiglobulin test, revealing immune-mediated coating
File:Coombs test schematic.png” by No machine-readable author provided. A. Rad~commonswiki assumed (based on copyright claims). is licensed under CC BY-SA 3.0.

At the heart of the DAT lies the unique property of RBCs – their negative surface charge. This charge acts like a built-in shield, repelling each other and preventing them from clumping together. However, when an antibody or complement protein binds to an RBC, it acts as a bridge, overcoming this repulsive force. This allows multiple RBCs coated with the same antibody or complement to clump together, a phenomenon called agglutination.

Antihuman globulin (AHG) is a specially designed antibody that recognizes and binds to the Fc portion of human antibodies and complement proteins. Essentially, AHG acts like a “double agent,” recognizing the immune cloak worn by the sensitized RBCs.

The DAT protocol revolves around this interaction. The DAT plays a crucial role in diagnosing conditions where in vivo coating of RBCs by antibodies or C3d occurs, leading to their premature destruction. Washing the cells eliminates freely circulating antibodies, allowing for the specific detection of those directly attached to the RBCs. Then, AHG is introduced. If the RBCs are coated with antibodies or complement, AHG binds to them, forming visible clumps. This agglutination signifies a positive DAT. This bound antibody-AHG complex formation reveals sensitized RBCs, providing valuable clues for the diagnosis of autoimmune hemolytic anemia, hemolytic disease of the newborn, drug-induced hemolysis, and other immune-mediated disorders. 

A negative DAT, on the other hand, suggests the absence of bound antibodies or complement. However, this doesn’t guarantee the absence of an immune reaction. Some antibodies, particularly weak ones, may not cause visible agglutination even though they are present. This is why the DAT has different sensitivity levels, utilizing polyspecific AHG for broad detection and monospecific AHG for identifying specific antibody types.

Materials

  • Patient’s EDTA-anticoagulated blood sample
  • Normal saline (0.9% NaCl)
  • Antihuman globulin (AHG) reagents: polyspecific, anti-IgG, and anti-C3d
  • Glass tubes
  • Centrifuge
  • Coombs control red cells – IgG coated red cell (CCC)

Protocol

  1. Prepare a red cell suspension by washing 5 mL patient RBCs with normal saline 3 – 4 times, discard the supernatant and resuspend RBCs to a 3-5% suspension in saline.
  2. Label four test tubes: Polyspecific, IgG, C3d, and Negative Control.
  3. Add one drop of RBC suspension to each tube.
  4. Add 2 drops of the respective AHG reagent to each tube (Polyspecific, IgG, C3d).
  5. Add 2 drops of saline to the Negative Control tube.
  6. Mix the suspension by flicking the tubes gently to thoroughly resuspend the cells.
  7. Centrifuge the tubes for 15-20 seconds at 1000-1500 RPM.
  8. Incubate Polyspecific and C3d tubes at 37°C for 5 minutes (optional).
  9. Gently resuspend cell buttons and observe for agglutination macroscopically and microscopically.
  10. Grade agglutination strength (0 to 4+) if present.
  11. In the absence of agglutination after initial centrifugation, incubate the mixture at room temperature for 5 minutes (if recommended by the manufacturer).
  12. Recentrifuge, resuspend, and re-assess for agglutination. 
  13. If still negative, add Coombs control cells (CCC) and centrifuge as per manufacturer instructions. Observe and record any agglutination with CCC, confirming test validity.

Interpretation

This image depicts the red cell agglutination test score. The agglutination patterns observed in the test tubes provide clear and distinct indicators presence of red blood cell antibodies.
This image showcases the red cell agglutination test score. The distinct agglutination patterns observed in the test tubes provide definitive clues to presence of antibodies.

Positive DAT

  • Agglutination of red blood cells (RBCs) in the test indicates the presence of antibodies or complement proteins bound to the RBC surface in vivo.
  • Strength of agglutination (graded 0 to 4+) can provide clues about the amount of bound antibody or complement.
  • Specific AHG reagent used (polyspecific, anti-IgG, anti-C3d) can help narrow down the type of immune involvement.

Negative DAT

  • No agglutination suggests the absence of significant RBC-bound antibodies or complement.
  • Does not completely rule out immune hemolysis, as weakly bound antibodies or complement may not be detected.
  • Clinical correlation with patient symptoms and other laboratory findings is essential.

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. 

Function of Coombs Control Cells (CCC)

  • Quality control measure to ensure the validity of the DAT reagents and technique.
  • Consists of RBCs pre-coated with antibodies in the laboratory.
  • Should agglutinate when mixed with AHG reagent, demonstrating its ability to detect antibody-coated RBCs.
  • Negative CCC reaction with AHG suggests a technical error or reagent issue for example AHG was not added or inadequate, the reagent has expired or the AHG has been neutralized by free antibodies due to insufficient washing of the red cells.

Possible Causes of Positive DAT

Algorithm for the main causes in positive direct antiglobulin test (DAT, or direct Coombs test).
Algorithm for the main causes in positive direct antiglobulin test (DAT, or direct Coombs test). “Algorithm in positive direct antiglobulin test (DAT, or direct Coombs test)” by Mikael Häggström, M.D. Author info – Reusing images- Conflicts of interest: None Mikael Häggström, M.D. is marked with CC0 1.0.
  • Autoimmune hemolytic anemia (AIHA): Immune system attacks and destroys its own RBCs.
  • Hemolytic disease of the newborn (HDN): Maternal antibodies attack fetal RBCs, causing hemolysis.
  • Drug-induced immune hemolytic anemia (DIIHA): Certain drugs can trigger antibody formation against RBCs.
  • Transfusion reactions: Incompatible blood transfusions can lead to antibody-mediated hemolysis.
  • Paroxysmal cold hemoglobinuria (PCH): Cold-induced destruction of RBCs by antibodies.
  • Myelofibrosis: Bone marrow disorder sometimes associated with positive DAT.
  • Evans syndrome: Combination of AIHA and immune thrombocytopenia (low platelets).
  • Infections: Some infections (e.g., cytomegalovirus, Epstein-Barr virus) can cause transient positive DAT.
  • Solid tumors and lymphomas: Rarely associated with positive DAT.
  • Allogeneic bone marrow transplantation: Immune reactions against donor RBCs can cause a positive DAT.

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.

References

  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. Bain BJ, Bates I, Laffan MA. Dacie and Lewis Practical Haematology: Expert Consult: Online and Print 12th Edition (Elsevier). 2016.

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