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 direct antiglobulin test (DAT)

At the heart of the direct antiglobulin test (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 direct antiglobulin test (DAT) protocol revolves around this interaction. The direct antiglobulin test (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 direct antiglobulin test (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 direct antiglobulin test (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 direct antiglobulin test (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

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

Symbol	Agglutination score	Description
4+ / Complete (C)	12	Macroscopically visible cell button with a clear supernatant.
3+	10	Macroscopically visible large clumps of cell button with a clear supernatant.
2+	8	Macroscopically visible small clumps of cell button with a clear supernatant.
1+	5	Just macroscopically visible fine granular clumps of the cell button and the supernatant is turbid and reddish.
or weak (w)	3	Only microscopically visible fine granules of the cell button and the supernatant is turbid.
0	0	No agglutination seen. The supernatant is clear and reddish in color.
MF (mixed field)	MF	A mixture of agglutinated and unagglutinated red cells seen.
H		Complete 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

• 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.

Frequently Asked Questions (FAQs)

What is a direct antiglobulin test (DAT) test used for?

The DAT (Direct Antiglobulin Test) is a laboratory test used to diagnose conditions related to the destruction of red blood cells (RBCs) by the immune system. Causes of positive DAT has been listed in the article above.

What is the difference between DAT and antibody test?

The key difference between a DAT (Direct Antiglobulin Test) and an antibody test lies in what they are looking for and how they approach detection.

• DAT (Direct Antiglobulin Test): This test specifically focuses on red blood cells (RBCs). It checks for antibodies that are already attached to the surface of RBCs. A positive DAT indicates that these antibodies are binding to and potentially destroying the RBCs.
• Antibody Test (Broader Term): This is a broader term encompassing various tests that can detect different types of antibodies in the blood, not just those targeting RBCs. It can include tests like:
  • Indirect Antiglobulin Test (IAT): This test, also known as the indirect Coombs test, looks for free antibodies in the blood plasma that have the potential to bind to RBCs. Unlike the DAT, these antibodies aren’t attached yet. A positive IAT indicates a risk of problems during a blood transfusion or in a newborn with hemolytic disease.
  • Infectious Disease Antibody Tests: These tests detect antibodies produced by the immune system in response to a specific infection, such as HIV or hepatitis.
  • Autoimmune Antibody Tests: These tests identify antibodies directed against the body’s own tissues, which can be helpful in diagnosing autoimmune diseases like lupus or rheumatoid arthritis.

What does Coombs positive mean for a baby?

A Coombs positive test result for a baby, also known as a positive direct antiglobulin test (DAT), indicates that antibodies are attached to the baby’s red blood cells (RBCs). This doesn’t necessarily mean there’s a serious problem, but it does require further investigation to determine the cause and potential risks.

Possible Reasons for Positive Coombs Test

• Rh Incompatibility: This is a major concern when a Rh-negative mother carries an Rh-positive baby. The mother’s immune system might develop antibodies against the baby’s Rh-positive RBCs, which can lead to hemolytic disease of the fetus and newborn (HDFN). HDFN can cause anemia and other complications in the baby.
• ABO Incompatibility: This is a less concerning scenario where the mother’s blood type (A, B, or AB) is incompatible with the baby’s blood type (usually O). However, unlike Rh incompatibility, ABO incompatibility rarely causes severe problems for the baby.
• Maternal Antibodies: In some cases, the mother might have pre-existing antibodies from a previous pregnancy or blood transfusion that can target the baby’s RBCs.

What Happens Next

• Further Testing: Doctors will likely perform additional tests to determine the cause of the positive DAT and assess potential risks. This might include:
  • Antibody Identification: This test pinpoints the specific antigens targeted by the antibodies in the mother’s blood.
  • Evaluation of Mom’s Blood Type and Rh Status: This confirms the mother’s blood type and Rh factor.
  • **Fetal Blood Typing (if possible): **This test, depending on the pregnancy stage and medical technology available, can determine the baby’s Rh factor and blood type, if possible.
  • Amniocentesis (in some cases): This prenatal diagnostic test involves collecting a small amount of amniotic fluid to analyze fetal cells and potentially determine the baby’s Rh status. However, this is usually a later-stage option due to its invasive nature.
  • Bilirubin Levels: Monitoring the baby’s bilirubin levels can help assess the severity of any potential breakdown of red blood cells.

Potential Outcomes

• Close Monitoring: If the cause is ABO incompatibility or the baby shows no signs of anemia, close monitoring of the baby’s bilirubin levels and health might be sufficient.
• Rh Incompatibility Management: If Rh incompatibility is confirmed and the baby is at risk for HDFN, doctors can take steps to prevent complications. This might involve administering Rh immunoglobulin (RhoGam) to the mother at specific intervals during pregnancy. RhoGam helps suppress her immune response and protect the baby’s red blood cells.
• Treatment for Anemia: If the baby develops anemia due to RBC destruction, they might require phototherapy treatment to help break down bilirubin or even blood transfusions in severe cases.

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.