Direct Antiglobulin Test (DAT) and Indirect Antiglobulin Test
Direct and indirect antiglobulin tests:
The direct and indirect antiglobulin tests are two important tests in blood bank and a strong understanding of each will make your life easier.
Direct antiglobulin test (DAT):
The direct antiglobulin test can also be called the direct Coombs test, and it detects if antibody (IgG) or complement is bound to patient red cells. The test utilizes a reagent called antihuman globulin (AHG) which can also be referred to as antiglobulin. AHG is an antibody to an antibody. Reagent AHG is created by adding human serum to an animal such as a rabbit, and the rabbit makes antibodies to the antibodies in the serum (whoa an antibody to an antibody!?). AHG works by binding the fc portion of IgG molecules creating a cross linking which allows agglutination of RBCs with IgG attached. Unlike IgM, IgG antibodies cannot crosslink and agglutinate without the help of AHG. AHG should be added immediately after the last wash to prevent dissociation of antibody bound to RBCs. Polyspecific AHG will also have antibodies to the C3 portion of complement. This will cause RBCs coated with complement to agglutinate.
There are four main groups of clinical applications that the DAT is used for. Let’s break them down.
1. Hemolytic disease of the newborn (HDN)
a. We briefly learned earlier about the D antigen and how its presence can cause a strong transfusion reaction in a patient without the D antigen (Rh-negative). Another important reason to test for D antigen is pregnancy. Every person receives one copy of the RHD gene from mom and dad. In some cases dad will be Rh-positive and mom Rh-negative. Dad will pass the RH gene that codes for the D antigen and this will cause the baby to be Rh-positive. Baby’s blood can cross the placenta and it’s possible the mother will then make an antibody (IgG) to the baby’s blood. This can create a dangerous scenario because IgG antibodies are small enough to cross the placenta (IgM cannot cross). The IgG antibodies then attack the baby’s red cells causing a hemolytic reaction. The DAT can be used to detect maternal antibodies on cord RBCs.
2. Hemolytic transfusion reaction (HTR)
a. There are two main causes of this condition. The first is a patient’s own antibodies coat donor red cells.
b. The second is donor antibodies coat a patient’s own red cells. Either of these conditions can cause a positive DAT.
3. Autoimmune hemolytic anemia (AIHA)
a. There are three main causes of this condition. The first is warm autoimmune hemolytic anemia (WAIHA). Typically an IgG autoantibody (see warm antibodies below) and/or C3 complement protein binds to the patient’s red cells leading to red cell destruction.
b. The second is the activation of IgM and complement. IgM autoantibodies bind to red cells in the peripheral circulation, and complement protein binds to IgM when returning to higher temperatures in the body leading to red cell destruction.
c. The third is paroxysmal cold hemoglobinuria (PCH). Paroxysmal means intermittent, so it basically is hemoglobin in the urine intermittently (when it’s cold). IgG autoantibodies bind to red cells in colder parts of the body. The binding of the IgG causes complement protein to bind to the red cell leading to red cell destruction. PCH is associated with anti-P.
4. Drug induced autoantibodies
a. There are three main types of drug induced autoantibodies. The first is a hapten dependent antibody. A hapten is a small molecule that when bound to a larger molecule can cause an antibody response. The drug binds to the red cell and stimulates an antibody to be created to the hapten or hapten RBC complex.
b. The second is a drug causes an antibody to be produced against the RBC. The mechanism is unknown. The drug does not have to be present for the antibody to function.
c. The third is a drug causes an antibody to be produced against the RBC only when the drug is present. The mechanism is also unknown.
False positive DATs are commonly associated with clotted specimens where complement has coated the RBCs in vitro.
False negative DATs can be caused by failure to add AHG, or delayed addition of AHG. As outlined above AHG should be added immediately after the last wash to prevent dissociation of antibody bound to RBCs. Under centrifugation can also result in residual unbound antibody that can bind reagent AHG when AHG is added.
Ok, let’s jump into the indirect antiglobulin test (IAT).
Indirect antiglobulin test (IAT):
The indirect antiglobulin test is similar to the DAT except the IAT uses patient serum (for the most part), and the DAT uses patient RBCs. The most important distinctions between the two tests is that in the DAT the antibodies are attached already in the blood sample (in vivo); whereas, in the IAT patient serum or reagent antibodies are added to reagent red cells or donor red cells followed by an incubation period to see if antibodies attach in vitro. The IAT is used for many reasons in the lab. Here are a few.
1. Compatibility testing
a. For someone who is going to receive blood from a donor, the donor blood is tested with the patient serum to make sure the patient (recipient) antibodies don’t bind with the donor red cells.
2. Type and screen
a. The screen portion of the type and screen is used to detect unexpected antibodies in the patient serum. The type and screen doesn’t involve the need for donor blood so donor blood is not wasted and used efficiently.
3. The IAT can be used on an antibody panel for antibody identification.
4. The IAT can be used for weak D antigen testing. Weak D testing is performed on pregnant women if they test Rh-negative; it’s also used on donor red cells that initially test as Rh-negative. Let’s take a deeper dive into weak D and another important concept, partial D.
a. Weak D is defined as red cells that have the complete D antigen but just in lower quantity. Red cell antigens are complex structures that weave in and out of the red cell membrane. This low quantity can go undetected due to a lack of anti-D binding in regular Rh typing and can cause issues. For example, a weak D donor who was typed as Rh-negative could cause a transfusion reaction in a recipient who is Rh-negative.In weak D testing, donor RBCs are reacted with reagent anti-D. AHG is added and if there is agglutination the blood will be typed Rh-positive. If there is no agglutination check the control. The control should be donor RBCs and AHG only (no anti-D). If there is agglutination in the control, there is a non-specific immunoglobulin coating the donor RBCs. If the control is negative, use check cells, which are reagent red cells coated with IgG to confirm the test was properly setup. If there is no agglutination, the control is negative and the check cells are positive, the donor RBCs are Rh-negative.
b. Partial D is defined as red cells that don’t have the complete set of D-antigen epitopes. The D antigen has many different binding sites or epitopes and a mutation here or there in the RHD gene can cause a person’s red cells to not have the full set. There are a few problems that can arise from partial D. If a donor is typed as Rh-negative but is actually partial D, the epitopes the donor does have can cause a transfusion reaction in the Rh-negative recipient. Conversely, if a partial D recipient is typed as Rh-positive and receives Rh-positive blood, that patient will not recognize the complete D antigen and create antibodies to the epitopes it doesn’t have. This can cause a transfusion reaction as well.
False positive IATs can be caused by saline contaminated with bacteria and over centrifuged tubes.
False negative IATs can be caused by errors like not adding AHG or not adding patient serum (human error).
Ok so now we’ve covered the basics of ABO, Rh, and a few of the common tests used in the lab. Next we’ll look at more antibodies. There are a lot!