Direct antiglobulin test (DAT or direct Coombs' test)

A. General.

A purple top (EDTA) tube is required to avoid false positive reactions. Testing takes 10 to 15 minutes, working without interruption. The DAT looks for immunoglobulin or complement on the surface of circulating RBCs. The first step uses patient red cells mixed with polyspecific Coombs' reagent as a screening step. Polyspecific Coombs' reagent in the PMH lab looks only for IgG and C3d. It does not look for IgM, IgA, C3b or C4. If this step is positive, a second step is performed using anti-human IgG and anti-human C3d separately.

B. Evaluation of a positive DAT.

The resident will be called to investigate a positive DAT if:

  1. it is a new finding (there is no previous history, or the DAT was previously negative)
  2. it is stronger than it was previously.

YOU ARE TO DECIDE WHETHER ADDITIONAL SEROLOGICAL WORK NEEDS TO BE DONE NOW, PRIOR TO THE ISSUE OF BLOOD. Usually the additional work will be to perform a red cell elution. We are worried that the positive DAT represents a new auto- or alloantibody to the patient's RBCs that may present a threat to future transfusions. It is possible that a newly emerging antibody will be present in such low levels that it is all bound to the RBCs and is not detectable free in the serum.

  1. First, find out the urgency of the transfusion request. If blood is emergently needed for a life-threatening bleed, issue blood right away with a "blue card" and proceed with the work-up as soon as possible. Normally, we try to resolve the problem of a positive DAT before issuing blood for transfusion, but we must not obstruct necessary patient treatment.
  2. Next, find out if this is really a positive DAT, and not just a positive auto-control (see section below on autocontrol) or testing performed on a clot specimen only. Sometimes the staff get careless with their terminology and tell you it's a positive DAT, when in fact a purple top tube has not been obtained yet. Testing of a clot specimen can result in a false positive test for complement because of in vitro artifact. Therefore, if a purple top tube has not been tested yet, especially if the finding is a positive result for complement, ask for DAT testing on an EDTA specimen.
  3. Assess the serological findings. How strong is the DAT? The stronger the DAT, the more likely it is to be clinically significant. A 2+ or stronger result should be investigated very seriously. Weaker reactions may or may not represent a clinically significant finding. How has the DAT changed from previous testing? If a prior DAT has been weakly positive over weeks to months without any change, the current weak DAT is unlikely to be clinically important to the issue of blood. Is there IgG, C3d, or both on the patient's RBCs? If only C3d is present, an eluate is not likely to yield any useful information and should not be performed in most cases. Is the antibody screen positive? If it is, your suspicion should be high that the positive DAT may be identifying a clinically relevant (possibly new) antibody. Remember that patients who have formed one RBC alloantibody are at high risk for forming additional antibodies (e.g. they are "responders"). However, a negative antibody screen does not rule out that significant antibody is bound only to the RBCs and cannot be detected free in serum.
  4. Assess the patient's history. The most important question to ask is: Has there has been a transfusion or pregnancy in the last three months? The positive DAT is unlikely to represent a red cell allo-antibody if there is no recent exposure to foreign RBCs. (The foreign RBCs would have to still be circulating in the body for the patient's antibody coating them to cause a positive DAT). Without any history of transfusion or pregnancy, the patient is unlikely to have a clinically significant alloantibody. The positive DAT could still be an auto-antibody, however. Ask about medications. Appendix 6 in the paper supplement lists drugs that can cause a positive DAT. Always ask about antibiotics, especially penicillin and cephalosporins, alpha-methyldopa (Aldomet), anti-lymphocyte or anti-thymocyte globulin (ALG or ATG), and intravenous gamma globulin injections. If the patient has a weakly positive DAT that can be explained by a medication, further work-up may not be necessary. Is there a history of a positive DAT in the past? If so, do we know why it was positive? A patient with a weakly positive DAT of unknown etiology which has been present for months or more is not likely to have a clinically important DAT now unless the strength is increased or the clinical picture has changed (e.g. new evidence of hemolysis). But don't be fooled -- if there is a recent transfusion history, the weak DAT may actually now represent a new alloantibody. Is there a history of autoimmune disease? Patients with one auto-immune process may be prone to develop another, therefore, in a patient with systemic lupus, for example, be highly suspicious of a warm autoantibody to RBCs.
  5. Assess the clinical situation. Is there evidence of hemolysis?
    Look at what lab values are available to you that are indicators of hemolysis:

    The last two indicators of hemolysis are generally only present when you have fairly brisk hemolysis occurring, such as intravascular hemolysis. They may be absent from extravascular hemolysis, such as present with Rh, Duffy, Kell and Ss antibodies. If the lab studies have not been done, ask the clinician for their impression. If they don't have a clue, perhaps they should order some labwork to look for hemolysis, if time allows. What is the patient's diagnosis? This may shed some light on their ability to make antibodies. Are they immunosuppressed? Do they have an autoimmune disease already? Do they have another reason for hemolysis? (Examples: patient on ECMO, new heart valve, etc.) Patients with lymphocytic leukemias, for example, rarely make red cell alloantibodies, but patients with sickle cell disease frequently do.

  6. Now, put together all of your available information. If there is a recent history of transfusion, and the DAT has just become positive, be very suspicious of a new alloantibody and order an elution. If the DAT is stronger than 2+, be very suspicious. On the other hand, if the DAT is weak, there is no recent transfusion history, and no evidence of hemolysis, then don't order an elution, especially if the patient has another reason (e.g. a drug) to have a positive DAT. Since elutions are very time-consuming, please don't order them unless they are really necessary. If you're not sure what to do, call the fellow or faculty for advice.

IMPORTANT: please come by the blood bank at your earliest convenience and fill out the back of the green worksheet with the pertinent information that led to your decision.

MOST IMPORTANT OF ALL: When you make your decision about whether or not to do an eluate, sound confident. The technologists will be less upset about doing the extra work if they think you know what you're talking about!

C. Autologous control cell ("Autocontrol").

This is a phase of the antibody screen where the patient's own RBCs are reacted with their serum. The autocontrol is not performed routinely in pretransfusion testing. It is only performed in selected circumstances. A positive result may indicate that the patient has a positive DAT. Generally, the techs will follow-up a positive autocontrol by asking the clinicians for a purple top tube and then do direct antiglobulin testing on the patient's RBCs. However, once in awhile, they will call you and ask you to investigate the positive autocontrol, assuming the DAT is positive without actually performing it. It is best to go ahead and do the DAT if possible.

D. Red cell elution.

This procedure removes IgG immunoglobulin from the surface of RBCs and concentrates it to some degree in an eluate which can then be tested against reagent RBCs to determine the specificity of the antibody. Ordinarily, an elution should not be done if the DAT was negative for IgG (see further discussion below).

Our technique of choice at PMH is the acid elution, using Gamma Biological's Elukit®. For ABO antibodies, the Lui freeze technique may be preferable. The supervisors in the lab should be able to advise you if a question arises. REMEMBER: if you are looking for anti-A or anti-B in your eluate, please make sure the techs know that. The routine panel against which the eluate is tested is composed entirely of group O cells. Therefore, an anti-A or anti-B will be missed. They will have to select a sample of group A and/or group B RBCs to find these antibodies in the eluate. A red cell eluate takes a minimum of one hour to perform.

Request to perform an eluate despite a negative DAT. Occasionally, the eluate will show a clinically significant antibody at very low levels that was missed on the DAT. This is because the eluate does concentrate the antibody to some degree for further testing. In most cases, however, the eluate will be a waste of time. Therefore, please limit eluates following a negative DAT to cases with a very high level of suspicion for immune hemolysis. A common request in the past at PMH was to do an eluate for newborns with suspected HDN due to ABO incompatibility who had a negative DAT. If an eluate is performed on such newborns, it often is positive for anti-A,B. However, the literature strongly suggests that the presence of this antibody in the eluate is clinically meaningless when the DAT is negative. These infants do not have hemolysis due to the extremely low levels of anti-A,B present. Another cause of hemolysis should be sought. Fortunately, it is rare to get such a request now.

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