Autoimmune Hemolytic Anemia (AIHA): Direct and Indirect Coombs' Test

by Carlo Raj, MD

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    00:01 and that would be warm though.

    00:01 Let’s begin at the top.

    00:01 Let’s take a look at the protocol for direct Coombs test.

    00:05 Now, the Coombs test is indirect and direct.

    00:08 I’ll give you the indications as to when you would want to conduct which type and briefly just going to identify or kind of look at the steps of some of these Coombs tests.

    00:20 So let’s say you have an RBC with an IgG antibody.

    00:24 And right off the bat, I’m going to go ahead and tell you, well, we have a state called Georgia.

    00:28 And, well, for the most part the state of Georgia tends to remain warm, but whatever, let's go with stereotypes in generalities, please.

    00:37 So it’s warm in Georgia.

    00:39 The G in IgG if you wish to use is warm type of autoimmune hemolytic anemia.

    00:48 Is that clear? You need to find a way in which you differentiate warm and cold.

    00:51 I’m going to keep reinforcing this so that you know as to what type we’re dealing with.

    00:57 Warm is only dealing with IgG.

    00:59 So here, you have an RBC in your patient that is stuck with IgG.

    01:06 How? We’ll come to that later.

    01:07 What are we going to do? We’re going to then identify the pathologic complex.

    01:11 This is in the patient.

    01:13 This complex right here.

    01:16 So what are we going to use? We’re going to use an anti-IgG antibody.

    01:22 That’s the Coombs’ reagent.

    01:24 Make sure you're familiar with this.

    01:25 So what is it going to do, do you think? If you use an anti-IgG antibody, it’s going to attack or not so much attack, but really bind the IgGs.

    01:33 What do you end up forming? A positive direct Coombs test.

    01:38 What have you identified with this test? Oh, I’ve identified an “antigen” which is the RBC in this case or viewed as such by the body and the antibody.

    01:49 Which is which one here? IgG.

    01:51 And what type would that be for you? Warm or cold? Warm.

    01:55 I haven’t given you any associations.

    01:58 I haven’t given you any causes.

    01:58 But what I have given to you, at this point, is the protocol that is quick and easy for you to understand the direct Coombs test, direct.

    02:08 Now, looking for antibody and/or C3 on surface of a patient’s RBC is your objective if you take a look at the bottom of this page.

    02:18 So what about the indirect? How do you use this? Now, this one, we’re not going to use so much with our autoimmune hemolytic anemia.

    02:24 However, this is an important test.

    02:28 Well, as I said, I’ll give you the indications and I’m not going to go through all the immunology.

    02:31 However, I’ll give you the basics.

    02:33 Step 1: You add test blood group.

    02:36 Let’s say O.

    02:37 What does O mean to you? Universal donor, right? And the reason that it’s universal donor is because as you know, the O type RBC refers to the fact there are no antigens on the membrane of that RBC.

    02:55 Do you remember that from immunology? So if there is no antigens on that RBC, remember it’s very easy for you to O, do-nate your RBC to the recipient that requires a transfusion.

    03:07 But this is the problem, right? Is the O type of RBC able to receive any blood? No.

    03:17 So it’s not a universal recipient because the O type blood/person/individual has antibodies for both A and B.

    03:28 So therefore, it cannot accept any A type of RBC because, oh my goodness, you’ll have a transfusion reaction and the patient is going to die, transfusion reaction.

    03:37 Why? Because the O type has antibody attacking the A RBC from the donor.

    03:42 But then also has B antibodies.

    03:46 So if I’m the O type RBC patient and I’m receiving B type blood, my antibody is going to then attack the B type RBC.

    03:57 Can’t have that either.

    03:57 That’s a transfusion reaction.

    04:00 What can I give? Yes, universal donor.

    04:03 I’m extremely generous, but all I do is give, give, give, but I never receive anything back.

    04:07 Does that sound like your relationship? That’s a topic for another day.

    04:10 But anyhow, add test blood type O RBC to the test tube to bind to what? The IgG antibody.

    04:19 So now what happens? There you go.

    04:22 At this point, you have now created some type of coating of your IgG.

    04:26 Next, what are you going to do? You’re going to do step 2, which is adding that Coombs reagent, which is what again? It’s an anti-IgG antibody.

    04:36 You have agglutination.

    04:36 But then, "So Dr. Raj, this doesn’t look any different from the direct." Well, it’s completely different.

    04:45 I want you to go back to that first step there.

    04:45 Do you see those O RBCs there? Those are not from the patient.

    04:52 Go back and compare this to direct.

    04:53 In direct, those RBCs were from the patient that already had a pre-existing IgG to it.

    05:01 Is that clear? Then, what you end up doing is you get these IgG antibodies and then they will bind to the O type test RBCs.

    05:11 So this is the one that you may want to spend a little bit more time with so that your step here, what are you looking for? You’re looking for the antibody in the patient’s serum.

    05:20 That’s what indirect does.

    05:23 Indirect is going to then identify your antibodies.

    05:27 Therefore, indirect Coombs test is a? Read the top.

    05:32 Antibody screen.

    05:32 This is not what we’re going to use in autoimmune hemolytic anemia.

    05:36 Why might you ask? Because in the previous discussion of direct, in autoimmune hemolytic anemia, we’re going to find IgG already bound to the RBC.

    05:48 And that is what's in the patient.

    05:50 And what we’re going to do is then confirm that there is such a complex taking place and we’ll find agglutination.

    05:58 If you’re still unclear about this, well, it’s okay.

    05:59 For now, you fully understand direct and you keep coming back to this explanation for indirect.

    06:05 Then trust me, you’ll get it, you’ll get it.

    06:08 Let's continue.

    About the Lecture

    The lecture Autoimmune Hemolytic Anemia (AIHA): Direct and Indirect Coombs' Test by Carlo Raj, MD is from the course Hemolytic Anemia – Red Blood Cell Pathology (RBC).

    Included Quiz Questions

    1. Blood group O
    2. Blood group AB
    3. Blood group B+
    4. Blood group A
    5. Blood group B
    1. IgG
    2. IgD
    3. IgA
    4. IgM
    5. IgE
    1. The Coomb’s reagent is an antibody against the patient’s IgG antibody.
    2. The Coomb’s reagent is an antibody against the patient’s RBC undergoing hemolysis.
    3. The Coomb’s reagent is an antibody against the ‘O’ glycoprotein on the surface of RBC.
    4. The Coomb’s reagent is an antibody against a donor-derived test IgG antibody.
    5. The Coomb’s reagent is an antibody against either the ‘A’ or ‘B’ glycoprotein on the surface of RBC.
    1. Direct Coombs test utilizes donor-derived O RBCs.
    2. In the direct Coombs test, the antibody coating the surface of the RBC is patient-derived.
    3. A direct Coombs test is performed to diagnose autoimmune hemolytic anemia.
    4. The indirect Coombs test utilizes donor-derived O RBCs.

    Author of lecture Autoimmune Hemolytic Anemia (AIHA): Direct and Indirect Coombs' Test

     Carlo Raj, MD

    Carlo Raj, MD

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