Cold Autoantibodies

[roseyrevisited]

Hi, my name is Kim and I'm a newbie here lol I am in the MLT program at my school and we are studying blood banking and I had a question,

in our text book it has an IAT with just SC I,II and no autocontrol, both of them had a +2 reaction at IS RT but was negative when incubated, The DAT was positive for C3 but negative for IGG, my question is how can you tell its an cold autoantibody without the autocontrol? this is what i am having problems digesting.. any help would be greatly appreciated

[David Saikin]

You can't . . . however, when you run the auto at IS and it reacts like the screening cells (same strength), you can be fairly certain you have a cold auto. A few decades ago, many institutions decided to stop running the auto ct with their absc as a cost saving measure . . . Also, not many places run their absc at IS or RT because we really don't want to find those cold abs . . . they are a nuisance. As is seen in your scenario, after 37C, the ab is no longer detected. HOWEVER, if you had to do an IS xm on this patient (without an IS absc, you would have been surprised to get the xm's incompabile . . . this would be another hint that the patient has a cold reacting ab (whether it has a specificity would then need to be determined), Decades ago, when I was newbie in the BB, we ran absc at IS and 15 min RT incubation (neat and with enzymes) - it was not unusual to run 5 abid's on a shift just because of cold abs. Hope I haven't confused you.

[L106]

I agree with everything David just said.

The only think I'd like to add is that we don't always see the patient's Auto Control reacting on Immediate Spin. So if we suspect that the patient has a Cold Auto Antibody, we set up the Screening Cells and an Auto Control and incubate it in the refrigerator (usually 15-20 minutes.) A Cold Auto Antibody will usually show up 3+ or 4+. (Pretty quick and easy.)

[David Saikin]

A further comment - I think that with 15-20 minutes at 4C you will find most everyone has some type of cold ab. I like to set up a quick cold screen with my screening cells, auto ct, and a couple of ABO compatible cord bloods. (also, if the person is group A I will run A1 and A2 cells). I read these at IS, 5 min at RT, and 5 min at 4C. Someone with a potent cold ab will react at one of these phases. If the auto is negative, then I'm going to look for some specificity, unless I feel good about anti-I.

[roseyrevisited]

Thank you both very much, The reason I asked which I will bring this problem up with my instructor, is our text book has a few big mistakes in it and I am not confident with its text, my text states that with a positive DAT for C3 is indicative of an autoantibody because it shows an antigen and antibody attachment, so it says based on just the DAT it can be concluded that it is an autoantibody, is this correct?

[Yanxia]

I don't think this conclusion is correct, because some disease can get the C3 DAT pos result ,such as DIC .

[Abdulhameed Al-Attas]

I am afraid I opt to disagree with shily but agree with Rosey. A positive C3d DAT is adequate surrogate indicator of autosensitiztion of the red cells by antibodies whether diseased-induced or otherwise is another issue.

Can you give me an example of a disease that causes C3d attachment onto the red cells that is not mediated by antigen-antibody interaction?

Thank you.

Edited March 22, 2009 by Abdulhameed Al-Attas

[adiescast]

As far as the original question, you can tell that your results are likely to be a cold antibody because they are present at immediate spin and absent at 37 and Coombs. The DAT and the autocontrol can be considered to give equivalent information. The fact that the DAT is positive for C3 points convincingly toward a cold autoantibody.

In terms of the DAT that is positive for C3, C3 is a complement component and not an antibody. The complement cascade can be set off by many processes. One of the possibilities (the classic activation pathway) does include antigen/antibody interaction. The alternative pathway for complement activation includes exposure to particular polysaccharides and lipopolysaccharides on cell membrane surfaces (such as bacteria or cancer cells) and does not require antigen/antibody interaction. Both pathways lead to active C3.

Given the screen results and the DAT results, you can be fairly certain that you have cold autoantibody activity in your patient. We usually confirm this by performing a cold screen, but it would not be necessary.

Edited March 23, 2009 by adiescast
Added last paragraph

[nagaraju_palumaru]

Confirming with auto control is best option

2ndly because antibody is reacting at Immediate spin technique it showing agglutination after incubation cold antibodies may show weaker/no reaction. In addition C3 is a complement which is reactive at colder temperature that why DAT +ve for C3

thank you

Do contact by email- nagaraju_palumaru@yahoo.com

[Yanxia]

I am afraid I opt to disagree with shily but agree with Rosey. A positive C3d DAT is adequate surrogate indicator of autosensitiztion of the red cells by antibodies whether diseased-induced or otherwise is another issue.

Can you give me an example of a disease that causes C3d attachment onto the red cells that is not mediated by antigen-antibody interaction?

When we test a coagulate blood sample , we may get the positive C3d DAT result, this is not because the antigen and antibody reactive ,but because the blood coagulation is crosslinked with complement activation.

In this question, I think all the positive result with screening cells and panel cells in RT with the same degree and neg in 37 degree C, we can get the conclusion that the antibodies is to a kind of high frequent antigen ,but not the autoantigen ,and the C3d DAT pos result can't give us the conclusion to autogentigen, too. Though lots of situation the conclusion is right,but I still think we need the autocontrol , it is necessary.

[Liz]

Regarding heart surgery using the pump where the blood drops to 10 C or so, cold auto/allo antibodies can be a problem. Should one run the cold phase routinely, does anyone? We use the gel method, and so we skip the cold phase in all tests: abscreen, abid, Cxm.

Any thoughts are greatly appreciated.

Thanks

Liz

[galvania]

If I am reading the last couple of posts correctly, I think there are a few confusions about Complement that maybe need clearing up:

2ndly because antibody is reacting at Immediate spin technique it showing agglutination after incubation cold antibodies may show weaker/no reaction

All antibodies showing up in an immediate spin technique would give stronger reactions after incubation at room temperature or 4°C. Incubation at 37°C may well cause the antibody to disappear, however

In addition C3 is a complement which is reactive at colder temperature that why DAT +ve for C3

Complement is NOT reactive at colder temperatures. Complement activation occurs optimally at 37°C. However, if the cause of complement activation is an antibody-antigen reaction, then the best types of antibody to trigger this are usually IgM antibodies, which react best at cold temperatures. Once Complement activation has been triggered, it can either go all the way to C9 activation, which leads to haemolysis of the red cells, or is blocked by inhibitors at the C3d stage. The C3d coated red cells usually live normal lives, but the fact that we can find the C3d on the red cells in the DAT indicates that an antibody-antigen reaction may have taken place at some time in the recent past. The complement activation can remain for some time after the antibody has 'quietened down'. This is a bit of a simplification, I admit.

When we test a coagulate blood sample , we may get the positive C3d DAT result, this is not because the antigen and antibody reactive ,but because the blood coagulation is crosslinked with complement activation

With clotted blood, the problem is not really that there is cross-reaction. EDTA used in anticoagulated blood actually blocks complement activation in vitro. As EDTA is not present in clotted blood, in vitro complement activation can (and does) occur, giving false positive DAT results

I also have a question for Shily. I have not heard that DIC causes a positive DAT due to C3. Do yopu have a reference?

[Malcolm Needs ☆]

The answer is that you can't, but you can make a very educated guess.

The fact that the agglutination disappears once the tests are incubated strongly suggests the presence of a "cold" reacting antibody, and the fact that the DAT reacts only with anti-C3 also suggests a "cold" antibody. There is a very strong chance, therefore, that the antibody is auto-anti-H or auto-anti-HI, but without performing an auto control (which takes very little time and will not be that expensive) an auto-antibody cannot be proved.

That having been said, the chances of the antibody being clinically significant (in terms of a transfusion) are disappearingly small.

[irshadaad]

its not only cold anti bodies will bind c3,even some warm antibodies are compliment binding

[Malcolm Needs ☆]

its not only cold anti bodies will bind c3,even some warm antibodies are compliment binding

Actually, many "warm" are complement binding, but the question was about "cold" antibodies.