Gel DAT

[johna]

Was wondering how gel users are handling DATs. Since there is no anti-C3 card are you simply repeating all polyspecific positives by a tube method or as one individual suggested assuming that a positive poly and a negative anti-IgG was a positive anti-C3?

Another issue is that I have heard reports that a positive DAT by gel does not always correlate with a positive tube test. How are you handling this? Default to tube method? Thanks!

[Seveets]

When we started with Gel we did the IgG part in gel and the C3 in tube.

Our doctors (are suppose to) realize the sensitives issue of gel versus tube so thats not a problem but we test the eluate in gel as well and try to stay away from tubes.

We now use the combo IgG/C3 gel cards to do a DAT - if negative great.

If positive we then have to go back to the previous method of repeating the test with the IgG only gel and the C3 tube.

[janet]

Diamed makes a gel card that has an IgG, a C3d and a control well in each card (x2 TO make up the 6 wells). We get it through our Ortho supplier. It is wonderful, the rare time it is on back-order and we have to revert back to tube C3d testing the staff realize how lucky we are to have it!

[johna]

Diamed makes a gel card that has an IgG, a C3d and a control well in each card (x2 TO make up the 6 wells). We get it through our Ortho supplier. It is wonderful, the rare time it is on back-order and we have to revert back to tube C3d testing the staff realize how lucky we are to have it!

Thanks for the info Janet but I'm not sure if this card is licensed in the States! I'll check with Ortho.

[johna]

When we started with Gel we did the IgG part in gel and the C3 in tube.

Our doctors (are suppose to) realize the sensitives issue of gel versus tube so thats not a problem but we test the eluate in gel as well and try to stay away from tubes.

We now use the combo IgG/C3 gel cards to do a DAT - if negative great.

If positive we then have to go back to the previous method of repeating the test with the IgG only gel and the C3 tube.

Thanks for the response. The correlation problem that I mentioned is that the poly gel card is positive and the tube test with poly, anti-IgG & anti-C3 is negative. Is anyone seeing this?

[galvania]

Gel pos and tube neg - can happen as gel is more sensitive for Coombs than most tube methods. There is also a card (but as with the IgG/C3d card, I'm not sure if it's licensed in the States) that can differenciate between C3c (actually C3b) and C3d). Sometimes the pos gel test can be due to non-specific uptake of immune complexes. Usually washing a couple of times before putting on gel gets rid of these.

[johna]

Gel pos and tube neg - can happen as gel is more sensitive for Coombs than most tube methods. There is also a card (but as with the IgG/C3d card, I'm not sure if it's licensed in the States) that can differenciate between C3c (actually C3b) and C3d). Sometimes the pos gel test can be due to non-specific uptake of immune complexes. Usually washing a couple of times before putting on gel gets rid of these.

Thanks Galvania! I guess the next question would be: What is the clinical significance of a positive gel and a negative tube test?

I believe that most blood bankers here in the States perform tube DATs without the aid of a microscope. Would these gel+/tube- DATs be evident in the tube if they were examined microscopically? Has anyone looked at this? Thanks again for your response.

[galvania]

I always think it's so difficult to determine the clinical significance of a pos DAT unless you know the details surrounding the person whose DAT is positive. For example a weak pos DAT in someone with malaria and due only to the malaria - well not really very significant on its own (not compared to the malaria, I mean) - the same thing post transfusion might be the beginnings of a transfusion reaction....And as for tube Coombs (I did Coombs tubes for years when I was in the UK) is that it's so difficult to standardise them - both carrying out and shaking and reading. One person's microscopic only could easily be someone else's 1+

[OPUS104]

We are unfortunately beginning to defer donors on a regular basis due to positive DATs found at crossmatch with Gel cards at the hospitals we supply. The usual difference we find between tube and Gel is usually a factor of 2.(i.e. 1+tube=3+Gel) These donors (many of them VERY regular, long time donors) have no tube results at all.....but when the hospital Gel crossmatches the unit it is 1+ positive. This is very frustrating to both us and the donors. Many of them become upset over being defered and want us to explain IN DETAIL what the DAT means. You can imagine the long confusing conversations from here on....... the most important question is the one you posed, is it clinically significant?????????? We of course don't know so the donor gets defered.

[johna]

We are unfortunately beginning to defer donors on a regular basis due to positive DATs found at crossmatch with Gel cards at the hospitals we supply. The usual difference we find between tube and Gel is usually a factor of 2.(i.e. 1+tube=3+Gel) These donors (many of them VERY regular, long time donors) have no tube results at all.....but when the hospital Gel crossmatches the unit it is 1+ positive. This is very frustrating to both us and the donors. Many of them become upset over being defered and want us to explain IN DETAIL what the DAT means. You can imagine the long confusing conversations from here on....... the most important question is the one you posed, is it clinically significant?????????? We of course don't know so the donor gets defered.

This is exactly my concern. By reporting these gel results my guess would be that we are waving red flags that needn't be waved and as in your case deferring donors that needn't be deferred. Fortunately I am in a situation which does not involve crossmatching. In our case I would have a tendency to defer to tube DAT results. Tube was apparently good enough before the advent of gel; why wouldn't it be good enough now?

It would be interesting to see if Galvania's suggestion concerning washing the cells prior to use would clear up these gel crossmatch problems in your hospitals.

[bbbirder]

I have been considering switching to gel DATs but have been hesitant to, for the reasons discussed above. We have been running a few both ways and occasionally get positive in gel but neg in tube. What does this mean? For these patients, if they have a normal haptoglobin, LDH, etc. they probably don't have any hemolysis.

We had another patient not long ago with obvious severe, hemolytic anemia (low haptoglobin, elevated bili and LDH, etc.) and negative DAT (tube and gel). DAT was positive using cold LISS wash technique.

So... you can have hemolytic anemia with negative DAT and positive DAT without hemolytic anemia. I am hesitant to routinely use a more sensitive method that might have more positives, but these might really be more false positives.

LF

[galvania]

Hi b-birder - I was interested in your case of severe HA with a negative DAT. Could it be that all the cells with antibody attached had actually haemolysed? Or was this a severe cold HA or PCH?

As for the positives that are negative in tube. It's a very difficult question in regards to donors. I know that it has been reported that there is a proportion of healthy donors who have positive DATs for no reason whatsoever - but on the other hand, they might of course signal the beginning of some form of haematological disorder. We had a cluster of about 40 samples sent to us to look at recently, from India, many of them giving quite strong positive results in gel; all the strong (<2+) ones were IgG or IgG + C3d. We still don't know why or whether they are significant results. I have often wondered whether they aren't due to some sort of non-prescription drug or herbal tea or suchlike, as with drug-induced + DATs. They probably aren't significant at all, but they will, of course, cause problems if you do a Coombs crossmatch. This is a research project begging to be done!!!

[bbbirder]

The patient had negative cold screen with adult, cord and auto cells, and the Donath-Landsteiner test was negative. Specimen was also sent out for flow testing for IgA on red cells. Seems like we did some other testing, but I don't remember everything...

Because the DAT using cold LISS wash was strongly positive (3-4+ IgG), I don't think hemolysis of cells was an issue. It pretty much fits the descriptions given of "DAT negative AIHA associated with Low-Affinity IgG autoantibodies" described by Petz & Garraty in their wonderful book on Immune Hemolytic Anemias.

This patient did not respond to steroids or IVIG and required a splenectomy.

Linda Frederick

[galvania]

Thanks Linda. Did the auto-antibody actually have a specificity?

Anna

[bbbirder]

Nope, no specificity for this antibody.

LF

[David Saikin]

I validated anti-C3b,-C3d using buffered gel cards. We use 0.8% patient cells and we make the commercial Complement Check cells 0.8% too. We spin after a 5 minute incubation at room temperature. The check cells are always 3-4+. As for the sensitivity problems being discussed - negative tube vs + gel . . . why are you using gel? Up front we all know it is more sensitive. I don't think you can ignore a + gel result just because you repeat it in tubes and it is negative. My ped docs are enthralled with the increased DAT sensitivity. I have found 3 anti-Jka in gel that were negative with tube/PeG testing. Are they clinically significant? I can't ignore them.

[johna]

I validated anti-C3b,-C3d using buffered gel cards. We use 0.8% patient cells and we make the commercial Complement Check cells 0.8% too. We spin after a 5 minute incubation at room temperature. The check cells are always 3-4+. As for the sensitivity problems being discussed - negative tube vs + gel . . . why are you using gel? Up front we all know it is more sensitive. I don't think you can ignore a + gel result just because you repeat it in tubes and it is negative. My ped docs are enthralled with the increased DAT sensitivity. I have found 3 anti-Jka in gel that were negative with tube/PeG testing. Are they clinically significant? I can't ignore them.

I think when the situation arises in which you are deferring an unusually high number of apparently normal donors (as indicated in the OPUS104 post) one has to weigh the the value of a positive gel DAT when the tube test is negative against depleting your donor population. The question remains as to whether any of the DAT-negative patients/donors who were tested before gel existed suffered any pathological consequences because no one knew that their gel test would have been positive. In any case David your comment about the anti-C3 gel card is certainly useful information. Thanks!

[Mabel Adams]

David, I assume you run the complement check cells as a parallel positive control in another microtube. You aren't using them as "check cells" somehow in gel are you?

[Mabel Adams]

I think different decisions might prevail in different circumstances. Cord blood DATs, vs. testing donors, vs. transfusion reaction workups, vs. antibody ID in the recently transfused, vs. possible AIHA. In some cases the extra sensitivity would be good and in some cases not so good.

[David Saikin]

Yes Mabel, I run the check cells in a parallel microtube at the same time. I also run both the patient and the check cells with Dil 2+ as a negative control. I just feel better that way; due to the increased sensitivity of the gel, I wouldn't want some non-specific agglutination giving me a false +.

[momie]

I have been considering switching to gel DATs but have been hesitant to, for the reasons discussed above. We have been running a few both ways and occasionally get positive in gel but neg in tube. What does this mean? For these patients, if they have a normal haptoglobin, LDH, etc. they probably don't have any hemolysis.

We had another patient not long ago with obvious severe, hemolytic anemia (low haptoglobin, elevated bili and LDH, etc.) and negative DAT (tube and gel). DAT was positive using cold LISS wash technique.

So... you can have hemolytic anemia with negative DAT and positive DAT without hemolytic anemia. I am hesitant to routinely use a more sensitive method that might have more positives, but these might really be more false positives.

LF

Hi....i am Lab tech student.....not smart enough to answer but i have read into my book that positive hemolysis with negative Dat is associated with patient in sickle cell crises,

thalassemia or G6PD deficiency patient or the unit is over heated or frozen or if all cells are hemolysed ......as it is possible that Dat may be negative if all incompatible cellls are destroyed; ABO antibodies rapidly activate complement leadingto lysis....and u r saying it was severe hemolysis then it might be the case of all cells hemolysed.

[momie]

Hi....i am Lab tech student.....not smart enough to answer but i have read into my book that positive hemolysis with negative Dat is associated with patient in sickle cell crises,

thalassemia or G6PD deficiency patient or the unit is over heated or frozen or if all cells are hemolysed ......as it is possible that Dat may be negative if all incompatible cellls are destroyed; ABO antibodies rapidly activate complement leadingto lysis....and u r saying it was severe hemolysis then it might be the case of all cells hemolysed.

[Malcolm Needs ☆]

Hi....i am Lab tech student.....not smart enough to answer but i have read into my book that positive hemolysis with negative Dat is associated with patient in sickle cell crises,

thalassemia or G6PD deficiency patient or the unit is over heated or frozen or if all cells are hemolysed ......as it is possible that Dat may be negative if all incompatible cellls are destroyed; ABO antibodies rapidly activate complement leadingto lysis....and u r saying it was severe hemolysis then it might be the case of all cells hemolysed.

You are correct in what you say momie (so don't put yourself down - not even the world's most expert "expert" knows everything)! There are many occasions when there may be haemolysis in conjunction with a negative DAT. Certainly, hyperhaemolysis in sickle cell patients is one of these, as are the other situations that you cite. Others to think about are some types of drug-induced AIHA and bacterial infection of a blood component that has been transfused, gangrene, and, of course rare cases of AIHA, where the DAT is negative.

Good luck as a Lab Tech student. You have taken a giant stride towards your future by joining this site.

[momie]

Well thank you so much for encouraging me!! Being a student, i really need it.

I am a MLT student, doing my hematology rotations currently at ETMC, gilmer TX.

By joing this website, i think u all will be a great help for me.

Thanks once again,

Momie

[Transfusion98]

On 10/8/2007 at 9:40 AM, johna said:

Was wondering how gel users are handling DATs. Since there is no anti-C3 card are you simply repeating all polyspecific positives by a tube method or as one individual suggested assuming that a positive poly and a negative anti-IgG was a positive anti-C3?

Another issue is that I have heard reports that a positive DAT by gel does not always correlate with a positive tube test. How are you handling this? Default to tube method? Thanks!

Could you share your "hybrid" policy?