Hi everyone,

Need some help here. I have been pulling my hairs on this for days, wondering where could have gone wrong in my preparations. Need some advice/help here to gurus who prepare 0.2 M DTT for testing. Peharps some tips on how to get it right (maybe have "tweaked" a little during make up of the solutions?). Any help is appreciated!!!

Emergency room

Sorry Emergency room, but please could you post a few more details about what exactly went wrong? It is difficult to comment with out such details.

Oops, Sorry Malcolm, I should have explained more in details. Few months ago, I prepared 0.2 M DTT using DTT powder and dissolved them in buffered saline of pH 7.3-7.4. I read this from the Red Cross Immunohematolgy methods and procedure book. After preparations, I treated three cell screen with the 0.2M DTT and ran the cells against anti-k antisera. It clearly showed that the k antigens have been denatured as the reactions were negative with anti-k. However, when I tested the treated cells with a known patient with anti-k, the reactions were positive ( the reaction did decreased from 3 to 1+-weak positive). So last week, I thought well maybe the pH of the buffered saline wasn't right, so I experimented by modifying the pH of the saline with phosphate buffer. Unfortunately, it still didn't work at all, worst were no reductions of reactivity and anti-k control came out positive. Any thoughts?

Emergency room

When you did your first treatment on your 3 cell screen and your testing with k antisera came out negative, did you run the known patient anti-k on the same cells or on a separate 2nd treatment of those cells? If you did a second treatment on a different aliquot of cells, then ran the patient anti-k on those, it may be possible you didn't treat them long enough to completely denature the anitgens. It could also be possible that your patient with the known anti-k also has another antibody whose corresponding antigen is not affected by DTT, and that's the remaining weak reactivity.

Edited May 20, 201312 yr by Emwilson7

I did wonder about that myself Emwilson7.

The other thing you could try is ZZAP emergencyroom.

Hi, Emwilson can you tell me where did u get your DTT powder from? In fact I treated 2 sets of screen cells, subjected to the same DTT treatment process. Is that possible that one set worked and the other did not? You probably right about using patient's sample.

Malcolm, I cannot use zzap because i cannot even get my DTT work. Isn't zzap consists of DTT?

Emergency room

Good point Emergency room! I'll keep quiet until I learn to think before I type!!!!!!!!!!!!!!!!!!!!!!!

:imslow::imslow::imslow::imslow::imslow::surrender:surrender:surrender:surrender:surrender

Edited January 20, 201312 yr by Malcolm Needs

DTT podwer comes with expiration date. I thought ARC was using two different concentration 1) for destroying kell system & others & 2) one for IgM ...Do not kill me if I am wrong!!!

Hi, Emwilson can you tell me where did u get your DTT powder from? In fact I treated 2 sets of screen cells, subjected to the same DTT treatment process. Is that possible that one set worked and the other did not? You probably right about using patient's sample.

Malcolm, I cannot use zzap because i cannot even get my DTT work. Isn't zzap consists of DTT?

Emergency room

No, you are not wrong. One is 0.02M and the other is 0.01M.

Exactly what Malcolm said about concentrations.

 

When you treated the 2 sets of cells, did you follow the exact same procedure for both? (incubation time, temp, amount of DTT solution added, same concentration of DTT, number of washes, etc.) If there was any differences between the 2 sets, maybe that could account for the kell antigens not being destroyed.

Try treating a larger aliquot of those cells at once, splitting them into 2 sets of tubes after treatment, then run one set against your anti-k reagent and the other against your patient anti-k. If your k antisera is still negative, and the patient anti-k is still positive, then your treatment works fine and you'll know there's some other antibody contaminate in your patient anti-k sample.

Edited May 20, 201312 yr by Emwilson7

No, you are not wrong. One is 0.02M and the other is 0.01M.

For destroing Kell you need 0,2M. a typing error I think

We have the same problem. We use the powder to make 0,01 and 0,2. The 0,01 is fine but the 0,2 is not destroing the Kell antigen completely. It is going from 3+ tot 1+ but not completely gone. We use a strong anti k reagent to test but it is nog working as we wanted. We have planed tot test also other antigens (other Kell and other systems) tot see how that goes.

Peter

I'll be honest Peter; it wasn't so much a typing error as brain freeze!!!!!!!!!!!!!!

Has this thread always been about reagents? Is there something in the reagent that is 'protecting' the antibody? What happens if you use a 'real' antibody?

Did you run an autocontrol when you tested the patients serum? I think it might be possible you are bringing up some kind of auto after the DTT treatment.