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DTT-Treatment of Screening Cells (Daratumumab/Darzalex)

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I am looking to bring DTT treatment to our small lab.  This forum has been very helpful, but I have a few more questions.   

It looks like Judds Methods in Immunohematology is using 0.2M DTT and the Technical Manual is using 0.1M DTT.  Is anyone noticing a difference in testing with 0.1M vs 0.2M?

I researched some of the reagents.  It appears the powder form is considerably cheaper.  I was wondering what you use for a solvent?

Once you put it into a liquid form can you freeze it up to 5 times for up to a year or is that just with the pre-liquid reagent? (when does it expire)?

How long does it take to work up a patient... I have heard 2 hours for negative screen after DTT treatment?

Thank you for your help

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On ‎3‎/‎15‎/‎2017 at 11:58 AM, tkakin said:

I am looking to bring DTT treatment to our small lab.  This forum has been very helpful, but I have a few more questions.   

It looks like Judds Methods in Immunohematology is using 0.2M DTT and the Technical Manual is using 0.1M DTT.  Is anyone noticing a difference in testing with 0.1M vs 0.2M?

I researched some of the reagents.  It appears the powder form is considerably cheaper.  I was wondering what you use for a solvent?

Once you put it into a liquid form can you freeze it up to 5 times for up to a year or is that just with the pre-liquid reagent? (when does it expire)?

How long does it take to work up a patient... I have heard 2 hours for negative screen after DTT treatment?

Thank you for your help

I found out from one of the manufacturers that sterile saline is used to reconstitute the powder form of DTT

They said it was better to reconstitute for each use then to freeze aliquots.....they didn't say no :) 

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We are a little late to the ball game.  But there's some confusion in the lab about what RBC's are being DTT Treated.    Reagent cells, patient cells, donor cells?   

Can anyone shine some light for us. 

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1 hour ago, amym1586 said:

We are a little late to the ball game.  But there's some confusion in the lab about what RBC's are being DTT Treated.    Reagent cells, patient cells, donor cells?   

Can anyone shine some light for us. 

Reagent cells.  The cells are used to try to see if there are any alloantibodies underlying the monoclonal anti-CD antibodies.

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1 hour ago, amym1586 said:

We are a little late to the ball game.  But there's some confusion in the lab about what RBC's are being DTT Treated.    Reagent cells, patient cells, donor cells?   

Can anyone shine some light for us. 

Most typically Screening Cells will be treated. If that screen is negative, then no further action is required (except perhaps antigen-typing the patient). If the DTT-treated screen is positive, it may be necessary to DTT-treat a reagent red cell panel (or selected cells).

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Has anybody tried DTT treating reagent red blood cells in MTS gel method? If so are you concentrating your cells before treating or have adjusted your ratio of DTT to the .8% suspension? TIA.

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On 10/18/2017 at 3:12 PM, BloodbankZ said:

Has anybody tried DTT treating reagent red blood cells in MTS gel method? If so are you concentrating your cells before treating or have adjusted your ratio of DTT to the .8% suspension? TIA.

We are looking to bring DTT treatment in house as well.  Still working out the details and hoping to have the same question answered - does anyone test the treated cells in MTS-Gel?  It seemed to work well for me when I experimented with it.  

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When I tried to run it in gel I never could get nice clean reactions. Their would always be particulate at the top of the wells. I have seen several tube procedures that are just using so many drops of 4% cell suspensions instead of concentrating their cells and then getting an exact 4:1 ratio with DTT. I have since went to doing it in tube. What kind of steps are you performing?

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I performed this study of long term stability (14 days) of panel cells treated in bulk as my SBB project. I also used regular blood bank saline vs the 8.0 from the technical manual. I was able to publish my study and findings in the Immunohematology journal. I would be happy to share if you leave your email address. Happy to help those interested. 

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If patient on DARA how soon they can develop pan-reactive antibody? We had our first patient who is on DARA had negative  antibody screen before treatment after 36 hours of treatment antibody screen is positive in all 3 cell line. Patient's antibody panel  shows some pan-reactive reaction with 8 out of 11 cells .But auto is negative . Is it due to DARA or patient is developing some allo antibody?

 

Edited by gagpinks

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On ‎3‎/‎10‎/‎2018 at 4:30 AM, gagpinks said:

If patient on DARA how soon they can develop pan-reactive antibody? We had our first patient who is on DARA had negative  antibody screen before treatment after 36 hours of treatment antibody screen is positive in all 3 cell line. Patient's antibody panel  shows some pan-reactive reaction with 8 out of 11 cells .But auto is negative . Is it due to DARA or patient is developing some allo antibody?

 

I would say it is due to the DARA. DTT treated cells will eliminate the DARA reactivity and let you be able to rule out everything besides mainly Kell system which DTT denatures. We perform a baseline type and screen and DNA HEA typings on patients before they start. If DTT screen is negative we give K negative cells if patient is antigen negative.

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Further follow up.  We did purchase the HemoBioScience premade DTT - very nice and really not too expensive for a small hospital - saves all the worry over the manufacture of an in-house DTT reagent. 

Works well to use 8-10 drops of your 3-4% RBC reagent cells to make one drop of "Packed" RBCs to treat with 4 drops of DTT.  Once they are treated and washed, you remake to a 3% solution and get 8-10 drops of treated cells to work with.  As hard as the DTT treatment is on the reagent cells - I don't think they are going to last very long for a small hospital - maybe a day or two for a reference lab??  (didn't see the results of that validation study.)

We treat the reagent cells (I, II, III) and the first RBCs requested for crossmatch every 72 + hours (new specimen), but if the antibody screen was negative - we just continue with K neg , immed spin, type specific RBCs for the 72 hours (same specimen) for any add-on units. 

If the antibody screen is positive - a reference lab is going to get the workup.  We only have one liquid panel, so I can't see us being able to work up an underlying allo antibody in-house.

Dr still is not happy that it takes someone 3-4 hours to work one of these pts up - go figure.  It is amazing that even an Oncology specialist thinks that just because we did an antibody screen 3 months ago - we should be fine with giving them "some of that least incompatible stuff"! 

oh well - will attach my SOP for DTT for anyone it might help.  We can survive the simple pts with this procedure here - don't look forward to the 1st pt that forms an allo-antibody on top of the DARA.

DTT SOP.pdf

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20 minutes ago, cswickard said:

Dr still is not happy that it takes someone 3-4 hours to work one of these pts up - go figure.  It is amazing that even an Oncology specialist thinks that just because we did an antibody screen 3 months ago - we should be fine with giving them "some of that least incompatible stuff"! 

Don't you just love 'em?  Doctors know everything!

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