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DTT-Treatment of Screening Cells (Daratumumab/Darzalex)

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Is anyone batch-DTT-treating screening cells for DARA patients? (As opposed to individually DTT-treating panel cells?)

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We are validating that now.  We were going to add in a K+ cord cell, but those were hemolyzing, so we'll type the recipient instead and if needed give them K-.

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14 hours ago, Cliff said:

We are validating that now.  We were going to add in a K+ cord cell, but those were hemolyzing, so we'll type the recipient instead and if needed give them K-.

Cliff,

If you wouldn't mind, please explain what you are validating - not specifically how, but what elements are you evaluating.

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I'm not certain what "batch treatment" means..... if it refers to treating several cells for testing at the same time, then Yes - we are treating screening cells (1-3) and K-negative donor red cells for compatibility testing to be used on a current sample.  If it refers to treating a panel of cells to keep for an extended period of time (like longer than 12 hours) to use for future patient sample testing, then No - and my limited experience with this says that the reagent red cells (especially - donor cells fared slightly better)  become fragile after treatment even when washed 4-6x and lyse over time rendering them useless for testing.

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7 hours ago, exlimey said:

Cliff,

If you wouldn't mind, please explain what you are validating - not specifically how, but what elements are you evaluating.

Sure, here is our validation plan, we are still validating the process.  I took out names and links to documents.

DTT.pdf

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11 hours ago, applejw1 said:

I'm not certain what "batch treatment" means..... if it refers to treating several cells for testing at the same time, then Yes - we are treating screening cells (1-3) and K-negative donor red cells for compatibility testing to be used on a current sample.  If it refers to treating a panel of cells to keep for an extended period of time (like longer than 12 hours) to use for future patient sample testing, then No - and my limited experience with this says that the reagent red cells (especially - donor cells fared slightly better)  become fragile after treatment even when washed 4-6x and lyse over time rendering them useless for testing.

Applejw1,

Sorry for the ambiguity. I did mean treating the screening cells to store for an extended period of time. We were looking into validating a method in which we are pre-treating Screening Cells 1 and 2, so when a known DARA patient comes for a subsequent Type/Screen, we will just run the primary screen and if it comes up negative, no further workup needs to be done and K-negative RBCs will be XM'd and issued.

 

 

5 hours ago, Cliff said:

Sure, here is our validation plan, we are still validating the process.  I took out names and links to documents.

DTT.pdf

 

Thank you, Cliff!

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17 hours ago, Cliff said:

Sure, here is our validation plan, we are still validating the process.  I took out names and links to documents.

DTT.pdf

Thank you, Cliff. That's very interesting. I was wondering if you planned to do comprehensive phenotyping, and you are.

One comment: Super-strong, FDA-licensed reagents may not allow detection/demonstration of weakening of antigens. Perhaps a better way to detect weakening of antigens would be to use a weak and/or diluted reagent. You could even do comparative titrations and add up scores....

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Human source commercial antisera like anti-Fyb have shown weakening antigens after DTT treatment in our hands .  Others have not found this.  We wish we could figure out why our reagent cells sometimes lose their Fyb antigens.  Donor units that we have treated have not seemed to lose reactivity.

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Does anyone know if any of the reagent vendors has a commercial DTT product that can be purchased and used to eliminate the dilution prep?

 

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16 minutes ago, Marianne said:

Does anyone know if any of the reagent vendors has a commercial DTT product that can be purchased and used to eliminate the dilution prep?

 

I think I remember someone here saying Hemobioscience has it and it looks like perhaps that is the case? http://www.hemobioscience.com/Products/Specialist-Solutions It's listed as a 2mL liquid vial. Unfortunately I couldn't find a package insert so I'm not 100% sure, I have not used this product myself.

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11 hours ago, Marianne said:

Does anyone know if any of the reagent vendors has a commercial DTT product that can be purchased and used to eliminate the dilution prep?

 

Hemobioscience does sell 0.2M DTT.  I have used it.  It works fine.  A bit labor intensive.  The pkg insert comes with a procedure.

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I am curious to know how this validation is coming along. We do several DARA patients a month, and I'm all for saving my techs time. We have also experienced issue when we try to hold on to treated RBCs longer than a couple of hours.

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On ‎1‎/‎24‎/‎2017 at 11:31 PM, David Saikin said:

Hemobioscience does sell 0.2M DTT.  I have used it.  It works fine.  A bit labor intensive.  The pkg insert comes with a procedure.

Does anyone have an SOP for this specific product that they would be willing to share?

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How many of you who treat with DTT do comprehensive phenotyping including Kpa, Kpb, Jsa and Jsb?  When I talked to our reference lab they indicated that would be a good idea.  If we send our specimens out to the reference lab and get a negative antibody screen in return, how many of you would feel comfortable with electronic crossmatching? If the patient's antibody screen has always been negative, it should be eligible.  Because otherwise we wouldn't get a compatible crossmatch, since we don't use DTT here.  We just got our first patient and I did a screen and antigen type for K.

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How many of you who treat with DTT do comprehensive phenotyping including Kpa, Kpb, Jsa and Jsb?  When I talked to our reference lab they indicated that would be a good idea.  If we send our specimens out to the reference lab and get a negative antibody screen in return, how many of you would feel comfortable with electronic crossmatching? If the patient's antibody screen has always been negative, it should be eligible.  Because otherwise we wouldn't get a compatible crossmatch, since we don't use DTT here.  We just got our first patient and I did a screen and antigen type for K.

Also, what is your experience with how frequently patients on DARA need transfusions?  Weekly, every other week?  I'm concerned because we have to Fedex ours to the reference lab, but they usually can fax us results within 36 hours.

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I have never done DTT treated cells so bare with me. I have been looking at the Dithothreitol 0.2M that Hemo bioscience offers. Whe you get the product is it in powder or liquid form? How much is in the bottel? If its a liquid to do thaw it and aliquot it out into smaller volumes? When you treat the cells to you mix 1 ml of DTT with 25 mL of reagent cells?

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I work at a reference lab and I have personally worked up many Dara patients. I have had to DTT treat reagent cells only once to remove the reactivity caused by it.  Every other instance I have been able to complete rule-outs/rule-ins in LISS.  Maybe as these patients are on the drug for longer we will have to start DTT treating cells more but as of now it hasn't been as big of an issue as it was thought to be last year when this all started(at least for us).  Has anyone else noticed this too?  Could be we are just lucky with our patients.

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I work at a reference lab as well and we get roughly 3-4 DARA patients a month. More often than not, half are repeat customers. It seems strength varies from patient to patient, but definitely increases as the treatment continues, but nothing more than a 2+. We treat screening cells with DTT every time. We also treat a second set with trypsin to help rule out Kell group system antibodies. So far we haven't had much viability with cells lasting longer than a full shift.

Edited by BloodBankBlake

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On ‎1‎/‎30‎/‎2017 at 10:21 AM, BloodBankBlake said:

I work at a reference lab as well and we get roughly 3-4 DARA patients a month. More often than not, half are repeat customers. It seems strength varies from patient to patient, but definitely increases as the treatment continues, but nothing more than a 2+. We treat screening cells with DTT every time. We also treat a second set with trypsin to help rule out Kell group system antibodies. So far we haven't had much viability with cells lasting longer than a full shift.

Where do you get your Trypsin?  Could you share that SOP?  Since it destroys far fewer antigens (correct?) - would it be ok to use just that for DARA patients?  Is it commercially pre-made or do you have to "make it up" also like the DTT?

Or does the Hemo-Bioscience DTT come "pre-made"?  I know someone else already asked that, but I don't see an answer yet?  If it is "pre-made", can you aliquot it to small volumes so you could just treat one set of cells at a time for use that day?  Will it last that way?  Someone asked if you use 1 ml of DTT to treat 25 ml of reagent cells - how do you even get that much reagent test cells?

Our DARA pts have varied widely in how often they have come in.  Our first 2 were frequent fliers for a while and even the Dr was surprised and dismayed at how long we could still pick up the DARA - 3-6 months after discontinuing the drug, according to current literature.  Our 3rd DARA pt we have only seen once - it all might depend on where they are in the disease process before they start the drug.  Newer cases will get a chance to start on the drug earlier and might not be as sick to start with as our 1st pt was. 

Any answers would be appreciated.  This is not fun for smaller facilities.  Drs hate waiting for reference lab workups, but they don't think it necessary to even notify us that they have a new pt on DARA either, so we could start a workup earlier - they just send a Type and Cross for "TODAY!"

 

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On ‎1‎/‎27‎/‎2017 at 3:48 PM, jshafer said:

I have never done DTT treated cells so bare with me. I have been looking at the Dithothreitol 0.2M that Hemo bioscience offers. Whe you get the product is it in powder or liquid form? How much is in the bottel? If its a liquid to do thaw it and aliquot it out into smaller volumes? When you treat the cells to you mix 1 ml of DTT with 25 mL of reagent cells?

it comes in liquid and is kept frozen.  Can be thawed up to 5 times.  You don't need much to treat reagent rbcs.  You'd have to work out the volume you'd want to use.

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The Hemo Bioscience 0.2 M DTT comes in 2ml vials - the ratio listed in the Technical Manual is a 1:4 ratio - 1 drop of packed red cells to 4 drops of 0.2M DTT. Incubate at 37 and then wash at least 4 times with 0.9% saline.

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6 hours ago, applejw1 said:

The Hemo Bioscience 0.2 M DTT comes in 2ml vials - the ratio listed in the Technical Manual is a 1:4 ratio - 1 drop of packed red cells to 4 drops of 0.2M DTT. Incubate at 37 and then wash at least 4 times with 0.9% saline.

I would definitely suggest using 2 drops of cells to 8 drops of DTT.  You will lose cells in the washing process.

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10 hours ago, Vikman said:

I would definitely suggest using 2 drops of cells to 8 drops of DTT.  You will lose cells in the washing process.

If one does as suggested (by AABB TM and Hemo) - 1 drop of PACKED CELLS to 4 drops of DTT, you should be able to make at least 15 - 20 drops of a 3% cell suspension after treatment and washing.

To make 1 drop of packed cells from a commercial red cell suspension, you typically need to centrifuge down 20 - 25 drops.

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On Tuesday, January 31, 2017 at 3:25 PM, cswickard said:

Where do you get your Trypsin?  Could you share that SOP?  Since it destroys far fewer antigens (correct?) - would it be ok to use just that for DARA patients?  Is it commercially pre-made or do you have to "make it up" also like the DTT?

Or does the Hemo-Bioscience DTT come "pre-made"?  I know someone else already asked that, but I don't see an answer yet?  If it is "pre-made", can you aliquot it to small volumes so you could just treat one set of cells at a time for use that day?  Will it last that way?  Someone asked if you use 1 ml of DTT to treat 25 ml of reagent cells - how do you even get that much reagent test cells?

Our DARA pts have varied widely in how often they have come in.  Our first 2 were frequent fliers for a while and even the Dr was surprised and dismayed at how long we could still pick up the DARA - 3-6 months after discontinuing the drug, according to current literature.  Our 3rd DARA pt we have only seen once - it all might depend on where they are in the disease process before they start the drug.  Newer cases will get a chance to start on the drug earlier and might not be as sick to start with as our 1st pt was.

Any answers would be appreciated.  This is not fun for smaller facilities.  Drs hate waiting for reference lab workups, but they don't think it necessary to even notify us that they have a new pt on DARA either, so we could start a workup earlier - they just send a Type and Cross for "TODAY!"

 

Sorry for the delay in response. We make up our Trypsin that we get from Sigma Aldrich. It seems to work just as well as DTT, but we treat it with both since the "standard" seems to be DTT. I've attached our Trypsin testing SOP.

Trypsin Testing.docx

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Because the technical Manual does state packed cells we now use donor cells for DTT treatment. We have found that we get better results than the DTT treated reagent cells. The reagent cells seem to lyse more and we have had to repeat testing a lot because of that.

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