Preparation of DTT for treating RBCs

[Kathy]

Does anyone have a recipe for making the DTT solution for treating RBCs?  The AABB Technical Manual says to "prepare 0.2M DTT by disolving 1 g of DTT powder in 32 ml of phosphate buffered saline (PBS), pH 8.0".  I cannot find a source of PBS with a pH of 8.0.  Do I need to buy phosphate buffer with a pH of 8.0, add it to saline, and then add the DTT powder?

[Bb_in_the_rain]

Do you consider readjusting pH of blood bank saline using NaOH? 

Edited December 30, 2015 by dothandar

[Cliff]

I found an older SOP for making PBS in our reference lab.

 

Preparation of Phosphate Buffered Saline Solutions.docx

[jill]

Are you referring to 0.2M DTT or 0.01M DTT?

There may be a procedure for making them up in the Methods

section of the AABB Technical Manual.   

 

[Kathy]

Thanks.  I need to make 0.2M DTT, but I need to make the PBS first.  I ended up ordering this PBS with pH 7.3:

http://www.lonza.com/products-services/bio-research/cell-culture-products/reagents/buffered-saline/pbs-without-ca-mg-or-phenol-red.aspx

and this DTT:

http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11093&catalogId=10101&productId=22063&storeId=11787&langId=-1

One of my coworkers found an SOP that calls for adding 1 g of DTT to 32 ml PBS.  When the chemicals arrive I will try that and check the pH.  Unfortunately, we don't have a high-accuracy scale for weighing dry chemicals, so I needed to order the everything as much pre-measured as possible.  Stay tuned....

[jill]

We dissolve 7.7g of DTT into 250 mL phosphate buffered saline

made from a cube of saline that had a bottle of pHix(from Immucor) added to it.

 

 

[Carina]

Cathy how did you solve the PBS pH 8.0 issue? I have the same problem today. I have DDT power to prepare the reagent to treat RBCs, however no PBS at the 8.0 pH and no solutions to adjust pH. I cannot find any supplier for the PBS at that pH.

[David Saikin]

It seems more prudent to purchase already prepared.  Not overly expensive.  Unless of course you are doing a tremendous volume.

[natalynn]

Quote

 

Dissolve 1 gram DTT into 32 ml PBS.  Aliquot into 2 ml samples and store at -20ºC or below.

Expiration is 1 year from preparation.

 

http://www.sigmaaldrich.com/catalog/product/sial/d0632?lang=en&region=US

 

^ you can buy it pre packaged in 1g amounts. (the price has gone up! ha supply and demand I guess)

[exlimey]

51 minutes ago, natalynn said:

Dissolve 1 gram DTT into 32 ml PBS.  Aliquot into 2 ml samples and store at -20ºC or below.

Expiration is 1 year from preparation.

 

http://www.sigmaaldrich.com/catalog/product/sial/d0632?lang=en&region=US

 

^ you can buy it pre packaged in 1g amounts. (the price has gone up! ha supply and demand I guess)

You performed stability testing on your home-made material ?

[catchmenow51]

If anyone is using PBS (made with pHix) you can adjust your pH with pHix to meet the 8.0 criteria or you could you the base buffering solution from an elution kit. We add this to already prepared PBS until the pH reaches 8.0.  It only takes a few drops in 32mLs.

[amym1586]

We went and bought the BIO-RAD DTT

http://www.bio-rad.com/en-us/sku/1610611-dithiothreitol-dtt?parentCategoryGUID=796138a1-8821-4b35-872f-0c682b293e0b

I started looking for a package insert and now I see it says For research only.

 

Can we not use this for patient testing?

[Quality_Lady]

On ‎6‎/‎20‎/‎2017 at 6:05 AM, catchmenow51 said:

If anyone is using PBS (made with pHix) you can adjust your pH with pHix to meet the 8.0 criteria or you could you the base buffering solution from an elution kit. We add this to already prepared PBS until the pH reaches 8.0.  It only takes a few drops in 32mLs.

Use of either pHix or Buffering solution as described is outside of IFU and would require extensive in-house validation to support their use.  Not only would you have to provide "in-use" testing, you would also have to determine stability data.

[exlimey]

On 6/20/2017 at 1:54 PM, amym1586 said:

We went and bought the BIO-RAD DTT

http://www.bio-rad.com/en-us/sku/1610611-dithiothreitol-dtt?parentCategoryGUID=796138a1-8821-4b35-872f-0c682b293e0b

I started looking for a package insert and now I see it says For research only.

 

Can we not use this for patient testing?

In my opinion: Yes, you can. That statement on chemicals is meant to tell you that you can't use it in a medical or nutritional fashion.

You will not find a package insert for any raw chemical - the supplier has absolutely no idea what the buyers are going to do with the materials. If you buy plain old sodium chloride (NaCl), it doesn't have a package insert, exactly for the reasons stated above.

Anything anyone is doing with DTT and/or other exotic chemicals in the Blood Bank realm is completely out of the typical regulated environment. These chemicals assist in a complex investigation,  they are not making a diagnosis. Sometimes it is necessary to go beyond the use of licensed, registered, validated reagents to best serve a patient.

That being said, the DARA issue has brought DTT use into some routine labs. Complex serological investigations should be left to experts - the high level Reference Laboratories, who understand the pros, cons and limitations of the specialized reagents they use.

End of rant.☺

[exlimey]

19 hours ago, Quality_Lady said:

Use of either pHix or Buffering solution as described is outside of IFU and would require extensive in-house validation to support their use.  Not only would you have to provide "in-use" testing, you would also have to determine stability data.

What do you mean by "extensive in-house validation" ? What is "in-use testing" ?

[catchmenow51]

Don't reference labs/hospital labs use many things outside of IFU?  These things are used to aide in the identification of antibodies. They are not used solely for ID, as that would be ridiculous. Just like using expired panels, only, to ID antibodies.  Expired panels are only used to help rule in/out.  It seems that validation would be the fact that DTT works when the controls work.

 

Edited June 22, 2017 by catchmenow51

[Quality_Lady]

Exlimey - What I was referring to is the confirmation / validation that the use of the materials as described in the post works in the manner they are being used, provides equivalent results, and there is parallel testing with accepted methods (e.g., buffering with NaOH) showing that the use of either solution gives comparable results in the hands of the lab..

Catchnenow51 - Did not intend to hit your hot button, just wanted to give you food for thought before an inspector asked the same questions.  I came from medical devices so I have heightened expectations of regulatory impact.  Plus there is no data to support the expiration date and stability of the PBS prepared in this fashion.

[exlimey]

14 hours ago, Quality_Lady said:

Exlimey - What I was referring to is the confirmation / validation that the use of the materials as described in the post works in the manner they are being used, provides equivalent results, and there is parallel testing with accepted methods (e.g., buffering with NaOH) showing that the use of either solution gives comparable results in the hands of the lab..

Catchnenow51 - Did not intend to hit your hot button, just wanted to give you food for thought before an inspector asked the same questions.  I came from medical devices so I have heightened expectations of regulatory impact.  Plus there is no data to support the expiration date and stability of the PBS prepared in this fashion.

Bottom line: If a facility "manufactures" a reagent, it is obliged to prove that it functions as designed and can be made repeatedly/reliably. However, the rules and regulations that apply to licensed and registered reagents do not necessarily apply to "home-brew" materials.

Providing the material will only be used in-house and not distributed, there is no need for "parallel testing" of any other formulation, no matter how crude the manufacturing process appears to be. Such testing is only required for licensed or registered reagents and devices (under the umbrella of Design Control).

I'm assuming (correct me if I'm wrong) that the PBS used in preparation of the DTT is made immediately before use, and therefore the stability of it is irrelevant. Stability of the finished reagent may be important if the "manufacturers" wish to assign an expiration date. This may not be necessary, since in the case of DTT, controls are included EVERY time it is used - the operator has a current and immediate indication of whether the reagent worked or not.

Validation is essential to provide confidence when it is impossible to directly prove that a process has worked. Think of vaccine manufacture: One cannot test every vial/dose to see if it meets specifications - all of the product would be compromised. When integral testing proves that a process works each time, validation requirements are minimized.

[Bb_in_the_rain]

Hello, 

Great discussion. I just set up a comparison study using DTT made with different pH buffer and here is the data (a copy of poster to be posted at 2017 AABB meeting). Hope it help answering some questions here. 

to print DTT poster AABB 2017.pdf

[carolyn swickard]

We purchased the HemoBioScience DTT - premade also.  It worked very well in parallel testing and seemed to completely decrease the reaction of DARA with the treated cells.  It is frozen and can be thawed up to 3 times (I think), but you will use it up before that as only    2 mls comes in each tube.  Much faster than a send out.  Gives you a clean screen and then you can just crossmatch treated K neg units.  (barring the production of anti-k, that is!)

Used a combination of the AABB procedure (recommendations and limitations) and the manufacturer's procedure to get a clean procedure that worked here.

I can not get a file copy of our procedure from our system (Policy Stat) to this system but would be able to email a copy if anyone cares for it.  If anyone notices a problem - please let me know.  This was a scary stretch for us too.