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David Saikin

0.2m DTT and cord blood

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So, My reference lab tells me that the DTT treatment for anti-CD38 therapy does not destroy the Kell system ags on cord cells.  Is this valid?

Is there a difference between cord Kell ags and adult Kell ags?  What am I missing here?  They told me the patient doesn't have anti-K so I don't 

 need Kell negtaive.  I told them I still need Kell neg since they have been unable to type him.

Any thoughts anyone?

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The Kell glycoprotein contains many (16) Cysteine residues, which results in it being highly folded through the resultant disulphide bonds.

DTT is a reducing agent that will destroy these disulphide bonds, and this is why Kell antigens are denatured by DTT.

I can see absolutely no logical reason why DTT should not denature the Kell antigens on cord blood any less thoroughly than on adult red cells.

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Supposedly cord blood doesn't react with anti-CD38.  I suppose if they have K pos cord cells they could have ruled out anti-K but you can't get them commercially anymore.  Where does a reference lab get them?  Sounds like someone has mixed up some information or has miscommunicated (and it's not you).

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On 9/17/2016 at 5:16 PM, Mabel Adams said:

Supposedly cord blood doesn't react with anti-CD38.  I suppose if they have K pos cord cells they could have ruled out anti-K but you can't get them commercially anymore.  Where does a reference lab get them?  Sounds like someone has mixed up some information or has miscommunicated (and it's not you).

Is that the scenario, CD38 is not well developed on cord cells?

Thanks Malcolm, that is what I thought.

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On 9/17/2016 at 5:16 PM, Mabel Adams said:

Supposedly cord blood doesn't react with anti-CD38.  I suppose if they have K pos cord cells they could have ruled out anti-K but you can't get them commercially anymore.  Where does a reference lab get them?  Sounds like someone has mixed up some information or has miscommunicated (and it's not you).

I agree - mixed-up information. If CD38 is only poorly expressed on cord cells, it would make sense that they would be less likely to react with the therapeutic anti-CD38 antibodies.

Many workers believe that antibodies to Lutheran system antigens (also carried by CD38) rarely cause HDFN partly due to their poor expression on cord cells. This seems to parallel one of the mixed concepts above - no antigens on cord cells, no reactivity. If this is true, then K+ cord cells could, perhaps, maybe used to exclude anti-K in Dara patients. Not quite sure how you'd put that in a report.

However, having access to a large number of cord cells and the ability to mine them on regular basis for K+ examples seems like a pie-in-the-sky situation. It might be possible in a large, competent Reference Laboratory that has a good working relationship with a large maternity hospital. Might be some ticklish Informed Consent issues, but.......at least its feasible.

As to typing the patient - monoclonal IgM anti-K reagents are available. This makes the DAT moot.

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Our first experience with Darzalex was with a patient who was receiving an "experimental drug" for MM.  The tech ran ton of cells, 4 of which were cord cells.  All 4 cord cells reacted similarly to the other cells tested.

I've read somewhere where it is thought that anti-CD38 didn't react with cord cells, but we don't support that position.

In regards to the .2M DTT treatment of cord cells not having an impact on Kell Sys. antigens, I've never come across any literature that would support that position.

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16 hours ago, exlimey said:

I agree - mixed-up information. If CD38 is only poorly expressed on cord cells, it would make sense that they would be less likely to react with the therapeutic anti-CD38 antibodies.

Many workers believe that antibodies to Lutheran system antigens (also carried by CD38) rarely cause HDFN partly due to their poor expression on cord cells. This seems to parallel one of the mixed concepts above - no antigens on cord cells, no reactivity. If this is true, then K+ cord cells could, perhaps, maybe used to exclude anti-K in Dara patients. Not quite sure how you'd put that in a report.

However, having access to a large number of cord cells and the ability to mine them on regular basis for K+ examples seems like a pie-in-the-sky situation. It might be possible in a large, competent Reference Laboratory that has a good working relationship with a large maternity hospital. Might be some ticklish Informed Consent issues, but.......at least its feasible.

As to typing the patient - monoclonal IgM anti-K reagents are available. This makes the DAT moot.

This was actually studied.

Schmidt, A.E., Kirkley, S., Patel, N., Masel, D., Bowen, R., Blumberg, N., & Refaai, M.A. (2015). An alternative method to dithiothreitol treatment for antibody screening in patients receiving daratumumab. Transfusion, 55(9), 2292-3. doi: 10.1111/trf.13174

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19 hours ago, exlimey said:

Many workers believe that antibodies to Lutheran system antigens (also carried by CD38) rarely cause HDFN partly due to their poor expression on cord cells.

There is no doubt that the Lutheran antigens are not expressed as strongly on cord red cells as they are on most adult red cells (with the exception of the In(Lu) phenotype, the X-linked Lu(a-b-) type and the genuine amorphic Lu(a-b-) type), but that is not the only reason that the antibodies very, very rarely cause clinically significant HDFN.  The Lutheran antibodies can be essentially IgM, a mixture of IgM and IgG or IgG (but IgG only is most unusual), and, of course, IgM antibodies would not be expected to pass through the placenta.  However, even if there is an element of IgG Lutheran antibody in the maternal circulation, foetal Lutheran glycoprotein is expressed on placental tissue, and so these ectopic (for want of a better way of putting it) Lutheran antigens will largely adsorbed out the maternal Lutheran antibodies.

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4 hours ago, Malcolm Needs said:

The Lutheran antibodies can be essentially IgM, a mixture of IgM and IgG or IgG (but IgG only is most unusual), and, of course, IgM antibodies would not be expected to pass through the placenta.  However, even if there is an element of IgG Lutheran antibody in the maternal circulation, foetal Lutheran glycoprotein is expressed on placental tissue, and so these ectopic (for want of a better way of putting it) Lutheran antigens will largely adsorbed out the maternal Lutheran antibodies.

I agree completely. Hence why I wrote "partly due to their poor expression". And, don't forget, if antibodies to Lutheran system antigens are in part IgG, they are often they wrong subclass to readily cross the placenta (IgG2 and IgG4).

Edited by exlimey
Clarification

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Sounds like poor advice from the IRL, I think what they meant to say was that the CD38 antigen is weakly expressed on Cord cells.  The Kell system is completely removed by DTT treatment so you should give K neg blood as you have no way to exclude anti K, BTW that also means JSa, Jsb, Cellano etc. also Lu null cells do not express CD38 either.

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