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Samples were referred to us (the reference laboratory) and were found to contain a pan reactive antibody detected by all IAT techniques available to us (including two gel technologies, and tube techniques perfomed with a range of different media). The 'Auto' and DAT were negative. An elution of the patient's red cells was also 'negative'. The patient had been transfused a single red cell unit, about 6 weeks prior to referral (no previous transfusion history), when their IAT red cell antibody screen was negative. Reactivity with test cells was now a, relatively, uniform positive grade 3 (on a scale of 1-5), but some stronger and weaker reactions were noted. Reaction strength increased when an enzyme IAT was performed, using papain treated red cells. We initially investigated for the possible presence of an antibody to a higher frequency antigen, but found no negative results with, for example, Csa-, Lan-, Vel-, Lub-, Kpb-, CR1-, Yta-, k-, red cells. The plasma hade a titre of 2048 against each of the red cells of a 3 cell antibody screening 'set'. We were then informed that the patient had started treatment with anti-CD38, immediately after their transfuson, 6 weeks ago. We treated a standard red cell antibody identification panel with DTT, and found that this failed to react with the sample. This result, in combination with results from an extended phenotype, allowed us to rule out most significant antibodies (other than those with antigens that are sensitive to DTT treatment and that the patient lacked) and be fairly confident taht the antibody we were detecting was the anti-CD38.

 

What I want to know is why are the patient's 'Auto' and DAT negative, where has their CD38 antigen gone? Also, has anyone who has encountered this antibody come up with any other methods for 'dealing' with it?

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I saw a papers about this recently - see below abstract.

 

BACKGROUND: Daratumumab (DARA), a promising novel therapy for multiple myeloma, is an IgG1 monoclonal antibody that recognizes CD38 on myeloma cells. During routine compatibility testing, we observed

that the plasma of five of five DARA-treated patients demonstrated a positive antibody screen and panreactivity on red blood cell (RBC) panel testing. We hypothesized that the observed panreactivity reflected

DARA binding to CD38 on reagent RBCs, and we investigated methods to prevent this binding.

STUDY DESIGN AND METHODS: DARA binding to CD381 or CD38– HL60 cells was assessed by flow cytometry. To remove cell surface CD38, cells were incubated with dithiothreitol (DTT) or trypsin. Soluble

CD38 or anti-DARA was used to neutralize DARA in solution. Routine blood bank serologic methods were used to test samples from DARA-treated patients and normal plasma samples spiked with DARA and/or alloantibodies.

RESULTS: Normal plasma samples spiked with DARA (0.1-10 mg/mL) and incubated with reagent RBCs recapitulated the interference observed with samples from DARA-treated patients. Flow cytometry experiments

confirmed DARA binding to CD381 HL60 cells, but not to CD38– controls. DTT treatment of CD381 HL60 cells reduced DARA binding by 92% by denaturing cell surface CD38. Treating DARA-containing plasma with soluble CD38 or anti-DARA idiotype also inhibited DARA binding.

CONCLUSION: DARA causes panreactivity in vitro by binding to CD38 on reagent RBCs. Treating reagent RBCs with DTT is a robust method to negate the DARA interference, enabling the safe provision of blood to

DARA-treated patients. Because DTT denatures Kell antigens, K– units are provided to these patients.

 

Resolving the daratumumab interference with blood compatibility testing, Chapuy et al. TRANSFUSION 2015;00;00–00

 

As to why the patient's DAT and auto were neg, my guess would be that the treatment had already neutralised, maybe even destroyed???? their own CD38, so that their cells are no longer reacting 'normally' in that respect.  I don't know.  I have no actual experience with this at all.

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It's odd; nothing in the literature suggest that the antigen is destroyed, only that the (white) cells that express it are destroyed. A few theories as to the nature of the destruction are postulated.

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We got our first one yesterday - antibody panel in gel a uniform weak, but nonetheless clear, reaction with all panel cells, negative auto and DAT (like John's patient).  Antibody in tube (ratio 4 to 1 with no LISS) gave moderate reactions with all cells.  We DTT treated the red cells and the antibody then disappeared.  Exciting.  Felt like doing REAL science!

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We got our first one yesterday - antibody panel in gel a uniform weak, but nonetheless clear, reaction with all panel cells, negative auto and DAT (like John's patient).  Antibody in tube (ratio 4 to 1 with no LISS) gave moderate reactions with all cells.  We DTT treated the red cells and the antibody then disappeared.  Exciting.  Felt like doing REAL science!

 

I'm jealous! Almost...

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And now it is FDA approved in the US.

 

Multiple myeloma drug from J&J receives FDA approval
Johnson & Johnson has won FDA approval for the use of Darzalex, or daratumumab, in multiple myeloma patients who have received at least three lines of standard therapies. The decision was backed by data from two trials, including a 42-patient study showing that tumors were completely or partially reduced in 36% of patients who took the drug.
Reuters (11/16)

 

 

Do all patients taking the drug show the effects outlined on this thread or do some have a weaker or undetectable effect?  I need to have a discussion with our oncologists (among other things).

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A colleague in Montana suggested that we do an antibody screen and K type any patient before they are started on the drug so we have a baseline antibody screen.  Her oncologists have said they will be using this drug.  I figured advance testing also would give us time to send out k typing on any K pos patient since we don't keep that antiserum.  Of course I still need to find out if this is going to be a rarely used drug or if the patients on it will have a reduced need for transfusion.  Other questions like half-life and how often it is given and for how long may help determine how likely we are to see a case in the BB.  I heard at AABB that cord cells don't react with it but would there be any way to validate a process for using cord cells for this?  We might have to get in some DTT and write up a protocol for dealing with this antibody if it is going to be used much in our area.

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Does anyone know what would happen if someone mistook this situation for a warm auto and did a PEG autoadsorption on the sample?  Or maybe an alloadsorption?  Can this antibody be adsorbed out?

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The failure of adsorption is probably a mix of the high titre of the antibody (in the 'thousands' if a the high dose is given) and a lowi density of cd38 on red cells used for adsorption. Maybe if you spent a week adsorbing the same sample you'd get rid of it!  As for how common the phenomenon is; I would have thought you'd find it every time you looked for it, so maybe it's just that the test isn't requested after therapy starts, in a lot of the patients. I still wonder why the auto/DAT is negative in some patients; maybe the DAT positive ones already had a positive DAT before the therapy started?

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The oncologist referred to a "positive Coombs" so I don't know if we were actually talking about the same problem.  When they say "Coombs" they usually mean a direct antiglobulin test not a screen.  I wonder if some of his buddies have been ordering DATs on these patients, finding them negative and concluding that there isn't much of a problem--until they need to crossmatch them anyway.  At least we have begun the conversation.

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6 hours ago, Mabel Adams said:

The oncologist referred to a "positive Coombs" so I don't know if we were actually talking about the same problem.  When they say "Coombs" they usually mean a direct antiglobulin test not a screen.  I wonder if some of his buddies have been ordering DATs on these patients, finding them negative and concluding that there isn't much of a problem--until they need to crossmatch them anyway.  At least we have begun the conversation.

Ah, the need to use correct terminology rears its ugly head again!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

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What you say, Mabel, could well be the case. The (limited) published work implies that the patients will be DAT positive, but my (again, limited) experience is that they are DAT negative (and remain so). Could the apparent lack of cd38 on patient red cells be the effect of a prior therapy (the use of gel cards (and  other results) rules out 'insufficient washing' , and 'prozone' doesn't 'fit')?

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I wonder if it might be that any patient red cells that express cd38 strongly get coated with antibody and removed from circulation thereby selecting for rbcs with lower expression of cd38.  Maybe this even happens to precursor cells (assuming they express the antigen) so that fewer strong cd38 cells get released from the marrow.  Just speculating.

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I wondered this too, but the patients don't seem to be particularly anaemic, which is what I'd expect if red cells were being destroyed. Maybe antigen is being removed without red cell destruction? Just had a new 'cd38' case and this one has a positive DAT', with therapy starting only a week or so ago. DAT wasn't done before therapy started. Titre of the antibody is almost 4000.

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I have recently done an oral presentation about our research project on anti-CD38(Daratumumab) during this recent AABB meeting in Anaheim. I have some abstract references that I have attached here. Hope it helps. 

Also a full article here. http://onlinelibrary.wiley.com/doi/10.1111/trf.13069/full

3 of 8 patients(i have seen to date) has positive DAT and 2 of them were on IVIG. We found that this antibody is non-reactive with In(Lu) cells by tube method. By tube method, DTT treated cells were non reactive, cord cells were weakly reactive to non-reactive.(not enough cord cells were tested yet to find pattern) 

Our flow cytometry data wiith florusecnt labeled anti-CD38 shows that DTT treated cells has very low percentage of CD38 (2%-4%), cord cells has about 21%-24% and positive control varies from (53-82%), In(Lu) cells has about >20%. These data were presented orally during AABB meeting but not yet been published. 

 

SKMBT_75115100213120.pdf

NEJM Aug 2015 editorial on Dara.pdf

cord rule out.pdf

abstract-ARC and NYBC.pdf

abstract-ARC and NYBC.pdf

Edited by dothandar

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