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Thanks for the heads up.  Now if only we could get someone to tell us or tell the patient to tell us that they are on these drugs.  If you have any "pull" with someone in Seattle, feel free to pass along the request.  :)

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On 4/15/2019 at 3:04 PM, Mabel Adams said:

We just got our first anti-CD47 out here in the sticks on a patient who went to Seattle for a clinical trial.  That drug is Hu5F9-G4.  No other name yet.  It interfered with her reverse as well as all gel testing.  We did a 30 minute saline screen and it was 4+ at 37C but negative at IAT using Immucor's anti-IgG which doesn't react with IgG4.  She was antigen typed before starting treatment so we got that information and gave her K and Fya negative units (lucky she is positive for most antigens).  We called them incompatible because we have not validated the Immucor anti-IgG as our test of record, the screen was 4+ at 37C and because the drug causes the patient's H&H to drop so I wasn't sure that the units would be certain to have normal survival.  I didn't expect to get one of these for a few more years since we aren't in a big teaching hospital region.  It would have been nice if the big center had sent her home with information that she was on this and instructions to tell the blood bank. We lucked out finding clues in Epic's Care Everywhere so we called the Seattle blood bank.

Mabel was kind enough to share this sample with our lab as we had not had the pleasure of seeing one of these.  The sample was limited and it technically was not a referral, so my work was centered on getting to know anti-CD47.  The patient forward typed as an A with no issues (I've heard the forward type can at times be affected), but reverse typed as an O, with 2+ reactivity in the A1 and A2 reverse cells.  The patient typed Rh positive; no reactivity noted in the Rh control.  The DAT was micro positive with poly specific AHG, heavy-chain IgG (HC) and C3, but was not interpret-able due to a micro positive saline control.  Warm washing x4 with 37C saline resolved the positive saline control and still demonstrated micro reactions in the previously mentioned AHG sources.  Interestingly, the reactivity was actually slightly stronger than the initial testing.

Initial antibody testing was 2+ reactive in saline at initial spin, 3+ in saline at 37C spin (post 30 minute incubation) and 3-4+ reactive in saline at IAT with HC IgG, 3-4+ reactive in PEG at IAT (4+ reactions with the Rh negative cells tested as compared to 3+ reactivity with Rh positive cells; consistent with reports in literature), and 4+ reactive with papain treated cells (carryover reactivity was noted as a "no spin read" was 3+ and agglutination was noted prior to the addition of HC AHG).  Testing with Immucor's murine monoclonal IgG Gamma Clone in PEG at IAT was macro negative, micro positive.

Alloadsorption x2 (using 2 volumes of cells to one volume of plasma) onto papain-treated R1R1 and rr cells of known phenotypes (we usually do R1, R2 and r cells, but with the limited sample I dropped the R2 adsorption) removed the initial spin reactivity and confirmed the patient was group A.

Alloadsorptions x3 were 1+ reactive in saline at 37C, and 3+ reactive at IAT using the HC IgG.  x4 adsorptions yielded the same results.  Out of curiosity, I did run a D-- cell with the x4 and it was nonreactive at 37C, but 2+ reactive at IAT.

For fun, I performed titration studies on both the R1 and rr x4 adsorptions (saline, 60 minutes 37C incubation).  Macro reactivity at 37C was noted at 1:32 in both samples, but technically they had a titer of 4 (1st observed 1+ reaction).  At IAT, both samples had a titer of 16,384.... AFTER  4 adsorptions!  A recent report in IMMUNOHEMATOLOGY stated that using equal volume (cells:plasma) adsorptions onto papain-treated cells x3 and x4 resulted in "the majority" of the samples being nonreactive in saline at initial spin and in PEG at IAT.

Lastly, I did perform EGA treatment of the patient's red cells to see its affect on the positive DAT.  The micro reactivity was removed following a 1min. 30sec treatment of the patient's cells, but since the sample was way beyond the manufacturer's  specimen recommendation, I'm not totally confident in the observed results.

Net result:  It looks like alloadsorptions will be needed to resolve any observed ABO discrepancies due to reverse cell issues and PEG testing with Immucor's murine monoclonal Gamma Clone, with a macro only read,  would resolve any the issue of detecting underlying alloantibodies.

Thanks much Mabel for sharing this sample with us!

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Posted (edited)

Brilliant post StevenB.  I just out of curiosity, you said the after-adsorption plasma reactived with IAT, and had a  titer of 16,384 , which is very high, but non reactive with PEG IAT. So interesting.

If my understading of English is right:)

Edited by yan xia

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Thanks yan....the macro negative reaction in PEG at IAT was with the unadsorbed plasma and was tested with Immucor’s murine monoclonal Gamma Clone IgG.  As Mabel pointed out, this reagent does not detect IgG4 and the anti-CD47 is....evidently.... an IgG4 antibody.  In my hands, it was microscopic reactive so I believe in the future we will drop that read when faced with these patients. 

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8 hours ago, StevenB said:

Thanks yan....the macro negative reaction in PEG at IAT was with the unadsorbed plasma and was tested with Immucor’s murine monoclonal Gamma Clone IgG.  As Mabel pointed out, this reagent does not detect IgG4 and the anti-CD47 is....evidently.... an IgG4 antibody.  In my hands, it was microscopic reactive so I believe in the future we will drop that read when faced with these patients. 

Then why not drop it for everyone?  Surely, Peter Issitt was correct all those years ago when he wrote that reading tests under a microscope is a step too far, he was talking about ALL patients, not just those where you get an "inconvenient" weak reaction, like these patients on anti-CD47?

 

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Lol...I like the way you think, Malcom, but I don't have control over those decisions.  Being a reference lab though, we push our efforts when testing referred samples.  Micro reads are part of our routine if the patient has been transfused or pregnant within the last 3 months.  Testing in PEG however is optional and PEG is notorious for revealing micro reactivity which often can not be identified.  Having said that, I have on many occasions identified micro only, clinically significant antibodies in PEG.  Would I recommend a hospital blood bank read micro?  No, I wouldn't. 

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On ‎4‎/‎15‎/‎2019 at 3:04 PM, Mabel Adams said:

We just got our first anti-CD47 out here in the sticks on a patient who went to Seattle for a clinical trial.  That drug is Hu5F9-G4.  No other name yet.  It interfered with her reverse as well as all gel testing.  We did a 30 minute saline screen and it was 4+ at 37C but negative at IAT using Immucor's anti-IgG which doesn't react with IgG4.  She was antigen typed before starting treatment so we got that information and gave her K and Fya negative units (lucky she is positive for most antigens).  We called them incompatible because we have not validated the Immucor anti-IgG as our test of record, the screen was 4+ at 37C and because the drug causes the patient's H&H to drop so I wasn't sure that the units would be certain to have normal survival.  I didn't expect to get one of these for a few more years since we aren't in a big teaching hospital region.  It would have been nice if the big center had sent her home with information that she was on this and instructions to tell the blood bank. We lucked out finding clues in Epic's Care Everywhere so we called the Seattle blood bank.

Hi Mabel,

I was wondering how to handle the screen due to the positive reactivity at 37.  Are you reporting out a positive tube screen then?

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On ‎4‎/‎30‎/‎2019 at 7:48 AM, tkakin said:

Hi Mabel,

I was wondering how to handle the screen due to the positive reactivity at 37.  Are you reporting out a positive tube screen then?

We called the screen positive (went ahead and reported the gel screen) because we wanted the computer to prevent electronic crossmatch if they wanted more blood while that specimen was still good on a later shift that hadn't heard about her and didn't read her comments.  We were giving her units negative for the 2 antigens that she lacked (good thing that she was heterozygous for most of our "usual suspects") so we didn't want random units to be issued inadvertently.  Also, I read that patients on these drugs sometimes have a drop in H&H at first dosing so I wasn't really willing to go out on a limb and call them compatible and imply that they would have normal red cell survival.

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On 4/9/2019 at 2:47 AM, Veejay said:

What ethnic descent is the patient. Could the antibody be anti-U?

Sorry I missed this one. U antigen is ficin-resistant, therefore this cannot be anti-U 

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