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Posted (edited)

Lets do a muck case study just for fun. Here we go.

Background- 31 year old Hispanic Male was admitted to hospital with GI bleed. Patient blood type is O Pos, C+E-c-e+K-S-s+Fy(a-b+)Jk(a+b-). Antibody screen showed all 3 cells positive (2+) and autocontrol was negative 

All 18 cells tested in antibody panel showed positive (2+) reaction, including C+E-c-e+K-S-s+Fy(a-b+)Jk(a+b-) cells. Ficin-treated panel cells were all negative and DTT-treated panel cells were all positive. 

What would you do next? 

** I am trying to tease some brains from transfusion services. If reference lab folks are reading this, please do not give away the answer** 

Edited by dothandar

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Off to reference lab...LOL  We are very limited with what we can do.  2 panels...Enhancement only

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Posted (edited)

What is the Fya and S phenotyping for the 3 cell screen?

Test some more Fya neg, S neg RBCs as the enzyme panel was non reactive but IAT did react, also the cell tested that matches the patient's phenotype is reacting so something else is there too. Maybe it is anti-Fya+/- Anti-S + something else?

Send to red cell reference lab.

Edited by Veejay
extra info

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17 hours ago, Malcolm Needs said:

Can the Reference people make suggestions yet?

Alright, since most transfusion service agreed to send it off the reference lab, maybe reference folks can shed some light on this case?

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5 hours ago, R1R2 said:

Clarifying question - Is it Friday afternoon or Monday morning?  

Lets say it is Monday morning. Your night shift tech was in a very good mood, finished all the QC and patient works. Also, there was no more patient work up coming in on Morning morning rush, it is an unusually quiet morning. So, you got all the time you need to solve that case! 

Also lets say your antibody panel vendor happened to be so generous that they will provide all kinds of cells that you want. 

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Posted (edited)
2 hours ago, Veejay said:

What is the Fya and S phenotyping for the 3 cell screen?

Test some more Fya neg, S neg RBCs as the enzyme panel was non reactive but IAT did react, also the cell tested that matches the patient's phenotype is reacting so something else is there too. Maybe it is anti-Fya+/- Anti-S + something else?

Send to red cell reference lab.

Very well done!! You were right that there was something corresponding to an enzyme-sensitive antigen or antigens. Now here is more information in order for you to sort this out. 

In your antibody panel,

Fy(a-b+) S-s+ cell was negative 

Fy(a+b-) S-s+ cell was negative

Fy(a-b+) S+s- cell was negative

Fy(a+b-) S+s- cell was negative .

What do you think now? 

Edited by dothandar

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6 hours ago, Bb_in_the_rain said:

Very well done!! You were right that there was something corresponding to an enzyme-sensitive antigen or antigens. Now here is more information in order for you to sort this out. 

In your antibody panel,

Fy(a-b+) S-s+ cell was negative 

Fy(a+b-) S-s+ cell was negative

Fy(a-b+) S+s- cell was negative

Fy(a+b-) S+s- cell was negative .

What do you think now? 

Right then.  The first thing I would say is that I am simply amazed that these cells were found to be negative, and yet these types were NOT represented in any way in the original screening cells or the 18 panel cells.  The first thing I would do, therefore, is to change my reagent supplier as a matter of urgency.  It would be equally amazing for all four of these red cell samples to be uniquely negative for the same antigen.

I noticed is that the reactions are all 2+, which suggests that there is a single specificity in the plasma, directed against a high prevalence antigen.

2+ reactions are pretty weak, which suggests the presence of what used to be called high titre, low avidity (HTLA) antibodies - I am STILL confused as to what we are supposed to call them these days, but hey! - so the first thing I would do is to titre the antibody to see if the end point is unexpectedly high, compared with what is expected for the reaction strength.

The other thing I would do is to see if the antibody can be inhibited 1:2 with pooled human plasma, using the patient's own plasma diluted 1;2 in saline as the negative control.

If the antibody is inhibitable as above, the probability is that the antibody is either anti-Ch or anti-Rg.  Neither specificity is clinically significant, and so many people would go no further, but one thing that could be easily done these days is to try to inhibit the plasma with recombinant red cell protein (rRCP) that is specific for the C4d molecule where these antigens are expressed.  Inhibition with this will prove the specificity.

I, however, being a purist, would try to inhibit the plasma with plasma from a Ch-, Rg+ individual, and a Ch+, Rg- individual to prove the more exact specificity.

If none of this works, I would have to have another think!

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All the DTT treated cells were still positive so that should rule out Darazalex.  I wonder about the new one anti-CD47?  Has anyone run into it yet and do we have any way of coping with it yet?  Does it have a name yet?

I was thinking anti-Fy3 or anti-U because of the ficin testing results, but the phenotyping is wrong for that, isn't it?

 

 

 

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43 minutes ago, cswickard said:

I was thinking anti-Fy3 or anti-U because of the ficin testing results, but the phenotyping is wrong for that, isn't it?

The reactions of both anti-Fy3 and anti-U are enhanced, rather than ablated, by the use of ficin-treated red cells.

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

Right then.  The first thing I would say is that I am simply amazed that these cells were found to be negative, and yet these types were NOT represented in any way in the original screening cells or the 18 panel cells.  The first thing I would do, therefore, is to change my reagent supplier as a matter of urgency.  It would be equally amazing for all four of these red cell samples to be uniquely negative for the same antigen.

I noticed is that the reactions are all 2+, which suggests that there is a single specificity in the plasma, directed against a high prevalence antigen.

2+ reactions are pretty weak, which suggests the presence of what used to be called high titre, low avidity (HTLA) antibodies - I am STILL confused as to what we are supposed to call them these days, but hey! - so the first thing I would do is to titre the antibody to see if the end point is unexpectedly high, compared with what is expected for the reaction strength.

The other thing I would do is to see if the antibody can be inhibited 1:2 with pooled human plasma, using the patient's own plasma diluted 1;2 in saline as the negative control.

If the antibody is inhibitable as above, the probability is that the antibody is either anti-Ch or anti-Rg.  Neither specificity is clinically significant, and so many people would go no further, but one thing that could be easily done these days is to try to inhibit the plasma with recombinant red cell protein (rRCP) that is specific for the C4d molecule where these antigens are expressed.  Inhibition with this will prove the specificity.

I, however, being a purist, would try to inhibit the plasma with plasma from a Ch-, Rg+ individual, and a Ch+, Rg- individual to prove the more exact specificity.

If none of this works, I would have to have another think!

Woops!! I totally messed this one up! I meant to say they are positive!! 

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Posted (edited)
20 hours ago, cswickard said:

All the DTT treated cells were still positive so that should rule out Darazalex.  I wonder about the new one anti-CD47?  Has anyone run into it yet and do we have any way of coping with it yet?  Does it have a name yet?

I was thinking anti-Fy3 or anti-U because of the ficin testing results, but the phenotyping is wrong for that, isn't it?

 

 

 

CD47 is not effected by ficin treatment. There are currently several different clones in clinical trial. So there are so many names!! Fy3 and U antigens are not destroyed by ficin as well, even though Fya, Fyb, S and s are destroyed by ficin. 

Edited by Bb_in_the_rain

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Posted (edited)
1 hour ago, simon hook said:

Are the missing MN groups a clue? Anti-Ena?

My thought when I made up this case was missing/weak glycophorins. So anti-Ena would be one of them. What else are the possibilities? 

Edited by Bb_in_the_rain

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

Right then.  The first thing I would say is that I am simply amazed that these cells were found to be negative, and yet these types were NOT represented in any way in the original screening cells or the 18 panel cells.  The first thing I would do, therefore, is to change my reagent supplier as a matter of urgency.  It would be equally amazing for all four of these red cell samples to be uniquely negative for the same antigen.

I noticed is that the reactions are all 2+, which suggests that there is a single specificity in the plasma, directed against a high prevalence antigen.

2+ reactions are pretty weak, which suggests the presence of what used to be called high titre, low avidity (HTLA) antibodies - I am STILL confused as to what we are supposed to call them these days, but hey! - so the first thing I would do is to titre the antibody to see if the end point is unexpectedly high, compared with what is expected for the reaction strength.

The other thing I would do is to see if the antibody can be inhibited 1:2 with pooled human plasma, using the patient's own plasma diluted 1;2 in saline as the negative control.

If the antibody is inhibitable as above, the probability is that the antibody is either anti-Ch or anti-Rg.  Neither specificity is clinically significant, and so many people would go no further, but one thing that could be easily done these days is to try to inhibit the plasma with recombinant red cell protein (rRCP) that is specific for the C4d molecule where these antigens are expressed.  Inhibition with this will prove the specificity.

I, however, being a purist, would try to inhibit the plasma with plasma from a Ch-, Rg+ individual, and a Ch+, Rg- individual to prove the more exact specificity.

If none of this works, I would have to have another think!

Lets say the antibody is not inhibited by Pooled plasma or rRCP. What would be next? 

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Antibody reacts consistently with all cells tested, including a phenotypically "matched" cell, but is nonreactive with the patient's autocontrol is indicative of an antibody to a high frequency antigen.  Is initial testing in Gel, solid phase, LISS, PEG?   The reason I ask is that in my experience, HTLA antibodies tend to show variable reactivity in tube, but can react more uniform in Gel testing (just guessing the same would be true for solid phase).

Chido/Rodgers are possibilities; U is not as the patient is s+.  Ena is a possibility as are Ge:2 and Ge:4.  

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18 hours ago, StevenB said:

Antibody reacts consistently with all cells tested, including a phenotypically "matched" cell, but is nonreactive with the patient's autocontrol is indicative of an antibody to a high frequency antigen.  Is initial testing in Gel, solid phase, LISS, PEG?   The reason I ask is that in my experience, HTLA antibodies tend to show variable reactivity in tube, but can react more uniform in Gel testing (just guessing the same would be true for solid phase).

Chido/Rodgers are possibilities; U is not as the patient is s+.  Ena is a possibility as are Ge:2 and Ge:4.  

Yes!! Well done!!! 

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Are you saying it is anti-Ena, or anti-Ge2/anti-Ge4?

The reason I ask is because not all examples of anti-Ena are ficin sensitive, some are ficin resistant (see page 110 of Reid ME, Lomas-Francis C, Olsson ML. The Blood Group Antigen FactsBook.  3rd edition, 2012, Academic Press), but you also said that the antibody was not inhibited with rBGP, and yet there are Gerbich specificities available that would inhibit these antibodies (Schawalder A, Reid ME, Yazdanbakhsh K.  Recombinant glycophorins C and D as tools for studying Gerbich blood group antigens,  Transfusion 2004; 44: 567-574) and could even distinguish between the two specificities, as Ge:2 is on glycophorin D and Ge:4 on glycophorin C.

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1 hour ago, Malcolm Needs said:

Are you saying it is anti-Ena, or anti-Ge2/anti-Ge4?

The reason I ask is because not all examples of anti-Ena are ficin sensitive, some are ficin resistant (see page 110 of Reid ME, Lomas-Francis C, Olsson ML. The Blood Group Antigen FactsBook.  3rd edition, 2012, Academic Press), but you also said that the antibody was not inhibited with rBGP, and yet there are Gerbich specificities available that would inhibit these antibodies (Schawalder A, Reid ME, Yazdanbakhsh K.  Recombinant glycophorins C and D as tools for studying Gerbich blood group antigens,  Transfusion 2004; 44: 567-574) and could even distinguish between the two specificities, as Ge:2 is on glycophorin D and Ge:4 on glycophorin C.

I am sorry I apologize for  failing to realize that there may be more than one blood group recombinant proteins available for use in immunohematology. We may be talking about different recombinant proteins. The protein that I was thinking about is soluable CR1 recombinant protein (Mould JM, et al, Neutralization of Knops system antibodies using soluable complement receptor 1), which may be different from that of recombinant glycophorin protein by Schawalder A et al.  Therefore, when I made up the result that "rRBG-treated plasma was positive", I meant to exclude antibodies to Knops and Ch/Rg blood group proteins. 

I also failed to mention that the suspect would be anti-EnaFS (but not Anti EnaFR or anti-EnaTS, which I almost have forgotten about since I have not seen those before). In this case, I suppose we can throw in anti-Pr to the mix of possibilities (if the patient's cell is glycophroin-deficient, with autocontrol negative, long shot??). 

Thank you very much for an opportunity for further learning. Awesome as always!!! 

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On 4/10/2019 at 11:23 AM, cswickard said:

  I wonder about the new one anti-CD47?  Has anyone run into it yet and do we have any way of coping with it yet?  Does it have a name yet?

 

 

 

 

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.

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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.

I have heard a couple of different clones coming from Seattle Hospitals (Hu5F9-G4 and ALX148). Watch out for the clone numbers when you work on patients on anti-CD47. It might make a big difference whether or not you can use Immucor anti-IgG to rule out your underlying alloantibodies. 

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