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Posted (edited)

Ok Lets try the first one here. 

Background - A 31 year old African American male, Bpos, C-E+c+e+K-S-s+Fya+Fyb-Jka+Jkb+. This patient was not previously transfused or pregnant. 

Plasma reactions

tested with 18 cells antibody panel and the reactions are....... 3+ reactions with C+e+ cells, 1+ reaction with C-e+ cells, negative with e- cells AutoControl was negative. 

Reactivity in DTT-treated cells were similar to that of untreated cells. 

The reactivity with ficin-treated cells were enhanced, C+e+cells 4+, C-e+cells 3+, negative with e- cells

Eluate

reacted 1+ with D- cells, 3+ with D+ cells.

non-reactive with DTT-treated cells

cord cells yielded similar reactivity as that of the adult.

Panagglutination 4+ was observed with ficin-treated cells. 

 

What are the possible antibodies that the patient can have?

 

If genomic testing is warranted, I will be back next week to tell you what genomic testing the results are.

Lets give a chance for the transfusion service folks to work on it before reference lab folks share their wisdom regarding this case. 

Edited by Bb_in_the_rain

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I have an idea what it might be, but am bound by the rules of the game (fair enough).

Please can you remind the likes of me when we can "put our oar in"?

Thanks.

P.S.  It looks really good Bb_in_the_rain!

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1 hour ago, Malcolm Needs said:

I have an idea what it might be, but am bound by the rules of the game (fair enough).

Please can you remind the likes of me when we can "put our oar in"?

Thanks.

P.S.  It looks really good Bb_in_the_rain!

Will sure do!

This look so good because it is not a real-life case that I just worked on. (cough cough) 

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21 hours ago, Bb_in_the_rain said:

Ok Lets try the first one here. 

Background - A 31 year old African American male, Bpos, C-E+c+e+K-S-s+Fya+Fyb-Jka+Jkb+. This patient was not previously transfused or pregnant. 

Plasma reactions

tested with 18 cells antibody panel and the reactions are....... 3+ reactions with C+e+ cells, 1+ reaction with C-e+ cells, negative with e- cells AutoControl was negative. 

Reactivity in DTT-treated cells were similar to that of untreated cells. 

The reactivity with ficin-treated cells were enhanced, C+e+cells 4+, C-e+cells 3+, negative with e- cells

Eluate

reacted 1+ with D- cells, 3+ with D+ cells.

non-reactive with DTT-treated cells

cord cells yielded similar reactivity as that of the adult.

Panagglutination 4+ was observed with ficin-treated cells. 

 

What are the possible antibodies that the patient can have?

 

If genomic testing is warranted, I will be back next week to tell you what genomic testing the results are.

Lets give a chance for the transfusion service folks to work on it before reference lab folks share their wisdom regarding this case. 

Correction: Eluate is reactive with DTT-treated cells. 

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I guess there are a mixture of alloantibodies anti-C and anti-hrs.

What puzzled me is the autocontrol is neg, but the eluation result is pos.  As for the eluation result, it maybe anti-LW.

 

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Posted (edited)
On 5/26/2019 at 3:38 PM, yan xia said:

I guess there are a mixture of alloantibodies anti-C and anti-hrs.

What puzzled me is the autocontrol is neg, but the eluation result is pos.  As for the eluation result, it maybe anti-LW.

 

Very well done! I would suspect something along this line as well. You are very very close!! Lets hear from reference lab folks to see what their thoughts are. I will share more information on this patient's work up later. 

Edited by Bb_in_the_rain

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On 5/24/2019 at 12:05 PM, Malcolm Needs said:

I have an idea what it might be, but am bound by the rules of the game (fair enough).

Please can you remind the likes of me when we can "put our oar in"?

Thanks.

P.S.  It looks really good Bb_in_the_rain!

Ok It has been a while since this case was posted, I think we have given enough time for transfusion services folks to participate. 

Lets hear from the reference folks! I am so excited to see what your thoughts are! 

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Okay, so let's have a think.

My first reaction is that I am glad this "31 year old African American male" has not been pregnant!!!!!!

On the face of it, the patient has an anti-C (or, more likely anti-Ce), but has almost certainly also got an anti-hrB (rather than an anti-hrS), as anti-hrB mimics an anti-C+e, reacting more strongly with the C antigen, than the e antigen, whereas anti-hrS mimics an anti-ce (anti-f).  To resolve this once and for all, the plasma should be tested against known hrB Negative and hrS Negative red cells.  However, many such red cells have been mis-typed (even those used by reputable Reference Laboratories, as the antibodies made by individuals  with a partial e antigen are a heterologous lot in terms of actual specificity, and so, these days the RHCE gene should be sequenced to ensure it is known what partial e is actually present (and, come to that, the C and c antigens are also often partial in nature).  Obviously, the patient's own RHCE gene should also be sequenced.

Like my friend yan xia, I was a little flummoxed by the auto-control being negative, but the eluate reacting weakly with D Negative red cells, but quite strongly with D Positive red cells.  I mean, if the gentleman has never been transfused (and has not been pregnant!), one wonders why an elution was performed in the first place, but then I thought, if his Hb and hct are low (information not given), it could be that he has a WAIHA that has a negative DAT (see Sachs UJH, Röder L, Santoso S, Bein G.  Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases.  British Journal of Haematology 2006; 132: 651-661).  Like yan xia, I think this could be an auto-anti-LW (it is highly unlikely to be an allo-anti-LW) (see, for example, Giles CM, Lundsgaard A.  A complex serological investigation involving LW.  Vox Sanguinis 1967; 13: 406-416).  This can be "proved" by reacting the eluate with group O, D Negative cord red cells, as D Negative cord red cells express the LW antigen strongly.  However, I then remembered that all three of the LW antigens are sensitive to DTT, and so, as you corrected the original to say that the antibody in the eluate reacted with DTT-treated red cells, it cannot be an auto or an allo-anti-LW.  I am, therefore, stumped at present, as to the specificity of the antibody/antibodies in the eluate.

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Posted (edited)
5 hours ago, Malcolm Needs said:

Okay, so let's have a think.

My first reaction is that I am glad this "31 year old African American male" has not been pregnant!!!!!!

On the face of it, the patient has an anti-C (or, more likely anti-Ce), but has almost certainly also got an anti-hrB (rather than an anti-hrS), as anti-hrB mimics an anti-C+e, reacting more strongly with the C antigen, than the e antigen, whereas anti-hrS mimics an anti-ce (anti-f).  To resolve this once and for all, the plasma should be tested against known hrB Negative and hrS Negative red cells.  However, many such red cells have been mis-typed (even those used by reputable Reference Laboratories, as the antibodies made by individuals  with a partial e antigen are a heterologous lot in terms of actual specificity, and so, these days the RHCE gene should be sequenced to ensure it is known what partial e is actually present (and, come to that, the C and c antigens are also often partial in nature).  Obviously, the patient's own RHCE gene should also be sequenced.

Like my friend yan xia, I was a little flummoxed by the auto-control being negative, but the eluate reacting weakly with D Negative red cells, but quite strongly with D Positive red cells.  I mean, if the gentleman has never been transfused (and has not been pregnant!), one wonders why an elution was performed in the first place, but then I thought, if his Hb and hct are low (information not given), it could be that he has a WAIHA that has a negative DAT (see Sachs UJH, Röder L, Santoso S, Bein G.  Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases.  British Journal of Haematology 2006; 132: 651-661).  Like yan xia, I think this could be an auto-anti-LW (it is highly unlikely to be an allo-anti-LW) (see, for example, Giles CM, Lundsgaard A.  A complex serological investigation involving LW.  Vox Sanguinis 1967; 13: 406-416).  This can be "proved" by reacting the eluate with group O, D Negative cord red cells, as D Negative cord red cells express the LW antigen strongly.  However, I then remembered that all three of the LW antigens are sensitive to DTT, and so, as you corrected the original to say that the antibody in the eluate reacted with DTT-treated red cells, it cannot be an auto or an allo-anti-LW.  I am, therefore, stumped at present, as to the specificity of the antibody/antibodies in the eluate.

Haha. I copied that info right out the demographic sheet and typed "not pregnant or transfused" not thinking much about his gender. opps! 

I am surprised by this positive eluate as well. I was just "shooting in the dark" and performed the eluate blindly when I saw these reactions with e+ cells (since I saw way too much warm autoantibodies with relative anti-e specificity). 

Further serology results are- 

this antibody is weakly reactive w+ with 1 of 3 hrB- cells tested (all of them are C-e+) 

it is weakly reactive with 1 of 2 hrS- cells tested (both of them are C-e+) 

Genomic sequencing results in RHD gene (sequenced 1-10 exons)

the following heterozygote changes were observed- c.410C>T, c.455A>C, c602C>G, c667T>G and 819G>A - predicted to be RHD*DIIIa or RHD*DAR3.01 

A normal D gene was also observed. So the prediction was a normal D gene in Trans position to RHD*DIIIa heterozygote. 

Since RHD gene was associated with altered RHCE gene, RHCE sequencing was reflexed (sequenced 1-10 exons and some intronic flanking regions) 

variants - c.48G>C, c.676G>C, c733C>G, c.1006G>T - predicted to be RHCE*ceVS.03 in trans with RHCE*cE  (because serologic phenotype is D+C-E+c+e+) 

So when I looked up RHCE*ceVS.03, I found it to be associated to V-VS+hrB-. 

So your suspicion is right on the spot! It is most likely anti-hrB!! At this point anti-C was not excluded but the transfusion recommendation was R2R2 blood, so we are ok here. 

In terms of eluate, it most likely is warm auto antibody with relative anti-D specificity due to the presence of normal D gene in hetrozygote expression. I am still puzzled by a negative auto-control. However since the antibody was eluated out of his untransfused red cells, so I can accept that it is most likely an autoantibody. 

Please let me know what your thoughts are and any further results that you may need. Hope this is a good case study! 

 

 

 

Edited by Bb_in_the_rain

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38 minutes ago, Ensis01 said:

Did you test the eluate against DAT negative patient cells?

No I did not do that. Great idea!! If I can find my eluate, I should...

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Please let me know if you would like me to do more "mock-up cases" with RHCE variants. I can look for some good ones.  I think it is fun to interact with case studies here. (I mean it is quite fun to pick Malcolm's brain and learn from him... cough cough). 

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