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Posts posted by Bb_in_the_rain
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On 9/18/2019 at 8:06 AM, SMILLER said:
The patient is long gone. But the same method -- manual gel -- was used for both the screening cells and the panel cells. As I mentioned, the reagent cells were both all R1R1 (but from different donors)--yet only the cells from the one screening set was negative.
Had to be something wrong with that particular cell and that particular patient.
Scott
Maybe if you have ficin or papain, you can treat the C+ cells that were originally negative to see if your strange "anti-C" pops up? If the patient is an African American, I would consider the presence of variant RHCE gene.
- Yanxia and Malcolm Needs
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On 8/6/2019 at 3:11 PM, butlermom said:
Malcolm, thanks so much for the article. It was very helpful. As it turned out, we sent mom's sample to our reference lab for MMA testing, and we also antigen typed her 2 brothers and her father. One of the brothers matched her Duffy and Kidd antigen types and was Coombs crossmatch compatible with her. He donated two units of packed red cells (at one donation) and was also confirmed to be Diego b negative. The patient's anti-Dib came back as clinically significant based on the MMA test. She did have a C-section after all and did not require any blood! The baby had a negative direct coombs so there were no issues there either!
Please be sure to recruit the brother as blood donor
as he is a valuable donor. - AMcCord, butlermom and Malcolm Needs
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On 7/26/2019 at 7:18 AM, SMILLER said:
We had a patient, 30-year-old female, B pos, who showed up last week with an anti-C along with a few equivocals. When it was time to repeat the T&S (and antibody ID) a few days later, we went ahead and set up a panel along with the screening cells, since we already knew there was an atypical antibody that would show up. To our surprise, the screen was negative (Ortho manual gel). The panel reacted as expected, pretty good 1+ reactions for the C, with a few equivocals.
The second specimen's screen was with a different lot of screening cells. We also tested with the original specimen's lot number of screen cells, and 3% tube screening cells -- those reacted as expected for an anti-C.
Kinda odd. The newer screening cells were checked with another patient who makes anti-C - that reacted as expected also. Its just the one patient with the one lot of screening cells that did not react. Kinda odd.
Scott
Are you using solid phase by any chance? We have seen solid phase does that, since a lot of our hospital used solid phase method. When that happened, we usually look for antibodies using PeG Tube method or by testing ficin-treated cells. We were usually able to find the suspected antibod(-ies) except for Kidd antibodies. You may be looking at some method-dependent antibody? Anti-C in your 2nd sample may be stronger than that in your first sample?
What is the patient's ethinicity? Is the patient e- or e+? I am also thinking about anti-Ce like antibodies if you see this anti-C reacting stronger with e+ cells than e- cells.
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Is Rh(D) a correct terminology to describe D antigen? I saw this notation time and time and make me wonder "why not Rh(C), Rh(E)..etc.." It does not seem to be fair that only the D antigen get this "Rh" terminology in front of the antigen but not the whole other 40+ antigens in the Rh system
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You were right about "although I have my doubts about that being a coincidence." It was not a coincidence. The Sanger sequencing on this lady indicated that the patient is cisAB.01/O.01.
In Geoff Daniel's Human Blood Group text book, page 43, described that sera from cis AB people almost always contain weak anti-B. This is a great learning case indeed!
Have anybody tried testing acidified anti-B with known cisAB cells? I am wondering if acidified anti-B would be non-reactive with cisAB in addition to A(B)?
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On 7/8/2019 at 4:03 AM, butlermom said:
Our Reference Lab has informed us that a patient's sample we sent to them has anti-Dib (Diego b), anti-Fya, and anti-Jka! The patient is pregnant and due the end of July. All 3 antibodies are capable of causing HDFN, although usually mild from what I have read. My concern is if we have to transfuse the mother. The prospect of getting blood is problematic. Most likely the units would be frozen and our local supplier would have them shipped in already thawed and deglycerolized, plus the time of flying them here is a challenge with flight schedules to our area. From my reading the Diego antibodies are more commonly associated with HDFN than transfusion reactions, and anti-Dib typically causes mild HDFN. Therefore, my strategy is to have units that are at least antigen negative for Fya and Jka for the mom in case we cannot obtain units that are negative for all 3 antigens. If we can get units that are antigen negative for all 3 that's great, but the patient is not a scheduled C-section so the timing will be next to impossible. Any thoughts or ideas for a different strategy?
Thanks for any input!
We usually try to
- collect autologous unit if she can donate.
- If mom cannot donate, you can also send out the sample for monocyte mono layer assay (MMA) to see if anti-Dib is clinically significant or if you can transfuse this patient Di(b+), had she bleed during her C section. (This test is a test that predict hemolytic potential, just like reminiscence assay performed in Europe as the journal that Malcolm has describe above. Here in the US, we used MMA assay instead of CLT).
- We also perform titer on the antibodies to predict potential HDFN, which is supervised by our Medical Director. (titer value more than 2 tubes difference in consecutive sample collected within a month is considered critical value here).
Hope this is helpful. -
On 7/8/2019 at 3:16 AM, Matthew Kim said:
Today, We performed Kell antigen phenotyping, revealing k-, Kpb-, Jsb- (genotypically predicted as positive using SNP
Now, I believed his phenotype is Ko.
I consider further genotyping using Sanger sequencing.
Thank you for your help.
Matthew Kim
Wow! things must come in a bundle. This week, we just had a case with a broadly specific antibody, non reactive with K0 cells. K-k-Kpa-Kpb- and SNP genotyping predicted a presence of KEL gene. We are on the same page with you on our patient as well, in the process of sequencing.. It will be very interesting to see the sequencing results. Please keep us posted when your sequencing is done. Our patient is a bleeder, so we gave one Ko unit. yike!!
- Ward_X and Malcolm Needs
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On 7/1/2019 at 8:31 AM, SMILLER said:
We currently have a 50 year old male in house that had an accident that damaged his foot 3 weeks ago. He arrived septic and has had to have an amputation.
His ABO/Rh gives a B pos with a 4+ anti-D. His gel screen and panel give 1+ results that match up with an anti-D (all others rules out). His autocontrol was positive at 1+ by IgG, neg for compliment. The eluate results matched the original antibody ID. Presently this patient's specimen is on its way to our reference lab. Previous history at another facility lists him as B Pos, screen negative. As far as we know, he has never been transfused.
What are the possibilities (for what appears to be an D auto antibody), and how should he be treated?
Scott
Hello, Scott,
I have a few questions about this case.
1) What is the eluate result that "matched the original antibody ID"? Is it panagglutination or anti-D in eluate?
2) How many sources of anti-D did you use to type your patient? We use up to 3 or 4 sources in this lab if we have such problem in this lab.
Here are the things that I usually do to figure out if anti-D is that of auto- or allo- ..
1) exclude the possibility of RhoGm or IVIG adminstration
2) Perform D typing on the patient's red cells using different sources of anti-D. (we usually have 4 or 5 sources). If D reactivity is found variable positive and negative reactions with different clones, perform genomic testing to exclude partial/variant D antigen.
3) If the patient is elderly male and recently transfused with D+ blood (or if you see mixed field in the D typing), perform cell separation by density centrifugation, perform D typing and auto control using retic-rich cells.
4) If the patient is DAT positive, perform EGA or CDP treatment, test EGA, CDP treated cells with plasma and eluate.
5) Lastly, test plasma and/or eluate (wherever you are seeing anti-D) with DTT-treated red cells and cord cells to exclude or confirm anti-LW.
Please let me know if there is anything else to add to this list. Hope this is helpful.
- Yanxia, Malcolm Needs and Ward_X
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17 hours ago, yan xia said:
It seems like an acquired-B to me. Maybe you can try to lower the pH to 4.5 to see the lady's cells react with anti-B reagents.
13 hours ago, Malcolm Needs said:An Acquired-B isn't a true type of polyagglutination in the way that is, say, a T or Tn, but is, rather, a deacetylation of the immunodominant N-acetyl-D-galactosamine of the group A antigen, which makes the immunodominant sugar residue so close to the D-galactose of the B antigen, that some anti-B reagents will react with it.
Because this is a "phenotype" type of thing, I don't think genotyping would help, but sending a sample to a good Reference Laboratory (and, as yan xia says, lowering the pH of the anti-B) should help.
I have tested the patient's cells with 2 sources of acidified anti-B as your guidance. After 5 minute incubation, the patient's cells were non-reactive with one source of anti-B and 1+ with the other source. I used AB donor cells as control and they both remained 4+. I think I can accept that this is an A(B).
My question is
Why do we see this in the prenatal patient without signs of infection? I thought acquired B was found in those with cancer and infections? Does it mean that she has an abnormal galactosyl transferase?
Thank you very much for your guidance and walking me through this case. I should have listened when the experts said it was acq-B!!! Thank you so much again for your guidance!!
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10 hours ago, yan xia said:
It seems like an acquired-B to me. Maybe you can try to lower the pH to 4.5 to see the lady's cells react with anti-B reagents.
6 hours ago, Malcolm Needs said:An Acquired-B isn't a true type of polyagglutination in the way that is, say, a T or Tn, but is, rather, a deacetylation of the immunodominant N-acetyl-D-galactosamine of the group A antigen, which makes the immunodominant sugar residue so close to the D-galactose of the B antigen, that some anti-B reagents will react with it.
Because this is a "phenotype" type of thing, I don't think genotyping would help, but sending a sample to a good Reference Laboratory (and, as yan xia says, lowering the pH of the anti-B) should help.
Thank you very much for your guidance and advises. I would like to try acidified sera using 1M HCl to repeat typing of this patient's cells. I am wondering what source of anti-B would you acidify? I assume I would acidify the clones that was previously reactive (including the ones with ESO4 clone and polyclonal anti-B)?
So the purpose of testing with acidified anti-B is to re-acetylate the A(B) into A antigen. Is that correct? So I should expect clones if anti-B that were previously reactive to be negative with the patient's cells after being acidified.
I think sending it to another Reference is a good option; however, the submitting hospital has decided to give Group A blood and not to further investigate that. So we are left with the tools that we have here in this lab. I hope the acidified anti-B will give me a clue to whether it is A(B) or not.
I will keep you guys updated after I am done with the test.
With regard to genotyping, if (hypothetically) speaking (and I do not mean to be picky), if the gene that transcribed B transferase is detected, it could mean this is a subgroup of B rather than a A(B)?
I might be listening to horsehoof and thinking of Zebra. Somehow, I am not convinced that this is an Acquired-B... The diagnostic does not make sense and also one out of 2 polyclonal anti-B is moderately reactive, and both ES4 and B0001 clones were strongly reactive (3+). It is not quite adding up in my mind. I thought I should see somewhat of the difference in reactivity between ES4 and other clones.
- Yanxia and Malcolm Needs
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3 minutes ago, Bb_in_the_rain said:
If we see cases of CAD with titer <64 that is reactive at 30C with or without albumin, that would be a perfect opportunity to start a conversation with ordering physician with references to Garratty G et al, The correlation of cold agglutinin titrations in saline and albumin with haemolytic anemia, BrJ Haemat 1977;35, along with the paper that you have cited above (which I am printing out and filing it in my "good hemolytic anemia reference" folder now)
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3 hours ago, Malcolm Needs said:
I am not going to be "precious" about this, but what would you tell the physicians if you had a case where there was an antibody of wide thermal amplitude (reacting at, at least 30oC), but had a low titre (given that most cases, but by no means ALL cases of CHAD involve an antibody with a titre above 512)? In our own case, see above, we found that antibody had a titre of 256 at 4oC, the anti-I had a titre of 16 at 23oC, the anti-i a titre of 4 at 23oC, the anti-I reacted only in neat plasma at 30oC and the anti-i was not detected at 30oC. On the other hand Professor Sir John Dacie (a nice chap if ever there was one) stated that, in general, naturally occurring cold-reactive auto-antibodies have a titre below 64 at 4oC and have a thermal amplitude of less than 20oC (Dacie J V. The haemolytic anaemias, congenital and acquired, part II. The auto-immune haemolytic anaemias. 2nd edition, 1962, Churchill, London, 460), whereas Petz and Garratty. world experts in auto-immune disease, both of whom have been given numerous awards throughout the world, state, "In general, anti-I is commonly found in patients with CHD, and the auto-antibody titre is much higher (>1, 000) at 4oC (Petz LD, Garratty G. Immune Hemolytic Anemias. 2nd edition, 2004, Churchill-Livingstone).
I agree though, it is incredibly difficult to change the minds of clinicians who have bee told dubious "facts" during their education, however famous are the references.
If we see cases of CAD with titer <64 that is reactive at 30C with or without albumin, that would be a perfect opportunity to start a conversation with reference to Garratty G et al, The correlation of cold agglutinin titrations in saline and albumin with haemolytic anemia, BrJ Haemat 1977;35, along with the paper that you have cited above (which I am printing out and filing it in my "good hemolytic anemia reference" folder now)
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3 hours ago, Malcolm Needs said:
To be honest, I think we need a little more information, such as the underlying pathology of the patient, including whether or not they have recently had a bacterial infection.
In addition, we need to know if an anti-B is still detectable in the reverse grouping.
I can put my hand on my heart and say that I am simply AMAZED that a monoclonal anti-B is still available that contains ES4 as a clone, following the publication of the paper by Garratty G, Arndt P, Co A, Rodberg K, Furmanski M. Fatal hemolytic transfusion reaction resulting from ABO mistyping of a patient with acquired B antigen detectable only by some monoclonal anti-B reagents. Transfusion 1996; 36(4):351-357.
The reason you can't find the clonal numbers for the polyclonal anti-B reagents is almost certainly because they are human-derived, and so are quite literally polyclonal, and nobody would have a clue of the clones involved!
This case sounds (from the distance of the entire Atlantic Ocean, and not seeing the actual results of the tests with my own eyes) very much like a case of an acquired-B (if there is anti-B in the patient's plasma, it would NOT react with the patient's own cells and, indeed, would not react with another individual's red cells who has an acquired-B antigen, caused by the same bacterial strain).
Opps (again), my apology for incomplete information. The patient is pregnant and no infection was noted. The plasma was non-reactive with A cells and reactive (2+) with B cells.
Even though the serology initially "smells" to me like an acquired B, the diagnostic is pregnancy with no signs of infection.
Moreover, only 1 of the 2 known monoclonal anti-B is confirmed to include ES4 clone and the other includes B005 clone, yet both of them were reactive strongly (3+). So I am really puzzle as to whether it is A(B) or simply a subgroup of B. It will be nice to have an example of cells from an individual with acquired-B to test it out, but we do not have these.
If the reactivity is due to tartrazine in anti-B, I would see a positive reaction with all 7 anti-B that I have tested (as they certain are yellow and I assume they all have tartrazine in them). But that was not the case.....only 5 of 7 anti-B were reactive.
My question is
As I saw AcqB characterized as a polyagglutination in Issitt and Anstee's textbook (table 42-3), does it mean that I can exclude Acq-B based on the non-reactivity with Fresh AB plasma?
Would that be a good idea to send it to genomic lab to see if there is any B-transferase present in this patient?
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5 minutes ago, Malcolm Needs said:
But the titre tells you nothing (it is about as useful as either the specificity or a chocolate tea pot! See Win N, Needs M, Rahman S, Gold P, Ward S. An unusual case of an acute haemolytic transfusion reaction caused by auto-anti-I. Immunohematology 2011; 27 (3): 101-103, amongst others).
We just go with the thermal amplitude, but we do agree that albumin should be included (as per Petz and Garratty, as above).
I agree with titer value. As the physicians prefer seeing a numerical value, it is hard to turn down an order for titer. I think there were other publication out there eliciting the association of Mycoplasma Pneumonae infection to anti-I titer and Infectious Mono associated with anti-i titer, etc. it is really really hard to not perform titer when physician comes to the hospital labs with such requests. I rather offer titer and thermal amplitude as one test so that both will be performed.
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6 minutes ago, Tympanista said:
Thank you for the references. I'm always curious to know what is being done in other labs. We don't currently perform thermal amplitude testing here, so that may end up being a sendout test. I've been tracking all of the cold agglutinin titer orders we've gotten over the past few months and they all seem to be coming from one physician who is ordering them as part of an autoimmune panel for patients with Raynaud's. I will have my Medical Director speak with him after reviewing the reference you provided and we may be able to convince the physician to request thermal amplitude testing along with, or instead of the cold agglutinin titers.
It may be more efficient workflow to perform titer at 4 different temperature, 22C, 30C, 37C and read them after 1 hour incubation. That way, you get your titer and thermal amplitude done in one shot and also see the titer difference between your 3 different temperature. Also, your order physician does not have a choice but to perform titer and thermal amplitude as they are offered as one test.
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Patient was historically typed as A postive in Korea.
Red cells typing
Anti-A= 4+, anti-B=3 ( same strength with 2 different sources of antisera)
One of the Anti-B includes ES4 clone and one of the Anti-A includes MHO4 clone.
Tested with 3 more sources of monoclonal and 2 more sources of polyclonal anti-B. (I cannot find the clone numbers on them)
2 of 3 monoclonal anti-B reacted 2+ and 1 of 3 monoclonal anti-B was negative.
1 of 2 poly anti-B reacted 2+, and the other was negative.
The patient cells were also tested with 2 sources of fresh AB plasma (10 minute incubation, room temperature) to exclude polyagglutination or B(A) ; they were both negative
Autocontrol was also negative (10 minute incubation, room temperature).
Please let me know what you think about this case.
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On 6/9/2019 at 6:19 AM, Tympanista said:
Would anyone be willing to share their cold agglutinin titer procedure with me? I am a relatively new Blood Bank supervisor and, though I have many years of Blood Bank experience, I have no reference lab experience. The procedure at my new facility hasn't been revised since 1999 and has several glaring issues. For example, it says to incubate the tubes "overnight." Is that for 8 hours, 24 hours? I reviewed the procedure in the Technical Manual and it makes much more sense to me, but I was hoping I could get some examples of what is being done at other facilities. I would appreciate any information you all are willing to share. Thanks.
I much rather perform titer and thermal amplitude as outlined in Petz and Garratty's, Immune Hemolytic Anemias textbook. And also, Thermal Amplitude Test with albumin has much higher positive predictive value per Table 5-14 in the textbook. So if the test were to be overhauled, I would perform titer and thermal amplitude, and supplement thermal amplitude testing with album if necessary.
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Has anybody look in maternal breast milk for IgG antibody in cases of prolonged HDFN? I just come across this article and found it to be very interesting.
Leonard et al, "Identification of Red Cell Antibody in Maternal Breast Milk Implicated in Prolonged Hemolytic Disease of Fetus and Newborn"
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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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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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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!
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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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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.
as he is a valuable donor. 
Applied Blood Group Serology Reprinted.
in Education / Quality
Posted
Hello,
I just would like to share this great news with you all so you won't miss out.
http://blog.aabb.org/appliedbloodgroupserology/