mpmiola

Agree with Dr Blumberg. In an abstract from way back, 1996, Ann Church studied the value of an eluate, reference and abstract below (apologies for quality of print). 
A Church, S Nance, D Kavitsky.  Assessment of Elution Studies in Cases with 37OC Reactive Serum Autoantibodies (SA).  Transfusion 1996; 36:161S (Suppl)

Agree that in “not recently transfused”patients, eluates are of no clinical use unless you like seeing panagglutinins.😜 Negative eluates occur in some drug dependent examples but the clinical information is paramount in such situations in any case.

Many, many years ago now, when I was working at the old Westminster Hospital in London as a quite junior member of the Blood Transfusion staff, I spent quite a few hours working on a sample from a patient with a positive DAT, trying to determine the specificity, in order to see whether the antibody was an allo- or an auto-antibody.  This included the use of several very rare red cells that I had frozen down and also examining the eluate.  After many happy hours, I had got precisely nowhere, and so sent a sample to my former colleges at the International Blood Group Reference Laboratory.
I received a somewhat "spicy" report from my heroine Joyce Poole, who explained to me, in words of one syllable, that I had rather been wasting my time, rare cell collection and the laboratory's money as, in almost all cases, the specificity would be found to be an auto-anti-Rh17 or auto-anti-Rh18!!!!!
Since then, I have reverted to doing as little as possible on such samples and, when I retired 43 years later, and, as far as I know, none of the patients ever died as a result.  There was a close one once, when I was working on a sample on a Saturday on-call in the Red Cell Reference Laboratory at NHSBT-Tooting Centre.  I was working on a sample that was overtly a case of wAIHA.  After several allo-adsorptions, I was finally able to provide "suitable blood".  It was only at the last minute that the computers came back on after an unplanned downtime, and it appeared that the patient was known, from many years previously as having an allo-anti-Vel!!!!!!!  As you can imagine, I did several more tests before I released the blood (on Pathologist's orders), as there was absolutely no evidence of an anti-Vel (auto- or allo-) in the present sample - but it did give me a bit of a turn!!!!!!!!!!!!
I would recommend a thorough reading of Petz LD, Garratty G.  Immune Hemolytic Anemias.  2nd edition, 2004, Churchill-Livingstone. ISBN  978-0-443-08559-8.
Other than that and, possibly, looking at the patient's sample at a more molecular level (as noelrbrown suggests above), I really wouldn't do much else, except that I would put a little note with the blood to be transfused to remind the doctors and nurses on the ward to be vigilant with their patient observations.
ALL OF THE ABOVE HAVING BEEN SAID, I AM NOT, AND NEVER HAVE BEEN, MEDICALLY QUALIFIED!!!!!!

This is an interesting topic and something i worked on many years ago.  We would always do a DAT on suspected AIHA patients and would do the subclass DAT too to give us more information.  The data we obtained was combined with CR51 assay data and the Hematologic picture.  However usually and pretty quickly the DAT would max out at 4+ or 5+ depending on your facility scoring.  This made it difficult to assess the efficacy of the drug regimen i.e. is it improving red cell survival or not.  We used to quantitate IgG on the red cell using a radioligand assay to assess efficacy of drugs and guide the treatment.  is this still done now? I imagine there is another assay instead of radioligand that quantitates IgG bound to the red cell?

That's an awfully big question and I am going to "try" to hit the highlights. PLEASE feel free to add or clarity anything to keep my foot out of my mouth! I will start with the standard Dat is positive. You perform an eluate and it is negative. That points to the possibility of a drug based reaction. It's the policy of some facilities to not repeat the elution if the strength of the dat has not increased in strength or a new allo antibody has been created. The elution can also help you determine if it is truly a auto or an allo antibody by seeing if the eluate reacts with DAT negative auto cells (retic harvested if needed of course). Next, what about the times when the DAT is negative? The auto could be be directed at something that is surpressed or even negative. The antibody might even be a different class of antibody such as Iga for example or perhaps a subclass of IgG that your antisera doesn't pick up. These are just a few things that I will note, quickly. Perhaps we can get Mr Needs to make a few comments. 
 
Jason Temple 

Due to the recurrence of requests for Direct Antiglobulin Test (DAT) and, if positive, to perform the Eluate for patients with suspected Autoimmune Hemolytic Anemia (AIHA), I come here in the hope of obtaining answers or ideas from the specialists in this group.
I understand the purpose of performing DAT (IgG/IgA/IgM and C3) to confirm cases of clinical suspicion of AIHA and/or to monitor an ongoing hemolytic process. But I wonder:
What additional information can be obtained from the elution of red blood cells where IgG has already been detected? I understand the importance of the Eluate in the transfusion context, but knowing the methodological limitations and risks of false negatives, I cannot understand how doctors are interpreting and defining treatment based on this test.
Is it possible to confirm a clinical suspicion of AIHA using immunohematological tests without performing the eluate? My opinion is that yes, based on some references.
My interpretation is that the eluate would be important in the laboratory diagnosis of AIHA only if DAT is negative.
I appreciate contributions.

No problem Mabel, BUT, what I don't understand is why your Pathologist didn't tell the OB doctors "to get knotted" when they asked for the antibody to be titrated.  As far as I am aware, there has only ever been one peer-reviewed paper suggesting that a maternal anti-Le(a) has caused "clinically significant" HDFN (Carreras Vescio LA, Torres OW, Virgilio OS, Pizzolato M.  Mild hemolytic disease of the newborn due to anti-Lewisa.  Vox Sang 1993; 64: 194-195.  DOI: 10.1111/j.1423-0410.1993.tb05387.x.) and, from memory, I believe that this was not, how should I put it, universally accepted!
More to the point though, given that there a[[ears to be no detectable anti-Le(a) in the plasma at the moment, have you checked the lady's present Lewis phenotype?  It could be that she is now Le(a+b-), or even Le(a-b+).  Excuse me asking, as I am sure you probably know this, but there may be others reading this who do not, it has been known for many years that pregnant women may become transiently Le(a-b-) and may even produce Lewis antibodies.  It was originally thought that pregnant women produced less Lewis glycolipid, but it is now thought that this is not so.  It has been theorised that the increased incidence of the Le(a-b-) phenotype during pregnancy may be a result of increased concentration of plasma lipoproteins during pregnancy (Hammar L, Mansson S, Rohr T, Chester MA, Ginsburg V, Lundblad A, Zopf D.  Lewis phenotype of erythrocytes and Leb-active glycolipid in serum of pregnant women.  Vox Sang 1981; 40: 27-33.  DOI: 10.1111/j.1423-0410.1981.tb00665.x.).  In pregnant women, the ratio of lipoprotein to red blood cell mass increases more than fourfold, so that much more Lewis glycolipid is attached to plasma lipoprotein than is available for the red blood cell surface.
I shall now shut up and let others comment!!!!!!!!!!!!!!!!!!!!!!

I just answered this question.

My Score PASS  

I just answered this question.

My Score PASS  

I just answered this question.

My Score PASS  

I've had further thoughts upon this case (having told you not to worry about it - I live a sad life - NOT!).
It struck me that the patient has an Rh type of D+ C+ c+ E+ and e+, suggesting that the probability is that the patient has a genotype of DCe/DcE (R1R2), but this may not be the case.  She could have one of the rarer Rh genotypes, such as DCE/Dce (RzRo), DCE/dce (Rzr), Dce/dCE (Rory), etc, and this may be potentially important.
Some years ago, Joyce Poole explained to me that most grouping reagents labelled as anti-C are, in fact, a mixture of anti-c and anti-Ce, and this, she told me, included most monoclonal anti-C reagents (which surprised me, to be honest).  This is because the vast majority of the red cells transfused that stimulate an anti-C would have the haplotype of either DCe or dCe, or both, and will, therefore, also stimulate an anti-Ce.  As a result, these "hybrid" anti-C/anti-Ce reagents will react more strongly with red cells expressing the Ce compound Rh antigen (Rh7) and the C antigen (Rh2), than with red cells that only express the C (Rh2) antigen.
This would not, incidentally, explain the stronger than normal reaction with the e antigen.

However, if the patient does express one of the rarer Rh types mentioned above, say she is RzRo, she can actually produce an allo-anti-Ce, and most antibody panels only contain C+ red cells that are only Ce+ as well.  In other words, her antibody in the plasma MAY be identified as an anti-C, whereas it is actually a monospecific anti-Ce, which would neatly explain why she has an apparent anti-C.

Of course, she may also have an auto-anti-C, or a mimicking auto-anti-C (and, possibly, an allo-anti-Bg of some sort).  Sadly, for a nerd like me, I doubt if we will ever know!
I think it was John C Staley who once accused me of looking for zebras, when I hear horses hooves (I may be wrong, but I think it was John).  Anyway, this proves that he was absolutely correct about me!!!!!!!!!!!!!!!!!!!!!!!!

While it is infrequently referenced, universal leukoreduction is one strategy for minimizing pulmonary and cardiovascular adverse responses to transfusion (see attached).  When we instituted it in 2000 our rate of TRALI decreased by 80+ % and TACO decreased by 50%.  Probably mechanism is that white cells, DNA, histones and neutrophil extracellular traps (NETs) cause acute lung injury and inflammation when infused (good animal model data exist).  Thus the failure to implement universal leukoreduction in the USA during the last 23-25 years was a terrible and tragic mistake, and this fatal error persists to this day.
ULR TRALI TACO PMC version.pdf

In the UK, it is STANDARD practice in all laboratories that I know to use either the phrase "No Antibodies Detected", or, more frequently, "No Atypical Antibodies Detected", as the latter also includes such things as the iso-antibodies of the ABO and H Blood Group Systems.  Indeed, some go further still and use "No Atypical Allo-antibodies Detected", as this covers such findings as an auto-anti-H, auto-anti-I and auto-HI, as well as the ABO and H iso-antibodies.
These phrases do not mean that there are no atypical allo-antibodies detected.  It would be an incredibly rare set of screening cells and antibody identification panel cells that would both express, for example, the HJK antigen, or any other genuine low prevalence antigen.
In some cases, where an atypical allo-antibody IS detected, but it is known to be clinically-insignificant (such as anti-Kna), we may use the phrase "No Clinically-Significant Atypical Allo-antibodies were Detected" (or words to that effect).

One thing is for certain, and that is that a UK Reference Laboratory (and most hospital laboratories) worth their salt would report out as "Negative", or "No Antibodies", although, even using the phrases I've quoted above, occasionally the phrase, "All Clinically-significant Allo-antibodies have been Ruled Out using etc.", or words to that effect.

MIND YOU - you have to remember that I am RENOWNED for being a pedant - but I learned it from a few good sources; Peter Issitt, Carolyn Giles and Joyce Poole (to name but three).

Thanks for your return.
I'm glad they took on this condition.
However, I hope they are looking at ways to minimize this risk of ABO phenotyping failure, especially with recipient samples. Alternatives must exist, such as decreasing sample centrifugation time or speed...

We used the rate of transfused patients for each type of procedure. We carried out a survey of more than 5 years to identify the frequency of use of concentrated red blood cells for each procedure, including the immediate postoperative period (up to 48 hours). With this data, we define the reservation request guideline. When the doctor requests a reservation, he needs to select the type of procedure, and when doing so, the system fills the request according to the guidelines. For frequencies of use below 10%, zero red blood cell concentrate will appear and the blood therapy service will only perform T&S. We recommend that patients with requests a reservation whose frequency is greater than 10% have an ABO confirmation prior to the transfusion if they do not have at least two concordant ABO records in our system.

We used the rate of transfused patients for each type of procedure. We carried out a survey of more than 5 years to identify the frequency of use of concentrated red blood cells for each procedure, including the immediate postoperative period (up to 48 hours). With this data, we define the reservation request guideline. When the doctor requests a reservation, he needs to select the type of procedure, and when doing so, the system fills the request according to the guidelines. For frequencies of use below 10%, zero red blood cell concentrate will appear and the blood therapy service will only perform T&S. We recommend that patients with requests a reservation whose frequency is greater than 10% have an ABO confirmation prior to the transfusion if they do not have at least two concordant ABO records in our system.

Congratulations, Malcolm! Very well deserved!

I am enormously honoured to announce that I am going to be awarded the Gold Medal of the British Blood Transfusion Society at their Annual Scientific Meeting in Brighton this year.  It is awarded to an individual for their exceptional and long standing services to the Society and to the practice of blood transfusion in the UK.  Sorry if this sounds egocentric, but I am very excited.

Well, that's got rid of two of my possible theories in one fell swoop!

I was wondering either about loss of antigenicity due to some form of myeloid malignancy, or of adsorption of autologous secreted A substance on to the  donor group O red cell surface following a successful BMT or stem cell transplant, which may be seen with only some clones of anti-A (see, for example, Cripps K, Mullanfiroze K, Hill A, Moss R, Kricke S.  Prevalence of adsorbed A antigen onto donor-derived group O red cells in children following stem cell transplantation: A single-centre evaluation.  Vox Sang 2023; 118: 153-159.  DOI.10.1111/vox.13386., but I saw this phenomenon in adults many times when working at Westminster Hospital).

Oh well, back to having more thoughts!

I agree that the difference between cisAB and B (A) is serological and divergent. They coulding be one, but they respect the names given by the authors. I do not think that the ABO * cisAB.05 and ABO * BA.06 alleles are different. It must have been an ISBT mistake!
See a summary I made in 2019.
In general, the phenotype cisAB presents normal expression of antigen A, but similarly to phenotype A2, and weak expression of antigen B. On the other hand, B(A) presents a very weak expression of antigen A, but a normal expression of B.(1)
    The rare phenotype cisAB was first described in a case of mother AB with child O.(2) Its authors suggested that this phenotype was formed by the interaction of two genes, one A2 gene and another atypical B gene, located in the same locus. However, with the molecular characterization of the cisAB-1 allele (ABO*cisAB.01), it was observed that a sequence of the ABO*A1.02 allele containing an additional mutation at position 803G>C (Gly268Ala) was capable of synthesizing a GT with mixed activity. The cisAB-1 allele is more common in Asian populations and considering the four positions that differentiate alleles A and B, it can be described as AAAB.(3) In a study of 16 Korean blood donors heterozygous for the ABO*cisAB.01 allele, it was demonstrated that both GTA and GTB have clearly decreased activity. GTA activity was 29% of GTB was 27% compared to wild GTA encoded by the A1 allele. (4) 
    Phenotype B(A) was first detected when monoclonal ABO reagents became commercially available. This phenotype exhibits normal levels of antigen B and very low levels of antigen A in tests with some anti-A monoclonal reagents. (1) The GT of this phenotype has the ability to produce normal levels of antigen B, but also use The UDP-GalNAc as substrate to produce detectable levels of antigen A.
    The B(A) alleles are variants of allele B and the first of them (ABO*BA.01) was identified by Yamamoto and collaborators. (5) This allele is commonly referred to as BABB due to the aa of position 235 being the same as consensus A1. The second allele B(A) (ABO*BA.02) has the aa sequence of allele B, being referred to as BBBB, but contains an additional mutation at position 700C>G (Pro234Ala) which is close to aa 703, one of the four that differentiate the A allele from B.(6,7) 
    By the way, a normal GTB encoded by the consensus B allele has the ability to synthesize minimal amounts of antigen A which are detectable by some anti-A reagents. As well, GTA encoded by the A consensus alleles can also synthesize minimal amounts of antigen B, which are detectable by some anti-B reagents. These reagents were considered inappropriate for the ABO phenotyping routine,(8) for example, the anti-B monoclonal antibody (BS-85), reported by Voak et al. (9)
1. Daniels G. Human blood groups: Introduction. Oxford, UK: Wiley-Blackwell2013.
2. Seyfried H, Walewska I, Werblinska B. Unusual inheritance of ABO group in a family with weak B antigens. Vox Sang. 1964;9:268-77.
3. Yamamoto F, McNeill PD, Kominato Y, Yamamoto M, Hakomori S, Ishimoto S, et al. Molecular genetic analysis of the ABO blood group system: 2. cis-AB alleles. Vox Sang. 1993;64(2):120-3.
4. Cho D, Shin MG, Yazer MH, Kee SJ, Shin JH, Suh SP, et al. The genetic and phenotypic basis of blood group A subtypes in Koreans. Transfus Med. 2005;15(4):329-34.
5. Yamamoto F, McNeill PD, Yamamoto M, Hakomori S, Harris T. Molecular genetic analysis of the ABO blood group system: 3. A(X) and B(A) alleles. Vox Sang. 1993;64(3):171-4.
6. Haslam DB, Baenziger JU. Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family. Proc Natl Acad Sci U S A. 1996;93(20):10697-702.
7.  Yu LC, Lee HL, Chan YS, Lin M. The molecular basis for the B(A) allele: an amino acid alteration in the human histoblood group B alpha-(1,3)-galactosyltransferase increases its intrinsic alpha-(1,3)-N-acetylgalactosaminyltransferase activity. Biochem Biophys Res Commun. 1999;262(2):487-93.
8. Goldstein J, Lenny L, Davies D, Voak D. Further evidence for the presence of A antigen on group B erythrocytes through the use of specific exoglycosidases. Vox Sang. 1989;57(2):142-6.
9. Voak D, Sonneborn H, Yates A. The A1 (B) phenomenon: a monoclonal anti-B (BS-85) demonstrates low levels of B determinants on A1 red cells. Transfus Med. 1992;2(2):119-27.
 

Thanks Malcolm, it did answer my question.  Apparently while their blood is circulating these people maintain normal levels of K+.  It is only upon storage that they leak the K+ at higher levels than normal. It appears to be a genetic cell membrane "defect" 
"Familial pseudohyperkalaemia (FP) • dominantly inherited, asymptomatic • characterised by an increased rate of leakage of K+ from red cells at refrigerated temperatures • usually caused by the minor allele of a non-synonymous single nucleotide polymorphism (FP SNP; rs148211042; R723Q) in the transporter gene ABCB6 (ATP-Binding Cassette, subfamily B, member 6) • codes for a red cell membrane transporter protein • raw chip measurements from a screen of the UKBioBank suggested ~1:400 of the UK population have the FP SNP"
"Study – identified FP individuals • screening of the National Institute for Health Research Cambridge BioResource (NIHRCBR) – identified 16 out of 8712 individuals with the FP SNP. • 2 more individuals with the FP SNP were identified when clinical cases of unusually high K+ levels were reported in RCC units that they had donated – characterised in Bawazir WM, et al. 2014"
Thanks again, 

Hi John,

I am not absolutely sure, as this all happened just as I was retiring, but I have emailed a former colleague of mine who may know, but, if you put "NHSBT blood donors with high potassium levels" in to your search engine, you should get up a PowerPoint lecture that you can download with the long title of, "POTASSIUM LEAKAGE AND MEASURES OF THE RED CELL STORAGE LESION IN DONATIONS FROM INDIVIDUALS WITH FAMILIAL PSEUDOHYPERKALAEMIA", by Athina Meli, Maggie McAndrew, Amy Frary, Karola Rehnstrom, Christian J Stevens, Waleed M Bawazir, Joanna F Flatt, William Astle, Rekha Anand, Helen V New, Lesley J Bruce and Rebecca Cardigan, which may answer your question.

Once clinicians understand that ABO mismatched platelets not only do not provide hemostasis, but make bleeding more likely/worse, they will be less willing to accept infusion of ABO mismatched antibody/antigen.  Once blood transfusion services realize that infusing ABO mismatched platelets increases utilization by two fold, they will be more interested in making the effort to give ABO identical or remove incompatible plasma by washing.  Doing the right thing for patients is never the wrong answer to the question. Our current practices are convenient for us and minimize waste.  We need to prioritize clinical benefit over inventory control and waste reduction.  What we are doing now is providing little to no benefit and actually harming patients in many instances.  The bleeding rate in the PLADO (platelet dose study) in NEJM was 70%.  That's not exactly a clinical triumph.  Our bleeding rate is probably less than 5-10% employing ABO identical/washed platelets.  

Disciplinary action over product wastage sounds like it is perhaps well intentioned but ignorant bureaucrats, not health care workers running the show.  That's a big part of the problem in many institutions these days.
We are fortunate in that the senior decision makers in our hospital are all physicians, nurses, etc., including the CEO, CMO, COO.  Washing your hands before delivering babies turns out to be inconvenient but a better idea.  Universal leukoreduction and avoiding infusion of ABO incompatible antigen and antibody are also better ideas than what we have done for decades or longer.  These practices will save lives, reduce need for transfusions and actually save the system money overall, albeit at greater expense in the transfusion service.
You are no doubt correct that there will be pushback from transfusion service staff used to doing things the old, easy, but harmful way, and hospital administrators who prioritize the wrong things such as budgetary tunnel vision over reduced harm.  And blood centers are not likely to initially be all that interested in changing practices.  But when the data says patients do better with universal leukoreduction and ABO matching, hopefully,  in the long run the dogma will be replaced by data driven practices.  May take a while.

Cannot post the entire article due to copyright restrictions, but most institutions have access to NEJM through their library. If not, shoot me an email at neil_blumberg@urmc.rochester.edu and I'll send along the .pdf.
If you are transfusing 40-60 platelets a day, giving ABO identical to group O and A individuals should be relatively easy.  When patients are changing  ABO blood group it becomes more difficult. We avoid transfusion ABO antigen and/or antibody that is incompatible with either original recipient type or donor type.  Usually means washed group O red cells and platelets.  That's the bad news. It does require time and effort, and as you say, med techs are in short supply.  Here's the good news. If you transfuse ABO identical or washed compatible platelets you will use between 30-50% fewer platelets per patient, increasing your supply and decreasing your cost/problems. You will also use next to no HLA matched platelets (we used 3 out of 6,000 one recent year), you will have fewer febrile and allergic transfusion reactions, you will have fewer red cell as well as HLA antibodies made in recipients, and you may reduce TRALI and TACO.  Obviously you have to have universal leukoreduction to start with.  Selective leukoreduction misses about 50% of the  patients who will become refractory, probably due to missed or delayed diagnosis of hematologic malignancy, aplastic anemia, etc.  But the big attraction is you will have less bleeding, although that mainly affects the patients and the docs and nurses at the bedside.  

When you transfuse ABO major incompatible, which seems to be the default due to fear of hemolysis from minor incompatible, you don't get any increments, you use lots of platelets and the patients bleed more. (see references below)  Bleeding causes lots of harm, but also impacts the blood transfusion service for obvious reasons.  So figure out a way to start giving patients with aplastic anemia and acute myeloid leukemia who are newly diagnosed only ABO identical platelets and that will be a great start for the patients and the transfusion service.  Those patients will bleed less, need fewer platelet transfusions, have fewer transfusion reactions, will not have positive DATs, and will likely survive their hospitalization and disease at higher rates if our experience is typical.
And if you cannot give ABO identical or washed platelets free of incompatible cellular and soluble antigen and free of incompatible ABO antibody, start out with minor incompatible platelets (e.g., O to A) rather than ABO major incompatible (e.g., A to 0).  The risks of hemolysis are not negligible (about 1 in 800) but are less serious and severe than having life threatening bleeding or refractoriness which occur more rapidly with ABO major incompatible in all likelihood.  There's a ton of antibody that is incompatible with antigen transfused when we give A platelets to O recipients which means each antigen winds up with a ton of antibody making huge immune complexes.  When we  transfuse antibody incompatible we are transfusing a small amount of antibody into a recipient with huge amounts of antigen, so the size and number of immune complexes is probably smaller. These are my best guesses that we've been making exactly the wrong decision when we give ABO mismatched platelets. Best to avoid any, but major mismatched provides no hemostasis, minimal to no increment and is associated with increased bleeding mortality in the study from Columbia (David Roh and colleagues https://pubmed.ncbi.nlm.nih.gov/33649761/).  But ABO identical is not that hard for larger centers for the 85% of patients who are group O or A.  You just have to start small, get the hang of it, and then extend to other blood groups and other diseases than leukemia, MDS and aplastic anemia (including transplants, particularly allo--transplants). All those tables of how to select ABO mismatched platelets for transplant recipients are well intentioned but scientifically without evidence.  Avoid infusing incompatible antigen and antibody as much as possible, and delay transfusion when ABO identical will be available within hours.  Give priority to patient well being over inventory management. Give reduced doses, which work just as well.  Get a Terumo or Haemonetics washing device and wash with PAS. It's a big set of changes, but neither terribly expensive nor rocket science. The dogmas and expert opinion about universal leukoreduction and ABO matching of transfusions are now proven to be tragically mistaken.
 
https://www.ashclinicalnews.org/news/from-the-blood-journals/written-in-blood/outcomes-abo-incompatible-platelet-transfusions-patients-intracerebral-hemorrhage/
https://pubmed.ncbi.nlm.nih.gov/11399821/
https://pubmed.ncbi.nlm.nih.gov/21414009/