Yanxia

My greatest apology for leaving you all hanging. We've been so incredibly busy and short-staffed that I could not even think about anything else but trying to finish up my daily duties. Anyway, seems that the patient also had an impella device that had to be "adjusted" and I believe that corrected the hemolysis. I'm only reading the responses today (2 weeks later)☹️so hats off to you all who thought mechanical causes. I would have not thought that the device would be that far-off to cause the gross hemolysis we saw. We do see slight hemolysis with impella devices but not like this one. I guess never say never. Thank you all for your responses.

We have seen this phenomenon from time to time, albeit rarely. We use R1wR1 red cells with all of our antibody identifications, purely because it is always cell 1 in the panel! We also, however, use two R1R1 panel cells and an r'r panel cell.
I'm not too sure why we get the odd sample that reacts like that, because the C antigen on the R1wR1 panel cell is not that much weaker than the C antigen on the R1R1 panel cells, and certainly no weaker than that on the r'r panel cells.
Remember that the "w" of "Cw" stands for "Willis", and not "weak", as it was named after the donor who caused the immunisation to the antigen in the first example of anti-Cw described (something I forgot a few years back in an article I wrote, much to my embarrassment, if that is any solace to you) and that RH8 (RHCw) is allelic to both RH9 (RHCx) and RH51 (MAR), and not to either RH2 (RHC) or RH4 (RHc). Therefore, any weakening must be due to steric hinderance, or something similar.
:confuse::confuse:

At our facility, we have to do a cold adsorption IF the cold auto interferes with the reverse and/or ISXM. Since testing with "neat" plasma is our standard of practice, we would still report the XM as incompatible and send it out with a release stating the incompatibility was due to an autoantibody. We are not "allowed" to prewarm away IS reactions. 🥶 IF we prewarm - it's only if the cold has a high thermal amplitude and causes interference after 37deg incubation / antiglobulin phase...........but that's just us.........

Happy Friday everyone! 
First off, Thank you Malcolm 😊 for that excellent PPT lecture! 👌 With your permission, I'd love to share it with my students; it's exactly the kind of content that helps bring these complex concepts to life (and mildly melt brains in the best way possible).
Speaking of melted brains… let's talk Anti-G differentiation, shall we? While I agree that differentiation isn’t necessary for transfusion purposes because in cases where anti-G is suspected, the recommendation is the same (D and C antigen-negative PRBCs); in the US Reference Lab world, it's a whole different dance. Differentiating anti-D + anti-C from anti-G is essential in alloimmunized patients with anti-D + anti-C for Rh Immune Globulin (RhIg) prophylaxis administration indication. Medical teams want to know if a patient is a candidate for RhIG, and if it's still indicated, especially to avoid future medical and legal complications. And so begins the beautiful (read: painstaking) double adsorption and elution process. 😫
 
The presence/development of a real anti-D indicates the patient does not require RhIg administration, whereas the presence of anti-G indicates the need for RhIg prophylaxis. That way, RhIG is avoided in patients with a real anti-D (although clinical correlation is recommended to guide every decision).
In this case, we saw what looked/reacted like anti-D and anti-C (1). Adsorption with R2R2 cells showed anti-C in the adsorbed serum (2) and anti-D and/or G in the adsorbing R2R2 cell eluate (3). Usually, we would stop and call anti-C and anti-D, if we have no reactions on C Ag Pos cells, but since suspected anti-G was in the mix, we routinely differentiate/separate it. The anti-D was confirmed in (4) by adsorbing the eluate containing the suspected (anti-D + anti-G) with an r'r unit. So far, so good. However, when we tried to separate the anti-G by elution of the adsorbing r′r cells, expecting a reaction on D and C antigen-positive cells (5), we got a negative result (Cue dramatic music).
I know, I know...at this point you’re probably wondering if we’re still in the Blood Bank or if we’ve accidentally wandered into theoretical physics. Trust me, my brain also wobbles when explaining G differentiation. It's the kind of thing that makes you rethink your career choices... for about 5 minutes... and then roll up your sleeves and start prepping another adsorption. 😅
A couple of housekeeping: We ficin treat our adsorbing cells, and sometimes use PEG in adsorption for efficiency. After briefly considering a career in something less chaotic, we retraced our steps. No PEG this time, 60-minute incubation, followed up with a DAT after each adsorption to check for antibody coating... and voilà: finally got the positive reaction we were looking for. The patient has anti-D, anti-C, and anti-G.
I can only think of a possible weak DAT strength pending elution, or PEG interference when used in adsorption. As always, this is Blood Bank, we know a million things can go wrong, and often do. But in the end, science (and a lot of perseverance) wins. 
Thanks again to everyone for your input, patience, your brains, and your sense of humor. And again, Malcolm, thanks for your lecture, teaching, dedication and inspiration!
I wish everyone a calm weekend. May your panels be clear, your DATs negative, and your eluates informative. 😉
 

In our lab, we do 30 patients ABO typing daily in average. In those tests we will find out forward and reverse typing mismatch at least once daily. Maybe because we tested patients' sample, the incidence is higher than donors', but just as Malcolm said it is definitely necessary to do forward and reverse typing and make sure they are matching.

In the UK, we would test serum/plasma samples from pregnant patients to see if there was an anti-C + Anti-G, or an anti-G on its own, but if the tests showed an anti-D+C, we didn't go any further to see if there was an anti-G there as well.  I mean, what for?  What difference does it make?

I attach a PowerPoint lecture on the subject I wrote some years ago, but I think it is still pertinent.
The G Antigen and Anti G.pptx

In our lab, we do 30 patients ABO typing daily in average. In those tests we will find out forward and reverse typing mismatch at least once daily. Maybe because we tested patients' sample, the incidence is higher than donors', but just as Malcolm said it is definitely necessary to do forward and reverse typing and make sure they are matching.

In our lab, we do 30 patients ABO typing daily in average. In those tests we will find out forward and reverse typing mismatch at least once daily. Maybe because we tested patients' sample, the incidence is higher than donors', but just as Malcolm said it is definitely necessary to do forward and reverse typing and make sure they are matching.

Sorry Neil, but I have to point out that this is not completely accurate.  Any red cell antigens that are adsorbed onto the red cell surface, rather than being an integral part of the red cell membrane remain the type of the patient, rather than the donor.  This is true of the Lewis phenotype (for instance, if the recipient was Le[a+b-], and the donor was Le[a-b+], after the transplant, the red cells will group as Le[a+b-], and not as Le[a-b+]}.  This is also true of antigens within the Chido/Rodgers Blood Group System, and certain others.

If the recipient is a Secretor, they will continue to secrete ABO substance of the original ABO type, which, of course, will also be adsorbed onto the red cell surface (as well as being in the plasma, leading to the phenomenon of "accommodation", and this is why most recipients stay with a reverse group of "AB" after an ABO mis-matched stem cell/bone marrow transplant.

SORRY TO BE A PEDANT, PARTICULARLY AS I AGREE WITH EVERYTHING ELSE YOU HAVE WRITTEN!

The +s stands for strongly expressed.

The expression of the P1 antigen varies considerably from person to person, but the reaction strength with anti-P1 is an inherited trait (i.e. the strength of the expression on the red cell surface).

"I apologize for this dumb question."  BBnoob69, NO QUESTION IS A DUMB QUESTION, IF YOU DO NOT KNOW THE ANSWER.  If you don't know the answer, the dumb thing is to not ask the question in the first place.  NEVER be afraid to ask a question on here,

Hi Rich,

Yes you can, and, don't forget, under BSH Guidelines, you do not have to give blood that has been tested for the Cw antigen, if the unit is compatible by IAT with the patient's plasma/serum.  It is one of the few Rh antigens that can be given under these circumstances.

Be aware though, that I answer this in the knowledge that you are working in the Isle of Man (i.e. the UK).  This may not apply in other parts of the world (particularly Lithuania and Finland).

ALL Rh antibodies react with red cells treated with proteolytic enzymes, such as ficin, papain, trypsin and alpha-chymotrypsin (well, red cells that are expressing the cognate antigen, anyway), BUT, be careful because most monoclonal grouping reagents, including monoclonal anti-Cw, will often say to be used by either direct agglutination or by IAT, BUT NOT to be used with enzyme-treated red cells, because they can cause false positives.

Most of what I have written above can be found in Reid ME, Lomas-Francis C, Olsson ML.  The Blood Group Antigen FactsBook.  3rd edn, 2012. Academic Press.  ISBN: 978-0-12-415849-8.  The rest can be found in the manufacturer's insert, if the reagent is commercial.

Hope that helps, but feel free to get back if it doesn't.

when we see up the side reactions in gel (we call them train tracks) it usually ends up being a cold auto sort of antibody.
we would run a DAT - Poly, IgG and Complement.......
run a cold screen (IS, RT, 4C)
most times the explanation is there in this kind of testing...........
other reason could be rouleaux - 
either way we would switch to PEG here..........and notate in the patient's record to perform future testing in PEG.

reactivity "all over the place" describes my experience of anti-P1.  I have seen negative reactions from cells labelled as strong and cells labelled as weak have been positive. P1 substance (if you have it) to neutralize the anti-P1 provides an elegant resolution.   

I expected to see a stronger reaction with the P1 cells labeled as "strong", but it does react with only the P1+ cells.   Maybe P1 persists better on donor cells than reagent cells.

This is from Human Blood Groups, Deoff Daniels, the second edition.

Mabel, maybe there was an anti-P1 which was initially a  cold reactive one that warmed off by 30 min prewarm technique. But I can't explain why it didn't react with reagent cells after transfudion except there were antigen loss.

The H explanation seems plausible although we didn't see much difference between A and O donors in terms of reaction strengths.  I attached the original panel using Ortho 0.8% pre-diluted cells.  Maybe these keep their H antigen better than the 3% cells we converted to 0.8%.  The panel shown was only a few days from expiring.  The 3% cells expire May 9th. This seems backwards from what we would expect if the antigens were weakening in storage.  However, they are from different manufacturers and in different diluents.  The 3% cells were from Immucor.
 

Thanks Mabel for your explanation. I think we can exclude the protein factor.
1.In my work, I have noticed that the reagent cells express less H antigen than the donor cells do. Our screen and panel cells have 3-month shelf life, but our donor cells only have about 35 days. Even though we do our best to preserve the antigens on panel cells, there are still some losses. Of course, there are other antigen loss other than H.
2.I have read lewis antibodies may react with A type, sorry I can't recall it exactly, I will check it out after this work shift when I get home. If there is an anti-A Lewis antobody, it will react stronger with donor cells. As to the incompatible O donors, my bold guess is they express more Lewis antigen than reagent cells.
Sorry again for my imagination.

Thanks Mabel for your explanation. I think we can exclude the protein factor.
1.In my work, I have noticed that the reagent cells express less H antigen than the donor cells do. Our screen and panel cells have 3-month shelf life, but our donor cells only have about 35 days. Even though we do our best to preserve the antigens on panel cells, there are still some losses. Of course, there are other antigen loss other than H.
2.I have read lewis antibodies may react with A type, sorry I can't recall it exactly, I will check it out after this work shift when I get home. If there is an anti-A Lewis antobody, it will react stronger with donor cells. As to the incompatible O donors, my bold guess is they express more Lewis antigen than reagent cells.
Sorry again for my imagination.

Thanks Mabel for your explanation. I think we can exclude the protein factor.
1.In my work, I have noticed that the reagent cells express less H antigen than the donor cells do. Our screen and panel cells have 3-month shelf life, but our donor cells only have about 35 days. Even though we do our best to preserve the antigens on panel cells, there are still some losses. Of course, there are other antigen loss other than H.
2.I have read lewis antibodies may react with A type, sorry I can't recall it exactly, I will check it out after this work shift when I get home. If there is an anti-A Lewis antobody, it will react stronger with donor cells. As to the incompatible O donors, my bold guess is they express more Lewis antigen than reagent cells.
Sorry again for my imagination.

That's a good question.  The new sample was tested against Ortho 0.8% screen cells which were both positive due to her anti-D/C/G. Four C & D neg 3% panel cells were converted to 0.8% and run in gel with a 30-minute incubation. They were all negative.  Then the new specimen was also used to crossmatch about 10 units, and we found 3 were compatible.  I checked to see if she was getting TPN but don't see any.  Sometimes that fats and proteins in the nutrition IV cause strange reactions.  Usually, I have seen a positive DAT with it.   If you can further describe the sort of protein you are thinking of, I would appreciate it. 

We have a 42 y.o. Caucasian female with chronic anemia and cellulitis/sepsis needing debridement who has anti-D (2+) & anti-C (3+) by Ortho MTS gel.  She was transfused elsewhere in 2021 and here in 2022, all Rh neg units. Two units each time. Screens were negative then. She has a history that suggests she may have shared IV drug needles at some point. I don't think there is a pregnancy history but not ruled out. She is A neg. Her initial testing in Ortho gel was clearly anti-D with C (could include G) but she had some 1+ reactivity with 4 of 5 D and C negative panel cells. The cell that didn't react was E+, D-, C-. Fya+, Fyb-, heterozygous for Kidd and MNS, but Lea- & Leb-.  Auto control is negative. Three percent panel cells were then selected, diluted with Ortho diluent 2 to a 0.8% suspension and run in gel with a 30-minute incubation. By this method, we detected the anti-D and C antibodies in 2 cells that were D+, C- and 2 that were D-, C+ respectively. We were able to rule out all other typical specificities on 7 non-reactive cells and did not detect the weak reactivity previously found, suggesting that it was antibody to the pre-diluted 0.8% cells' diluent. One A neg, C neg unit was crossmatch compatible by gel that day and was transfused.  Only that unit was crossmatched that day. Two days later (today), they requested more blood. All antiglobulin crossmatches in gel were incompatible--some units were A neg, some O neg.  The reactions in gel were all 2+ or weaker with an atypical appearance of having one to two "stripes" from bottom to top, something we usually associate with cold reactive antibodies. Their strength appeared variable from weakly positive to 2+.  We tried washing the donor cells, prewarming cells/plasma before combining them, 30 min. incubation in gel, and performing PEG XM's in tube which did not help.  PEG was a bit weaker on the 2 units tested by that method (one the strongest reaction in gel and one the weakest), but still positive.  We redrew the patient and got the same results with the new specimen. We did not test reagent cells by PEG. We ended up crossmatching about 20 A neg and O neg RBC units by gel and there were 3 that were truly compatible (not including the one from the first day). She got her second unit of this visit in the past few hours with no problems.  What might cause reactions with AS-1 donor units but not with reagent red cells?  Both are made up in the same diluent and tested by the same process. Some of the donor units were O cells like the reagent cells. Is there anything we should be considering?  I expect she will be back for more blood in coming days to years (with our luck, probably with new alloantibodies since she is a responder who was transfused again).  Thanks for your wisdom.

As an aside, I would suggest that an anti-G is almost certainly present, indeed, it may ONLY be an anti-G, with no or very weak anti-D and/or anti-C present, as the antibody is reacting stronger with the C antigen, than the D antigen.  This is a common finding with anti-G.

Turning to your real question, we have found in the past that people can have a "naturally occurring" antibody to an antibiotic added to the cell preservative by the manufacturers.  This is NOT easy to wash off from the reagent red cells, as they seem to adsorb it onto their surface almost as strongly as the Lewis antigens are adsorbed.  It could be that this patient has made just such an antibody, but this is a very tenuous explanation, and other members will probably come up with something a lot more probable!