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!

I noticed that pattern that suggests anti-G also.  In transfusion, it doesn't matter whether it is anti-D/C or anti-G or a combination of them, right?  We would pursue it in pregnant patients.  Could she have been exposed to G without being exposed to either D or C? I feel like I've read that this is extremely rare, but not impossible. 

Yes, we find antibody to the diluent in Ortho's pre-diluted 0.8% reagent cells quite often.  I believe that is what we found in the original testing besides the anti-D/C/G.  When we make 0.8% suspensions from 3% cells, we use diluent 2 which lacks most of the antibiotics that are in the pre-diluted cells. It is a bit strange that the diluent antibody did not react with one cell. I have seen before where the antibody seems to have a predilection for some Rh antigens, and it seems like the antibiotic almost complexes with them on the RBC surface (maybe sterically).  I think an antibody to the gel diluent explains why the panel went to negative except for with the D or C positive cells, Unfortunately, the reaction difference causing my consternation is between 3% reagent cells and donor cells suspended in the same diluent. If there were antibodies to the diluent that the 3% reagent cells were originally suspended in, I would expect those cells to react in gel rather than the donor units. I wonder if there is something in AS-1 that coats the RBCs to which this patient has an antibody. The negative reactions would then be due to lower levels in some donors compared to those eliciting stronger reactions. I had was told years ago that 3% cells freshly converted to 0.8% were somewhat more sensitive in Ortho gel testing, but this is acting in reverse. Also, I have heard that units are preserved to maximize their function after transfusion, not to stabilize antigens, whereas reagent cells are preserved to maximize antigen expression. Again, this is behaving backwards. I wonder if we should have done more antibody testing in PEG. Some specificities "like" PEG better than gel. Maybe we would have identified a 3rd antibody.  It will be interesting to see if, in future testing, she makes another alloantibody.

Appreciate this and any other input.

2 hours ago, Mabel Adams said:

I noticed that pattern that suggests anti-G also.  In transfusion, it doesn't matter whether it is anti-D/C or anti-G or a combination of them, right?  We would pursue it in pregnant patients.  Could she have been exposed to G without being exposed to either D or C? I feel like I've read that this is extremely rare, but not impossible. 

You are completely correct about this Mabel.  In fact, this was how anti-G was discovered (Allen FH, Tippett PA.  A new Rh blood type which reveals the Rh antigen G.  Vox Sang 1958; 3: 321-330.  DOI:  10.1111/j.1423-0410.1958.tb04013.x.).

As for the rest of your answer, I will bow to your greater knowledge, as I retired in 2016 (although it sometimes now feels like 2006!!!!!!!!!!!!!!).

Nice case, by the way.

My qustion may sound stupid, but out of passion, I still ask out. I am sorry for this. Did you test the after transfusion specimen against the reagent cells? I guess the pos reaction to antigen neg donors maybe caused by protein in the patient's blood sample.

 

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. 

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.

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.

 

 

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

8 hours ago, Mabel Adams said:

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.

 

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.

Edited April 17Apr 17 by Yanxia

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.

On 4/17/2025 at 4:27 PM, Mabel Adams said:

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.

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.   

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.