We had an outpatient order for two units of blood. Three cell Gel antibody screen was 2+, 0, 2+. The patient had a few weak reactions on a panel back in June with no antibody specificity identified. A gel panel was 2+ with all cells. Auto control was negative, as was the DAT. A three cell PeG antibody screen was 0, w+, 0. Red cell typings showed patient to be R2R2 and negative for Fy(a). A selected cell panel of R2R2 cells was prepared and tested in gel. All cells reacted 2+. Ortho was called to see if they had any information on screening cell #2, the only negative cell we had. They did not. They suggested antibody to preservative but we had already tried washing the 0.8% cells without success. Peg crossmatches of R2R2 cells were compatible but crossmatches in gel using both saline suspended and Diluent 2 suspended donor cells were 2+. The antibody titered >2048 in gel. Plasma inhibition removed the antibody reactivity and the patient's plasma reacted with complement coated red cells at immediate spin but not with uncoated red cells. So, we are pretty certain we have a Chido/Rodgers.

Here is the dilemma: During the workup, we found out the patient had been to a hospital in a neighboring state last month and received three units of blood. We called the hospital and they said they had a negative antibody screen with gel. We repeated the e typing and read it microsopically and was hard pressed to even find a few cells shaking hands. Assuming the units transfused were little e positive, we would have expected to see some reactivity. Even though the DAT was negative, we prepared an eluate and tested it using gel. The eluate was negative.

So, would you say anti-e is present and continue giving little e negative red cells? Even though plasma inhibited the reactivity of the Chido/Rodgers and we did not see an underlying anti-e, could it be the anti-e is so weak that the 1:2 dilution of the plasma removed the anti-e reactivity?

The first thing I would do would be to test the patient's plasma/serum with papain-treated red cells.

Apart from some very rare cases of extremely avid antibodies, the Ch and Rg antigens are desroyed by the action of papain, but at the same time, papain will enhance the reaction between anti-e and the e antigen.

I would also type the patient for other antigens, M, N, S, s, Fy(a), Fy(, Jk(a) and Jk( for two reasons. Firstly to see if there are any mixed-field reactions, showing that some of the red cells from the transfused units are still present (it is extremely unlikely that the units given would be exactly the same phenotype as the patient). Secondly, to see what other antibodies the patient could make in the future.

Certainly, it is doubtful that an anti-Ch or an anti-Rg would have caused all three units to have "disappeared" from the patient's circulation (sporadic reports of "transfusion reactions" caused by these antibodies suggest more of an HLA-type reaction, rather than a delayed haemolytic transfusion reaction), but I wonder if there may be another cause for the units to have "disappeared" so quickly (if, indeed they have), such as a chronic bleed, compensated by an active marrow.

I wonder if you could tell us what the underlying pathology from which the patient is sufferring to require transfusions so often? This may give a clue.

All of that having been said, if we have a patient who has anti-Ch or anti-Rg, we would tend to give them Rh and K matched blood in any case, just so that they are not stimulated to make further atypical alloantibodies. I have recently read a paper that stated 20% of patients who have produced one atypical alloantibody go on to make further antibodies when transfused, but almost all of these had a specificity within the Rh Blood Group System (C, c, D, E or e) or were anti-K. Only a very small minority produced antibodies directed against antigens in other blood group systems.

I hope that helps in some small way.

:):)

Hi Malcolm,

We did do complete red cell antigen typings and noted no mixed field reactions. She was negative for C, e, Kell, and Fy(a). We do not have papain but did do a screen with ficin treated red cells. The Chido/Rodgers reactivity was gone and we did not see any evidence of anti-e.

The patient is diabetic with peripheral vascular disease and multiple gastrointestinal AVMs. Apparently, she had a GI bleed last month which brought her to the other hospital with a hemoglobin of 6. They transfused three units of red cells. She presented to us last Friday with a hemoglobin of 7.3. They are looking to identify the bleeding AVM and will treat accordingly. We will give R2R2, Kell negative blood and be on the lookout for anti-e should she require additional transfusions.

Hi Malcolm,

We did do complete red cell antigen typings and noted no mixed field reactions. She was negative for C, e, Kell, and Fy(a). We do not have papain but did do a screen with ficin treated red cells. The Chido/Rodgers reactivity was gone and we did not see any evidence of anti-e.

The patient is diabetic with peripheral vascular disease and multiple gastrointestinal AVMs. Apparently, she had a GI bleed last month which brought her to the other hospital with a hemoglobin of 6. They transfused three units of red cells. She presented to us last Friday with a hemoglobin of 7.3. They are looking to identify the bleeding AVM and will treat accordingly. We will give R2R2, Kell negative blood and be on the lookout for anti-e should she require additional transfusions.

Sounds good to me!

I don't think there is anything else you could do at the moment, except, as you say, give R2R2, K- as a precaution.

Ficin works just as well as papain for what I was saying.

:D:D:D

Dear Bmarooto and Malcolm,

Thank you for this very interesting post and response. I was wondering why we had what appears to be inconsistant reactivity with the 3 cell Ab screen, with and without the use of PEG. With Chido/ Rogers should we not have seen all three cells positive in both cases? Does the ABS reactivity have to do with the apparent newness (lack of better word) of the antibody? Also, has the Anti-e been clearly ruled out with the non-reactive Ficin Cells?

Just a couple of other questions. You state that the patient is diabetic with periferal vascular disease; does this combination, along with a more frequent need for blood products do to the GI bleed, provoke a more sensitive immune system similar to what is seen in patients with Sickle Cell disease?

And, when performing the antigen typing of the patient specimen, what woud be the significance of mixed field reactivity? Additionally, how relevant are the results obtained from the antigen typing, given the fact that the patient was transfused one month earlier? Could they be relevant at all?

Dear Bmarooto and Malcolm,

Thank you for this very interesting post and response. I was wondering why we had what appears to be inconsistant reactivity with the 3 cell Ab screen, with and without the use of PEG. With Chido/ Rogers should we not have seen all three cells positive in both cases? Does the ABS reactivity have to do with the apparent newness (lack of better word) of the antibody? Also, has the Anti-e been clearly ruled out with the non-reactive Ficin Cells?

Hi rravkin@aol.com,

No, the inconsistency of the antibody is not likely to be due to the apparent newness of the antibody; it is much more likely to be due to the specificity of the antibody and the nature of the antigen.

Although fully recognised as a Blood Group System by the ISBT, both Ch and Rg are antigens that are adsorbed onto the surface of the red cell membrane from the plasma, rather than being an integral part of the red cell membrane.

The Ch and Rg antigens are, in fact, C4B and C4A of the complement system, so that, if you are Ch- (as all good people should be!!!!!!!!!!!!!!), you lack C4A, and if you are Rg-, you lack C4B.

The strength of the Ch and Rg antigens can vary greatly from one individual to another (hence the apparent difference in reaction strengths with different red cells and the same plasma), but, in addition, Ch- and Rg- are not all true Ch- or Rg-! This seems a weird thing to say, but there are 6 different Ch antigens and 2 different Rg antigens (together with a WH antigen that also belongs to the system).

Therefore, the cell that only reacted weakly could be negative for one or more of the Ch antigens, but positive for the others, or negative for one of the two Rg antigens.

The negative results with ficin-treated red cells does rule out the presence of an anti-e (although there are extremely rare examples of some Rh antibodies in the literature that only react by IAT, and not with enzyme-treated red cells - I published a poster myself [with others] a few years ago at a BBTS Meeting concerning an anti-E that reacted in just this way).

I hope all that helps.

Malcolm

:):)

Just a couple of other questions. You state that the patient is diabetic with periferal vascular disease; does this combination, along with a more frequent need for blood products do to the GI bleed, provoke a more sensitive immune system similar to what is seen in patients with Sickle Cell disease?

And, when performing the antigen typing of the patient specimen, what woud be the significance of mixed field reactivity? Additionally, how relevant are the results obtained from the antigen typing, given the fact that the patient was transfused one month earlier? Could they be relevant at all?

The answer to your first question is, I don't know for certain, but I don't think so.

The significance of mixed-field reactions is that, if these were present, it would prove that at least some of the transfused red cells were still present in the patient's circulation.

It is highly unlikely that transfused blood would be of exactly the same types as the patient throughout from Rh to Kidd ( although it is not entirely impossible) and so one would expect to see some mixed-field reactions with some of the grouping reagents (although not Lewis, because transfused red cells take up the Lewis type of the recipient, as do Ch and Rg types come to that!).

The fact that there was no evidence of mixed-field reactions strongly suggests that the transfused red cells are no longer in the circulation (although this does NOT necessarily mean that they have been destroyed by an antibody in the patient's plasma), but it does mean that the antigen typing will be that of the recipient.

:):)

I agree with Malcolm's answers. If the patient continues to require transfusions, we will give R2R2, Kell negative. I will update this thread should she develop additional alloantibodies.

I agree with Malcolm's answers. If the patient continues to require transfusions, we will give R2R2, Kell negative. I will update this thread should she develop additional alloantibodies.

I am extremely pleased that you agree with my posts, especially the bit about all the best people being Ch-!!!!!!!!!!!!!!!!!!!!!!!!!!!!

:D:D:D:D

Hi rravkin@aol.com,

The negative results with ficin-treated red cells does rule out the presence of an anti-e (although there are extremely rare examples of some Rh antibodies in the literature that only react by IAT, and not with enzyme-treated red cells - I published a poster myself [with others] a few years ago at a BBTS Meeting concerning an anti-E that reacted in just this way).

I hope all that helps.

Malcolm

:):)

UUUGGGHHHH Malcolm,

You have once again disillusioned me with your knowledge. We have always abided by the premise that Rh system antibodies are enhanced by an enzyme-treated panel. :mad: Have we really been wrong all these years????

UUUGGGHHHH Malcolm,

You have once again disillusioned me with your knowledge. We have always abided by the premise that Rh system antibodies are enhanced by an enzyme-treated panel. :mad: Have we really been wrong all these years????

No, not really.

Rh antibodies that do not react with enzyme-treated red cells are really exceptionally rare (of the same order of rarity as an Rh antibody that stimulates the complement system - and I have only ever seen one of those, when I was working at the International Blood Group Reference Laboratory as a very Junior Technician).

I think that it is quite reasonable to assume that all but the rarest Rh antibody/antigen reactions are enhanced by enzyme treatment of red cells.

It's me talking about zebras again!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

:D:D:D:D

jcdaya...here is an example for you::

The patient was at a rehabilitation hospital that we provide Blood Bank services to. He had a negative gel antibody screen but had intravascular hemolysis during transfusion (they called the reaction durnig the second unit). Post transfusion Direct AHG was negative by tube and gel. We did an eluate anyway but it was negative. Repeat pre-tx and Post tx antibody screens were negative with gel, Peg, LISS, and enzyme. We sent a sample to our local Red Cross who also found nothing. We confirmed that only saline was running with the blood, and neither a blood warmer nor pressure infuser was used. The Rehab hospital sent the patient to us. We contacted the hospital the patient was at originally. They said he had a negative screen but had a "febrile" transfusion reaction before he was discharged. We tried a third unit a two days later...slowly...and collected a fingerstick sample every 15 minutes. After about 75 mL the patient developed fever, shaking chills and increased hemolysis was noted.

I phenotyped the patient and the three units. The patient appeared to be R2r. There was some microscopic agglutination with big C yet all three units were big C positive. In fact big C was the only thing all three units were positive for that the patient appeared to be negative for. His hct was dangerously low so we gave big C negative blood and the patient tolerated the transfusion with no additional hemolysis.

We sent a sample to George Garaitty's lab at the Los Angeles Red Cross for Polybrene testing. They were able to confirm the presence of anti-C reacting by manual Polybrene only.

I guess this antibody did not read the textbooks! :D :D

jcdaya...here is an example for you::

The patient was at a rehabilitation hospital that we provide Blood Bank services to. He had a negative gel antibody screen but had intravascular hemolysis during transfusion (they called the reaction durnig the second unit). Post transfusion Direct AHG was negative by tube and gel. We did an eluate anyway but it was negative. Repeat pre-tx and Post tx antibody screens were negative with gel, Peg, LISS, and enzyme. We sent a sample to our local Red Cross who also found nothing. We confirmed that only saline was running with the blood, and neither a blood warmer nor pressure infuser was used. The Rehab hospital sent the patient to us. We contacted the hospital the patient was at originally. They said he had a negative screen but had a "febrile" transfusion reaction before he was discharged. We tried a third unit a two days later...slowly...and collected a fingerstick sample every 15 minutes. After about 75 mL the patient developed fever, shaking chills and increased hemolysis was noted.

I phenotyped the patient and the three units. The patient appeared to be R2r. There was some microscopic agglutination with big C yet all three units were big C positive. In fact big C was the only thing all three units were positive for that the patient appeared to be negative for. His hct was dangerously low so we gave big C negative blood and the patient tolerated the transfusion with no additional hemolysis.

We sent a sample to George Garaitty's lab at the Los Angeles Red Cross for Polybrene testing. They were able to confirm the presence of anti-C reacting by manual Polybrene only.

I guess this antibody did not read the textbooks! :D :D

No offense to you intended...but I HATE THIS STORY!!!! It is scary!! Holy ****! I've never heard of such. Thanks for the info!

Although, I will say that a large number of antibodies don't "Read the Textbooks" otherwise, they wouldn't need us!

jcdaya...here is an example for you::

The patient was at a rehabilitation hospital that we provide Blood Bank services to. He had a negative gel antibody screen but had intravascular hemolysis during transfusion (they called the reaction durnig the second unit). Post transfusion Direct AHG was negative by tube and gel. We did an eluate anyway but it was negative. Repeat pre-tx and Post tx antibody screens were negative with gel, Peg, LISS, and enzyme. We sent a sample to our local Red Cross who also found nothing. We confirmed that only saline was running with the blood, and neither a blood warmer nor pressure infuser was used. The Rehab hospital sent the patient to us. We contacted the hospital the patient was at originally. They said he had a negative screen but had a "febrile" transfusion reaction before he was discharged. We tried a third unit a two days later...slowly...and collected a fingerstick sample every 15 minutes. After about 75 mL the patient developed fever, shaking chills and increased hemolysis was noted.

I phenotyped the patient and the three units. The patient appeared to be R2r. There was some microscopic agglutination with big C yet all three units were big C positive. In fact big C was the only thing all three units were positive for that the patient appeared to be negative for. His hct was dangerously low so we gave big C negative blood and the patient tolerated the transfusion with no additional hemolysis.

We sent a sample to George Garaitty's lab at the Los Angeles Red Cross for Polybrene testing. They were able to confirm the presence of anti-C reacting by manual Polybrene only.

I guess this antibody did not read the textbooks! :D :D

I have heard of this scary situation twice before in 37 years of my professional life; both involving Rh antibodies (although neither was proved by PEG because it wasn't around at the time, but only by the fact that the patient, in each case, tolerated antigen negative blood).

One was described in a US patient either by Malcolm Beck or George Garratty in a meeting in the UK (both were lecturing, and I can't remember which of them described the case), and one by Bill Chaffe in the UK. Both involved anti-E.

It really is incredibly worrying when one hears of these cases.

:eek::eek:

I have heard of this scary situation twice before in 37 years of my professional life; both involving Rh antibodies (although neither was proved by PEG because it wasn't around at the time, but only by the fact that the patient, in each case, tolerated antigen negative blood).

One was described in a US patient either by Malcolm Beck or George Garratty in a meeting in the UK (both were lecturing, and I can't remember which of them described the case), and one by Bill Chaffe in the UK. Both involved anti-E.

It really is incredibly worrying when one hears of these cases.

:eek::eek:

I think possibly that there are more cases like these described that are not noticed because they are never investigated to these levels. It might be good to do a survey (UK/ US) to ask labs how they specifically investigate Transfusion reactions and try to standardise these practices.

I think possibly that there are more cases like these described that are not noticed because they are never investigated to these levels. It might be good to do a survey (UK/ US) to ask labs how they specifically investigate Transfusion reactions and try to standardise these practices.

Nice idea, but exceedingly difficult to do. There are so many other causes of these kinds of reactions (e.g. hyperhaemolysis in patients other than sicklers) that it is usually impossible to identify such cases other than be trial and error (and the trail and error could be a really big error for the patient).

:confused::confused:

I would like to add something here...it might not belong in this thread.. I have been witness to an Anti-E being prewarmed away. Yes, apparently 2 TECHS prior to my working on the specimen thought the reactions they were seeing was just "junk". So they both did a prewarm and the reaction was gone. THEY MISSED AN ANTI-E.!!! Bad Bad Bad!

I would like to add something here...it might not belong in this thread.. I have been witness to an Anti-E being prewarmed away. Yes, apparently 2 TECHS prior to my working on the specimen thought the reactions they were seeing was just "junk". So they both did a prewarm and the reaction was gone. THEY MISSED AN ANTI-E.!!! Bad Bad Bad!

Ah, but was it truely an immune anti-E, or a "naturally occurring" anti-E, which are almost never clinically significant, and are almost alwasys mimicking anti-E?

:confused::confused:

Ah, but was it truely an immune anti-E, or a "naturally occurring" anti-E, which are almost never clinically significant, and are almost alwasys mimicking anti-E?

:confused::confused:

I've no clue for certain. What I do know is I pulled the segments that were previously transfused to this patient and tested them for E. 2 out of 6 were positive! We then sent the patient specimen to our local reference lab for a cell separation and antigen typing to be done. The patient was indeed E antigen negative! I don't think any further work was done. We transfused E negative units to this patient from then on. The need for transfusion seemed to decrease after we switched to E negative units.

Honestly, this occurred so many years ago, who knows if I have remembered my facts right! My memory is failing me quickly! I think it is called "Old Age"!:mad:

Hi rravkin@aol.com,

No, the inconsistency of the antibody is not likely to be due to the apparent newness of the antibody; it is much more likely to be due to the specificity of the antibody and the nature of the antigen.

Although fully recognised as a Blood Group System by the ISBT, both Ch and Rg are antigens that are adsorbed onto the surface of the red cell membrane from the plasma, rather than being an integral part of the red cell membrane.

The Ch and Rg antigens are, in fact, C4B and C4A of the complement system, so that, if you are Ch- (as all good people should be!!!!!!!!!!!!!!), you lack C4A, and if you are Rg-, you lack C4B.

The strength of the Ch and Rg antigens can vary greatly from one individual to another (hence the apparent difference in reaction strengths with different red cells and the same plasma), but, in addition, Ch- and Rg- are not all true Ch- or Rg-! This seems a weird thing to say, but there are 6 different Ch antigens and 2 different Rg antigens (together with a WH antigen that also belongs to the system).

Therefore, the cell that only reacted weakly could be negative for one or more of the Ch antigens, but positive for the others, or negative for one of the two Rg antigens.

The negative results with ficin-treated red cells does rule out the presence of an anti-e (although there are extremely rare examples of some Rh antibodies in the literature that only react by IAT, and not with enzyme-treated red cells - I published a poster myself [with others] a few years ago at a BBTS Meeting concerning an anti-E that reacted in just this way).

I hope all that helps.

Malcolm

:):)

Hi Malcolm! Hope you are doing well!

I have a question about Ch/Rg reactivity in LISS, PEG and gel. Joann Moulds, in a presentation called "Don't Call them HTLA's!" mentions that a characteristic of Ch/Rg is reactivity in gel and non-reactivity in LISS and PEG. I can't find a scientific explanation--is it because the Ch/Rg antigens are adsorbed onto RBCs and in LISS and PEG they disassociate?

Thanks!

Catherine

Hi Malcolm! Hope you are doing well!

I have a question about Ch/Rg reactivity in LISS, PEG and gel. Joann Moulds, in a presentation called "Don't Call them HTLA's!" mentions that a characteristic of Ch/Rg is reactivity in gel and non-reactivity in LISS and PEG. I can't find a scientific explanation--is it because the Ch/Rg antigens are adsorbed onto RBCs and in LISS and PEG they disassociate?

Thanks!

Catherine

Well Catherine, Ch and Rg are indeed adsorbed onto the red cell surface from the plasma/serum, as they are a series of antigens that are soluble in plasma/serum and are not intrinsic to the red cell membrane (for a proper explanation, see Geoff Daniels' excellent book "Human Blood Groups"e many papers on the subject by my old boss, Carolyn Giles).

They do tend to react more strongly in gel than in tube technique (I have NEVER actually performed PEG technique, believe it or not), but I have never come across an anti-Ch/Rg that has reacted by gel that has not reacted by LISS tube technique. What I have come across, on VERY rare occasions, however, are examples of anti-Ch that have reacted by enzyme technique as well as IAT (contrary to what you will see in text books, but confirmed by the International Blood Group Reference Laboratory).

I believe Joanne Moulds is saying "don't call them HTLA's" for the simple reason that not all of the antibodies are high titre, low avidity - some of them are surprisingly low titre and some of them are surprisingly high avidity!

It is true that it is sometimes difficult to group an individual as Ch+ or Rg+, but this is probably because the antigen is adsorbed from the plasma/serum in the first place. That having been said, if you treat the red cells with sucrose, they will react much stronger with the antibodies.

Going back to how they react in gel, compared to how they react in LISS and PEG, there is the probability that there is something to do with the shear forces that are there on washing the red cells before the addition of the AHG, which, of course, are not there for gel techniques (see an old paper by Douggie Voak [can't remember the full citation off the top of my head], and the fact that the washing of the tests may well dissociate the antibodies from the antigens when using these technologies.

I will, however, look for references over the next couple of days to see if I can find any, and get back to you.

Meanwhile, I hope that helps a little bit.

Best wishes,

Malcolm

:whew::whew:

Edited June 4, 201213 yr by Malcolm Needs