aafrin

If this is such a rare happening my suggestion is, if it ever does happen, simply do the best you can with what you have.  If you come up with some procedure then you will have to review and train with it on a regular basis and I would suggest that once per year is not enough.  Bottom line, you cannot prepare for every eventuality your fertile mind can come up with. The key in the rare circumstances is to not panic or get bullied into doing something you know is not correct.  You can drive yourself crazy with all the "what ifs".  I am all for preparation but it must be reasonable and realistic.  

Hi Rich,
I am not a clinician but as far as I know IVIG can be given to obstetrical patient in diff. conditions (autoimmune disorders, recurrent pregnancy loss, ...).
I thought about IVIG when I saw the DAT becoming positive plus additional reactions coming up over the time. Anti-A and Anti-B are indeed the most prevalent antibodies in plasma derived products but other specificities of low titre can be present sometimes such as anti-D, anti-K and a bunch of antibodies of undetermined specificity reacting with several to not say all RBCs. 
Just a thought that can be doublechecked with the clinician..?
Hereunder is a very great (not recent though) paper to be read and re-read again:
Problems Associated With Passively Transfused Blood Group Alloantibodies 
George Garratty, PhD, FRCPath American Journal of Clinical Pathology, Volume 109, Issue 6, 1 June 1998, Pages 769–777, https://doi.org/10.1093/ajcp/109.6.769

Old school thought was that you had to wait for 24 hours. I think that one to two hours is common practice.
I also remembered reading something in a CAP publication that discussed this and actually managed to find that in one of the threads here. There are references associated. Quoted below....................
Q. How long should you wait after a unit of blood has been transfused before drawing a complete blood count, or doing other lab work, to ensure accurate test results?
A. Optimum timing of post-transfusion phlebotomy is critical for ensuring meaningful laboratory testing results, and medical judgment is required in making this determination. Several factors must be considered, including the type and amount of blood product given, purpose of the test (that is, the question it is intended to answer), and clinical setting.
In general, it is best to perform phlebotomy when the patient’s circulatory system is in homeostasis. A patient who is bleeding or undergoing blood product transfusion, or both, is not in a steady state. Whenever possible, samples for laboratory testing should be postponed until bleeding has stopped and transfusion is complete. One obvious exception to this rule, however, would be the setting of massive transfusion, during which monitoring certain laboratory values, such as cell counts and coagulation parameters, is essential to guide ongoing therapy. Variables such as patient blood volume, cardiac output, renal function, and volume of blood products transfused affect how quickly homeostasis is achieved following transfusion.
For the evaluation of post-transfusion increments in hemoglobin, hematocrit, and platelet counts, a practical approach is to draw blood samples within 10 to 60 minutes after completing transfusion, as this time interval is aimed at measuring peak recovery.1 Results determined from blood samples drawn later than 60 minutes post-transfusion are increasingly affected by confounding conditions, such as splenic sequestration, sepsis, and consumption.1,2 If the intent is to determine the extent of such confounding processes on red cell and platelet counts, one should combine a 10-minute post-transfusion sample with sequential samples drawn at one hour and 24 hours post-transfusion.
Alterations in chemistry test results following transfusion are not usually a concern in the low-volume transfusion setting. However, assay results may be affected for varying periods following transfusion of large amounts of blood products, as seen in massive transfusion, red cell, or plasma exchange—particularly if the recipient has impaired hepatic or renal function. Banked storage of red cells results in elevated plasma levels of hemoglobin, potassium, LDH, and iron in the blood unit that may, particularly in the metabolically impaired patient, be reflected in the post-transfusion laboratory values. In addition, citrate anticoagulant present in blood products may result in transient hypocalcemia in the recipient.3 Therefore, following large-volume transfusions or exchanges, waiting 12 to 24 hours before drawing samples for chemistry assays will provide results that are more reflective of the patient’s underlying metabolic state.
References
Choo Y. The HLA system in transfusion medicine. In: McCullough J, ed. Transfusion Medicine. New York, NY: *McGraw–Hill Book Co;1998:401. Legler TJ, Fischer I, Dittman J, et al. Frequency and causes of refractoriness in multiply transfused patients. Ann Hematol. 1997;74:185–189. Brecher ME, ed. Technical Manual. 15th ed. Bethesda, Md.:AABB;2005;649–650.  
Rita A. Reik, MD
Pathology Consultants of South Broward
Medical Director/Transfusion Medicine Services
Memorial Healthcare System
Hollywood, Fla
 
Edited November 30, 2012 by Justina
Added CAP website find
Justina
Posted December 7, 2012 Articles:
Elizalde JI, et al.Early changes in hemoglobin and hematocrit levels after packed red cell transfusion in patients with acute anemia. Transfusion. 1997 Jun;37(6):573-6
Wiesen AR, et al. Equilibration of Hemoglobin Concentration after Transfusion in Medical Inpatients Not Actively Bleeding. Ann Intern Med. 1994;121:278-280
   
   

Our policy is to issue O Group PRCs and group AB FFPs to a neonate (<4 months age). The Rh of PRCs will depend on the Rh status of the neonate. Top-up PRC transfusions are usually from one single unit <28 days.

Definitely an A subgroup.   But clearly with an anti-A1 so best to transfuse group O.  Tube is actually MORE sensitive than gel for ABO

They are from different clones and titer.
We repeated the blood group using BioRad Newborn Card and it showed 2+ with anti-A,B but negative with anti-A.

When I was working in RCI at NHSBT-Tooting Centre, we used to store our liquid reagent cells in Cellstab, but wash them an resuspend them in Dil2 for use.  We found the reactions we got were much, much sharper (I don't mean that the reactions were more sensitive to detecting weak antibodies, although we believed that they were, but that it was far easier to "see" the reactions as clear reactions, rather than "fuzzy" reactions), and, in addition, it meant that we didn't detect reactions caused by antibodies directed against the preservatives in the Cellstab.  We were able to show this with multiple photographs.
Despite all the evidence, we were told that we couled not continue to do this, as we were not standardised with the other NHSBT RCI Laboratories (standardisation is everything these days, even if it means dumbing down, rather than bringing everyone up to an excellent standard, and because it was more expensive.  The only problem was that we were able to show that it was actually LESS expensive, because it meant less testing, and no testing for antibodies against preservatives.
This did not fit with management theory, however, and so we had to stop.  Since then we got "fuzzy" reactions, leading to many cases of repeat testing, and many cases of antibodies against preservatives and, hence, more expensive testing in terms of reagents, staff time and fairly simple investigations into moderate or even complex investigations, but hey, what did we know!  At least we are now standardised (and expensive)!

Well, one reason why anti-c is detected more often in the reverse group than is anti-e is simple that anti-c is a much more common antibody specificity than is anti-e.
There are two possible reasons for this.  Firstly, apart from the rarer c Negative Rh types, the most common c Negative Rh type is R1R1 (all the best people are R1R1!!!!!), and the R1R1 is not that rare, but, except in certain circumstances, it would be unusual to deliberately give an R1R1 individual R1R1 blood, unless they have already produced an anti-c.  This means that such individuals are often given blood that is c Positive (R1r, R2r, R1R2 and rr) and so their immune system is challenged.
Secondly, apart from the rarer e Negative Rh types, the most common e Negative Rh type is R2R2, and the R2R2 is comparatively rare, and, except in certain circumstances, it would be unusual to deliberately give an R2R2 individual R2R2 blood, unless they have already produced an anti-e, or anti-C.  This means that such individuals are often given blood that is e Positive (R1r, R2r, R1R1, R1R2 and rr) and so their immune system is challenged.
That having been said, anti-c is, as I said above, much more common than is anti-e, and so this also suggests that the c antigen is much more immunogenic than is the e antigen, and all of this serves to show why anti-c is detected with the reverse grouping cells, more often than is anti-e.  This is the kind of thing about which Geoff was writing.
As for your other question about E Positive units being given to patients with anti-E, you may be talking about patients with an auto-antibody that mimics anti-E, rather than someone with a genuine alloanti-E, but I would need a few more details to be certain.

Very true.

Reminds me of a donor we had once when I was a reference lab sup.  He had donated 12 times as O NEG.  The next time he donated, they picked up weak typing with Anti-A,B and with further testing, turns out he was a very weak subgroup of A!  Unbelievable.
I agree with A subgroup.  I see a lot of people want to automatically classify the subgroup....but without further testing, that is actually erroneous.  Best to just leave it at subgroup.
Brenda Hutson, MT(ASCP)SBB

Yes, but c.261delG characterizes deleterious O alleles and is not present in alleles A. If this mutation is homozygous, I would suspect a cross-reactivity.

There are MANY more mutation than this leading to O alleles.

I also agree with the subgroup of A. However, I would do other tests before considering the case as resolved.
1. Are the reagents you used from different manufacturers from the same clone? Do they have the same title? Differences in clone or titer may lead to differences in reactivity.
2. Tn antigens may be cross-reactive with some anti-A antibodies. Treatment of RBCs with the enzyme may help to exclude this interferant.
3. I would or would refer you to the molecular investigation to identify the deletion mutation at position 261 of exon 6, which characterizes O alleles.
4. Flow cytometry can also assist in the evaluation of the mixed field. Is it happening because of poor reactivity or chimerism?

I prefer to call it an A subgroup. Maybe some human anti-A can do help, since it is polyclonal, not monoclone as our reagent anti-A.

Irradiation does not "kill" T lymphocytes per se, which are the cells that cause TA-GvHD, but what it does is disrupt the DNA within the nucleus, and this disruption prevents them from cloning.  As a result, they are unable to "reproduce" (for want of a better way of putting it) and so, instead of being able to form a clone within the recipient, will be removed from the circulation by natural apoptosis.  Prior to this apoptosis, once they have been irradiated, the T lymphocytes are relatively benign.

Even docs don't understand BB. We had a pediatric registrar who was insisting on issue of O Rh negative FFP to a 3 yr old baby with B Positive blood group who had suddenly started bleeding at night and we didn't have any B group FFP in stock. He was refusing to accept AB group FFP which the tech had thawed. I had to step in & talk to consultant at 2 am & convince him for them to accept AB Group FFP.

Monoclonal anti-D is manufactured anti-D (particularly if it contains IgG and IgM, as it would be a blend).
 
Yes, all of the tests were performed at 4oC - but that is the point!  If the reagents were taken straight from the fridge, for an immediate spin test, they would still be close to 4oC when the test was performed.  As I said in an earlier post, if the IgM part of the blend had sensitised the I and/or i antigens on the red cells at this point, and the test was (gently) centrifuged, more and more of the antibody would agglutinate these red cells (which is the whole point of the centrifugation step - to bring the red cells into closer proximity with one another).  What I then went on to say, though, was that I did not, and still do not know, how quickly the antibody (in the shape of the V4-34 moiety) would dissociate from the red cells when the tests are then taken to the Weak D test - but I also mentioned that clinically insignificant anti-M, that does not react strictly at 37oC, can often be detected by IAT at the end of just such a technique, unless the reactants are only introduced to one another at 37oC, and washed with pre-warmed saline.  In the routine Weak D test, I doubt if the saline used for washing the test would be pre-warmed, and yet the centrifugation step would be at a much higher speed than would be used for the initial test.  If the antibody has not fully dissociated, these steps would enhance the reaction caused by the V4-34 moiety, and so the stronger reeaction could, quite possibly, be detected.
 
During the very short time in my professional life, when I worked in a haematology laboratory, rather than a blood transfusion or blood group serology laboratory, we occasionally received an EDTA sample that would not go through the Coulter Counter properly because of cold agglutinins.  We used to put these into a 37oC incubator for a minimum of an hour, to allow the cold agglutinin to dissociate from the red cells, prior to putting the sample through the machine again.  I was told by the Senior Chief Technician that the time of one hour had been chosen because experiments had shown that anything much shorter did not allow enough time for the dissociation of the antibody/antigen complex, and we would get rubbish results a second time.  Even then, on occasions, the removal of the sample from 37oC to ambient temperature was enough for the antibody to go back onto the red cells.  This incubation time of at elast an hour was, I would suggest, much longer than the incubation time used these days for an IAT.

Smiller - I don't think it's fair to say that the original result was a 'false' positive.  Until the actual reason for the discrepancy is known, then both results should simply remain as discrepant.
Another lesson to learn from this case - It is not good practice to give out a blood group on a previously unknown patient  when just one group has been done.  The best scenario is two separate samples.  If that really is not possible, then at least the 1 sample should be tested twice, from 2 separate cell suspensions and preferably with two sets of different reagents (i.e. 2 different cell lines)
Cases like this one really help to understand why blood banking sometimes has such strict rules about things.

Well, as a minimum, test with several different clones of anti-D but preferably test with molecular biology.  But, as I said before, this patient should have been treated as Dneg.  A 1+w is very weak indeed, regardless of what you got in the IAT phase.  For future cases, it might be a good idea to check this type of reaction under the microscope, for a mixed field agglutination - which would indicate that the mother's blood also contained a large amount of foetal cells.
About the point - at which strength do you treat as D+, my answer to that would be, it depends on how you are testing and who you are testing.  You have to bear in mind that a 2+ in tubes and a 2+ in gel do not represent the same 'true' strength, as gel is much more sensitive than tubes. In gel, anything less than a good 3+ should be treated with suspiscion of being a variant - either a weak or a partial.  does it matter?  Well, if it's a donor, they would all be treated as D+, no problem.  A man - also no problem, unless he's going to be regularly transfused for the rest of his life.  For 'old' women (I can say that - I am a dinosaur) - also, no problem.  However, for women with child-bearing potential, you have to be much more careful.  You need at all times to do your very best to prevent her from forming an anti-D which will cause horrible problems in her next pregnancy.  So anything less that a normal straightforward positive (4+ or 5+ depending on your scoring system) should be treated with utmost caution and fully investigating before deciding that it is safe to treat her as a D+.  And while waiting for those results, she should be treated as a Dneg.
It's honest of you to own up to using the reagents straight out of the fridge.  Reagents should never be used cold - and as Malcolm has said, that MIGHT be the reason for this discrepancy. whether it is or is not the reason, I hope there is a lesson here for everybody.  But could I ask again about how far pregnant this lady was?

Well, the reason I asked is because Thorpe et al1,2 have reported that monoclonal anti-D molecules possess a V4-34 moiety that is also present in anti-I and anti-i.  As a result, if these reagents are used straight from the fridge, rather than allowing them to come to room temperature, there is a chance of a false positive, as this moiety will bind to the I and/or i antigens expressed on the red cells, and so D Negative red cells can be mis-grouped as D Positive.
 
I do not know how long it would take for the antibody, under these circumstances, to dissociate from the I and/or i antigens at 37oC, but, certainly, in the case of a cold reacting anti-M (one that does not react strictly at 37oC), it takes longer than the incubation time, as can be seen if the reactants are not allowed to come to 37oC before they are mixed (hence a clinically insignificant anti-M can appear to be active at 37oC).
 
I am not, for one minute, saying that this is the answer to the problem, but I am saying that it could be a contributory factor, particularly if there was a substantial amount of the foetal red cells (which would be, essentially, I-, i+) in the maternal circulation, and, hence, in the sample you tested.
 
1.  Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellberg MB, Natvig JB, Thompson KM.  Cold agglutinin activity is common among human monoclonal IgM Rh system antibodies using the V4-34 heavy chain variable gene segment.  Transfusion 1997; 37: 1111-1116.
 
2.  Thorpe SJ, Ball C, Fox B, Thompson KM, Thorpe R, Bristow A.  Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both activities.  Transfusion 2008; 48: 930-940.

Another possibility - how far pregnant was she when she had the miscarriage?  It might be that there was actually a mixture of D- maternal blood and D+ foetal blood in the sample you had.  Of course, mixed fields are not always easy to see in tubes.  Then you could be back in the discussion we had a few weeks ago on this forum about where you sample from in a sample that has a mixture in it.
Having said that, I would never be happy about calling a young woman with a 1+w reaction in a single anti-D D+ without carrying out further tests (and I don't mean an IAT anti-D test) and if this were not possible I would have treated her as Dneg  (When in doubt.......)

Well, it is never easy to make a hard and fast rule for all cases.  However, thinking back to first principles can help.  Anti-D is to avoid at all costs in women of child-bearing age who can still have children (For example, I mean in a 25 year old woman who is having her uterus removed for cancer is of child bearing age, but can't have children).  You have finite stocks of D neg blood, and your young women should be the priority.  Your second priority should be your patients who are transfusion dependent for life - like sicklers or thalasssaemics. For other chronic transfusions, well, it depends what 'chronic' means, and how much blood you have available and how often the patient needs blood.  I would argue that probably for a 90-year old who is not likely to live more than 6 months who needs 2 units of blood every month, you could probably switch to D+ if you needed to without much of a problem.  I wouldn't do it on a 40 year old who was receiving blood regularly now but with hopes of remission.   Then you have to think that in cases of massive bleeding, the blood doesn't usually stay in the patient long enough for the immune system to 'see' it, so in cases of heavy bleeding it's better to give your D+ first and then switch to D- once the patient is stable.  But that's only my opinion.....

I'll have a go (but don't hold me to this being entirely correct)!
 
A cold auto-antibody is an auto-antibody that reacts against the patient's own red cells, but is not active at 30oC or above.  So, I could have, as a group A1 individual, and probably do have, an auto-anti-H or auto-anti-Hi, but it is clinically benign.  In other words, the cold auto-antibody is present in my plasma, but, unless tests are performed at temperatures below 30oC, I would never know, because the presence of this auto-antibody is not clinically significant.
 
If, on the other hand, I had CAS, my auto-anti-H or auto-anti-HI (in the example I have given) would be reactive at 30oC or greater (30oC is estimated to be the temperature to which the extremities - such as the toes, fingers, ears and nose - amongst other things - will drop in cold weather or other cold conditions), under which conditions the auto-antibody would be clinically significant, as I would start to destroy my own red cells.
 
The higher the thermal amplitude of the antibody, the more clinically significant the antibody becomes (irrespective of the specificity of the antibody, or the titre of the antibody, DESPITE what a lot of doctors would have you believe).
 
In addition, however, it also depends upon the turnover of your complement.  If you consume your complement quicker than you can produce complement, then your red cells will not be destroyed as quickly as you may think, particularly as they will be "covered" in C3dg, which protects the autologous red cells.  BUT, if the Hb drops to a dangerously low level ( and I do mean a DANGEROUSLY low level - not just what is traditionally thought of as a "low" level), then the patient may require a transfusion.  This transfusion may not be as successful as would be assumed, however, as the "virgin" allogeneic red cells will not be protected by the coating of C3dg, and so will be vunerable to complement coating to C3d level and beyond, and so be removed from the circulation quite quickly.  This may result in the requirement for a splenectomy, possibly leading to the chance of Evan's syndrome setting in.
 
I hope that explanation is of some use, but others may have other (valid) explanations, may disagree with what I have written, or may well disagree entirely with what I have written.

I would just query how "clinically significant" are the antibodies you are missing that are being detected by solid phase, but not by other techniques Terri.  As I have said many times on this site, I cannot remember us killing too many patients when we only had tube techniques, before the days of gel and solid phase.  It could be that we are detecting them earlier "in their life", but just how "clinically significant" are these antibodies?

Not to be a whippersnapper, but I have also never seen a case of ABO HDFN in a non-O mom in 24 years.