MinerJ

Hi Malcom,
As always, thank you kindly for your detailed explanation. I feel like I am over-reliant on electronic issue, that sometimes I forget that there are antibodies (which have the potential to be clinically significant) that is missed by the screening cells. 
I will have a read of the article you have referred to, for an extra boost.
Cheers!

No, there is a lot more to it than that.

Anti-A and anti-B are isoantibodies, rather than alloantibodies.  In other words, they are "naturally occurring" and do not have to be stimulated by either red cell transfusion or pregnancy.  They are usually stimulated by particles in the air (including human cells that have been shed into the air) that either express chemical compounds that mimic the A and/or B antigens or, in the case of shed human cells, actually do express these antigens (remember, the A, B and H antigens are histoantigens).
On the other hand, genuine alloantibodies (for example, let's say an anti-Jka) that are stimulated by transfusions and/or pregnancy, have, by definition, shown the individual to be a "responder".  It is by no means unusual for an individual who has produced a genuine alloantibody (such as the anti-Jka mentioned above) to produce an alloantibody of another specificity (or alloantibodies of other specificities).  Such other alloantibodies may not be easily detectable by routine serological techniques for various reasons.  Three of these are that the antibodies may not become serologically detectable at the same time (one may be detectable as early as the other, as not all antibodies "read the books"), that an antibody may be evanescent (or "disappears" from the circulation quite quickly - such as many Kidd antibodies - but these can remain clinically significant if re-stimulated), and thirdly, that the cognate antigen is not expressed on either the screening red cells or the red cells used in the antibody identification panel (for example, in the UK, to give two examples, the Jsa antigen and the Wra antigen, and both of these antibodies can be exceedingly clinically significant).

For this reason, it is very important that a serological cross-match is preformed (and found to be compatible), even if the blood provided is antigen negative for a known cognate antibody.

You may well ask, "Well, what about an anti-Wra (for example) that is present as a monospecific antibody?  Is that not clinically significant?", and the answer is "Yes"!  Indeed, there was a fatal case of an acute transfusion reaction caused by anti-Wra within the last decade in the UK, and the court decided that it was death by misadventure, because anti-Wra is known to be quite a common antibody, whereas the cognate antigen is sufficiently rare for the Law to recognise that it does not need to be expressed on screening cells (otherwise the Reference Laboratories would be overwhelmed with samples that have anti-Wra in their plasma/serum - and this is only one such specificity).  This may be one of the few times that our judiciary have used their brains (did I say that??????????!!!!!!!!!!!!!!!!!) and the decision may have been influenced by an editorial in Transfusion, written by the late, great Professor George Garratty (Garratty G.  How concerned should we be about missing antibodies to low incidence antigens?  Transfusion 2003; 43(7): 844-847.  DOI: 10.1046/j.1537-2995.2003.00492.x.).
SORRY THIS IS A BIT (VERY) LENGTHY!

The mechanisms of what have been termed TRALI (actually a subset of acute lung injury/acute respiratory distress syndrome) and TACO (actually something very common, congestive heart failure) have been widely misunderstood due to unjustified assumptions/dogma. There are many biologic mediators other than antibodies that can cause lung injury after venous infusion which directly subjects the lung vascular endothelium to these mediators (antibodies, activated cells, lipids, mediators such as sCD40L, DNA/histones).  Likewise there are many mediators that can cause or exacerbate cardiac failure after venous infusion (inflammatory mediators, excess volume).  Cardiac failure is not just volume overload, but can be caused by fever, inflammatory cytokines and vascular/myocardial muscle dysfunction.  The notion that these are distinct entities is also at variance with clinical experience.  Many patients have signs of both cardiac failure and pulmonary failure simultaneously.  So the definitions and pathophysiology used in reviews and texts are lacking in validity and just plain oversimplified and wrong, in my view.  There are compelling data to support these iconoclastic contentions for TRALI, and some for TACO.
Most germane (see attachment), when we introduced universal leukoreduction, we saw a sustained 83% drop in reports of TRALI and 50% in TACO over the following years.  This suggests that white cells/DNA/histones play a role in causing lung and heart inflammation and dysfunction.  This clinical observation was confirmed in animal studies from Denisa Wagner's lab at Harvard demonstrating that neutrophil extracellular traps (NETS) infused intravenously can cause acute lung injury (see attachment).  To me these observations are convincing evidence that leukoreduction alters cardiorespiratory injury and failure post-transfusion and represents one of the strongest arguments for universal leukoreduction.  Needless to say, this challenge to dogma has been ignored by the transfusion medicine community which continues, at least in the USA, to infuse deadly white cells and their degradation products (free DNA/histones) to patients, one of the great tragedies of the last 20 years in the USA blood bank field.  We got this entirely wrong and tens of thousands of patients have probably died unnecessarily due to complications of non-leukoreduced transfusions.
ULR TRALI TACO PMC version.pdf NETS and TRALI Wagner 2012.pdf

Hey Malcom,
Yes, you are absolutely right, meant to talk about the pH and not enzyme.
And thank you again, that does clarify a lot of my queries

Hi MinerJ,
We used to do something similar to you when I was in RCI at Tooting, except that we performed tube IAT strictly at 37oC, with warmed NISS for the washing process.  As far as this is concerned, I could never see any difference between having the red cells initially suspended in NISS or LISS, except, of course, that the incubation time was shorter with LISS (although we used to bring the reactants to 37oC before the initial mixing).  Again, like you, this was mainly for pregnant women.
After many experiments trying to get the CAT reaction chamber strictly up to 37oC BEFORE the addition of the reactants, and failing miserably, we abandoned the idea, as we could never quite get to 37oC.  Unfortunately, "cold reacting" antibodies can (and do) sensitise their cognate antigens extremely quickly, but are much slower to dissociate, so we were never quite sure whether the anti-M was reacting genuinely at 37oC or not.
I think you may have made a typo.  There are no enzymes involved, because of course, the M antigen is papain sensitive, by the columns have a slightly low pH, and this, together with centrifugation and the difficulty in keeping the reaction chambers at strictly 37oC all mitigate towards the possibility of a false positive at 37oC.
I hope this bit of rambling answers your question sufficiently, but, if not, do not hesitate to get back to me (or anyone else, come to that!).
Malcolm

Column Agglutination Technology (CAT) is superb at detecting IgG antibodies, and also at detecting certain "cold-reacting" antibodies (such as anti-I and (because of the pH of the column) anti-M, BUT the manufacturers themselves state quite freely, that they are not designed to detect all ABO mis-matches, because most ABO antibodies are IgM (anti-M are usually a mixture of IgM and IgG, but also react preferentially at a low pH).

The first thing to say is that the laboratory personnel cannot diagnose a transfusion reaction.  This may be a delayed haemolytic transfusion reaction, where the patient is clinically compromised, or it may be a delayed serological transfusion reaction, where the sample from the patient tests for a positive DAT and a "new" antibody specificity, that can be eluted from the red cells, but where the patient is not clinically compromised.  This can only be diagnosed by the physician looking after the patient.
Secondly, the anti-Jka may be a de novo specificity, or may be present in the circulation as a result of an anamnestic reaction.  Certainly, two weeks seems a bit quick for a de novo specificity to be detected, but it can happen (never say never in blood transfusion!), so it is more likely to be present as a result of an anamnestic reaction, although there must be a certain proportion of IgM immunoglobulin, as well as IgG.
As yan xia says, Kidd antibodies can cause complement fixation, but can only so do if there is some element of IgM present (anti-Jka that is pure IgG cannot fix complement), however, it is incredibly rare for Rh antibodies to fix complement (as far as I know, there are only two examples of anti-D described in the literature that have been able to fix complement - and just think how many millions of anti-D have been detected), so the complement on your patient's red cells is much more likely to be there as a result of the anti-Jka, than the anti-E.
Adding fresh serum does increase the sensitivity of the test (the so-called "two-stage IAT"), but treating the red cells with a protolytic enzyme, such as papain, and then performing the IAT is even more sensitive.  An eluate can be used to "concentrate" the antibody sensitising the patient's red cells, but, be careful, as, is you are using a commercial elution kit, this may go counter to the kit instructions.

Thanks everyone 
Clinician hasn't suspected transfusions reaction and hasn't asked for any investigations. This is something we noticed the results when they requested further blood unit.   just wanted to know causes of DAT to be positive in C3d therefore shared on discussions board.
 

There are one or two comments I would make, which you may find pedantic, but, that notwithstanding, they are true.  There is, and never has been, a blood group system named Rhesus.  Rhesus was an ancient king of Thrace.  The correct name for the blood group system is Rh.  So, it is the Rh Blood Group System, the two genes involved are RHD and RHCE, the carrier proteins are RhD and RHCcEe, but the antigen of which you are talking is just plain D, and not Rh D.
There is a very good reason why the instructions that come with the anti-D state that the tests should be read macroscopically.  Thorpe et al, in two papers, 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 papain-treated D- red cells are tested with such antisera, or untreated D- red cells are tested with such antisera that have not been brought to room temperature, they may agglutinate.  This could result in D- red cells being mistyped as D+ - a particular danger in females of child-bearing potential, and babies (Thorpe SJ, Boult CE, Stevenson FK, Scott ML, Sutherland J, Spellerberg 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, and 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).
In addition, if you do not follow the manufacturer's instruction, and something goes wrong, under UK (and EU) Law, you could well be liable, as described by Bob Doughty some 30 years ago now (Doughty RW.  Product liability in the medical laboratory.  Medical Laboratory Sciences 1989; 46: 68-71.
In the case that you describe, do you know the Weak D type of the baby (it may well be Weak D Type 2, which can be particularly weak) and, at any time, did you test the mother's red cells to see if she was also a Weak D of the same type?  If she was, then it is highly likely that anti-D immunoglobulin would not have been required anyway, although I am aware that this is not part of the BSH Guideline (White J, Qureshi H, Massey E, Needs M, Byrne G, Daniels G, Allard S and British Committee for Standards in Haematology.  Guidelines for blood grouping and red cell antibody testing in pregnancy.  Transfusion Medicine 2016; 26: 246-263.  doi: 10.1111/tme.12299).
My honest advice is that you do not read the tests under a microscope.  If you have ANY doubt, give the mother anti-D immunoglobulin anyway - it is not that expensive, and has a good safety record in terms of TTI.
.

I am going to be REALLY unpopular here, but I'm going to say it anyway (because I am a pedant)!!!!!!!!!!!
 
Antigens CANNOT be either heterozygous or homozygous; only genes can be heterozygous or homozygous.
 
An antigen can be described as either showing homozygous expression, or heterozygous expression.
 
That having been said, is a red cell sample that types as K+k- phenotypically, genotypically K/K or K/Ko, or even K/k, with a mutation within the Kell gene that prevents the k antigen being expressed and detected with all anti-k grouping reagents (just in case anyone doesn't believe me - we had one!).
 
That's got that off my chest.
 
Now then, there is NO doubt that there are some anti-K's around that only react with K+k- red cells (dosage), but they are fairly rare, however, how many people use antibody screening red cells that are K+k-?  I doubt if there are any.  Therefore, we are all ruling out anti-K using red cells with apparent K antigen heterozygous expression on every single sample that (apparently) has no atypical alloantibodies present.  Am I wrong about this?
 
It follows, therefore, that, over the years, there MUST have been occasions when a patient with a very weak anti-K (one that is only detected using red cells that are apparently showing homozygous expression) and who has been transfused with K+ blood (do the maths).  As far as I know, there are no papers within the literature that report a case of either a delayed or an acute transfusion reaction as a result of this.  Yes, this may cause the anti-K to become stronger (and, hence, be detectable using an apparent heterozygous red cell sample showing K+k+ expression), but then, if this happens, you give K- blood.
 
So, my considered answer is that you can exclude using K+k+ red cells.
 
I shall now go and lie down!!!!!!!!!!!!!

Does your laboratory staff have the necessary procedures to follow for this scenario?   I think flow charts work very well for things like this.  

I prefer the second scenario.  I have seen this quite often.

Have this patient been transfused ? If it had, then maybe the allogentibodies coated with transfused cells, then caused the pos DAT, which would show mixed field .
 Or the autoantidies are weak, they can only coated with red cells, no free autoantibodies showed on the circulation, then they will not interfere with the crossmatch and screen.

Hi All,
I have a question, but firstly good old story time for some context. I came across a patient who had positive antibody screen on all three screening cells used (BioRad). I was concerned this may be an auto and pan-reactive, and required units. Performed a monospecific DAT, showing a positive reaction to IgG only. By this time antibody panel finished cooking and showed the patient may have anti-Fya , but couldn't do phenotype. By this time I was nearing my shift so handed it over to my colleague and asked for some units to be crossmatched. However, he refused as DAT was positive and said he rather send the sample to reference laboratory for them to crossmatch. The next day I crossmatched units to verify if it could have been done in our laboratory (just because I am sad that way), and turn out the unit I crossmatched was compatible (which I wasn't surprised about)
Question
Why does positive DAT (or the cause of positive DAT) sometimes interfere with IAT techniques (such as antibody panel and crossmatch) and sometimes it does not? If both use AHG, then wouldn't positive DAT with IgG cause antibody panels shows pan-reactive with red cells? But obviously it doesn't, but I'm trying to figure out why, and I'm sure the answer is quite obvious. 
My laboratory seems very hesitant whenever they see anything regarding autoantibodies or positive DAT, and thinks that sample cannot be crossmatched in-house and needs to be sent off without even trying to investigate. Hopefully, by me asking this question, I can explain it back to my colleagues (but obviously take all the credit).
Cheers in advance,
Jermin
 

Hi Malcolm,
I agree with what you have to say. I have no problem relying on FDNA result, as accuracy is pretty damn high. The people who we are trying to convince/explain are the Midwives who don't understand why we are double checking something if you are meant to rely on it the initial result- and we kinda understand what they have to say. I will read the latest papers as you mentioned. I'm sure it would be helpful, 
Thanks,
Jermin

The results from both the study in the Netherlands and the UK have shown that the accuracy of the testing of cffDNA is extremely accurate (no test is ever 100% accurate).  In the case of the UK, the testing has been thoroughly studied by NICE (a tough lot of people to convince, believe me!), and if they recommend it, then I think you would be justified in getting fully behind it.  Yes, there will be the odd discrepancy, but these have been demonstrated to be very few and far between (and have also been explained).  I really would suggest you read some of the latest papers by Ellen van der Schoot and/or Kirstin Finning if you need convincing, and even, perhaps, the NICE Guidelines (but be aware that the nomenclature in the NICE Guidelines is appalling!).

Hi Jermin
We took up FDNA testing just over a year ago but still test all cord samples from D negative mothers to confirm the accuracy of the FDNA results.  We perform a retrospective comparison of results on a weekly basis and have found a number of discrepancies during that time.  All of these discrepancies have been carefully investigated by us and IBGRL, where appropriate.  Two of them were false positive FDNA results whereby the phenotype of the neonate didn't match the genotype...this scenario (D negative baby following a D positive genotype test result) will occur, albeit rarely, owing to the presence of a non-expressed RHD gene.  We have also had two false negative FDNA results due to insufficient foetal DNA in the maternal blood sample.  By testing the cord the risk to the Mum was minimized as she was able to receive post-natal anti-D immunoglobulin.  Rather more worrying, two discrepancies have highlighted poor practice on labour ward - a cord that was sampled from the sluice (yes, really) that IBGRL was able to show couldn't have belonged to the mother in question and twins who were initially both grouped as D negative but patient recall showed one of the them to be D positive!  By testing the cord and feeding-back any discrepancies to IBGRL it also helps to inform the accuracy of the test.
Although we group the cord samples, we stopped performing DATs on these samples many years ago and only perform a DAT in the presence of maternal antibodies or for clinical investigation of jaundice.
 
Hope this helps
 
Letty
 

I would TOTALLY agree with Letty's post.  Indeed, the reports from the IBGRL ALWAYS state that there may be discrepancies from time to time - not least because there may be insufficient cffDNA harvested from the maternal circulation, and because of mutant RHD genes.  They are few and far between, but if you read any paper by Kirstin Finning, who runs that laboratory, she never quotes 100% accuracy.
Most cases of ABO HDFN are sub-clinical (just need a bit of phototherapy) and, quite often, the DAT is negative at birth anyway, and "develops" a couple of days into life.  If the ABO HDFN is clinically significant, the doctors, nurses and midwives should easily be able to see the symptoms and then order a DAT.
In terms of tests on a positive DAT, we would only perform elutions if the baby is affected or we know that the mother has an alloantibody.

Cheers. I have the  Introduction to Transfusion Science Practice.  Robina Qureshi, and I will seek others when I get the chance.

That is no problem whatsoever Jermin.
In no way am I saying that there is anything wrong with Rodak's Haematology; very many people have used this excellent book and passed countless exams, but that does not mean that it is for everyone.  I would suggest, for example, that such "greats" as Geoff Daniels' book Human Blood Groups and "Mollison" are not necessarily everybody's "cup of tea".  In addition, I would suggest that, brilliant as it is, The Blood Group Antigen FactsBook" is not necessarily what you might need.
As a Band 5, may I suggest both Human erythrocyte antigens or blood groups” in Fundamentals of Biomedical Science, Transfusion and Transplantation Science, edited by Robin Knight for the IBMS.  1st Edition, Oxford University Press 2013 (ISBN 978-0-19-953328-2 (although the second edition is due out in [about] May of this year, and Introduction to Transfusion Science Practice.  Robina Qureshi.  6th Edition.  2015, British Blood Transfusion Society and, actually, come to think of it, Essential Guide to Blood Groups.  Geoff Daniels and Imelda Bromilow.  3rd Edition.  2014, Wiley-Blackwell, any oNOTr all of which may be of use to you, and all three of them, give or take the odd chapter, are a very easy read.
The other thing you must do, of course, is go to the BSH website and have a quick scan (NOT a thorough read) of their Guidelines.
Good luck, and do not despair - you WILL finish your Specialist, and you will pass easily, because you have bothered to seek advice; that shows application!  Well done and keep it up.

Which book are you using, and at what "stage" are you in your professional life?  The reason I ask is that I may be able to suggest an alternative (but do not want to advise you read the same one! - and I stress an alternative book, rather than a better book; each to their own), and I need to know where you are in your professional life, so that I do not suggest a book that is either too basic, or too advanced.

We preform our own acid elutions in-house, but this flowsheet may still be of use on whether or not to sendout for an elution.  I've attached our flowsheet here:
Stephanie
Elution Flowsheet COMPAT SER FS4.doc

In my opinion, this very much depends upon the underlying pathology.  For example, if the patient has an auto-immune haemolytic anaemia, the chances are very strong that the DAT will be positive before as well as after the transfusion, and that any eluate will be positive with all red cells tested (of normal type).  The chances of detecting a new antibody specificity on the DAT positive red cells under these circumstances is disappearingly small.
Therefore, if the sample is sent to a reference laboratory on a regular basis, your manager will be 1) showing a degree of ignorance that should be surprising, 2) will be upsetting the staff of the reference laboratory, as most have enough to do, without having to perform extra, unnecessary work, and 3) as you are in the UK, will be wasting the tax payer's money (and, as a UK tax payer, I feel very strongly about this).
If, on the other hand, the positive DAT is new, then a reference laboratory would be delighted to help out.
Of course, what your manager could do is to buy his/her own laboratory an elution kit, and train his/her staff to use it!!!!!!!!!!!!!!!!  This may bring the cost of the kits to meaningful results ratio to the overall pathology manager, which could be of interest!

Hi Jermin
I think there are a couple of points here that seem to me to be a bit confused.
1.  'The DiaMed reagents are, but not the NBS reagents. Thanks for that question, as it slipped my mind. '  You won't be using NBS reagents to do your Rh phenotype on the IH1000.  All you will be using are the cards, which are what you are using now, and diluent 2, which you are using now.
2.  I was planning on getting the reagent cells, centrifuge them to separate from the preservative it is in, then resuspend it in PBS.  No, Jermin you really cannot do this.  It is not the preservative that is the issue, but the concentration.  The samples you put on the analyser need to be packed cells - and you need enough packed cells for the instrument's needle to be able to aspirate the correct amount.  If you spin down reagents, even if they are at 3-5%, you will not have enough cells in the pellet; even less so if you them dilute them with PBS - which you should never use with the cards anyway as it can cause trailing.
3.  I wish I knew how I could do that.   I will need to probably check with the manufacturer's reagent sheet and see what sort of limitations and discrepancies there might be, otherwise it might be beyond me.   You are currently using the same cards and the same Diluent to carry out your manual testing as you will be using on the Instrument.  Any discrepancies you see will come about because of manipulation error only.
 
Can I suggest that you contact the local Biorad rep who installed the instruments and ask them to help you with this?

Yes I agree Malcolm.  I was thinking more of some of those reactions you get that are a bit 'iffy' in a panel in gel - which is extremely difficult to do at strictly 37 - and you just need an aid in identification