gagpinks

We had a smilliar scenario and the explaination is transfused cells are dense so upon centrifugation transfused cells settle at the bottom and analyser's probe aspirate  from bottom. And it's differ from  manual technique .

If you want to know more, there are two books I would recommend.
The first is Hadley A, Soothill P.  Alloimmune disorders of pregnancy.  1st edition, 2002, Cambridge University Press.  This is a bit old now, and may be difficult to obtain,, but it is still a good book.
The other is, of course, Klein HG, Anstee DJ.  Mollison's Blood Transfusion in Clinical Medicine.  12th edition. 2014, Wiley Blackwell, Chapter 12.

There have been several different methods used in an effort to "manage" the foetus over the years - most of which were aimed at controlling the maternal antibody levels.
One of these was the use of an Rh hapten and red cell stroma, but this met with very little success.
Promethazine hydrochloride was used, but, again, with very little success.
The use of IVIgG has been tried, and I did see this used with a certain amount of success myself.  I remember that it was a case where the maternal anti-D level was quite high (in the mid 30IUmL-1) fairly early in the pregnancy, and the mother was given IVIgG throughout the pregnancy (I can't remember why they did not give an IUT, but there was a reason) and, at the end of the pregnancy, the maternal anti-D level had fallen to around 15IUmL-1, and the baby was okay.  I believe I am correct in saying that this is a rare and expensive form of treatment (I certainly have only seen it used in one other case in my fairly long career.
Plasma exchange has been used to lower the maternal antibody levels (I remember Dr Cyril Levene using this technique on a pp lady with anti-PP1Pk, who had experienced several early miscarriages, and who produced a healthy baby), but it is very rarely used, as it is expensive, tedious and very uncomfortable for the pregnant lady, and requires replenishment of clotting factors.  It is claimed that the antibody levels can be reduced by 75%, BUT, it needs to be done several times during the pregnancy, and the antibody levels are prone to rebound, as the IgG antibody comes back into the circulation from the interstitial spaces.  It is rarely used.
Therefore, the chances are that, should the foetus require management, an IUT would be performed.

Looks like the patient is a potential Kidney Transplant candidate so we will likely be sending specimens to National ARC Reference Lab for Monoclonal Testing.  Stay tuned.....
Brenda

If the reverse typing use the same A and B cells, then the differ between gel and tube are caused by the methods, otherwise, it maybe caused by low antibodies against B cells in tube method. To verify it, we can change to another B cells to test in tube method.
I tend to agree it looks like an ABsubgroup, on my daily use of gel, I find it is not as sensitive to detect reverse reaction as tube method, I  guess that is the cause of no reverse reaction on gel but has reaction in tube.
 
 

We would not routinely perform eluates if the IgG DAT was negative but if we suspecting a delayed hemolytic transfusion reaction we would definitely consider doing this testing.  I have been able to elute IgG antibodies from DAT negative samples on several occasions.  Initially I thought this phenomenon was unique to the eluting of anti-A or –B from cord samples.  I think we’ve all experienced this, with mom group O and negative antibody screen, infant group A or B and negative DAT.  MD suspects ABO HDN and requests elution.  Generally you will be able to elute the maternal IgG ABO antibody despite the negative DAT.  As a med tech student we were always taught that this related to the way the infant’s ABO antigen developed, less branched structures.  Over the years we have seen that this happens with other antibodies too.  In several cases where mom had a known significant antibody, and infant DAT was negative but infant was positive for the antigen to which the antibody was directed.  This has happened with anti-c, anti-Jka and most recently with anti-Ge3. Most of these eluates reacted 2+ to 3+ despite the negative DAT.  Also think about Delayed Transfusion Reaction Investigations that you may have performed where the DAT may be weakly positive but the antibody in the eluate reacting quite strongly.  Another observation: Just recently I was preparing competency unknown samples for eluate testing by coating cells with various patient antibodies.  Some of the samples had DAT’s that were negative or only weakly positive yet the antibody was recovered in the eluates, again reacting 2+ to 4+.  And this was not even increasing the concentration of red cells as Malcolm suggested.  If you think about it, the process of preparing the eluate really does have a “concentrating” effect.  The eluate is prepared from a large mass of red cells where the packed red cell volume to eluate volume is nearly equal.  That eluate is then tested against a very diluted concentration of reagent red cells, e.g. 3-4%, compared to the very MUCH larger quantity of cells that it came from, thus you are greatly increasing the sensitivity of the test.

NB2...
From the article referenced by Malcolm above:
Two major conclusions can be drawn from our study. The DAT must be interpreted in conjunction with clinical and other laboratory data to avoid erroneous conclusions; it can be expected to be false-negative in up to 3% of AIHA patients. Elution of RBC antibodies is a valid additional procedure to clarify whether autoantibodies are present in DAT-negative patients.
Basically saying that around 3% of negative DAT reports are false negative even though the patient has AIHA.  I wonder how many physicians would consider ordering an eluate after seeing a negative DAT?
Scott

Hi Scott,
I'm back in the land of the living!
I think the key bit of your quote from the Technical Manual is:
"However. an eluate from the patient's red cells coated only with complement should be tested if there is clinical evidence of antibody-mediated hemolysis, for example, after transfusion.  The eluate preparation can concentrate small amounts of IgG that may not be detectable in routine testing of the patient's plasma.".
which, incidentally, is essentially what I said in an earlier post.
The other important phrase from the quote from the Technical Manual is:
"When the only coating protein is complement, the eluate is likely to be nonreactive."
The word "likely" is very important here, as it means that the eluate is not definitely going to be nonreactive.
Particularly in the case of a very weak antibody, such as one that is derived from an anamnestic response, it is not unknown for almost all of the said antibody to be sensitizing the red cells, and for very little, if any, of this antibody to be free in the plasma/serum - certainly not by normal serological techniques.  However, if an eluate is performed, and the eluate is made with less eluting fluid than normal (normally it is a 1:1 ratio with the washed packed red cells, but that ratio can be changed to, for example, two volumes of washed packed red cells to one volume of eluting fluid), then essentially, the concentration of the antibody being eluted from the red cells is doubled.
Such findings were described in Sachs UJH, Roder L, Santoso S, Bein G.  Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases.  British Journal of Haematology 2006; 132: 651-661.
As I have said in other, earlier threads, although this paper was designed to look at DAT Negative cases of AIHA, the authors also actually describe many cases of a delayed haemolytic transfusion reaction (possibly delayed sereological transfusion reactions) detected by eluting antibodies from the red cells of the patient, even though the DAT was negative and there was no free antibody in the plasma/serum that was detected by routine serological techniques.
So, the evidence from this is that the eluate may well be more reactive that the plasma/serum in terms of being able to identify the presence of, and elucidation of the actual antibody specificity.
You go on to say:
"And this from Malcolm: there are times when the causative antibody is an IgM.
But what is the likelihood that you are going to pick up an IgM antibody (significant or otherwise) with the anti-IgG reagents used for antibody detection?  And from an eluate no less, which I believe are notoriously weak anyway even if present?"
What I was driving at here was the fact that, if there is clinical evidence of antibody-mediated haemolysis, particularly after a transfusion, every effort should be made to identify the specificity of the antibody, to ensure that the cognate antigen is not expressed on the red cells of any units that may subsequently be transfused.  This effort may include the use of a clotted sample, rather than a sample anticoagulated with EDTA, as, of course, the EDTA would chelate the calcium, manganese and magnesium ions that are required for maximal initiation of the classical complement cascade.  In such a case, a monospecific anti-C3d reagent, or even a monospecific anti-IgM reagent, rather than a monospecific anti-IgG reagent would be used.  It was in this way that one of the red cell immunohaematology reference laboratories of the NHSBT was able to show the presence of an IgM anti-Vel in the circulation of a patient who had undergone an acute (sadly fatal) haemolytic transfusion reaction, where no IgG anti-Vel could be detected in the plasma.
The bit about the "strength" of the eluate I have dealt with above.
I am acutely aware that the ability for the normal hospital blood transfusion laboratory to be able to carry out such tests is unlikely, as they would not carry the necessary rare and expensive reagents, but in a case where there is clinical evidence of antibody-mediated haemolysis, samples should be sent to a reference laboratory for full investigation before further transfusion is attempted, unless the physician decides that withholding further transfusion is more dangerous than a possible further transfusion reaction.
Sorry for the over-long post.

Technical Manual, 18th edition, Chapter 17, DAT/Immune Hemolysis, page 428.
Test an eluate prepared from the DAT-positive red cells with reagent red cells to determine whether the coating protein has red cell antibody specificity. When the only coating protein is complement, the eluate is likely to be nonreactive. However, an eluate from the patient's red cells coated only with complement should be tested if there is clinical evidence of antibody-mediated hemolysis, for example, after transfusion. The eluate preparation can concentrate small amounts of IgG that may not be detectable in routine testing of the patient's plasma.

Yes, as there are times when the causative antibody is an IgM (such as anti-Vel), and, never forget, the titre of the anti-Vel may be VERY low, but the complEment system is an amplification system (i.e. one C1qrs complex will result in huge numbers of other activated complement molecules further down the line), but you can concentrate the eluate and be able to detect the antibody originally sensitising the red cells.  The antibody can also be a VERY weak IgG antibody (IgG1 or IgG3, or a mixture), and the same applies.

We do something extremely similar to your daily executive safety huddle.
We previously had two individuals for laboratory quality/compliance, one for all lab and the other for blood bank specifically. Now we just have the one individual who covers the whole lab.

We had graduated to doing ABO/Rh and DAT only on babies born to Rh neg moms. Then...........a new Family Medicine doc came to town and became the head of the OB committee and now we are doing ABO/Rh and DAT on babies born to all O moms as well. The new pediatrician head of the pediatric committee is perfectly OK with testing only the babies born to Rh neg moms - all our newborns are scanned for evidence of elevated bili before discharge, so a high bili is not going to be missed. We are hoping he can eventually win the day and we can go back to testing only the Rh neg babes. One category that we don't automatically get cord bloods on is the moms with clinically significant alloantibodies. I would like to see that change. If a pediatrician doesn't order a DAT on those babies, I find a hemo sample and run one - if it's positive, I would take that info to the medical director for followup with the attending.

We only routinely get cords on Rh negative moms & moms w/ a clinically significant antibodies.  Occasionally a physician will request one on an O mom who has had a previously affected infant, but it’s not very common.  If a baby didn’t have a cord performed, and there are concerns of jaundice later, we have a separate test order for the ABO/Rh/DAT  drawn via venipuncture or heel stick.

It would be interesting to hear exactly why the new paediatrician wants to go back to this testing regime, considering that it has been known for decades that the DAT in a case of ABO HDN can be negative for a couple of days from birth, and only then become positive.  May I respectfully suggest that this new paediatrician relies on his or her ability to look at the baby's symptoms, rather than his or her ability to read laboratory results.  This way, more babies may survive.

My personal opinion - no validation required.
You are switching from one FDA-licensed product to an equivalent. Unless you plan to use it in a fashion contrary to the manufacturer's instructions it's a business decision rather than one of quality or performance.
If you have an internal policy that directs you to "validate" in these situations, you should change that policy. Anything that an end user does to "validate" a commercial, FDA-licensed red cell panel is dwarfed by the process involved to get these products to the market. 
Perhaps more important is that the replacement product suit your facility's specific needs. The typical antigenic make-up of the panel you select should reflect your particular testing requirements. For example....if you have lots of patients with anti- D, a panel with lots of D+ cells my not be very useful.
 

I would advise A+, BUT, I am not a doctor.

It's interesting that you posted this because the supervisor and I reviewed our Meditech Antibody and Antigen dictionaries just recently. It hadn't been done in many years and whoever set it up previously configured antigen warnings and IAT XM for every antibody, so review/revision was a long time coming!
One difference from the discussion here that we decided upon was to retain the requirement of IAT XM for antibody of undetermined specificity (INC).

So, the easy questions first eh Mari?????!!!!!!!!!!!!!
Personally, I think the IT guy gave you a poisoned chalice.  The reason I say this is because we can all list antibodies that are not generally considered to be clinically significant, and then, all of a sudden, one comes along amongst these specificities that has not read the appropriate text books and goes ahead and causes a clinically significant reaction.  Then what happens is that the person who said "anti-X" is not clinically significant, and this single example of anti-X turns out to be clinically significant, and you have to defend this in court.
The real problem these days is that the technologies available to us are now much more sensitive than when I started (when cross-matches were recorded on a stone slab with a hammer and chisel) and many antibodies that were not clinically significant (because we just didn't detect them with the technologies available at the time) are now readily detectable - BUT, they are not necessarily detectable at strictly 37oC, as , for example, many examples of anti-M are now detected by "IAT", even though they do not really react (in real terms) at 37oC.  The real problem comes when, for example, an anti-M genuinely DOES react at 37oC, and it is treated as clinically insignificant, electronic issue is used, and one or more of the units is M+ and the patient has a severe reaction - who answers in court?
The worrying thing is that there have been papers published over the last few years quoting an anti-Leb as causing a transfusion reaction, and an anti-P1 causing a transfusion reaction (a certain Garratty G being a co-author on this one).
I would say, therefore, that the best thing to do is to read through the relevant parts of The Blood Group Antigen FactsBook, Human Blood Groups and Mollison's Blood Transfusion in Clinical Medicine (latest editions in each case), and use their experience, rather than your own (no insult intended) as the courts would probably take the authors as "experts" should you come across any of these clinically significant "outliers".
I wish you the very best of luck!

We consider Anti-DARA/CD-38 as a clinically INsignificant antibody.  Computer will allow ISXM/ELXM if current ABSC is negative and AB is insignificant and the workup is complete with no other underlying clinically significant antibodies.  We've had about 25 patients and transfused multiple times over the course of the year with no adverse events.

Well, that's me finished.  I am officially retired from work - but not from this wonderful site!

Interesting patient #2 this month.  A multiple myeloma patient who had no history had all testing positive in Gel, including the  Auto control.  Expecting a Warm auto we sent the specimen to the reference lab.  Again, they sent it further to the American red Cross.  They discovered a new medication on the med list was a medication that pretty much interfered with all blood bank testing except Immediate spin crossmatches.    Darzalex is the name of the drug.  The bulletin is below from the AABB.   So, while the patients are on this drug, our reference lab will have to perform the antibody screens for us. 
 
 
 
Association Bulletin #16-02
Date: January 15, 2016
To: AABB Members
From: Donna M. Regan, MT(ASCP)SBB—President
Miriam A. Markowitz—Chief Executive Officer
Re: Mitigating the Anti-CD38 Interference with Serologic Testing
Summary
A new class of therapeutic agents for multiple myeloma, CD38 monoclonal antibodies, can result in interference with blood bank serologic tests and thereby cause delays in issuing Red Blood Cell (RBC) units to patients receiving these agents. To minimize these delays, hospitals should set up procedures to inform the transfusion service when patients start receiving these agents. Considerations for the transfusion service, both before and after initiation of anti-CD38 therapy, are detailed below.
The AABB Clinical Transfusion Medicine Committee has developed this bulletin to provide background information and guidance to members regarding anti-CD38 interference with serologic testing. The bulletin includes recommendations for its prevention and treatment.
Association Bulletins, which are approved for distribution by the AABB Board of Directors, may include announcements of standards or requirements for accreditation, recommendations on emerging trends or best practices, and/or pertinent information. This bulletin contains information and recommendations. No new standards are proposed.
Background
CD38 monoclonal antibodies are a new treatment for multiple myeloma
CD38, an integral membrane protein that is highly expressed on myeloma cells, has been identified as an effective target antigen for monoclonal antibody therapies. In November 2015, the first therapeutic CD38 monoclonal antibody [daratumumab (Darzalex, Janssen Biotech, Horsham, PA)] was approved by the Food and Drug Administration.1 Other CD38 monoclonal antibodies are under development.
CD38 monoclonal antibodies interfere with blood bank serologic tests
CD38 is weakly expressed on red cells. Anti-CD38 binds to CD38 on reagent RBCs, causing panreactivity in vitro.2,3 Plasma samples from anti-CD38-treated patients consistently cause positive reactions in indirect antiglobulin tests (IATs), antibody detection (screening) tests, antibody identification panels, and antihuman globulin (AHG) crossmatches. Agglutination due to anti-CD38 may occur in all media (eg, saline, low ionic strength saline, polyethylene glycol),
1
and with all IAT methods (eg, gel, tube, solid phase). Agglutination reactions caused by anti-CD38 are usually weak (1+), but stronger reactions (up to 4+) may be seen in solid-phase testing. However, anti-CD38 does NOT interfere with ABO/RhD typing or with immediate-spin crossmatches.
Other notes on anti-CD38 serologic interference:
 Adsorptions using either untreated or ZZAP-treated cells fail to eliminate the interference.
 Anti-CD38 variably interferes with direct antiglobulin tests (DATs) and antibody identification panel autocontrols.
 Some rare Lu(a–b–) cells are not reactive in the presence of anti-CD38, potentially giving the false impression that the patient has a Lutheran-related antibody.4,5
 Positive IATs can be observed for up to six months after anti-CD38 is discontinued.1,3
 Anti-CD38 may cause a small decrease in hemoglobin in vivo (~1 g/dL), but severe hemolysis has not been observed among treated patients.3,6
Anti-CD38 interference can cause delays in issuing RBCs
If the transfusion service is unaware that a patient has received anti-CD38, the following scenario may occur when the patient’s sample is tested:
1. ABO/RhD typing: no issues.
2. Antibody detection (screening) test: all cells positive.
3. Antibody identification panel: all cells positive (autocontrol may be negative).
4. DAT: positive or negative.
5. AHG crossmatches: positive with all RBC units tested.
6. Adsorptions: panreactivity cannot be eliminated.
This leads to delays in issuing RBCs to the patient. In some cases, the anti-CD38 interference could mask the presence of a clinically significant alloantibody.
Recommendations
To avoid problems with transfusion, hospitals should set up procedures to inform the transfusion service whenever any patient is scheduled to begin taking anti-CD38.
BEFORE a patient begins taking anti-CD38:
 A baseline type and screen should be performed.
 In addition, a baseline phenotype or genotype is recommended.
AFTER a patient begins taking anti-CD38:
 ABO/RhD typing can be performed normally.
 For antibody detection (screening) and identification, dithiothreitol (DTT)-treated cells can be used to eliminate the interference.2,7
o Because DTT treatment destroys Kell antigens, K-negative units should be provided unless the patient is known to be K-positive.
o Antibodies against other DTT-sensitive blood group antigens (anti-k, anti-Yta, anti-Doa/Dob, etc) will not be detectable when the antibody screen with DTT-
2
treated cells is performed; such antibodies are encountered infrequently, however.
Crossmatch
 For patients with a negative antibody screen using DTT-treated cells, an electronic or immediate-spin crossmatch with ABO/RhD-compatible, K-matched units may be performed.
 For patients with known alloantibodies, phenotypically or genotypically matched RBC units may be provided.6,8
o As some typing antisera require the use of AHG, phenotyping should be performed before the patient receives anti-CD38.
o Genotyping can be performed either before or after the patient receives anti-CD38.
o AHG crossmatches with phenotypically or genotypically matched units will still be incompatible.
o Some clinically significant antibodies may be missed with the use of uncrossmatched phenotypically or genotypically matched units, although this will occur infrequently.
 Alternatively, an AHG crossmatch may be performed using DTT-treated donor cells.
 If an emergency transfusion is required, uncrossmatched ABO/RhD-compatible RBCs may be given per local blood bank practices.
Future/alternative approaches to mitigating the anti-CD38 interference
It is possible to neutralize anti-CD38 in plasma and eliminate the interference using either recombinant soluble human CD38 or daratumumab idiotype antibody.2,3 Neither reagent is widely available at this time, and additional validation would be needed. In principle, soluble CD38 could be used to neutralize any anti-CD38, while different idiotype antibodies would be needed to neutralize different CD38 therapeutic antibodies. Finally, antigen-typed cord cells have been used for the antibody screen as an alternative to DTT-treated cells.9
3
References
1. Darzalex package insert. Horsham, PA: Janssen Biotech, 2015. [Available at: http://www.darzalex.com/shared/product/darzalex/darzalex-prescribing-information.pdf (accessed January 7, 2016).]
2. Chapuy CI, Nicholson RT, Aguad MD, et al. Resolving the daratumumab interference with blood compatibility testing. Transfusion 2015;55(6pt2):1545-54.
3. Oostendorp M, Lammerts van Bueren JJ, Doshi P, et al. When blood transfusion medicine becomes complicated due to interference by monoclonal antibody therapy. Transfusion 2015;55(6pt2):1555-62.
4. Velliquette RW, Shakarian G, Jhang J, et al. Daratumumab-derived anti-CD38 can be easily Mistaken for clinically significant antibodies to Lutheran antigens or to Knops antigens (abstract). Transfusion 2015;55(3S):26A.
5. Aye T, Arndt PA, Leger RM, et al. Myeloma patients receiving daratumumab (anti-CD38) can appear to have an antibody with Lutheran-related specificity (abstract). Transfusion 2015;55(3S):28A.
6. Chari A, Satta T, Tayal A, et al. (2015, December) Outcomes and management of red blood cell transfusions in multiple myeloma patients treated with daratumumab (oral and poster abstract presented Monday, December 7, 2015, 6:00 PM-8:00 PM at 57th Annual American Society of Hematology meeting). Blood 2015;26(Suppl):Abstract 3571.
7. Chapuy CI, Aguad MD, Nicholson RT, et al. International validation of a dithiothreitol (DTT)-based method to resolve the daratumumab interference with blood compatibility testing (oral and poster abstract presented Monday, December 7, 2015, 6:00 PM-8:00 PM at 57th Annual American Society of Hematology meeting). Blood 2015;126(Suppl):Abstract 3567.
8. Hannon JL, Caruk B, Clarke G. Serological findings related to treatment with a human monoclonal antibody (daratumumab) in patients with advanced plasma cell myeloma (abstract). Transfusion 2014;54(2S):162A.
9. Schmidt AE, Kirkley S, Patel N, et al. An alternative method to dithiothreitol treatment for antibody screening in patients receiving daratumumab (abstract). Transfusion 2015;55(3S):2292-3.
4

I probably should not be saying this, as I am a member of the IBMS Special Advisory Committee for Transfusion Science, but I do so agree with you about your first point. I am somewhat surprised that the BBTS representatives on the committee did not kick up more of a fuss.
I totally agree with your comments concerning the Edinburgh MSc (especially so, as I lecture on this course!) and, personally, I think that the Bristol MSc is its equal.
On the face of it, I would agree with your comments in 3, but when you look closer, some of the recommendations could not possibly be complied with by the Reference part of the Red Cell Immunohaematology Departments of the NHSBT (although this does not apply to most antenatal work and grouping for the armed forces or the British Antarctic Expedition). If, for example, you look at bullet point 2.1, much of our work involves the investigation of auto-antibodies (or rather, what, if anything, is underlying the auto-antibodies). there is no way that full walkaway automation (or any other kind of automation) could be used to perform these investigations.
Almost al of the other reference samples contain at least one clinically significant atypical alloantibody, and so the use of electronic issue (bullet point 2.2) is a non-starter for us.
I think, though, that many of the general points raised in the Recommendations are already adhered to by the RCI Departments within the NHSBT. Certainly, nobody could work as a Biomedical Scientist during core hours, let alone during non-core hours, unless they were registered with the HPC.
Point 4 is well made. Presumably, anyone who is taken on in this fashion would have to show capability and be signed off as such by the most senior member of staff within the Laboratory (and they themselves would have to have qualifications in Blood Transfusion), but I do agree that this should have been made more explicit.
As far as I am concerned, funding is a matter for the CEO, and, as I said in an earlier post, they fail to give the correct funding at their own peril. It will only take one disaster to occur, where the CEO is implicated for not funding the requirements listed in the Recommendations, and I think that funding will suddenly be coming out of our ears!
:cool::cool:

I know exactly what you mean Rashmi, but in circumstances where CEO's do get a "talking to", particularly where extra finance for the Blood Transfusion Department is required, the MHRA are quite capable of taking things much further (and higher), and have recently done just that in one of their inspections.
In the particulr case of which I am thinking (not public knowledge yet, so I can't name names) the BBM was given extra budget, extra powers and was promoted a KSF Grade (so it can work to our advantage).
:D:D

For some hospitals however the the responsibility for ensuring compliance is forced down to the staff with the suggestions that if you can't cope with the regulations then you aren't suitable for the job. The 'resource' word isn't allowed. Goodness knows what will happen to staff at these places whene their CEO does actually get a talking to.
This UK collaborative document, which I do fully support however will potentially create an additional financial burden for BBMs on top of everything else- how much does it take for staff to finally give in?