Bb_in_the_rain

Thank you for the detailed explanation and laying out all the possible case scenarios. Amazing as always! 
 

In addition to the scenario that Yan has describe above, I would like to add awarm autoantibody, where the antibody is saturated on the patient's own cells but has not "spill" into the plasma, therefore DAT and autocontrol may be positive but non-reactive with reagent red cells. 

In addition to the scenario that Yan has describe above, I would like to add awarm autoantibody, where the antibody is saturated on the patient's own cells but has not "spill" into the plasma, therefore DAT and autocontrol may be positive but non-reactive with reagent red cells. 

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I dont think you need special state licenses for these states. CLIA (Clinical laboratory improvement amendment) defines most tests performed in hematology and immunohematology as moderate to high complexity testing, therefore needs specific education/experiences or accreditation. The easiest way to achieve it is to challenge accreditation exams conducted by ASCP (american society for clinical laboratory science). I have the link to their website below. You can go ahead and make an account free of charge and start looking through education/experience requirements and paperworks needed to challenges the exams. As you have 17 years of prior experience, recommend you to look into BB(work in blood bank), MLS (can work in every area of the lab except for cytology), H (can work in hematology)which are technoligist licenses. Also, specialized licenses SBB (specialist in blood bank, which majority of blood bank lab leadership personnel are certified for) or SH (specialist in hematology, which majority of hematology lab leaderships are certified for). 
https://www.ascp.org/content/board-of-certification/get-credentialed
Please do not hesitate to ask us questions here. We are all here to help you with your transition. 

Well, in the case about which I know, it was fresh EDTA plasma that reacted, but stored EDTA plasma that did not, so it couldn't have been the complement, as the Ca++. Mn++ and Mg++ required for the complement cascade had already been chelated in the fresh plasma.

I would love to have a national system where all transfusion/transplant patients are treated equally regardless of their genetic basis, here, in the US. Unfortunately here in the States, blood is often times is looked as a commodity and treated as such, as well as the services that goes with it. I do not even want to start talking about how deficient our healthcare system is. 

Yan xia- I have no idea why it does not react with a 6 month old frozen/thawed AB plasma but reacted with fresh AB plasma. I treated 3 more sources of cells and they reacted weaker (2+) with this same source of frozen/thawed AB plasma but strong (4+ blasted) with fresh AB plasma. May be anti-T diminishes with storage? 
Galvania- I think docs are suspecting an infection in this baby. 

Whats boring is American quality control departments? Cannot do anything without running into red tapes. :~

You can never charge two HCPCs codes for a single transfusion. You also can't individually charge testing/processing CPT codes if there's a bundled HCPCs code that describes it best.

I have heard of this kind of thing once before in 43 years of working (it was in Scotland), but, SORRY, I don't think anyone ever got to the bottom of the cause.  I realise that doesn't help much!

Well dothandar, the answer to your first question is "YES", because this phenomenon has been seen many times over many years.  In those earlier years, experiments were performed to determine the immunoglobulin molecules present, and in every case there was a mixture of IgM and IgG.  In the end, it was decided that performing such experiments was a waste of time and money, for very little return.  Why reinvent the wheel now?
Turning to question two, all immune antibodies start off as IgM as a result of primary sensitisation, but then quickly change to IgG, even from the primary sensitisation (and almost always from a secondary sensitisation), and, undoubtedly, some IgG antibodies can cause agglutination when no potentiating agents are present (for example, IgG ABO antibodies), however, this is not true of Rh antibodies.  In the case of Rh antibodies, it is not a function of the antibody, so much as a function of the number and proximity of the antigens.  This can be seen with certain examples of human-derived IgG anti-D, which never cause "saline agglutination" at 37oC with red cells of "normal" Rh types, but do so with red cells exhibiting the exulted-D types (such as D--/D--, DCw-/DCw-. etc, because the number of D antigen sites available, particularly towards the outer layer of the sialic acid cloud, allow for Rh IgG immunoglobulins to come close enough between two (or more) different red cells to cause agglutination.  However, even in such a situation, the incubation temperature needs to be 37oC, and not "room/ambient" temperature.

Folks, please read this carefully, I suggest he is saying it is acceptable to call him Malcolm, as compared to Mr. Needs - hence informal.  He did not suggest he would find it acceptable to refer to him as a tosser, wanker, or some other (really cool sounding British) pejorative. 
I need to move to England, if only for a short time.  American English is so boring.

and I still don't understand why they thought that this patient was polyagglutining  anyway!

why the frozen/thawed AB plasma not react with the treated cells?

Hello. sorry about the delayed reply. We worked further on this case and turned out the pooled AB plasma that we have frozen did not react with neuraminidase-treated cells (another aliquot of NeuNac treated cells) but fresh AB plasma did. We did test this patient's cells with fresh AB plasma and got a positive reaction. We have to try not to used frozen/thawed AB pooled plasma for this test in the future

What would be really helpful, and is quite simple, is to treat the plasma with 0.01M DTT (or ZZAP come to that), which will denature IgM molecules by breaking the J-chains.  This will show if the antibody is IgM or not.  Do not forget that some IgG antibodies can cause agglutination in the "cold" and the "warm", with no potentiating agents being present, so it is worth knowing, from the word "Go", with what you are dealing.

OK Malcolm, in that case you can exclude it.  I hadn't noticed the little English flag on the first post!
Has anyone thought to do an eluate?  That might be helpful

We do have some liquid monospecific anti-IgM (but I am now retired and cannot remember the name of the supplier - I will investigate), but the cards we use are those shown in the photographs in the Kidd HDN thread gagpinks started, which do have an anti-IgM column.

Yes,   you are completely right Yanxia.
Briefly, the patient's plasma and reagent red cells are incubated at 37oC, as for a normal tube IAT, to allow the antibody in the plasma to sensitise the antigens on the red cells.  The tests are then washed free of unbound antibody (as for the normal tube IAT), but then, instead of adding AHG at this stage, fresh ABO compatible serum (it has to be serum, rather than plasma, to ensure there is complement there to initiate the classical complement pathway), which is known not to contain any atypical antibodies (we used to use AB serum from a source that had been extensively tested and found to be free of any such antibodies) and mixed with the red cells.  The tests are then incubated again at 37oC, to allow for the complement cascade to be initiated, and then washed again, as for a normal tube IAT.  Lastly, monospecific anti-C3d is added, and the tests GENTLY centrifuged, and examined for agglutination.
A negative control, using the inert AB serum, rather than the patient's plasma, must be set up and tested in parallel.
Of course, such a technique can only be performed by tube, capillary, tile or liquid-phase microtitre plate techniques, as column agglutination and solid-phase microtitre plate techniques cannot be used.

Hi dothandar and Malcolm
These are the card we use in our lab as part of DAT protocol.  
Malcolm , I guess this finding is not clinically significant then. Is there any particular reason for DAT to be positive in C3d or cause is unknown?

I recently used this procedure to differentiate anti-Pr from anti-En(a), which is the only time I have used it. Since our lab refer possible Asub for genotyping lab, we have no chance of trying this out to catch weak subgroup of A. 

The patient takes loratadine. It doesn't look like brompheniramine or phyenyltoloxamine are ingredients and I see loratadine listed anywhere interfere with their reagents.
We diluted our 3% reagent RBCs to a 0.8% suspension with Grifols diluent and the gel screen is negative.

You could still be looking at an additive/preservative issue.
You state that Gel testing performed by your site was Grifols and the Reference Lab was Ortho Gel with Immucor reagent red cells. Immucor does not make reagent red cells for use in gel systems so the Reference lab would have had resuspend the 3% cells to 0.8% for use in gel. If I remember correctly that involves taking an aliquot of the red cells, adding saline to make them easier to decant to a "dry" button and then adding MTS Diluent. So you are essentially washing away the additive/preservative in the reagent cells and thereby removing that as a potential problem.
The fact that the crossmatch performed in Grifols gel was negative also points to the reagent cells as being the issue. I would suggest that you try rerunning the specimen in Grifols with "washed" reagent red cells to remove the additive/preservatives and resuspend them in the same diluent you use for the donor cells and see what you get.
As to the C3d being positive. Have you checked patient's meds list? Some meds cause the DAT to be Complement Positive e.g. antihistamines containing brompheniramine, phyenyltoloxamine