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Elution Studies


goodchild

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29 minutes ago, TreeMoss said:

One thing our reference lab mentioned was transfusing antigen-matched blood so you know no new antibodies will be formed.  In that situation, I don't think we would need to continue sending specimens to the reference lab every time the patient came in for transfusion.

Anne

There are over 400 antigens recognised by the ISBT,  Of course, many of these are low prevalence, and so you would be unlucky to come across a unit expressing such an antigen, when the cognate antibody is in the plasma (although, never forget that antibodies directed against low prevalence antigens tend to cross-react at best, or appear in the plasma as multiple different specificities), and many are high prevalence, so you would be unlucky to come across a patient who was negative for one of these (I did once - an anti-Vel under a warm auto-antibody, whilst on-call!  Thank goodness the anti-Vel was detected and recorded in our computer before the warm auto-antibody developed), but many are polymorphic.  It is these that I would worry about, as you never know when an antibody to such an antigen will "pop-up", and bite you (and the reference laboratory) on the behind, and there is no way that you(or they) could type the patient, or the units to be transfused  for all of these antigens.

I actually agree with the idea that you don't need to perform a full cross-match each time, but I disagree profoundly with the reason.  Yes, you want to avoid giving the patient blood expressing the most immunogenic antigens that they do not express themselves, but the real reason that I agree is that the patient's immune system will be acting at less than optimum, and so producing a de novo specificity is much less likely!  I would cross-match "properly" about monthly.

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  • 2 weeks later...
On ‎4‎/‎11‎/‎2016 at 11:57 AM, goodchild said:

Hi everyone,

Doing an informal survey for anyone willing to contribute. If you're willing to respond but not in the thread itself, please feel free to message me.

  1. When do you perform an elution? (e.g. all positive DATs, all positive DATS within 3 months of transfusion, IgG positive only)
  2. What method is utilized for the elution?
  3. What method is utilized for testing the eluate?
  4. How is the eluate tested? (e.g. screening cells, full panel, specially selected cells)
  5. Feel free to mention any special notes/criteria for which I may not have though to ask.

Thanks in advance to all participants!

1) IgG positive adult cells ( not Cord cells)

2) Acid Elution Method

3) Ortho Gel Card

4) Screening Cell 1 and 2, along with Last Wash supernatant as a control, and full panel if either screening cell is positive. It has been awhile but I think that we practice to perform the panel in tube; I will check up on it.

5) Our Acid Elution Kit comes with a Working Wash Concentrate which is diluted 1:9 with BB Saline and used for testing. Our method also gives the option of a total of five  or six washes with saline if suspecting a weak antibody that may be rendered inactive with the Working Wash Solution.

 

Edited by rravkin@aol.com
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On ‎4‎/‎11‎/‎2016 at 11:57 AM, goodchild said:

Hi everyone,

Doing an informal survey for anyone willing to contribute. If you're willing to respond but not in the thread itself, please feel free to message me.

  1. When do you perform an elution? (e.g. all positive DATs, all positive DATS within 3 months of transfusion, IgG positive only)
  2. What method is utilized for the elution?
  3. What method is utilized for testing the eluate?
  4. How is the eluate tested? (e.g. screening cells, full panel, specially selected cells)
  5. Feel free to mention any special notes/criteria for which I may not have though to ask.

Thanks in advance to all participants!

1. Positive DAT IGG within 3 mos transfusion, when we get a request for polybrene , neonate

2. Gamma Elukit 11 acid elution, , Lui Freeze for neonates

3. Capture solid phase, tube

4. Initial antibody screen cells, panel

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1.  We perform an elution on patients transfused within the last month, who develop a positive IgG DAT or have an IgG DAT which has increased in strength.

2. Gamma Elu-kit

3. Gel

4. Last wash gets a screen, Eluate gets a panel

That said, have any of you Elu-Kit/Gel users had issues with broad reactivity, usually fairly crappy looking reactions?  We have had a few later proven to be false positives when sent to our reference lab for followup.  Any suggestions?  Should we drop the wash with the Wash Solution?  Drop back to tube testing?

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  • 7 months later...
54 minutes ago, Tabbie said:

Does anybody have issues with eluate washing with saline which can remove low prevalence antibodies ?

or do most people use the low ionic strength solution?

Thanks

 

I'm sorry Tabbie, but I see no reason why washing with saline would wash off antibodies directed against low prevalence antigens any more than it does high prevalence antigens or polymorphic antigens.

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Just reading a the Gamma ELU-KIT 11 Immucor insert just says it may result in the dissociation of low-affinity antibody during the wash procedure, leading to an eluate containing no activity or less antibody than expected? It does describe final wash should be tested in parallel with eluate. I will read the references and get back to you with more info

Thanks

 

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10 hours ago, Tabbie said:

Just reading a the Gamma ELU-KIT 11 Immucor insert just says it may result in the dissociation of low-affinity antibody during the wash procedure, leading to an eluate containing no activity or less antibody than expected? It does describe final wash should be tested in parallel with eluate. I will read the references and get back to you with more info

Thanks

 

Sorry Tabbie, but low prevalence antigens are antigens that are only expressed on the red cells of a few individuals in a particular population, or even worldwide, whereas low affinity antigens are antigens that may be expressed on any number of individual's red cells, but which do not "fit" well with their cognate antibodies.  In other words, there is only a weak link between the antigen and antibody, and these links can also easily be broken.  This is because antibody/antigen reactions follow The Law of Mass Action.  In the case of a low affinity antigen, the reverse reaction (the dissociation between the antigen and antibody) is at least as quick and easy as is the forward reaction (the association between the antibody and the antigen).  In this case, YES, repeated washing with saline can wash away the antibody, and YES, the eluate may be unreactive, but this is very rare.  That notwithstanding, the commercial companies have to write this into their inserts, to cover themselves in law.

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We used to do eluates but with the infrequency of need and competency challenges we decided to send to our reference lab. We request eluates on patients who need a transfusion (or investigating a TRRX) and are DAT + and who have been transfused within the past 14 days to rule out newly forming antibodies that could be completely absorbed onto the donor RBCs and may not be found in the plasma.  Our reference lab has recently changed from 14 to 21 days post transfusion for eluate testing but we have not yet changed our rule.  I believe the thought process behind this is that after 14 (or 21) days the newly formed antibody has had sufficient time to spill over into the plasma and can be identified in the ABSC/ABID.  On patients that are transfused frequently we sometimes opt to give phenotypically similar blood for transfusion instead of sending out for an eluate with each transfusion event as this is time consuming and expensive. 

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  • 1 month later...
On 26/02/2018 at 7:28 AM, Malcolm Needs said:

Sorry Tabbie, but low prevalence antigens are antigens that are only expressed on the red cells of a few individuals in a particular population, or even worldwide, whereas low affinity antigens are antigens that may be expressed on any number of individual's red cells, but which do not "fit" well with their cognate antibodies.  

Good points I used the wrong description. Can you clarify the use of frequency and incidence are synonymous with prevelance. Assuming so and that the 700 series is different collection only because they do no belong to any known blood group system ?

Thanks

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5 minutes ago, Tabbie said:

Good points I used the wrong description. Can you clarify the use of frequency and incidence are synonymous with prevelance. Assuming so and that the 700 series is different collection only because they do no belong to any known blood group system ?

Thanks

Yes, frequency, incidence and prevalence are all interchangeable with either "high" or "low" before any of them.  For some reason (I know not why), low prevalence has suddenly become the "word of the day", but there is no particular reason for this (as far as I know).

Those low prevalence antigens within the 700 series, and, come to that, the high prevalence antigens within the 901 series, are, as you say, placed there as they do not belong to any known blood group system.  However, do not run away with the idea that all low prevalence antigens are in the 700 series, and all high prevalence antigens are in the 901 series.

The antigen KREP, of the Diego Blood Group System has only ever been reported in one Polish individual and one Slovakian individual, and yet it is not in the 700 Series of antigens.

Conversely, and as far as I know, DOLG of the Dombrock Blood Group System has only been found to be negative in one Sri Lankan woman, and yet is not in the 901 Series of antigens.

This is because, in each case, the chromosome and the particular loci have been identified, and they are mapped to known areas of the genes which encode each of these antigens. 

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In regard to the low affinity antigens it seems that DAT are usually negative as described because these antigens do not fit well with their cognate antibodies and HDFN/HTR is rare.

Can any distinction be made in regard to positive/negative DAT results for high prevalence antigens ? The 901 series seems to cause severe HDN/HTR (Anti-Vel) and MAM describes DAT + but no HDFN with IgG1 and IgG 3 ? 

Thanks

Edited by Tabbie
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11 minutes ago, Tabbie said:

In regard to the low prevalence antigens it seems that DAT are usually negative. I am making the assumption that this has been described as so because these antigens do not fit well with their cognate antibodies and HDFN/HTR is rare and usually detected because other antibodies are present.

Can any distinction be made in regard to positive/negative DAT results for high prevalence antigens ? The 901 series seems to cause severe HDN/HTR (Anti-Vel) and MAM describes DAT + but no HDFN with IgG1 and IgG 3 ? 

Thanks

Unfortunately, what you say is far from "universal".  Certainly, it is unusual to see an antibody directed against a low prevalence antigen causing either HDFN or an HTR, but it is by no means unknown.  For example, anti-Bea has caused very severe HDFN, and anti-Wra caused a fatal HTR only a couple of years back in the UK.

In terms of the 901 series (and, come to that, anti-U of the MNS Blood Group System) it is possible that it is to do with the IgG subclass, but it is more likely to do with them being (occasionally) IgG2 and/or IgG4, but, as far as I know, this remains a theory, rather than having been proven.

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Well, the thing is that the FcY receptors on the immune system leukocytes are few and far between, so such antibodies are often clinically insignificant.  As a result polyspecific (broad spectrum) and monospecific anti-IgG reagents often do not detect such antibodies (or do so ineffectively) and so these are often (deliberately) not detected.  In addition, IgG2 and IgG4 are not good at initiating the complement system (IgG4 doesn't at all).  Lastly, of course, some of these antibodies can be IgA and, again, they are, at most, of doubtful clinical significance.

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  • 1 year later...
On 7/16/2017 at 5:49 PM, Byfaith said:

That said, have any of you Elu-Kit/Gel users had issues with broad reactivity, usually fairly crappy looking reactions?  We have had a few later proven to be false positives when sent to our reference lab for followup.  Any suggestions?  Should we drop the wash with the Wash Solution?  Drop back to tube testing?

We have found that not adding enough buffer will cause false positives, especially in gel testing.  I say to add an extra drop or two after they already think it is blue enough to be sure.

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On 7/16/2017 at 8:49 PM, Byfaith said:

That said, have any of you Elu-Kit/Gel users had issues with broad reactivity, usually fairly crappy looking reactions?  We have had a few later proven to be false positives when sent to our reference lab for followup.  Any suggestions?  Should we drop the wash with the Wash Solution?  Drop back to tube testing?

Centrifuge your final eluate for 60 seconds and pipette it to a new tube. Check the old tube for fine red particulate matter adhered to the tubes sides, and if present repeat the process. 

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  1. When do you perform an elution? (e.g. all positive DATs, all positive DATS within 3 months of transfusion, IgG positive only) - Positive DAT with anti-IgG if elution has not been done within previous 30 days or performing a transfusion reaction investigation
  2. What method is utilized for the elution?  Elukit
  3. What method is utilized for testing the eluate?  Gel
  4. How is the eluate tested? (e.g. screening cells, full panel, specially selected cells) Panel
  5. Feel free to mention any special notes/criteria for which I may not have though to ask.  If the plasma antibody is very strong (particularly with cord blood eluates), I am very careful to change tubes between washes and test Last Wash before proceeding further with adding the acid to red cells.  Once eluate is prepared, at least two centrifugations in clean test tubes to make sure that any stroma is removed as it will cause major headaches when performing gel testing.
Edited by applejw
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  • 8 months later...
1 hour ago, AMcCord said:

The pH indicator used in the Buffering Solution would be specific for the desired pH range. If you see a blue color, the solution is in that fairly narrow range - chemistry at work. There is no requirement in the insert (or in standards that I'm aware of)  to check the pH by a method other than eyeballing the color.

In reading the package insert and other information, I found no need to be testing the pH, due to the blue indicator. I was curious what others were doing. Historically the facility I am at has tested the pH, as well as eyeballing the color. I was looking to remove testing the pH from our procedure if it is not necessary.

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On 12/24/2019 at 11:58 AM, Michelle L said:

Does anyone that uses Immucor's Elu-Kit II test the pH of their eluate once it turns blue? Or do you just go by the blue color indicator as being within the appropriate pH range?

1. Personally, I wouldn't do ANYTHING different/extra than the manufacturer recommends. You may inadvertently "modify" the process and find yourself in a corner that requires validation of your local modification. Yikes !

2. Typical eluate volumes are quite small - often too small to be measured with a pH probe. So I suspect, correct me if I'm off-base, that the pH has been checked using pH paper. If so, eyeballing the color of the pH paper is no better than eyeballing the color of the eluate.

And, as AMcCord suggests, while the blue color may vary, the differences in pH values of the different blues is very small. The manufacturer (Gamma/Immucor) has put a lot of effort into making the kit idiot-proof.:)

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20 hours ago, exlimey said:

1. Personally, I wouldn't do ANYTHING different/extra than the manufacturer recommends. You may inadvertently "modify" the process and find yourself in a corner that requires validation of your local modification. Yikes !

2. Typical eluate volumes are quite small - often too small to be measured with a pH probe. So I suspect, correct me if I'm off-base, that the pH has been checked using pH paper. If so, eyeballing the color of the pH paper is no better than eyeballing the color of the eluate.

 

I would agree completely with these statements (especially #1). If it was me I would remove the requirement to check the pH from your SOP.

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