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Adsorption/Absorption


Bertie

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Hello,I am a new tech in training in a Reference Lab in a Blood Center.I would like to know more about

absorptions.If anyone is good about explaining this process so that I could understand it.I know that

if I think my patient has an autoantibody and an alloantibody that I am supposed to adsorb it with an

allogenic specimen.Any opinions on this subject would be appreciated.Thanks,Bertie:confused:

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Hello,I am a new tech in training in a Reference Lab in a Blood Center.I would like to know more about

absorptions.If anyone is good about explaining this process so that I could understand it.I know that

if I think my patient has an autoantibody and an alloantibody that I am supposed to adsorb it with an

allogenic specimen.Any opinions on this subject would be appreciated.Thanks,Bertie:confused:

Hi Bertie, I'll do my best.

The very best way to adsorb out an auto-antibody is to use autologous red cells (i.e. auto-adsorption). Such an adsorption will take out any auto-antibody, leaving behind any alloantibody, however rare the specificity of the alloantibody may be. To put it another way, if the auto-antibody is panreactive, but underneath that is, say, an anti-Lan (an antibody directed against a very high frequency antigen), however many times you adsorb the patient's plasma with autologous red cells, you will not remove the anti-Lan because the patient is, by definition of the fact that the anti-Lan is an alloantibody, Lan Negative.

BUT, you can only perform an auto-adsorption if you have sufficient red cells (which would be unusual - as a patient with an auto-antibody would normally have a low haemoglobin and haematocrit) and if the patient has not been transfused within the previous 3 months (unlikely, because of the low Hb and hct), because some of the transfused red cells may be present, and may take out the alloantibody.

So, under such circumstances, you would have to go for a differential alloadsorption.

It is important to remember, at this stage, that most (but not all by any means) auto-antibodies are directed against Rh antigens. It is, therefore, vital that you choose three different cells, one of which is R1R1, one of which is R2R2 and one of which is rr. These must be fully typed, so that one is Jk(a+b-), whilst another is Jk(a-b+), one is K+, etc, etc, so that any alloantibody is adsorbed out by one (or two) of the cells, but not by all three.

The red cells used, initially anyway, should be enzyme-treated (to enhance any reaction between an Rh antibody/antigen). At this stage, of course, the MNS and Duffy type of the red cells does not matter, as these antigens will be destroyed by the action of the enzyme).

The plasma is split into three aliquots, and each is then adsorbed by the R1R1, R2R2 or rr red cells. These are incubated at 37oC (usually, but, if a "cold" auto-antibody, or a mixed "cold"/warm auto-antibody is suspected, 4oC) for about 20 minutes. The mixture is then centrifuged, and the plasma, if initially in R1R1 red cells, transferred to another aliquot of (packed) R1R1 red cells (if R2R2 then to the next aliquot of R2R2 cells, and, if initially rr red cells, to the next aliquot of rr red cells). The incubation is repeated, as is the centrifugation and the transfer to the next aliquot of red cells.

Eventually, the uto-antibody will be adsorbed out, and then all three (individual) plasma samples are panelled against your normal antibody identification panel. Any alloantibody will then become obvious (remembering, of course, that, if the alloantibody is, say, an anti-Jka, those cells used to adsorb the auto-antibody that are also Jk(a+b-) will have taken out the anti-Jka, but that those that are Jk(a-b+) will not have done so).

Rarely, enzyme-treated red cells will not adsorb out the auto-antibody, in which case (unfortunately) you have to start all over again with non-enzyme-treated red cells. This is a complete pain, but, nonetheless, vital.

Of course, either way, you will miss an antibody such as the anti-Lan I spoke about earlier, because the chances are that all three red cells will be Lan Positive (but you can't have everything.

I hope this helps.

By the way, the correct term is adsorption, rather than absorption. If you see water vapour condense onto a window (fogging it up), this is water vapour adsorbing onto the glass surface. If you see water going into a sponge, the water is being absorbed into the sponge. One is on the surface, whilst the other is in something. The antigens on a red cell are on the surface, rather than in the red cell cytosol, and so the antibody is adsorbed onto the surface of the red cell, rather than absorbed into the red cell.

See also References (at the top of the page), choose Document Library from the drop down list, then Educational Material, go to page 2 and choose the PowerPoint lecture entitled "Laboratory Investigation od Autoimmune Haemolytic Anaemia".

:):):):):)

Edited by Malcolm Needs
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What I meant to add, but forgot, is that if you are adsorbing with red cells that are not enzyme-treated, then you also have to take their MNS and Duffy types into account.

At least one of the three cells should be S+s- and at least another S-s+, and at least one of the cells should be Fy(a+b-) and at least another Fy(a-b+).

That's what comes of replying to a post after a couple of glasses of wine!!!!!!!!!!!!!!!!

:D:D:D:D:D

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Malcolm, that was wonderful! In 15 years of being a tech I never reallyunderstood why the procedure you described is an adsorption rather than an absorption! Thank you for the time and thoughtful answers you post on this site.

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Great explaination Malcolm.

Have always shied away from adsorptions, sending them to the referance lab, but will give it a go with our next auto patient.

Could you tell me what voulmes and concentrations of red cells and plasma you use when performing the adsorption?

Thanks

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Could you tell me what voulmes and concentrations of red cells and plasma you use when performing the adsorption?

Thanks

Therein lies the problem for a Hospital Blood Bank.

The actual mechanics of performing an alloadsorption are really easy, but finding sufficient red cells of the correct phenotype is really difficult. You would need a minimum of about 10mL of each of the packed red cells (and I do mean packed, otherwise there is a danger that any saline in which the red cells are suspended may dilute out any underlying alloantibodies) for 4 adsorptions, but, of course, double that if you take it to 8 adsorptions (rare, but with a stubborn auto-antibody this may be necessary), and you will need, as a minimum, 2 full EDTA tubes of patient's blood to get sufficient plasma.

You will always lose a little of the plasma during the adsorption process itself (and, don't forget, you will have to split whatever you have into three aliquots from the word go), then you will have to perform a panel on each of the aliquots of the adsorbed plasma, and, as like as not, perform a cross-match on each of the aliquots at the end.

The whole thing is pretty heavy on both reagents and time.

In the UK, such techniques are almost exclusively carried out by the Reference Laboratories of the Blood Services, rather than the Hospital Blood Transfusion Laboratories, simply because it is almost impossible to supply sufficient adsorption cells for each Hospital. Even for us, the Reference Laboratories are supplied by our Reagents Division to save on red cell usage.

I would never discourage anyone from trying new techniques and technologies, but before you do this, you have to take into account whether or not you will have sufficient red cell and staff resources.

.....but I hope I haven't put you off!!!!!!!!!!!!

:):):):):)

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  • 4 weeks later...

I very seldom post a reply on any of the bloodbanktalk threads but I very often read through them. I feel that I have way more to learn than to offer. This thread is a great example of that. Thank you, Malcolm, for a simple yet thorough explanation! I am a generalist (have been for 30 years). A few years ago I added lead tech in blood bank to my generalist duties. This forum has been a great in helping me to learn more. Thanks everyone!

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Thank you ever so much Malcolm what a great in depth explanation! That being said, you definitetly illustrated the exact reason that adsorptions will always be done in my reference lab. The most technical that I get at all is maybe a quick little lui freeze if I suspect an ABO on a cord or something.:)

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I very seldom post a reply on any of the bloodbanktalk threads but I very often read through them. I feel that I have way more to learn than to offer. This thread is a great example of that. Thank you, Malcolm, for a simple yet thorough explanation! I am a generalist (have been for 30 years). A few years ago I added lead tech in blood bank to my generalist duties. This forum has been a great in helping me to learn more. Thanks everyone!

Thanks Nancy L, but everybody has something to offer in the way of posts. I've learned an awful lot from this site too!

:D:D:D:D:D

Edited by Malcolm Needs
Guess what! Spelling!!!!!!!!!!!!
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The plasma is split into three aliquots, and each is then adsorbed by the R1R1, R2R2 or rr red cells. These are incubated at 37oC (usually, but, if a "cold" auto-antibody, or a mixed "cold"/warm auto-antibody is suspected, 4oC) for about 20 minutes. The mixture is then centrifuged, and the plasma, if initially in R1R1 red cells, transferred to another aliquot of (packed) R1R1 red cells (if R2R2 then to the next aliquot of R2R2 cells, and, if initially rr red cells, to the next aliquot of rr red cells). The incubation is repeated, as is the centrifugation and the transfer to the next aliquot of red cells.

What Malcolm describes here is how we did it at my reference lab. We made our own red cell stroma concoctions using 3 different cell types--the same as Malcolm described. Then after absorbing we would test the adsorbed plasma with our regular antibody panels.

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Wonderful explanation, Malcolm, as usual. May I add that if you can perform an autoadsorption (see Malcolm's discussion of when you should or shouldn't), the PEG autoadsorption procedure in the AABB Tech Manual is easier, quicker, more efficient, cheaper, and generally requires fewer adsorptions than using ZZAP, Immucor's Warm Autoadsorption Removal Medium, or other techniques I've tried. The procedure has a caveat about potential weakening of reactivity, but we did not see this with titers of spiked samples before and after the procedure. The procedure has a workaround as well to accomodate this potential.

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OK Malcolm..I'm taking a risk here....I have always been taught that the "process" was called absorption and the end result was called "adsorbed plasma". Have I been repeatedly misguided?

Well, from my point of view, and that of my English Dictionary, I'm very sorry to say YES!

:redface::redface::redface::redface::redface:

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Really? All these years I've been using incorrect terminology! How sad is that?!

So every step in the process is called aDsorption?

Well, this is only my opinion, but YES, and this is also backed up by the definitions in the post by wahaneebelly (post number 13 in this thread).

Antibodies adsorb onto the antigens on the surface of the red cell, rather than absorb into the red cell.

:):):):)

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  • 1 month later...
Malcom,

At what point as a general rule of thumb, at what point do you consider sufficient adsorption vs. dilution due to multiple adsorptions. You mention adsorbing 8x.

8 adsorptions is the absolute maximum we would perform. If the auto-antibody has not been adsorbed out by then, we would perform a quick dilution (as, by now, the auto-antibody should be, at least, weakened) to see if there is any alloantibody underneath that can be readily recognised, and if not, we would, under Clinician's advice, give ABO, Rh and K matched blood, slowly, whilst watching the patient like a hawk, and keeping our fingers and toes crossed! It is very rare for us to have to do this, but not totally unknown. Bit of an overlong sentence!

Normally, we can get away with 4 adsorptions for DiaMed, and maybe 2 for LISS tube IAT at 37oC.

Just doing the test by dilution, without the adsorptions, I am 100% against, but some people/gamblers do this.

I hope that helps.

:):):):):)

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Thank You Malcom,

I always worried about dilution factor. I mean we could potentially dilute the underlying allo, and therefore get a false negative? I currently recommend adsorptions with PEG, LISS, or WARM but not more than 4x, and still worry about missing something.

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