Auto adsorption Procedure

[kmmoton]

After an adsorptionx1. Do you add an equal amout of x1 plasma20 drops to 20 drops of red cells for the x2 adsorption or do you add 40 drops of adsx1 to 20 drops of red cells. What's your procedure?

[David Saikin]

what kind of absorption are you performing?  I use the PeG autoabsorption - there is a version in the AABB Technical Manual or you can find the original article in the Jan, 1999 issue of Transfusion.  There is also a WARM autoabsorption but I do not have that procedure.  I have found that the PeG works for most cases.

[Malcolm Needs ☆]

We use papain-treated red cells for autoadsorption (and differential alloadsorption come to that), with double the amount of plasma to that of red cells. We incubate for 20 minutes at either 37oC or 4oC (depending on whether we are working with a "warm" auto or a "cold" auto), or alternate these incubation temperatures when we are working with rare mixed warm and cold autoantibodies. We do not change these ratios for each adsorption cycle.

[Rh-fan]

• We use untreated cells and take 1 part of serum (600ul) and 2 parts of cells (1200ul). The first to times we incubate for 30 min, then go to 60 min.

After absorptiun we use 25ul of serum in the Birad/Diamed gel test.

[David Saikin]

Malcolm

 

What's the difference between using papain vs ficin treated cells for an absorption?   I would use enzyme treated cells if I didn't think the PeG was a good choice and I have ficin on hand..

[Eagle Eye]

It seems like reference labs prefers papain over ficin...I believe NY Blood center also uses papain.

Is it because Ficin has two step procedure???

[Eagle Eye]

After an adsorptionx1. Do you add an equal amout of x1 plasma20 drops to 20 drops of red cells for the x2 adsorption or do you add 40 drops of adsx1 to 20 drops of red cells. What's your procedure?

 I want to understand your question here.

If you are talking about second cycle, then it will be same amount of plasma you started with isn't it? If had 20 drops of plasma and 20 drops of cells in first cycle, you would transfer same plasma over to second aliquot of the red cells!

[Malcolm Needs ☆]

Hi David and Eagle Eye,

Actually, there is very little difference between papain and ficin for the adsorption, but we use papain because that is what we use to enzyme treat our red cells for serological work. When we use papain, it is still a two stage method, but we find we get much clearer results iwth the serology using papain, as ficin tends (in our hands anyway) to up the number of false positives considerably.

[David Saikin]

Is there a vendor for papain?  Does it come ready to use or do you need to "mix it up"?

thanks

 

 

Hi David and Eagle Eye,

Actually, there is very little difference between papain and ficin for the adsorption, but we use papain because that is what we use to enzyme treat our red cells for serological work. When we use papain, it is still a two stage method, but we find we get much clearer results iwth the serology using papain, as ficin tends (in our hands anyway) to up the number of false positives considerably.

[Malcolm Needs ☆]

We use that provided for us by NHSBT Reagents, which is ready to use and CE-marked.

Although we get it free, as we are part of NHSBT, I think that I am correct in saying that they do sell it commercially, although I don't know for how much (not a lot, I wouldn't think).

[kmmoton]

 I want to understand your question here.

If you are talking about second cycle, then it will be same amount of plasma you started with isn't it? If had 20 drops of plasma and 20 drops of cells in first cycle, you would transfer same plasma over to second aliquot of the red cells 

 

When the tech did the adsx1 she yieled 40 drops of supernatant (yes I'm perplexed) and proceeded to the adsx2 with those 40 drops of supernatant plus 20 drops of fresh red cells.  An SBB informed her this was correct.  That you should have double the amount of the adsx1 (40 drops) to 20 drops of fresh cells.//KMMOTON

[Eagle01]

Nationwide, is there any rate of false-positive and false-negative when using Papain versus Ficin? and what the AABB recomend between the two?

 

Thanks.

[Malcolm Needs ☆]

I'm afriad that I can't answer that one Eagle01, as I work on the other side of the big pond.

[David Saikin]

I am purchasing some papain to compare with ficin; if they react comparably, the papain is far less expensive.

[Eagle Eye]

I am purchasing some papain to compare with ficin; if they react comparably, the papain is far less expensive.

which company sells it? & how much for? 

[David Saikin]

which company sells it? & how much for? 

Immucor sells it as freeze dried/2mL vials 10 for ~$275. A lot less expensive than their ficin.  Cos may be dependent on your purchasing group.

Edited October 3, 2013 by David Saikin

[GinaL]

Both ficin and papain work well for adsorptions. We have routinely used ficin in our reference lab for decades, both for antibody ID and adsorptions. And both enzymes can be used as constituents of ZZAP. A couple of years ago I compared cysteine-activated papain and ficin as constituents of ZZAP, and the volume of 1%ficin used in ZZAP. In the AABB Technical Manual, the ZZAP recipe calls for twice as much 1% ficin as 1% papain without explanation. I found there is no need to use 2x the volume of ficin. I suspect there may have been some issues with the activity of ficin some years ago. We prepare and standardize our enzymes. Both the ficin and papain for this study were purchased from Sigman-Aldrich.  ref: Leger & Garratty. Comparison of papaina and ficin as constituents of ZZAP for adsorption using allogeneic RBCs to remove warm autoantibodies for detection of alloantibodies (abstract). Transfusion 2011;51(Suppl):173A. 

 

Gina Leger