Do you wash your screening cells? For rouleaux? Why?

[trisram]

I had this patient with multiple myeloma. Her antibody screen on gel card had rouleaux reactions for all 3 screening cells. I then washed the screening cells six times with warm saline. The screen was then negative.

Forgive me if this is dumb, but why wash the screen cells? Obviously it worked, but isn't rouleaux a plasma property? That is why we do saline replacement when doing tube cross matches.

I was just wondering if there is a mechanism or principle I am missing here. Thank you for time and reading.

[Malcolm Needs ☆]

I had this patient with multiple myeloma. Her antibody screen on gel card had rouleaux reactions for all 3 screening cells. I then washed the screening cells six times with warm saline. The screen was then negative.

Forgive me if this is dumb, but why wash the screen cells? Obviously it worked, but isn't rouleaux a plasma property? That is why we do saline replacement when doing tube cross matches.

I was just wondering if there is a mechanism or principle I am missing here. Thank you for time and reading.

I will start by admitting that I don't know the answer, but it could be that your patient has developed antibodies against one or more of the antibiotics that are in the liquid suspending the red cells (and which may even be coating the red cells), and that the act of washing the screening cells gets rid of the antibiotic.

This is only a thought, because I have seen patients with such antibodies that react with screening cells suspended in an Alsever's solution (never sure of the spelling - indeed, I'm never sure of any spelling!), but do not react with the same cells suspended in DiaMed Diluent2.

:confused::confused:

[mcgouc]

Is it acceptable to wash the cells with saline and then use the cells on gel? The insert for the 0.8% says to use the cells directly from the vials. If you are washing with saline, aren't you washing off the enhancement?

[janet]

We will wash cells for cases as Malcolm suggested (lloks like the patient is reacting with the anti-microbials/suspension medias in all the manufactured cells)....we always resuspend in the MTS Diluent because you need the LIS solution to use the small volumes and shortened incubation time!

I beleive others have spoken of doing this in another thread where we've been discussing alot of weak/unidentified antibodies lately with the 'new' formulation.

[mcgouc]

I would wash and resuspend with diluent 2. There is another thread about concentrating .8% cells and I posted I had switched from a .8% panel to the 3% panel. . One of the reasons I switched was we had a couple of cases where we were getting reactions with all our .8% screen and .8% panel cells and referring them to our reference lab. They didn't get anything. Since our autos suspended in diluent 2 were negative, I decided to try diluting a 3% panel with diluent 2. Whatever the reason, we have gotten a few patients where the screen cells are positive, but the diluted 3% panel is negative. I first noticed this problem after the new formulation for the .8% cells so I totally agree with Malcolm that it might be the antibiotic they use now in the .8% cells. On the ones where the diluted 3% panel was negative, I did try washing the screen cells with the diluent 2 and testing them and it made the reactions weaker. Since I had done a gel and PEG panel and there was no reactivity in either of those, we just called it a reagent problem with the .8% diluent.

[trisram]

Is it acceptable to wash the cells with saline and then use the cells on gel? The insert for the 0.8% says to use the cells directly from the vials. If you are washing with saline, aren't you washing off the enhancement?

what enhancement? I thought that was the clear liquid in the gel cards

[trisram]

I would wash and resuspend with diluent 2. There is another thread about concentrating .8% cells and I posted I had switched from a .8% panel to the 3% panel. . One of the reasons I switched was we had a couple of cases where we were getting reactions with all our .8% screen and .8% panel cells and referring them to our reference lab. They didn't get anything. Since our autos suspended in diluent 2 were negative, I decided to try diluting a 3% panel with diluent 2. Whatever the reason, we have gotten a few patients where the screen cells are positive, but the diluted 3% panel is negative. I first noticed this problem after the new formulation for the .8% cells so I totally agree with Malcolm that it might be the antibiotic they use now in the .8% cells. On the ones where the diluted 3% panel was negative, I did try washing the screen cells with the diluent 2 and testing them and it made the reactions weaker. Since I had done a gel and PEG panel and there was no reactivity in either of those, we just called it a reagent problem with the .8% diluent.

which one is diluent 2? is that the mts diluent?

[trisram]

Wow, if Malcolm doesn't know the answer, it probably doesn't exist

[David Saikin]

there are 2 diluents in the US ... MTS 2 and MTS 2+. The 2+ is for cells not collected in EDTA.

[Malcolm Needs ☆]

Wow, if Malcolm doesn't know the answer, it probably doesn't exist

It is what we use in Europe as one of the cell suspension media that is supplied by DiaMed. It is called DiaMed ID-Diluent 2. It is a modified LISS for red cell suspensions used in their gel system.

That is very flattering, but I don't know everything, believe me!

:D:D:D:D

[Ellie Ross]

At my facility I created an SOP called Anitbody Indentification Guide ever since we went to gel and had all these problems. What we do if we see Mixed Field in gel is repeat the antibody screen using 3.0% cells and continue on using tube method. i.e Saline replacement is used for determining Rouleaux, it is not a method for crossmatching. Or Cold agglutinins. BE CAREFUL about using WARM Saline for saline replacement, you could be washing away an antibody. ROULEAUX is due to the high proteins in the myeloma patient and is a nuisance but must be careful of your methods as not to miss antibodies. Patients who react in gel due to your discribed problem we tag them to be done by tube method. This has cut down on repeat problems each time they come back for transfusions.

[trisram]

At my facility I created an SOP called Anitbody Indentification Guide ever since we went to gel and had all these problems. What we do if we see Mixed Field in gel is repeat the antibody screen using 3.0% cells and continue on using tube method. i.e Saline replacement is used for determining Rouleaux, it is not a method for crossmatching. Or Cold agglutinins. BE CAREFUL about using WARM Saline for saline replacement, you could be washing away an antibody. ROULEAUX is due to the high proteins in the myeloma patient and is a nuisance but must be careful of your methods as not to miss antibodies. Patients who react in gel due to your discribed problem we tag them to be done by tube method. This has cut down on repeat problems each time they come back for transfusions.

Thanks but you misunderstood what I said. The washing with warm saline procedure was from the people who makes the gel cards. It wasn't for replacement, because there was no plasma to replace and no antibody to wash away. We add the plasma afterward.

Well, this is what happened:

1) We did the gel card Ab screen and it showed rouleaux in the gel.

2) we read manufacturer's procedure of ridding rouleaux by washing screening cells 6 times

3) after washing screening cells, we pipet them into the gel cards and finally add the patients plasma

4) incubate for 15 mins

5) centrifuge gel card

6) Rouleaux all gone

My question is , why wash the screen cells? what are we washing off?

forget the plasma. this is not replacement. we add the plasma after we wash our screen cells with warm saline? why warm saline? why not cold?

like I said, if Malcolm doesn't know the answer, chances are, nobody does

[trisram]

there are 2 diluents in the US ... MTS 2 and MTS 2+. The 2+ is for cells not collected in EDTA.

I don't care about diluents. This still doesn't answer my initial question. Dude, you're a SBB, do you know the answer?

[rravkin@aol.com]

Trisram,

Did you run a known antisera as a control after you washed the cells 6 times? I am asking this qestion because I am wondering if after six washes you may be denaturing most of the potential antigen sites contained on the screening cells despite manufacturer's recommendations. Also, there is a reaction buffering issue. MTS diluent buffers the reaction volume within a certain pH range while normal saline may not buffer to within the same range potentially causing missed reactions.

[mcgouc]

what enhancement? I thought that was the clear liquid in the gel cards

The OCD 0.8% Surgiscreen cells are suspended in a low ionic strength diluent according to the manufacturer's directions. The limitations of the procedure state "addition of other potentiators to the gel test card is not recommended and affect these test results." The anti-IgG in the gel cards is suspended in a "diluent and buffered gel solution".