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converting 0.8% to 3%


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We use the gel system for most all of our testing. Occassionally we have problem false positives that occur with the gel. When sending out "positive" antibody screens for identification the reference lab blood bank will recommend we re-screen the patient using tubes since they got negative reactions using tubes. Our problem is that we only have the 0.8% cells. We don't do a high volume of blood banking at our hospital and it would not be economical to keep the 3% cells on hand. Does anyone convert their 0.8% cells to 3%? If so, how? and is this acceptable?

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I am in the process of implementing the concentration of the cells to 3% as above. Do you need to do validation studies? Does this process get rid of positive reactions caused by antibodies to the preservative/antibiotic/unknown substance that can, but rarely happens with gels.

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It is not recommneded to convert the 0.8% red cells for gel test to 3% red cells for tube test. You may purchase another set of 3% for tube test or prepare it by yourself using donor blood.

CK Cheng, MSc, SBB(ASCP), CQA(ASQ)

Mar 9, 2010

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When we set up for gel (9 years ago), we were told that 0.8% cells should not be converted to 3% suspensions for tube testing. If you want the ability to go both ways, you could buy 3% cells and convert to 0.8%. We made a batch of 0.8% cells routinely every morning, QC'd them and then used them for 24 hours. The upside was that we stopped seeing most of the funky reactions like you are sending off to the reference lab that turn out to be nothing. Money saver!

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I called Ortho Technical Service for their advice on adjusting 0.8% to 3%. They do have a "procedure" as indicated above. Ortho recommends that this procedure should not become a "routine" process. Also, these cells need to be QC'd using tube method when used for patient testing.

DMR

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Yes, I agreed with AMcCord, you may prepare the 0.8% red cells for gel test from 3% red cells using the diluent provided by the manufacturer, like ID-2, but not the other way round.

CK Cheng, MSc, SBB(ASCP), CQA(ASQ)

Mar 9, 2010

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DMR, that is exacty the information that I got. The concentration of the cells would only be done on a rare ocassion due to a fasle positive due to cold agglutination, or panreactivity with the ortho cells. Warm autoantibody, etc. I defininately would not buy the overpriced ortho 3% cells. So having to revert to concentrating the cells once in 3-4 months would not be a routine process.

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Just wondering:

If the manufacturer has a process, why would it not be recommended?

On the flip side, why would the manufacturer then tell you not to use it 'routinely'?

(What constitutes 'routinely'?)

I have wondered this same thing (converting from 0.8 to 3), but we have just kept a set of 3% around for the Anti-Gel's. Daily diluting of screen cells was never an option-- too busy.

There are also some people that are Anti-Ortho's-- they don't react in gel when using Immucor (diluted) cells...

There's always somebody who doesn't like something!

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I have always understood its not good practise to do this as the 0.8 % cells are in a LISS diluent and then you are going from a LISS to a NIS ( Normal Ioonic strength) at 3% if you use blood bank saline as a diluent for the 3% cells. I understand its ok to go the other way i.e. NIS to LISS. it doesent have anything to do with the cell suspension just the diluent. There are some commercially available diluents that are LISS and could be used to go from 0.8 to 3% and keep a LISS diluent throughout but i cant mention them here.

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It is also NOT surprising that the reactions found in gel were not demonstrable in tubes . . . My experience is that gel reactions of 2+ or weaker are invariably negative in tube testing (even enzyme pretreated with PeG). This was one of the determining factors for me switching to gel . . . facilities I did reference work for were sending me antibody id's and I was finding no reactivity. Like AMcCord says, get 3% cells and dilute them. I don't get screening cells like this but I do purchase 2 panels that are this concentration.

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Actually all you have to do is add your enhancement solution once converting 0.8% to 3%. After concentration you can use what ever enhancement you want. 2 drops LISS, PEG, Albumin, NISS, so it is not about the enhancement, you add that to your cell solution. 2 drops patient plasma, 2 drops enhancement. Not to mention you can just add 1 drop LISS following decanting the 4 drops of washed cells.

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:)Centrifuge 4 drops of 0.8% for 30 seconds. Remove supernant. Respend with 1 drop of saline. We use this frequently as needed.

yes mary is right but LISS is better option to use

Edited by sona
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It is also NOT surprising that the reactions found in gel were not demonstrable in tubes . . . My experience is that gel reactions of 2+ or weaker are invariably negative in tube testing (even enzyme pretreated with PeG). This was one of the determining factors for me switching to gel . . . facilities I did reference work for were sending me antibody id's and I was finding no reactivity. Like AMcCord says, get 3% cells and dilute them. I don't get screening cells like this but I do purchase 2 panels that are this concentration.

Totally agree. We also buy the .8% screen cells and 3% panel. We originally got the .8% panel, but after several issues, I switched to an Immucor 3% panel. Although we have to dilute the cells for the gel panels, there are times when we want 3% cells. We still do some of the antigen types in tubes so we don't have to concentrate the cells for the positive and negative controls. We have had several weak reactions in gel panels where a quick selected cell panel at room temperature was a perfect anti-M. I can also do a selected cell tube panel when I have an ABO discrepancy faster than I can concentrate the screening cells.

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Surely a lot depends on exactly WHY you want to do this in the first place. I sometimes recommend that labs concentrate up their ABO cells so that they can do a tube reverse group if they had unexpected negatives with the reverse group in gel. By repeating in tube, they can increase the volume of plasma used to a significant degree, and incubate for hours at 4°C if necessary. But I don't see how this would help with your antibody screen. If you think the problem is due to the buffer present in the cells, then just concentrating them up is not going to help. If you think the gel is just too sensitive - then yes, you'll probably get less positives in tube - especially if you shake the tubes REALLY hard before you read them.................

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Before we send out any workups to a reference lab we first run a gel panel. If the auto control is negative and all allo-antibodies are ruled out, with one or more positive cells, we will call it a non specific antibody, so full crossmatching is done, a patient safety issue. I know gel has been an issue with, a lot of non specific reactions, but keep in mind gel is much more sensitive than tube method so you cannot just dismiss a positive gel result by getting a negative tube result. Then there are patients in which everything is positive in gel so we tag them to use tube method only, this may be due to an antibody to the gel itself. We do alot of workups. Cold antibodies do get detected in gel. If we do get Mixed field reactions we convert to tube method to rule out colds and rouleaux. If we get a positive auto control in gel, we repeat it by tube method and if still positive in tube we then send it out to a reference lab for warm auto antibody workup. These are just a few things we do. I have a whole SOP just dedicated to working ABI in gel. Even though it may be some cost we do keep 3% screen cells and 1 3% panel on hand. This has saved us the more costly sendouts to a ref lab.

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It is always a good idea to have at least one set of 3% screening cells. Even if you are only buying one set a month. You can always dilute to 0.8% and use in Gel testing system to avoid waste. It is even a good idea to have the 3% set from a different vendor. It allows more flexibility and is worth the cost in our experience.

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An advantage to purchasing the 3% solutions and diluting to 0.8% daily ... avoid contaminating the primary source of screening cells. Our usage was so close to using up a set of 0.8% reagents each month we ran the risk of running out of reagent cells before the next standing order shipment arrived. We also had some contamination issues of the primary bottles of reagent. Switching to the 3% solutions allowed us the breathing room we needed, provides a ready 3% solution for our tube backup method, and allows the primary reagent vials to be placed back into the refrigerator instead of remaining on the bench for extended periods. If the daily suspension becomes suspect, pitch it and dilute a fresh set, perform QC, and continue. Many advantages to starting with a 3% solution for our facility.

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  • 4 years later...

I'm not sure I would agree with this. I have seen all positive reactions in gel testing but negative in say "solid phase". Could be a method dependent antibody but not necessarily an auto. (ie media used in diluent, gel cards, etc.)

Everything positive in gel, is often due to an autoantibody.

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Ortho has laminated documents they can give you that have specific "formulas" on them for converting from 0.8% to 3%; or 3% to 0.8%.  But the initial instructions given above are accurate...

But when it comes to making 0.8% from 3%, I follow a more exact formula (place 1 ml Diluent 2 in a test tube; in a separate tube, centrifuge a few drops of your 3% cells; then using a 10ul pipette, take 10ul of the packed cells from this centrifugation and add it to your 1ml). Also just an "obsessive/compulsive" note when doing this.....10ul is a very small amount; so I remind staff that they should wipe off the outside of the pipette tip before dispensing the cells into the tube of diluent.

Brenda Hutson

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Another method to concentrate 0.8% to 3% is to use  the MTS pipet set to deliver 0.5uL.  Pipet 6 - 0.5uL parts of the 0.8% in a glass test tube.  Centrifuge for 30-60 seconds.  Decant all of the supernatent.  Add 2 drops (maybe 1 depending on how "fat" the drop) of saline until it is 2-5% by appearance. We only concentrate 0.8% cells when using expired panel cells for antibody ruleouts and it is necessary to verify the antigen expression is acceptable.  The panel cell must be 3% when testing one drop with an antisera to the specific antigen desired.  Our acceptible minimum agglutination is +2 for the cell to be used to ruleout an antibody.

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Another method to concentrate 0.8% to 3% is to use  the MTS pipet set to deliver 0.5uL.  Pipet 6 - 0.5uL parts of the 0.8% in a glass test tube.  Centrifuge for 30-60 seconds.  Decant all of the supernatent.  Add 2 drops (maybe 1 depending on how "fat" the drop) of saline until it is 2-5% by appearance. We only concentrate 0.8% cells when using expired panel cells for antibody ruleouts and it is necessary to verify the antigen expression is acceptable.  The panel cell must be 3% when testing one drop with an antisera to the specific antigen desired.  Our acceptible minimum agglutination is +2 for the cell to be used to ruleout an antibody.

 

We use the GEL method and carry both Ortho and Immucor Panels.  So if we want to use some of the Immucor cells for antibody ID with GEL, we have to make them 0.8%.  Also, used for Autocontrol on panel....also if performing GEL crossmatch manually (which we rarely do; we usually put in on the ProVue).

Brenda

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  • 3 weeks later...

Just wondering:

If the manufacturer has a process, why would it not be recommended?

On the flip side, why would the manufacturer then tell you not to use it 'routinely'?

(What constitutes 'routinely'?)

I have wondered this same thing (converting from 0.8 to 3), but we have just kept a set of 3% around for the Anti-Gel's. Daily diluting of screen cells was never an option-- too busy.

There are also some people that are Anti-Ortho's-- they don't react in gel when using Immucor (diluted) cells...

There's always somebody who doesn't like something!

The reason the method is not recommended is because the manufacturer does not have sufficient data to support the use and what they are telling you is essentially an off label use for their product.  So legally they cannot make an official recommendation but they can tell you what is technically possible and that you need to validate it or QC it yourself.  Even then I would be shocked to see them put this into writing.

 

Undoubtedly, this works fine but manufacturers are constrained by regulations as to what they can recommend as the use for their products.

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