Problem With Gel And Typing In Units

[DBRITT]

We use ABD/Reverse gel cards to type in units that we recieve from ARC. Last week we started having a problem. On the wells that are negative, we see a group of cells that are negative and then about a 2+ reaction above them. We see it mostly in the B well and the control well. I've tried everything I can think of. Tried different centrifuge, different box of cards (same shipment), a new lot of cards seemed to be a little better until our last shipment. New MTS2 diluent, new bottle MTS2 diluent without using dispenser. I pulled units off the shelf that we had previously typed and 2 of the 3 had problems. Nothing we were using was new all had been open for 7-14 days with no problem. 3 different techs have seen this problem, so it's not one person. Qc works fine and patients work fine we only have a problem with units. We even tried spinning the segs down before we use them. Is anyone else seeing a problem? Does anyone have any suggestions? All will be greatly appreciated!

[Yanxia]

Maybe it is because bacteria contamination. Had you change the reverse type cells?

[ovrwkd]

You should not see this type of mixed field reaction with forward types on donor units. Are you performing reverse type on donor segments as this is not only donor plasma? Is this the same lot number of cards you have used in the past or is there any indication you have a different lot of anti-B in the cards?

You could wash the segments 1X before dilution with diluent if MFI allows (I believe this is permissible).

If all else fails call the Ortho hotline, they're open 24/7

[ovrwkd]

I also noted you indicate using diluent 2. Isn't diluent 2plus for ABO?

[Eagle Eye]

Are you using ABD reverse cards or ABD/ABD cards? If you are typing donors, you really do not need to use ABD reverse cards!!!

[David Saikin]

2+ diluent is indicated for specimens not obtained in EDTA, which would include donor retypes. I don't know if this is your solution, but it may help to use the correct diluent (unless you validated yous the dil 2 for all your work).

[larevalo]

Our Antibody identification in gel method(Diamed) shows an exact pattern of Anti-S.So the technician proceeds to antigen typing for S.But the phenotyping results was positive.We checked all our reagents QC,expiration and Patient's ID, all were OK.So the next step was to check for RF(rouleaux formation)There was strong RF and to asked for another sample.With the second sample the staff used tube method(Immucor) .Immediate spin shows weak reactions on some cells but negative on 4,5,7,8,15.But at 37C and AHG Phase all reactions were positive including auto control.DAT was negative, this patient received blood transfusion last 2004. Antibody screening was negative.

Because there was RF we used immediate spin saline replacement technique: the results was negative.

Is there any other technique to solved the problem?

[Melanie]

Rouleaux can really be a problem if you are using Peg enhancement with your tube testing technique. If so, I recommend you choose another enhancement reagent like LISS or make sure that you do extra washes prior to addition of IgG. Although it is said that no rouleauz is detected at the coombs phase, by adding PEG to a patient with abnormal proteins you can cause red cell agglutination without having an antibody present. Extra washes usually will do the trick.

[David Saikin]

Larevalo - how did you f/u you IS saline replacement? Did you add serum for 37C/AHG testing?

[larevalo]

Hi David,the Immediate spin technique for rouleaux formation that we do is by adding 2 drops of serum and LISS to screening cells I ,II, then centrifuge and read for hemolysis.Then we take off the serum and LISS and add 2 drops of saline.Centrifuge and record the results.

[galvania]

Do I get the impression that there are two separate threads that have got mixed up here - one dealing with mixed field reactions in the ABO/Reverse card, and one with rouleaux and anti-S?

For the ABD card, it sounds to me like a problem with your pipetting technique with some anti-A being carried over into the B well and so on. Check the cards. Sometimes during transport, if they are shaken about, small droplets of antiserum can migrate into the well above the gel; then when you pipette the cells in the tip only needs to touch this antisera very slightly, and you have exactly the phenomenon you describe. The other thing is that sometimes the drops form, through condensation on the underside of the aluminium cover. When you remove the aluminium, the drops can 'jump' from well to well. If you know what you're looking for it's quite easy to see. Centrifuging the cards before use usually solves the problem. Hope this helps