DAT/C3d performed in gel

[Peggy]

Curious...Anybody performing DAT/C3d using gel? Currently we are using gel combo card(IgG/C3d) when performing DAT's. When positive, we perform an IgG gel DAT and tube test C3d. I have been doing correlation of tube method C3d vs gel. C3d gel is done with typical gel methodology using a buffered gel card, adding cells and anti-c3d, spin. The controls and patient results are good, and gel results are an easy read. We use the Coombs complement control cells for our positive control and selectogen for our negative control. I believe a weak C3d is easily missed in tube testing and using gel prevents this. Any thoughts or current users???

[David Saikin]

I use the diluent as my negative control (with the patient ceills). I originally ran the C check cells with diluent too but am removing that after 12 months of experience. I like the complement testing with the buffered gel card.

[Peggy]

Thanks David, glad to see others are using gel for complement testing. By dropping the negative control and complement coombs control cells, are there any issues with CAP? How do you control?

[LaraT23]

I am curious about the complement control cells also, or do you mean you just don't do the C3d testing now?

[David Saikin]

I still run the complement control cells. I used to run my patient and control cells with just the diluent also (so that I would feel better in case there was something non-specific going on). I am still kind of new to gel. However, after a year of doing so I am no longer running the control cells with the diluent. My complement DAT is pt and control cells with anti-C3b,-C3d and my patient with diluent only (negative control).

[NedB]

We have been using gel a good while, and do our AHG antigen typing on gel which is still not endorsed by Ortho; but I don't know about using gel for C3 activity as you describe. Do you incubate 5 minutes first, or is the 10 minute centrifugation your incubation? Do you follow the reading with addition of EC3d cells to control for false negatives? We do tube testing for our DATs because our experience has been that a positive DAT by tube correlates well with being able to elute antibodies from the cells, whereas a positive autocontrol on gel does not.

[David Saikin]

Ned - we are talking anti-Complement activity here. I run the patient and the C check cells at the same time in the buffered gel. I do a 5 minute incubation prior to the centrifugation step. I also use the anti-IgG card for that portion of the DAT. We stopped running an auto control with our routine absc. The only time we run an auto is if we are doing an antibody ID. I have had good luck with eluates following + IgG DAT in gel.

[Peggy]

Thanks Ned, The non endorsment by Ortho was of concern to me. For that reason we performed correlation studies for about one year prior in order to validate the test system. The results were good. We do incubate for 5 minutes prior to spinning. We do not add cells to the negatives, in exchange, we run a positive and negative control with each patient test(s) set up. Prior to DAT/C3d in gel, we had already been performing the DAT IgG/C3d in gel using the Ortho 'combo' card. We have performed DAT/IgG in gel since about 1995. We have had gel for a long time. Currently, we do not do antigen typing in gel. Did you go through a validation process?

[Peggy]

We also have had good luck with eluates using DAT gel as the indicator to perform them

[David Saikin]

Peg - we have validated antigen typing in gel using either the IgG or buffered gel cards (depending on the type of antisera used). Only the anti-Lea did not work, probably becuae it was outdated for a few years.

[NedB]

When we validated antigen-typing in gel we used Patient samples and tested with "heterozygous' cells. At that time we validated Anti-Jka, -Jkb, -Fya, -Fyb, -Lea, -Leb, -S and -s. Since then Ortho changed the Anti-Jka to a non-AHG protocol; and we no longer antigen-type for Lea and Leb. I can't see the expense of using gel when the protocol does not require AHG. For antigen-typing, sensitivity is not an issue; you will verify with an AHG crossmatch anyway. For our positive controls we insist the Techs use a 'heterozygous' cell. One inspector asked us to stop using our screen cells as controls, so now we try to rotate among the panel cells as much as we can.

[SMW]

OK.....maybe it's just because it's Friday morning but I can't keep my fingers off the keyboard on this one. I'm curious what agency the inspector was representing, but more curious about what possible rationale or standard did the "inspector" provide for not using the screening cells as your controls (providing the screening cells had a "heterozygous" representation of the antigen being tested)?

[Mary**]

Why didn't the inspector like using the screening cells?

[NedB]

I was working the evening shift in those days, now I am in a position to write the rules, so to speak. What was relayed to me was that he said we proved the screening cells work, but what about the rest of the cells we get. It didn't seem reasonable at the time, but those in charge made it policy. Over time it became in-grained; then when we switched to gel and stopped getting 3% screen cells, but continued to get at least one 3% panel, that panel became the most-used. Now I tend to stay away from screen cells because we've gotten out of the routine of refrigerating them during the day and some of the antigens may have leached. Not entirely logical, I know - so I probably should no longer blame that poor inspector, but he started it, as my kids used to say.

Actually I should apologize for an error in my original reply. We used commercial antisera, not Patient samples to validate the methods. We used panel cells and had each run by more than one Tech on a particular day, a day we happened to need to run a particular antigen for a particular patient. We wanted this spread out over the whole staff and all shifts.

[SMW]

Seems like the topics (and intent) are getting a little muddy here. Remember, the purpose of running controls when performing antigen typings is to confirm that the antisera is reacting as expected....It is not meant to be a control of the panel/screening cells.