I am going to attempt to validate anti-C3 testing in gel. I will be using Immucor anti-C3 in Ortho's buffered gel cards. I believe others have successfully validated this procedure. Anyone interested in sharing your procedure with me?

If you are lucky enough to have C+DAT it is easy. I used 50uL of 0.8% cells and 25uL of

anti-C3b,C3d. 5 minute incubation at RT; spin for 10 minutes. I also use the Immucor C control cells - make them 0.8%. They are ALWAYS 4+ in gel. Oops - I use the buffered gel card. AND I now use the Ortho anti-Compelment reagent not Immucor. If you need to make C+ cells, I believe there is a method in the technical manual (or somewhere - I bet Malcolm knows). You just compare your tube testing results with your gel results and decide if the gel method is valid.

It just so happens...., but I must confess that this is taken directly from Peter D Issitt Applied Blood Group Serology 3rd edition, 1985, Montgomery Scientific Publications.

1. Take 10mL of a 10% solution of sucrose in distilled water and add either 2 to 3 drops of whole fresh blood (e.g. from a finger *****) or 1 drop of washed packed red cells and 1 to 4 drops of fresh normal serum or 1mL of whole blood collected in ACD or CPD.

2. Mix well and incubate at 37oC for 15 minutes.

3. Wash the cells two or three times in physiological saline.

This makes C3b/C4b-coated red cells. To convert these into C3d (and C4d)-coated red cells.

4. For each drop of packed complement-coated red cells, add 1 drop of 0.1% trypsin in pH7.3 buffered saline.

5. Incubate at 37oC for 30 minutes.

6. Wash the cells three times in physiological saline.

7. Resuspend the cells to an appropriate strength for testing.

:):):)

You never cease to amaze Malcolm!

Edited April 29, 201015 yr by Deny Morlino
Happy fingers

You never cease to amaze Malcolm!

Thank Peter Issitt, not me!

:D:D

David, thanks for the advice. How do you convert the Immucor C Control cells to 0.8%? Are you using Ortho's procedure?

Yes, but I take 50uL of cells, wash 1x with MDP2+ buffer and resuspend with 100uL of buffer. I also run the pt's 0.8% red cells soln with the buffer, just to make certain that I am not seeing any non-specific agglutinination.

We validated Ortho's Anti-C3b/d in gel. Very simple: 12.5 mcg patient's 4% cell suspension (in Diluent-2) plus 25mcg Anti-C3b/d in a buffer card. For patient control, add 12.5mcg patient cells into another well without adding the Anti-C3b/d.

We do make up our own Complement Coated cells using an old Red Cross Procedure Manual. It's easy and it works!

For QC, we run the Complement Coated Cells each morning as a positive control. We run a reagent cell as a negative control.

Thanks again David! I think I have all the information that I need.

So I got some buffered gel cards to run anti-C3b C3d for complement testing in a DAT.  How many samples do I have to do to validate this method?  I ran the last DAT survey from CAP and it worked out perfectly.

For those of you who use this method, do you run controls with each sample, since you have to remember to add the antibody?  Or do you run a patient control with each sample?  I'm trying to get rid of my poly reagent and complement check cells.

Appreciate any help!

34 minutes ago, mollyredone said:

I'm trying to get rid of my poly reagent and complement check cells.

This is brilliant. Following this thread now.

On 7/21/2016 at 2:02 PM, mollyredone said:

So I got some buffered gel cards to run anti-C3b C3d for complement testing in a DAT.  How many samples do I have to do to validate this method?  I ran the last DAT survey from CAP and it worked out perfectly.

For those of you who use this method, do you run controls with each sample, since you have to remember to add the antibody?  Or do you run a patient control with each sample?  I'm trying to get rid of my poly reagent and complement check cells.

Appreciate any help!

How did your validation work out?