C3 DAT using buffered gel cards

[griffsh]

I have noticed on the CAP DAT surveys from 2013 that about 12 or so facilities are using buffered gel cards when performing their C3 DAT using anti-C3b/C3d reagent.  We are in the process of validating this method and was wondering if anyone out there who is doing this has a validation/ comparison and SOP to share with us.  We just want to make sure we are on the right track.   We especially want to know what is used for controls.  Thank you!

[David Saikin]

I validated making C3 coated cells (pain in the butt) - you can find a method in any good blood bank book, and Compl Control cells.  I did not run very many samples.  I always run the pt cells with diluent with my complement testing just to r/o spurious agglutination (not required).  I use 50uL of cells and 25uL of antiserum.  One thing is that for a heavily complement coated survey specimen (CAP) the IgG gel card may be a weak 1+.  You know that CAP wanted it to be negative but you need to report your results.  The survey specimens have been getting better in this regard.

[David Saikin]

For controls I run a purchased complement coated cell - usually 1+ in tubes, always 3-4+ in gel.  You have to make them 0.8%.  I run the pt cells with diluent as stated in the previous post.  I qc my anti-C3b,-C3d with a positive and  negative cell prior to use.

[cthherbal ☆]

We do 3-part DATs in gel only. Polyspecific, IgG and Buffer cards. Beautiful reactions, clear cut.

For the buffer cards we do 50 uL cells (0.8%) and 25 uL of the anti-C3b, C3d (Immucor).

Pos QC = Complement Control Cells

Neg QC = A2 cells.

We normally use Immucor Complement Control Cells but with the recent shortage, we validated Hemobioscience which works just as well.

No problems with CAP surveys.

[Cathy]

We use the same volumes as above.  Positive control is Complement Check Cells diluted to 0.8%; negative control is a 0.8% screening cell. We let the card sit 5-10 minutes prior to spinning.  If the AHG, IgG and complement reactions are all positive, we run a patient control in the buffer card.  We require the positive and negative controls to be run with each complement test in the buffer card since we have to add the reagent in that card. 

[griffsh]

We use the same volumes as above.  Positive control is Complement Check Cells diluted to 0.8%; negative control is a 0.8% screening cell. We let the card sit 5-10 minutes prior to spinning.  If the AHG, IgG and complement reactions are all positive, we run a patient control in the buffer card.  We require the positive and negative controls to be run with each complement test in the buffer card since we have to add the reagent in that card. 

 

We do 3-part DATs in gel only. Polyspecific, IgG and Buffer cards. Beautiful reactions, clear cut.

For the buffer cards we do 50 uL cells (0.8%) and 25 uL of the anti-C3b, C3d (Immucor).

Pos QC = Complement Control Cells

Neg QC = A2 cells.

We normally use Immucor Complement Control Cells but with the recent shortage, we validated Hemobioscience which works just as well.

No problems with CAP surveys.

Thank you so much for your response! I'm glad to hear this method is working so well for you!  I have a few questions if you don't mind answering.  

1) We were wondering why you would use a Polyspecific card in addition to the IgG and Buffer card.  

2) Could a 0.8% Selectogen be used instead of A2 cells for your Neg QC?  

3) Do you run a patient control if all are positive?  Do we need to worry about spontaneous agglutination in the buffered gel card?

4) When you validated did you make up your own complement coated cells or did you use the Immucor Complement Control cells.  Did you make dilutions to represent different reaction strengths?  How many did you run? 

[griffsh]

I validated making C3 coated cells (pain in the butt) - you can find a method in any good blood bank book, and Compl Control cells.  I did not run very many samples.  I always run the pt cells with diluent with my complement testing just to r/o spurious agglutination (not required).  I use 50uL of cells and 25uL of antiserum.  One thing is that for a heavily complement coated survey specimen (CAP) the IgG gel card may be a weak 1+.  You know that CAP wanted it to be negative but you need to report your results.  The survey specimens have been getting better in this regard.Thank

Thank you for your response.  Did you make varied strengths of C3 coated cells? Which blood bank book did you find a method for making them?  I checked the Technical Manual and didn't see the method for making them (but I could have missed it).