33-P : A NEW WAY TO QC DTT

Aim Our laboratory uses DTT treated sera for flow crossmatches because of possible IgM interference with the crossmatches. We always QC’ed the DTT serologically using IgM and IgG reagents we purchased commercially. However, when we phased out serologic crossmatches we were faced with the challenge of effectively QC’ing the DTT without the use of serology. Methods DTT works by breaking the disulfide bonds of the IgM molecule and separating it into five subunits that are no longer able to bind. Using this principle, we decided to try an inhibition study to test for the presence and then absence of the IgM molecule. We purchased rat anti-human CD52 IgM Campath and rat anti-human CD52 FITC conjugated IgG. We treated the IgM Campath with DTT for 30 minutes at 37°C. We then performed a flow crossmatch comparing untreated cells with cells incubated with Campath and cells incubated with DTT treated Campath. We measured the amount of FITC bound to each population of cells to determine whether the Campath was still intact and was taking up the binding sites or if it had been destroyed by the DTT. Results The amount of FITC bound to the Campath treated cells was very low, indicating that Campath was bound to the CD52 receptors on the cells, leaving the FITC unable to bind. The amount of FITC bound to the untreated cells was similar to the amount of FITC bound to the DTT treated Campath cells, indicating that the DTT had broken the disulfide bonds of the Campath and inactivated the molecule.[figure1] Conclusions The use of CD52 IgM Campath and CD52 FITC conjugated IgG has proven to be a simple and effective way to QC DTT without the use of serologic crossmatches.