P054 EDTA treatment of serum eliminates interference in flow cytometric crossmatches

Abstract

Aim A recurrent problem in our flow crossmatch (FXM) is a skewing, or shift to the left, of the T cell population, indicating interference by some component of patient serum. The suspected interfering agents are high levels of complement proteins or IgM antibodies. Previously, we have tried to correct this problem using heat inactivation and spin column treatment of the serum, or running the FXM with nonpronase treated cells. However, each of these options had drawbacks and did not consistently provide accurate results. Using nonpronase cells reduces sensitivity and causes occasional false positive results. Ethylenediaminetetraacetic acid (EDTA) has been shown to eliminate interfering substances, specifically complement proteins, in patient serum that causes a prozone or blocking effect during antibody testing, especially those patients with high titer antibodies (Schnaidt M, et. al. Transplantation, 2011). We report the use of EDTA as a pretreatment of patient serum to remove interfering agents in the T cell FXM. Methods Three color FXM using the Halifax protocol were performed with pronase treated (15 min/37 °C) donor cells. For FXM showing skewed T cells, sera were pretreated with EDTA (95 μl serum + 5 μ l EDTA) and then added to cells as in standard FXM. Results Twelve patient serum samples showed significant T cell skewing in FXM against seven different donor cells, but this shift to the left was eradicated by EDTA treatment in all of these FXM. For example, in one FXM there was a reduction in gated CD3 + T cells from 80% in the negative control serum compared to 15% in the neat, untreated patient serum – but when this patient serum was treated with EDTA, the CD3 + T cell population was restored to 80%. Furthermore, in this particular FXM, there was a difference in FITC median fluorescence intensity (MFI) of 120 channels when comparing the neat to the EDTA treated patient serum. EDTA treatment did not affect negative and positive control sera as the CD3 + T cells and FITC MFI levels were unchanged in FXM comparing EDTA to untreated serum controls. Conclusion EDTA treatment of serum is an easy, quick, inexpensive and effective way to remove elevated levels of complement proteins, IgM and possibly other proteins from patient sera which interfere with T cell FXM. EDTA treatment provides accurate interpretation of the T cell FXM.