P259 Comparison of two cell preparation methods: Standard Ficoll hypaque vs. Easysep for flow and CDC crossmatching

Abstract

Aim Background: Preparation of lymphocytes from living related and deceased donor for flow and CDC crossmatching is labor intensive and occasionally difficult. It is not uncommon that the number of donor cells and cell viability are inadequate, which makes it difficult to obtain valid crossmatch results prior to transplant. Aim: The aim of our study was to determine efficacy of cell isolation methods using Ficoll hypaque vs. Easy Sep technique. Methods Lymphocytes were isolated from living and/or deceased donor peripheral blood, spleen and lymph node samples using Ficoll hypaque density gradient method and EasySep. The volume of initial samples used for each method was same. We compared cell yield, purity, initial/ final viability, we also evaluated reaction pattern and strength of CDC and flow crossmatches tests using lymphocytes isolated using these two methods. Results The cell yield from peripheral blood using Ficoll hypaque method was twofold higher but less pure by 30–40%. The cell yield and purity from spleen and lymph node were the same for both techniques. The number of T and B cells separated by Dyna Beads using cells prepared by both methods for CDC crossmatch were similar. The strength of reaction for cells prepared by Easy Sep was the same as the current method. However, the reactions were easier to read using cells isolated by the EasySep method due to reduction in number of granulocytes. The initial and final cell viability were the same at 95–98% for both techniques. EasySep enrichment procedure provided more concentrated T and B cells population for flow crossmatch testing. This resulted in better gating of T and B cells for analysis and faster acquiring of the 20,000 events required per well. Conclusions EasySep method is a faster and less labor-intensive method to isolate lymphocytes for the flow and CDC crossmatch assays. The high quality of lymphocyte preparation, with less platelets and granulocytes, makes results easy to interpret. The EasySep method uses negative selection technique to deplete unwanted cells from the cell suspension resulting in no injury or manipulation of the lymphocytes required by the CDC and Flow crossmatch assays. This process can be automated by EasySep bio-robot.