P009 : REDUCING THE COST OF A ROSETTING METHOD WHICH IMPROVES FLOW CROSSMATCH SENSITIVITY BY REMOVING NEUTROPHILS AND MONOCYTES

Aim To reduce the cost of using RosetteSepTM (RS) Human Total Lymphocyte Enrichment Cocktail (Stemcell Technologies), which improves Flow crossmatch (FC) sensitivity by removing neutrophils and monocytes. Often deceased donor blood neutrophils and monocytes are not completely removed by Ficoll Hypaque (FH) density gradient centrifugation. These cells can compete for patient HLA antibodies. Although RS can remove contaminating cells, the reagent traditionally costs $136.00/FC, this study evaluated adding 85% less RS. Methods Total lymphocyte isolation from Deceased Donor blood using less RS: 1. Use 3–4 ACD tubes, spin for Buffy Coat (BC) at 2000 rpm for 10 min. 2. Add 200 μL of RS to BC tube, instead of 50 μL/1 mL of whole blood. 3. Then follow the FH SOP. Results Seven donor samples (living related, autologous and cadaveric) were used for this study. The Median channels (MC) were unaffected for Negative controls and 0% CPRA patient sera, for example the Neat MC averages were T cell = 6 and B cell  = 13 vs. RS MC averages were T cell = 6 and B cell  = 14. However, MCs were significantly higher for all Positive controls where the Neat MC averages were T cell  = 301 and B cell  = 805 vs. RS MC averages were T cell  = 956 and Ave B cell  = 2842. The dot plots showed a significant reduction in cell contaminants such as Granulocytes. There is a 85% cost reduction compared to the recommended RS procedure, 50 μL RS/1 mL with whole blood vs. 6 μL RS/1 mL of using BC. Conclusions We have demonstrated that adding 1/7 of the normal amount of RS to the FH procedure removes a significant amount of cell contaminants and increases FC sensitivity. This protocol saves the laboratory approximately $115.60/FC (4 ACDs). Further study is being evaluated for high CPRA sera. Table 1. This table shows the comparisons in Median Channel results for 2 donor samples between FH and RS + FH. ID ∗ T CELL ∗ B CELL ∗∗ T CELL ∗∗ B CELL NC1_1 5 6 5 7 PC1_1 260 686 563 2049 Pt1_1 4 6 4 8 NC2_1 6 21 7 18 PC2_1 554 1696 1008 2688 Pt2_1 6 9 7 10 ∗  = Ficoll Hypaque. ∗∗  = RosetteSep.