ISHAGE-based single-platform flowcytometric analysis for measurement of absolute viable T cells in fresh or cryopreserved products:: CD34/CD133 selected or CD3/CD19 depleted stem cells, DLI and purified CD56+CD3- NK cells

Considering the growing use of immunotherapeutic strategies in paediatric stem cell transplantation associated with risk of graft-versus-host disease, an accurate method for the enumeration of residual T cells/kg recipient's body weight is of paramount importance. Therefore, we propose a multi colour-flow cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was determined to be at 0.7 +/- 0.5 CD3(+) cells/mu l with a minimum of 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3(+) T cell number of 221 x 10(3) (0.09%, CD34 selected grafts, n = 187), 900 x 10(3) (0.004%, CD3/CD19 depleted grafts, n = 15) and 283 x 10(3) (0.012%, CD3 depleted/CD56 enriched NK-cells, n = 14), respectively. The results differed of those from conventional T cell measurement in cell products after extensive manipulation. Our method provides reliable residual T cell enumeration even at extremely low concentrations.