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/microl 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.
Koehl
U
U
Pediatric Hematology and Oncology, Laboratory of Stem Cell Transplantation and Immunotherapy, University Hospital Frankfurt, Frankfurt, Germany. koehl@em.uni-frankfurt.de
Bochennek
K
K
Esser
R
R
Brinkmann
A
A
Quaritsch
R
R
Becker
M
M
Soerensen
J
J
Bader
P
P
Schwabe
D
D
Klingebiel
T
T
Fischer
J
J
Zimmermann
S Y
SY
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000302:isolation & purification; D018952:Antigens, CD34 / Q000302:isolation & purification; D015925:Cryopreservation; D005434:Flow Cytometry / Q000379:methods*; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D016219:Immunotherapy, Adoptive; D007694:Killer Cells, Natural; D018655:Lymphocyte Count / Q000379:methods*; D017710:Lymphocyte Transfusion; D010455:Peptides / Q000302:isolation & purification; D016176:T-Lymphocyte Subsets
D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides