P32

Trypan Blue accurately defines the viability of CFU-GM cells in Cord Blood Units

Sofia Ulrika Frändberg1,*, Eva Anghem2, Inger Ögärd2, Lars Palmqvist2

1Stem cell laboratory, CLINICAL IMMUNOLOGY AND TRANSFUSION MEDICINE SAHLGRENSKA UNIVERSITY HOSPITAL GÖTEBORG, 2Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine Sahlgrenska Academy, Göteborg, Sweden

Introduction: The Colony forming unit assay is a cultivation assay used to approximate progenitor cell content in donated cord blood units (CBU).

Objectives: Investigation of the accuracy of the viability measurement by TB prior to cultivation.

Methods: CBU were processed to buffy coat (CBB) using the Sepax (Biosafe) and 0. 5 ml of CBB was frozen in a vial in liquid nitrogen. The CBB was thawed and viability measured by 0.1% TB (T8154, Sigma), 200 cells were counted by microscopy and the fraction of stained cells was determined. For each CBB two cultivation protocols were performed in duplicate; one with 50.000 mononuclear (MNC) cells (cNoT) and the other with 50.000 viable MNC as adjusted for the fraction of viable cells measured by TB (cWiT) per ml of medium (Methocult GF H84434, Stem Cell Technologies) 37 CBU were investigated and calculations performed using SPSS (IBM) version 21.

Results: After 14 days the growing Granulocyte-Monocyte colonies (CFU-GM) in both protocols were counted by microscopy. Since only viable CFU-GM form colonies, the number of growing colonies in the cNoT protocol was expected to be lower compared to the cWiT protocol relative the fraction of viable cells in the corresponding sample given that the viability measurement by TB was accurate. We therefore calculated the number of expected colonies (cCalc) in the cNoT protocol as given by; cCalc CFU-GM colonies = (fraction of viable cells by TB * cWiT CFU-GM colonies) The cCalc results were correlated to the number of actually growing colonies in the cNoT protocol. The Pearson correlation between the cCalc colonies and the cNoT colonies was 0.93 (p<0.0001)

Conclusion: An accurate viability measurement is necessary to obtain correct CFU-GM assay results. Herein we show that the TB stain is an accurate method to determine viability of MNC prior to cultivation and thus also CFU-GM cells in CBU prior to release for transplantation and therefore eliminating the need to implement more expensive and time-consuming methodology such as flow cytometry.

 

Keywords: Blood, Method