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Comparison of volumetric and bead-based counting of CD34 cells by single-platform flow cytometry

Published

Author(s)

Luisa Saraiva, Lili Wang, Martin Kammel, A. Kummrow, E Atkinson, J Y. Lee, B Yalcinkaya, M Akgoz, A Ruf, A Engel, Yu-Zhong Zhang, O O''Shea, M P. Sassi, C Divieto, T Lekishvili, J J. Campbell, Y Liu, J Wang, R Stebbings, Adolfas Gaigalas, P Rigsby, J Neukammer, S Vessillier

Abstract

Background: Over 2000 people a year in the UK need a bone marrow or blood stem cell transplant. It is important to accurately quantify the haematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, in recent years, flow cytometers have become equipped with the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations. Method: In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated. Results: The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method, although separate analysis of each instrument in the study showed 4 out of 9 cytometers returned slightly lower concentrations than the bead-based method. We discuss sources of uncertainty specific to each method that can contribute to discrepant readings. Conclusions: Overall, our study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement.
Citation
Cytometry

Keywords

Heamatopoietic stem cells, CD34 cell concentration, single-platform flow cytometry, volumetric counting method, reference bead-based counting, result comparability, measurement uncertainty.
Created February 20, 2019, Updated January 8, 2020