Validation of the single-platform ISHAGE protocol for enumeration of CD34+ hematopoietic stem cells in umbilical cord blood in a Brazilian center

Abstract

Background

This study aims to validate the single-platform method for enumeration of CD34+ cells, by comparing the performance of two different commercial kits, as well as to evaluate the efficiency of the Accuri^TM C6 cytometer in providing direct counts of absolute cell numbers.

Method

We evaluated 20 samples from umbilical cord blood (UCB), comparing the two different methodologies for enumeration of CD34+ cells: single and dual-platform. For the assessment of the single-platform, Procount and SCE kits were used, both of which use fluorescent beads as a counting reference to obtain absolute CD34+ cells numbers. Moreover, after the acquisition of samples in flow cytometer Accuri^TM C6, following the protocol established for each kit, the number of CD34+ cells was recalculated, considering the cell count provided by the Accuri^TM C6.

Main Results

In our analysis, the results showed a strong correlation between the number of CD34+ cells/μL (r² = 0.77) when comparing the SCE kit and the current dual-platform method. On the other hand, the comparison between Procount kit and dual-platform results showed a moderate correlation for the number of CD34+/μL cells (r² = 0.64).

Conclusion

Our results showed that the Accuri^TM C6 flow cytometer can be used safely, applying both the dual and single platform analysis strategy. Considering the ISHAGE protocol-based single-platform approach, as the most appropriate methodology for CD34+ cells enumeration, our results demonstrated that the SCE kit has great potential for national standardization of UCB samples analysis methodology.

Keywords
Stem cell CD34+; ISHAGE; Flow cytometry; Dual-platform; Single-platform