Abstract

Background & Aim Enumeration of viable CD34+ cells provides critical information for bone marrow transplant physician. Single-platform ISHAGE protocol is the most reliable method currently available to quantitate accurately this important subset of cells. FACS Canto II clinical software is the IVD cleared and accepted as a reference method because is more streamlined and automated . Agreement between white blood cells (WBC) count obtained from hematology analyzer and absolute CD45+ count is important to accuracy . However, in thawed cryopreserved samples this agreement cannot be achieved because there is an increase in the number of dead cells due to cryopreservation.We attempted to compare the CD34+ cell numbers based stem cell enumeration ISHAGE templates on BD FACS Canto Clinical Software and BD FACSCanto™ Diva Software in fresh and thawed samples. Methods, Results & Conclusion Method During the study period, CD34+ cells enumeration was performed on total of 162 samples granulocyte–colony-stimulating factor (G-CSF)-mobilized peripheral blood. Fresh and post thawed samples were stained using stem cell enumeration kit then acquired and analyzed with Clinical software as well as with Diva Software using ISHAGE template on BD FACS Canto II flow cytometry. Results Comparison of results showed that there was no statistical difference between fresh samples run with SCE kit-based assays with either Diva or Clinical software regarding CD34+ cell viability and count. Mean results for Clinical Software were 1360 cells/µl, 99.2% and for Diva software were 1348 cells/ µl, 99.4 % respectively. Pair-wise comparison of data showed no statistically significant difference and all correlation coefficients had an coefficient R2 > 0.97. However, there was a significant increase in the mean of CD34+ cells viability in the thawed samples that were analyzed using Diva software 76.4% comparing with samples analyzed with Clinical software 73.1%. Moreover, the total number of CD45+ cells in the thawed samples showed an agreement with total white blood cells count obtained from hematology analyzer with Conclusion FACS Canto II Clinical and Diva software generated very similar data on a range of fresh samples. However, for the thawed samples, using Diva Software with more flexible gating feature allowing optimization of the debris exclusion gate during analysis improves the CD34+ cells viability outcome.

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