Overcoming the deceptively low viability of CD45+ cells in thawed cord blood unit segments

Abstract

Background and objectives: There is no standard methodology for post-thaw sample preparation for viability analysis of umbilical cord blood units (CBU). A common challenge faced by CB bank is for their product to meet the post-thaw cell viability threshold for CD45⁺ cells set at 40% by NetCord-FACT. The objective of this work was to improve the post-thaw staining method to maximize CD45⁺ cell viability so that clinically valuable samples meet the NetCord-FACT threshold criteria for CD45⁺ and CD34⁺ cell viabilities.

Materials and methods: Samples of CBU buffy coats and CBU segments were thawed and taken for staining. Various parameters were evaluated on CD45⁺ and CD34⁺ cell viability as measured by 7-actinomycin D (7-AAD) staining.

Results: The results revealed that initiating the staining at 20 min post-thaw instead of 30, shortening the red cell lysis treatment, or performing lysis on ice and removing this step all together, all improved the viability of CD45⁺ cells. Using CBU segments, it was shown that the most effective approach in increasing the viability of CD45⁺ cells was the complete omission of red cell lysis step. However, removal of the lysis step can create technical artefacts during flow cytometry acquisition that results in an underestimation of the viability of CD34⁺ cells. This can be avoided and CD34⁺ cell viability restored with additional thresholding on CD45 signal.

Conclusion: CB CD45⁺ cells are sensitive to red cell lysis treatment post-thaw; omission of this step provides the best viability and ultimately better reflects the quality of cells used for transplantation.

Keywords: CD45+ and CD34+ cells; ammonium chloride lysis; cord blood; cryopreservation; viability.