Abstract
Background and objectives: The aim of this study was first, to analyze the post-thaw progenitor assays usually performed on peripheral blood stem cell autografts and second, to achieve standardization with improved flow cytometric and CFU-GM assays.
Materials and methods: In the first part of the study (n=79), recovery and Intraclass Correlation Coefficient (ICC) of total nucleated cells, CD34 and CFU-GM were analyzed before and after cryopreservation. In the second part (n=20), evaluation methods were modified : the washing step was suppressed in the flow cytometric method and 500 CD34 were plated compared to 4×104 total nucleated cells in the CFU-GM assay. The recovery rates were analyzed and the CFU-GM results were regarded as reliable when 30–100 colonies were observed, according to the manufacturer recommendation.
Results: The analysis of the first part showed an ICC that was perfect for total nucleated cells (0.93), substantial for CD34 (0.67) and fair for CFU-GM (0.25). Median CD34 recovery was 112.6% (29.9–222%). The CFU-GM median recovery was 31.7% (0.19–142%) leading to reliable results for 27 grafts. In the second part, the median CD34 recovery was 85.75% (54–99%). No recovery over 100% was observed. The CFU-GM assay led to 18 out of 20 evaluable autografts when 500 CD34 were seeded, compared to 10 out of 20 when total nucleated cell were seeded.
Conclusion: Avoiding cell washing in the flow cytometric method limited the overestimate of the CD34 percentage. Plating 500 thawed CD34 improved reliability of the results and allowed a better standardization of the assay.