EVALUATION OF METHODS FOR PREPARING AND THAWING CRYOPRESERVED CD34- CELL-LINES FOR USE AS REAGENTS IN FLOW-CYTOMETRY OF HEMATOPOIETIC PROGENITOR CELLS( AND CD34)

Background: Flow cytometry is used to quantitate CD34+ hematopoietic p rogenitor cells for transplantation. The present study evaluates metho ds for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as flow cytometry reagents. Study Design and Methods: The huma n myeloid leukemic cell lines KG1a(CD34+) and K562 (CD34-) were grown in culture under standard conditions and then prepared on ficoll gradi ents of different densities to determine which gave the component that was most reproducible. After ficoll preparation, the cells were froze n in standard cryopreservation media and four methods of thawing were examined. Determination of the method that gave the cell component tha t was most reproducible was based on viability, percentage of cell rec overy, and maintenance of CD34 antigenicity status. Results: Ficoll gr adient preparation improved the ease of flow cytometry analysis when o riginal viability was low, and it produced a more uniform cell populat ion. However, it resulted in significant cell loss for both cell lines . While the cell recovery for K562 cells was not significantly differe nt with any of the densities of ficoll, recovery was significantly bet ter for KG1 a cells with ficoll at a specific gravity of 1.077. Of the thawing methods examined, all three that involved a rapid thaw at 37 degrees C were statistically equivalent to each other and were better than thawing at 4 degrees C. Conclusion: With a standardized method of preparing cell lines as reagents for quality control purposes, data c omparison among cell processing laboratories may more readily be initi ated. Such cell lines could also be useful as a teaching tool for flow cytometry and in proficiency testing.