Colony Forming Unit (CFU) Assay with High Resolution Digital Imaging (HRDI): a Reliable, Standardized Technology for Evaluation of Cord Blood Potency Before and After Cryopreservation.

The Total Nucleated Cell dose (TNC) is the graft characteristic most commonly used as indicator of engraftment potential for CB unit selection. Even thought well standardized, TNC does not reflect the functional state of hematopoietic progenitor cells, an important determinant of their engraftment ability, and therefore, of the CB unit quality. In contrast, CFU is a better graft characteristic since it measures the functional state, repopulation capacity and number of hematopoietic progenitor cells of a CB graft. However, it has not yet been standardized, so that it can be used for comparison of CB unit quality among different CB banks, or for evaluation of cryopreserved units prior to release for transplantation. There is therefore, an urgent need of a potency assay that could allow transplant physicians to select the highest quality unit for their patients.

We sought to evaluate whether our new technology that combines the CFU assay with High Resolution Digital Imaging and colony staining (CFU-HRDI) [ASH 2008;2306], would allow for a better standardization of the traditional CFU assay and the testing of a large number of CB samples in a ”High Throughput” approach. Further, we examined whether the CFU-HRDI can be a reliable method for determination of CBU quality before release for transplantation.

CFU growth was assessed in a total of 7,447 CBU over a period of 14 months, by using the HRDI technology. Samples were obtained after CB processing (AXP® system) and all were evaluated in duplicates. The HRDI system achieves a resolution of 7.6 μM per pixel allowing a clear view of all colonies in the dish with their barcoded IDs. A short one-step colony staining procedure provides an even better definition of CFU-GM/E and CFU-GM by bestowing a specific color on each type. A good correlation was observed after comparison of the CFU-HRDI against traditional microscopy enumeration (R²= 0.95; p<0.001). The number of CFU (average: 3109 10×3 +/- 2100 CFU/CBU) correlated strongly with total mononuclear cell count-MNC (R²=0.66, p<0.001); TNC (R²=0.64, p<0.001), nucleated red blood cells-NRBC (R²=0.44, p<0.001) and especially with CD34+ cell content (R²=0.81, p<0.001) of the CBU.

To evaluate the reproducibility obtained by the use of HRDI, CB samples (n=3) were plated multiple times each (n=24 times) and five operators with different levels of expertise counted and classified CFU in all dishes. CFU-HRDI allowed excellent reproducibility for counting and classification (CFU-GM and CFU-Mixed-Red; R²≥0.9; p<0.001; n=72). Importantly, correlation was good even when HRDI showed CFU growth of several sizes and shapes for different CB units, a feature that increases the complexity of CFU classification and enumeration. Furthermore, to evaluate the effect of cryopreservation on CB quality, a total of 186 attached segments were assessed after thawing for CFU, CD34 and viability (7-AAD). Among those units, n=43 were split in two bags after processing and thus, the respective segments could be compared (R²=0.70, p<0.001). All segments showed good viability (95 +/- 5%; range 71-100). Additionally, the CFU and CD34 cell content of the unit pre-cryopreservation were well correlated when compared to the results of their respective segments (R²= 0.57, p<0.001 and R²= 0.59, p<0.001 respectively; n=58).

Our new strategy (CFU-HRDI) shows reliability and reproducibility and importantly, supports high-throughput implementation as shown by the assessment of more than 7,000 CBU/year. CFU can be objectively classified and enumerated and thus CFU-HRDI can be standardized to be used as a potency determinant of CBU. Digital images can be stored for future review or further classification of CFU colonies if needed for their association with transplant engraftment. The feasibility of CFU growth testing from thawed segments and its correlation with post-processing CFU counts strengthens its use as a practical quality control measure for the cryopreservation process prior to a CBU release for transplantation.

No relevant conflicts of interest to declare.