The Colony-Forming Assay (CFA)
The colony-forming assays have been used for basic and clinical research for 40 years. With the exception of recombinant growth factors and serum-free conditions, the basic assay procedure has not changed.
Most laboratories use the macro assay shown in the diagram below. In 1982, a mini assay, now called CAMEO™-4, utilizing a 35mm Petri dish, incorporating 4 wells each containing 100µl of culture reagents, was introduced. In addition, cultures of all colony-forming populations were incubated under low oxygen tension, which reduces oxygen toxicity and increases plating efficiency. The mini assay was also performed in quadruplicate. This miniaturization of the colony-forming assay laid the foundation for development of the HALO® Platform.
Despite its worldwide use in basic research, the colony-forming cell assay suffers from a number of drawbacks.
- The assay is NOT a measure of direct proliferation potential; it detects differentiation potential. If detection of a proliferative response is required, the traditional colony-forming assay is NOT the assay of choice.
- There is a complete lack of standardization in colony enumeration procedures, despite the availability of colony atlases.
- Manual enumeration of colonies is highly subjective.
- Lineage and species comparisons are difficult to perform, as are studies involving large numbers of test compounds or other samples because of,
- Low-throughput of the assay procedure.
- Due to manual enumeration of colonies, the assay is time consuming.
- Due to the lack of standardization and subjectivity of the manual enumeration process, a high degree of technical expertise is required involving between 6 to 12 months of training.
- The time and personnel costs required for manual enumeration means that the assays are expensive to perform.
To rely on a subjective, non-standardized, non-validated assay for toxicity, safety and/or risk assessment purposes was unacceptable to HemoGenix® and should be unacceptable at any stage of drug development and particularly as a stem and progenitor cell quality control assay.
It is for this reason why all of these drawbacks and disadvantages were taken into account during the development of the HALO® Platform and why HemoGenix® has also developed and introduced the first standardized colony-forming cell assay procedure called CAMEO™-96 STD.
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Compare different HALO® Formats with the Colony-Forming Assay
