Step 2 — Development of an assay and assay refinement
- Define the specificity of the assay, i.e., what the assay is designed to measure.
- Establish initial optimum conditions of assay, e.g. types of specimens, fixation, antigen retrieval, reagents/components of the assay system, detection system, positive and negative controls, etc. Such assay characteristics have to be established for any proposed technique including molecular techniques.
- Define a scoring procedure and how the data will be reported.
- Assess the consistency of various characteristics of the assay, including expression in different types of specimens (including different organs, normal, diseased), staining patterns (membrane, nucleus, etc.) reproducibility of PCR or other molecular analyses, positive and negative controls, etc.
- Demonstrate intra-laboratory reproducibility under initial optimum assay conditions.
- Estimate prevalence of the marker on an expanded collection of targeted specimens. These specimens should be of the same type (fixed, frozen, etc.) for which the assay is being proposed, and the patient characteristics of this pilot set should be similar to the target patient group. Tissue reference sets would facilitate this step. In some cases, these reference sets can be prepared as tissue micro-arrays.
- If the marker is being evaluated as a prognostic indicator, correlation between the new marker and standard prognostic variables should be reported. This provides a suggestion as to whether the marker is likely to serve as an independent indicator. If it is highly correlated with a known marker, it may add information, but it may also not be of value. If the marker provides equivalent information about the disease but is easier to measure or has better assay characteristics, it may replace the older marker. If it does not correlate, it may provide new information about the disease.
- There should be a preliminary proposal of a clinical question for which the marker might be useful. Some pilot data should be presented supporting this (although statistical significance may not be attainable at this point).