In addition, proficiency panel projects have demonstrated that even after an experiment was done and spots were counted, considerable variation can occur due to the interpretation of raw data from ELISPOT assays (25). As various methods for response determination lead to variable outcomes (36), harmonization of ELISPOT assays has to include the harmonization of response determination as addressed in Chapter 15 in this book.
The assay harmonization efforts conducted over the past 5 years led to the identification of several critical experimental process steps based on the analysis of large, representative data sets. Obviously, any published report of ELISPOT experiments should include sufficient information on critical test variables and process steps (see Note 5). To this end, the MIATA project was launched which addresses the minimal information that needs to be published when reporting results from T-cell assays (see Note 6) (24,37).
4. Integration of Assay Harmonization into the Regular Workflow of Assay Progression
The typical evolution of assay development can be divided into six subsequent steps (Fig. 4):
1) Development
2) Optimization
3) Standardization
4) Prevalidation
5) Validation
6) Implementation
Assay development begins even prior to the first experiment by defining the actual assay (what will it measure, how it will be measured) and the first selection of reagents, materials, and protocol variables. The initial assay test runs are typically followed by systematic benchmarking studies for all (or the most critical) assay variables/steps. Results from internal benchmarking studies can be used to further optimize the protocol. Obviously, investigators in these early stages of assay evolution can benefit considerably from integrating recommendations and guidelines deduced from harmonization efforts (Fig. 4).
Protocol optimization is followed by standardization which is typically achieved by generating and implementing SOPs. Once a working SOP is in place, assay qualification and validation can be tackled which is supported by first describing the purpose and design of planned validation studies and how each of the critical parameters is addressed in detail (validation plan). The prevalidation stage establishes the parameters for qualifying the assay by performing a series of exploratory experiments addressing each of the defined validation parameters. The validation stage involves conducting a series of experiments to determine whether the specifications established during the prevalidation stage can be consistently met.
The organizers of proficiency panels acknowledge that the most advanced labs generally contribute best to harmonization efforts as they can generate robust data sets. Nevertheless, labs with newly developed and nonvalidated assay protocols can also achieve outstanding test sensitivity and performance and thus contribute valuable data sets (Fig. 4).
Obviously, an investigator who has already validated an ELISPOT assay might prefer not to change any assay component as this would ask for time-consuming revalidation of the new protocol. However, in more than one instance, panel participation was regarded as an eye opener and has led to modifications in even long-established protocols (see Note 7).
The increased comparability of results generated across institutions that can be reached by harmonization efforts represents a clear advancement for the scientific community. Without doubt, even investigators who use validated assays within clinical studies can value the possibility to better compare own results with data sets that were generated by peers who use similar antigens and drug formats and treat similar patient groups. In addition, it seems reasonable to argue that participation in proficiency panels can benefit a lab’s assay development independent of its stage. During assay evolution, one constantly needs to compare assay performance to a reference standard. Proficiency panels can offer an alternative reference standard, as described earlier, thus providing a solution to the lack of a true gold reference standard in ELISPOT. Further, participation in proficiency testing projects allows the performance comparison with the field at each step of assay evolution. This contributes to enhanced confidence in optimization and standardization procedures and the actual performance of appointed staff members.
In summary, the output from harmonization activities can help to develop and optimize an assay at early stages of assay evolution. By repetitively comparing the performance of many different protocols, large data sets are generated which can be used to define typical and extreme performance characteristics for the ELISPOT assay. This knowledge can be used to set specifications for assay validation. Finally, even experienced and validated labs can profit from participating in harmonization activities due to the feedback of performance they obtain, which would expose the quality of their assay performance.
注意事项
1. For assays for which no accepted gold standard exists, the feedback may also be expressed as test performance relative to the performance of other panel participants.
2. However, the actual number of antigen-reactive cells remains to be determined.
3. While standardization across the immune monitoring field would be desirable, it has to be recognized that this is not feasible due to a variety of circumstances and testing requirements, and not at last by the ever-present question of which standard is the “best” standard.
4. These refinements are based on the outcome of new panels, during which guidelines are investigated in detail where applicable to provide further guidance to the field.
5. Nevertheless, reports which lack considerable parts of the critical information can frequently be found in the published literature.
6. The immune monitoring field can actively contribute to shaping these guidelines by participating in the public consultation process which can be accessed at the project-oriented Web site (37).
7. The experience from previous proficiency panels revealed that even labs that were effectively using an assay for several years had to face the fact that certain protocol steps used in the field, but not addressed in their own SOP, could induce less background spot production in medium controls and a higher number of antigen-specific spots in the experimental wells.
首页
