Justifying the lack of incurred sample reproducibility in a study Perspective
ISR must also rely on the other aspects discussed in this article.
The other ISR data obtained in the same laboratory Since the assessment of ISR has been a norm for a number of years now, laboratories often have in-house ISR data for a specific analyte. As mentioned in the EMA Q&A paper, this additional data can be used to demonstrate the reproducibility of the assay even if it was not generated for the same study. However, before using this data, one must look at the assay with which it was generated. With the years, the requirements of regulated bio-
analysis have expanded; more validation parameters are required and therefore, less recent assays must be modified to be more sensitive, specific, precise and accurate. The same can be said for technologies. The standards for instrumentation are in constant progres- sion; the assay used for a study performed earlier might have thus been modified over the years for the use of a new type of chromatographic system for example. So, in such a situation, when is the recent in-house
ISR data usable for an older study and when is it not? The answer to this question is not black or white. It all depends on the modifications that were brought to the assay. Modifications that do not alter the storage, extraction or injection conditions should be consid- ered minor. For example, ISR data generated with an assay where only the analytical range is slightly dif- ferent could be used to show that the method used at the time was reproducible. This particular scenario will, for example, happen if the method was validated and used for an older study but then had its analytical range modified and used for more recent unpublished studies. However, any changes that could affect the analyte (or its metabolites) stability, recovery or ion- ization, for example, makes, in our opinion, the use of the recent ISR data inadequate to support studies conducted with the older version of the assay. Figure 1 below illustrates the evaluation process of the in-house ISR data.
The data from repeat analysis There can be three main types of repeats in a bioana- lytical study that can demonstrate the reproducibility of the analysis:
Disregarded repeats As described in the EMA Q&A document, there are the repeats that are automatically generated when, for a multi-analyte assay, samples are repeated for one of the compounds only. In such cases, values are generated for all the analytes quantified by the assay,
future science group
required or not. For the purpose of this paper, we will call this type of values ‘disregarded repeats’. The data obtained from disregarded repeats are the
best substitute to regular ISR data. These results were, in most cases, obtained from fully acceptable analyti- cal runs and thus can be considered as good as any ISRs. It should be noted that, similarly to the ISR obtained from other studies, the disregarded repeats generated during the conduct of a different analyti- cal project (studies conducted for the same program for example) can also be used to demonstrate the reproducibility of the assay. However, disregarded repeats are not too frequent.
As mentioned in the EMA Q&A paper, due to the nature of the reanalysis, other types of repeats may not be reliable and are thus unusable as ISRs. On the other hand, certain of them may be used to as supportive data as detailed below.
Analytical repeats We will call ‘analytical repeats’, the samples for which the initial analysis was judged unacceptable due to measurable factors, for example, the internal standard response, the quality of the chromatography or the limit of the analytical range used. Laboratories are required to have a criterion to
define an acceptable internal standard response. However, this criterion is often established in an arbi- trary manner and does not always truly illustrate a limit of the assay. As mentioned in the Global CRO Council white paper published in Bioanalysis in 2011 [5], a variable IS response may still be part of a method that is delivering accurate and precise results whereas more stable IS responses can be associated with an assay that has issues. With the common use of sta- ble isotope as internal standards, variation in their response is accompanied by an equivalent variation in the analyte response. In such cases, the analyte/inter- nal standard ratio and consequently the concentra- tion obtained is not affected. Therefore, the samples repeated because the IS response in the first analysis was not within the preset margins could be used to show the assay reproducibility in the absence of ISRs. However, it is advisable to explain in the response to the deficiency, why these values are considered as an adequate support. Repeats due to chromatography that did not meet
the laboratory’s standards can also be examined as, in some cases, the chromatographic anomaly may not always affect the exactitude of the quantitation. In all, analytical repeats for which a measurable value was obtained upon the first analysis may be used as a support provided that the rationale behind the use of these analytical repeats is explained.
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