Statistical assay control

It is assumed that before the analyst has considered using the assay for patient samples, it has been analytically validated (i.e. the recovery, sensitivity, linearity and reproducibility have been determined) and that the method is able to reproduce results, under ideal conditions, so that its variation is sufficiently predictable to apply statistical quality control. 

Scintillation proximity assay Statistical process control... 

D. Rodbard, Statistical quality control and routine data processing (or radioimmunoassays and immunoradiometric assays, C/in. Chem., 20 1255-1270 (1974). 

Unfortunately this type of acceptance sampling is not always applied. One reason is without doubt that just stating the content alone as requirement is not sufficient. A detailed procedure for the assay method is required from which the margins follow or a tolerance interval is given from which the details of the assay method can be derived. Mixing up of these two approaches leads to an unclear situation. In the following subsections the second approach will be followed as it is mostly used in the Statistical Quality Control (SQC) and Statistical process control (SPC) community. 

The specification development process is a data-driven activity that requires a validated analytical method. The levels of data needed include assay precision, replicate process results (process precision), and real-time stability profiles. A statistical analysis of these data is critical in setting a realistic specification. Most often, aggregation and fragmentation degradation mechanisms are common to protein and peptide therapeutics. Therefore, the SE-HPLC method provides a critical quality parameter that would need to be controlled by a specification limit. 

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

The criterion employed for a positive response in this assay is a reproducible statistically significant increase in mutation frequency (weighted mean for duplicate treated cultures) over the concurrent vehicle control value (weighted mean for four independent control cultures). Ideally, the response should show evidence of a dose-response relationship. When a small isolated significant increase in mutation frequency is observed in only one of the two duplicate experiments, then a third test should be carried out. If the third test shows no significant effects, the initial increase is likely to be a chance result. In cases where an apparent treatment-related increase is thought to be a result of unusually low variability or a low control frequency, comparison with the laboratory historical control frequency may be justified. 

The test material, test cells, used, method of treatment, harvesting of cells, cytotoxicity assay, and so on, should be clearly stated as well as the statistical methods used. Richardson et al. (1989) recommend that comparison be made between the frequencies in control cells and at each dose level using Fisher s Exact Test. 

Zhang X. D, (2007) A pair of new statistical parameters for quality control in RNA interference high-throughput screening assays. Genomics 89 552-561... 

Incubate at least four series, cells with three or more different concentrations of the preparation to be examined and the reference preparation in a microtitre plate and include in each series appropriate controls of untreated cells. Choose the concentrations of the preparations such that the lowest concentration produces some protection and the largest concentration produces less than maximal protection against the viral cytopathic effect. At a suitable time add the cytopathic virus to the wells with the exception of a sulScient number of wells in ah series, which are left with uninfected control cells. Determine the cytopathic effect of virus quantitatively with a suitable method. Calculate the potency of the preparation to be examined by the usual statistical methods for a parallel line assay. 

Statistical thinking and practice is very much determined by the regulatory guidelines that are in place. Primarily it is ICH E9 Statistical Principles for Clinical Trials , published in 1998, which sets down the broad framework within which we operate. In 2001 we saw the publication of ICH ElO Choice of Control Group which contained advice on the appropriate choice of concurrent control group and in particular first introduced the concept of assay sensitivity (see Section 12.5) in active control, non-inferiority trials. 

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