Reproducibility, protocol testing

In all antiseptic testing, it is recognized that skin and mucous membranes to which products ate appHed cannot be disinfected or sterilized but it is possible to significantly reduce the population of transient and resident pathogenic bacterial flora. AH in vivo test methods requite a deterrnination of the bacteria on the skin before and after treatment. Because of the normal variation in bacterial population of the skin of different people, a number of people must be tested in order to make a statistical analysis of the results. Different parts of the body are used for different tests. In aH of the tests the details of the protocol ate extremely important and must be strictly adhered to in order to obtain reproducible results. 

Dunkel VC, Zieger E, Brusick D, et al. 1984. Reproducibility of microbial mutagenicity assays 1. Tests with Salmonella typhimurim and Escherichia coli using a standardized protocol. Environ Mutagen 6 (Suppl. 2) 1-254. 

Figure 5.1 Diagrammatic explanation of standardization of IHC via AR and test battery to achieve a maximal retrieval level by an optimal protocol of AR. The intensity of IHC (axis y) is inversely correlated with the time of formalin fixation (axis x) as indicated by a reduced slope. Three arrows indicate a potential maximal retrieval level that may equalize the intensity of IHC to a comparable result for routinely processed, paraffin-embedded tissues with various time of fixation. Reproduced with permission from Shi et alHistotechnol. 1999 22 177-192.

The precision values referred to in 1 (iii) shall be obtained from a collaborative trial which has been conducted in accordance with an internationally recognised protocol on collaborative trials (e.g. International Organisation of Standardization Precision of Test Methods )17 The repeatability and reproducibility values shall be expressed in an internationally recognised form (e.g. the 95% confidence intervals as defined by ISO 5725/1981). The results from the collaborative trial shall be published or be freely available. 

The combination of proteolytic enzyme digestion and heat-based treatments has been reported however, this increases the susceptibility of the tissues for disintegration. Significant shortening of the digestion time and reduction of the enzyme concentration should be undertaken when testing such protocols. Furthermore, reproducible proteolytic enzyme treatments require optimal conditions for individual tissues, which make these protocols difficult to perform. 

A simplified approach to assess MU is the JUriess-for-purpose approach, defining a single parameter called the fitness function. This fitness function has the form of an algebraic expression u=f(c) and describes the relationship between the MU and the concentration of the analyte. For example, = 0.05c means that the MU is 5% of the concentration. Calculation of the MU will hereby rely on data obtained by evaluating individual method performance characteristics, mainly on repeatability and reproducibility precision, and preferably also on bias [21,40,41]. This approach can more or less be seen as a simplification of the step-by-step protocol for testing the MU, as described by Eurachem [14]. 

If a stepped ramp is used, the number of points and a point-time interval should be entered. This is calculated to take the same time as above (e.g., 120 to 300 points at one point per second or 240 to 600 points at two points per second). The exact timing of the test is irrelevant unless the experiment is intended to reproduce work done previously, in which case the previous protocol should be adhered to as closely as possible. 

The equilibrium flow test in Basic Protocol 2 is very useful for measurement of time-dependent samples. The nonequilibrium tests in Basic Protocol 1 are much quicker, but can give results that are hard to reproduce if the sample is time dependent. The equilibrium flow test is far longer but may only need to be performed once. 

The design of the validation testing and the composition of the protocol reflect the circumstances under which the study is conducted. For retrospective validation the test may be statistical analysis of batch release data, such as assay, pH, physical appearance, residual moisture, reconstitution time, and constituted solution appearance. This retrospective process validation would be intended to demonstrate that the product is of consistent quality. A critical review of the processing conditions in a retrospective validation may consist of a test comparing actual processing conditions during lyophilization with ideal parameters. This not only shows adherence to the defined processing conditions, but also demonstrates process reproducibility. 

In general, ELISA tests have been implemented in meso-scale instrumentation based on the microtitre plate format, which has become a standard, very widely spread configuration. The analyses are usually performed with a protocol that enables the thermodynamic equilibrium of the immunoreaction to be reached at each step of the assay. In this manner, the capture efficiency is optimised and the obtained results are often very satisfactory in terms of sensitivity and reproducibility. In order to further increase the performances and throughput of these tests, fully automatic robotised stations have been developed, thereby reducing manipulation errors such as dilution or pipetting imprecision, for instance. 

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