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The following section is published for information.
The majority of nucleic acid amplification analytical procedures are qualitative (quantal) tests for the presence of nucleic acid with some quantitative tests (either in-house or commercial) being available. For the detection of HCV RNA contamination of plasma pools, qualitative tests are adequate and may be considered to be a limit test for the control of impurities as described in the Pharmeuropa Technical Guide for the elaboration of monographs, December 1999, Chapter III "Validation of analytical procedures". These guidelines describe methods to validate only qualitative nucleic acid amplification analytical procedures for assessing HCV RNA contamination of plasma pools. Therefore, the two characteristics regarded as the most important for validation of the analytical procedure are the specificity and the detection limit. In addition, the robustness of the analytical procedure should be evaluated. However, this document may also be used as a basis for the validation of nucleic acid amplification in general.
For the purpose of this document, an analytical procedure is defined as the complete procedure from extraction of nucleic acid to detection of the amplified products. Where commercial kits are used for part of or the complete analytical procedure, documented validation points already covered by the kit manufacturer can substitute for the validation by the user. Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user (e.g. detection limit, robustness, cross contamination).
Specificity is the ability to unequivocally assess nucleic acid in the presence of components which may be expected to be present.
The specificity of nucleic acid amplification analytical procedures is dependent on the choice of primers, the choice of probe (for analysis of the final product) and the stringency of the test conditions (for both the amplification and detection steps).
When designing primers and probes, the specificity of the primers and probes to detect only HCV RNA should be investigated by comparing the chosen sequences with sequences in published data banks. For HCV, primers (and probes) will normally be chosen from areas of the 5' non-coding region of the HCV genome which are highly conserved for all genotypes.
The amplified product should be unequivocally identified by using one of a number of methods such as amplification with nested primers, restriction enzyme analysis, sequencing or hybridisation with a specific probe. In order to validate the specificity of the analytical procedure, at least 100 HCV RNA-negative plasma pools should be tested and shown to be non-reactive. Suitable samples of non-reactive pools are available from the European Directorate for the Quality of Medicines. The ability of the analytical procedure to detect all HCV genotypes will again depend on the choice of primers, probes and method parameters. This ability should be demonstrated using characterised reference panels. However, in view of the difficulty in obtaining samples of some genotypes (e.g. genotype 6), the most prevalent genotypes (e.g. genotype 1 and 3 in Europe) should be detected at a suitable level.
The detection limit of an individual analytical procedure is the lowest amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value.
The nucleic acid amplification analytical procedure used for the detection of HCV RNA in plasma pools usually yields qualitative results. The number of possible results is limited to two, either positive or negative. Although the determination of the detection limit is recommended, for practical purposes, a positive cut-off point should be determined for the nucleic acid amplification analytical procedure. The positive cut-off point (as defined in the General Chapter (2.6.21)) is the minimum number of target sequences per volume sample which can be detected in 95 per cent of test runs. This positive cut-off point is influenced by the distribution of viral genomes in the individual samples being tested and by factors such as enzyme efficiency and can result in different 95 per cent cut-off values for individual analytical test runs.
In order to determine the positive cut-off point, a dilution series of a working reagent or of the hepatitis C virus RNA for NAT testing BRP, which has been calibrated against the WHO HCV International Standard 96/790, should be tested on different days to examine variation between test runs. At least 3 independent dilution series should be tested with a sufficient number of replicates at each dilution to give a total number of 24 test results for each dilution to enable a statistical analysis of the results.
For example, a laboratory could test 3 dilution series on different days with 8 replicates for each dilution, 4 dilution series on different days with 6 replicates for each dilution, or 6 dilution series on different days with 4 replicates for each dilution. In order to keep the number of dilutions at a manageable level, a preliminary test (using, for example, log dilutions of the plasma pool sample) should be done in order to obtain a preliminary value for the positive cut-off point (i.e. the highest dilution giving a positive signal). The range of dilutions can then be chosen around the predetermined preliminary cut-off point (using, for example, a dilution factor of 0.5 log or less and a negative plasma pool for the dilution matrix). The concentration of HCV RNA which can be detected in 95 per cent of test runs can then be calculated using an appropriate statistical evaluation.
These results may also serve to demonstrate the intra-assay variation and the day-to-day variation of the analytical procedure.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
The evaluation of robustness should be considered during the development phase. It should show the reliability of the analytical procedure with respect to deliberate variations in method parameters. For NAT, small variations in the method parameters can be crucial. However, the robustness of the method can be demonstrated during its development when small variations in the concentrations of reagents (e.g. MgCl2, primers or dNTP) are tested. To demonstrate robustness, at least 20 HCV RNA negative plasma pools (selected at random) spiked with HCV RNA to a final concentration of 3 times the previously determined 95 per cent cut-off value should be tested and found positive.
Problems with robustness may also arise with methods which use an initial ultracentrifugation step prior to extraction of the viral RNA. Therefore, to test the robustness of such methods, at least 20 plasma pools containing varying levels of HCV RNA, but lacking HCV specific antibodies, should be tested and found positive.
Cross contamination prevention should be demonstrated by the accurate detection of a panel of at least 20 samples consisting of alternate samples of negative plasma pools and negative plasma pools spiked with high concentrations of HCV (at least 102 times the 95 per cent cut-off value or at least 104 IU/ml).
I Human plasma pools for NAT validation BRP are suitable for use as a negative control.
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