Calculation

Calculate an assay document once you have completely set it up. Results of the calculation are added to the assay document.

Tip: Set up suitability tests to check the validity of calculations every time they are performed.

The following figure shows the steps that are performed when calculating an assay document and the document elements you use to define the calculation logic:

Common steps in the calculation of Dichotomous assay documents
Figure 1. Common steps in the calculation of Dichotomous assay documents

Data transformation

Specify the logarithm base you want to use for transformation of the dose or response. The following logarithmic transformations are available:

  • Binary logarithm (base 2)

  • Natural logarithm (base e)

  • Decimal logarithm (base 10)

Regression analysis

Regression analysis runs two regressions using a full and a restricted model. The difference between full and restricted model is usually used for suitability testing.

  • The full regression model finds the best fit for the system. Regression parameters are defined separately for the Standard and each Test sample.

  • The restricted regression model assumes perfect similarity/parallelism and is used to estimate the relative potency. The restricted model approximates the Standard sample and Test samples together. Only the regression parameter used to determine relative potency is assumed to differ. Other regression parameters are assumed to be equal in the Standard and Test samples.

PLA 3.0 currently supports the probit and the logit models.

Tip: Use simultaneous regression, if you want to address incomplete data in individual assay elements. Dichotomous assays normally perform all assay regressions one by one, that is, all regression is done in pairs. In the case of simultaneous regression, all regressions are performed together in a single model.

Analysis of variance

Use Analysis of variance (ANOVA) to determine sources of variation in statistical models. ANOVA has several methods to estimate variance in the data. You can define a value for the theoretical variance other than 1, if you require.

Potency calculation

The primary goal of dichotomous assays is to calculate relative potencies of Test samples compared to a Standard sample. You can also determine absolute potencies if you provide information on stock solutions or raw materials. In this case, assays still calculate relative potencies first and then perform calculations to determine absolute potencies.

Note: To correctly calculate stock solutions, you have to provide the predilution factors of all assay elements.
The dilution model follows the path depicted in the following figure:
Dilution model applied to a Dichotomous assay document
Figure 2. Dilution model applied to a Dichotomous assay document

Definitions of raw materials and stock solutions are optional and not always provided. PLA 3.0 can perform various calculations to derive absolute potency. The type of calculation PLA 3.0 uses depends on the potency information you provide.

The following tables show the most common use cases.

No absolute potency calculation

No absolute potencies are calculated if you provide a stock solution or raw material potency for the Test sample but not the Standard sample:

Standard sample Test sample
Raw material potency Stock solution potency Raw material potency Stock solution potency
- - - -
- - - yes
- - yes -

Calculation of absolute potency for stock solution

Absolute potencies are calculated for stock solutions if you provide one of the following combinations of potency information:

Standard sample Test sample
Raw material potency Stock solution potency Raw material potency Stock solution potency
- yes - *
- yes - yes
- yes yes -
yes - - *
yes - - yes

* If you provide potency information on the raw material or stock solution of the Standard sample and do not provide any potency information on the raw material or stock solution of the Test sample, the stock solution potency of the Test sample is assumed to be 1.

Calculation of absolute potency for raw material

Absolute potencies are calculated for raw materials if you provide a raw material potency for both the Standard and Test sample:

Standard sample Test sample
Raw material potency Stock solution potency Raw material potency Stock solution potency
yes - yes -