Use Concentration to select a curve fitting method and configure concentration and response formulas for standards (Figure 7-23).
Figure 7-23Configuring Concentration Parameters
To configure a standard curve and standards:
1. To use a stored Standard Curve select Use stored Standard Curve. Click
Select to select the standard curve. Using the Standard Curve Record Selection Form dialog select the specific record to use as the standard curve or Always use the most recent record from the Database. Click Next
to continue. OR
To use the standard curve definition from another protocol select Copy Standard curve definition from another protocol to this protocol. Click Select
to select the protocol to copy the standard curve from. Click Next to continue.
OR
Select a Curve Type: Point to Point, Linear Regression, Cubic Spline, Four Parameter Fit, or Polynomial. See Table 7-5 more information about curve fitting methods.
Note:Concentration is available for configuration only when selected in General Settings for an Analysis or Quantitation protocol. See Configuring General Settings on page 148.
Note:Standards may be deleted or inserted with a special key stroke combination. Use CTRL+Y to delete a standard and CTRL+N to insert a standard.
Creating and Running Protocols
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Table 7-5Curve Fitting Methods
Method Description Example
Linear regression Construction of a straight line using the least squares method with the highest possible approximation to all standard points. Requires a minimum of 3 standard points.
Point to Point Direct connection of all standard points. Requires a minimum of 3 standard points.
Cubic Spline All standard points are connected by the best fitting curve.
Can only be used for nonlinear and nonsigmoid functions.
Requires a minimum of 3 standard points.
4 Parameter Fit This procedure can be used only to characterize sigmoid curves. The curve is calculated according to the formula:
a = zero dose response (upper asymptote) d = infinite dose response (lower asymptote) c = dose level which results in a response midway between a and d
b = slope factor
Requires a minimum of 3 standard points. The X and Y axes are fixed:
X = logarithm Y = linear
Polynomial Calculates the least squares fit through points using the formula:
Requires a minimum of n+1 standard points, where n is the order of the polynomial.
Multi-Mode Analysis Software User Guide
2. Select the Number of standards to configure. Up to 12 standards may be configured in a protocol. Standards configured in the labware layout are automatically added to Concentration.
3. In Extrapolation, enter a percentage value to extrapolate the standard curve above and below the highest and lowest standard points in the curve, if desired. Extrapolation may be configured for all curve fitting methods.
4. In Y-Axis, select the Base for the axis.
Reduction: the reduced data from the protocol run.
Transformation: the value calculated using the transformation formula configured in Transformation. This option is available only when Transformation is configured in the protocol. See Configuring a Transformation Formula on page 177.
5. In Y-Axis, select the Type of scale for the Y-Axis: linear or logarithmic.
6. In X-Axis, enter a Name for the axis, if desired.
7. In X-Axis, select the Type of scale for the X-Axis: linear or logarithmic.
8. Click Report Options and select the concentration information and data included in printouts:
Definition: curve type, parameters, and statistics, such as intercept and slope.
Graph: the standard curve.
Print in Matrix: the transformed result calculated for each sample will display in a measurement results matrix corresponding to the plate layout.
Print in List: the transformed result calculated for each sample will display in a list of measurement results.
Print Status: status indicating whether or not the well was read successfully.
9. In the Graph Setup tab, edit each Response Formula and the corresponding Concentration. Response formulas may contain any controls, standards, or variables defined in the test, as well as
numerical constants and mathematical operators. A response formula is often simply the value of a measured standard, which is expressed as STD1, STD2, or STD3.
Note:Transformation is the default name for transformation formulas. If a different name is entered in Transformation, that name appears in Base. See Configuring a Transformation Formula on page 177.
Note: Type is not available when configuring a four parameter fit curve.
Note: Type is not available when configuring a four parameter fit curve.
Note:See Mathematical Operators and Functions on page 249 for detailed information about mathematical operators supported by the software.
Creating and Running Protocols
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10.If configuring a linear regression curve fitting method, select the
Validation tab to validate the protocol based on acceptable coefficient of correlation, if desired (Concentration Options Tab on page 183).
Figure 7-24Configuring Standard Curve Validation Parameters
11.Select Linear Regression Check Correlation to enable validation.
12.In Linear Regression Min Correlation, enter the minimum correlation percentage value for the test to be valid.
13.In the Options tab, it is possible to configure an algebraic formula that is applied to the calculated concentration of every well (Figure 7-28). The formula shall include X, and may include mathematical operators, but not relational or logical operators. For detailed information about mathematical operators supported by the software, see Mathematical Operators and Functions on page 249.
Note:The Validation tab appears only when configuring a linear regression curve fitting method.
Note:X represents the result value of the concentration calculation. For example: X/DF => divides the concentration values of every well by the dilution factor (DF), which is entered in the plate layout settings.
Multi-Mode Analysis Software User Guide
Figure 7-25Concentration Options Tab