Standard Curve Form

The standard curve form controls details of fitting the standard curve.  Any changes made apply only to the selected experiment (the one whose graphs are shown).

The first item ("Generate standard curves") causes curves to be fit and the data interpolated.  If un-checked, the raw data in MFI is presented instead.  The checkbox can be toggled to switch between raw and interpolated data.

The second item ("Subtract blank wells...") determines if background subtraction is turned on.   The default is always off.   Background subtraction should be used with caution; negative data must be discarded on a log scale and cannot be used for fitting.   The only strong justification for background subtraction is if the negative control probes show high background levels, indicating non-specific binding of analytes and/or antibodies to FirePlex particles.

The third item ("Show all curves in plots") controls whether all the probes or only the selected probes are shown in the individual and overlay plots.   Normally on, the checkbox can be useful with a 36-plex or 72-plex panel, where the number of plots becomes overwhelming.   When off, the plots are restricted to whatever subset of probes is selected.

Curve fitting is normally done in log space to give equal weight to the low end and the high end of the standard curve.   It is possible to fit in linear space with weights to compensate for the small numerical values at the bottom of the curve; the usual weight is the inverse square of the concentration.  (No weights are needed for log fitting.)

The fifth item "Calculate RoQ from recovery" defines how the region of quantitation is defined.  Normally it is expected that all dilutions above the limit of detection are quantitive, but another commonly used definition is the region where recovered values are within 25% of nominal values. 

For non-immuno assay, e.g. microRNA copy count, the units can be defined in the next field.

Standard Curve Form

The lower part of the form controls how outliers and detection are defined.

The first item "reject points below detection" causes dilutions which are below the detection level not to be used in fitting the curve.

Outliers are either those points whose deviation is 3 standard deviations away from the mean of the other deviations, or those points which are more than a specified percentage from the standard curve.  If the threshold is set as a number > 1, it is assumed to mean a number of standard deviations.   If the threshold is to to a number < 1, it is assumed to mean a fractional deviation.  For instance, a value of 3 would mean outliers are rejected if they are 3 deviations from mean deviation, while a value of 0.2 would mean outliers are rejected if they deviate more than 20% from the curve.

Water samples are used to define the MDD.  If there are many water samples (often 6-8 are used) some analytes may show unusually high values due to accidental contamination.  When there are more than 6 water samples and a measured value has less than a 1% chance of belonging to the same distribution as the other water samples, it is considered an outlier.   The outlier test is applied to each analyte separately.

Some organizations used the water value itself as a point on the curve, with a fictive concentration below the last dilution.  If desired, this type of curve fitting can be turned on by setting a value of, e.g. 10 in the fictive dilution box, indicating water samples should be treated as if they are 10X lower in protein concentration than the last dilution.

Alternatively, the water values can be used to fix the bottom asymptote of the curve (which corresponds to infinite dilution) and only the other three parameters are fitted (the top of the curve, the 50% concentration, and the slope parameter).   While rarely used, this option can be useful if there are no dilutions close to the lower asymptote.

See Also

Standard Curve
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