Methods for threshold determination in multiplexed assays
Methods for determination of threshold values of signatures comprised in an assay are described. Each signature enables detection of a target. The methods determine a probability density function of negative samples and a corresponding false positive rate curve. A false positive criterion is established and a threshold for that signature is determined as a point at which the false positive rate curve intersects the false positive criterion. A method for quantitative analysis and interpretation of assay results together with a method for determination of a desired limit of detection of a signature in an assay are also described.
1. A method for determination of threshold values of signatures composing an assay, each signature enabling detection of a target, the method comprising:
providing an assay, said assay comprising signatures, said signatures enabling detection of a target, and for each signature:
determining a probability density function (PDF) of negative samples in the assay;
based on the determined PDF of negative samples, determining a false positive rate curve;
generating a calibration curve of assay response parameter values versus concentration;
generating a plot of false positive rate versus the limit of detection (LOD);
selecting a LOD such that a desired false positive rate is obtained; and
determining the threshold corresponding to the selected LOD.
2. The method of claim 1 , wherein the threshold values are MFI threshold values.
3. The method of claim 1 , wherein the signatures are the primer-probe sets comprising a forward primer, a reverse primer, and a probe.
4. The method of claim 1 , wherein the target is indicative of a disease.
5. The method of claim 1 , wherein the assay is a multiplexed array assay.
6. The method of claim 1 , further comprising:
assigning to each signature the determined threshold as a distinct threshold value;
providing a sample; and
for each signature, comparing the sample with that signature to generate positive samples
if a sample fluorescence intensity-related parameter is higher than the threshold value for that signature and negative samples if the sample fluorescence intensity-related parameter is lower than the threshold value for that signature.
7. The method of claim 6 , wherein the sample fluorescence intensity-related parameter is a median fluorescence intensity (MFI) parameter.
8. The method of claim 6 , wherein providing the sample comprises generating a sample response through flow cytometry.
9. The method of claim 6 , wherein the target is indicative of a disease.
10. The method of claim 6 , wherein the assay is a multiplexed array assay.
11. The method of claim 1 , further comprising:
assigning to each signature the determined threshold as a distinct threshold value;
for each signature, generating a calibration curve of fluorescence intensity-related parameter values versus concentration; and
determining a desired LOD of the signature as the intersection value between the calibration curve of the signature and the threshold value of the signature.
12. The method of claim 11 , further comprising determining a second LOD in addition to the desired LOD.
13. The method of claim 11 , wherein the fluorescence intensity-related parameter values are median fluorescence intensity (MFI) values.
14. The method of claim 1 , wherein the false positive rate curve is plotted as false positive rates as a function of threshold values.
15. The method of claim 1 further comprising:
establishing a false positive criterion; and
determining a threshold for the signature as a point at which the false positive rate curve intersects the false positive criterion.