A system, comprising: a test device comprising (a) a label pad (16, 104) comprising: (i) a first population of mobilizable detectable particles for specific binding to an analyte in a sample; and (ii) a second population of mobilizable detectable particles for specific binding to a non-test analyte; and (b) a first detection line (24, 138, 544) at a first position comprising a first binding member configured to capture detectable particles in the first population that have bound to the test analyte; and (c) a reference detection line (26, 142, 470, 538) at a second position that is separate from the first position, the reference detection line comprising a second binding member configured to capture detectable particles in the second population that have bound to the non-test analyte; an analyzer configured to receive the test device, the analyzer comprising an optical system for detection of a signal generated from the first population of particles at the first position and the second population of particles at the second position when each population reaches a specific and separate position on the test device, wherein the analyzer is configured to determine a cut-off value for the analyzer using signal from all or a portion of the second population of particles to determine whether signal from all or a portion of the first population of particles indicates presence of analyte in the sample and to render analyzer detection of signal from all or a portion of the first population of particles insensitive to incubation time over a time period of 3-10 minutes, or wherein the analyzer is configured to determine a cut-off value for the analyzer using a ratio of signal detected from all or a portion of the second population of particles to signal from all or a portion of the first population of particles to determine presence or absence of analyte in the sample. a test device comprising (a) a label pad (16, 104) comprising: (i) a first population of mobilizable detectable particles for specific binding to an analyte in a sample; and (ii) a second population of mobilizable detectable particles for specific binding to a non-test analyte; and (b) a first detection line (24, 138, 544) at a first position comprising a first binding member configured to capture detectable particles in the first population that have bound to the test analyte; and (c) a reference detection line (26, 142, 470, 538) at a second position that is separate from the first position, the reference detection line comprising a second binding member configured to capture detectable particles in the second population that have bound to the non-test analyte; (a) a label pad (16, 104) comprising: (i) a first population of mobilizable detectable particles for specific binding to an analyte in a sample; and (ii) a second population of mobilizable detectable particles for specific binding to a non-test analyte; and (i) a first population of mobilizable detectable particles for specific binding to an analyte in a sample; and (ii) a second population of mobilizable detectable particles for specific binding to a non-test analyte; and (b) a first detection line (24, 138, 544) at a first position comprising a first binding member configured to capture detectable particles in the first population that have bound to the test analyte; and (c) a reference detection line (26, 142, 470, 538) at a second position that is separate from the first position, the reference detection line comprising a second binding member configured to capture detectable particles in the second population that have bound to the non-test analyte; an analyzer configured to receive the test device, the analyzer comprising an optical system for detection of a signal generated from the first population of particles at the first position and the second population of particles at the second position when each population reaches a specific and separate position on the test device, wherein the analyzer is configured to determine a cut-off value for the analyzer using signal from all or a portion of the second population of particles to determine whether signal from all or a portion of the first population of particles indicates presence of analyte in the sample and to render analyzer detection of signal from all or a portion of the first population of particles insensitive to incubation time over a time period of 3-10 minutes, or wherein the analyzer is configured to determine a cut-off value for the analyzer using a ratio of signal detected from all or a portion of the second population of particles to signal from all or a portion of the first population of particles to determine presence or absence of analyte in the sample.