A diagnostic method and associated test kit for detecting an analyte residing in a test sample is provided. A sample membrane is utilized having a collection region and a detection region, the collection region having a known saturation volume for the intended test sample. A barrier is defined between the collection region and the detection region. The collection region is saturated with the test sample having a volume of less than about 100 microliters so that a known volume of the test sample is contained in the collection region. The barrier is removed from between the collection region and detection region of the membrane and a diluent is supplied to the collection region of the membrane to facilitate flow of the test sample from the collection region to the detection region of the membrane.