A method for preparing a calibration system comprising at least two calibrators for use in an analyte detection assay instrument, the method comprising the steps of: preparing a first calibrator, the first calibrator having a first concentration of an analyte of interest present in a patient's liquid test sample, wherein the first concentration is higher than an average threshold concentration of the analyte of interest present in the patient's liquid test sample; and preparing a second calibrator, the second calibrator having a second concentration of the analyte of interest present in the patient's liquid test sample, wherein the second concentration is lower than the average threshold concentration of the analyte of interest present in the patient's liquid test sample, wherein the patient's liquid test sample is whole blood, the analyte of interest is selected from the group consisting of total hemoglobin, glycated hemoglobin, and combinations thereof, the average threshold concentration of the glycated hemoglobin is from 4% to 8% of a concentration of the total hemoglobin present in the patient's liquid test sample, the first concentration of the glycated hemoglobin is equal to or greater than 8.1%, and the second concentration of the glycated hemoglobin is equal to or less than 3.9%, wherein the second concentration of the second calibrator is decreased below the average threshold concentration of the glycated hemoglobin by a method selected from the group consisting of chemical modification, utilization of at least one solid phase-bound enzyme, utilization of at least one dye, and combinations thereof, wherein at least one of: the chemical modification comprises oxidation of the glycated hemoglobin with periodate; or the at least one solid phase-bound enzyme comprises fructosyl peptide oxidase bound to magnetic particles, or the at least one dye absorbs at a wavelength(s) used to measure total hemoglobin in the assay, but does not absorb at a wavelength(s) used to measure either glycated hemoglobin or the assay blank. preparing a first calibrator, the first calibrator having a first concentration of an analyte of interest present in a patient's liquid test sample, wherein the first concentration is higher than an average threshold concentration of the analyte of interest present in the patient's liquid test sample; and preparing a second calibrator, the second calibrator having a second concentration of the analyte of interest present in the patient's liquid test sample, wherein the second concentration is lower than the average threshold concentration of the analyte of interest present in the patient's liquid test sample, wherein the patient's liquid test sample is whole blood, the analyte of interest is selected from the group consisting of total hemoglobin, glycated hemoglobin, and combinations thereof, the average threshold concentration of the glycated hemoglobin is from 4% to 8% of a concentration of the total hemoglobin present in the patient's liquid test sample, the first concentration of the glycated hemoglobin is equal to or greater than 8.1%, and the second concentration of the glycated hemoglobin is equal to or less than 3.9%, wherein the second concentration of the second calibrator is decreased below the average threshold concentration of the glycated hemoglobin by a method selected from the group consisting of chemical modification, utilization of at least one solid phase-bound enzyme, utilization of at least one dye, and combinations thereof, wherein at least one of: the chemical modification comprises oxidation of the glycated hemoglobin with periodate; or the at least one solid phase-bound enzyme comprises fructosyl peptide oxidase bound to magnetic particles, or the at least one dye absorbs at a wavelength(s) used to measure total hemoglobin in the assay, but does not absorb at a wavelength(s) used to measure either glycated hemoglobin or the assay blank. the chemical modification comprises oxidation of the glycated hemoglobin with periodate; or the at least one solid phase-bound enzyme comprises fructosyl peptide oxidase bound to magnetic particles, or the at least one dye absorbs at a wavelength(s) used to measure total hemoglobin in the assay, but does not absorb at a wavelength(s) used to measure either glycated hemoglobin or the assay blank.