Hemoglobin Colorimetric Detection Kit

ValiDATion DATA Sensitivity and limit of Detection Sensitivity was calculated by comparing the OD's for twenty wells run for each of the zero and standard #7. The detection limit was determined at two (2) standard deviations from the zero along the standard curve. Sensitivity was determined as 0.021 g/dl for the Regular format and 0.020 mg/mL (0.0020 g/ dl) for the High Sensitivity format. The Limit of Detection for the assay was determined in a similar manner by comparing the OD's for twenty replicates for each of the zero standard and a low concentration diluted human sample. limit of Detection was determined as 0.021 g/dl for the Regular format and 0.033 mg/mL (0.0033 g/dl) for the High Sensitivity format.

Standard Preparation - Regular Format Label glass test tubes as #2 through #7. Briefly vortex and spin the vial of standard in a microcentrifuge to ensure contents are at bottom of vial. The Hemoglobin Standard supplied in the kit is standard 1. Pipet 50 μL of Sample Diluent into tubes #2 to #7. Carefully add 50 μL of the Hemoglobin Standard provided to tube #2 and vortex completely. Take 50 μL of the Hemoglobin solution in tube #2 and add it to tube #3 and vortex completely. Repeat the serial dilutions for tubes #4 through #7. The concentration of Hemoglobin in the Hemoglobin Standard vial and tubes #2 through #7 will be 16, 8, 4, 2, 1, 0.5 and 0.25 g/dL. Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Sample Diluent Volume (μl) - 50 50 50 50 50 50 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Volume of Addition (μl) - 50 50 50 50 50 50 Final Conc (g/dl) 16 8 4 2 1 0.5 0.25 Standard Preparation - High Sensitivity Format Label glass test tubes as #1 through #7. Briefly vortex and spin the vial of standard in a microcentrifuge to ensure contents are at bottom of vial. Pipet 525 μL of Sample Diluent into tube #1, and 250 μL into tubes #2 to #7. Carefully add 75 μL of the Hemoglobin Standard provided to tube #1 and vortex completely. Take 250 μL of the Hemoglobin solution in tube #1 and add it to tube #2 and vortex completely. Repeat the serial dilutions for tubes #3 through #7. The concentration of Hemoglobin in tubes #1 through #7 will be 20, 10, 5, 2.5, 1.25, 0.625 and 0.313 mg/mL. Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std 7 Sample Diluent Volume (μl) 525 250 250 250 250 250 250 Addition Stock Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Volume of Addition (μl) 75 250 250 250 250 250 250 Final Conc (mg/ mL) 20 10 5 2.5 1.25 0.625 0.313 Use all Standards within 2 hours of preparation

For Regular Format Whole blood Whole blood must be diluted ≥ 1:2 with Hemoglobin Sample Diluent prior to running in the kit. Red blood Cell/erythrocytes RBC samples should be lysed with Hemoglobin Sample Diluent prior to running in the kit. For High Sensitivity Format Serum and Plasma Serum and plasma samples should be run in the High Sensitivity format without any dilution. Hemolyzed samples can be read in the Regular format. Any samples with hemoglobin concentrations above the standard curve range should be diluted further with Hemoglobin Sample Diluent to obtain readings within the standard curve. Use all samples within 2 hours of dilution.

Allow the kit reagents to come to room temperature for 30 minutes. We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine hemoglobin concentration. Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit. Regular Format 1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. 2. Pipet 10 μL of samples or standards into wells in the plate. Pipet 10 μL of Sample Diluent into the zero standard wells. 3. Add 100 μL of the DetectX® Hemoglobin Detection Reagent to each well, using a repeater pipet. Tap the plate to mix. 4. Incubate at room temperature for 30 minutes. 5. Read the optical density generated from each well in a plate reader capable of reading at 560-580 nm. See spectra on Page 6 for details. 6. Use the plate reader's built-in 4PLC software capabilities to calculate Hemoglobin concentration for each sample. High Sensitivity Format 1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification. 2. Pipet 100 μL of samples or standards into wells in the plate. Pipet 100 μL of Sample Diluent into the zero standard wells. 3. Add 100 μL of the DetectX® Hemoglobin Detection Reagent to each well, using a repeater pipet. Tap the plate to mix. 4. Incubate at room temperature for 30 minutes. 5. Read the optical density generated from each well in a plate reader capable of reading at 560-580 nm. See spectra on Page 6 for details. 6. Use the plate reader's built-in 4PLC software capabilities to calculate Hemoglobin concentration for each sample.

Average the duplicate OD readings for each standard and sample. Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean OD's for the Zero standard. The sample concentrations obtained should be multiplied by the dilution factor to obtain neat values. TyPiCAl DATA - RegUlAR FoRmAT Sample net oD Hemoglobin Conc. (g/dl) Zero 0 0 Standard 1 1.993 16 Standard 2 0.870 8 Standard 3 0.426 4 Standard 4 0.199 2 Standard 5 0.113 1 Standard 6 0.057 0.5 Standard 7 0.028 0.25 Sample 1 0.844 7.64 Sample 2 0.133 1.35