Drabkins Reagent For Hemoglobin Quantification

Protocol Overview

Set the spectrophotometer wavelength to 540 nmand the absorbance to zero using water as thereference.2. Set up a series of labeled test tubes for Blank andTests.3. To all tubes, add 5.0 mL of the Drabkin’s Solution.4. To each tube labeled Test, add 20 mL of the wholeblood sample, rinsing the pipette 3–4 times withreagent. Mix well and allow to stand for at least15 minutes at room temperature (18–26 °C).Note: Samples with appreciable carboxyhemoglobincontent may require a longer reactiontime under these reaction conditions. Alternatively,it has been suggested that in those cases,warming the reaction mixture at 56 °C for 3–5 minutes with gentle mixing will bring the reactionto completion.5. Read and record absorbance (A) of each Testversus the Blank as the reference at 540 nm in thesame instrument used to prepare the calibrationcurve.6. Determine the total hemoglobin concentration(mg/mL) of each Test directly from the calibrationcurve. Color is stable for several hours.Calibration Curve1. Prepare a Cyanmethemoglobin Standard Solutioncontaining 180 mg/mL of appropriate hemoglobinprepared in Drabkin’s Solution. Store tightlycapped and refrigerated (2–8 °C) in the dark.2. Prepare a Dilute Cyanmethemoglobin StandardSolution by adding 40 mL of the preparedCyanmet-hemoglobin Standard Solution (step 1)to 10.0 mL of the Drabkin’s Solution.3. Prepare working standards by pipetting and mixingthe solutions. (0 to 180 mg/mL, in 60 mg/mL increments)4. Read absorbance of Tubes 2–4 versus Tube 1 asthe reference at 540 nm.5. Record the absorbance values.6. Plot a calibration curve of absorbance valuesversus the cyanmethemoglobin concentration(mg/mL). The curve is linear, passing through theorigin.