method. This is the internationally-recommended method for determining hemoglobin. Principle Blood is diluted in the reagent solution containing potassium cyanide and potassium therysyanide. The latter converts hemoglobin to methemoglobin, which is converted to cyan methemoglobin by potassium cyanide. The absorbance of the solution is then measured in a spectrophotometer at a wavelength of 540 nanometers or in a calorie meter using a yellow green filter. Equipment, pipette. Coloury meter or spectrophotometer, the principle applied for the estimation of hemoglobin is the same in both. The wavelength is available for the estimation differ. Reagents required. Drapkins solution. The pH of the solution must be checked every month and should be maintained between 7 to 7.4. The solution is unstable if exposed to light and can be stored at room temperature in brown borosilicate bottles for several months. The solution should be clear and pale yellow in colour. When measured against water as a blank in a spectrometer at a wavelength of 540 nanometers, the absorbance must be adjusted to 0. If the room temperature is higher than 30 degree centigrade, the solution should be stored in a refrigerator but brought to room temperature before use. The solution must never be frozen. Discard the solution if found to be turbot, if pH is outside range, do not pipette drabkins solution by mouth. Hemoglobin standard solution. This is available commercially at a specific concentration or strength. It is stored in a brown bottle as it is photo sensitive. Exposure to light causes deterioration in the strength of the sand dirt. The concentration of the standard used in this demonstration is 14.8 gram per cent. Sample. Edta whole blood venous sample or capillary sample. Procedure. Take 5 milliliters of drabkins solution in a test tube. Mix the blood sample by gentle inversion and draw 0.02 milliliters of blood into the pipette. Wipe the outer surface of the pipette with a tissue paper to remove excess blood. Place the pipette into the tube containing drabkins solution and slowly expel the blood into the solution. Mix well. And let it stand undisturbed for 15 minutes. Measure the absorbance of this solution at 540 nanometers in a spectrophotometer after adjusting the optical density at 0 by using drabkins solution as blank. Calculate the hemoglobin concentration in the sample by using this formula or a standard curve. Concentration of hemoglobin in sample is equal to absorbance of sample divided by absorbance of standard multiplied by concentration of standard. Preparation of a standard curve will be discussed shortly. Concentration of standard will be available on the kit insert or the vial of a standard solution. Measure its absorbance or OD against a blank of drabkins solution. Preparation of standard curve for hemoglobin estimation by sine methemoglobin method. Let's learn to prepare a calibration curve for hemoglobin estimation by sine methemoglobin method. In a laboratory where several samples are tested in a day, this is an important exercise to standardize the test method. For this you will require drabkins solution and a hemoglobin standard. The concentration of the standard used in this demonstration is 14.8 gram percent. A W. H.O. International reference sine hemoglobin standard is also available commercially as 10 milliliters sealed ampules and is stable for years. Make several dilutions of the standard solution with drabkins solution. In the first test tube take 5 milliliters of drabkins solution in the second test tube mix 1 milliliters of standard with 4 milliliters of drabkins solution. The dilution of the standard is thus 1 in 5 or 0.2. Let's call this the dilution factor. In the third test tube mix 2 milliliters of standard with 3 milliliters of drabkins solution. The dilution factor here will be 0.4. In the fourth test tube mix 3 milliliters of standard with 2 milliliters of drabkins solution. The dilution factor is 0.6. In the fifth test tube mix 4 milliliters of standard with 1 milliliters of drabkins solution. Here the dilution factor is 0.8. In the sixth test tube take 5 milliliters of standard solution only. As this is pure standard dilution factor can be taken as 1. As a strength of the standard is known that is 14.8 gram per desilita. The value of hemoglobin in each dilution can be calculated by multiplying the dilution factor with the strength of the standard. Thus these values will be 0, 2.9, 5.9, 8.9, 11.8, 14.8, round off to the nearest decimal point. Take the test tube with neat drabkins solution and transfer the solution to the qubit. Place the qubit on the spectrophotometer and set the ODE to 0 at a wavelength of 540 nanometers. Now measure the ODE of each dilution in the spectrophotometer against the blank of drabkins solution taking a clean qubit for each dilution. Record the ODE values in a table as shown. This table shows volume of standard in each dilution. Volume of drabkins solution. Dilution factor ODE values and value of hemoglobin in each dilution of standard. This information can be plotted on a graph with the concentration of hemoglobin in grams per desilita in standard plotted on the horizontal axis and corresponding absorbance values plotted against the vertical axis. This graph can be plotted on an excel sheet or manually on a graph paper. The points should be in a straight line that pass through the origin. After ODE of the sample is taken, the corresponding hemoglobin value can be directly read by plotting on the graph. For example, if the ODE of a test sample is 0.32, after plotting it on the graph, the corresponding hemoglobin concentration is 14.8 grams per desilita. A new calibration curve must be prepared whenever the test method, colorimeter or qubit type reagent lot is changed. Advantages of sine methemoglobin method. All forms of hemoglobin except sulfhemoglobin are converted to sine methemoglobin. Visual error is eliminated as no color matching is required. A reliable and stable reference standard is available from WHO for direct comparison. This advantages the test. Potassium cyanide is a poisonous substance and that is the reason why drabkin solution must never be pitted by mouth. The rate of conversion of blood containing carboxyhemoglobin is slowed considerably, prolonging the reaction time to 30 minutes can overcome this problem. Abnormal plasma proteins cause turbidity when blood is diluted with drabkin solution. A high leukocyte count also causes turbidity on dilution of blood. Centrifuging the diluted blood can help overcome the turbidity.